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1
Choma, Christin Teresa. "Structural characterization of the insecticidal protein from Bacillus thuringiensis." Thesis, University of Ottawa (Canada), 1990. http://hdl.handle.net/10393/5624.
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During sporulation, Baccilus thuringiensis subsp. kurstaki produces a crystalline inclusion body which is toxic upon ingestion by susceptible Lepidopteran larvae. The major component of crystals from Lepidopteran-specific subspecies of B. thuringiensis is a 130-kDa protein, protoxin. Following ingestion by susceptible larvae, protoxin is proteolyzed to yield a 58-70 kDa toxic fragment, toxin. In the present study, a simplified procedure was used for isolating and purifying toxin generated by the tryptic digestion of protoxin from B. thuringiensis subsp. kurstaki HD-73. Characterization of this toxin showed that it is derived from the N-terminal half of the protoxin molecule. The toxin is insoluble at neutral pH values but is moderately soluble at alkaline values above pH 9. Application of several spectroscopic and theoretical procedures to the purified toxin showed that the protein is composed of approximately equal amounts of a $\alpha$-helix, $\beta$-sheet and random coil structures. The tertiary structure of toxin was shown to be comprised of two primary domains; these domains correspond to the toxic and specificity (or binding) domains predicted from analysis of protoxin gene nucleotide sequences. Evidence was obtained that at least one additional domain is present as a structural component of the C-terminal specificity domain. Both the toxic moiety within the protoxin molecule and free toxin were found to be unusually resistant to unfolding by chemical denaturants and to proteolysis. In contrast, the C-terminal half of protoxin could be readily unfolded and was extremely susceptible to proteolytic digestion. The unfolded protoxin and unfolded toxin were shown to refold rapidly into their native and biologically active conformations. Evidence was obtained that the conformation of the toxic moiety of protoxin is very similar to the conformation of toxin. Chemical modification of the cysteine and lysine residues in the protoxin did not affect the biological activity of the protein. However, the introduction of positive, negative or neutral groups onto these residues had a large effect on the solubility of the protein. These results, along with the results obtained from the unfolding/folding, studies, strongly indicate that the primary function of the C-terminal half of the protoxin molecule is to promote the formation of a stable crystal.
2
Bietlot, Henri P. "Characterization of the insecticidal crystal protein from Bacillus thuringiensis." Thesis, University of Ottawa (Canada), 1990. http://hdl.handle.net/10393/5668.
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Bacillus thuringiensis produces a crystalline inclusion body composed of a 130-kDa protein which is rendered toxic upon ingestion by lepidoteran larvae. It was shown that proteinases adsorb on the surface of the crystalline body lead to proteolysis of the protein crystal especially on solubilization in alkali. Extensive washing of the protein crystal was shown to remove these proteinases and give a stable preparation. Exposure of the protein crystal to simulated sunlight results in a loss of toxicity and in the destruction of the side-chains of tryptophan, histidine, tyrosine and methionine. Destruction of amino acid side-chains is not the primary cause of the photo-inactivation of the protein crystal. The finding that all the disulfide linkages in the protein crystal are interchain and symmetrical accounts for its alkaline lability and for the high degree of conservation in the primary structure of the cystine-containing regions of the protein from various subspecies. (Abstract shortened by UMI.)
3
Ahmad, Wasim. "Genetics and biochemistry of Bacillus thuringiensis insecticidal protein [?]-endotoxin." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306309.
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Bietlot, Henri P. "Characterization of the insecticidal protein from Bacillus thuringiensis: The importance of DNA-protein interactions." Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/6598.
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Many strains of Bacillus thuringiensis produce a crystalline inclusion during sporulation which is toxic to insect larvae. The major component of crystals toxic to lepidopteran larvae is a 130-kDa protein, the protoxin. Following ingestion by susceptible insect larvae, protoxin is proteolysed by larval gut proteinases to yield a 58-70 kDa toxic fragment, toxin. A procedure was developed to prepare purified toxin for chemical characterization. Toxin generated by bovine trypsin was shown to be composed of the amino acid residues that span position 29-623 of the protoxin. The results obtained from competitive labelling experiments on the protoxin show that the functional groups of the lysine and tyrosine residues do not exhibit regular titration behaviour over the pH range of 7 to 10. These results indicate that the majority of these groups are not free in solution but are involved in inter and intra molecular interactions. During purification by ion exchange chromatography of the bovine generated toxin, it was discovered that the toxin could be separated into two components. One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (T1). Only one major protoxin component was observed, and it was found to be associated with DNA. The DNA from the T2 toxin varied in size from 100 to 300 base pairs, whereas the crystal and the solubilized protoxin contain 20-kilobase DNA as the major DNA component. DNase treatment converted the T2 toxin to the DNA-free T1 toxin. In contrast, the DNA in the crystal and the solubilized protoxin was resistant to DNase digestion and was not dissociated from the protein by 1.5 M NaCl. The protoxin and DNA appeared to elute as a complex with a molecular mass of greater than $2\times 10\sp6$ Da on gel-filtration chromatography. No toxin was generated from the protoxin with trypsin after extensive digestion of the protoxin with DNase or dissociation of the DNA by succinylation of the lysine residues. It is proposed that DNA binds to the carboxyl terminal half of the crystal protein and is essential for maintaining the conformational integrity required for crystal formation and generation of toxin.
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Yamagiwa, Masashi. "THE MODE OF ACTION OF INSECTICIDAL PROTEIN PRODUCED BY BACILLUS THURINGIENSIS." Kyoto University, 2000. http://hdl.handle.net/2433/181060.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第8437号
農博第1121号
新制||農||801(附属図書館)
学位論文||H12||N3394(農学部図書室)
UT51-2000-F341
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 天知 輝夫, 教授 桒原 保正, 教授 加藤 暢夫
学位規則第4条第1項該当
6
Truong, Hung Phuc. "Fate of Cry Toxins from Bacillus thuringiensis in soil." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS210.
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Les propriétés insecticides du Bacillus thuringiensis, découvert par ShigentaneIshiwatari, ont été utilisées pendant des décennies comme biopesticides et cette utilisation a augmenté rapidement en raison de préoccupations au sujet des effets environnementaux négatifs des pesticides chimiques. Actuellement, la toxine Bt dans la forme de biopesticides et des plantes transgéniques Bt peut compléter ou remplacer les pesticides chimiques. Il y a peu d’indication que la toxine Bt a un effet nocif pour l'environnement ou la santé humaine. Néanmoins, il ya des préoccupations que les cultures transgéniques commerciales peuvent avoir des effets néfastes sur l'environnement. Après son introduction dans le sol l'exsudation racinaire et la dégradation des résidus végétaux, la toxine Bt interagit avec les particules de sol. Les interactions de la toxine Bt avec des particules de sol influencent sa mobilité, sa biodisponibilité, sa persistance et sa toxicité.Dans cette étude, nous visons à établir l'importance relative des facteurs biologiques et physico-chimiques dans la détermination de la dynamique des protéines Cry détectables dans les sols, de clarifier si la protéine adsorbée conserve ses propriétés insecticides et d'identifier les propriétés du sol qui déterminent le devenir des protéines Cry dans le sol. Les résultats montrent que les protéines Cry ont une forte affinité sur la surface du sol. Cependant, il y avait peu de relation entre l'affinité pour le sol ou le rendement d'extraction et les propriétés du sol, y compris la teneur en argile, teneur en carbone organique et le pH du sol. Il y avait peu de rapport entre l'affinité et le rendement d'extraction. Les protéines diffèrent à la fois dans leur affinité pour les sols et leurs rendements d'extraction.Une évaluation du rôle du sol et des facteurs environnementaux dans le sort des protéines Cry de la formulation de biopesticides commerciale a montré un déclin rapide de la protéine Cry détectable soumise aux rayons du soleil sous la condition de laboratoire, alors que peu d'effet a été observé dans des conditions de terrain. La demi-vie des protéines dans le sol dans des conditions naturelles était d'environ 1 semaine. Des effets de la température forts ont été observés, mais ils diffèrent pour les biopesticides et la protéine purifiée, indiquant différentes étapes limitantes. Pour le biopesticide, la baisse observée était ralenties par des facteurs biologiques, y compris éventuellement sporulation. En revanche pour des protéines purifiées, augmentation de la température améliorée des changements conformationnels de la protéine adsorbée du sol, conduisant à une fixation et, par conséquent diminué efficacité d'extraction qui a diminué avec le temps. En outre, l'étude de la persistance de diverses protéines Cry dans les sols contrastés a été réalisée par immuno-détection et dosage biologique a montré que la toxine extractible diminue avec incubation allant jusqu'à quatre semaines. L'activité insecticide était toujours maintenue à l'état adsorbé, mais a disparue après deux semaines d'incubation à 25°C. La baisse de la protéine extractible et la toxicité était beaucoup plus faible à 4°C à 25°C. La stérilisation du sol n'a pas eu d'effet significatif sur la persistance de la toxine Cry indiquant que le déclin observé était provoqué par la fixation en fonction du temps de la protéine adsorbée ce qui diminue la quantité de toxine Cry extractable, la dégradation de la protéine par l’activité microbienne jouant un rôle plus mineur.L’exposition des insectes aux protéines Cry sous la forme adsorbé pourrait avoir un impact significatif sur les insectes cibles et même les insectes non cibles, et devrait être plus étudiée afin de déterminer son impact potentielThe insecticidal properties of Bacillus thuringiensis, discovered by Shigentane Ishiwatari, have been used for decades as biopesticides and this use has been increasing rapidly because of concerns about the negative environmental effects of chemical pesticides. Currently, Bt toxin in the form of both biopesticides and Bt transgenic plantsmay supplement or replace chemical pesticide. There is little evidence to demonstrate that Bt toxin has any harmful effect to the environment or to human health. Nevertheless, there are concerns that commercial transgenic crops may have harmful impacts on the environment. After release into soil via root exudation and breakdown of plant residues, Bt toxin interacts with soil particles. The interactions of Bt toxin with soil particles influence its mobility, its bioavailability, its persistence and its toxicity. In this study, we aim to establish the relative importance of biological and physicochemical factors in the determination of the dynamics of detectable Cry proteins in soils, to clarify if adsorbed protein maintains its insecticidal properties and to identify the soil properties that determine the fate of Cry proteins in soil. The results show that Cry proteins have strong affinity on soil surface. However, there was little relationship between affinity for soil or the extraction yield and soil properties including clay content, organic carbon content and soil pH. There was little relationship between the affinity and the extraction yield. The proteins differ in both their affinity for soil and their extraction yields.An assessment of role of soil and environmental factors in the fate of Cry protein from commercial biopesticide formulation showed a rapid decline of detectable Cry protein subjected to direct sunlight under the laboratory condition, whereas, little effect was observed under field conditions. The half-life of proteins in soil under natural conditions was about one week. Strong temperature effects were observed, but theydiffered for biopesticide and purified protein, indicating different limiting steps. For biopesticide, the observed decline was due to biological factors, possibly including sporulation. In contrast for purified proteins, increased temperature enhanced conformationalchanges of the soil-adsorbed protein, leading to fixation and hence extraction efficiency decreased that decreased with time. Moreover, the study of persistence of various Cry proteins in contrasting soils was carried out by immuno-detection and bioassay showed that extractable toxin decreased with incubation of up to four weeks. Insecticidal activity was still retained in the adsorbed state, but lost after two weeks of incubation at 25°C. The decline in extractable protein and toxicity was much lower at 4°C than 25°C. There was no significant effect of soil sterilization to persistence of Cry toxin indicating that decrease in detectable Cry toxin in soil may be time-dependent fixation of adsorbed protein as well as decreasing solubilization in larva midgut, but not microbial breakdown.Exposition to Cry in the adsorbed form could have a significant impact on target and even non target insects and should be investigation to determine the potential impact
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Liu, Yilin. "Investigating insect molecular responses to two plant defense proteins and characterizing a novel insecticidal protein from Arabidopsis." Texas A&M University, 2005. http://hdl.handle.net/1969.1/4855.
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The molecular interaction between plants and insects is dynamic and
multifaceted. We are interested in understanding the molecular mechanism that insects
utilize to overcome plant defense proteins, as well as discovering novel plant insecticidal
proteins. Three projects were developed. First, we evaluated the effects of soybean
cysteine protease inhibitor (soyacystatin N, scN) on the growth and development in
southern corn rootworm. Both subtractive suppressed hybridization (SSH) and cDNA
microarray analyses were used to uncover the changes of gene expression profiles in
southern corn rootworm under the scN challenge. The counterdefense-related genes were
identified, suggesting that southern corn rootworm deployed several regulatory
mechanisms to overcome the dietary scN. Second, to identify and confirm insecticidal
properties of vegetative storage protein 2 in Arabidopsis (AtVSP2), the gene was cloned
and expressed in E.coli. This protein showed acid phosphatase activity. Feeding assay
indicated that AtVSP increased the mortality and delayed the development of two
coleopteran and one dipteran insects. Third, to identify the molecular mechanism of this novel insecticidal protein, P element mutagenesis was utilized to generate AtVSP
resistant mutants (VRs). Two balanced VR mutants and their revertants were generated,
and can be used to further characterize the genetic loci of P element inserted in the
mutants.
8
Clairmont, François. "Structure of the insecticidal crystal protein from Bacillus thuringiensis var. kurstaki HD-73." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0015/NQ48094.pdf.
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Audtho, Mongkon. "Mode of action of Cry2Aa, a Bacillus thuringiensis dual active insecticidal crystal protein /." The Ohio State University, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=osu1486397841221052.
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Wilson, F. Douglas, and Hollis M. Flint. "Field Performance of Cotton Genetically Modified to Express Insecticidal Protein from Bacillus thuringiensis." College of Agriculture, University of Arizona (Tucson, AZ), 1991. http://hdl.handle.net/10150/208376.
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Article is abstract onlyFive transgenic lines of cotton, Gossypium hirsutum L., carrying the delta-endotoxin gene from Bacillus thuringiensis Berl., and two control cultivars, Coker 312 (the parent stock) and MDS1N (an adapted nectoriless line) were evaluated at the Maricopa Agricultural Centerfor resistance to attack by several insect pests and for agronomic properties. The transgenic lines were highly resistant to pink bollworm (PBW), Pectinophora gossypiella (Saunders), as shown by 90% fewer rosetted blooms, 96% fewer PBW recovered from incubated bolls, and 92% less seed damage than in the control cultivars. The transgenic lines were highly resistant to saltmarsh caterpillar, Estigmene acres (Drury), and beet annyworm, Spodoptera exigua (Hbn.), as shown by minimal damage to transgenic leaves and almost complete defoliation of control leaves. The transgenic lines were virtually immune to cotton leafperforator, Bucculatrix thurberiella Busch as shown by no apparent damage to transgenic leaves, and many mines, "horseshoes", and feeding areas on the control leaves. Compared to Coker 312, one transgenic line yielded more lint, and one yielded less. Four transgenic lines had higher lint percentages and all five had smaller bolls and were later maturing than Coker 312. Compared to MD51N, no transgenic line yielded more lint and one yielded less. All five transgenic lines had lower lint percentages, three had smaller bolls, and three were earlier maturing than MDS1N (USDA, ARS, Western Cotton Research Laboratory in cooperation with Monsanto Co. and Arizona Agricultural Experiment Station).
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Nair, Manoj S. "Mechanism of action of insecticidal crystal toxins from Bacillus thuringiensis biophysical and biochemical analyses of the insertion of Cry1A toxins into insect midgut membranes /." Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1218558470.
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Nair, Manoj Sadasivan. "Mechanism of Action of Insecticidal Crystal Toxins from Bacillus thuringiensis: Biophysical and Biochemical Analyses of the Insertion of Cry1A Toxins into Insect Midgut Membranes." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1218558470.
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Lee, Sarah Caroline. "Characterisation of the insecticidal protein toxin complex from Xenorhabdus nematophila PMF1296 : structural and biophysical analysis of the XptA1 component." Thesis, Coventry University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420164.
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Smith, Susan K. "Parasitoid fitness and Cry1Ab : does the insecticidal protein Cry1Ab derived from Bacillus thuringiensis affect the beneficial parasitoid Cotesia marginiventris?" Thesis, University of Southampton, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445492.
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Marucci, Suzana Cristina [UNESP]. "Seleção e caracterização de novos genes vip3A: genes inseticidas de segunda geração de Bacillus thuringiensis." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/92703.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Como uma alternativa para diminuir as agressões constantes que o ecossistema vem sofrendo, devido à grande quantidade de produtos químicos utilizados no controle de pragas, pesquisas envolvendo microrganismos capazes de promover o controle biológico tem se intensificado. Dentre estes microrganismos a bactéria Bacillus thuringiensis tem se destacado. Essa bactéria caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos. Em particular, as proteínas Vip3A, estão em amplo estudo devido a sua especificidade, alto potencial ativo e como alternativa para o controle da resistência de insetos às proteínas Cry. Diante disto, o objetivo deste trabalho foi selecionar, a partir de 1080 isolados de diferentes regiões brasileiras, aqueles portadores de genes vip3A e obter a sequência de nucleotídeos completa dos mesmos. As linhagens padrão B. thuringiensis var. kurstaki HD1, B. thuringiensis var. tolworthi HD125 e o isolado I187 tiveram seus DNAs amplificados com oligonucleotídeos baseados na sequência de genes vip3A, descritos no banco de dados de B. thuringiensis e, a partir dos amplicons obtidos, a sequência completa de nucleotídeos dos mesmos foi determinada, utilizando-se da estratégia de “primer walking”. A proteína Vip3Aa43 (GenBank: [HQ594534]) da linhagem HD1 demonstrou ser 100% idêntica às proteínas Vip3Aa já descritas. Já as proteínas Vip3Aa42 (GenBank: [HQ587048]) da linhagem HD125 e Vip3Ag5 (GenBank: [HQ542193]) do isolado I187 demonstraram similaridade de 99% com as proteínas descritas Vip3Aa35 e Vip3Ag2, respectivamente, demonstrando serem duas novas proteínas Vip3A, devido às substituições de aminoácidos ocorridas. Os três genes vip3A obtidos poderão ser utilizados na produção de plantas Bt, piramidadas ou não, visando ao manejo da resistência dos insetos praga
As an alternative to decrease the constant aggressions that the ecosystem has suffered due to the large amount of chemical products used in pest control, researches involving microorganisms able to promoting biological control have been intensified. Among these microorganisms the bacterium Bacillus thuringiensis has been stood out. This bacterium is characterized by the production of toxic proteins to representatives of several insect orders. In particular, the Vip3A proteins are in large study due to its specificity, and high active potential as an alternative to control of insect's resistance to Cry proteins. According to this, the aim of this work was to select from 1080 isolates in different Brazilian regions, those carrying vip3A genes and obtain the complete nucleotide sequence of the same. The standard strains B. thuringiensis var. kurstaki HD1, B. thuringiensis var. tolworthi HD125 and the isolate I187 had their DNA amplified with primers based on vip3A gene sequence described in database of B. thuringiensis, and from the amplicons obtained, the full sequence of nucleotides was determined, by the use of the strategy of primer walking. The protein Vip3Aa43 (GenBank: [HQ594534]) of HD1 strain showed to be 100% identical to Vip3Aa proteins already described. However, the proteins Vip3Aa42 (GenBank: [HQ587048]) of HD125 strain and Vip3Ag5 (GenBank: [HQ542193]) of the isolate I187 showed 99% similarity with the Vip3Aa35 and Vip3Ag2 proteins described, respectively, showing been two new Vip3A proteins due to amino acid substitutions occurred. The three vip3A genes obtained can be used in the production of Bt crops, pyramidal or not, aiming to resistance management of pest insects
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Duarte, Boaventura Debora [Verfasser]. "Characterization of mechanisms of resistance in Spodoptera frugiperda to synthetic insecticides and insecticidal proteins / Debora Duarte Boaventura." Bonn : Universitäts- und Landesbibliothek Bonn, 2021. http://d-nb.info/1231911123/34.
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Lohmann, Tiago Rodrigo [UNESP]. "Interações da proteína Vip3Aa20, Diatraea saccharalis (Fabricius) e seus parasitóides, Cotesia flavipes (Cameron) e Trichogramma galloi Zucchi." Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/91374.
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O objetivo do presente estudo foi avaliar o efeito da proteína Vip3Aa20, originária da bactéria Bacillus thuringiensis Berliner, sobre a broca-do-colmo Diatraea saccharalis (Fabricius) e dois de seus parasitóides: o parasitóide larval Cotesia flavipes (Cameron) e o parasitóide de ovos Trichogramma galloi Zucchi. D. saccharalis mostrou-se suscetível à proteína, apresentando efeitos letais e subletais. Foram afetadas pela proteína as características mortalidade larval, duração do período larval, número de ínstares larvais e peso de larvas, enquanto que a mortalidade pupal e a duração do período pupal não foram afetadas e o peso de pupas apresentou resultados divergentes entre os bioensaios conduzidos. Para os parasitóides, avaliaram-se os efeitos da exposição direta (ingestão da proteína pelos adultos) e indireta (ingestão da proteína por D. saccharalis e posterior parasitismo). Em C. flavipes, não foram observados efeitos pela exposição direta, enquanto que na exposição indireta ocorreu efeito negativo sobre as características peso da massa de casulos e peso do adulto. Estes efeitos podem ser associados ao efeito mediado pelo hospedeiro. Em T. galloi, não foram observados efeitos da proteína Vip3Aa20 sobre os parasitóides, tanto na exposição direta como na indireta
The aim of this study was to evaluate the effect of Vip3Aa20 protein, originating from the Bacillus thuringiensis Berliner bacterium, on sugarcane borer Diatraea saccharalis (Fabricius) and two of its parasitoids: larval parasitoid Cotesia flavipes (Cameron) and egg parasitoid Trichogramma galloi Zucchi. D. saccharalis was susceptible to protein, with lethal and sublethal effects. Larval mortality, larval period, number of instars and larval weight were affected by the protein, while pupal mortality and pupal period were not affected and pupal weight presented discrepant results between bioassays conducted. For the parasitoids, direct (protein ingested by adults) and indirect (protein ingested by sugarcane borer with later parasitism) exposure were evaluated. In C. flavipes, no effects were observed by direct exposure, while in indirect exposure negative effects occurred on the cocoons weight and adult weight. These effects may be associated with the effect mediated by the host. No effects were verified on T. galloi when this species was direct or indirectly exposed to Vip3Aa20 protein
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Back, Emma Jane. "Insecticidal fusion proteins for the control of Coleopteran pests." Thesis, Durham University, 2011. http://etheses.dur.ac.uk/3283/.
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Fusion proteins containing a toxin fused to a carrier domain which directs transport across the insect gut epithelium have been shown to be effective orally active insecticides. Expression of functional recombinant fusion proteins comprising of snowdrop lectin (Galanthus nivalis agglutinin; GNA) fused to toxins from Indian red scorpion (Mesobuthus tamulus toxin; ButaIT) and Blue Mountains funnel-web spider (Hadronyche versuta toxins; ω-ACTX-Hv1a (ω-ACTX); κ-ACTX-Hv1c (κ-ACTX)) was carried out in both yeast (Pichia pastoris) and plant (Arabidopsis thaliana) expression systems. Addition of purification tags, altering the design of assembly of the fusion protein and point mutation of toxin sequence were all investigated to improve yield and reduce proteolytic cleavage during expression and purification. Recombinant proteins were assayed for oral toxicity against T. castaneum as a model coleopteran species. Fusion proteins incorporating ButaIT and ω-ACTX toxins showed toxicity ranging from complete mortality when fed at 1mg g-1 ((his)6-GNA-ω-ACTX and ω-ACTX-GNA-(his)6) to 65% mortality when fed at 2mg g-1 (ButaIT-GNA-(his)6). Fusion proteins incorporating κ-ACTX and GNA were shown to be non-toxic despite individual components being functional. Lack of toxicity was due to high proteolytic cleavage in the insect gut environment. Data was obtained to support the use of Tribolium as a model for wireworm (Agriotes spp.), serious pests of potatoes in the UK. Selected fusion proteins were expressed in transgenic Arabidopsis. Expression for ButaIT-GNA as a fusion polypeptide was readily detectable in transformants with estimated levels of expression of approx. 0.15% total soluble protein in leaf tissue. When plants expressing ButaIT-GNA fusion protein were fed to larvae of the tomato moth (Lacanobia oleracea) the fusion protein was shown to be fully functional with levels of toxicity comparable to that seen in previous artificial diet bioassays. ω-ACTX based constructs expressed in Arabidopsis were subject to high levels of proteolytic cleavage in planta and so were not assayed for toxicity.
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Lohmann, Tiago Rodrigo. "Interações da proteína Vip3Aa20, Diatraea saccharalis (Fabricius) e seus parasitóides, Cotesia flavipes (Cameron) e Trichogramma galloi Zucchi /." Jaboticabal : [s.n.], 2011. http://hdl.handle.net/11449/91374.
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Orientador: Odair Aparecido FernandesBanca: Celso Omoto
Banca: Ricardo Antonio Polanczyk
Resumo: O objetivo do presente estudo foi avaliar o efeito da proteína Vip3Aa20, originária da bactéria Bacillus thuringiensis Berliner, sobre a broca-do-colmo Diatraea saccharalis (Fabricius) e dois de seus parasitóides: o parasitóide larval Cotesia flavipes (Cameron) e o parasitóide de ovos Trichogramma galloi Zucchi. D. saccharalis mostrou-se suscetível à proteína, apresentando efeitos letais e subletais. Foram afetadas pela proteína as características mortalidade larval, duração do período larval, número de ínstares larvais e peso de larvas, enquanto que a mortalidade pupal e a duração do período pupal não foram afetadas e o peso de pupas apresentou resultados divergentes entre os bioensaios conduzidos. Para os parasitóides, avaliaram-se os efeitos da exposição direta (ingestão da proteína pelos adultos) e indireta (ingestão da proteína por D. saccharalis e posterior parasitismo). Em C. flavipes, não foram observados efeitos pela exposição direta, enquanto que na exposição indireta ocorreu efeito negativo sobre as características peso da massa de casulos e peso do adulto. Estes efeitos podem ser associados ao efeito mediado pelo hospedeiro. Em T. galloi, não foram observados efeitos da proteína Vip3Aa20 sobre os parasitóides, tanto na exposição direta como na indireta
Abstract: The aim of this study was to evaluate the effect of Vip3Aa20 protein, originating from the Bacillus thuringiensis Berliner bacterium, on sugarcane borer Diatraea saccharalis (Fabricius) and two of its parasitoids: larval parasitoid Cotesia flavipes (Cameron) and egg parasitoid Trichogramma galloi Zucchi. D. saccharalis was susceptible to protein, with lethal and sublethal effects. Larval mortality, larval period, number of instars and larval weight were affected by the protein, while pupal mortality and pupal period were not affected and pupal weight presented discrepant results between bioassays conducted. For the parasitoids, direct (protein ingested by adults) and indirect (protein ingested by sugarcane borer with later parasitism) exposure were evaluated. In C. flavipes, no effects were observed by direct exposure, while in indirect exposure negative effects occurred on the cocoons weight and adult weight. These effects may be associated with the effect mediated by the host. No effects were verified on T. galloi when this species was direct or indirectly exposed to Vip3Aa20 protein
Mestre
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Powell, Kevin Steven. "Antimetabolic effects of plant proteins on homopteran insect pests." Thesis, Durham University, 1993. http://etheses.dur.ac.uk/5757/.
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Homopteran insect pests can cause severe economical damage to crop plants by both direct physical means and as vectors of plant viral diseases. They are notoriously difficult insects to control by conventional methods, primarily due to their ability to evolve resistance-breaking biotypes within a relatively short time period. The production of genetically modified crop plants, expressing insecticidal genes, offers a novel method of control for a wide range of insect species. Once suitable gene products, such as plant- derived proteins, have been identified as having insecticidal effect against specific insects in vitro, their effect can be determined in vivo by expressing the relevant gene in transgenic plants. Insect feeding trials were carried out to determine the effects of incorporating a range of plant-derived proteins into artificial diets fed to planthopper, leafliopper and aphid pests and to aphids in planta. The lectins Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), and the enzyme soybean lipoxygenase (LPO) were shown to exhibit significant antimetabolic effects towards first and third instar nymphs of rice brown planthopper (Nilaparvata lugens Stal) when incorporated into artificial diet at 0 1% {w/v}, 0-1% (w/v) and 0 08% {w/v} levels respectively. The lectin GNA was also shown to exhibit a significant antimetabolic effect towards third instar nymphs of the rice green leafhopper (Nephottetix cinciteps Uhler) and the peach potato aphid {Myzus persicae Sulzer). A number of inert proteins, lectins, protein inhibitors and enzymes also tested showed relatively little or no effect towards both insects. The mechanism of action of all three effective proteins was examined using BPH as a model insect. As judged by honeydew production, the proteins all had a deterrent effect on insect feeding. However, subsequent toxic effects are also indicated. When fed sub-optimal concentrations of effective proteins in combination no synergistic or additive effects were observed, indicating that pyramiding the genes of these effective proteins would be of no advantage in protecting the crop against BPH.
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Jabeur, Rania. "Identification et caractérisation de protéines ayant des propriétés entomotoxiques contre deux principaux ravageurs du maïs." Thesis, Université de Montpellier (2022-….), 2022. http://www.theses.fr/2022UMONG004.
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Du fait de leur grande adaptabilité aux méthodes de lutte, les insectes demeurent une menace constante pour les plantes cultivées. Le maïs (Zea mays L.) est la troisième culture la plus importante dans le monde après le riz et le blé. La chrysomèle des racines du maïs (WCR) Diabrotica virgifera virgifera (LeConte, 1868) et la chenille légionnaire d'automne (FAW) Spodoptera frugiperda (J.E. Smith, 1797) sont parmi les ravageurs les plus importants du maïs et sont responsables de fortes pertes de rendement chaque année. Le maïs Bt exprimant des protéines insecticides provenant de Bacillus thuringiensis a été pendant longtemps une bonne alternative aux pesticides. Cependant, leur utilisation massive a conduit à l'apparition de populations de ravageurs résistantes, d'où la nécessité de trouver de nouvelles molécules et stratégies biotechnologiques ou de contrôle biologique pour gérer durablement ces insectes. Mes travaux de thèse s’inscrivent dans cet objectif et visent à trouver de nouvelles solutions contre WCR et FAW. Dans la première partie, je me suis particulièrement intéressée à une protéine binaire appelée GDI0005A/GDI0006A identifié dans un criblage contre des larves de WCR. Ces protéines ont été isolées de la bactérie Chryseobacterium arthrospharae et ont montré une activité insecticide, lorsqu'elles sont exprimées sous forme de protéines recombinantes, contre des population de WCR sensibles et résistantes aux protéines commerciales Cry3Bb1 et Gpp34Ab1/Tpp35Ab1. Ces résultats suggèrent que ces nouvelles protéines insecticides pourraient avoir des modes d’action différents de ceux des protéines Bt actuellement déployées et donc un nouveau mode d'action. Le deuxième membre de la protéine binaire (GDI0006A) est une lipoprotéine membranaire que j’ai purifiée partiellement. Cette propriété a été un facteur limitant pour évaluer le niveau d'activité de la protéine GDI0005A/GDI0006A. Nous avons essayé d'exprimer la protéine binaire en plante en utilisant deux systèmes d'expression : transitoire et stable. Les deux ont montré une faible accumulation de GDI0006A entraînant une perte de l'activité par rapport au essais au labo avec des lysats bactériens. Nous avons également démontré que des cultures de la bactérie d’origine C. arthrosphaerae avait une activité contre WCR ce qui en fait un agent de biocontrôle potentiel contre les chrysomèles.La deuxième partie a été consacrée à l'étude de l’éventuelle synergie entre Junonia coenia densovirus (JcDV) et une protéine insecticide Bt Cry2Ab2 dans une perspective de les combiner pour des applications de biocontrôle. Les deux sont connus pour infecter FAW et perturber la perméabilité intestinale de ce ravageur. Cependant, lorsqu’ils ont été testés ensemble avec des approches ex vivo et in vivo, nous avons plutôt remarqué un antagonisme suggérant une compétition avec les mêmes récepteurs intestinaux. Nous avons aussi vérifié la faisabilité de l'utilisation de VP4, la protéine majoritaire de la capside de JcDV, dans une approche biotechnologique pour améliorer l'activité d'autres protéines insecticides qui ciblent l’intestin. Les résultats préliminaires ont montré une expression importante de VP4 dans les feuilles de Nicotiana benthamiana sans aucun signe de phytotoxicité. Nous avons aussi mis en évidence la capacité de VP4 à s'auto-assembler en pseudo-particules virales (VLP) dans la plante. Ces VLP-VP4 ont été capables de pénétrer dans les cellules intestinales de FAW lorsqu'elles ont été testées ex vivo. Cette preuve de concept sera utile pour explorer les synergies possibles avecdes protéines insecticides qui affectent l’intestin dans le but de créer de nouvelles variétés de maïs transgéniques résistants à FAWDue to their great adaptability to management strategies, insect pests are a constant threat to crops. Maize is the third most important staple crop in the world after wheat and rice. The western corn rootworm (WCR) Diabrotica virgifera virgifera (LeConte, 1868) and the fall armyworm (FAW) Spodoptera frugiperda (J.E. Smith, 1797) are among the most serious pests of maize and are responsible for massive yield losses every year. Bt maize expressing insecticidal proteins from Bacillus thuringiensis has long been a good alternative to chemicals. However, their overuse has led to the development of resistant populations. Hence, the need to find new compounds and strategies for sustainable control of these insects. In this PhD project, we aimed to develop biotech and biocontrol solutions for both WCR and FAW. In the first part, I was particularly interested in the study of binary insecticidal proteins named GDI0005A/GDI0006A identified by screening bacterial genomes and test against WCR larvae. These proteins were identified in Chryseobacterium arthrosphaerae genome. Heterologous expression of the two components was performed in E. coli system. One of the two components (GDI0006A) was a membrane lipoprotein, and resulted in low amounts successfully purified. Nonetheless, the combination of the two proteins was active against WCR including WCR resistant to the currently marketed Cry3Bb1 and Gpp34Ab1/Tpp35Ab1 proteins. These results suggest that the new binary toxin may have different binding sites and thus a distinct mode of action compared to commercial toxins. The binary proteins were then expressed successfully in plant expression systems. However, the expression at low level of GDI0006A lead to no significant activity in planta. Finally, we tested the bacteria C. arthrospharae and show that it had activity in vitro against WCR, making of it a potential BCA against WCR.The second part was devoted to studying the possible synergy between Junonia coenia densovirus (JcDV) and a Bt insecticidal protein Cry2Ab2 for biocontrol applications. Both are known to infect FAW and disturb the midgut permeability. However, when tested together either with ex vivo or in vivo approaches, we have noticed an antagonism suggesting a competition for the same midgut receptors. We also checked the feasibility of using VP4, the major capsid protein of JcDV in a biotech approach to enhance the activity of other gut binding insecticidal proteins. The preliminary results have shown a good expression of VP4 in Nicotiana benthamiana leaves without any sign of phytotoxicity. We demonstrated the ability of VP4 to self-assemble into virus-like particles (VLPs) in plant. When tested ex vivo, VLP-VP4 were able to penetrate the FAW intestinal cells. This proof of concept will be useful to explore the possible synergies with gut-binding insecticidal proteins to create new generations of transgenic maize resistant to FAW
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Batista, Adelina Braga. "Potential fungicidal and insecticidal proteins present in seeds Dioclea megacarpa Rolfe." Universidade Federal do CearÃ, 2009. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=3789.
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CoordenaÃÃo de AperfeiÃoamento de NÃvel SuperiorDioclea megacarpa Rolfe is the correct synonym for D. relexa var. grandiflora. This species belongs to Fabaceae, the legume family. Previous studies, realized in our research group, showed the presence of active proteins for several phytopathogenic fungi in the seeds of this species, among of them Aspergillus niger. Thus, the present work was proposed with the objective of determining the bioactivity of protein(s) from D.megacarpa seeds against fungi, leading to its/their purification and partial characterization and further investigation of its/their action mechanism. Another approach of this work it was analyze the insecticidal potential of glucose/mannose-specific lectin, isolated from D.megacarpa seeds (Moreira et al., 1983), against the cowpea bruchid Callosobruchus maculatus. For this, seed flour was placed in contact with 0.15 M NaCl (1:5, w/w), followed by stirring for 3 h, filtration through a nylon cloth, re-extraction for 1 h, centrifugation at 11,500 x g, for 30 min, at 4 oC. The supernatant obtained, named total extract, showed antifungal activity against A. niger and presented several bioactive proteins, including lectin (129.27 UH/mgP, using trypsinized rabbit erythrocytes), trypsin inhibitor (18.91 mg de tripsina inibida/gF), urease (47.50 U/gF), toxin (LD50 119.60 mgP/Kg mice body weight ), chitinase (1.66 nKat/mgP) e β-1,3-glucanase (0.55 nKat/mgP). On the other hand, peroxidasic and proteolytic activities were not detected. For purification of antifungal principle, several chromatographies were performed on Sephadex G-50, Chitin and Resource Q, this last connected to an FPLC system. The purified antifungal protein, named Dm-PAF, with apparent molecular mass of 67-68 kDa (SDS-PAGE), did not show any haemagglutinating or chitinolytic activity and presented its NH2-terminal sequence blocked. Dm-PAF, at a very low concentration (0.015 ÂgP/ÂL), it was able to inhibit the growth of Saccharomyces cerevisiae and Candida tropicalis yeasts. The investigation of the antifungal action mechanism excluded the possibility of interaction between Dm-PAF and H+-ATPase pumps. In addition, the glucose/mannose-specific lectin, obtained from Sephadex G-50 column, exhibited a potent insecticidal activity against C. maculatus, interfering in important parameters related to life cycle of this insect. These data show to be the D. megacarpa seeds a rich source of biologically interesting proteins, possibly involved in the defense mechanism of plants
Dioclea megacarpa Rolfe à usada como sinonÃmia de D. relexa var. grandiflora, uma espÃcie pertencente à famÃlia Fabaceae (Leguminosae). Estudos prÃvios, realizados por nosso grupo de pesquisa, demonstraram a presenÃa em suas sementes de proteÃnas ativas contra fungos fitopatogÃnicos, dentre esses Aspergillus niger. Assim, o presente trabalho foi proposto no intuito de avaliar a bioatividade de proteÃnas de sementes de D. megacarpa contra fungos, conduzindo à sua purificaÃÃo e caracterizaÃÃo parcial, bem como à investigaÃÃo de seu mecanismo de aÃÃo. Outro objetivo deste trabalho foi examinar o potencial inseticida da lectina com especificidade por glucose-manose, isolada de sementes de D. megacarpa (Moreira et al., 1983), contra o bruquÃdeo do feijÃo-caupi Callosobruchus maculatus. Para tanto, farinha de sementes foi posta em contato com NaCl 0,15 M (1:5, p/v), seguida de agitaÃÃo contÃnua por 3 h, filtraÃÃo em pano de trama fina, re-extraÃÃo por 1 h e centrifugaÃÃo a 11.500 x g, 30 min, 4 oC. O sobrenadante obtido, denominado de extrato total, se mostrou ativo contra A. niger e apresentou vÃrias proteÃnas bioativas, compreendendo lectina (129,27 UH/mgP, com eritrÃcitos tripsinizados de coelho), inibidor de tripsina (18,91 mg de tripsina inibida/gF), urease (47,50 U/gF), toxina (DL50 119,60 mgP/Kg de peso corpÃreo de camundongo), quitinase (1,66 nKat/mgP) e β-1,3-glucanase (0,55 nKat/mgP). Por outro lado, atividades peroxidÃsica e proteolÃtica nÃo foram detectadas. Para purificaÃÃo da proteÃna antifÃngica, foram realizadas cromatografias em matrizes de Sephadex G-50, Quitina e Resource-Q, essa Ãltima acoplada ao sistema de FPLC. A proteÃna antifÃngica purificada de sementes de D. megacarpa, denominada de Dm-PAF, com massa molecular aparente de 67-68 kDa (PAGE-SDS), nÃo mostrou atividades hemaglutinante e quitinÃsica e apresentou sua seqÃÃncia NH2-terminal bloqueada. Dm-PAF, em concentraÃÃo baixÃssima (0,015 ÂgP/ÂL), se mostrou capaz de inibir o crescimento das leveduras Saccharomyces cerevisiae e Candida tropicalis, cuja investigaÃÃo do mecanismo de aÃÃo nÃo revelou envolvimento dessa proteÃna com bombas de H+-ATPase. Em adiÃÃo, a lectina ligante a glucose-manose, obtida na cromatografia em Sephadex G-50, mostrou potente atividade inseticida contra C. maculatus, interferindo em parÃmetros importantes relacionados ao ciclo de vida do inseto. Os dados apresentados mostram as sementes de D. megacarpa como uma rica fonte de proteÃnas interessantes, possivelmente envolvidas no mecanismo de defesa das plantas
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Knight, Peter J. K. "The biochemistry and molecular biology of insecticidal proteins and their cellular receptors." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388536.
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Yang, Sheng. "Exploitation of small cysteine-rich spider protein toxins as bio-insecticides." Thesis, Durham University, 2015. http://etheses.dur.ac.uk/11035/.
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Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered bio-pesticides. Here, some small cysteine-rich protein toxins were evaluated as insecticides, including δ-amaurobitoxin-Pl1a (Pl1a) from tangled nest spider (Pireneitega luctuosa), ω-atracotoxin-Hv1a (Hv1a) from funnel web spider (Hadronyche versuta) and κ-theraphotoxin-Ec2a (Ec2a) from Eucratoscelus constrictus, which target insect voltage-gated sodium channels, calcium activated potassium channels and voltage-regulated potassium channels, respectively. Recombinant proteins were produced using the yeast Pichia pastoris as expression host, by combining the coding sequences of the toxin with that of snowdrop lectin ("carrier"), that can deliver these toxins to the central nervous system of the target pest. Experimental results showed the toxins alone had limited or even no activities without being fused to the N-terminal of snowdrop lectin "carrier". Further, fusion of toxins to proteins other than snowdrop lectin also gave products with low or no biological activity. The absence of biological activity suggested that the toxin protein was not folding properly when expressed without fusion to the snowdrop lectin carrier, which meant GNA could not only direct transport of the toxins across the insect gut as a carrier, but also can help toxins to achieve correct folding. For example, the toxin Pl1a and a Pl1a/GNA fusion protein both caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, but the Pl1a/GNA fusion protein was approximately 6 times as effective as recombinant Pl1a on a molar basis. Pl1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the Pl1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th - 6th instar larvae. To attempt to further improve the folding of recombinant fusion proteins, the predicted Pro-regions of toxins, between the signal peptide and the final mature sequence of the protein were examined. Inclusion of the Pro-region in the expression construct was hypothesised to result in improved folding of the toxin when expressed in P. pastoris. The results proved that the new type fusion protein (Pro-region/toxin/GNA) had much higher biological activity than toxins alone and higher activity than toxin/GNA fusion proteins. In addition, the Pro-region was successfully removed from the Pro-region/toxin/GNA proteins after expression. For example, the LD50 of Pro-Hv1a/GNA was decreased by 12 fold compared to Hv1a/GNA when injected into Mamestra brassicae larvae of different stages of development. Increased biological activity of Pro-Hv1a/GNA when compared to Hv1a/GNA was also observed when the proteins were injected into slugs. The increased biological activity of Pro-Hv1a/GNA on injection was also observed as increased oral toxicity of the fusion protein to insects. A single dose (20 μg) of fusion protein Hv1a/GNA caused no mortality to 5th instar larvae of M. brassicae, or 30% mortality to 3rd instar larvae; in contrast, 20 μg Pro-Hv1a/GNA caused 30% mortality to 5th instar larvae, and 90% mortality to 3rd instar larvae. Fusion proteins have the potential to be a new class of bio-pesticides for commercial application and have potential uses in complementing or replacing existing pesticides. Insecticide-resistant strains of peach potato aphid (Myzus persicae), designated "kdr", "super-kdr" and "kdr+super-kdr" contain mutations in the voltage-gated sodium channel (NaCh). Pl1a/GNA and Pro-Hv1a/GNA fusion proteins have the LC50 values of 0.35 and 0.19 mg ml-1 when fed to wild-type M. persicae. For insecticide-resistant aphids, the LC50 for the Pl1a/GNA fusion protein, which targets NaCh, was increased by 2-6 fold correlating with pyrethroid resistance (wild-type < kdr < super-kdr < kdr+super-kdr strains). In contrast, the LC50 for the Pro-Hv1a/GNA, which targets calcium channels, showed limited correlation with pyrethroid resistance. Therefore, mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium channel-specific toxin, despite differences in ligard-channel interactions. This may be because changes to the spatial structure of domain II as a result of these mutations presumably also disturb the binding of Pl1a to receptor site 4, in domain II of sodium ion channel. However, mutations in the sodium channel do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could delay resistance development in M. persicae.
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Mitchell, Judith K. "Effects of ultraviolet and visible radiation on the insecticidal activity of the spores and crystals of Bacillus thuringiensis." Thesis, Cranfield University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278753.
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Ahmed, Sohail. "Intracellular proteases and mechanisms of insecticide resistance in strains of Musca domestica L." Thesis, University of Newcastle Upon Tyne, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299632.
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Alves, Meire de Cássia [UNESP]. "Identificação e caracterização de genes cry3, vip1, vip2 E vip1/vip2 em isolados de Bacillus thuringiensis E toxicidade em larvas de Anthonomus grandis (Boheman, 1883) (Coleoptera: curculionidae)." Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/92689.
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O controle biológico utilizando microrganismos vem sendo estudado como uma alternativa ao uso de agroquímicos. Estes pesticidas poluem o ambiente, além de serem tóxicos a diversas espécies vegetais e animais. Diante disso, a bactéria Bacillus thuringiensis é um importante entomopatógeno que vem sendo utilizado na agricultura pelo fato de secretar proteínas que são tóxicas a insetos pertencentes a diversas ordens. Dentre os diversos genes de B. thuringiensis que codificam proteínas tóxicas, as classes vip1, vip2 e cry3 destacam-se como alternativa para o controle de insetos-praga da ordem Coleoptera. O presente trabalho objetivou a identificação e caracterização molecular, por meio da técnica de PCR-RFLP, dos genes cry3, vip1, vip2 e vip1/vip2 em uma coleção de B. thuringiensis, quanto a possíveis polimorfismos existentes, procurando relacioná-los à toxicidade em larvas de Anthonomus grandis. Da análise de 1078 isolados de B. thuringiensis, foram encontrados 151 isolados positivos para os genes em estudo e 14 perfis polimórficos, indicando a presença de subclasses destes genes. Os resultados obtidos nos ensaios de toxicidade indicam que os polimorfismos gênicos podem apresentar interferência na toxicidade das proteínas, sendo que a toxina Cry3 apresentou uma maior efetividade na mortalidade em larvas de A. grandis. Os resultados também evidenciaram que a PCR-RFLP mostrou-se apropriada para a detecção da variabilidade genética em B. thuringiensis, permitindo a identificação de haplótipos
Biological control using microorganisms is being studied as an alternative to the use of agrochemicals. These pesticides pollute the environment, being toxic to many species of plants and animals. For these reasons, the bacterium Bacillus thuringiensis is an important entomopathogen that has been used in agriculture, once it produces proteins that are toxic to many target insect orders. Among several genes from B. thuringiensis that encode toxic proteins there are vip1, vip2 and cry3 classes that stand as an alternative for controlling insect pests belonging the Coleoptera order. The objective of this work was to identify and characterize molecularly, through PCR-RFLP, the genes cry3, vip1, vip2 and vip1/vip2 in a collection of B. thuringiensis, for possible polymorphisms exist, trying to relate them to the toxicity in Anthonomus grandis larvae. Analysis of 1078 isolates of B. thuringiensis, were found 151 positive isolates for the genes under study and 10 polymorphic profiles, which indicate the presence of subclasses of the genes. These results obtained in tests toxicity indicate that polymorphisms gene may have interference with the toxicity of the protein, and the toxin Cry3 showed a greater effectiveness in mortality in A. grandis larvae. These results also showed that the PCR-RFLP proved to be suitable for the detection of genetic variability in B. thuringiensis, allowing the identification of haplotypes
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Saville, Giles P. "Improving the efficacy of baculovirus insecticides by genetic manipulation of the AcMNPV Chitinase gene." Thesis, Oxford Brookes University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364878.
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Bughio, Fazalullah M. "Resistance in rice grains and feeding by insecticide-resistant and susceptible strains of Tribolium and Sitophilus species." Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327274.
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Van, Jaarsveld Martha Johanna. "Geographic susceptibility of Helicoverpa armigera (Lepidoptera: Noctuidae) to insecticidal proteins in Bt-cotton in South Africa." Thesis, Rhodes University, 2004. http://hdl.handle.net/10962/d1005387.
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Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) (African bollworm) is a typical noctuid with a very catholic taste in food plants and whose larvae feed on a wide range of cultivated and wild plants. It has been identified as the most polyphagous and injurious pest in South Africa. Helicoverpa armigera is also a key pest of cotton in many parts of the world. This key pest requires extensive control as it adversely effects yield and has built up resistance to synthetic pyrethroid insecticides. Cotton is an important crop produced by commercial and small-scale farmers in South Africa. The local demand for cotton has not been exceeded yet, but to satisfy a demanding market, pest control costs play an important role in cotton production. The threat of an insect pest that has already shown resistance prompted the present study to investigate the possibility of resistance to Bt-cotton. Genetically engineered or Bt-cotton was introduced commercially in 1996 in South Africa. All Bt-cotton plants contain one or more foreign genes derived from the soil-dwelling bacterium, Bacillus thuringiensis (Berliner), which produces protein crystals. These crystals were isolated and transferred into the genome of a cotton plant resulting in the plant producing it’s own protein insecticide. In 1998, Monsanto (Pty) Ltd requested research into the geographic susceptibility of H. armigera to the insecticidal proteins in Bt-cotton in SA. Laboratory reared and field sampled populations of H. armigera were exposed to a diet mixed with various baseline concentrations of the Bt-gene Cry1Ac freeze dried protein. This study also determined the performance of H. armigera and Spodoptera littoralis (Boisduval) on different Bt-cotton field cultivars containing different Cry-protein genes. Results obtained indicated a significant difference in susceptibility in two field populations of H. armigera to the Bt-protein Cry1Ac, even though the LD50,s in the 2003 season did not indicate resistance. Bt-cotton cultivar 15985 BX controlled H. armigera and S. littoralis larvae, the best followed in descending order by cultivar 15985 X, 15985 B and DP50 B. Results on H. armigera also indicated that the Cry-proteins in the plant parts of the different cultivars did not diminish as the season progressed. The Bt-cotton cultivars induced retarded growth of larvae, due to either a repellent effect or lack of feeding by larvae. Widespread adoption of Bt-cotton by South African farmers led to regional declines in bollworm populations, reduced insecticide use, and increased yields. Genetically modified crops therefore contribute to a cost effective, sustainable, productive and efficient form of agriculture, with a resultant positive impact on the environment. As the market for commercial Bt-cotton in South Africa expands, it is recommended that a monitoring programme for potential resistant genes in H. armigera should be implemented at least every 2 - 3 years. This will ensure that effective resistance management strategies are utilised. Coupled with this are the Biosafety Risks regarding the effect of new proteins expressed in transgenic plants, which require further studies.
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Howe, David S. "Biochemical basis of insect resistance in winged bean (Psophocarpus tetragonolbus) : characterisation of insecticidal proteins and their encoding genes." Thesis, Durham University, 1991. http://etheses.dur.ac.uk/6102/.
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Many pulses and beans grown for human comsumption are susceptible to insect attack. Winged bean, a high protein crop of the tropics, yield seeds which appear to be immune to infestation by the storage bruchid Callosobruchus maculatus. In this thesis the biochemical basis of this resisitance was investigated. Insect bio-assays were carried out in which protein fractions from seeds of winged bean were incorporated at a range of concentrations into artificial seeds, and their effects upon the development of C.maculatus determined. Both albumin and globulin fractions were toxic to the developing larvae and their toxicity correlated with their haemagglutination activity. Assay of psophocarpin fractions A, B and C found the fraction psophocarpin B to be most insecticidal. On further purification this fraction yielded two lectin fractions and a protease inhibitor fraction. Purified basic lectin was highly insecticidal to C. maculatus larvae with an LC(_50) value of 0.35%. The physiological level of this protein in winged bean seeds is sufficient to account for their resistance to attack by C maculatus. Winged bean trypsin inhibitor was also purified and tested in artificial seeds against C maculatus. However, even at concentrations in excess of twice the physiological concentration it had no deleterious effects upon development. Winged bean protein fractions, incorporated in artificial diets, proved toxic to the Lepidopteran pests Heliothis virescens and Spodoptera littoralis in bio-assays, but it appeared that the basic lectin was not responsible for toxicity towards these insects. Attempts to clone the gene encoding the winged bean basic lectin were made by constructing cDNA and genomic libraries, and heterologous lectin genes from soybean and Phaseolus were investigated as possible probes for the basic lectin gene. Purification of the basic lectin B3 and sequencing of 44% of its primary protein structure, along with comparisons with other legume lectin sequences allowed the synthesis of oligonucleotide primers for use in polymerase chain reaction experiments. However, all the PGR products obtained were shown to be the result of non-specific amplification. Further work needed to obtain the basic lectin gene is discussed.
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Haider, Syed Tanveer. "Host-range specificity of a Bacillus thuringiensis (Bt) toxin as analyzed by protein engineering /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/5269.
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Lemes, Ana Rita Nunes [UNESP]. "Proteínas Cry1 e Vip3A de Bacillus thuringiensis: sinergismo e efeito sub-letal no controle de Heliothis virescens." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/92666.
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A bactéria Bacillus thuringiensis (Bt) possui a capacidade de produzir inclusões protéicas (proteína Cry) e proteínas vegetativas (Vip). Estas proteínas podem ser tóxicas para insetos e por meio de transgenia, a expressão em plantas, podem também proporcionar controle de importantes pragas agrícolas. Nesse sentido, esta pesquisa teve por objetivo avaliar o potencial de controle das proteínas Cry1Aa, Cry1Ac, Cry1Ca, Vip3A(1), Vip3A(2) e Vip3A(3) em uma população brasileira da lagarta-da-maçã, Heliothis virescens (Lepidoptera: Noctuidae), bem como o efeito subletal e efeito sinérgico entre estas proteínas e a proteólise pelo suco do intestino do inseto praga em estudo. Para tanto, clones de Escherichia coli recombinantes portadores de genes únicos foram cultivados em meio para a indução das proteínas e os lisados obtidos foram utilizados para as análises de toxicidade por meio de bioensaios. Diferentes concentrações protéicas foram utilizadas para conduzir os bioensaios. A mortalidade foi avaliada e obteve-se a CL50. Desta forma, observou-se que, dentre as proteínas testadas, Cry1Ac (CL50 39,89 ng.cm-2), Vip3A(2) (CL50 945,77 ng.cm-2) e Vip3A(3) (CL50 874,45 ng.cm-2 ) foram as mais tóxicas e houve correlação negativa entre a concentração de proteínas e o peso das lagartas. Nos ensaios referentes ao efeito sinérgico das proteínas, ativadas e não ativadas com tripsina comercial, foram encontradas possíveis combinações eficientes no controle da praga em estudo destacando-se Vip3A(2)/Cry1Aa, Vip3A(1)/Cry1Aa, Vip3A(1)/Cry1Ac, e Vip3A(2)/Cry1Ac. Os resultados referentes à interação das enzimas digestivas do intestino de H. virescens com as toxinas Cry1 e Vip3A permitiram constatar que as proteínas são ativadas
Bacillus thuringiensis (Bt) produces protein inclusions (Cry proteins) and vegetative proteins (Vip). Such proteins may act as toxic to some insects and through transgenesis plants they may be able to control important agricultural pests. Thus this work aimed to evaluate the control potential of Cry1Aa, Cry1Ac, Cry1Ca, Vip3A(1), Vip3A(2) and Vip3A(3) proteins in a Brazilian population of Heliothis virescens (tobacco budworm) (Lepidoptera: Noctuidae), as well as to analyze the sublethal and the synergic effects among these proteins and the proteolysis on the intestinal juice of this pest. In order to do this, Escherichia coli clones expressing each one of the above mentioned proteins were induced to produce them and the obtained lysates were used to determine the level of toxicity through bioassays with neonatal larvae of H. virescens. Different protein concentrations were used to carry out the bioassays. The mortality was evaluated and it was possible to detect the CL50. It was possible to observe that among the tested proteins, Cry1Ac (CL50 39.89 ng.cm-2), Vip3A(2) (CL50 945,77 ng.cm-2) and Vip3A (3) (CL50 874,45 ng.cm-2) were the most toxic and showed a negative correlation between the protein concentration and larvae weight. During the bioassays concerning the synergistic effect of these proteins, which were either previously activated or not using commercial trypsin, there we found efficient combinations for the control of the pest under study in this work, being that the combinations Vip3A(2)/Cry1Aa, Vip3A(1)/Cry1Aa, Vip3A(1)/Cry1Ac and Vip3A(2)/Cry1Ac were considered the best ones. The results with reference to the interaction of the digestive enzymes from the intestine of H. virescens larvae when using the toxins Cry1 and Vip3A allowed us to detect that these proteins are activated
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Lemes, Ana Rita Nunes. "Proteínas Cry1 e Vip3A de Bacillus thuringiensis : sinergismo e efeito sub-letal no controle de Heliothis virescens /." Jaboticabal : [s.n.], 2012. http://hdl.handle.net/11449/92666.
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Orientador: Janete Apparecida DesidérioCoorientador: Odair Aparecido Fernandes
Banca: Maria Inês Tiraboschi Ferro
Banca: Juliana Regina Vieira da Costa
Resumo: A bactéria Bacillus thuringiensis (Bt) possui a capacidade de produzir inclusões protéicas (proteína Cry) e proteínas vegetativas (Vip). Estas proteínas podem ser tóxicas para insetos e por meio de transgenia, a expressão em plantas, podem também proporcionar controle de importantes pragas agrícolas. Nesse sentido, esta pesquisa teve por objetivo avaliar o potencial de controle das proteínas Cry1Aa, Cry1Ac, Cry1Ca, Vip3A(1), Vip3A(2) e Vip3A(3) em uma população brasileira da lagarta-da-maçã, Heliothis virescens (Lepidoptera: Noctuidae), bem como o efeito subletal e efeito sinérgico entre estas proteínas e a proteólise pelo suco do intestino do inseto praga em estudo. Para tanto, clones de Escherichia coli recombinantes portadores de genes únicos foram cultivados em meio para a indução das proteínas e os lisados obtidos foram utilizados para as análises de toxicidade por meio de bioensaios. Diferentes concentrações protéicas foram utilizadas para conduzir os bioensaios. A mortalidade foi avaliada e obteve-se a CL50. Desta forma, observou-se que, dentre as proteínas testadas, Cry1Ac (CL50 39,89 ng.cm-2), Vip3A(2) (CL50 945,77 ng.cm-2) e Vip3A(3) (CL50 874,45 ng.cm-2 ) foram as mais tóxicas e houve correlação negativa entre a concentração de proteínas e o peso das lagartas. Nos ensaios referentes ao efeito sinérgico das proteínas, ativadas e não ativadas com tripsina comercial, foram encontradas possíveis combinações eficientes no controle da praga em estudo destacando-se Vip3A(2)/Cry1Aa, Vip3A(1)/Cry1Aa, Vip3A(1)/Cry1Ac, e Vip3A(2)/Cry1Ac. Os resultados referentes à interação das enzimas digestivas do intestino de H. virescens com as toxinas Cry1 e Vip3A permitiram constatar que as proteínas são ativadas
Abstract: Bacillus thuringiensis (Bt) produces protein inclusions (Cry proteins) and vegetative proteins (Vip). Such proteins may act as toxic to some insects and through transgenesis plants they may be able to control important agricultural pests. Thus this work aimed to evaluate the control potential of Cry1Aa, Cry1Ac, Cry1Ca, Vip3A(1), Vip3A(2) and Vip3A(3) proteins in a Brazilian population of Heliothis virescens (tobacco budworm) (Lepidoptera: Noctuidae), as well as to analyze the sublethal and the synergic effects among these proteins and the proteolysis on the intestinal juice of this pest. In order to do this, Escherichia coli clones expressing each one of the above mentioned proteins were induced to produce them and the obtained lysates were used to determine the level of toxicity through bioassays with neonatal larvae of H. virescens. Different protein concentrations were used to carry out the bioassays. The mortality was evaluated and it was possible to detect the CL50. It was possible to observe that among the tested proteins, Cry1Ac (CL50 39.89 ng.cm-2), Vip3A(2) (CL50 945,77 ng.cm-2) and Vip3A (3) (CL50 874,45 ng.cm-2) were the most toxic and showed a negative correlation between the protein concentration and larvae weight. During the bioassays concerning the synergistic effect of these proteins, which were either previously activated or not using commercial trypsin, there we found efficient combinations for the control of the pest under study in this work, being that the combinations Vip3A(2)/Cry1Aa, Vip3A(1)/Cry1Aa, Vip3A(1)/Cry1Ac and Vip3A(2)/Cry1Ac were considered the best ones. The results with reference to the interaction of the digestive enzymes from the intestine of H. virescens larvae when using the toxins Cry1 and Vip3A allowed us to detect that these proteins are activated
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Abdulganiyyu, Ibrahim A. "A single AKH neuropeptide activating three different fly AKH-receptors: an insecticide study via computational methods." Doctoral thesis, Faculty of Science, 2021. http://hdl.handle.net/11427/33621.
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Flies are a widely distributed pest insect that poses a significant threat to food security. Flight is essential for the dispersal of the adult flies to find new food sources and ideal breeding spots. The supply of metabolic fuel to power the flight muscles of insects is regulated by adipokinetic hormones (AKHs). The fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis all have the same AKH that is present in the blowfly, Phormia terraenovae; this AKH has the code-name Phote-HrTH. Binding of the AKH to the extracellular binding site of a G protein-coupled receptor causes its activation. In this thesis, the structure of Phote-HrTH in SDS micelle solution was determined using NMR restrained molecular dynamics. The peptide was found to bind to the micelle and be reasonably rigid, with an S 2 order parameter of 0.96. The translated protein sequence of the AKH receptor from the fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis were used to construct two models for each receptor: Drome-AKHR, Sarcr-AKHR, and Bacdo-AKHR. It is proposed that these two models represent the active and inactive state of the receptor. The models based on the crystal structure of the β-2 adrenergic receptor were found to bind Phote-HrTH with a predicted binding free energy of –107 kJ mol–1 for Drome-AKHR, –102 kJ mol–1 for Sarcr-AKHR and –102 kJ mol–1 for Bacdo-AKHR. Under molecular dynamics simulation, in a POPC membrane, the β-2AR receptor-like complexes transformed to rhodopsin-like. The identification and characterisation of the ligand-binding site of each receptor provide novel information on ligand-receptor interactions, which could lead to the development of species-specific control substances to use discriminately against these pest flies.
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Alves, Meire de Cássia. "Identificação e caracterização de genes cry3, vip1, vip2 E vip1/vip2 em isolados de Bacillus thuringiensis E toxicidade em larvas de Anthonomus grandis (Boheman, 1883) (Coleoptera: curculionidae) /." Jaboticabal : [s.n.], 2011. http://hdl.handle.net/11449/92689.
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Resumo: O controle biológico utilizando microrganismos vem sendo estudado como uma alternativa ao uso de agroquímicos. Estes pesticidas poluem o ambiente, além de serem tóxicos a diversas espécies vegetais e animais. Diante disso, a bactéria Bacillus thuringiensis é um importante entomopatógeno que vem sendo utilizado na agricultura pelo fato de secretar proteínas que são tóxicas a insetos pertencentes a diversas ordens. Dentre os diversos genes de B. thuringiensis que codificam proteínas tóxicas, as classes vip1, vip2 e cry3 destacam-se como alternativa para o controle de insetos-praga da ordem Coleoptera. O presente trabalho objetivou a identificação e caracterização molecular, por meio da técnica de PCR-RFLP, dos genes cry3, vip1, vip2 e vip1/vip2 em uma coleção de B. thuringiensis, quanto a possíveis polimorfismos existentes, procurando relacioná-los à toxicidade em larvas de Anthonomus grandis. Da análise de 1078 isolados de B. thuringiensis, foram encontrados 151 isolados positivos para os genes em estudo e 14 perfis polimórficos, indicando a presença de subclasses destes genes. Os resultados obtidos nos ensaios de toxicidade indicam que os polimorfismos gênicos podem apresentar interferência na toxicidade das proteínas, sendo que a toxina Cry3 apresentou uma maior efetividade na mortalidade em larvas de A. grandis. Os resultados também evidenciaram que a PCR-RFLP mostrou-se apropriada para a detecção da variabilidade genética em B. thuringiensis, permitindo a identificação de haplótiposAbstract: Biological control using microorganisms is being studied as an alternative to the use of agrochemicals. These pesticides pollute the environment, being toxic to many species of plants and animals. For these reasons, the bacterium Bacillus thuringiensis is an important entomopathogen that has been used in agriculture, once it produces proteins that are toxic to many target insect orders. Among several genes from B. thuringiensis that encode toxic proteins there are vip1, vip2 and cry3 classes that stand as an alternative for controlling insect pests belonging the Coleoptera order. The objective of this work was to identify and characterize molecularly, through PCR-RFLP, the genes cry3, vip1, vip2 and vip1/vip2 in a collection of B. thuringiensis, for possible polymorphisms exist, trying to relate them to the toxicity in Anthonomus grandis larvae. Analysis of 1078 isolates of B. thuringiensis, were found 151 positive isolates for the genes under study and 10 polymorphic profiles, which indicate the presence of subclasses of the genes. These results obtained in tests toxicity indicate that polymorphisms gene may have interference with the toxicity of the protein, and the toxin Cry3 showed a greater effectiveness in mortality in A. grandis larvae. These results also showed that the PCR-RFLP proved to be suitable for the detection of genetic variability in B. thuringiensis, allowing the identification of haplotypes
Orientador: Janete Apparecida Desidério
Coorientador: Odair Aparecido Fernandes
Banca: Maria Inês Tiraboschi Ferro
Banca: José Roberto Postali Parra
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Koganemaru, Reina. "Reduced cuticular penetration as a contributor to insecticide resistance in the common bed bug, Cimex lectularius L." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/73497.
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The Common bed bug, Cimex lectularius L., suddenly reappeared in developed countries in the past 15 years. The factor contributing to the sudden resurgence of the bed bugs is insecticide resistance. In this study, we investigated the mechanisms of reduced cuticular penetration type insecticide resistance in bed bugs. First, we determined and compared the lethal dosage (LD50) of a pyrethroid insecticide using topical and injection application. The resistant strain not only had significantly greater resistance ratios, but also demonstrated significantly greater penetration resistance ratios. This provided the evidence of the reduced cuticular penetration in bed bugs. Second, we determined the levels of gene transcription (CPR-type cuticle protein genes) using real-time quantitative polymerase chain reaction (qRT-PCR). We identified 62 putative bed bug cuticle protein-encoding contigs based on the presence of the Chitin-binding 4 (CB4) domain. Based on the qRT-PCR analysis of the mRNAs, we found many of the genes were up-regulated in the resistant strain suggesting thickening of the cuticle or increasing the cuticular proteins might be involved in the reduced cuticular penetration. Third, we identified and described the cuticular proteins using the matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometry (MALDI-TOF/TOF). The total of 265 peptides were identified, among which 206 belonged to one of 50 confidently identified proteins. We identified the CPRL, CPF, CPFL, TWDL, and CPAP1 family proteins. The profile of the cuticular proteins between the resistant and the susceptible strains bed bugs were almost identical. Fourth, we determined and compared the cuticular thickness using Scanning Electron Microscopy (SEM). We found statistical differences of the cuticular thickness among different strains (populations), however, correlation between the levels of insecticide resistance and cuticular thickness were not found. Finally, we identified and described bed bug cuticular hydrocarbon profiles using Gas-Chromatography and Mass-Spectrometry (GC-MS). The total of 87 compounds in addition to n-alkanes were extracted and identified. There were no correlation found with the concentration and the levels of insecticide resistance. However, several additional compounds exhibited the correlation between the concentration of the compounds and the levels of insecticide resistance. Overall, we found three lines of evidence to support reduced cuticular penetration as a mechanism of insecticide resistance in some bed bug populations. This study provides additional evidence of the reduced cuticular penetration type resistance in bed bugs.Ph. D.
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Marucci, Suzana Cristina. "Seleção e caracterização de novos genes vip3A : genes inseticidas de segunda geração de Bacillus thuringiensis." Jaboticabal : [s.n.], 2010. http://hdl.handle.net/11449/92703.
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Orientadora: Janete Apparecida DesidérioBanca: Agda Paula Facincani
Banca: Irlan Leite de Abreu
Resumo: Como uma alternativa para diminuir as agressões constantes que o ecossistema vem sofrendo, devido à grande quantidade de produtos químicos utilizados no controle de pragas, pesquisas envolvendo microrganismos capazes de promover o controle biológico tem se intensificado. Dentre estes microrganismos a bactéria Bacillus thuringiensis tem se destacado. Essa bactéria caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos. Em particular, as proteínas Vip3A, estão em amplo estudo devido a sua especificidade, alto potencial ativo e como alternativa para o controle da resistência de insetos às proteínas Cry. Diante disto, o objetivo deste trabalho foi selecionar, a partir de 1080 isolados de diferentes regiões brasileiras, aqueles portadores de genes vip3A e obter a sequência de nucleotídeos completa dos mesmos. As linhagens padrão B. thuringiensis var. kurstaki HD1, B. thuringiensis var. tolworthi HD125 e o isolado I187 tiveram seus DNAs amplificados com oligonucleotídeos baseados na sequência de genes vip3A, descritos no banco de dados de B. thuringiensis e, a partir dos amplicons obtidos, a sequência completa de nucleotídeos dos mesmos foi determinada, utilizando-se da estratégia de "primer walking". A proteína Vip3Aa43 (GenBank: [HQ594534]) da linhagem HD1 demonstrou ser 100% idêntica às proteínas Vip3Aa já descritas. Já as proteínas Vip3Aa42 (GenBank: [HQ587048]) da linhagem HD125 e Vip3Ag5 (GenBank: [HQ542193]) do isolado I187 demonstraram similaridade de 99% com as proteínas descritas Vip3Aa35 e Vip3Ag2, respectivamente, demonstrando serem duas novas proteínas Vip3A, devido às substituições de aminoácidos ocorridas. Os três genes vip3A obtidos poderão ser utilizados na produção de plantas Bt, piramidadas ou não, visando ao manejo da resistência dos insetos praga
Abstract: As an alternative to decrease the constant aggressions that the ecosystem has suffered due to the large amount of chemical products used in pest control, researches involving microorganisms able to promoting biological control have been intensified. Among these microorganisms the bacterium Bacillus thuringiensis has been stood out. This bacterium is characterized by the production of toxic proteins to representatives of several insect orders. In particular, the Vip3A proteins are in large study due to its specificity, and high active potential as an alternative to control of insect's resistance to Cry proteins. According to this, the aim of this work was to select from 1080 isolates in different Brazilian regions, those carrying vip3A genes and obtain the complete nucleotide sequence of the same. The standard strains B. thuringiensis var. kurstaki HD1, B. thuringiensis var. tolworthi HD125 and the isolate I187 had their DNA amplified with primers based on vip3A gene sequence described in database of B. thuringiensis, and from the amplicons obtained, the full sequence of nucleotides was determined, by the use of the strategy of "primer walking". The protein Vip3Aa43 (GenBank: [HQ594534]) of HD1 strain showed to be 100% identical to Vip3Aa proteins already described. However, the proteins Vip3Aa42 (GenBank: [HQ587048]) of HD125 strain and Vip3Ag5 (GenBank: [HQ542193]) of the isolate I187 showed 99% similarity with the Vip3Aa35 and Vip3Ag2 proteins described, respectively, showing been two new Vip3A proteins due to amino acid substitutions occurred. The three vip3A genes obtained can be used in the production of Bt crops, pyramidal or not, aiming to resistance management of pest insects
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Charles, Jean-François. "Bacillus thuringiensis sérotype H 14 et bacillus sphaericus : sporulation, biogenèse des cristaux larvicides et cytopathologie sur larves de moustiques (diptères; culicidae)." Paris 6, 1987. http://www.theses.fr/1987PA066303.
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Peng, Cheng-Hung, and 彭成弘. "Protein factors involved in crystallization of insecticidal crystal protein in Bacillus thuringiensis." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/51065500478320599945.
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碩士國立陽明大學
生物化學研究所
90
Bacillus thuringiensis produces large amounts of various pesticide proteins during the stationary phase. Production of crystal proteins in various Bt strains is wildly different. Todate, mechanism for production of pesticide proteins is focusing in transcriptional levels; However, factors involved in crystallization of insecticidal crystal protein of the Bt strains are still unknown. Therefore, in this study, attempt has been made to use affinity chromatography to fish any protein interacting with Cry protein followed by proteomic approach to identify any possible protein involved in the crystallization process. Over 200 B.t. isolates, which produced phase bright inclusions, have been isolated from soil samples from Taipei, Taichung and mountain area of Yang Ming Mountain, Yushan and Shishan in Taiwan. Among these isolates, we found that one isolate, designated as YM11 isolate, produce bigger size of phase bright inclusions than B. thuringiensis subspecies kurstaki HD-1( a Bt stardard strain). In order to identify any protein interacting with Cry protein, three distinct domains of Cry protein with his-tag have been constructed.Using these constructs as bait, number of proteins have been identified from BT cell extracts interacting with the baits. These proteins were analysed by 2-D gel electrophoresis and these protein spots were picked for further identification by MALDI-TOF mass spectrometry. One of these proteins was found related to MecB (ClpC) of Bacillus subtilis, which is a member of the ClpC ATPase family involved in a pleiotropic regulator controlling competence gene expression. Futhermore, MecB also triggers the autolysins through the sigma factor D to enhance the mother cell lyses. We speculate that YM11 may express less MecB than the Bt HD-1 strain, thus the degress of mother cell lysis of YM11 isolate may be much less than that of the wild type strain, which may allow longer time for accumulating large Cry protein shock? In conclusion, the proteomic approach of this study may shed light on revealing the overall factors affecting the formation of Cry protein of the Bt strain.
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CHEN, HONG-WEN, and 陳鵬文. "Isclation and application of insecticidal crystal protein genes from bacillus thuringiensis." Thesis, 1992. http://ndltd.ncl.edu.tw/handle/90937569926935871029.
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YANG, HUI-FEN, and 楊惠芬. "Agrobacterium-mediated transformation of insecticidal crystal protein gene in tomato (lycopericon esculentum)." Thesis, 1992. http://ndltd.ncl.edu.tw/handle/70660245419585467171.
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Lin, Yung-Di, and 林詠迪. "Molecular Cloning and Purification of an Insecticidal Protein from Pseudomonas sp. TKU015." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/60717748403664689902.
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碩士淡江大學
生命科學研究所碩士班
97
The chitinase, chitosanase and nattokinase producing strain, Pseudomonas sp. TKU015, was isolated from the soil in North Taiwan. The shrimp shell waste powder was used as sole carbon / nitrogen source. The optimized condition for nattokinase production was found when the culture was shaken at 30℃for 2 days in 100mL of medium contain 1% SSP, 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH7). The molecular mass of the nattokinase determined by SDS-PAGE was approximately 21 kDa. The nattokinase was inhibited completely by PMSF, and more activated by Fe2+. The most sensitive substrate for nattokinase was N-succinyl-Ala-Ala-Pro-Phe-pNA. Therefore, nattokinase was considered to be a chymotrypsin-like serine protease. The results of peptide mass mapping showed that two tryptic peptides of the nattokinase were identical to a chitin binding protein from Bacillus cereus ATCC 14579 (GenBank accession number gi30020946) with 23% sequence coverage. Designing the suitable primer, by way of a succession of polymerase chain reaction, discovered the insecticidal protein gene sequence fragment. The sequence was similar that amino acid sequence comparison with Pseudomonas entomophila L48, then designing that suitable that, by way of a succession of polymerase chain reaction, found the C end of insecticidal protein gene. The all insecticidal protein gene of 2079 base pair was revealed that used GenomeWalkerTM and polymerase chain reaction. Cloning insecticidal protein gene from Pseudomonas sp. TKU015 in the pET-32 Xa/Lic vector, and transferred to the Escherichia coli XL1-Blue host. The plasmid constructed completely because of the host characteristic. Afterward purification plasmid was extracted and then expression of transferred the Escherichia coli BL21 host. Inducing the performance of 94.7kDa the protein product with IPTG when the culture was shaken at 37℃for 1 day. The purification of insecticidal protein used HisTaq, and then desalted. Finally, the 77 kDa of goal protein was separated by using factor Xa protease. The protein was to be the biological activity test against larva of fruit fly.
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Chang, Hsin-Tzu, and 張興慈. "Molecular Cloning and Characterization of an Insecticidal Protein from Pseudomonas taiwanensis TKU015." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/15846454645009648146.
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碩士淡江大學
生命科學研究所碩士班
98
The availability of effective and cheap insecticides heralded an agricultural revolution. The insecticides not only help farmers increase yields, but also lower food prices. However, use of these insecticides is detrimental since some of these persist in the environment and accumulate in living organisms, causing various fatal diseases and are also toxic to nontarget species. In time new resistant strains of insects emerge, requiring increased doses of insecticides and introduction of new insecticides. Recently, bioinsecticides are being used as an alternative to the insecticides. Bioinsecticides are certain types of pesticides derived from natural materials including animals, plants, and bacteria. The toxic action of bioinsecticides is often specific to a single group or species of insects, does not hazardous to non-target species. They are also degraded in sunlight. The most widely used bioinsecticides are microbial pesticides. Pseudomonas taiwanensis TKU015 was isolated from Taiwan soil and was found that the amino acid sequence of one of its insecticidal proteins was similar to Pseudomonas entomphila L48, so the primers was designed to clone this insecticidal protein gene in further studies. By using polymerase chain reaction method, the 2-kb DNA fragments encoding insecticidal toxin was obtained as described previously. By using Genome WalkerTM kit, the 3-kb fragments encoding full-length insecticidal toxin was obtained. The insecticidal toxin genes were subcloned into the pET-32 Xa/Lic vector and then transformed into E. coli BL21. The recombinant insecticidal toxins were expressed by the transformed E. coli BL21 by induction with IPTG at 37 degrees overnight. The target proteins fused with His-tag were purified and de-salted. Finally, the purified insecticidal proteins were analyzed its insecticidal effect on Drosophila larvae.
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Liao, Chunyan. "The toxicology of Bacillus thuringiensis Cry1Ac insecticidal crystal protein in Helicoverpa armigera." Phd thesis, 1999. http://hdl.handle.net/1885/148096.
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Yeu-Yen, Chen, and 陳彥宇. "Studies on the Expression of the Insecticidal Crystal Protein us thuringiensis in Transgenic Plants." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/14409589955794540045.
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Lin, Cheng-Yi, and 林正義. "Cloning and expression of the vegetative insecticidal protein （vip3Aa25） gene of Bacillus thuringiensis from Taiwan." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/28149907133541717639.
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碩士朝陽科技大學
應用化學系碩士班
96
Nine Bacillus thuringiensis（B.t.）isolates were screened by polymerase chain reaction（PCR）to detect the presence of vegetative insecticidal protein gene（vip）. The vip3Aa25 gene was amplified from B.t. G10-01a by PCR using vip28F and vip26R primers. The vip3Aa25 gene was cloned into vector pET29b and pαHY300 to from recombinant expression plasmid pET29b-vip3Aa25 and pαHY300-vip3Aa25. By transformation and electroporation, the recombinant expression plasmid were cloned into Escherichia coli BL21 and Cry－B respectively to form recombinant strains of pET29b-vip3Aa25 and pαHY300-vip3Aa25. The pET29b-vip3Aa25 was induced by IPTG and pαHY300-vip3Aa25 was purified by ammonium sulfate to produce a protein of approximately 88.5 kDa. Moreover, by MS/MS analysis, the nucleotide sequence was determined to predict a protein of 789 amino acid residues. Bioassay showed that VIP3Aa25 protein was toxic to Trichoplusia ni, Spodoptera exigua, Helicoverpa armigera, S. litura and Plutella xylostella and caused the reduction of larval weight from 1st~3rd instar.
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Bowen, David J. "Characterization of a high molecular weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus liminescens." 1995. http://catalog.hathitrust.org/api/volumes/oclc/34778012.html.
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Huang, Wen-Chung, and 黃文忠. "Expression of insecticidal crystal protein genes from Bacillus thuringiensis in Escherichia coli, Erwinia herbicola and plants." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/39672996891831598689.
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碩士國立中興大學
植物學系
81
Genes for insecticidal crystal protion (cry genes) were isolated form various Bacillus thurgiensis strains by polymerase chain reaction (PCR) technology. There cry genes were constructed in different promoter containing E.coli expression vectors, pUNG and pTrc99A respectivly. The pUNG vector contain the promoter of lacZ operon and the pTrc99A vector contains the trc promoter. Results of insecticidal activity assay indicated that the cry gene in pTrc99A has better expression than that of in pUNG. However,these changed did not affect the insecticidal activity to tested insects. A cry gene modified (BTM) to increase its expression in plaht was also tested in E.coli. This maybe due to the codons of BTM gene were not well recognized in E.coli. After transforming cry genes , carried by a plasmid vector, into Erwinia herbicola, the transformed E.herbicola could express insecticidal crystal protein and show insecticidal activity to tested insects. These transformed E.herbicola could retain on the leave of cabbage at the natural environmental condition for about 7 day. To test the stability of cry gene containing plasmid in E. herbicol,a series of subculture in ampicillin free LB medium was performed. The results indicated that the plasmid will be excluded form the E.herbicola within few days in the ampicillin free culture condition. Due to the low transformation efficiency of Brassica plants, only five kanamycin resistant Brassica plants were obtained and three of them werw detected to be cry gene containing transgenic plants by PCR. However, only one plant was further confirmed by the southern analysis.
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LI, LEI, and 黎蕾. "Characterization and cloning of an insecticidal crystal protein gene from bacillus thuringiensis YMB 96 strain isolated from Taiwan." Thesis, 1991. http://ndltd.ncl.edu.tw/handle/40612641254710577551.
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