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1
Bai, Xue Liang, Dan Wang, Ning Ning Liu, et al. "Construction of Two Vectors for a Bypass of Monocotyledon Plants Photorespiration." Advanced Materials Research 340 (September 2011): 351–56. http://dx.doi.org/10.4028/www.scientific.net/amr.340.351.
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In order to modify the photorespiration of monocotyledonous crops, we aimed to construct vectors that will be used to introduce a bypass to the native photorespiration pathway. Firstly, we cloned the encoding sequences of glyoxylate carboligase (GCL) and tartronic semialdehyde reductase (TSR) fromE. coli, glycolate dehydrogenase (GDH) fromArabidopsis thalianaand chloroplast transit peptide (cTP) from rice. Then we constructed a universal vector pEXP harboring the encoding sequence of cTP for targeting a protein into chloroplast. By insertion of these three encoding sequences into the universal vector pEXP, we obtained the expression cassettes for GCL, TSR and GDH, respectively. Finally, we inserted the cassettes for GCL and TSR in tandem into the binary vector pCAMBIA 1301, and for GDH into another binary vector, pPGN, to obtain our plant expression vectors pCAMBIA 1301-TG and pPGN-GDH, respectively. These two expression vectors possess different selection resistance and can be used to transform monocots together, to introduce the bypass pathway of photorespiration. By this way, the transgenic plants can recycle glycolate, the by-product of photosynthesis in C3plants, within the chloroplast, simultaneously, save energy and avoid the loss of ammonia, which will contribute to improved growth.
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Fang, Le, Xin-Yu Wei, Ling-Zhi Liu, et al. "A tobacco ringspot virus-based vector system for gene and microRNA function studies in cucurbits." Plant Physiology 186, no. 2 (2021): 853–64. http://dx.doi.org/10.1093/plphys/kiab146.
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Abstract Cucurbits are economically important crops worldwide. The genomic data of many cucurbits are now available. However, functional analyses of cucurbit genes and noncoding RNAs have been impeded because genetic transformation is difficult for many cucurbitaceous plants. Here, we developed a set of tobacco ringspot virus (TRSV)-based vectors for gene and microRNA (miRNA) function studies in cucurbits. A TRSV-based expression vector could simultaneously express GREEN FLUORESCENT PROTEIN (GFP) and heterologous viral suppressors of RNA silencing in TRSV-infected plants, while a TRSV-based gene silencing vector could knock down endogenous genes exemplified by PHYTOENE DESATURASE (PDS) in Cucumis melo, Citrullus lanatus, Cucumis sativus, and Nicotiana benthamiana plants. We also developed a TRSV-based miRNA silencing vector to dissect the functions of endogenous miRNAs. Four representative miRNAs, namely, miR159, miR166, miR172, and miR319, from different cucurbits were inserted into the TRSV vector using a short tandem target mimic strategy and induced characteristic phenotypes in TRSV-miRNA-infected plants. This TRSV-based vector system will facilitate functional genomic studies in cucurbits.
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Xia, Xun-Li, Guang-Xiao Yang, and Guang-Yuan He. "Design of tandem genes cluster and expression vector for biosynthesis of soybean isoflavones." Physiology and Molecular Biology of Plants 15, no. 1 (2009): 99–102. http://dx.doi.org/10.1007/s12298-009-0011-0.
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Pinneh, Elizabeth C., Dolleweerd Craig J. van, Kathrin Göritzer, Pascal M. W. Drake, Julian K.-C. Ma, and Audrey Y.-H. Teh. "Multiple gene expression in plants using MIDAS-P, a versatile type II restriction-based modular expression vector." Biotechnology and Bioengineering 119 (March 3, 2022): 1660–72. https://doi.org/10.1002/bit.28073.
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MIDAS-P is a plant expression vector with blue/white screening for iterative cloning of multiple, tandemly arranged transcription units (TUs). We have used the MIDAS-P system to investigate the expression of up to five genes encoding three anti-HIV proteins and the reporter gene DsRed in Nicotiana benthamiana plants. The anti-HIV cocktail was made up of a broadly neutralizing monoclonal antibody (VRC01), a lectin (Griffithsin), and a single-chain camelid nanobody (J3-VHH). Constructs containing different combinations of 3, 4, or 5 TUs encoding different components of the anti-HIV cocktail were assembled. Messenger RNA (mRNA) levels of the genes of interest decreased beyond two TUs. Coexpression of the RNA silencing suppressor P19 dramatically increased the overall mRNA and protein expression levels of each component. The position of individual TUs in 3 TU constructs did not affect mRNA or protein expression levels. However, their expression dropped to non-detectable levels in constructs with four or more TUs each containing the same promoter and terminator elements, with the exception of DsRed at the first or last position in 5 TU constructs. This drop was alleviated by co-expression of P19. In short, the MIDAS-P system is suitable for the simultaneous expression of multiple proteins in one construct.
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Hong, Yiguo, John Stanley, and Rene van Wezel. "Novel System for the Simultaneous Analysis ofGeminivirus DNA Replication and Plant Interactions in Nicotianabenthamiana." Journal of Virology 77, no. 24 (2003): 13315–22. http://dx.doi.org/10.1128/jvi.77.24.13315-13322.2003.
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ABSTRACT The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based on Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replication-associated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R2, R5, R7 and K11 or H56, L57 and H58 prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.
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Blokhina, Elena A., Eugenia S. Mardanova, Anna A. Zykova, et al. "Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope." Plants 12, no. 11 (2023): 2228. http://dx.doi.org/10.3390/plants12112228.
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Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15–30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant “universal” vaccine against influenza A produced in plants.
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Püth, Nils, Franziska Ersoy, Ulrich Krings, and Ralf G. Berger. "Sesquiterpene Cyclases from the Basidiomycete Cerrena unicolor." Catalysts 11, no. 11 (2021): 1361. http://dx.doi.org/10.3390/catal11111361.
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Hundreds of terpenoids have been isolated from Basidiomycota, among them are volatile mono- and sesquiterpenes with amazing sensory qualities, representing a promising alternative to essential oils from endangered plant species. Sesquiterpene synthases (STS) appear to be an abundant class of enzymes in these fungi. The basidiomycete Cerrena unicolor, a known sesquiterpene producer, was in silico screened for sesquiterpene cyclases via homology Basic Local Alignment Search Tool searches. Cyclase genes identified were cloned and heterologously expressed in Escherichia coli Bl21 using pCOLD I as the expression vector. Ten cyclases were successfully produced and purified, and their identity was confirmed using amino acid sequencing of tryptic peptides by nano-liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry. Gas chromatography/mass spectrometry analysis was applied to characterize these cyclases according to the formation of sesquiterpene hydrocarbons and oxidized terpenoids. Bioinformatic characterization and phylogenetic determination allowed for the classification of these diverse fungal enzymes. A representative single and a multi-product STS, respectively, were further analyzed for their dependency from divalent metal cations as a cofactor for the catalytic activity.
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Blokhina, Elena A., Eugenia S. Mardanova, Liudmila A. Stepanova, Liudmila M. Tsybalova, and Nikolai V. Ravin. "Plant-Produced Recombinant Influenza A Virus Candidate Vaccine Based on Flagellin Linked to Conservative Fragments of M2 Protein and Hemagglutintin." Plants 9, no. 2 (2020): 162. http://dx.doi.org/10.3390/plants9020162.
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The development of recombinant influenza vaccines with broad spectrum protection is an important task. The combination of conservative viral antigens, such as M2e, the extracellular domain of the transmembrane protein M2, and conserved regions of the second subunit of hemagglutinin (HA), provides an opportunity for the development of universal influenza vaccines. Immunogenicity of the antigens could be enhanced by fusion to bacterial flagellin, the ligand for Toll-like receptor 5, acting as a powerful mucosal adjuvant. In this study, we report the transient expression in plants of a recombinant protein comprising flagellin of Salmonella typhimurium fused to the conserved region of the second subunit of HA (76–130 a.a.) of the first phylogenetic group of influenza A viruses and four tandem copies of the M2e peptide. The hybrid protein was expressed in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff up to 300 µg/g of fresh leaf tissue. The intranasal immunization of mice with purified fusion protein induced high levels of M2e-specific serum antibodies and provided protection against lethal challenge with influenza A virus strain A/Aichi/2/68(H3N2). Our results show that M2e and hemagglutinin-derived peptide can be used as important targets for the development of a plant-produced vaccine against influenza.
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Ma, Lulin, Wenjie Jia, Qing Duan, et al. "Heterologous Expression of Platycodon grandiflorus PgF3′5′H Modifies Flower Color Pigmentation in Tobacco." Genes 14, no. 10 (2023): 1920. http://dx.doi.org/10.3390/genes14101920.
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Flavonoid-3′,5′-hydroxylase (F3′5′H) is the key enzyme for the biosynthesis of delphinidin-based anthocyanins, which are generally required for purple or blue flowers. Previously, we isolated a full-length cDNA of PgF3′5′H from Platycodon grandiflorus, which shared the highest homology with Campanula medium F3′5′H. In this study, PgF3′5′H was subcloned into a plant over-expression vector and transformed into tobacco via Agrobacterium tumefaciens to investigate its catalytic function. Positive transgenic tobacco T0 plants were obtained by hygromycin resistance screening and PCR detection. PgF3′5′H showed a higher expression level in all PgF3′5′H transgenic tobacco plants than in control plants. Under the drive of the cauliflower mosaic virus (CaMV) 35S promoter, the over-expressed PgF3′5′H produced dihydromyricetin (DHM) and some new anthocyanin pigments (including delphinidin, petunidin, peonidin, and malvidin derivatives), and increased dihydrokaempferol (DHK), taxifolin, tridactyl, cyanidin derivatives, and pelargonidin derivatives in PgF3′5′H transgenic tobacco plants by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) analysis, resulting in a dramatic color alteration from light pink to magenta. These results indicate that PgF3′5′H products have F3′5′H enzyme activity. In addition, PgF3′5′H transfer alters flavonoid pigment synthesis and accumulation in tobacco. Thus, PgF3′5′H may be considered a candidate gene for gene engineering to enhance anthocyanin accumulation and the molecular breeding project for blue flowers.
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Liu, Chang, Kangyu Wang, Ziyi Yun, et al. "Functional Study of PgGRAS68-01 Gene Involved in the Regulation of Ginsenoside Biosynthesis in Panax ginseng." International Journal of Molecular Sciences 24, no. 4 (2023): 3347. http://dx.doi.org/10.3390/ijms24043347.
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Ginseng (Panax ginseng C. A. Meyer) is a perennial herb from the genus Panax in the family Araliaceae. It is famous in China and abroad. The biosynthesis of ginsenosides is controlled by structural genes and regulated by transcription factors. GRAS transcription factors are widely found in plants. They can be used as tools to modify plant metabolic pathways by interacting with promoters or regulatory elements of target genes to regulate the expression of target genes, thereby activating the synergistic interaction of multiple genes in metabolic pathways and effectively improving the accumulation of secondary metabolites. However, there are no reports on the involvement of the GRAS gene family in ginsenoside biosynthesis. In this study, the GRAS gene family was located on chromosome 24 pairs in ginseng. Tandem replication and fragment replication also played a key role in the expansion of the GRAS gene family. The PgGRAS68-01 gene closely related to ginsenoside biosynthesis was screened out, and the sequence and expression pattern of the gene were analyzed. The results showed that the expression of PgGRAS68-01 gene was spatio-temporal specific. The full-length sequence of PgGRAS68-01 gene was cloned, and the overexpression vector pBI121-PgGRAS68-01 was constructed. The ginseng seedlings were transformed by Agrobacterium rhifaciens-mediated method. The saponin content in the single root of positive hair root was detected, and the inhibitory role of PgGRAS68-01 in ginsenoside synthesis is reported.
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Su, Hong-Gang, Bo Li, Xin-Yuan Song, et al. "Genome-Wide Analysis of the DYW Subgroup PPR Gene Family and Identification of GmPPR4 Responses to Drought Stress." International Journal of Molecular Sciences 20, no. 22 (2019): 5667. http://dx.doi.org/10.3390/ijms20225667.
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Pentatricopeptide-repeat (PPR) proteins were identified as a type of nucleus coding protein that is composed of multiple tandem repeats. It has been reported that PPR genes play an important role in RNA editing, plant growth and development, and abiotic stresses in plants. However, the functions of PPR proteins remain largely unknown in soybean. In this study, 179 DYW subgroup PPR genes were identified in soybean genome (Glycine max Wm82.a2.v1). Chromosomal location analysis indicated that DYW subgroup PPR genes were mapped to all 20 chromosomes. Phylogenetic relationship analysis revealed that DYW subgroup PPR genes were categorized into three distinct Clusters (I to III). Gene structure analysis showed that most PPR genes were featured by a lack of intron. Gene duplication analysis demonstrated 30 PPR genes (15 pairs; ~35.7%) were segmentally duplicated among Cluster I PPR genes. Furthermore, we validated the mRNA expression of three genes that were highly up-regulated in soybean drought- and salt-induced transcriptome database and found that the expression levels of GmPPR4 were induced under salt and drought stresses. Under drought stress condition, GmPPR4-overexpressing (GmPPR4-OE) plants showed delayed leaf rolling; higher content of proline (Pro); and lower contents of H2O2, O2− and malondialdehyde (MDA) compared with the empty vector (EV)-control plants. GmPPR4-OE plants exhibited increased transcripts of several drought-inducible genes compared with EV-control plants. Our results provided a comprehensive analysis of the DYW subgroup PPR genes and an insight for improving the drought tolerance in soybean.
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Li, Donghui, Alison M. Ashby, and Keith Johnstone. "Molecular Evidence that the Extracellular Cutinase Pbc1 Is Required for Pathogenicity of Pyrenopeziza brassicae on Oilseed Rape." Molecular Plant-Microbe Interactions® 16, no. 6 (2003): 545–52. http://dx.doi.org/10.1094/mpmi.2003.16.6.545.
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Recent evidence has suggested that cutinase is required for cuticular penetration and, therefore, is essential for pathogenicity of Pyrenopeziza brassicae, the causal organism of light leaf spot disease of oilseed rape and other brassicas. In order to acquire molecular evidence for the role of cutinase in pathogenesis, the single-copy P. brassicae cutinase gene Pbc1 was disrupted by a transformation-mediated approach. Southern hybridization analysis revealed that in one mutant, NH10-1224, the disruption was due to a tandem insertion of two copies of the disruption vector into the 5′ coding region of Pbc1. In contrast to the wild type, no expression of Pbc1 was detected during in planta growth or in cutin-induced mycelium of NH10-1224 and no cutinase activity was detected in culture supernatants from NH10-1224 using pnitrophenyl butyrate as substrate. Scanning electron microscopy of Brassica napus cotyledons infected with wild-type P. brassicae confirmed that entry into the host is by direct penetration of the cuticle. In contrast, the cutinase-deficient mutant NH10-1224 failed to penetrate the cuticular layer and was unable to develop disease symptoms. This evidence is consistent with the hypothesis that Pbc1 is required for P. brassicae to penetrate the plant cuticle. Demonstration that complementation of NH10-1224 with the Pbc1 wild-type gene restores both cutinase activity and pathogenicity will be required to definitively establish that cutinase is required for successful pathogenesis of brassicas by P. brassicae.
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He, Sijun, Weining Li, Ruirui Zhang, Hao Nan, Wangcheng Song, and Xiaodong Xu. "Improving the production of baculovirus expression vector by overexpression of IE0/IE1 through tandem promoter." PLOS ONE 20, no. 3 (2025): e0320182. https://doi.org/10.1371/journal.pone.0320182.
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The baculovirus expression vector system, known for its protein production in insect cells and has been criticized for its relatively low expression capacity. IE0/IE1, acknowledged vital early regulators of baculovirus, are indispensable for the virus proliferation and regulate the expression of various genes within the virus. Prior research has reported a substantial rise in exogenous gene expression upon overexpression of IE01. In this study, to mitigate the risk of generating defective viruses due to homologous recombination, we introduced an additional promoter in vivo within the viral genome, thus overexpressing IE0/IE1. The research outcomes demonstrate that the expression of exogenous proteins is notably enhanced without the homologous regions sequence for enhancement. In parallel, they still indicate that the upregulation of IE0/IE1 not only boosts viral titers but also enhances apoptosis within cellular populations. In sum, we successfully constructed a novel baculovirus expression vector that significantly enhances the expression of exogenous genes, presenting a new perspective for optimizing the baculovirus expression vector system.
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Hefferon, Kathleen. "Plant Virus Expression Vector Development: New Perspectives." BioMed Research International 2014 (2014): 1–6. http://dx.doi.org/10.1155/2014/785382.
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Plant made biologics have elicited much attention over recent years for their potential in assisting those in developing countries who have poor access to modern medicine. Additional applications such as the stockpiling of vaccines against pandemic infectious diseases or potential biological warfare agents are also under investigation. Plant virus expression vectors represent a technology that enables high levels of pharmaceutical proteins to be produced in a very short period of time. Recent advances in research and development have brought about the generation of superior virus expression systems which can be readily delivered to the host plant in a manner that is both efficient and cost effective. This review presents recent innovations in plant virus expression systems and their uses for producing biologics from plants.
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Jacob, S. S., R. Vanitharani, A. S. Karthikeyan, Y. Chinchore, P. Thillaichidambaram, and K. Veluthambi. "Mungbean yellow mosaic virus-Vi Agroinfection by Codelivery of DNA A and DNA B From One Agrobacterium Strain." Plant Disease 87, no. 3 (2003): 247–51. http://dx.doi.org/10.1094/pdis.2003.87.3.247.
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Agroinfection of bipartite geminiviruses is routinely done by mixing two Agrobacterium strains that independently harbor partial tandem repeats of DNA A and DNA B. We report here an improved agroinfection method for bipartite geminiviruses that utilizes one strain of Agrobacterium that harbors DNA A and DNA B partial tandem repeats on two compatible replicons. A cointegrate vector, pGV2260∷pGV1.3A, with the partial tandem repeat of Mungbean yellow mosaic virus-Vi (MYMV-Vi) DNA A and a binary vector, pGA1.9B, with the partial tandem repeat of MYMV-Vi DNA B gave an agroinfection efficiency of 24% when harbored in two Agrobacterium strains and an efficiency of 61% when harbored in one Agrobacterium strain. A combination of binary vectors, pGA1.9A with MYMV-Vi DNA A partial tandem repeat and pGA1.9B with DNA B partial tandem repeat, gave an agroinfection efficiency of 74% when harbored in two strains. But pGA1.9A and pPZP1.9B (a partial tandem repeat of DNA B), when present in the same Agrobacterium strain, gave 100% agroinfection. Accumulation of viral DNA was shown by Southern blotting. The single-strain method using two compatible replicons consistently gave 100% agroinfection efficiency.
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Schoggins, John W., Jason G. D. Gall, and Erik Falck-Pedersen. "Subgroup B and F Fiber Chimeras Eliminate Normal Adenovirus Type 5 Vector Transduction In Vitro and In Vivo." Journal of Virology 77, no. 2 (2003): 1039–48. http://dx.doi.org/10.1128/jvi.77.2.1039-1048.2003.
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ABSTRACT Altering adenovirus vector (Ad vector) targeting is an important goal for a variety of gene therapy applications and involves eliminating or reducing the normal tropism of a vector and retargeting through a distinct receptor-ligand pathway. The first step of Ad vector infection is high-affinity binding to a target cellular receptor. For the majority of adenoviruses and Ad vectors, the fiber capsid protein serves this purpose, binding to the coxsackievirus and adenovirus receptor (CAR) present on a variety of cell types. In this study we have explored a novel approach to altering Ad type 5 (Ad5) vector targeting based on serotypic differences in fiber function. The subgroup B viruses bind to an unidentified receptor that is distinct from CAR. The subgroup F viruses are the only adenoviruses that express two distinct terminal exons encoding fiber open reading frames. We have constructed chimeric fiber adenoviruses that utilize the tandem fiber arrangement of the subgroup F genome configuration. By taking advantage of serotypic differences in fiber expression, fiber shaft length, and fiber binding efficiency, we have developed a tandem fiber vector that has low binding efficiency for the known fiber binding sites, does not rely on an Ad5-based fiber, and can be grown to high titer using conventional cell lines. Importantly, when characterizing these vectors in vivo, we find the subgroup B system and our optimal tandem fiber system demonstrate reduced liver transduction by over 2 logs compared to an Ad5 fiber vector. These attributes make the tandem fiber vector a useful alternative to conventional strategies for fiber manipulation of adenovirus vectors.
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Ohkawa, J., N. Yuyama, Y. Takebe, S. Nishikawa, and K. Taira. "Importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus." Proceedings of the National Academy of Sciences 90, no. 23 (1993): 11302–6. http://dx.doi.org/10.1073/pnas.90.23.11302.
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The kinetic behavior of ribozymes derived from two types of multiple-ribozyme expression vector were examined. In some cases, multiple ribozymes were expressed as a single RNA molecule and all the ribozymes were simply connected in tandem (connected type). In other cases, multiple ribozymes were flanked by cis-acting ribozymes at both their 5' and 3' ends so that, upon transcription, multiple ribozymes were trimmed at both their 5' and 3' ends, with resultant liberation of multiple independent ribozymes (shotgun type). When levels of ribozyme expression were examined for the shotgun-type vector, the level of the ribozyme transcript was found to be proportional to the number of units (n) connected in tandem. Accordingly, the activities of the shotgun-type ribozymes, in terms of the cleavage of HIV-1 RNA in vitro, were also found to be proportional to the number of units connected in tandem (n). By contrast, the activities of the connected-type ribozymes reached plateau values at around n = 3. These results indicate that, when the shotgun-type expression system is used, it is theoretically possible to generate various independent ribozymes, each specific for a different target site, without sacrificing the activity of any individual ribozyme.
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Wang, Gaoyan, David C. Manns, John J. Churey, and Randy W. Worobo. "Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361." Applied and Environmental Microbiology 80, no. 12 (2014): 3576–84. http://dx.doi.org/10.1128/aem.00433-14.
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ABSTRACTThurincin H is an antimicrobial peptide produced byBacillus thuringiensisSF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1,thnA2, andthnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designatedB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter,thnA1, and a Cry protein terminator into theEscherichia coli-B. thuringiensisshuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in thethnA1gene, were generated and separately transformed intoB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities ofB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3carrying different pGW133 variants against three different indicator strains were subsequently compared.
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Browne, Quinton Ambrose, David P. Puthoff, and Rebekah Taylor. "Cloning ospA of Borrelia Burgderferi for Plant Expression." Journal of Immunology 208, no. 1_Supplement (2022): 181.01. http://dx.doi.org/10.4049/jimmunol.208.supp.181.01.
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Abstract Borrelia burgdorferi (Lyme disease) is a vector-borne disease that is both very common in the Mid-Atlantic region and which causes long term negative health effects. It is primarily spread to humans by deer ticks, and because one of the main ways that it is passed between ticks is by previously infected mouse blood, our goal is to design an oral vaccine which decreases infection rates in mice. Our goal is to do this by inserting outer surface protein A (ospA) of B. burgdorferi into a plant vector: pCambia 1201. Agrobacterium tumefaciens was used as a means of introducing the vector (bearing B. burgdorferi ospA) into Arabidopsis thaliana. The pCambia vector was introduced into A. tumefaciens competent cells after being purified from E. coli colonies which had undergone a similar transformation process, as E. coli is more suitable for cloning. The successfully electroporated A. tumefaciens was then plated and colonies were picked and grown as liquid cultures. Those liquid cultures transformed with both the ospA bearing pCambia and a control pCambia were used for floral dip of Arabidopsis thaliana (both ospA bearing and pCambia only control plants). These dipped plants’ seeds have been tested for successful transformation, and one plant has been found to contain the sequence for ospA. Continued floral dips will be undertaken to increase the number of positive plants, and to develop a proper control for testing efficacy as a vaccine. Any additional seeds of positive plants will be planted and crossed to ensure use of only plants which express pCambia in later testing, and to ensure that the process has been successful. Supported by Frostburg State University Department of Biology and FSU Foundation
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Peretz, Yuval, Rita Mozes-Koch, Fuad Akad, Edna Tanne, Henryk Czosnek, and Ilan Sela. "A Universal Expression/Silencing Vector in Plants." Plant Physiology 145, no. 4 (2007): 1251–63. http://dx.doi.org/10.1104/pp.107.108217.
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Read, Jeremiah D., Paul A. Colussi, Mehul B. Ganatra, and Christopher H. Taron. "Acetamide Selection of Kluyveromyces lactis Cells Transformed with an Integrative Vector Leads to High-Frequency Formation of Multicopy Strains." Applied and Environmental Microbiology 73, no. 16 (2007): 5088–96. http://dx.doi.org/10.1128/aem.02253-06.
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ABSTRACT The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.
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MR, Fibi, J. Kunz, T. Weimer, et al. "Status and transcriptional activity of a bovine‐papillomavirus‐I‐based expression vector in a recombinant production cell line." Biotechnology and Applied Biochemistry 23, no. 1 (1996): 83–93. http://dx.doi.org/10.1111/j.1470-8744.1996.tb00367.x.
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We have analysed the status and transcriptional activity of the bovine papillomavirus‐I (complete BPV‐I genome)‐based expression vector pCES in CI27i‐cell‐line‐derived 3TI cells used for the industrial production of recombinant human erythropoietin (rhuEpo). Complete tandem head‐to‐tail integration of about 600 vector copies at a single site of the cellular genome was observed. Deletions, insertions or rearrangements of pCES‐specific sequences or extrachromosomal copies of vector sequences were not detected. Transcriptional analyses demonstrated that rhuEpo was abundantly expressed. BPV‐I early transcription was detected, as expected from a BPV‐I‐transformed cell line; however, compared with the mouse metallothionein‐I‐promoter‐driven rhuEpo‐specific transcription, it was very weak. Late BPV‐I transcription was also not detected in 3TI cells under conditions of large‐scale rhuEpo production. Therefore this expression system proved to be safe and suitable for the production of rhuEpo.
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Slightom, Jerry L. "Custom polymerase-chain-reaction engineering of a plant expression vector." Gene 100 (April 1991): 251–55. http://dx.doi.org/10.1016/0378-1119(91)90376-m.
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Picart-Picolo, Ariadna, Stefan Grob, Nathalie Picault, et al. "Large tandem duplications affect gene expression, 3D organization, and plant–pathogen response." Genome Research 30, no. 11 (2020): 1583–92. http://dx.doi.org/10.1101/gr.261586.120.
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Dobinson, Katherine F. "Genetic transformation of the vascular wilt fungusVerticillium dahliae." Canadian Journal of Botany 73, no. 5 (1995): 710–15. http://dx.doi.org/10.1139/b95-076.
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To facilitate genetic analysis of pathogenicity of Verticillium dahliae, a vascular wilt pathogen, a DNA-mediated transformation system has been developed. Resistance to hygromycin B was obtained by transforming spheroplasts with the cosmid vector pAN7-2. Transformation efficiencies ranged between 3 and 5 transformants/μg vector DNA. The transforming DNA was integrated into the V. dahliae genome, in single and multiple copies and in tandem array. In several multicopy transformants, minor alterations in the integrated DNA sequences were evident following extensive vegetative growth in the absence of hygromycin B. Electrophoretic karyotype analysis also provided direct evidence of chromosome rearrangements in two transformants. The availability of a transformation system for V. dahliae will facilitate the cloning and characterization of genes that are important for pathogenicity and development. Key words: Verticillium wilt, fungal transformation, electrophoretic karyotype, hygromycin B resistance, chromosome rearrangement.
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Lan, Tianying, Tanya Renner, Enrique Ibarra-Laclette, et al. "Long-read sequencing uncovers the adaptive topography of a carnivorous plant genome." Proceedings of the National Academy of Sciences 114, no. 22 (2017): E4435—E4441. http://dx.doi.org/10.1073/pnas.1702072114.
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Utricularia gibba, the humped bladderwort, is a carnivorous plant that retains a tiny nuclear genome despite at least two rounds of whole genome duplication (WGD) since common ancestry with grapevine and other species. We used a third-generation genome assembly with several complete chromosomes to reconstruct the two most recent lineage-specific ancestral genomes that led to the modern U. gibba genome structure. Patterns of subgenome dominance in the most recent WGD, both architectural and transcriptional, are suggestive of allopolyploidization, which may have generated genomic novelty and led to instantaneous speciation. Syntenic duplicates retained in polyploid blocks are enriched for transcription factor functions, whereas gene copies derived from ongoing tandem duplication events are enriched in metabolic functions potentially important for a carnivorous plant. Among these are tandem arrays of cysteine protease genes with trap-specific expression that evolved within a protein family known to be useful in the digestion of animal prey. Further enriched functions among tandem duplicates (also with trap-enhanced expression) include peptide transport (intercellular movement of broken-down prey proteins), ATPase activities (bladder-trap acidification and transmembrane nutrient transport), hydrolase and chitinase activities (breakdown of prey polysaccharides), and cell-wall dynamic components possibly associated with active bladder movements. Whereas independently polyploid Arabidopsis syntenic gene duplicates are similarly enriched for transcriptional regulatory activities, Arabidopsis tandems are distinct from those of U. gibba, while still metabolic and likely reflecting unique adaptations of that species. Taken together, these findings highlight the special importance of tandem duplications in the adaptive landscapes of a carnivorous plant genome.
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Li, T., J. K. Sun, Z. H. Lu, and Q. Liu. "Transformation of HBsAg (hepatitis B surface antigen) gene into tomato mediated by Agrobacterium tumefaciens." Czech Journal of Genetics and Plant Breeding 47, No. 2 (2011): 69–77. http://dx.doi.org/10.17221/5/2011-cjgpb.
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The plant expression vector pBRSAg was constructed as suitable for transformation via Agrobacterium-mediated approach. It contains all elements for plant expression, such as CaMV 35S promoter, both left and right border sequence for transferred DNA (T-DNA) in Agrobacterium, plant reporter gene gus, and plant selection marker gene hpt. The recombinant binary vector pBRSAg was transformed into Agrobacterium tumefaciens strains by using the freeze-thaw method. Tomato cotyledon explants were transformed by A. tumefaciens and plants were regenerated on selection medium. GUS staining, PCR and PCR-Southern analysis of the plants were positive. It was for the first time shown that the HBsAg (hepatitis B surface antigen) gene was introduced into tomato plants. The expression of transgene is under investigation.
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Kaur, Navneet, Daniel K. Hasegawa, Kai-Shu Ling, and William M. Wintermantel. "Application of Genomics for Understanding Plant Virus-Insect Vector Interactions and Insect Vector Control." Phytopathology® 106, no. 10 (2016): 1213–22. http://dx.doi.org/10.1094/phyto-02-16-0111-fi.
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The relationships between plant viruses and their vectors have evolved over the millennia, and yet, studies on viruses began <150 years ago and investigations into the virus and vector interactions even more recently. The advent of next generation sequencing, including rapid genome and transcriptome analysis, methods for evaluation of small RNAs, and the related disciplines of proteomics and metabolomics offer a significant shift in the ability to elucidate molecular mechanisms involved in virus infection and transmission by insect vectors. Genomic technologies offer an unprecedented opportunity to examine the response of insect vectors to the presence of ingested viruses through gene expression changes and altered biochemical pathways. This review focuses on the interactions between viruses and their whitefly or thrips vectors and on potential applications of genomics-driven control of the insect vectors. Recent studies have evaluated gene expression in vectors during feeding on plants infected with begomoviruses, criniviruses, and tospoviruses, which exhibit very different types of virus-vector interactions. These studies demonstrate the advantages of genomics and the potential complementary studies that rapidly advance our understanding of the biology of virus transmission by insect vectors and offer additional opportunities to design novel genetic strategies to manage insect vectors and the viruses they transmit.
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Catto, Michael A., Habibu Mugerwa, Brendon K. Myers, Sudeep Pandey, Bhabesh Dutta, and Rajagopalbabu Srinivasan. "A Review on Transcriptional Responses of Interactions between Insect Vectors and Plant Viruses." Cells 11, no. 4 (2022): 693. http://dx.doi.org/10.3390/cells11040693.
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This review provides a synopsis of transcriptional responses pertaining to interactions between plant viruses and the insect vectors that transmit them in diverse modes. In the process, it attempts to catalog differential gene expression pertinent to virus–vector interactions in vectors such as virus reception, virus cell entry, virus tissue tropism, virus multiplication, and vector immune responses. Whiteflies, leafhoppers, planthoppers, and thrips are the main insect groups reviewed, along with aphids and leaf beetles. Much of the focus on gene expression pertinent to vector–virus interactions has centered around whole-body RNA extraction, whereas data on virus-induced tissue-specific gene expression in vectors is limited. This review compares transcriptional responses in different insect groups following the acquisition of non-persistent, semi-persistent, and persistent (non-propagative and propagative) plant viruses and identifies parallels and divergences in gene expression patterns. Understanding virus-induced changes in vectors at a transcriptional level can aid in the identification of candidate genes for targeting with RNAi and/or CRISPR editing in insect vectors for management approaches.
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Tue, Nguyen Hoang, Nguyen Ngoc Luong, and Nguyen Quang Duc Tien. "Cloning an RBD-T4-LINKER-C5a sequence encoding the SARS-CoV-2 antigen into a plant expression vector." Hue University Journal of Science: Natural Science 132, no. 1D (2023): 73–78. http://dx.doi.org/10.26459/hueunijns.v132i1d.7075.
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This study aims to make a plant expression vector with an RBD-T4-Linker-C5a sequence that codes for the SARS-CoV-2 antigen and an A3Dsp signal peptide from the rice 3D amylase gene located before the RBD. Methods of molecular cloning were applied in this study. The plant expression vector pNHL22 harboring the RBD-T4-Linker-C5a sequence was successfully established and conjugated into Agrobacterium tumefaciens LBA4404 by triparental mating. Bacteria A. tumefaciens containing the RBD-T4-Linker-C5a sequence are now ready for genetic transformation into the Nicotiana benthamiana plant for future applications.
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Chung, Sang-Min, Ellen L. Frankman, and Tzvi Tzfira. "A versatile vector system for multiple gene expression in plants." Trends in Plant Science 10, no. 8 (2005): 357–61. http://dx.doi.org/10.1016/j.tplants.2005.06.001.
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Kobayashi, Hiroshi, Takeshi Yoshida, and Masayori Inouye. "Significant Enhanced Expression and Solubility of Human Proteins in Escherichia coli by Fusion with Protein S from Myxococcus xanthus." Applied and Environmental Microbiology 75, no. 16 (2009): 5356–62. http://dx.doi.org/10.1128/aem.00691-09.
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ABSTRACT Protein S is a major spore coat protein of Myxococcus xanthus, consisting of two homologous domains, the N-terminal domain (NTD) and the C-terminal domain, both of which contain a Ca2+-binding site. Protein S tightly binds to myxospores in a Ca2+-dependent manner. Here, we constructed a novel expression vector, pCold-PST, encoding two tandem repeat NTDs (PrS2). By using this vector, a number of human proteins that were expressed at low levels or in insoluble forms by a pET vector were expressed not only at high levels but also in soluble forms. We also demonstrated that an Escherichia coli protein tagged with PrS2 fully retained its function, indicating that it is folded independently from the tag. This technology not only allows simple, one-step protein purification using myxospores, but can also be used for the identification of proteins interacting with a protein of interest and will prove immensely useful for structural studies of proteins which are difficult to produce or are insoluble.
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Sen, Đào Thị, Nguyễn Chi Mai, Lê Quỳnh Liên, Trần Mỹ Linh, Chu Hoàng Hà, and Nguyễn Tường Vân. "Vector construction to enhance GP5 antigen expression of virus PRRS in plant cells." Vietnam Journal of Biotechnology 14, no. 3 (2016): 491–97. http://dx.doi.org/10.15625/1811-4989/14/3/9863.
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Plant vaccine is a new tool to enrich available vaccine resources for protection of human and animal health. Plant vaccine offers several advantages over current conventional vaccines, including that storage and transportationare convenient after lyophilization, production costs are low, and the contamination of mammalian pathogens is avoided. However, the low expression of foreign genes in plant still exists that limits the application of this kind of vaccine. Presently, plant virus-based expression vectors represent a technology that enables high levels of recombination proteins to be produced efficiently in plant cells. In this study, a modified TMV-based vector to elevate glycoprotein GP5 of PRRSV in plant cells without negative effects in the development of plant cells was developed. Gene encoded GP5 was replaced CP of TMV and the whole was regulated by the heat shock protein Hsp 18.2 promoter. Transgenic BY-2 cells carrying pHsp-TMV-GP5 showed the normal development compared to the ones harbouring pCB-35S-TMV-GP5. The results showed that the Hsp-TMV-GP5 construct do not have a negative effect on viability of transgenic plant cells. A further study will be carried out to investigate the expression of GP5 in stable transgenic plant cells to confirm the benefit of the modified expression vector in development of rapid and cost-effective antigens in plant cell suspension culture.
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Yi, Jian-Zhong, Ming-Qiu Liu, Cai-Zhu Zhu, et al. "Recombinant Bivalent Vaccine against Foot-and-Mouth Disease Virus Serotype O/A Infection in Guinea Pig." Acta Biochimica et Biophysica Sinica 36, no. 9 (2004): 589–96. http://dx.doi.org/10.1093/abbs/36.9.589.
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Abstract In this study, two DNA fragments encoding amino acid (141– 160)-(21–40)-(141–160) of the VP1 of FMDV (foot-and-mouth disease virus) serotype O and (138–160)-(21–40)-(138–160) of the serotype A FMDV were chemically synthesized. These two tandem-repeat fragments were ligated and transfected into prokaryotic expression vector pTrcHis A to construct pTH-O-A. The other vector called pTH-O-scIgG-A was constructed similarly only that the two tandem-repeat DNA fragments were linked by the bovine-IgG heavy chain coding sequence. Guinea pigs immunized with the two bivalent vaccines pTH-O-A and pTH-O-scIgG-A showed both specific antibody activity and T cell proliferation responses. FMDV challenge tests showed that 85% and 70% of guinea pigs vaccinated twice with 200 μg of the fusion protein of pTH-O-A were protected from FMDV serotype O and serotype A infection respectively. 70% and 57% of the guinea pigs immunized with the fusion protein of pTH-O-scIgG-A were protected from FMDV serotype O and serotype A infection respectively.
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Bossé, Janine T., Andrew L. Durham, Andrew N. Rycroft, J. Simon Kroll, and Paul R. Langford. "New Plasmid Tools for Genetic Analysis of Actinobacillus pleuropneumoniae and Other Pasteurellaceae." Applied and Environmental Microbiology 75, no. 19 (2009): 6124–31. http://dx.doi.org/10.1128/aem.00809-09.
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ABSTRACT We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of Actinobacillus pleuropneumoniae and other members of the Pasteurellaceae. A tandem reporter plasmid, pMC-Tandem, carrying promoterless xylE and gfpmut3 genes downstream of a multiple-cloning site (MCS), can be used for identification of transcriptional regulators and conditions which favor gene expression from different cloned promoters. The ability to detect transcriptional regulators using the tandem reporter system was validated in A. pleuropneumoniae using the cloned rpoE (σE) promoter (P). The resulting plasmid, pMCrpoEP, was used to identify a mutant defective in production of RseA, the negative regulator of σE, among a bank of random transposon mutants, as well as to detect induction of σE following exposure of A. pleuropneumoniae to ethanol or heat shock. pMCsodCP, carrying the cloned sodC promoter of A. pleuropneumoniae, was functional in A. pleuropneumoniae, Haemophilus influenzae, Haemophilus parasuis, Mannheimia haemolytica, and Pasteurella multocida. Two general expression vectors, pMK-Express and pMC-Express, which differ in their antibiotic resistance markers (kanamycin and chloramphenicol, respectively), were constructed for the Pasteurellaceae. Both plasmids have the A. pleuropneumoniae sodC promoter upstream of the gfpmut3 gene and an extended MCS. Replacement of gfpmut3 with a gene of interest allows complementation and heterologous gene expression, as evidenced by expression of the Haemophilus ducreyi nadV gene in A. pleuropneumoniae, rendering the latter NAD independent.
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García, J. A., C. Lucini, B. García, J. M. Alamillo, and J. J. López-Moya. "The use of Plum pox virus as a plant expression vector." EPPO Bulletin 36, no. 2 (2006): 341–45. http://dx.doi.org/10.1111/j.1365-2338.2006.01012.x.
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Varrelmann, Mark, Laszlo Palkovics, and Edgar Maiss. "Transgenic or Plant Expression Vector-Mediated Recombination of Plum Pox Virus." Journal of Virology 74, no. 16 (2000): 7462–69. http://dx.doi.org/10.1128/jvi.74.16.7462-7469.2000.
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ABSTRACT Different mutants of an infectious full-length clone (p35PPV-NAT) of Plum pox virus (PPV) were constructed: three mutants with mutations of the assembly motifs RQ and DF in the coat protein gene (CP) and two CP chimeras with exchanges in the CP core region ofZucchini yellow mosaic virus and Potato virus Y. The assembly mutants were restricted to single infected cells, whereas the PPV chimeras were able to produce systemic infections inNicotiana benthamiana plants. After passages in different transgenic N. benthamiana plants expressing the PPV CP gene with a complete (plant line 4.30.45.) or partially deleted 3′-nontranslated region (3′-NTR) (plant line 17.27.4.), characterization of the viral progeny of all mutants revealed restoration of wild-type virus by recombination with the transgenic CP RNA only in the presence of the complete 3′-NTR (4.30.45.). Reconstitution of wild-type virus was also observed following cobombardment of different assembly-defective p35PPV-NAT together with a movement-defective plant expression vector of Potato virus X expressing the intact PPV-NAT CP gene transiently in nontransgenic N. benthamiana plants. Finally, a chimeric recombinant virus was detected after cobombardment of defective p35PPV-NAT with a plant expression vector-derived CP gene from the sour cherry isolate of PPV (PPV-SoC). This chimeric virus has been established by a double recombination event between the CP-defective PPV mutant and the intact PPV-SoC CP gene. These results demonstrate that viral sequences can be tested for recombination events without the necessity for producing transgenic plants.
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Weng, Qiaoyun, Jihong Xing, Zhiyong Li, Zhiping Dong, and Jingao Dong. "Expression analysis of RUS1 and construction of RUS1 plant expressing vector." Frontiers of Agriculture in China 4, no. 1 (2010): 31–36. http://dx.doi.org/10.1007/s11703-010-0099-6.
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Nagai, Atsuko, Kenji Suzuki, Eric Ward, et al. "Overexpression of plant histidinol dehydrogenase using a baculovirus expression vector system." Archives of Biochemistry and Biophysics 295, no. 2 (1992): 235–39. http://dx.doi.org/10.1016/0003-9861(92)90512-u.
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Lakshmeesha, R, Nagesha, N, Natesha, J, and J. Harish. "Expression Studies of BmNPV Antiviral Proteins Serine Protease and Lipase in Mulberry Plant." Journal of Advances in Biology & Biotechnology 27, no. 3 (2024): 112–20. http://dx.doi.org/10.9734/jabb/2024/v27i3726.
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Sericulture is a significant agricultural cottage enterprise. The silkworm Bombyx mori L. is used in the commercial manufacture of silk. Silkworms are prone to fungal, bacterial, viral, and protozoan infections. Bombyx mori nuclear polyhedrosis virus (BmNPV) causes grasserie, a viral illness in silkworm that is a severe economic loss to the sericulture business. The current study focuses on the transformation of antiviral genes, serine protease, and lipase into mulberry leaves by agro infiltration. Antiviral proteins from BmNPV were cloned into the plant expression vector pBI121. The plant expression vector pBI121 was effectively transferred into Agro-bacterium cells for mulberry agroinfiltration investigations. PCR amplification and SDS-PAGE analysis validated the gene integration and production of antiviral proteins, which were discovered to be 885 bp of lipase gene with protein size of 29 Kda and 855 bp of serine protease gene with protein size of 24 Kda. Lipase and serine protease antiviral protein genes were cloned into the bacterial expression vector pET32a for bioassay experiments.
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Montero-Astúa, Mauricio, Dorith Rotenberg, Alexandria Leach-Kieffaber, et al. "Disruption of Vector Transmission by a Plant-Expressed Viral Glycoprotein." Molecular Plant-Microbe Interactions® 27, no. 3 (2014): 296–304. http://dx.doi.org/10.1094/mpmi-09-13-0287-fi.
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Vector-borne viruses are a threat to human, animal, and plant health worldwide, requiring the development of novel strategies for their control. Tomato spotted wilt virus (TSWV) is one of the 10 most economically significant plant viruses and, together with other tospoviruses, is a threat to global food security. TSWV is transmitted by thrips, including the western flower thrips, Frankliniella occidentalis. Previously, we demonstrated that the TSWV glycoprotein GN binds to thrips vector midguts. We report here the development of transgenic plants that interfere with TSWV acquisition and transmission by the insect vector. Tomato plants expressing GN-S protein supported virus accumulation and symptom expression comparable with nontransgenic plants. However, virus titers in larval insects exposed to the infected transgenic plants were three-log lower than insects exposed to infected nontransgenic control plants. The negative effect of the GN-S transgenics on insect virus titers persisted to adulthood, as shown by four-log lower virus titers in adults and an average reduction of 87% in transmission efficiencies. These results demonstrate that an initial reduction in virus infection of the insect can result in a significant decrease in virus titer and transmission over the lifespan of the vector, supportive of a dose-dependent relationship in the virus–vector interaction. These findings demonstrate that plant expression of a viral protein can be an effective way to block virus transmission by insect vectors.
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LI, Jun, Chang-rong LÜ, Lin DOU, and Zhong-ying DOU. "Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and Its Expression." Agricultural Sciences in China 6, no. 4 (2007): 487–92. http://dx.doi.org/10.1016/s1671-2927(07)60073-x.
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Christel, Carl J., Patricia Schmied, Verena Jagusch, et al. "Versatile Viral Vector Strategies for Postscreening Target Validation and RNAi ON-Target Control." Journal of Biomolecular Screening 20, no. 8 (2015): 976–84. http://dx.doi.org/10.1177/1087057115581803.
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Our approach aims to optimize postscreening target validation strategies using viral vector–driven RNA interference (RNAi) cell models. The RNAiONE validation platform is an array of plasmid-based expression vectors that each drives tandem expression of the gene of interest (GOI) with one small hairpin RNA (shRNA) from a set of computed candidate sequences. The best-performing shRNA (>85% silencing efficiency) is then integrated in an inducible, all-in-one lentiviral vector to transduce pharmacologically relevant cell types that endogenously express the GOI. VariCHECK is used subsequently to combine the inducible knockdown with an equally inducible rescue of the GOI for ON-target phenotype verification. The complete RNAiONE–VariCHECK system relies on three key elements to ensure high predictability: (1) maximized silencing efficiencies by a focused shRNA validation process, (2) homogeneity of the RNAi cell pools by application of sophisticated viral vector technologies, and (3) exploiting the advantages of inducible expression systems. By using a reversible expression system, our strategy adds critical information to hot candidates from RNAi screens and avoids potential side effects that may be caused by other, irreversible genomic manipulation methods such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas). This approach will add credibility to top-hit screening candidates and protect researchers from costly misinterpretations early in the preclinical drug development process.
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Liu, Yun-Jun, Fu-Ping Song, Kang-Lai He, et al. "Expression of a Modified Cry1Ie Gene in E. coli and in Transgenic Tobacco Confers Resistance to Corn Borer." Acta Biochimica et Biophysica Sinica 36, no. 4 (2004): 309–13. http://dx.doi.org/10.1093/abbs/36.4.309.
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Abstract The wild-type Cry1Ie gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Cry1Ie gene (designated as Cry1Iem) was cloned into prokaryotic expression vector pET28b and its expression in E. coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E. coli revealed that Cry1Iem protein had a similar toxicity to corn borer as wild-type Cry1Ie. Cry1Iem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. Transgenic tobacco plants carrying Cry1Iem showed insecticidal activity against corn borer.
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Guniganti, Devendar, and Kumar K. Raj. "DEVELOPMENT OF DOUBLE GENE MAMMALIAN EXPRESSION VECTOR." Biolife 2, no. 3 (2022): 731–37. https://doi.org/10.5281/zenodo.7219727.
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<strong>ABSTRACT</strong> Recombinant vectors are valuable tools in the biopharmaceutical industry with a number of novel vectors being developed every day. These Antibodies are often expressed in mammalian cells by co-transfecting light chain and heavy chain containing plasmids. However, co-transfection can lead to variable in the copy number of both heavy chain and light chain, there by affecting the protein productivity. Double gene expression vectors can overcome these problems. In the current design The first transcriptional unit comprises sequences for the CMV promoter, multiple cloning site (MCS) and a Polyadenylation Sequence and the second transcriptional unit comprises sequences for the SV40 promoter, MCS and polyadenylation sequence. The double gene expression vector (pUB-C-S) was constructed by ligating the BglII and BamHI fragment (974bp) form pSI plasmid with BglII digested pCI plasmid. The presence of two independent transcriptional units in the recombinant plasmid was confirmed by colony PCR. <strong>Key Words:</strong> Recombinant vectors, biopharmaceutical industry, co-transfection, promoter, multiple cloning site. <strong>REFERENCES</strong> Johnston K., Clements A., Venkataramani. R.N., Trienvel R.C., and Marmorstein.R., 2000. Coexpression of proteins in bacteria using T7-based expression plasmids: Expression of heteromeric cell-cycle and transcriptional regulatory complexes. Protein Expr. Purif. 20: 435-443. Rucker. P., Torti F.M., and Torti S.V., 1997. Recombinant ferritin: Modulation of subunit stoichiometry in bacterial expression system. Protein Eng. 10: 967-973. Susan K. Eszterhas<sup>1</sup> Eric E. Bouhassira,<sup>2</sup> David I. K. Martin,<sup>3</sup> and Steven Fiering<sup>1*</sup>, Transcriptional Interference by Independently Regulated Genes Occurs in Any Relative Arrangement of the Genes and Is Influenced by Chromosomal Integration Position. Mol Cell Biol. 2002 January; 22(2): 469–479. Bizily SP, Rugh CL, Meagher RB.,Phytodetoxification of hazardous organomercurials by genetically engineered plants., Nat Biotechnol. 2000 Feb;18(2):213-7.. Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leple JC, Boerjan W, Ferret V V, De Nadai V, Jouanin L., Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping. Plant Physiol. 1999 Jan;119(1):153-64. Chen L, Marmey P, Taylor NJ, Brizard JP, Espinoza C, D'Cruz P, Huet H, Zhang S, de Kochko A, Beachy RN, Fauquet CM., Expression and inheritance of multiple transgenes in rice plants., Nat Biotechnol. 1998 Nov;16(11):1060-4. Goderis IJ, De Bolle MF, François IE, Wouters PF, Broekaert WF, Cammue BP., A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol. 2002 Sep;50(1):17-27. Ashton, G. (2001) Growing pains for biopharmaceuticals. Nature Biotechnology <strong>19</strong>, 307-311 Walsh, G. (2004) Second-generation biopharmaceuticals. European Journal of Pharmaceutics &Biopharmaceutics<strong>58</strong>, 185-196 Current protocols in molecular biology, vol 1, 2005 Sambrook J and Russell D W, Molecular cloning a laboratory manual, Vol 1, 2001. Baneyx, F. (1999) Recombinant protein expression in Escherichia <em>coli</em>. Current Opinion in Biotechnology <strong>10</strong>, 411-421 Swartz, J. R. (2001) Advances in Escherichia <em>coli</em> production of therapeutic proteins. Current Opinion in Biotechnology <strong>12</strong>, 195-201 Kjaerulff, S. and Jensen, M. R. (2005) Comparison of different signal peptides for secretion of heterologous proteins in fission yeast. Biochemical & Biophysical Research Communications <strong>336</strong>, 974-982 Daly, R. and Hearn, M. T. W. (2005) Expression of heterologous proteins in Pichiapastoris: a useful experimental tool in protein engineering and production. Journal of Molecular Recognition <strong>18</strong>, 119-138 Blanchard V, Gadkari RA, George AV, Roy S, Gerwig GJ, Leeflang BR, Dighe RR, Boelens R, Kamerling JP., High-level expression of biologically active glycoprotein hormones in Pichiapastoris strains-selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated (15)N-labeled phCG. Glycoconj J. 2008 Feb 15; [Epub ahead of print] Forsburg SL., Overview of Schizosaccharomycespombe. CurrProtocMol Bio, 2003, Chapter 13, unit 13.14. Rodney E. Kellems, Gene amplification in mammalian cells. 1992. Paulina Balbas and Francisco Bolivar, Design and Construction of Expression Plasmid Vectors in Eschericha<em>coli</em>.., Methods in Enzymology, 185:14-37, 1990. Makrides, S.C. (1996). Strategies for achieving high-level expression of genes in Escherichia <em>coli</em>. Microbiol Rev <em>60</em>, 512-538. Lehman IR, DNA ligase: Structure, mechanism and function. Vol. 186, no. 4166, pp. 790-797, 1974.
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Gong, Qi, Bin Wang, Xubiao Lu, et al. "Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research." Plants 9, no. 9 (2020): 1090. http://dx.doi.org/10.3390/plants9091090.
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Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.
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Rajabi Memari, Hamid, Ramakrishnan Ramanan та Arbakariya Ariff. "Comparison of expression systems for the production of human interferon-α2b". Open Life Sciences 5, № 4 (2010): 446–55. http://dx.doi.org/10.2478/s11535-010-0036-y.
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AbstractThe production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 µg/L of culture, while the amount of nuclear expression in the plant was 4.46 µg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform.
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Katoh, Yohei, Saki Michisaka, Shohei Nozaki, et al. "Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system." Molecular Biology of the Cell 28, no. 7 (2017): 898–906. http://dx.doi.org/10.1091/mbc.e17-01-0051.
Full textAbstract:
The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.
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Reinar, William B., Vilde O. Lalun, Trond Reitan, Kjetill S. Jakobsen, and Melinka A. Butenko. "Length variation in short tandem repeats affects gene expression in natural populations of Arabidopsis thaliana." Plant Cell 33, no. 7 (2021): 2221–34. http://dx.doi.org/10.1093/plcell/koab107.
Full textAbstract:
Abstract The genetic basis for the fine-tuned regulation of gene expression is complex and ultimately influences the phenotype and thus the local adaptation of natural populations. Short tandem repeats (STRs) consisting of repetitive DNA motifs have been shown to regulate gene expression. STRs are variable in length within a population and serve as a heritable, but semi-reversible, reservoir of standing genetic variation. For sessile organisms, such as plants, STRs could be of major importance in fine-tuning gene expression as a response to a shifting local environment. Here, we used a transcriptome dataset from natural accessions of Arabidopsis thaliana to investigate population-wide gene expression patterns in light of genome-wide STR variation. We empirically modeled gene expression as a response to the STR length within and around the gene and demonstrated that an association between gene expression and STR length variation is unequivocally present in the sampled population. To support our model, we explored the promoter activity in a transcriptional regulator involved in root hair formation and provided experimentally determined causality between coding sequence length variation and promoter activity. Our results support a general link between gene expression variation and STR length variation in A. thaliana.
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Xuan, Hoang Thi Lan, Nguyen Binh Anh Thu, Nguyen Bao Thien Phuc, and Nguyen Phuong Thao. "Construction of GmNAC085 vector for future development of drought-tolerant crops." Vietnam Journal of Biotechnology 14, no. 4 (2018): 645–52. http://dx.doi.org/10.15625/1811-4989/14/4/12297.
Full textAbstract:
Various members of NAC transcription factor family have been shown to play important roles in regulating plant responses to abiotic stresses, such as drought, cold and salinity. Our previous research on differential expression patterns of twenty three soybean NAC genes (GmNACs) by realtime quantitative PCR suggested a correlation between inducible expression of GmNAC085 and the drought tolerance degree in DT51 and MTD720 soybean cultivars, which presented for the drought-tolerant and the drought-sensitive, respectively. Therefore, the gene has been proposed as a potential candidate for engineering in order to produce new varieties with better drought stress tolerance. However, functional studies of GmNAC085 should be carried out to identify how this transcriptional factor can contribute in the plant stress-responsive pathway. Herein, this paper presented that we have successfully developed a recombinant binary vector carrying full-length cDNA of GmNAC085 and expression of this gene is placed under the control of constitutive promoter CaMV 35S. The generated construct was firstly transformed into E.coli for sequencing the target gene and then the identified genuine construct was transformed into Agrobacterium tumefaciens. This can be used for plant transformation mediated by Agrobacterium in serving for GmNAC085 - related studies using in planta system such as the model plant Arabidopsis and also serving for the development of drought-tolerant crops by genetic engineering. Additionally, results from sequence alignment analysis revealed that GmNAC085s of DT51 and MTD720 had identical nucleotide sequence, thus supporting our hypothesis that difference in GmNAC085 gene expression levels, not the variation in GmNAC085 protein sequence or structure, might cause the difference in plant resistance degree to drought stress in these two soybean varieties.