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1
Wanitchakorn, Raktham. "Gene functions in banana bunchy top virus." Thesis, Queensland University of Technology, 1999.
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2
Burns, Thomas Michael. "Genome organisation of banana bunchy top virus." Thesis, Queensland University of Technology, 1994.
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3
Dugdale, Benjamin. "Promoter activity associated with the intergenic regions of banana bunchy top virus." Thesis, Queensland University of Technology, 1999. https://eprints.qut.edu.au/37001/6/37001_Digitised_Thesis.pdf.
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4
Hafner, Gregory. "Replication of banana bunchy top virus : mechanisms and interference." Thesis, Queensland University of Technology, 1998.
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5
Beetham, Peter Ronald. "Transcription of mRNAs from the banana bunchy top virus genome." Thesis, Queensland University of Technology, 1998.
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6
Soko, Misheck Mica Mafeni. "Evaluation of transgenic RNAi banana and plantain lines for resistance to banana bunchy top disease." Thesis, Queensland University of Technology, 2022. https://eprints.qut.edu.au/228515/1/Misheck%20Mica%20Mafeni_Soko_Thesis.pdf.
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This project evaluated genetically modified Cavendish bananas and plantains for field and glasshouse resistance to banana bunchy top virus in Malawi in Africa, over a three-and-a-half-year period. The study identified several GM lines with significant resistance to the virus and provided a rare insight into virus-vector relations and the climate. The research showed that rigorous field assessment of GM plants for disease resistance is critical and that immunity to this virus will likely only be achieved using a multi-faceted resistance strategy.
7
Karan, Mirko. "Sequence diversity of DNA components associated with banana bunchy top virus." Thesis, Queensland University of Technology, 1995.
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8
Horser, Cathryn Louise. "Characterisation of the putative satellite DNAs associated with banana bunchy top virus." Thesis, Queensland University of Technology, 2000.
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9
Becker, Douglas Kenneth. "The transformation of banana with potential virus resistance genes." Thesis, Queensland University of Technology, 1999. https://eprints.qut.edu.au/37023/6/37023_Digitised_Thesis.pdf.
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One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV).
A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated.
Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed.
The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter.
Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.
10
Hastie, Marcus Lachlan. "In vitro activities of BBTV-REP." Thesis, Queensland University of Technology, 2001. https://eprints.qut.edu.au/37077/1/37077_Digitised%20Thesis.pdf.
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11
Herrera, Valencia Virginia Aurora. "Molecular characterisation of the intergenic regions of banana bunchy top virus." Queensland University of Technology, 2006. http://eprints.qut.edu.au/16216/.
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Banana bunchy top virus (BBTV) is a circular, single-stranded (css) DNA virus that belongs to the genus Babuvirus in the family Nanoviridae. BBTV is responsible for the most devastating virus disease of banana known as "bunchy top", for which conventional control measures are generally ineffective. Genetically engineered resistance appears to be the most promising strategy to generate BBTV-resistant bananas but the success of this strategy is largely dependent upon the molecular characterisation of the target virus and knowledge of the virus life cycle, particularly the replication strategy. This PhD study was aimed at the molecular characterisation of the intergenic regions of BBTV, in order to complement the molecular information currently available and to potentially contribute to the development of transgenic resistance strategies against BBTV in banana. Three putative iterative sequences (iterons; GGGAC) previously identified in the BBTV intergenic regions were initially characterised. In order to determine their role in the binding of the master BBTV replication initiation protein (M-Rep), the putative iterons (F1 and F2 in the virion sense, and R in the complementary sense) were independently mutated in a BBTV DNA-6 greater-than-genome-length clone (1.1 mer). The DNA-6 1.1 mers (native and mutants) and the M-Rep-encoding component (DNA-1) were co-bombarded into banana (Musa spp. cv."Lady finger") embryogenic suspension cells and transient replication was evaluated by Southern hybridisation. Analysis of the DNA-6 replicative forms showed a significant decrease of approximately 41% for the F1 iteron mutant and 61% for the R iteron mutant in comparison with native levels. However, the mutation in the F2 iteron caused the most dramatic effect, decreasing replication to levels barely detectable by Southern hybridisation. These results suggest that the three iterons all play a role in BBTV replication, most likely as recognition and binding sites for the M-Rep, but that the F2 iteron appears to be the most important in replication. Following the observation that all BBTV isolates sequenced to date have identical iteron sequences, the extent to which the M-Rep would recognise, bind and initiate replication of heterologous components from geographically diverse BBTV isolates (the South Pacific and the Asian groups) was evaluated. Cross replication assays revealed that heterologous M-Reps from Fiji, Hawaii (South Pacific group) and Vietnam (Asian group) were able to initiate replication of the coat protein-encoding component (DNA-3) from the Australian BBTV isolate (South Pacific group). However, replication of DNA-3 from the Vietnamese isolate was not initiated by heterologous M-Reps from the two South Pacific isolates tested (Australia and Hawaii). These results suggest that a broad-range transgenic resistance strategy based on replication using Australian BBTV intergenic regions may be successful as this region will be recognised by the M-Reps from both Asian and South Pacific BBTV isolates. However, a Rep protein-mediated resistance strategy will more likely be specific to geographical isolates and, therefore, less suitable as a broad-range control strategy. To further characterise the BBTV intergenic regions and to gain a better understanding of the BBTV transcription process, the 5' untranslated regions (UTRs) of the major open reading frames (ORFs) associated with each of the six BBTV DNA components were mapped. In all cases, the transcription start sites were located 3' of a putative TATA box and the 5' UTRs varied in length from 23 nucleotides (DNA-6) to 5 nucleotides (DNA-3). Two potential transcription start sites (nt 84 and 87) were mapped for DNA-1, but whether these represent the transcription start sites of the two genes associated with DNA-1 remains to be determined. Two start sites were also associated with DNA-2 which is thought to be monocistronic. Whether one of these start sites is an artefact or whether they are due to natural sequence variability of BBTV is unknown. These results now enable us to define the transcribed regions of each BBTV DNA component and accurately predict their promoter regions in an attempt to gain a fundamental understanding of BBTV gene expression patterns.
12
Herrera-Valencia, Virginia Aurora. "Molecular characterisation of the intergenic regions of banana bunchy top virus." Thesis, Queensland University of Technology, 2006. https://eprints.qut.edu.au/16216/1/Virginia_Herrera-Valencia_Thesis.pdf.
Full textAbstract:
Banana bunchy top virus (BBTV) is a circular, single-stranded (css) DNA virus that belongs to the genus Babuvirus in the family Nanoviridae. BBTV is responsible for the most devastating virus disease of banana known as "bunchy top", for which conventional control measures are generally ineffective. Genetically engineered resistance appears to be the most promising strategy to generate BBTV-resistant bananas but the success of this strategy is largely dependent upon the molecular characterisation of the target virus and knowledge of the virus life cycle, particularly the replication strategy. This PhD study was aimed at the molecular characterisation of the intergenic regions of BBTV, in order to complement the molecular information currently available and to potentially contribute to the development of transgenic resistance strategies against BBTV in banana.
Three putative iterative sequences (iterons; GGGAC) previously identified in the BBTV intergenic regions were initially characterised. In order to determine their role in the binding of the master BBTV replication initiation protein (M-Rep), the putative iterons (F1 and F2 in the virion sense, and R in the complementary sense) were independently mutated in a BBTV DNA-6 greater-than-genome-length clone (1.1 mer). The DNA-6 1.1 mers (native and mutants) and the M-Rep-encoding component (DNA-1) were co-bombarded into banana (Musa spp. cv."Lady finger") embryogenic suspension cells and transient replication was evaluated by Southern hybridisation. Analysis of the DNA-6 replicative forms showed a significant decrease of approximately 41% for the F1 iteron mutant and 61% for the R iteron mutant in comparison with native levels. However, the mutation in the F2 iteron caused the most dramatic effect, decreasing replication to levels barely detectable by Southern hybridisation. These results suggest that the three iterons all play a role in BBTV replication, most likely as recognition and binding sites for the M-Rep, but that the F2 iteron appears to be the most important in replication.
Following the observation that all BBTV isolates sequenced to date have identical iteron sequences, the extent to which the M-Rep would recognise, bind and initiate replication of heterologous components from geographically diverse BBTV isolates (the South Pacific and the Asian groups) was evaluated. Cross replication assays revealed that heterologous M-Reps from Fiji, Hawaii (South Pacific group) and Vietnam (Asian group) were able to initiate replication of the coat protein-encoding component (DNA-3) from the Australian BBTV isolate (South Pacific group). However, replication of DNA-3 from the Vietnamese isolate was not initiated by heterologous M-Reps from the two South Pacific isolates tested (Australia and Hawaii). These results suggest that a broad-range transgenic resistance strategy based on replication using Australian BBTV intergenic regions may be successful as this region will be recognised by the M-Reps from both Asian and South Pacific BBTV isolates. However, a Rep protein-mediated resistance strategy will more likely be specific to geographical isolates and, therefore, less suitable as a broad-range control strategy.
To further characterise the BBTV intergenic regions and to gain a better understanding of the BBTV transcription process, the 5' untranslated regions (UTRs) of the major open reading frames (ORFs) associated with each of the six BBTV DNA components were mapped. In all cases, the transcription start sites were located 3' of a putative TATA box and the 5' UTRs varied in length from 23 nucleotides (DNA-6) to 5 nucleotides (DNA-3). Two potential transcription start sites (nt 84 and 87) were mapped for DNA-1, but whether these represent the transcription start sites of the two genes associated with DNA-1 remains to be determined. Two start sites were also associated with DNA-2 which is thought to be monocistronic. Whether one of these start sites is an artefact or whether they are due to natural sequence variability of BBTV is unknown. These results now enable us to define the transcribed regions of each BBTV DNA component and accurately predict their promoter regions in an attempt to gain a fundamental understanding of BBTV gene expression patterns.
13
Mware, Benard Ouma. "Development of banana bunchy top virus resistance in bananas: RNAi approach." Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/95736/1/Benard%20Ouma_Mware_Thesis.pdf.
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Bunchy top, caused by banana bunchy top virus (BBTV), is the most important viral disease affecting banana worldwide, for which control is very difficult. In this thesis, a molecular approach was used to generate genetically modified banana plants, some of which were resistant to BBTV infection. Further, sources of natural resistance to BBTV were identified in a range of different banana cultivars which may be exploited in future control strategies. The results from this study provide a strong platform for the future development of BBTV-resistant banana plants.
14
Tsao, Theresa Tsun-Hui. "Towards the development of transgenic banana bunchy top virus (BBTV)-resistant banana plants : interference with replication." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/17031/1/Theresa_Tsao_Thesis.pdf.
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Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions.
A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated.
The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos.
The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV.
In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.
15
Tsao, Theresa Tsun-Hui. "Towards the development of transgenic banana bunchy top virus (BBTV)-resistant banana plants : interference with replication." Queensland University of Technology, 2008. http://eprints.qut.edu.au/17031/.
Full textAbstract:
Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions.
A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated.
The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos.
The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV.
In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.
16
Bolton, Clair Louise. "Development of a Rep-inducible, BBTV-based expression system in banana." Thesis, Queensland University of Technology, 2009. https://eprints.qut.edu.au/25939/1/Clair_Bolton_Thesis.pdf.
Full textAbstract:
Banana bunchy top is regarded as the most important viral disease of banana, causing significant yield losses worldwide. The disease is caused by Banana bunchy top virus (BBTV), which is a circular ssDNA virus belonging to the genus Babuvirus in the family Nanoviridae. There are currently few effective control strategies for this and other ssDNA viruses. “In Plant Activation” (InPAct) is a novel technology being developed at QUT for ssDNA virus-activated suicide gene expression. The technology exploits the rolling circle replication mechanism of ssDNA viruses and is based on a unique “split” gene design such that suicide gene expression is only activated in the presence of the viral Rep. This PhD project aimed to develop a BBTV-based InPAct system as a suicide gene strategy to control BBTV.
The BBTV-based InPAct vector design requires a BBTV intergenic region (IR) to be embedded within an intron in the gene expression cassette. To ensure that the BBTV IR would not interfere with intron splicing, a TEST vector was initially generated that contained the entire BBTV IR embedded within an intron in a β-glucuronidase (GUS) expression vector. Transient GUS assays in banana embryogenic cell suspensions indicated that cryptic intron splice sites were present within the IR. Transcript analysis revealed two cryptic intron splice sites in the Domain III sequence of the CR-M within the IR. Removal of the CR-M from the TEST vector resulted in an enhancement of GUS expression suggesting that the cryptic intron splice sites had been removed.
An InPAct GUS vector was subsequently generated that contained the modified BBTV IR, with the CR-M (minus Domain III) repositioned within the InPAct cassette. Using transient histochemical and fluorometric GUS assays in banana embryogenic cells, the InPAct GUS vector was shown to be activated in the presence of the BBTV Rep. However, the presence of both BBTV Rep and Clink was shown to have a deleterious effect on GUS expression suggesting that these proteins were cytotoxic at the levels expressed. Analysis of replication of the InPAct vectors by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector through the nicking/ligation activity of BBTV Rep. However, Rep-mediated episomal replicons, indicative of rolling circle replication of the released circularised cassettes, were not observed.
The inability of the InPAct cassette to be replicated was further investigated. To examine whether the absence of Domain III of the CR-M was responsible, a suite of modified BBTV-based InPAct GUS vectors was constructed that contained the CR-M with the inclusion of Domain III, the CR-M with the inclusion of Domain III and additional upstream IR sequence, or no CR-M. Analysis of replication by Southern hybridisation revealed that neither the presence of Domain III, nor the entire CR-M, had an effect on replication levels. Since the InPAct cassette was significantly larger than the native BBTV genomic components (approximately 1 kb), the effect of InPAct cassette size on replication was also investigated. A suite of size variant BBTV-based vectors was constructed that increased the size of a replication competent cassette to 1.1 kbp through to 2.1 kbp.. Analysis of replication by Southern hybridisation revealed that an increase in vector size above approximately 1.5 - 1.7 kbp resulted in a decrease in replication.
Following the demonstration of Rep-mediated release, circularisation and expression from the InPAct GUS vector, an InPAct vector was generated in which the uidA reporter gene was replaced with the ribonuclease-encoding suicide gene, barnase. Initially, a TEST vector was generated to assess the cytotoxicity of Barnase on banana cells. Although transient assays revealed a Barnase-induced cytotoxic effect in banana cells, the expression levels were sub-optimal. An InPAct BARNASE vector was generated and tested for BBTV Rep-activated Barnase expression using transient assays in banana embryogenic cells. High levels of background expression from the InPAct BARNASE vector made it difficult to accurately assess Rep-activated Barnase expression. Analysis of replication by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector but no Rep-mediated episomal replicons indicative of rolling circle replication of the released circularised cassettes were again observed.
Despite the inability of the InPAct vectors to replicate to enable high level gene expression, the InPAct BARNASE vector was assessed in planta for BBTV Rep-mediated activation of Barnase expression. Eleven lines of transgenic InPAct BARNASE banana plants were generated by Agrobacterium-mediated transformation and were challenged with viruliferous Pentalonia nigronervosa. At least one clonal plant in each line developed bunchy top symptoms and infection was confirmed by PCR. No localised lesions were observed on any plants, nor was there any localised GUS expression in the one InPAct GUS line challenged with viruliferous aphids.
The results presented in this thesis are the first study towards the development of a BBTV-based InPAct system as a Rep-activatable suicide gene expression system to control BBTV. Although further optimisation of the vectors is necessary, the preliminary results suggest that this approach has the potential to be an effective control strategy for BBTV. The use of iterons within the InPAct vectors that are recognised by Reps from different ssDNA plant viruses may provide a broad-spectrum resistance strategy against multiple ssDNA plant viruses. Further, this technology holds great promise as a platform technology for the molecular farming of high-value proteins in vitro or in vivo through expression of the ssDNA virus Rep protein.
17
Bolton, Clair Louise. "Development of a Rep-inducible, BBTV-based expression system in banana." Queensland University of Technology, 2009. http://eprints.qut.edu.au/25939/.
Full textAbstract:
Banana bunchy top is regarded as the most important viral disease of banana, causing significant yield losses worldwide. The disease is caused by Banana bunchy top virus (BBTV), which is a circular ssDNA virus belonging to the genus Babuvirus in the family Nanoviridae. There are currently few effective control strategies for this and other ssDNA viruses. “In Plant Activation” (InPAct) is a novel technology being developed at QUT for ssDNA virus-activated suicide gene expression. The technology exploits the rolling circle replication mechanism of ssDNA viruses and is based on a unique “split” gene design such that suicide gene expression is only activated in the presence of the viral Rep. This PhD project aimed to develop a BBTV-based InPAct system as a suicide gene strategy to control BBTV.
The BBTV-based InPAct vector design requires a BBTV intergenic region (IR) to be embedded within an intron in the gene expression cassette. To ensure that the BBTV IR would not interfere with intron splicing, a TEST vector was initially generated that contained the entire BBTV IR embedded within an intron in a β-glucuronidase (GUS) expression vector. Transient GUS assays in banana embryogenic cell suspensions indicated that cryptic intron splice sites were present within the IR. Transcript analysis revealed two cryptic intron splice sites in the Domain III sequence of the CR-M within the IR. Removal of the CR-M from the TEST vector resulted in an enhancement of GUS expression suggesting that the cryptic intron splice sites had been removed.
An InPAct GUS vector was subsequently generated that contained the modified BBTV IR, with the CR-M (minus Domain III) repositioned within the InPAct cassette. Using transient histochemical and fluorometric GUS assays in banana embryogenic cells, the InPAct GUS vector was shown to be activated in the presence of the BBTV Rep. However, the presence of both BBTV Rep and Clink was shown to have a deleterious effect on GUS expression suggesting that these proteins were cytotoxic at the levels expressed. Analysis of replication of the InPAct vectors by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector through the nicking/ligation activity of BBTV Rep. However, Rep-mediated episomal replicons, indicative of rolling circle replication of the released circularised cassettes, were not observed.
The inability of the InPAct cassette to be replicated was further investigated. To examine whether the absence of Domain III of the CR-M was responsible, a suite of modified BBTV-based InPAct GUS vectors was constructed that contained the CR-M with the inclusion of Domain III, the CR-M with the inclusion of Domain III and additional upstream IR sequence, or no CR-M. Analysis of replication by Southern hybridisation revealed that neither the presence of Domain III, nor the entire CR-M, had an effect on replication levels. Since the InPAct cassette was significantly larger than the native BBTV genomic components (approximately 1 kb), the effect of InPAct cassette size on replication was also investigated. A suite of size variant BBTV-based vectors was constructed that increased the size of a replication competent cassette to 1.1 kbp through to 2.1 kbp.. Analysis of replication by Southern hybridisation revealed that an increase in vector size above approximately 1.5 - 1.7 kbp resulted in a decrease in replication.
Following the demonstration of Rep-mediated release, circularisation and expression from the InPAct GUS vector, an InPAct vector was generated in which the uidA reporter gene was replaced with the ribonuclease-encoding suicide gene, barnase. Initially, a TEST vector was generated to assess the cytotoxicity of Barnase on banana cells. Although transient assays revealed a Barnase-induced cytotoxic effect in banana cells, the expression levels were sub-optimal. An InPAct BARNASE vector was generated and tested for BBTV Rep-activated Barnase expression using transient assays in banana embryogenic cells. High levels of background expression from the InPAct BARNASE vector made it difficult to accurately assess Rep-activated Barnase expression. Analysis of replication by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector but no Rep-mediated episomal replicons indicative of rolling circle replication of the released circularised cassettes were again observed.
Despite the inability of the InPAct vectors to replicate to enable high level gene expression, the InPAct BARNASE vector was assessed in planta for BBTV Rep-mediated activation of Barnase expression. Eleven lines of transgenic InPAct BARNASE banana plants were generated by Agrobacterium-mediated transformation and were challenged with viruliferous Pentalonia nigronervosa. At least one clonal plant in each line developed bunchy top symptoms and infection was confirmed by PCR. No localised lesions were observed on any plants, nor was there any localised GUS expression in the one InPAct GUS line challenged with viruliferous aphids.
The results presented in this thesis are the first study towards the development of a BBTV-based InPAct system as a Rep-activatable suicide gene expression system to control BBTV. Although further optimisation of the vectors is necessary, the preliminary results suggest that this approach has the potential to be an effective control strategy for BBTV. The use of iterons within the InPAct vectors that are recognised by Reps from different ssDNA plant viruses may provide a broad-spectrum resistance strategy against multiple ssDNA plant viruses. Further, this technology holds great promise as a platform technology for the molecular farming of high-value proteins in vitro or in vivo through expression of the ssDNA virus Rep protein.
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Kleidon, Jennifer. "Development of an excisable selectable marker system for banana." Thesis, Queensland University of Technology, 2019. https://eprints.qut.edu.au/134478/1/Jennifer_Kleidon_Thesis.pdf.
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This study describes the development of a recombinase-based platform for the removal of selectable marker genes from transgenic bananas. Using a steroid-inducible recombinase enzyme and dual selection vector containing the green fluorescent protein reporter gene, a protocol based on Agrobacterium-mediated transformation of Cavendish banana embryogenic cells was established. The system was then practically applied to generate the first ever marker-free banana plants with potential resistance to banana bunchy top virus. This platform will provide an effective means of improving this economically important crop into the future and is a major step towards public acceptance of genetically modified bananas.
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Hermann, Scott Richard. "Transgene expression in banana." Thesis, Queensland University of Technology, 2001.
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Kato, Maiko. "Molecular strategies for resistance to circular single-stranded DNA viruses." Thesis, Queensland University of Technology, 2019. https://eprints.qut.edu.au/134475/1/Maiko_Kato_Thesis.pdf.
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This thesis describes how the "In Plant Activation" (INPACT) expression platform was adapted into a molecular resistance strategy against circular single-stranded DNA viruses. The system was first optimized using a model geminivirus in the dicot host tobacco then applied to a commercially relevant pathosystem. When used in conjunction with RNAi, the platform provided immunity to the devastating bunchy top virus in the elite banana cultivar "Cavendish". This work shows that a multi-faceted approach to engineered resistance may be a simple means of generating immunity to this important family of viruses.
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Anhalt, Mandy D. "Transmission characteristics of banana bunchy top virus to banana Pentalonia nigronervosa Coq." Thesis, 2007. http://hdl.handle.net/10125/20596.
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Yeh, Hsin-Hung, and 葉信宏. "Cloning and Molecular analysis of Banana bunchy top virus." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/29210634628985811488.
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碩士國立臺灣大學
植物病蟲害學系
81
Large quantity of double and single-stranded（ds and ss） viral DNAs have been purified by using an improved purification method .Compared with viral genome（ss DNA） and hybridization analysis ,it is proved that the ss DNA is the viral DNAs contain both linear and circular form. Two ds viral DNAs have been cloned. Sequencing analysis of the ds DNA clones revealed that the viral ds DNA is circular with a size of 1094 bp. The sequence is part- ialy homologous to the genome of coconut foliar decay virus 1990）and the genome of BBTV published by Dale（1993）,but both sequence homology are not significant.Several ss viral DNAs were also cloned. DNA hybridization tests revealed that there were several forms of viral genome with different degrees of sequence homology. I conclude from these results that BBTV is a new group of circular ss DNA virus, the ds DNA may be the replication intermediate of viral genome ,and the virus contains at least 3 DNA components.51
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Liu, Yu-Tzu, and 劉于滋. "Genome characterization of strains of Banana bunchy top virus." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/33816872750366187490.
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Lin, Chia-Hua, and 林家華. "Genome characterization of the mild strain of Banana bunchy top babuvirus." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/05621904844042226126.
Full textAbstract:
碩士國立臺灣大學
植物病理與微生物學研究所
93
The causal agent of Banana bunchy top disease is Banana bunchy top babuvirus (BBTV). The BBTV-infected banana plants show symptoms of dwarf, bunchy top, leaf atrophy, vein clearing, dark-green streak on pseudostem, and were unable to produce fruits in severe cases. BBTV is a complex circular single-stranded DNA virus consist of six genomic integral components, DNA 1 ~ 6. DNA 1 encodes master replication initiation protein (master Rep) for viral genomic replication. Besides master Rep, components encode additional replication initiation protein (additional Rep) with the self-only-replication function could also be isolated from some BBTV-infected banana plants. BBTV strains can be classified into five types, type I ~ V, defined by symptoms, and could be differentiated by polymerase chain reaction (PCR)-based assay using C1, S and SR primer pairs. Type V is a mild strain, and its complete genome organization has yet been characterized. In this study, primer pairs based on conserved and specific regions of BBTV components were designed. The designed primer pairs and total nucleic acids extracted from each type V isolate was used in PCR reaction. The PCR products were cloned to construct PCR-libraries. Clones from each PCR-library were analyzed by restriction fragment length polymorphisms (RFLP). Randomly selected clones from each RFLP group were sequenced. Based on result of analysis, five integral components, DNA 1 ~ 5, were identified from isolates of type V, V-1 and V-2. However we were unable to obtain DNA 6 from both isolates even two primer pairs designed from DNA 6 conserved region. No signal could be detected from total nucleic acids extracted of V-1 or V-2 isolates by Southern blot analysis using DNA 6 open reading frame as probes. However we were also unable to detect DNAs in the nucleic acids by Southern blot analysis. It indicates that the amounts of BBTV genomic DNA are much lower in the mild strains. Real-time quantitative PCR were performed to quantify the amounts of DNA 1 and DNA 3. The result indicated the amount of DNA 1 and DNA 3 in mild strain isolates were fewer than in severe strain isolates by more than 1000 folds. These data suggest that DNA 6 was absent or had some sequence changes in mild strain. Besides, mild strain had a ca. 0.5 kb PCR product amplified by S primer pairs. After cloning and sequencing of these fragments, we found that they are defective forms of master Reps, and they could be classified into five subgroups by sequence homologies and genome organizations. The result of immunocapture PCR suggests these defective Reps are incapsidated. In addition to the 0.5 kb defective master Reps, another 0.7 kb defective master Rep could also be identified from V-2 isolate. Further studies are needed to resolve if the absence of ordinary DNA 6 and/or the presence of defective Rep could account for symptoms amelioration of mild strain virus infection.
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WU, RUI-YU, and 吳瑞鈺. "Characterization and monoclonal antibodies of the virus causing banana bunchy-top." Thesis, 1987. http://ndltd.ncl.edu.tw/handle/86755465601508651020.
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Chang, Hsin-Yuan, and 張馨元. "The methylation analysis and comparison between Banana bunchy top virus and Banana streak Mysore virus." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/31029277439922695005.
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碩士國立臺灣大學
植物病理與微生物學研究所
99
Banana bunchy top virus (BBTV) and Banana streak Mysore virus (BSMysV) are DNA viruses that causing Banana bunchy top disease and Banana streak virus disease in bananas (Musa spp.), respectively. Generally, BSMysV presents as a free virus, an episome, in hosts. However, BSMysV has integrated into the genome of M. balbisiana, thus, many banana cultivars contain non-infectious BSMysV (eBSMysV). Endogenous pararetrovirus sequences may be activated to become infectious due to stress. BSMysV have an impact on the banana production because of not only its episomal form infecting bananas directly but also eBSMysV which is likely to become infectious. In plants, cytosine methylation is one of the ways to regulate the level of activation of DNA. Some DNA viruses were shown to be methylated by host plants, and among these viruses, the levels of methylation in several viruses are related to the symptom severity. In this study, to determine whether DNA viruses in the banana were transcriptionally silenced, we analyzed the methylation patterns of BBTV and BSMysV by bisulfite sequencing. The methylation levels of DNA-R and DNA-M of BBTV were quite low, and it indicated that BBTV did not induce strong transcriptional gene silencing. By comparing the average percentages of methylation between severe strain and mild strain of BBTV, it showed that the methylation levels of BBTV did not reflect the symptom severity. The difference in the level of methylation between the heart leaf and the old leaf was not statistically significant, so it suggested that the methylation levels of BBTV did not change drastically in different pathogenesis stages. For BSMysV, episomal BSMysV is hypomethylated and doesn’t induce strong transcriptional gene silencing. On the contrary, eBSMysV is hypermethylated, and the methylation rate is significantly higher in symmetric (CG and CHG) than in asymmetric (CHH) cytosines. It indicated that eBSMysV may be transcriptionally silenced. However, there was almost no methylation observed in a partial region of ORF III in eBSMysV, so we speculated that eBSMysV is probable to be activated.
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Yang, Wen-Wen, and 楊文雯. "Studies on genetic transformation of banana for resistance to banana bunchy top virus using Agrobacterium tumefaciens." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/23987718051650668837.
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碩士國立臺灣大學
園藝學研究所
90
The development of a regeneration system from banana (Musa spp. cv. ‘Pei Chiao’, Cavendish, AAA group) hand primordia on male flowers is described. The most important factors affecting embryogenic callus initiation were the source of explant and the composition of the culture medium. The closer the hand primordia is situated to the inflorescence apical meristem, the stronger is the regenerative capability. Improved callus initiation was obtained on culture medium supplemented with 1 mg/l picloram and 4 mg/l 2,4-D. Somatic embryogenesis was observed on callus cultures subcultured consecutively to a culture medium containing 1 mg/l picloram, 4 mg/l 2,4-D, 1 mg/l IAA, and 1 mg/l NAA. Somatic embryo germination and plantlet development was obtained using established protocols. A protocol for the production of transgenic banana (Musa spp. cv. ‘Pei Chiao’, Cavendish, AAA group) was developed via Agrobacterium-mediated genetic transformation of hand primordia. Two disarmed Agrobacterium tumefaciens strains, A 281 and LBA 4404, both carrying the binary plasmid pBIRC1 with the nptⅡ gene were evaluated as vector systems. A number of parameters were tested with respect to maximizing transformation efficiency. While wounding was inhibitory, the pre-culture (12 days), acetosyringone treatment (200 μM), bacterial growth phase (optical density; OD600 = 1.0), co-cultivation period (2 days) had positive effects on transformation. Following co-cultivation, hand primordia were placed on multiplication medium with cefotaxime for 2 weeks then transfered onto the same medium and stressed with geneticin (50 mg/l) for 1 month. Further selection occurred in the medium at an elevated geneticin level (100 mg/l). A number of geneticin-resistant microadventitious buds were multiplied in vitro. Banana plants transformed with the banana bunchy top virus (BBTV) antisense replicase gene were generated and twenty-one independently transformed plant lines were analyzed for resistance to BBTV. Three different responses were obtained. Some of the transgenic plants showed a pre-established, complete, and highly resistant phenotype since no viral symptoms were observed, and no virus detected. Some of the transgenic plants showed no viral symptoms before winter, but showed a delayed viral symptom in the next spring. The remaining plants from these plants showed a rapid disease symptom. Furthermore, the transformants resistant to BBTV genotypeⅠwere secondary challenged with BBTV genotypeⅡ. Transformants exhibited two different responses. One transgenic plant still showed complete, highly resistant phenotype since no viral symptoms were observed, and no virus detected. The other two transformants showed disease symptom after 2 months of inoculation.
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Liu, hsiang-cheng, and 劉向正. "Strains of Banana Bunchy-Top Virus and Transmission Natures of Vector Aphids." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/43800996693926666404.
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碩士國立臺灣大學
植物病理學研究所
88
Single pure isolates of banana bunchy top virus (BBTV) strains were obtained through transmission by vector aphid( Pentalonia nigronervosa ). The BBTV was differentiated into such four strains as severe (S), intermediate (I), mild (M) and latent (L) strains according to symptom expression on Cavendish banana in Taiwan.S strain induced severe typical symptoms including stunted, chlorotic margin and narrow, erect new leaves, bunchy top, vein clearing and dark green dot-dash streaks on petioles. I strain caused mild stunt, slight leaf atrophy and distinct vein clearing. M strain-infected banana plants produced no distinct symptoms except dash-like vein clearing scarcely. No visible symptoms was developed in L-strain-infected plants which were looked like healthy plants. The four strains were differentiated into six genotypes by polymerase chain reaction ( PCR ) amplification with 3 primer pairs (C1-CR, S-CR, SR-CR) followed by electrophoresis analysis. The monoclonal antibodies ( McAbs ) developed against BBTV were recognizing the common epitopes of Taiwan strains, therefore the selected McAb ( 2H6 ) reacted with the all strains of BBTV in Taiwan. However, the 2H6 McAb did not react with a severe isolate of BBTV from Sarawak, Malaysia.The virus is transmitted by the banana aphid in the persistent manner. Although the adult aphids reared on infected banana plants were BBTV positive in PCR tests, none of the offspring of the viruliferous adults were BBTV positive. This fact indicated that BBTV was not transmitted through axeual reproduction. BBTV was transmitted by non-alate adult banana ahpids transmission rate more than alate aphids. The minimum acquisition feeding period of banana aphid is 2 hours. BBTV may multiply in a aphids vectors in way of propagative persistence. The PCR genotypes of BBTV strain in banana identified by PCR tests with the three primer pairs. In direct fluorescence detection in thloem with on UV microscope, the 4 strains showed different degrees of specific fluorescence ( SF ), S strain produced largest amount of SF, L strain produced least SF. At 40℃or 50℃BBTV in tissue dried preserved at 4℃maintained serological activity for at least one year. Appication of systemic insecticides showed insecticidal efficacy for prenention BBTV transmission by banana aphid. No symptom appeared on banana plantlets treated with soil application of comfidor 2GR( 7.5g/plant ) before inoculation by viruliferous aphids.
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ZHONG, YU, and 鍾瑜. "Ecological studies on banana bunchy top virus:virus strains, intermediate hosts and vectorship." Thesis, 1989. http://ndltd.ncl.edu.tw/handle/39616167580381228743.
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Tsao, Li-Yu, and 曹麗玉. "Biologiacl and molecular characterization of banana bunchy top virus strains and their ecology." Thesis, 1998. http://ndltd.ncl.edu.tw/handle/90069743342335214354.
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博士國立臺灣大學
植物病蟲害學系
86
The banana bunchy top virus (BBTV) was differentiated into such four strains as severe (S), intermediate (I), mild (M) and latent (L) strains according to symptom expression in Taiwan. S strain induced severe typical symptoms including dwarfing, slender atrophy leaf with marginal chlorosis, bunchy top, vein clearing and green streaks on petioles. I strain caused mild stunt, slight leaf atrophy and distinct vein clearing. M strain- infected banana plants produced no distinct symptoms except dash-like vein clearing scarcely. No visible symptoms were developed in L strain-infected plants which were looked like healthy plants. The banana plants were frequently infected with strain complex of different strains. Single pure isolates of different BBTV strains were obtained through transmission by vector aphid (Pentalonia nigronervosa). Monoclonal antibody, 2H6 reacted with the all strains, however did not differentiate the strains of Taiwan. The four strains were differentiated into six genotypes by polymerase chain reaction (PCR) amplification with 3 primer pairs (C1-CR, S-CR, and SR-CR) followed by electrophoresis analysis. The foreign isolates of severe strain were mainly identical with Taiwan severe S-3 isolate belonging to PCR genotype II showing amplification pattern, +/+/- with the 3 primer pairs. Only the S-My-3 isolate of Malaysian severe strain was found as distinct as no reaction with 2H6 monoclonal antibody, and PCR amplification pattern of genotype Ⅲ (-/+/-). The isolates of Taiwan severe strain belonged to each half of genotype I and Ⅱ. BBTV was detected in such non-Mussa hosts of vector aphid as garland flower (Hedychium coronarium), Canna spp. and Alpinia formosana by ELISA and PCR assays. The virus was transmitted by aphid between banana plants and alternative host plants reciprocally. The virus distributed quite unevenly in different portions of bananas and alternative host plants. The virus titer was higher in the leaves near by spindle leaf and lower toward outer older leaves. The highest content of virus titer was detected in the petioles and midribes. The incubation periods (lp) of BBTV infecting tissue culture (TC) plantlets of 10 to 50 ㎝ height, were about 1 to 5 months after infection. It was over a lifespan in banana plants over 70 ㎝ high during infection, which developed BT symptoms in the following suckers from the mother plants. The severe strain was transmitted at highest rate of aphid transmission (100%), which the intermediate and mild strains showed 40 to 44% of transmission rates. The banana plants in the field were commonly mix- infected by BBTV and CMV causing banana mosaic. The interaction between the two viruses in a banana plant, affected virus replication and symptom expression. BBTV usually dominated CMV by suppressing development of mosaic symptom. The streak symptoms appeared ahead the bunchy-top symptoms in a same plant inoculates with BBTV and banana streak badnavirus (BSV) simultaneously. Each of the two viruses replicated without counter interaction between the two viruses. Consequently, the symptoms of the two viruses appeared later on, and the BT symptoms dominated finally. The BSV could not be inoculated into a plant infected with BBTV ahead. However, BBTV was able to be inoculated into BSV- infected plants, and replicated normally. The BT symptoms were developed well along with the BS symptoms in the mix-infected plants.
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Shen, Tang-Long, and 沈湯龍. "Detection of banana bunchy top virus using cloned DNA probes and PCR technique." Thesis, 1994. http://ndltd.ncl.edu.tw/handle/34307304823480160273.
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碩士國立臺灣大學
植物病蟲害學系
82
Recently,three different BBTV-DNA components had been cloned and sequenced. The three cloned BBTV-DNA components labeled with biotin were used as DNA probes (Probe C-1, Probe C-2, and Probe C-3) for disease diagnosis. Four pair of oligonucleotides (c-1a/a-1b,c-2a/c-2b,c-3a/c-3b,and s6a/s6b) sythesized according to their sequences,were used as primer pairs in polymerase chain reactions(PCR).Using these BBTV- specific DNA probes in dot hybri dizations,positive results were observed with 30 ∼ 8pg of purified BBTV-DNA extracted from severe strain, and 60 ∼ 15pg from mild strain, except component 1. Cross dot hybridization among BBTV-DNA components showed that distinct cross hybridiza- tion between C-2 and C-3 components,slight dot signal between C-1 and C-1 or C-3,while C-s6 did not show dot signal in corssing with the other 3 components. The detectable end- points of DNA extracted as template in PCR test were found to be 1pg using c-1a/c-1b,1/10pg with c-2a/c-2b, and 1/1000pg with in c-3a/c-3b primer pairs. A simple and rapid method was developed for prepa- ring total DNA from BBTV- infected plants, and was applied to detection and analysis of BBTV-DNA components in different strains, tissues and vector aphids.
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Chen, Jun Hong, and 陳俊宏. "Genomic components of banana bunchy-top virus and genomic analysis of different sympton-type strains." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/00104250278872462552.
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Huang, Yi-Chi, and 黃奕錡. "Biological and molecular characterization of additional replicase encoded components associated with Banana bunchy top virus." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/85788306315155538658.
Full textAbstract:
碩士臺灣大學
植物病理與微生物學研究所
95
Banana is one of the most important fruit crops around the world. Banana bunchy top disease caused by Banana bunchy top virus (BBTV) is a destructive disease for banana. BBTV is isometric and belongs to the genus Babuvirus, in the family Nanoviridae. BBTV consists of six circular single-stranded integral DNA components, DNA 1-6. In some isolates, however, in additional to the integral components other replication competent components (additional Reps) are also associated with BBTV infection. Currently, 8 different additional Reps, Y, W1, W2, W3, W4, S1, S2, and S3, have been reported. To understand the roles of BBTV additional Reps in BBTV infection, we collected samples from field and using primer pairs specific to each BBTV additional Reps to detect the additional Reps. The samples can be grouped into five groups by the presence of additional Reps in a single infected plant. One isolate were selected from each group and use as source to inoculate healthy bananas by using of banana aphids (Pentalonia nigronervosa). Briefly, BBTV associated with S1, or Y plus S2 induced the most severe symptoms, and BBTV associated with Y induced comparatively slight severe symptoms. In addition, BBTV with no additional Reps association induced less severe symptoms. Phylogenetic analysis of additional Reps ORFs (open reading frames) encoded by Nanoviridae reveled that BBTV additional Reps evolved from a common ancestor and later evolved to two directions and form 6 monoclade. And we find that SRCR (stem-loop right site common region; a specific region conserved among BBTV additional Reps) within ORF evolved with a strong selection pressure; which suggest this region contains an unidentified protein motif. To further study the interaction between integral components and additional Reps, we construct BBTV integral components DNA 1-6 and additional Reps Y and S1. Each infectious clone consists of greater-than-unit (range 1.2 to 1.6 mers). We have demonstrated the replication of BBTV DNA 1 infectious clones in banana suspension cells.
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Liu, Chien-wei, and 劉建瑋. "Preparation of antibodies against bacteria expressed coat protein of Banana bunchy top virus and their application on virus detection." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/07642338410126209071.
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碩士朝陽科技大學
生物技術研究所
93
Banana bunchy top virus (BBTV) is one of the most devastating banana diseases (Musa spp.) around the world. Its genome consists of single-stranded DNA and it is transmitted in a persistent manner by vector aphid (Pentalonia nigronervosa). The symptoms induced by BBTV include chlorotic margin, narrow erect and witches’ broom-like leaves. The infected plants are usually evidently stunted and their yield and quality of banana fruits are significantly affected. Unlike the conventional means of propagation by vegetative suckers, bananas are presently propagated mainly by tissue culture plantlets. Screening for virus-free banana mother plants for mass production of tissue culture plantlets is a crucial and necessary step in modern banana production system. Therefore, to development a sensitive and efficient virus detection technique is always an attractive research topic. In the past decades, serological methods are widely and routinely used for the detection of BBTV. Monoclonal antibodies are normally used as detection kits for BBTV because the preparation of polyclonal antibodies by conventional means is very difficult comparing with other viruses diseases. This is because purification of BBTV virus particles from infected banana tissues is highly difficult. In this research, we took the approach of cloning and expressing the BBTV CP gene in bacteria and using the bacteria expressed CP as immunogen for antiserum preparation. We designed a set of primer (BBTV-up/ BBTV-dw) according to BBTV’s coat protein (CP) gene sequences documented in the GenBank and successfully amplified a 1023 bp DNA product by polymerase chain reaction (PCR) from banana specimens collected from Taichung County. The PCR product was later cloned and sequenced and found that it contained the coat protein gene of BBTV. In order to clone the CP gene into the bacterial expressing vector plasmid, pET-28b(+), we designed three types of construction of BBTV CP expression cassette. Using three sets of primers (BBTV-up1/ BBTV-dw1, BBTV-up1/ BBTV-dw2 and BBTV-up2/ BBTV-dw3) designed in this study, the complete or part of BBTV CP gene were successfully cloned and directionally constructed into pET-28b(+), allowing the bacteria (E. coli strain Rosetta) to express three different types of BBTV CP related fusion proteins. The three different constructs, namely FL-2, FL and CR, can synthesize fusion proteins with molecular weights of 21 kDa, 19 kDa and 14 kDa, respectively. These three proteins were shown serologically reacted to an antiserum to BBTV purchased from Agdia Inc., USA, indicating that they were originated from BBTV CP gene. Three antisera with the code numbers of #115, #123, and #121 were subsequently produced by immunizing three rabbits with the aforementioned fusion proteins synthesized by constructs FL-2, FL and CR, respectively. In SDS-immunodiffusion tests, these three antisera were found not feasible to detect infected banana plant antigens. However, they all can be used for the detection of BBTV CP in Western blotting tests. In ELISA, only the antiserum #115 was confirmed to be useful in the detection of BBTV infected plants in the field. The other two antisera need further experiments to confirm their feasibility to be applied in ELISA tests. Using antiserum #115, higher percentage of BBTV detection was always obtained by taking midribs as sample for ELISA indexing than taking the interveinal tissues. Of 23 midrib sampling from different banana plants with severe marginal necrosis symptoms, 20 of them were positively detected in ELISA. For those three samples non-detected were subsequently confirmed as BBTV-infected ones by PCR, indicating some severely BBTV-infected plants might accumulate too low of virus concentration in the midribs to be detected by ELISA. On the contrary, when taking the midribs from banana leaves with mild or no symptoms for ELISA indexing, 100% of detection was obtained. This result indicates that the antiserum #115 against bacteria expressed BBTV CP is feasible to be applied in ELISA for the detection of BBTV in banana fields.
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Su, Yi-Rong, and 蘇怡蓉. "Studies on the expression of Banana bunchy top virus coat protein (CP) through the help of a leader sequence from N-terminal region of Cymbidium mosaic virus CP gene." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/74952327050024758393.
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碩士朝陽科技大學
生化科技研究所碩士班
100
Banana bunchy top virus (BBTV) is taxonomically belonging to the genera of Babuvirus of Nanoviridae family. It infects banana systemically and induces symptoms of stunting, shortening of internodes, small and upward leaves and witches’ broom like of apical stem. Dark greenish lines can be observed on the diseased leaves, midribs, and petioles and the leaf margins turn frequently from yellowing into necrotic that weaken significantly the vigor of the infected plants. Besides aphid transmission, the virus can also be easily transmitted by vegetatively propagated suckers. Nowadays, since industrialized production of banana depends mainly on tissue cultured plantlets as growing materials, the virus indexing of mother plants before tissue culture cloning becomes highly important. Traditionally, detection of BBTV can be approached by both serological and molecular based techniques. However, production of antibody against BBTV is always confined by the difficulty of purifying virions from infected banana tissues. Our laboratory once tested the possibility of producing recombinant coat protein (CP) of BBTV by bacterial expressing technology, but the result was not satisfactory due to low expression of its CP in pET system. In a separate study, we found that a construction of pET-28b(+) expression cassette, namely CyOrN, using a 177 bp sequence encoding 59 amino acid residues from the N-terminal region of coat protein (CP) gene of Cymbidium mosaic virus (CymMV) as upstream leader sequence followed by partial sequences of Odontoglossum ringspot virus (ORSV), could generate significant quantity of expressed recombinant viral proteins. In this study, we took the advantage of this unique expression cassette, CyOrN, by maintaining the sequence of CymMV but replacing the following ORSV sequences with the complete or partial CP gene sequences of BBTV and tested the differential accumulation of different recombinant BBTV coat proteins. The CP gene of BBTV was obtaine by amplification and cloning in pGEM-T Easy vector using primer pairs designed in this study. To facilitate later directional cloning into expressing cassette of CyOrN for replacement of ORSV sequences, we designed the primers by creating a Pst1 site at the 5’-terminus and Xho1 site at the 3’-terminus of the amplified BBTV CP genes. After amplification using the designed primers, the BBTV amplicons were then digested with Pst 1 and Xho 1 restriction enzymes and directionally cloned into CyOrN expression plasmid separately. Three constructions of pET-28b expression plasmids, namely CyBTFCP, CyBTNCP and CyBTCCP, expressing full length (541 bp), N-terminal (304 bp), and C-terminal (198 bp) of BBTV CP gene sequences were made and tested, respectively. These plasmids were then separately transformed into host bacteria, E. coli strain Rosetta, and analyzed its protein expression by induction the bacteria cultures with IPTG. In agarose gel electrophoresis, recombinant proteins with expected sizes of 26 kDa, 17 kDa and 13 kDa were located in the bacterial lysates containing expression plasmids of CyBTFCP, CyBTNCP and CyBTCCP constructs, respectively. These proteins were confirmed by Western blotting tests all reacting positively with antisera against CymMV. The expression levels of these three recombinants were all significantly higher than the control clone of pET-28b expressing only BBTV CP sequence. Two of the expression constructs (CyBTFCP and CyBTNCP) were selected for mass production of their recombinant proteins in IPTG induced bacteria cultures. After purification the recombinant proteins by preparative PAGE, they were used as immunogens to produce their respective antisera (anti-CyBTFCP and anti-CyBTNCP) in rabbits. Both antisera were shown in SDS-immunodiffusion and Western blotting tests to react strongly and specifically with their homologous recombinant proteins and to CymMV CP, indicating the recombinant proteins had indeed raised antibodies against epitopes from CymMV CP sequences. Therefore, these recombinant proteins should theoretically be able to induce antibodies against BBTV CP due to the co-expression of BBTV and CymMV CP sequences in the same protein. This was later confirmed by the revelation of reactivity of both antisera with bacteria expressed BBTV CP in Western blotting test. In the later experiments, we tested the reactivities of both antisera in indirect ELISA with BBTV-infected bananas and CymMV-infected orchids and found that the antiserum against full length BBTV CP (anti-CyBTFCP) could only strongly react with CymMV-infected orchids but not with BBTV-infected bananas. However, antiserum against N-terminal sequences of BBTV CP (anti-CyBTNCP) could give positive reactions against both CymMV-infected and BBTV-infected banana tissues, although the reactivities with BBTV were not as high as those against CymMV. Altogether, we have shown in this study that by the use of CymMV leader sequence in the same pET-28b expression vector, different size of BBTV CP can be expressed and the expression level was significantly higher than the control pET-28b plasmid expressing only BBTV CP sequence. We also showed that the expressed recombinant proteins of CymMV and BBTV can induce both antibodies against CymMV and BBTV. Furthermore, at least one of the antiserum against N-terminal sequence of BBTV CP can be used in indirect ELISA and positively detects BBTV infection in bananas.