Genetic analyses of bamboo mosaic potexvirus (BaMV) symptom determinant and construction of BaMV Vector

• Main Authors: Jia-Teh Liao, 廖家德
• Other Authors: Yau-Heiu Hsu
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/80524813208497500857

博士 === 國立中興大學 === 農業生物科技學研究所 === 88 === We have constructed the full-length infectious cDNA clone of bamboo mosaic potexvirus (BaMV) and studied the potential use of BaMV as a gene expression vector in plants. BaMV has a 6.4-kb single stranded positive-sense RNA genome with five conserved open reading frames. In contrast to previously described BaMV strain O (BaMV-O) which causes symptomless infection on Nicotiana benthamiana and small necrotic local lesions on Chenopodium quinoa, a mutant derived from BaMV-O, denoted as BaMV strain S (BaMV-S), causes severe yellow mosaic symptom on N. benthamiana and necrotic local lesions on C. quinoa. The full-length infectious cDNA clones of BaMV-S were constructed downstream of the T7 RNA polymerase promoter by reverse transcriptase-polymerase chain reaction (RT-PCR). Two cDNA clones were sequenced and compared. The specific infectivity of capped BS 2-8 transcripts produced in vitro was similar to viral RNA in N. benthamiana and C. quinoa. The sequence comparison between BaMV-O and BaMV-S (BS 2-8) revealed 94 nucleotide changes evenly distributed in the RNA genome that resulted in 24 mutated amino acids. The sequence identities of BS 2-8 cDNA were 98.46% and 89.51% when compared with BaMV-O and BaMV-V, respectively. BS 2-19 transcripts, another BaMV-S cDNA clone, showed smaller necrotic local lesions on C. quinoa and did not infect N. benthamiana systemically which were distinct to authentic virus. There were twenty and eight amino acids changes found in BS 2-19 when compared with BaMV-O and BS 2-8 respectively. To evaluate the infectivity differences between BaMV-S (BS 2-8) and BaMV-O, a series of clones with different poly(A) tail length were constructed and examined on C. quinoa. The infectivity was found to reflect poly(A) tail length in both BaMV-S and BaMV-O genomes. These results suggest that the infectivity differences between BaMV-S and BaMV-O were related to the ability of replication and accumulation. In order to examine the symptom determinant on BaMV genome, chimeric viruses between the two strains were constructed and inoculated onto C. quinoa and N. benthamiana plants. The results showed that the 3'' region from nt 5318-6366 plays an essential role on symptom expression. PCR based mutagenesis revealed that the single amino acid difference in coat protein gene did not confer the BaMV symptom expression on C. quinoa and N. benthamiana. To test the use of BaMV as a gene expression vector, the green fluorescent protein (GFP) gene was inserted into BaMV cDNA clone with duplicated region encompassing the putative CP promoter at three different positions: between ORF1 and ORF2, between ORF4 and CP gene, and behind CP gene. These clones were denoted as BV2-GFP, BV-GFP, and BV3-GFP, respectively. The most stable expression cassette was BV-GFP. Systemic expression of foreign protein (GFP) on monocots barley (cv. Larker) and rice (Taichung Native 1) was demonstrated by Western blot analysis.