花生條斑病毒Ts 系統基因序列之解讀與分析

Abstract

在台灣田間的感病花生植株中可以分離出花生條斑病毒(Peanut stripe virus, PStV) 嚴重型(Ts) 和普通型(Tc) 兩種系統。PSt V-Tc 在落花生上所引起之病徵，主要為沿葉脈上出現深綠色條斑(stripe) 或斑塊(blotch)，感病葉片有凹凸不平現象，嚴重者扭曲變形，植株矮化，生長不良。嚴重型分離株(PSt V-Ts)，除引起上述之病徵外，葉片下表面主脈上還會出現壞疽(necrosis)情形，隨病勢之進展壞疽還會延伸至葉柄與莖部，感病植株極度矮化生長不良，產量損失可達67% 以上。本研究將感染PSt V- Ts 之紅豆病葉萃出液經polyethylene glycol (PEG) 沈降及硫酸銫等密度離心後可得純化之病毒顆粒，病毒RNA 萃取後經電泳顯示其長度均約10 kb。針對Ts系統進行基因序列之定序，近全長度基因體共含10009 個核啟酸(Acc. No.：AY968604)，其大小和其它potyviruses 相似。基因體RNA 只含一個轉譯架構，由第87 個核啟酸起轉譯至第9755 止，共轉譯出一個含3222 個胺基酸的複合蛋白。將所完成之P S t V- Ts 近全長度核酸與本實驗室已譯讀完成之黑眼豇豆嵌紋病毒台灣系統(Blackeye cowpea mosaic virus Taiwan strain,B l C M V-TW) 及國外之花生條斑病毒(Peanut stripe virus blotch strain, PStV-B), 菜豆普通嵌紋病毒R 系統(Bean common mosaic viru s R strain, BCMV-R) 和Y 系統( B C M V- Y )、比較各基因核啟酸序列與胺基酸序列相同度，各基因之核啟酸與胺基酸除P 1、P3 及6K2 基因外，皆高於8 0 %；尤其P S t V- Ts 與國外發表之P S t V-B 相同度最高，除P1 外其餘基因的相同度都在90% 以上。這幾種病毒皆保有coat protein (CP) 基因N 端與蚜蟲傳播有關的DAG motif，在NIb 蛋白質基因亦保留了核心複製瓷活性部位GDD motif。在helper component protease (HC-Pro) 蛋白皆保留了KITC 與PTK motifs。比較各基因核啟酸序列與胺基酸序列相同度，結果顯示這幾種病毒確為 菜豆普通嵌紋病毒(BCMV) 之不同系統。 Peanut stripe virus ( PStV ) is a member of genus P o t y v i ru s in the family of P o t y v i r i d a e. It is an important virus infecting peanut and legume crops in Taiwan. PStV was found in Taiwan, Japan, China, Thailand, Malaysia and America. Virons of PStV Ts strain (PStV- Ts) was purified from leaves of infected asparagus bean by polyethylene glycol precipitation followed by a Cs2S O4 i s o p y c n i c centrifugation. Electrophoretic analysis showed that PStV- Ts contained a 10 kb viral RNA. The cDNAs of PStV- Ts genes were synthesized by using cDNA synthesis technology and polymerase chain reaction (PCR). Nearly full length of viral genome was sequenced. PStV- Ts contained 10009 nucleotides including the 5' and 3'-nontranslated region. The base composition of PStV- Ts RNA (Acc. No. AY968604) shows similar to those of other potyviruses. The genomic RNA of PStV- Ts contains an open reading frame which starts at 87th position and terminates at 9752th position, encoding a polyprotein of 3222 amino acid residues. Comparison of the nucleotide and amino acid sequences of genes with Blackeyes cowpea mosaic virus TW strain (BlCMV-TW) and those reported for B e a n common mosaic viru s R strain (BCMV-R) and Y strain (BCMV-Y) and Peanut stripe virus blotch strain (PStV-B) showed that BCMV- Y, BCMV-R, PStV-B and BlCMV-TW shared over 80% identities, excluding P1, P2 and 6K2 genes. Comparison of the nucleotide and amino acid sequences of genes between PStV- Ts and PStV-B showed that both shared over 90 % identities, excluding the P1 gene. DAG motif involving in the aphid transmission, the conserved active site GDD motif of the core replicase, and the KITC and PTK motifs were found in the N-terminal of CP gene, the NIb gene, and the Hc-Pro gene of PStV- Ts, BCMV- Y, BCMV-R, PStV-B and BlCMV- T W, respectively. The comparison of multiple aligments of nucleotide and amino acid sequences among BCMV- Y, BCMVR, PStV-B and BlCMV-TW confirmed that they are different strains of BCMV.