【摘要】 通过RT-PCR获得了玉米粗缩病病毒湖北分离物(Hbm)基因组片段S8和S9的全长cDNA克隆,并测定了它们的全序列。S8全长1936bp,其编码链只含1个ORF,编码1个由591个氨基酸组成、分子量约为68kD蛋白;S9全长1900bp,含有两个非重叠的ORF,分别编码由347个和209个氨基酸组成、分子量分别约为40kD和24kD蛋白。序列分析表明,Hbm-S8和S9与水稻黑条矮缩病毒(RBSDV)中国不同分离物之间的核苷酸和氨基酸序列同源性分别为94.4%~98.9%和96.0%~99.0%,且与日本RBSDV的序列同源性高于与玉米粗缩病毒相应片段的序列同源性,进一步证明了引起我国玉米粗缩病的病原为RBSDV。SDS-PAGE分析表明,经IPTG诱导,RBSDV8和9编码的蛋白可在大肠杆菌(Escherichiacoli)中高效表达。以这些蛋白为抗原,制备了抗血清。用Westernblot方法在感病的玉米(Zeamays)植株中可检测到RBSDV-HbmS8和S9ORF1编码的蛋白。更多还原

【Abstract】 The full-length cDNAs of genomic segments 8 (S8) and 9 (S9) from a virus isolate (Hbm) causing maize rough dwarf disease in Hubei Province were cloned by RT-PCR and their complete sequences were determined. Hbm-S8 and S9 consisted of 1 936 and 1 900 bp, respectively. Hbm-S8 had a single open reading frame (ORF) and encoded a 68 kD polypeptide containing 591 amino acids. Hbm-S9 contained two non-overlapping ORFs encoding 347 (ORF1) and 209 (ORF2) amino acids. Sequence analysis showed that Hbm-S8 and S9 shared 94.4%~98.9% nucleotide sequence identities and 96.0%~99.0 % amino acid sequence identities with the different Chinese isolates of Rice black-streaked dwarf virus (RBSDV). Furthermore, the sequence identities to the Japanese RBSDV were higher than those to the corresponding segments of Maize rough dwarf virus at nucleotide or amino acid level. These results further supported that the pathogen responsible for maize rough dwarf disease in China was RBSDV. The proteins encoded by S8 and S9 were expressed as fusion proteins in Escherichia coli at high level. Each antibody to the expressed RBSDV-Hbm S8 ORF and S9 ORF1 products detected a single protein of the predicted size in total protein extracts from infected maize(Zea mays) leaves using Western blot.更多还原