水稻矮缩病毒基因组第五号片段编码区的cDNA克隆及序列分析

cDNA CLONING AND SEQUENCE ANALYSIS OF RICE DWARF VIRUS GENOME S5

水稻矮病毒(RDV)基因组第五号片段S5编码病毒的外层外壳蛋白(outer layer coatprotein)。从RDV中国福建分离物中分离出S5的双链RNA,反转录成cDNA,再利用PCR扩增了编码区cDNA,将扩增产物克隆到载体pBluescript上。经限制性内切酶分析,进而进行亚克隆和序列测定。结果表明,克隆片段全长2449bp,包含一个编码801个氨基酸的完整开放阅读框架。与RDV日本分离物S5相比,核苷酸和由此推出的氨基酸的同源率分别为94.5％和97.5％。与同组伤瘤病毒(WTV)相比,核苷酸和氨基酸的同源性分别高达58.1％和51.1％,S5是RDV与WTV基因组中同源性最高的片段。

cDNA of Rice Dwarf Virus (RDV) genome S5 which encodes the viral outer layer coat protein was synthesized and amplified by polymerase chain reaction (PCR). The product was then cloned into pBluescript (SK) and was identified by restriction enzyme digestion. Furthermore , nine subclones which covered the entire coding region were constructed and the complete nucleotied sequence was determined by Sanger's dideoxy-mediated chain-termination method. The cloned fragment is 2449bp in length and contains a large single open reading frame of 2403bp. It shares 97. 5% and 94. 5% dentity with RDV S5 of Japanese isolate in terms of amino acid and nucleotide sequences,respectively. Compared with Wound Tumor Virus (WTV) which is the type member of the group,the homologies on amino acid and nucleotide levels are 58. 1% and 51. I % respectively. Therefore, S5 is the fragment with the highest identity between RDV and WTV.