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本研究采用 PCR点突变的方法 ,对豇豆退绿斑驳病毒 (CCMV) RNA3的 c DNA克隆进行突变 ,突变体 TP7是在外壳蛋白基因之前突变形成一个独特的 Sal I酶切点 ;TP8是在 3a基因之前突变形成一个独特的 Bam H I酶切点 ;AG 1是在外壳蛋白基因之后突变形成一个独特的 Not I酶切点。TP7、TP8、AG1体外转录产物分别与 CCMV RNA1、RNA2的 c DNA克隆的体外转录产物混和后接种豇豆 ,结果表明 ,接种后第 6 d TP7植株首先表现出症状 ,9d后 TP8、AG1和野生型 CCMV接种的植株表现出症状 ,TP7的症状发展最快。采用 TP7、TP8、AG1接种 2 0 d的毒源植株 ,按 TP7+ TP8、TP7+ AG1、TP8+ AG1等量混合汁液接种豇豆 ,接种 2 0 d,通过 RT- PCR分析接种植物 ,在 TP7+ TP8和 TP7+ AG1混合侵染中 ,TP7在混合侵染中占优势 ,在 TP8+ AG1的混合侵染中 ,TP8和 AG1的种群十分接近。
In this study, PCR mutation was used to mutate c DNA clone of cowpea green mottle virus (CCMV) RNA3. Mutant TP7 was mutated before the coat protein gene to form a unique Sal I enzyme cleavage site. Genes prior to mutation form a unique Bam HI cleavage site; AG 1 is mutated after the coat protein gene to form a unique Not I enzyme cleavage site. The TP7, TP8 and AG1 in vitro transcripts were inoculated with the in vitro transcripts of the c DNA clones of CCMV RNA1 and RNA2, respectively, and then inoculated with cowpea. The results showed that the TP7 plants displayed symptom first on the 6th day after inoculation. After 9 days TP8, AG1 and wild type CCMV inoculated plants show symptoms and the symptoms of TP7 are the fastest growing. The plants were inoculated with TP7, TP8 and AG1 for 20 days. The cowpea was inoculated into equal volume of TP7 + TP8, TP7 + AG1, TP8 + AG1 and inoculated for 20 days. The plants were inoculated with TP7 + TP8 and TP7 + AG1 In mixed infection, TP7 predominates in mixed infection. In the mixed infection with TP8 + AG1, the populations of TP8 and AG1 are quite close.