【摘要】 以克隆水稻矮缩病毒(Ricedwarfvirus,RDV)基因组片段S11、S12为例,报道一种克隆植物dsRNA病毒基因组的方法。具体过程为:利用T4RNA连接酶将5′磷酸、3′氨基修饰的引物Primer1连接到RDV病毒基因组第11、12片段dsRNA的3′OH端,经逆转录、退火、补齐形成全长双链cDNA,使用单一的互补引物Primer2进行PCR扩增,扩增产物克隆在pMD18T载体上,对重组子进行两次限制性内切酶分析,结合序列测定分离鉴定S11、S12。结果表明,这种方法能同时克隆RDV基因片段S11、S12,是一种有效实用的dsRNA病毒基因组克隆方法。更多还原

【Abstract】 Based on the cloning of RDV(Rice dwarf virus) gene segment 11,12, single primer amplification, a method for the cloning of viral dsRNA genomes, was introduced.A single amino-linked modified oligonucleotide (primer 1) was ligated to either end of dsRNA genome segments by using T4 RNA ligase. Reverse transcription was done by PCR using a single complementary oligonucleotide (primer 2). The amplified cDNA was cloned into the pMD 18-T vector. And then RDV S11,S12 were determined by restricting the recombinant plasmids with endonuclease and gene sequencing. The results showed:S11 and S12 segments of RDV could be simultaneously cloned. The method for cloning viral dsRNA genomes is practicable and valid.更多还原