【摘要】 以克隆水稻矮缩病毒(Rice dwarf virus.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 T4 RNA 连接酶将5′-磷酸、3′-氨基修饰的引物 Primer 1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 cD-NA,使用单一的互补引物 Primer 2进行 PCR 扩增,扩增产物克隆在 pMD 18-T 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 intro- duced.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 re- sults showed:S11 and S12 segments of RDV could be simultaneously cloned.The meth- od for cloning viral dsRNA genomes is practicable and valid.更多还原