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从典型的马铃薯卷叶病毒病株中直接提取出植物总RNA ,根据PLRV外壳蛋白基因序列 ,设计合成了一对寡核苷酸引物 ,经RT -PCR法扩增出全长的cDNA片段 ,将其克隆到质粒pGEM - 3Zf (+)上 ,并进行了序列分析。结果表明 ,克隆的cDNA片段由 6 2 7个核苷酸组成 ,编码 2 0 8个氨基酸组成的理论分子量为2 3k的蛋白 ,与PLRV外壳蛋白的大小一致 ;此外克隆片段还含有一个不同于外壳蛋白基因的阅读框架 ,该框架由 46 5个核苷酸组成 ,编码 15 5个氨基酸组成的理论分子量为 17k的蛋白 ,与PLRV的 17k蛋白大小一致。与国外报道的几个株系相比 ,PLRV分离物的外壳蛋白基因及 17k蛋白基因的核苷酸同源率分别高达97 5 %～ 99 7%和 96 5 %～ 99 6 % ,由此推测的氨基酸同源率高达 97 6 %～ 99 5 %和 96 1%～ 99 4% ,说明所克隆的PLRV外壳蛋白基因和 17k蛋白基因具有高度的保守性 ,本研究克隆了PLRV分离物外壳蛋白和17k蛋白基因的序列 ,为进一步进行抗PLRV基因工程研究奠定基础
Total RNA was extracted directly from the typical potato leaf roll virus strain. A pair of oligonucleotide primers were designed and synthesized according to the PLRV coat protein gene sequence. The full-length cDNA fragment was amplified by RT-PCR, It was cloned into plasmid pGEM - 3Zf (+) and sequenced. The results showed that the cloned cDNA fragment consisted of 627 nucleotides and encoded a protein of 280 kD in theoretical molecular weight of 23k, which was consistent with the size of PLRV coat protein. In addition, the cloned fragment also contained a different protein This frame consists of 465 nucleotides and encodes a protein of 15k amino acids with a theoretical molecular weight of 17k, which is consistent with the size of 17k protein of PLRV. Compared with several foreign reported strains, the nucleotide homology of the coat protein gene and 17k protein gene of PLRV isolates were as high as 97.5% -99.7% and 96.5% -99.6%, respectively The amino acid homology of the PLRV isolates was as high as 97 6% -99 5% and 96 1% -99 4%, indicating that the cloned PLRV coat protein gene and the 17k protein gene were highly conserved. In this study, 17k protein gene sequence, laid the foundation for further anti-PLRV genetic engineering