马铃薯卷叶病毒分离株外壳蛋白基因和17k蛋白基因的克隆及序列分析

The cloning and sequencing of coat protein and 17k protein genes for an isolate of potato leafroll virus

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 。

Total RNA of potato leafroll virus (PLRV) was extracted from virus-infected potato leaves collected from fields in Zhangjiakou of Hebei. A pair of DNA primers were synthesized based on the nucleotide sequence of the PLRV coat protein (CP) gene and the cDNA was amplified by reverse-transcription polymerase reaction (PCR). The unique amplified product was then cloned into the pGEM-3Zf(+) vector and sequenced. The results showed that the cloned segment was 627bp and contained two open reading frames (ORFs).The first ORF is 627bp and encodes 208 amino acid residues with relative molecular weight of 23 000 which is the length of the PLRV CP,the second ORF is 465bp which is different from the CP gene and encodes 155 amino acid residues with relative molecular weight of 17 000. Comparison of the nucleotide sequence between the CP and the 17k protein genes and those of several foreign PLRV isolates showed that the homology of are 97.5%～99.7% and 96.5%～99.6% respectivelyi the homology of the putative amino acid sequence are 97.6%～99.5% respectively. It was suggested that the CP gene of PLRV isolate is a relatively conserved sequence within the PLRV genome.This will lay a foundation for breeding the transgenic potato resistance to PLRV.