摘要
本文报道应用聚合酶链式反应(PCR)技术,在体外扩增马铃薯 Y 病毒外壳蛋白基因及其克隆和序列分析的结果。病毒 RNA 从马铃薯 Y 病毒感染的烟草叶片中提取,用合成的PCR 3引物及 AMV 逆转录酶合成了单链的 cDNA。利用 PCR 技术,经30个循玎的扩增。得到了一特异的0.8kb 片段。克隆后对此片段进行了限制性内切酶物理图谱分析,并测定了其全序列。实验结果证明,我们克隆到的是完整的马铃薯 Y 病毒的外壳蛋白基因。与国外报道的马铃薯 Y 病毒 N 株相比,其核苷酸序列及推测的氨基酸序列的同源率分别为97.8%和97%。将该基因导入马铃薯以期获得抗 Y 病毒马铃薯的工作正在进行。本文还对 PCR 技术用于扩增植物 RNA 病毒的方法以及用基因工程方法培育抗病毒作物新品种的可行性等进行了讨论。
The results of cloning and sequencing of the gene encoding coat protein(CP)of potato virus Y(PVY)with PCR technique are reported in this paper.The virus RNA was extracted from tobacco leaf infected with PVY.The first strand of cDNA was synthesized from viral RNA template primed with synthetic 3' PCR primer using AMV reverse transcriptase.A DNA fragment with 0.8 kb was obtained after 30 PCR amplification circles.The restriction map of the DNA fragment has been analyzed and its whole DNA sequence has been determin- ed.The results show that the entire gene encoding the coat protein of PVY has been cloned. The homologies of the DNA sequences and the deduced amino acid sequence between the PVY shown here and the N-strain of PVY published abroad are 97.8% and 97% respectively.The work of transferring the CP gene into potato to obtain transgenic plants that resist to PVY infection is in progress.
关键词
克隆
外壳蛋白基因
马铃薯Y病毒
Potato virus Y
Virus coat protein gene
Plant genetic engineering of Virus-resistance
PCR
