葡萄抗霜霉病基因同源序列的克隆与分析

Cloning and Analysis of Downy Mildew Resistant Gene Analogues in Grapevine

依据抗病基因在保守区域序列同源的原理,针对已知抗病基因NBS保守结构区域设计9条简并引物,以高抗霜霉病的葡萄品种贝达为材料,人工接种葡萄霜霉病菌15d后,应用PCR技术从叶片cDNA中分离出2条抗霜霉病基因同源序列VR5和VR16,并提交到Genbank,其中VR5含有507bp,登录编号为JQ763391;VR16含有562bp,登陆编号为JQ763392。2条序列均含有P-环(GGVGKTT)、kinase-2(VLDD)、kinase-3(GSRII)及HD(即GLPL)等植物抗病基因中的保守结构。聚类分析结果表明,VR5与亚麻抗病基因L6聚为一类,VR16与拟南芥抗病基因RPS2聚为一类,编码氨基酸序列同源性分别为38%和56%。

Based on the NBS conserved sequences of known resistance genes, nine degenerated primers were designed to be used for amplifying grape downy mildew resistance gene analogs （RGAs）. In this study, 2 RGAs, were obtained from eDNA in Beta grape （high resisitance to downy mildew） that had been infected by downy mildew for 15 days and registered in the Genebank, VR5 （Accession number： JQ763391） has 507bp and VR]6 （Accession number：JQ763392） has 562bp. Sequence analysis indicated that the two cloned RGAs both have four motifs of NBS conserved regions, including P-Ioop（GGVGKTF）, kinase-Z（VLDD）, kinase-3（GSRII） and membrane spanning region（GLPL）. Cluster analysis results showed that VR5 and VRI6 were grouped with L6 （disease resistance gene in Linum） and RPS2 （disease resistance gene in A rabidopsis）, the amino acid sequence homology was 38% and 56%, respectively.