依据抗病基斟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,以高抗霜碡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VRl6,并提交到Genbank,其中VR5含有507bp,登录编号为JQ763391;VRl6含有562bp,登陆编号为JQ763392。2条序列均含有P-环(GGVGKTr)、kinase。(VLDD)、kinase。(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,J6聚为一类,VRl6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%$N56%。
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.