用改进的SSAP方法克隆抗甘薯茎线虫病相关的RGA  

Cloning of RGA Related to Stem Nematode Resistance of Sweetpotato (Ipomoea batatas (L.) Lam.) with Modified SSAP

作　　者：柳哲胜[1] 刘庆昌[1] 翟红[1] 玉萍 

机构地区：[1]中国农业大学农学与生物技术学院,北京100094

出　　处：《分子植物育种》2005年第3期369-374,共6页Molecular Plant Breeding

基　　金：国家杰出青年科学基金(30225028);国家863(2004AA241180);教育部教学科研奖励计划资助项目资助。

摘　　要：甘薯新品系农大603是从感茎线虫病品种徐薯18的辐照后代中获得的一个抗茎线虫病的突变体。根据已报道的植物线虫抗性基因的核苷酸结合位点(NBS)保守氨基酸序列设计兼并引物,用自行改进的SSAP(modified sequence-specific amplification polymorphisms)方法,对农大603和徐薯18的基因组进行PCR扩增,从农大603中扩增出含有抗性基因NBS保守序列的差异片段2个(片段54和片段72)。在GenBank上序列搜索和比对发现,克隆序列与已报道的植物抗病基因之间同源性较低。根据克隆片段的DNA序列设计引物,以抗、感茎线虫病的甘薯品种的基因组DNA为模板进行PCR扩增,结果显示由片段72设计的引物在抗、感品种上没有扩增出多态性带;由片段54设计的引物在抗病品种和感病品种之间扩增出多态性带,推测片段54是与甘薯抗茎线虫病有关的RGA。A mutant, Nongda 603, resistant to sweetpotato stem nematode was obtain ed from the gamma-irradiated progenies of sweetpotato cv. Xushu 18 susceptible t o the stem nematode. Degenerate primers were designed from the conserved NBS (nu cleotide-binding site) motifs of nematode resistance genes isolated from other c rops, and used in modified SSAP (sequence-specific amplification polymorphisms) PCR amplification with the temple of Nongda 603 and Xushu 18. Two fragments (the fragments 54 and 72) with NBS conserved domain of resistance genes were amplifi ed from Nongda 603, not from Xushu 18. DNA and peptide-sequence similarity and h omology between the sequence of fragments cloned and the resistance genes report ed in other crops were determined by blast analyses through GenBank. The results showed that these two sequences had low homology to the resistance genes report ed. Primers designed from the sequences of the two fragments cloned were used in PCR amplification with the templates of resistant and susceptible sweetpotato v arieties. No polymorphic band was amplified by the primers from the fragments 72 , however, polymorphic bands were obtained by the PCR with the primers from the fragment 54. Thus, it is thought that the sequence 54 is a RGA related to sweetp otato stem nematode resistance.

关 键 词：甘薯茎线虫病 RGA 克隆 核苷酸结合位点 GenBank PCR扩增 相关 植物抗病基因 基因组DNA 徐薯18 序列设计 抗性基因 植物线虫 保守序列 序列搜索 甘薯品种 感病品种 抗病品种 多态性 片段 引物 突变体 新品系 氨基酸 NBS 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]