小麦抗病基因同源序列(RGAs)的克隆与分析

RGA(抗性基因同源序列)法是克隆植物抗性基因的一种经济有效的方法,成为近年来的研究热点。本实验综合分析了拟南芥,西红柿,水稻,烟草等植物已克隆的抗性基因,并以这些抗性基因的NBS(核酸结合位点),LRR(富含亮氨酸重复),STK(丝氨酸/苏氨酸激酶)保守结构域设计并合成了几十对RGA引物,对小麦抗条锈病材料进行PCR扩增,获得以Xal-NBS为引物的R88RGA片段,经克隆和序列比对分析,发现该片段与逆境条件下植物抗病信号传导相关,与蛋白激酶同源性达到96%。此项研究对抗病机理的研究和基因的发掘有重要的指导意义。

The Cloning and Sequencing of Resistance Gene Analogues in Wheat

RGA analysis is an effective and economical approach to clone plant resistance genes and widely studied in recent years. Tens of RGA primers were designed according to serine/threonine protein kinase (STK) and NBS-LRR con-served motifs from disease resistance genes in Arobidopsis, tomato, rice and tobacco, etc in this study. These primers were used as PCR primers to amplify RGAs in wheat cultivars R88 and lines9N, 10N, 12Y with high resistance of stripe rust. Wheat leaves were inoculated with strip rust spores by manual smearing at seedling duration, and were cut at 0 hr, 24 hr, 48 hr, 72 hr after inocubating, respectively. Amplified RGA fragments in R88 (primer: Xal-NBS) were cloned and sequenced. It was found that the identity between phosphotase and the cloned fragments was 96%, and that it was likely related to the transformation of resistance singals under reverse circumstances. This study is of great significance for discovering its resistance mechanism and cloning new genes.