VIGS技术高通量筛选棉花抗黄萎病相关基因

High Throughput Screening of Verticillium dahliae Resistance Genes by VIGS Technology in Cotton

黄萎病是严重影响棉花生产的一种土传性维管束真菌病害,长期以来,因缺乏行之有效的手段,有关棉花对黄萎病菌抗性分子机制的研究难度很大,进展缓慢.病毒诱导基因沉默(VIGS)这一反向遗传学技术的建立和应用,为研究和阐明棉花基因的功能提供了新的契机.本研究挑选可能与棉花抗黄萎病相关的基因,通过VIGS方法进一步明确和验证这些基因在棉花抗黄萎病菌中的作用.共成功构建了62个基因的VIGS载体,通过重复筛选实验,最终筛选出一些与棉花抗黄萎病相关的基因,其中乙烯信号途径正调控因子EIN2,植物病原应答诱导蛋白基因DIR,木葡聚糖内转糖苷酶/水解酶(XTH)被沉默后,沉默棉花植株对黄萎病菌的抗性明显减弱,表明这些基因可能通过不同的信号途径参与棉花对黄萎病菌的抗性.

Cotton verticillium wilt, the most severe disease in cotton, is a soil-born vascular spreading fungus disease caused by Verticillium dahlia Kleb. Due to limitations of research materials and lack of research techniques, it is difficult to explore the molecular mechanism of cotton resistance to verticillium wilt at the genetic level. VIGS is a technology based on the concept of loss-of-function in genes, it provides a new opportunity to reveal functions of genes that participate in the cotton-Verticillium dahliae interaction. We selected 106 genes that are possibly involved in cotton resistance to verticillium wilt, then studied function of these genes through VIGS technology. Sixty-two vectors that silenced the target genes were successfully constructed. After screening for three times in independent experiments, we found three important genes related to cotton＇s resistance： a positive regulatory factor（EIN2） in ethylene signaling pathways, DIR（dirigent-like protein gene） in lignification, and XTH（xyloglucan endotransglucosylase/ ydrolase）. Silencing these genes significantly increased the susceptiblity to V. dahlia, indicating that these genes may be involved in resistance to verticillium wilt via different signaling pathways.