拟南芥AtSEC14基因反义RNA植株的获得及抗病性检测

Obtainment and analysis of antisense RNA transgenic plants of AtSEC14 from Arabidopsis thaliana

为确定拟南芥抗灰霉病相关基因AtSEC14的功能,本试验构建了AtSEC14基因的反义RNA载体;通过农杆菌介导的遗传转化方法,将其转化拟南芥野生型Col-0中;利用潮霉素抗性筛选和PCR检测,获得了阳性转基因植株。利用半定量RT-PCR技术,在转基因株系中未检测到AtSEC14基因的表达,说明该基因反义RNA载体的转入能特异影响AtSEC14基因的表达,表明试验所获得的转基因植株是AtSEC14基因的反义RNA转基因植株。对所获得的反义RNA转基因植株进行抗病性鉴定,发现反义RNA转基因植株对灰葡萄孢的敏感性增强,表明AtSEC14基因在拟南芥抗灰葡萄孢过程中起正调控的作用。

To identify the function of Arabidopsis AtSEC14 gene in the defense response to Botrytis cinerea ,the antisense RNA vector of the AtSEC14 gene was constructed and transferred into Arabidopsis wild-type Col-0 by Agrobacterium-mediated method. Positive trans- genic line was obtained through hygromycin B screening and PCR assays. The expression of AtSEC14 in positive transgenic line was obviously reduced comparing with that of Col-0 by semi-quantity RT-PCR analysis, suggesting that the antisense RNA vector has been inserted into the genome of Arabidopsis and induced the decrease of AtSEC14 gene expres- sion. Antisense RNA transgenic plants of AtSEC14 were distinguishable from Col-0 in their response to B. cinerea. Antisense RNA transgenie plants exhibited obviously susceptibility to B. cinerea, while Col-0 exhibited obviously resistance to B. cinerea. These results indicated that the AtSEC14 gene played a positive role in Arabidopsis resistance to B. cinerea.