拟南芥AtSEC14基因反义RNA植株的获得及抗病性检测

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。

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

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 trans-ferred 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 transgenic 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.