稻瘟病抗性基因Pi36原核表达载体构建·表达与鉴定  

Prokaryotic Expression Vector Construction,Expression and Identification of the Blast Resistance Gene Pi36

作　　者：王玲[1] 张向明[2] 刘新琼[2] 

机构地区：[1]华南农业大学资源环境学院,广东广州510642 [2]中南民族大学生命科学学院生物技术国家民委重点实验室,湖北武汉430074

出　　处：《安徽农业科学》2010年第21期11059-11060,共2页Journal of Anhui Agricultural Sciences

基　　金：国家转基因生物新品种培育重大专项(2009ZX08009-023B);国家自然科学基金青年项目(30600400)

摘　　要：[目的]研究稻瘟病抗性基因Pi36的结构和功能。[方法]利用双酶切构建Pi36基因CC、NBS结构域的原核表达载体,通过菌落PCR鉴定和测序验证阳性克隆。然后分别收集不同浓度IPTG诱导,不同温度30和37℃分别培养的大肠杆菌细胞提取蛋白,利用SDS-PAGE电泳检测目的蛋白的表达情况。[结果]当IPTG浓度为1mmol/L、30℃培养3h,目的蛋白表达量最大。[结论]为进一步研究稻瘟病抗性基因Pi36的抗病机理奠定基础。[Objective] The aim was to further study the structure and function of the gene Pi36,which encodes a coiled-coil nucleotide binding site-leucine rich repeat（CC-NBS-LRR）.[Method] Prokaryotic expression vector of the gene CC,NBS domain was constructed,and the positive clones were confirmed by colonies PCR and sequencing.The E.coli cells were cultured and collected to extract protein during different concentration of IPTG under 30 and 37 ℃,respectively,and SDS-PAGE was used to detect expression of target protein.[Result] The results showed that the interesting protein expressed successfully,and have the highest expression of level when concentration of IPTG was 1 mmol/L under 30 ℃ for three hours.[Conclusion] The study laid a foundation for research for resistance mechanism of the Pi36.

关 键 词：稻瘟病 抗性基因 原核表达载体 基因克隆 

分 类 号：S435.111.41[农业科学—农业昆虫与害虫防治]