棉花GhCOMT3基因的原核表达、纯化及鉴定  

Prokaryotic Expression,Purification and Identification of Caffeic acid-3-O-methyltransferase Gene from Gossypium hirsuturm L.

作　　者：李波[1] 倪志勇[1] 范玲 

机构地区：[1]新疆农业科学院核技术生物技术研究所,新疆乌鲁木齐830091

出　　处：《华北农学报》2010年第B08期12-16,共5页Acta Agriculturae Boreali-Sinica

基　　金：国家"863"项目(2006AA10Z184);农业部转基因重大专项课题(2009ZX08005-011B);新疆自治区高技术研究发展计划项目(200611101);国家自然科学基金项目(30660088)

摘　　要：为了深入研究GhCOMT3基因的功能,将GhCOMT3基因构建到原核表达载体pET-28a中,并转化到大肠杆菌BL21(DE3)中。用0.2mmol/L IPTG诱导融合蛋白表达,结果发现,16℃诱导12h、30℃诱导3h和37℃诱导3h后融合蛋白均以包涵体的形式表达;30℃诱导的蛋白表达量最大,之后对包涵体进行变性溶解,进行SDS-PAGE检测,再经过蛋白标记亲和层析柱(His TrapTMHP)纯化得到变性的pET-28a-GhCOMT3融合蛋白,用SDS-PAGE和Western blotting检测纯化效果,鉴定表达产物,目的蛋白相对分子量约为39.119kDa,检测结果与预期一致。结果表明,pET-28a-GhCOMT3蛋白在大肠杆菌中获得了高效表达产物,为在蛋白水平上研究GhCOMT3基因功能奠定了基础。To investigate the function of the GhCOMT3 gene,the GhCOMT3 gene was insert into the pET-28a vector to construct fusion vector pET-28a-GhCOMT3,and transformed into E.coli BL21（DE3） cells.The fusion protein could be induced to express by 0.2 mmol/L IPTG,in the form of inclusion body at 16℃for 4 h,30℃for 3 h or 37℃for 3 h.The most high expression quantity was induced at 30℃for 4 h.Then to dissolved inclusion body,the supernatants were analyzed by SDS-PAGE and the results were identified with expecting.Then the protein was purified using His Trap TM HP to get degenerated protein,SDS-PAGE and Western blotting were then employed to identify target protein,the results were identified with expecting.The prokaryotic expression system of pET-28aCOMT3 is successfully constructed in this experiment.The fusion protein was positive by Western blotting.So we can say that pET-28a-GhCOMT3 protein was expressed in E.coli.It can be used for further investigation on the function of the GhCOMT3 gene in protein level.

关 键 词：GhCOMT3 原核表达 蛋白纯化 WESTERN BLOTTING 

分 类 号：Q78[生物学—分子生物学]