棉花GhDHAR2基因克隆、功能序列分析及原核表达  

Cloning,Functional Sequence Analysis and Prokaryotic Expression of Cotton GhDHAR2 cDNA

作　　者：田大鹏[1] 葛娟[1] 石峰[1] 李鸿彬[1,2] 

机构地区：[1]石河子大学生命科学学院,石河子832003 [2]石河子大学农业生物技术重点实验室,石河子832003

出　　处：《生物技术通报》2012年第7期65-69,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30860031);农业部转基因专项(2009ZX08005-027B);兵团博士基金项目(2008JC01)

摘　　要：通过RT-PCR方法从棉花纤维组织中克隆得到脱氢抗坏血酸还原酶基因GhDHAR2的cDNA,该基因开放阅读框为639 bp,编码212个氨基酸的蛋白质。同源性序列对比分析显示,GhDHAR2蛋白具有较高的保守性,具有典型的功能结构域,包括GST-N家族和GST-C-DHAR家族的功能结构域;进化树分析显示GhDHAR2和拟南芥AtDHAR2在进化关系上较近。将GhDHAR2基因连接到原核表达载体pET-28a中,将重组载体pET28a-GhDHAR2转入到表达菌株BL21(DE3)中,通过IPTG诱导表达出重组GhDHAR2蛋白,SDS-PAGE凝胶电泳分析显示重组蛋白大小约为28 kD,诱导表达的重组蛋白具有较高的DHAR活性。首次克隆了棉花GhDHAR2基因,通过结构域分析其可能的作用,并成功进行蛋白体外表达及酶活性分析。A cotton GhDHAR2 cDNA, encoding a 639 bp open reading frame and protein of 212 amino acid residues, was cloned from fiber tissue using RT-PCR method. Homology sequence blast indicated that GhDHAR2 protein contained typical conserved functional domains including GST N-terminal family and GST C-terminal DHAR subfamily. Phylogenetic tree analyses showed that GhDHAR2 belonged to the Arabidopsis AtDI-IAR2 protein family. Prokaryotie expression recombinant pET28a-GhDHAR2, constructed by enzyme digestion of GhDHAR2 and pET-28a and successive ligation, was transformed of bacteria BL21 （ DE3 ） , and a 28 kD recombinant protein was obtained by IPTG induction and SDS-PAGE gel analysis, and GhDHAR2 recombinant protein expressed high DHAR enzyme activity. All these data are helpful to understand GhDHAR2 gene biological function and mechanism of participating cotton fiber cell elongation.

关 键 词：棉花 脱氢抗坏血酸还原酶基因克隆 原核表达 DHAR酶活力 

分 类 号：S562[农业科学—作物学]