棉花GhDHAR2基因克隆、功能序列分析及原核表达

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。

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

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 AtDHAR2 protein family.Prokaryotic 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.