棉花GhAO3基因的克隆、功能序列分析及原核表达

Cloning,Functional Sequence Analysis and Prokaryotic Expression of Cotton GhAO3 cDNA

通过RT-PCR方法从处于快速伸长发育时期(开花后15 d)的棉花纤维组织中克隆得到棉花抗坏血酸氧化酶3(Ascorbate oxidase 3,GhAO3)基因的全长开放读码框,该基因全长读码框为1 758 bp,编码包含585个氨基酸的蛋白质。通过序列功能分析和同源序列比对,GhAO3蛋白具有较高的保守性,包括3个多铜氧化酶结构域以及跨膜信号序列,进化树比对结果显示GhAO3与大豆GmAO的亲缘关系较近。将GhAO3基因与原核表达载体pET32a相连构建重组载体pET32a-GhAO3,把重组载体转入大肠杆菌BL21(DE3)中,用IPTG诱导重组菌,经SDS-PAGE电泳分析,获得分子量约为64 kD的重组GhAO3蛋白。

A cotton ascorbate oxidase（GhAO3）full-length open reading frame（ORF）cDNA was cloned from elongating fiber tissue（15 DPA）by RT-PCR method,GhAO3ORF contains 1 758 bp nucleotides and codes a protein of 585 amino acids.Through sequence function analysis and homology sequence alignment,GhAO3 protein includes three multi-copper oxidase domain and transmembrane signal sequence with a high conservation.Phylogenetic tree analysis showed that GhAO3 has a similar relationship with GmAO protein.Prokaryotic expression vector pET32a-GhAO3 was constructed and transformated into E.coliBL21（DE3）.Recombinant GhAO3 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD,and had a higher enzymatic activity.