陆地棉纤维中GhGMPase2基因克隆、序列分析及原核表达

Cloning,Sequence Analysis and Prokaryotic Expression of Cotton Fiber GhGMPase2 cDNA

从陆地棉纤维中克隆GDP-甘露糖焦磷酸化酶(GhGMPase2)基因,并进行序列分析及原核表达。利用RT-PCR技术进行基因克隆,构建原核表达载体pET28a-GhGMPase2并转化大肠杆菌BL21(DE3)进行蛋白体外诱导表达,通过SDS-PAGE检测目的蛋白的表达。研究结果表明,从陆地棉花纤维中克隆得到棉GhGMPase2基因,该基因的开放读码框为1 086bp,编码包含362个氨基酸的蛋白质,分子质量约为40ku。序列分析结果表明,GhGMPase2蛋白具有较高的保守性,具有典型的糖酰基转移酶转移酶A家族(GT-A)结构域和左手平行的β-的螺旋(LbetaH或LBH)域,它们分别属于糖酰基转移酶家族2(GT-2)超家族和LBH超家族。进化树分析的结果显示,棉花GhGMPase2与烟草NtGMPase在进化上亲缘关系较近。经过原核表达载体pET28a-GhGMPase2的诱导表达和SDS-PAGE分析显示,获得分子质量约为40ku的重组GhGMPase2蛋白。GMPase2基因的克隆、序列分析及原核表达为进一步研究其参与棉花纤维发育过程中的重要功能奠定基础。

A cotton GhGMPase2 full-length cDNA was cloned from elongating fiber tissue based on RT-PCR method. A series of bioinformatics software were used to analyze sequence characteristics; construction of prokaryotic expression vector pET28a-GhGMPase2 was performed; Recombinant Gh- GMPase2 protein was obtained after transformation into E. coli BL21 （DE3） and induction. SDS PAGE was used for further protein identification. The cotton GhGMPase2 gene contains a 1 086 bp open reading frame and codes an about 40 ku protein of 362 amino acids. GhGMPase2 protein includes typical Glycosyltransferase family A （GT-A） domain and Left-handed parallel beta-Helix （LbetaH or LBH） domain, which belongs to Glycosyltransferase superfamily 2 （GT-2） and LBH superfamily re spectively. Phylogenetic tree analysis showed that GhGMPase2 has closer similar relationship with NtGMPase. Prokaryotic expression vector pET28a-GhGMPase2 was constructed and confirmed, and recombinant GhGMPase2 protein with the molecular mass of 40 ku was obtained after induction and SDS-PAGE analysis . These results establish the foundation in researching its further functions and e-lucidating molecular mechanisms of GhGMPase2 involved in cotton fiber cell development.