棉花GhGGPase基因克隆、序列分析及原核表达

Cloning,Sequence Analysis and Prokaryotic Expression of Cotton GhGGPase cDNA

【目的】棉花GDP-L-半乳糖磷酸化酶(GhGGPase)基因的克隆、功能序列分析及原核表达。【方法】利用RT-PCR技术从棉花纤维中克隆棉花GhGGPase基因,进行生物信息学分析,构建原核表达载体pET28a-GhGGPase,转化大肠杆菌BL21(DE3)并进行体外诱导表达,通过SDS-PAGE检测目的蛋白的表达。【结果】从陆地棉纤维组织中扩增得到L-半乳糖磷酸化酶基因的cDNA开放读码框为1 350 bp,为包含450个氨基酸的蛋白质,理论分子质量为49 kDa。对氨基酸序列进行生物信息学分析,GhGGPase蛋白具有组氨酸三聚体(HIT)蛋白超家族的主要特性的结构域。进化树分析的表明棉花GhGGPase与柑橘CuGGPase在进化上亲缘关系上较近。成功构建了pET28a-GhGGPase原核表达载体;获得分子质量约为49 kDa左右的重组蛋白GhGGPase。半定量RT-PCR的组织表达特异性分析表明GhGGPase基因与棉纤维的快速伸长发育密切相关。【结论】GhGGPase基因的克隆、序列分析和重组蛋白的诱导表达为深入研究该基因在棉纤维发育中的作用奠定了基础。

[ Objective] To clone a cotton GhGGPase gene from fiber tissue, analyze the sequence and express the recombinant GhGGPase protein. [ Method ] A GhGGPase gene was cloned from fiber tissue through RT- PCR method. A series of bioinformatics software were used to analyze sequence characteristics; Prokaryotic expression vector pET28a- GhGGPase was constructed; Recombinant protein was expressed after being transformed into E. Coli BL21 （ DE3 ） and induction. SDS - PAGE was used for further protein diction. [ Result]A cotton GhGGPase full -length cDNA was cloned from fiber tissue. GhGGPase cDNA contains a 1350bp open reading frame and codes a 49 kDa several typical conserved HIT functional domain. protein of 450 amino acids. GhGGPase protein contains a Phylogenetic tree analysis showed that GhGGPase belonged to the Citrus CuGGPase protein family. A 49 kDa recombinant protein was obtained by SDS - PAGE gel analysis. [ Conclusion] GhGGPase cloning, functional sequence analysis and recombinant protein expression establish a basis for its further research into cotton fiber development.