陆地棉GhPLDα基因的克隆、序列分析及表达  

Cloning,Sequence Analysis and Expression of Cotton GhPLDα Gene

作　　者：谭兰[1] 李榕[1] 袁哈利 贺怡[1] 梁卓[1] 谢全亮 李鸿彬[1,2] 

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

出　　处：《新疆农业科学》2014年第5期792-800,共9页Xinjiang Agricultural Sciences

基　　金：兵团种质资源创新专项(2012BB050);国家自然科学基金(31260039);石河子大学杰出青年项目(2012ZRKXJQ03)

摘　　要：【目的】棉花磷脂酶D(GhPLDα)基因的克隆、序列功能结构域分析及表达。【方法】以陆地棉胚珠和纤维为材料提取总RNA,利用RT-PCR技术扩增得到PLDα基因的cDNA片段,将其构建到原核表达载体pET-28a中,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,利用分光光度法检测酶活性,采取RT-PCR方法检测基因表达。【结果】从发育的棉花胚珠和纤维混合材料中克隆得到的磷脂酶基因的开放读码框为2 421 bp,编码包含807个氨基酸的蛋白质,分子量约为88 kDa。通过同源序列比对和功能结构域分析,GhPLDα具有典型的磷脂酶D家族(HKD)结构域,同时还存在蛋白激酶C结构域2(C2结构域)。构建了pET28a-GhPLDα原核表达载体;获得分子量约为88 kDa左右的重组蛋白GhPLDα。半定量RT-PCR结果表明GhPLDα基因可能参与棉花胚珠与纤维发育过程。【结论】GhPLDα基因的克隆、序列分析和表达为进一步研究棉花GhPLDα基因在胚珠和纤维发育中过程的功能奠定了基础。[ Objective ] This project focuses on cloning, functional sequence domain analysis and expres- sion of cotton GhPLDα gene. [ Method] A cotton phospholipase D （GhPLD） gene was cloned by RT- PCR method using cotton ovules and fibers as material, and the recombinant pET28a - GhPLDα was constructed and transformed into E. Coli BL21 （ DE3 ） in succession in vitro induction and went through SDS - PAGE analysis, and the recombinant GhPLDa protein enzyme activity was determined by spectrophotometer. GhPLDα gene expression analysis was performed by semi - quantitative RT - PCR method. [ Result ] A cotton GhPLDα full -length cDNA was cloned from cotton ovules and fibers with a 2 421 bp open reading frame code which contained protein of 807 amino acid and molecular weight was about 88 kDa. Sequence alignment and function structure domain analysis showed that GhPLDα had a typical family of phospholipase D （HKD） domain and C2 domain. The pET28a- GhPLDα prokaryotic expression vector was constructed, and a - 88 kDa recombi- nant protein was obtained by SDS - PAGE analysis which expressed a high enzyme activity. Semi - quantita- tive RT - PCR analysis indicated that the GhPLDα gene was closely related to the process of cotton ovule and fiber development. [ Conclusion ] Cloning, functional sequence analysis and expression of GhPLDα established the basis for further researching GhPLDα functions in cotton ovule and fiber development.

关 键 词：棉花 GhPLDα基因 表达 

分 类 号：S562[农业科学—作物学] S503.53