GhCyt b5基因的克隆及其蛋白参与电子传递功能的分析  

Cloning and Expression Analysis of Cyt b5 Gene Involved in Electron Transfer in Gossypium hirsuturm

作　　者：刘金芝[1,2] 司怀军[1] 张宁[1] 吴家和[2] 

机构地区：[1]甘肃农业大学生命科学技术学院,兰州730070 [2]国家植物基因组学重点实验室中国科学院微生物研究所,北京100101

出　　处：《中国生物工程杂志》2013年第4期68-73,共6页China Biotechnology

基　　金：中国科学院院地合作资助项目(XBXJ-2010-002)

摘　　要：从陆地棉品种中棉所35盐胁迫EST文库中筛选到与其它植物高度同源的细胞色素b5蛋白(Cyt b5)基因片段,利用RACE技术,获得Cyt b5基因的cDNA序列,命名为GhCyt b5,基因全长810bp,最大阅读框402 bp,编码由134个氨基酸组成的蛋白质,分子量约为17kDa。将该基因的编码序列插入到原核表达载体pET32a中,构建重组质粒为pET32a-Cyt b5,对不同条件下进行蛋白诱导表达分析,发现在28℃和1 mmol/L IPTG条件下能够获得可溶性的GhCyt b5蛋白。利用Ni2+柱亲和层析纯化和SDS-PAGE鉴定分析表明获得重组蛋白为目的蛋白。通过提取棉花细胞物质为反应介质,进行体外电子传递功能分析,发现GhCyt b5能够从还原态变为氧化态,参与电子的传递功能。On the basis of salt-stress related EST library from Gossypium hirsuturm L. A partial sequence fragment of cytochrome b5 proteins （Cyt b5 ） encoding gene was obtained through homologous comparison with other plants. The Cyt b5 gene was isolated by the 3＇ ,5＇- RACE technology from G. hirsuturm, named GhCyt b5. The full-length cDNA of GhCyt b5 is 810 bp, containing a 402 bp ORF which encodes 134 amino-acid peptide. The relative molecular weight of GhCyt b5 protein is 17 kDa. The coding sequence fragment was amplified by PCR and cloned into vector pET32a to generate the pET32a-GhCyt b5 expression vector, which was transformed into BL21 （DE3） for expression of recombinant protein. On the base of optimization of different inducible conditions, the results showed that soluble GhCyt b5 protein under the 28 ℃ and 1 mmol/L IPTG conditions could be obtained. The expression product of GhCyt b5 was purified by Ni2~ affinity column and identified by SDS- PAGE. Using extracted cotton cell material as reaction medium for in vitro electron transfer analysis, it can be found the GhCyt b5 involved in the electron transfer system and accepted electron to become oxidation state from reducing state. Ultimately, the Cyt b5 gene for the first time from G. hirsuturm was cloned and its participating in electron transfer system was confirmed, which provide a base for further dissecting function in biotic and abiotic resistance.

关 键 词：棉花 细胞色素b5蛋白 原核表达 电子传递 

分 类 号：Q819[生物学—生物工程]