毛竹丙酮酸磷酸双激酶调节蛋白基因克隆、原核表达及纯化  

Cloning, expression and purification of pyruvate, orthophosphate dikinase regulatory proteins from Phyllostachys edulis

作　　者：王超莉[1] 张智俊[1] 屈亚平[1] 王蕾[1] 

机构地区：[1]浙江农林大学亚热带森林培育国家重点实验室培育基地,浙江临安311300

出　　处：《浙江农林大学学报》2015年第5期749-755,共7页Journal of Zhejiang A&F University

基　　金：国家自然科学基金资助项目(31270715);浙江省自然科学基金资助项目(LY14C160009)

摘　　要：丙酮酸磷酸双激酶调节蛋白(pyruvate,orthophosphate dikinase regulatory proteins,RP)是通过调控丙酮酸磷酸双激酶(pyruvate,orthophosphate dikinase,PPDK)参与C4途径光合碳循环途径。毛竹Phyllostachys edulis作为一种重要的经济竹种,分析克隆RP基因对于竹类植物光合作用的研究具有重大理论和应用价值。通过逆转录实时聚合酶链式反应(RT-PCR)成功克隆得到毛竹Pe RP1,该基因c DNA全长1 275 bp,编码425个氨基酸;经生物信息学预测,该蛋白属于kinase-PPPase超家族,主要含有UDF 299保守结构域;经多序列比对发现毛竹Pe RP1蛋白与C3植物中RP1亲缘关系较近,而与C4植物亲缘关系较远。为了研究Pe RP1的蛋白质结构,我们将Pe RP1蛋白进行原核表达,利用Ni-NTA树脂亲和层析结合分子筛(SEC)层析的方法纯化得到了Pe RP1重组蛋白。SEC纯化的结果表明:Pe RP1蛋白在溶液中主要以多聚体形式存在,二聚体及单体含量较少,推测Pe RP1蛋白可能以多聚体形式参与调控PPDK蛋白。这为今后研究该蛋白的结构与功能打下了良好基础。Pyruvate orthophosphate dikinase regulatory protein （PPDK-RP） is a key protein in C4 photosynthe- sis and can modified enzyme activity of PPDK. For Phyllostachys edulis, an important economic bamboo species, cloning PPDK-RP gene and studying its functions had vital theoretical value and application for bam- boo photosynthesis research. Firstly PeRP1 was successfully cloned by reverse transcription polymerase chain reaction （RT-PCR）, and afterward a multiple sequence alignment and phylogenetic analysis was conducted. Then for further study the crystal structure of the PeRP protein, the recombinant expression pe-SUMO vectors of PeRP were constructed and the protein was expressed in E. coli and purified by nickle beads column and size column. Results showed that the PeRP gene contained a 1.275 kb open reading frame （ORF） coding a 425 amino acid polypeptide, which contained the typical domain of UDF299 and was predicted as a number of ki- nase-PPPase superfamily. The multiple sequence alignment and phylogenetic analysis of PeRP revealed that PhyUostachys edulis was a typical C3 monocotyledon. Expressed soluble PeRP1 protein had three forms--poly- mer, dimmers, and monomers in water soultion. These provided a foundation for the structure and function of RP. [Ch, 7 fig. 15 ref.]

关 键 词：植物学 毛竹 丙酮酸磷酸双激酶调节蛋白 基因克隆 原核表达 蛋白纯化 

分 类 号：S718.3[农业科学—林学]