Cloning, expression and purification of pyruvate, orthophosphate dikinase regulatory proteins from Phyllostachys edulis
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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PeRP1,该基因cDNA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PeRP1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PeRP1的蛋白质结构,我们将PeRP1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PeRP1重组蛋白。SEC纯化的结果表明:PeRP1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PeRP1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。图7参15Abstract: Pyruvate orthophosphate dikinase regulatory protein (PPDK-RP) is a key protein in C4 photosynthesis 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 bamboo 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 kinase-PPPase superfamily. The multiple sequence alignment and phylogenetic analysis of PeRP revealed that Phyllostachys edulis was a typical C3 monocotyledon. Expressed soluble PeRP1 protein had three forms--polymer, dimmers, and monomers in water soultion. These provided a foundation for the structure and function of RP.[Ch, 7 fig. 15 ref.]
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https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2015.05.014
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