毛竹阿拉伯糖-5-磷酸异构酶的基因克隆、原核表达及纯化  

Gene cloning, expression, and purification of the arabinose-5-phosphate isomerase from Phyllostachys edulis

作　　者：屈亚平[1] 张智俊[1] 王超莉[1] 王蕾[1] 吴林军[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》2016年第6期928-934,共7页Journal of Zhejiang A&F University

基　　金：浙江省自然科学基金资助项目(Y14C160039)

摘　　要：毛竹Phyllostachys edulis阿拉伯糖-5-磷酸异构酶(Pe Kds D)是2-酮-3-脱氧辛糖酸(KDO)生物合成途径的第1个关键酶。采用逆转录实时聚合酶链式反应(q RT-PCR)克隆得到毛竹Kds D基因。该基因c DNA全长1 038 bp,编码346个氨基酸;对不同物种来源的Kds D氨基酸序列进行比对和系统进化树分析。结果表明:毛竹的Kds D与玉米Zea mays等植物来源的Kds D有很高的序列一致性,而与微生物来源的Kds D序列一致性较低。毛竹不同组织实时荧光定量聚合酶链式反应(q RT-PCR)结果表明:Pe Kds D基因在叶中表达量远远高于其他组织。在大肠埃希菌Escherichia coli原核表达系统中获得了Kds D的可溶性高表达蛋白,将大量表达的重组蛋白经过Ni-NTA亲和层析和分子筛层析(SEC)进行纯化,发现Kds D在溶液(30 mmol·L^(-1)三羟甲基氨基甲烷pH 8.0,200 mmol·L^(-1)氯化钠)中以多种聚体的形式存在;酶学性质测定的结果表明:毛竹Kds D酶最适pH值为pH 8.5,最适作用温度为37℃。此结果为后期研究植物Kds D蛋白的结构与功能奠定了基础。Phyllostachys edulis arabinose-5-phosphate isomerase （PeKdsD） is the first key enzyme in thebiosynthesis of 3-deoxy-D-manno-octulosonate （KDO）. The full length cDNA of the KdsD gene （1 038 bp with 346 amino acids） was cloned using reverse transcription polymerase chain reaction （qRT-PCR） followed by protein Basic Local Alignment Search Tool （BLAST） and phylogenetic tree analysis of the amino acid se-quence. Then a qRT-PCR analysis of diverse tissues in Ph. edulis was conducted, and a mass of soluble KdsD protein was obtained through a prokaryotic expression system in Escherichia coli. The recombinant protein wasfurther purified through Ni-NTA affinity and size exclusion chromatography （SEC） in a buffer of 30 mmol·L^-1 Tris-HCl pH 8.5, 200 mmol·L^-1 NaCl to determine enzymatic properties. Results of the amino acid sequence among different species indicated that the PeKdsD isomerase had a high sequence similarity with KdsD in Zea mays but was low in microorganisms. The qRT-PCR analysis of diverse tissues in Ph. edulis revealed that PeKdsD had its highest expression level in the leaf. SEC results showed that the recombinant KdsD protein existed as protein polymers, and for enzymatic properties of PeKdsD the optimum reaction temperature was 37 % with a pH of 8.5. This work provides a basis for determining the structure and function of plant KdsD. [Ch, 7 fig. 17 ref.]

关 键 词：林木育种学 毛竹 阿拉伯糖-5-磷酸异构酶 基因克隆 原核表达 蛋白纯化 

分 类 号：S722[农业科学—林木遗传育种]