茶树胞质型苹果酸脱氢酶的原核表达及生物信息学分析  

Prokaryotic Expression and Bioinformatics Analysis of Cytosolic Malate Dehydrogenase from Camellia sinensis(Theaceae)

作　　者：陈露露[1] 翟羽佳[1] 王鹏[1] 王晖[1,2] 郝家胜[1,2] 朱国萍[1,2] 

机构地区：[1]安徽师范大学生命科学学院分子进化与生物多样性重点实验室,安徽芜湖241000 [2]生物环境与生态安全安徽省高校重点实验室,安徽芜湖241000

出　　处：《云南植物研究》2010年第1期32-40,共9页Acta Botanica Yunnanica

基　　金：国家自然科学基金(30500300;30870062);教育部新世纪优秀人才支持计划(NCET-06-0558);教育部留学回国人员启动基金;安徽省优秀青年科技基金(06043089;08040106811);重要生物资源保护与利用研究安徽省重点实验室资助

摘　　要：利用RT-PCR及cDNA末端快速扩增法,获得了完整的茶树细胞质苹果酸脱氢酶(cMDH)基因Cs-cMDH(GenBank登录号为GQ845406)。该基因全长1235bp,编码332个氨基酸,分子量约为35.5kD。含重组质粒pGEX-MDH的E.coli Rosetta经0.5mmol·L-1IPTG于32℃诱导3h后可以获得大量可溶性的61.5kD融合蛋白。NCBI的BLAST结果显示,Cs-cMDH与高等植物cMDH的氨基酸序列一致性高达88%～93%。通过基于蛋白质结构的多序列比对,预测Cs-cMDH为二聚体,每个亚基包含13个β-折叠及13个α-螺旋。Cs-cMDH包含典型的MDH"指纹"(fingerprint)序列G12AAGQIG18,其氨基酸残基D43在所有NAD-MDH中都很保守。Cs-cMDH还包含一些与其它NAD-MDHs同源的保守序列单元,如NAD+结合位点、催化模体及底物结合位点。而且Cs-cMDH还包含在所有植物NAD-cMDHs中都相当保守的6个Cys,因此我们推断Cs-cMDH为茶树细胞质NAD-MDH。茶树基础代谢相关基因cMDH的克隆和原核表达为Cs-cMDH的功能研究奠定了基础。The complete gene of cytosolic malate dehydrogenase （cMDH） from Camellia sinensis,called Cs-cMDH,was obtained by RT-PCR and rapid amplification of cDNA ends （GenBank accession number GQ845406）. This gene was 1 235 bp in length,encoding a protein of 332 amino acids with the putative molecular weight of 35.5 kD. The E.coli Rosetta （DE3） harboring pGEX-MDH was induced by 0.5 mmol·L^-1 IPTG at 32℃ for 3 hours,and a 61.5 kD glutathione Stransferase （GST）-fused MDH was obtained in soluble form. The results of NCBI-BLAST revealed that Cs-cMDH shared 88%-93% of amino acid sequence identity with other cMDH from different higher plants. According to the multiple sequence alignment based on the three-dimensional structure of protein,Cs-cMDH was predicted to be a dimer with thirteen β-sheet and thirteen α-helix of each subunit. Cs-cMDH contains typical fingerprint sequence （G^12AAGQIG^18） as all MDHs. The amino acid D^43 in Cs-cMDH is conserved in all NAD-MDHs. Cs-cMDH also has some conserved sequence units homologous to other NAD-MDHs,such as NAD^＋ binding sites,catalytic motif and substrate binding sites. Moreover,Cs-cMDH contains six Cys which are highly conserved in all plant NAD-cMDHs. Therefore,Cs-cMDH was inferred to be NAD-dependent cMDH. The present study may provide the fundament for the further functional characterization of Cs-cMDH.

关 键 词：茶树 胞质型苹果酸脱氢酶 RT-PCR cDNA末端快速扩增法 序列分析 二级结构 

分 类 号：S571.1[农业科学—茶叶生产加工]