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Plant Diversity ›› 2010, Vol. 32 ›› Issue (01): 32-40.DOI: 10.3724/SP.J.1143.2010.09182

• 研究论文 • 上一篇    下一篇

茶树胞质型苹果酸脱氢酶的原核表达及生物信息学分析

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   

  1. 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
  • 收稿日期:2009-09-14 出版日期:2010-02-25 发布日期:2010-02-25
  • 通讯作者: 朱国萍

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

CHEN LuLu1, ZHAI YuJia1, WANG Peng 1, Wang Hui1,2, HAO JiaSheng1,2, ZHU GuoPing1,2   

  1. 1 The Key Laboratory of Molecular Evolution and Biodiversity, College of Life Sciences, Anhui Normal University, Wuhu 241000, China; 2Anhui Key Laboratory of Biotic Environment and Ecological Security, Wuhu 241000, China
  • Received:2009-09-14 Online:2010-02-25 Published:2010-02-25
  • Contact: ZHU GuoPing

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1 235 bp,编码332个氨基酸,分子量约为355 kD。含重组质粒pGEXMDH的Ecoli Rosetta经05 mmol·L1 IPTG于32℃诱导3 h后可以获得大量可溶性的615 kD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。

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;

Abstract: 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 (G12AAGQIG18) as all MDHs. The amino acid D43 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.

Key words: Camellia sinensis

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