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    崔喜艳, 秦思余, 刘忠野, 高瑜, 蒋载阳, 郭继勋. 羊草CDPK基因全长cDNA的克隆及原核表达[J]. 华南农业大学学报, 2015, 36(4): 123-128. DOI: 10.7671/j.issn.1001-411X.2015.04.022
    引用本文: 崔喜艳, 秦思余, 刘忠野, 高瑜, 蒋载阳, 郭继勋. 羊草CDPK基因全长cDNA的克隆及原核表达[J]. 华南农业大学学报, 2015, 36(4): 123-128. DOI: 10.7671/j.issn.1001-411X.2015.04.022
    shu
    CUI Xiyan, QIN Siyu, LIU Zhongye, GAO Yu, JIANG Zaiyang, GUO Jixun. Cloning and prokaryotic expression of the full-length cDNA of CDPK gene from Leymus chinensis[J]. Journal of South China Agricultural University, 2015, 36(4): 123-128. DOI: 10.7671/j.issn.1001-411X.2015.04.022
    Citation: CUI Xiyan, QIN Siyu, LIU Zhongye, GAO Yu, JIANG Zaiyang, GUO Jixun. Cloning and prokaryotic expression of the full-length cDNA of CDPK gene from Leymus chinensis[J]. Journal of South China Agricultural University, 2015, 36(4): 123-128. DOI: 10.7671/j.issn.1001-411X.2015.04.022
    shu

    羊草CDPK基因全长cDNA的克隆及原核表达

    Cloning and prokaryotic expression of the full-length cDNA of CDPK gene from Leymus chinensis

      摘要
    • 摘要:
      目的 通过对羊草Leymus chinensis CDPK基因进行克隆及原核表达，为进一步研究CDPK基因的功能，探讨羊草适应生境分子机制提供理论基础.
      方法 用RT-PCR结合RACE技术克隆了羊草钙依赖蛋白激酶(CDPK)基因，命名为Lc-CDPK; 构建了羊草CDPK基因的原核表达载体，转化到大肠埃希菌Escherichia coli BL21(DE3)，异丙基硫代半乳糖苷(IPTG)诱导后，经SDS-PAGE分析CDPK蛋白的表达情况.
      结果和结论 羊草CDPK基因全长cDNA序列为1 704 bp，包括1个1 647 bp的开放阅读框，编码548个氨基酸，从第81~339个氨基酸构成了具有丝氨酸/苏氨酸蛋白激酶催化活性的S-TKc结构域，有4个保守的EF手型结构域; 编码区核苷酸序列与其他禾本科作物比对后发现，与小麦CDPK基因的相似性最高，相似度为96%; IPTG诱导表达出相对分子质量为61 350的融合蛋白，表达产物大小与预计理论值相符.诱导7 h蛋白表达量最高，表达量占菌体总蛋白的49.1%.

       

      Abstract:
      Objective Cloning and prokaryotic expression of CDPK gene from Leymus chinensis would help to further study the function of it, which could probe into its molecule mechanism how L.chinensis adapts itself to the saline-alkali habitat and provide a theoretical basis.
      Method CDPK gene of L.chinensis was cloned by RT-PCR and RACE technology, which was named Lc-CDPK.By constructing prokaryotic expression vector of CDPK gene and transforming it into Escherichia coli BL21 (DE3) with IPTG induction, the expression of CDPK protein was analyzed by SDS-PAGE.
      Result and conclusion The full-length cDNA sequence of CDPK gene was 1 704 bp with a 1 647 bp ORF (open reading frame) encoding 548 amino acids, and the amino acids from 81 to 339 formed the S-TKc structural domains possessing catalytic activity of serine/threonine protein kinase and having four conservative EF hand structural domains.A comparison with the nucleotide sequence of coding region of L.chinensis and those of other graminaceous crops showed that it has a close similarity with CDPK gene of wheat, sharing at identity of 96%. The relative molecular mass of prokaryotic expressed fusion protein was 61 350 by IPTG induction, which matched up with the anticipated theoretical value.The induced protein reached the highest level at 7 hours of induction, accounting for 49.1% in the total bacterial proteins.

       

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