玉米ZmCIPK31基因原核表达及启动子序列分析
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国家“863”计划项目 (2006AA10Z188);转基因专项(2008ZX08003-004)


Prokaryotic Expression and Promoter Region Analysis of ZmCIPK31 Isolated from Maize
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    摘要:

    与钙传感器类钙调磷酸酶B蛋白(calcineurin B-like protein, CBL)互作的蛋白CIPK(CBL-interacting protein kinase)在植物特定的生长发育和应答胁迫过程中起重要作用。对前期研究得到的玉米ZmCIPK31基因构建原核表达重组载体,进行原核表达分析,转化重组质粒的大肠杆菌BL21菌株能够诱导出期望的目的蛋白。蛋白可溶性分析表明,它是可溶性的蛋白,通过淀粉树脂柱对其进行纯化,为下一步激酶活性分析提供了有活性的目的蛋白。同时,克隆了ZmCIPK31基因起始密码子ATG上游包括2189bp的启动子区段,顺式作用元件预测分析表明此区段不仅具有TATA-box和CAAT-box等启动子共有序列,还具有光应答、胁迫应答、发育相关、激素相关及其他功能未知的调控结构域。聚乙二醇(PEG)胁迫下,ZmCIPK31基因的诱导表达进一步表明其能够应答胁迫,且在地上部和根中的表达模式不同。

    Abstract:

    CIPK (CBL-interacting protein kinase) interacts with calcium sensor CBL (calcineurin B-like protein). It was proved to play important roles in responding to stresses and regulating certain developmental processes in plants. Here, a recombinant plasmid was constructed for prokaryotic expression analysis for ZmCIPK31 obtained in this study. The results showed that the predicted target protein could be induced in the recombinant plasmid transformed E. coli BL21 strain cells. Protein solublility analysis proved the target protein was soluble. Thus, the purified soluble target protein was obtained by being loaded onto an amylose resin column, which can be used for further protein kinase assay. Meanwhile, a 2189 bp DNA fragment, upstream of the putative translation initiation site (ATG) of the gene, was obtained as ZmCIPK31 promoter region. A promoter motif search of this region showed that there were not only TATA-box and CAAT-box, but also motifs related to light, stress, development, auxin and others. Under polyethylene glycol (PEG) stress, the expression of ZmCIPK31 was induced. Moreover, the expression pattern of ZmCIPK31 in shoots was different from that in roots.

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张成伟,刘颖慧,石云素,等.玉米ZmCIPK31基因原核表达及启动子序列分析[J].植物遗传资源学报,2010,11(2):186-191.

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