• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

木薯MebZIP27基因的克隆与原核表达分析

Cloning and Prokaryotic Expressions of Cassava MebZIP27

    摘要
  • 摘要:
    目的 碱性亮氨酸拉链（basic leucine zipper, bZIP）蛋白是真核生物中一类独特的转录因子，在植物的生长、发育和生理代谢调控中发挥着关键作用。特别是A类bZIPs通常负责调控携带ABA依赖元件的基因，进而引发ABA响应或实现环境适应。深入研究A类木薯bZIPs转录因子的功能，可进一步了解其调控ABA表达的分子机理。
    方法 通过RT-PCR技术克隆MebZIP27基因，并运用生物信息学方法对其进行基因和蛋白质水平的分析。此外，通过荧光定量PCR检测MebZIP27基因的时空表达模式及其对逆境的响应，并尝试通过原核表达获得MebZIP27蛋白。
    结果 MebZIP27基因位于木薯第5号染色体上，编码区全长1251 bp，具备典型的bZIP结构域特征。该基因与麻风树（Hevea brasiliensis）和橡胶树（Jatropha curcas）的A类bZIP序列一致性分别高达80.00%和85.41%，表明其属于A类bZIP亚族成员。亚细胞定位结果显示，MebZIP27基因位于细胞核。qRT-PCR分析结果表明，MebZIP27基因在木薯储藏根中表达最为显著。在甘露醇、NaCl和低温处理下，MebZIP27基因未表现出显著诱导表达，但在ABA和H2O2处理下表现出显著诱导。通过原核表达成功获得了MebZIP27基因编码的蛋白，并通过谷胱甘肽琼脂糖珠微球分离技术对GST-MebZIP27蛋白进行了纯化。
    结论 木薯中的MebZIP27基因属于A类bZIP基因家族，定位于细胞核，在木薯储藏根中的表达最为显著。此外，MebZIP27基因在ABA和H2O2处理下表现出显著诱导。这些结果为深入研究MebZIP27基因在ABA信号调控中的作用提供了参考依据。

     

    Abstract:
    Objective The Class A basic leucine zipper protein(bZIP), which regulates genes carrying ABA-responsive elements associated with the environmental adaptations as well as growth, development, and physiological metabolism of plants, in cassava was studied.
    Methods The Class A bZIP in cassava, MebZIP27, was cloned using RT-PCR technology to determine the functions of the gene transcription factors. Bioinformatic methods were applied to analyze properties of the gene and protein, fluorescence quantitative PCR employed to examine spatiotemporal expressions and responses to stress, and prokaryotic expression used to produce MebZIP27 protein.
    Results MebZIP27 was in the 5th chromosome of cassava with a coding region of 1251 bp and characteristic bZIP domain features. It had the highest homology with the Class A bZIP sequences from Hevea rubber at 80.00% and Jatropha curcas at 85.41%, as a member of the A-bZIP subfamily. Located in nucleus, the gene had the highest expression found in the roots. Treatments of mannitol, NaCl or low temperature did not cause significant induction, but ABA and H2O2 did on it. Via the prokaryotic expression, MebZIP27 could be produced and purified using glutathione agarose bead separation techniques.
    Conclusion Located in nucleus and belonged to the Class A bZIP gene family, MebZIP27 expressed most significantly in the storage roots of a cassava plant and by an ABA or H2O2 induction.

     

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