茶树CsCIPK11基因克隆、表达和互作蛋白分析

    Cloning, Expression, and Protein Interaction Analysis of CsCIPK11 in Camellia sinensis

    • 摘要:
      目的 探讨茶树CsCIPK11基因在茶树不同组织中的表达特征及其在逆境条件下的生物学作用。
      方法 以茶树‘龙井43’为试验材料,运用RT-PCR技术克隆CsCIPK11基因的编码序列,并进行生物信息学分析;采用实时荧光定量PCR技术探究该基因在茶树各组织及多种非生物胁迫条件下的表达模式;并通过酵母双杂交系统验证与之相互作用的蛋白。
      结果 CsCIPK11基因编码区全长1 299 bp,编码433个氨基酸。CsCIPK11蛋白相对分子量约为48 818.29 Da,不含信号肽和跨膜区域,表现出亲水性质,拥有29个磷酸化位点,预测其定位在细胞质中。此外,CsCIPK11基因启动子区域内含有响应光照(如Box 4、G-box)、激素(如ABRE、CGTCA-motif/TGACG-motif、P-box)、环境压力(如STRE)及低氧条件(如ARE)等作用元件。CsCIPK11基因在茶树根部表达最丰富,在花和老叶次之,嫩叶中表达量最低;在叶片发育过程中,该基因在茶芽生长出第1片真叶时表达量达到峰值,随叶片成熟其表达水平趋于稳定。进一步研究发现,低温、干旱、高盐及MeJA处理均可诱导CsCIPK11上调;酵母双杂交试验结果表明CsCIPK11能与CsMYC4-like蛋白互作。
      结论 CsCIPK11基因对茶树生长发育及适应不利环境因素具有重要作用,研究结果可为解析茶树应对逆境胁迫的作用机理提供基础。

       

      Abstract:
      Objective This study aimed to explore the expression characteristics of CsCIPK11 gene in different tissues of tea plant and its biological function under stress conditions.
      Method Using 'Longjing 43' as experimental material, the coding sequence of CsCIPK11 gene was cloned by RT-PCR, followed by bioinformatics analysis. Real-time quantitative PCR was used to investigate the expression patterns of this gene in various tissues and abiotic stress conditions. The interacting proteins of CsCIPK11 were verified by yeast two-hybrid system.
      Result The coding region of CsCIPK11 gene was 1 299 bp in length, encoding 433 amino acids. CsCIPK11 protein has a relative molecular weight of 48 818.29 Da. It does not contain signal peptides and transmembrane regions, exhibits hydrophilic properties, possesses 29 phosphorylation sites, and is predicted to be located in the cytoplasm. In addition, the promoter region of CsCIPK11 gene contains action elements in response to light (e.g. Box 4, G-box), hormones (e.g. ABRE, CGTCA-motif/TGACG-motif, P-box), environmental stress (e.g. STRE), and hypoxic conditions (e.g. ARE). The expression of CsCIPK11 gene was highest in tea roots, followed by flowers and old leaves, and lowest in young leaves. During leaf development, the expression of this gene peaked when the tea bud grew the first true leaf, and then stabilized as the leaf matured. Further studies showed that CsCIPK11 gene was up-regulated under low temperature, drought, high salt and MeJA treatment. At the same time, it was confirmed that CsCIPK11 could interact with CsMYC4-like protein by yeast two-hybrid assay.
      Conclusion In summary, CsCIPK11 gene plays an important role in the growth and development of tea plants and the adaptation to adverse environmental factors, and the study provides a basis for analysis of the mechanism of tea plants in response to adversity stress.

       

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