棉花GhCIPK6的克隆及其调控棉花抗旱的分子机理研究

Cotton (Gossypium hirsutum L.) shows strong drought tolerance to drought stress. However, the watering limitation of cotton cultivated areas remains an increasing threat to cotton production through impacts on cotton growth and limits fiber/oil quality. Thus, understanding the molecular mechanism of drought tolerance and exporation drought resistance genes has become important goals for drought resistance breeding programs in cotton. Somatic embryogenesis, during which many signaling pathways were involved, was considered as a stress-response developmental process. In our previous study, a CBL interaction of protein kinase (CIPK) were cloned and documented during cotton SE using RNA-Seq, defined as GhCIPK6. qRT-PCR analysis revealed that it was up-regulated under drought-stress. Ectopic expression of the GhCIPK6 enhances drought resistance in transgenic Arabidopsis. In the present project, to investigate the functional role of a drought-induced GhCIPK6 in cotton, we expressed its constitutively active form and RNAi vectors in cotton, to compare the growth index, physiological and biochemical index for transgenic plants and controls under drought stress. The interaction proteins of GhCIPK6 will be identified by Yeast two hybrid (H2Y) and Bimolecular fluorescence complementation (BiFC), and its downstream regulation genes will be investigated by RNA-Seq and qRT-PCR. Moreover, the expression and regulation of its promoter will be studied using transgene and Yeast one hybrid. Then the biological function and the related regulation pathways of GhCIPK6 during drought tolerance in cotton will be illuminated. This study will provide candidate gene and theoretical basis for drought resistance breeding programs in cotton.

棉花是一种抗旱性较强的作物，但干旱仍是影响棉花生产的重要逆境因素之一。了解抗旱机制和发掘抗旱基因已成为棉花抗旱育种的重要目标。体胚发生是一个逆境响应的过程，涉及到多种抗逆信号途径。本课题组前期从棉花体胚发生的表达谱中分离到一个与CBL互作的蛋白激酶并命名为GhCIPK6。qRT-PCR表明该基因在棉花干旱胁迫中上调表达，异源表达该基因能增强转基因拟南芥的抗旱性。本项目我们拟在棉花中抑制和超表达GhCIPK6基因，比较干旱胁迫下转基因植株与非转基因植株的生长情况及生理生化指标；通过酵母双杂和BiFC等确定其互作蛋白；进一步利用RNA-Seq筛选其可能的靶标基因，并利用qRT-PCR技术验证可能靶标基因和已知抗旱功能基因的表达，确定该基因调控的下游基因；研究启动子的表达特性及调控机制，从而明确GhCIPK6在调控棉花干旱响应过程中的生物学功能和调控途径，为棉花的抗旱育种提供候选基因及理论依据。

棉花是一种抗旱性较强的作物，但干旱仍是影响棉花生产的重要逆境因素之一。协调棉花水分利用，营养吸收及基础代谢的关系，是提高棉花生产效益的保障。基因组学的发展为挖掘棉花优异农艺相关基因带来了机遇，挖掘和鉴定抗旱基因，对关键信号通路的研究，深入解析不同信号下的内在联系，有望实现协调棉花生长和抗逆之间的矛盾。体胚发生是是一个生长与逆境响应相互协调的过程，涉及到多种抗逆信号途径。本项目前期从棉花体胚发生的表达谱中分离到一个与 CBL 互作的蛋白激酶并命名为 GhCIPK6。qRT-PCR 表明该基因在棉花干旱胁迫中上调表达，异源表达该基因能增强转基因拟南芥的抗旱性。我们进一步通过cDNA文库的构建及表达谱分析，筛选到CIPK家族一些其他成员也参与逆境响应；我们通过同源分析获得陆地棉中所有CIPK基因家族成员，并通过RT-PCR对棉花中的所有CIPK基因进行干旱诱导表达分析，得到了一些在干旱胁迫下差异表达的家族成员；我们在棉花中抑制和超表达 GhCIPK6基因，发现超表达株系失水速度较野生型快，干旱条件下存活率也较低，但其蒸腾速率显著低于对照，在正常条件下能够通过减缓蒸腾而显著提高水分利用效率；进一步研究发现超表达材料积累更多的可溶性糖，而干涉株系可溶性糖含量降低，但在离子吸收等方面存在一定的缺陷；通过酵母双杂、磷酸化测序、BiFC 等研究找到一些与GhCIPK6存在互作的蛋白质，VIGS研究发现CBL1和GhT2分别沉默后能将降低野生型糖含量，在超表达株系中进行干涉能师葡萄糖含量部分恢复至野生型，研究结果表明GhCIPK6通过GhCBL1-GhCIPK6-GhT2途径调控棉花可溶性糖的积累。