小麦TaCIPK2基因的表达分析及其在转基因烟草中抗旱功能的研究

发布时间：2019-06-12 02:59

【摘要】：干旱、高盐、低温等非生物胁迫严重制约植物生长发育,使作物的的产量及品质下降。植物在长期进化中,形成了一套精细的调控机制来响应各种非生物胁迫,以适应多变的环境。研究表明,Ca2+感受器CBL蛋白与其相互作用的蛋白激酶CIPK形成的CBL-CIPK复合体,在植物响应非生物逆境胁迫中起重要作用。目前,在重要粮食作物小麦中有关CBL-CIPK的研究报道十分有限。本文在已克隆的一个小麦CIPK—TaCIPK2的基础上,对该基因的表达模式及生物学功能进行了研究,主要研究结果如下:(1)利用实时荧光定量qRT-PCR技术,分析了小麦不同组织及经NaCl、PEG、H2O2和ABA处理下TaCIPK2基因的表达模式。发现在所检测的组织中,TaCIPK2基因表达水平存在差异,在叶和茎中表达量最高。在叶中,除NaCl处理外,PEG、ABA、H2O2处理均能明显上调TaCIPK2的表达;而在根中,各处理无明显变化。(2)利用农杆菌介导遗传转化法,将真核表达载体pBI121-TaCIPK2-GFP导入到小麦叶片细胞中进行亚细胞定位分析,结果显示,TaCIPK2为全细胞定位。(3)将构建的酵母表达载体pGADT7-TaCIPK2与pGBDT7-TaCBLs共转入到酵母细胞中,发现TaCIPK2能与TaCBL1,TaCBL2,TaCBL3和TaCBL4相互作用。(4)采用农杆菌介导的遗传转化法,将表达载体pBI121-TaCIPK2-GFP成功转化烟草,通过筛选获得了TaCIPK2过表达转基因烟草。干旱胁迫下表型分析结果表明,TaCIPK2转基因过表达株系对干旱处理的耐受性显著增强;生理生化分析结果表明,与对照株系相比,TaCIPK2过表达转基因株系具有更低的IL、MDA、H2O2含量及高的抗氧化酶活性。外源ABA存在下种子的发芽率更低,说明TaCIPK2转基因植株对外源ABA更加敏感。气孔开度实验结果显示,渗透胁迫下TaCIPK2过表达转基因株系气孔关闭的更快。以上研究结果表明,TaCIPK2作为一个正调控因子,在植物响应干旱胁迫中发挥重要作用。
[Abstract]:Abiotic stresses such as drought, high salt and low temperature seriously restrict the growth and development of plants and decrease the yield and quality of crops. In the long-term evolution, plants have formed a set of fine regulatory mechanisms to respond to various abiotic stresses in order to adapt to the changing environment. Studies have shown that the CBL-CIPK complex formed by Ca2 receptor CBL protein interacting with its protein kinase CIPK plays an important role in plant response to abiotic stress. At present, the research reports on CBL-CIPK in wheat, an important grain crop, are very limited. In this paper, the expression pattern and biological function of the gene were studied on the basis of a cloned wheat CIPK-TaCIPK2. The main results were as follows: (1) the expression patterns of TaCIPK2 gene in different wheat tissues and treated with NaCl,PEG,H2O2 and ABA were analyzed by real-time fluorescence quantitative qRT-PCR. It was found that the expression level of TaCIPK2 gene was different in the detected tissues, and the expression level was the highest in leaves and stems. In leaves, except NaCl treatment, PEG,ABA,H2O2 treatment could significantly up-regulate the expression of TaCIPK2. In the root, there was no significant change in the treatments. (2) the eukaryotic expression vector pBI121-TaCIPK2-GFP was introduced into wheat leaf cells for subcellular localization by Agrobacterium tumefaciens mediated genetic transformation. The results showed that TaCIPK2 was the whole cell localization. (3) the constructed yeast expression vector pGADT7-TaCIPK2 and pGBDT7-TaCBLs were co-transferred into yeast cells, and it was found that TaCIPK2 and TaCBL1,TaCBL2, could be co-transferred into yeast cells. The interaction between TaCBL3 and TaCBL4. (4) the expression vector pBI121-TaCIPK2-GFP was successfully transformed into tobacco by Agrobacterium tumefaciens mediated genetic transformation, and the transgenic tobacco with overexpression of TaCIPK2 was obtained by screening. The results of phenotypic analysis under drought stress showed that the tolerance of TaCIPK2 transgenic lines to drought treatment was significantly enhanced, and the results of physiological and biochemical analysis showed that TaCIPK2 overexpressed transgenic lines had lower IL,MDA,H2O2 content and higher antioxidant enzyme activity than the control lines. The germination rate of seeds in the presence of exogenous ABA was lower, which indicated that TaCIPK2 transgenic plants were more sensitive to exogenous ABA. The results of stomatal opening test showed that the stomatal closure of TaCIPK2 overexpressed transgenic lines was faster under osmotic stress. The above results suggest that TaCIPK2, as a positive regulator, plays an important role in plant response to drought stress.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S512.1

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