小麥TaCIPK2基因的表達分析及其在轉基因煙草中抗旱功能的研究
發(fā)布時間：2019-06-12 02:59
【摘要】：干旱、高鹽、低溫等非生物脅迫嚴重制約植物生長發(fā)育,使作物的的產量及品質下降。植物在長期進化中,形成了一套精細的調控機制來響應各種非生物脅迫,以適應多變的環(huán)境。研究表明,Ca2+感受器CBL蛋白與其相互作用的蛋白激酶CIPK形成的CBL-CIPK復合體,在植物響應非生物逆境脅迫中起重要作用。目前,在重要糧食作物小麥中有關CBL-CIPK的研究報道十分有限。本文在已克隆的一個小麥CIPK—TaCIPK2的基礎上,對該基因的表達模式及生物學功能進行了研究,主要研究結果如下:(1)利用實時熒光定量qRT-PCR技術,分析了小麥不同組織及經NaCl、PEG、H2O2和ABA處理下TaCIPK2基因的表達模式。發(fā)現(xiàn)在所檢測的組織中,TaCIPK2基因表達水平存在差異,在葉和莖中表達量最高。在葉中,除NaCl處理外,PEG、ABA、H2O2處理均能明顯上調TaCIPK2的表達;而在根中,各處理無明顯變化。(2)利用農桿菌介導遺傳轉化法,將真核表達載體pBI121-TaCIPK2-GFP導入到小麥葉片細胞中進行亞細胞定位分析,結果顯示,TaCIPK2為全細胞定位。(3)將構建的酵母表達載體pGADT7-TaCIPK2與pGBDT7-TaCBLs共轉入到酵母細胞中,發(fā)現(xiàn)TaCIPK2能與TaCBL1,TaCBL2,TaCBL3和TaCBL4相互作用。(4)采用農桿菌介導的遺傳轉化法,將表達載體pBI121-TaCIPK2-GFP成功轉化煙草,通過篩選獲得了TaCIPK2過表達轉基因煙草。干旱脅迫下表型分析結果表明,TaCIPK2轉基因過表達株系對干旱處理的耐受性顯著增強;生理生化分析結果表明,與對照株系相比,TaCIPK2過表達轉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存在下種子的發(fā)芽率更低,說明TaCIPK2轉基因植株對外源ABA更加敏感。氣孔開度實驗結果顯示,滲透脅迫下TaCIPK2過表達轉基因株系氣孔關閉的更快。以上研究結果表明,TaCIPK2作為一個正調控因子,在植物響應干旱脅迫中發(fā)揮重要作用。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2;S512.1
本文編號：2497675
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2;S512.1
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