【摘要】：干旱、高鹽、低溫等非生物脅迫嚴(yán)重制約植物生長(zhǎng)發(fā)育,使作物的的產(chǎn)量及品質(zhì)下降。植物在長(zhǎng)期進(jìn)化中,形成了一套精細(xì)的調(diào)控機(jī)制來響應(yīng)各種非生物脅迫,以適應(yīng)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(fù)合體,在植物響應(yīng)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(guān)CBL-CIPK的研究報(bào)道十分有限。本文在已克隆的一個(gè)小麥CIPK—TaCIPK2的基礎(chǔ)上,對(duì)該基因的表達(dá)模式及生物學(xué)功能進(jìn)行了研究,主要研究結(jié)果如下:(1)利用實(shí)時(shí)熒光定量qRT-PCR技術(shù),分析了小麥不同組織及經(jīng)NaCl、PEG、H2O2和ABA處理下TaCIPK2基因的表達(dá)模式。發(fā)現(xiàn)在所檢測(cè)的組織中,TaCIPK2基因表達(dá)水平存在差異,在葉和莖中表達(dá)量最高。在葉中,除NaCl處理外,PEG、ABA、H2O2處理均能明顯上調(diào)TaCIPK2的表達(dá);而在根中,各處理無明顯變化。(2)利用農(nóng)桿菌介導(dǎo)遺傳轉(zhuǎn)化法,將真核表達(dá)載體pBI121-TaCIPK2-GFP導(dǎo)入到小麥葉片細(xì)胞中進(jìn)行亞細(xì)胞定位分析,結(jié)果顯示,TaCIPK2為全細(xì)胞定位。(3)將構(gòu)建的酵母表達(dá)載體pGADT7-TaCIPK2與pGBDT7-TaCBLs共轉(zhuǎn)入到酵母細(xì)胞中,發(fā)現(xiàn)TaCIPK2能與TaCBL1,TaCBL2,TaCBL3和TaCBL4相互作用。(4)采用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化法,將表達(dá)載體pBI121-TaCIPK2-GFP成功轉(zhuǎn)化煙草,通過篩選獲得了TaCIPK2過表達(dá)轉(zhuǎn)基因煙草。干旱脅迫下表型分析結(jié)果表明,TaCIPK2轉(zhuǎn)基因過表達(dá)株系對(duì)干旱處理的耐受性顯著增強(qiáng);生理生化分析結(jié)果表明,與對(duì)照株系相比,TaCIPK2過表達(dá)轉(zhuǎn)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(zhuǎn)基因植株對(duì)外源ABA更加敏感。氣孔開度實(shí)驗(yàn)結(jié)果顯示,滲透脅迫下TaCIPK2過表達(dá)轉(zhuǎn)基因株系氣孔關(guān)閉的更快。以上研究結(jié)果表明,TaCIPK2作為一個(gè)正調(diào)控因子,在植物響應(yīng)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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2;S512.1

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本文編號(hào)：2497675