【摘要】：干旱脅迫是影響植物在自然環(huán)境中正常生長的重要因素之一。我國干旱半干旱地區(qū)面積高達(dá)30%,干旱嚴(yán)重影響著我國糧食和經(jīng)濟(jì)作物的產(chǎn)量。與此同時(shí),我們也發(fā)現(xiàn)在我國中西部地區(qū)常年種植著一些雜糧作物,這些雜糧作物不僅能夠在干旱條件下正常生長,而且產(chǎn)量極少受干旱影響,谷子就是能夠適應(yīng)干旱環(huán)境的雜糧作物之一。作為C4植物,谷子具有耐旱、抗病、抗倒伏等優(yōu)良性狀。本研究選取“晉谷34”這一優(yōu)良品種作為研究對象,通過比較“晉谷34”正常生長和干旱脅迫下表達(dá)譜數(shù)據(jù)中基因響應(yīng)干旱上調(diào)或下調(diào)幅度的差異,選出一個(gè)能夠響應(yīng)干旱的基因,命名為SiUPF3,該基因編碼谷子中的Upf3蛋白。相關(guān)文獻(xiàn)顯示,Upf3蛋白參與的無義介導(dǎo)降解途徑是真核生物中的主要的mRNA降解途徑之一。截止目前,植物無義介導(dǎo)降解途徑相關(guān)研究要遠(yuǎn)遠(yuǎn)少于動物和酵母,植物NMD機(jī)制的研究方法也相對落后。本文在綜述部分對植物NMD機(jī)制進(jìn)行了深入的分析和探討,并對植物無義介導(dǎo)降解機(jī)制給出相對全面合理的解釋。為了進(jìn)一步探究谷子中參與NMD途徑的耐旱基因SiUPF3的功能,我們將篩選獲得的耐旱基因SiUPF3分別進(jìn)行生物信息學(xué)分析、干旱和植物激素脫落酸誘導(dǎo)下基因表達(dá)模式分析,并對其進(jìn)行分子克隆,構(gòu)建了SiUPF3的植物表達(dá)載體,再通過植物轉(zhuǎn)基因的方法對SiUPF3進(jìn)行模式作物煙草的過表達(dá),最后通過耐旱生理指標(biāo)檢測來驗(yàn)證SiUPF3的耐旱功能。結(jié)果顯示,谷子SIUPF3基因的起始密碼子上游存在著脫落酸響應(yīng)元件和干旱響應(yīng)元件等一系列與逆境調(diào)節(jié)相關(guān)的順式作用元件。在干旱脅迫和植物激素脫落酸誘導(dǎo)下谷子中的SiUPF3基因相對表達(dá)量均表現(xiàn)為顯著上調(diào)。成功克隆SiUPF3基因后我們構(gòu)建了該基因的植物表達(dá)載體和帶有綠色熒光蛋白標(biāo)簽的載體分別進(jìn)行煙草轉(zhuǎn)基因和瞬時(shí)表達(dá)亞細(xì)胞定位,通過亞細(xì)胞定位發(fā)現(xiàn)SiUPF3蛋白主要存在于植物細(xì)胞核,而利用農(nóng)桿菌介導(dǎo)法進(jìn)行轉(zhuǎn)基因操作后我們獲得S1、S2、S3共三株過表達(dá)株系。最終,抗旱生理指標(biāo)檢測結(jié)果顯示：相比于野生型煙草,干旱處理后的SiUPF3基因過表達(dá)株系SOD酶活性明顯高于野生型,丙二醛的含量低于野生型,而脯氨酸含量無明顯差異,證明SiUPF3具有一定的耐旱功能。
[Abstract]:Drought stress is one of the important factors affecting the normal growth of plants in the natural environment. The area of arid and semi-arid areas in China is as high as 30. The drought seriously affects the yield of grain and cash crops in China. At the same time, we also found that in the central and western regions of our country, there are some crops planted all the year round, which can not only grow normally under drought conditions, but also have very little effect on the yield of crops affected by drought. Millet is one of the crops that can adapt to drought. As a C4 plant, millet has good characteristics such as drought tolerance, disease resistance, lodging resistance and so on. In this study, "Jingu 34", a fine variety, was selected as the research object, and the difference of up-regulation or down-regulation of gene response to drought was compared between the normal growth of "Jingu 34" and the data of expression profile under drought stress. Select a gene that can respond to drought, named SiUPF3, which encodes the Upf3 protein in millet. The related literatures show that the pathway of Upf3 protein involved in mRNA degradation is one of the major mRNA degradation pathways in eukaryotes. Up to now, the research of plant mediated degradation pathway is much less than that of animals and yeast, and the research methods of plant NMD mechanism are relatively backward. In this review part, the mechanism of plant NMD is analyzed and discussed in depth, and the mechanism of plant senseless degradation is explained in a relatively comprehensive and reasonable way. In order to further explore the function of drought tolerance gene SiUPF3 involved in the NMD pathway in millet, the selected drought tolerance gene SiUPF3 was analyzed by bioinformatics, and the gene expression pattern induced by drought and plant hormone abscisic acid was analyzed. The plant expression vector of SiUPF3 was constructed by molecular cloning, and SiUPF3 was overexpressed by plant transgenic method. Finally, the drought tolerance function of SiUPF3 was verified by the detection of drought tolerance physiological index. The results showed that there were a series of cis-acting elements related to stress regulation such as abscisic acid response element and drought response element in the upstream of the initiation codon of millet SIUPF3 gene. The relative expression of SiUPF3 gene in millet was significantly up-regulated under drought stress and plant hormone abscisic acid induction. After the successful cloning of SiUPF3 gene, we constructed the plant expression vector of the gene and the vector with green fluorescent protein tag for tobacco transgenic and transient expression subcellular localization, respectively. The subcellular localization revealed that SiUPF3 protein mainly existed in plant nuclei. After transgene manipulation by Agrobacterium tumefaciens, we obtained three overexpression lines of S1OS2OS3. Finally, the results of drought resistance physiological index test showed that compared with wild type tobacco, the SOD enzyme activity of SiUPF3 gene overexpression lines after drought treatment was significantly higher than that of wild type, the content of malondialdehyde was lower than that of wild type, but the content of proline had no significant difference. It is proved that SiUPF3 has the function of drought tolerance.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2

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