耐旱基因SiUPF3的生物信息学和功能分析

发布时间：2018-12-31 18:05

【摘要】：干旱胁迫是影响植物在自然环境中正常生长的重要因素之一。我国干旱半干旱地区面积高达30%,干旱严重影响着我国粮食和经济作物的产量。与此同时,我们也发现在我国中西部地区常年种植着一些杂粮作物,这些杂粮作物不仅能够在干旱条件下正常生长,而且产量极少受干旱影响,谷子就是能够适应干旱环境的杂粮作物之一。作为C4植物,谷子具有耐旱、抗病、抗倒伏等优良性状。本研究选取“晋谷34”这一优良品种作为研究对象,通过比较“晋谷34”正常生长和干旱胁迫下表达谱数据中基因响应干旱上调或下调幅度的差异,选出一个能够响应干旱的基因,命名为SiUPF3,该基因编码谷子中的Upf3蛋白。相关文献显示,Upf3蛋白参与的无义介导降解途径是真核生物中的主要的mRNA降解途径之一。截止目前,植物无义介导降解途径相关研究要远远少于动物和酵母,植物NMD机制的研究方法也相对落后。本文在综述部分对植物NMD机制进行了深入的分析和探讨,并对植物无义介导降解机制给出相对全面合理的解释。为了进一步探究谷子中参与NMD途径的耐旱基因SiUPF3的功能,我们将筛选获得的耐旱基因SiUPF3分别进行生物信息学分析、干旱和植物激素脱落酸诱导下基因表达模式分析,并对其进行分子克隆,构建了SiUPF3的植物表达载体,再通过植物转基因的方法对SiUPF3进行模式作物烟草的过表达,最后通过耐旱生理指标检测来验证SiUPF3的耐旱功能。结果显示,谷子SIUPF3基因的起始密码子上游存在着脱落酸响应元件和干旱响应元件等一系列与逆境调节相关的顺式作用元件。在干旱胁迫和植物激素脱落酸诱导下谷子中的SiUPF3基因相对表达量均表现为显著上调。成功克隆SiUPF3基因后我们构建了该基因的植物表达载体和带有绿色荧光蛋白标签的载体分别进行烟草转基因和瞬时表达亚细胞定位,通过亚细胞定位发现SiUPF3蛋白主要存在于植物细胞核,而利用农杆菌介导法进行转基因操作后我们获得S1、S2、S3共三株过表达株系。最终,抗旱生理指标检测结果显示：相比于野生型烟草,干旱处理后的SiUPF3基因过表达株系SOD酶活性明显高于野生型,丙二醛的含量低于野生型,而脯氨酸含量无明显差异,证明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.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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