番茄果实中特异性表达的SlSWEET14基因启动子的克隆与功能分析

发布时间：2017-12-27 02:34

本文关键词：番茄果实中特异性表达的SlSWEET14基因启动子的克隆与功能分析　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

更多相关文章：番茄 SlSWEET14 启动子 GUS 表达模式

【摘要】：SWEETs基因家族是新发现的膜转运蛋白,其可以参与蔗糖、果糖、葡萄糖等糖类的转运。前期研究表明番茄中SISWEET14为果实中特异性表达的基因,其参与转运糖类,但其具体作用方式尚不明确。在调控基因表达的过程中启动子起到关键调控作用,所以通过对基因启动子及其顺式作用元件的生物学功能的探究是对基因表达进行调控的重要手段。本研究以番茄(Solanum lycopersicum)Micro-Tom为试验材料,通过常规PCR的方法克隆得到SlSWEET以基因启动子,进一步通过5'端缺失的方法,通过结合Gateway技术与pBGWFS7,0表达载体融合,驱动GUS基因的表达,运用GUS组织化学染色分析和GUS荧光定量分析,明确启动子表达模式,从而为全面阐述SlSWEET14基因的功能奠定实验基础,并且也为SWEETs基因家族的研究和番茄优质品种育种提供依据。本研究主要结果如下:1,对SlSWEET14启动子进行生物信息学分析,表明SlSWEET14基因启动子除含有高等植物通用核心启动子顺式作用元件TATA-BOX、CAAT-BOX,还含有与激素响应有关的顺式作用元件如:响应水杨酸的顺式作用元件TCA-element、响应赤霉素的顺式作用元件TATC-box、响应生长素的顺式作用元件AuxRR-core、响应乙烯的顺式作用元件ERE;与胁迫相关的顺式作用元件如:响应热胁迫的顺式作用元件HSE、与干旱诱导有关的顺式作用元件MBS;与胚乳发育相关的顺式作用元件Skn-1_motif;与光响应有关的顺式作用元件GT1-motif、BoxI、Box4、AE-box等。2,利用农杆菌介导的果实注射法分析了启动子的瞬时表达活性,运用GUS组织化学染色分析和GUS荧光定量分析结果表明绿熟期果实注射后3天,随着启动子长度的逐渐变短,其驱动GUS基因的表达活性逐渐降低。3,通过对转基因拟南芥组织特异性GUS染色分析,发现三个启动子截断片段均能驱动GUS基因在拟南芥角果果柄处表达,但最短片段驱动GUS基因在拟南芥根和花中也有大量的表达,即启动子最短片段失去了组织特异性,在SlSWEET14启动子上游-1466bp～-767bp处含有决定果实特异性表达的顺式作用元件。
[Abstract]:The SWEETs gene family is a newly discovered membrane transporter, which can be involved in the transport of sugar, fructose, glucose and other sugars. Previous studies have shown that SISWEET14 is a specific gene expressed in fruit, and it is involved in transshipment of sugars, but its specific function is not clear. In the process of regulating gene expression, promoters play a key regulatory role. Therefore, exploring the biological functions of gene promoters and cis acting elements is an important way to regulate gene expression. In this study, tomato (Solanum lycopersicum Micro-Tom) as test materials, obtain SlSWEET in gene promoter by cloning method of conventional PCR method, further through the 5'terminal deletion, through the combination of Gateway technology and pBGWFS7,0 fusion expression vector and expression of GUS gene driven by GUS, histochemical staining and GUS fluorescence quantitative analysis, a clear start the sub expression pattern, so as to lay the foundation for a comprehensive exposition of the function of SlSWEET14 gene, and also provides the basis for the research and breeding of high quality varieties of tomato SWEETs gene family. The main results of this study are as follows: 1, the promoter of SlSWEET14 bioinformatics analysis showed that SlSWEET14 gene promoter in higher plants contain common core promoter cis element TATA-BOX, CAAT-BOX, also contains the cis acting elements such as hormone response: salicylic acid TCA-element cis element, gibberellin response TATC-box cis element, cis acting elements in response to auxin response AuxRR-core, ethylene ERE cis element; stress-related cis elements such as: heat stress response cis acting elements of HSE, and drought induced cis elements related to MBS; and the development of endosperm related cis element Skn-1_motif; and light responsive cis elements related to GT1-motif, BoxI, Box4, AE-box etc.. 2, using the fruit injection method of Agrobacterium mediated transient expression analysis of promoter activity by GUS histochemical staining and GUS fluorescence quantitative analysis results showed that 3 days after the injection of mature green fruit, with promoter length becomes shorter, the expression of GUS gene driven activity decreased gradually. 3, the transgenic Arabidopsis tissue specific GUS staining analysis found that three promoter fragments were truncated GUS gene expression in Arabidopsis can drive angle fruit handle office, but the short fragment of GUS gene driven are abundantly expressed in roots and flowers of Arabidopsis thaliana, namely the promoter short lost in tissue specificity. The SlSWEET14 promoter -1466bp ~ -767bp which determines the fruit specific expression of the CIS elements.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S641.2

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