拟南芥三个ABS3亚家族MATE转运蛋白的基因功能研究

发布时间：2017-12-26 22:24

本文关键词：拟南芥三个ABS3亚家族MATE转运蛋白的基因功能研究　出处：《西北农林科技大学》2016年硕士论文　论文类型：学位论文

更多相关文章： 拟南芥 MATE转运蛋白 ABS3亚家族

【摘要】：MATE(Multidrug and Toxic Compound Extrusion)家族转运蛋白是生物体内广泛存在的一种可以转运多种药物及毒素化合物的转运蛋白。在植物中,MATE转运蛋白帮助植物外排代谢产物、抗生素、阳离子化合物等物质,并参与调节植物体内多种物质的分布、积累和平衡,从而维持植物正常的生长发育过程。本实验室前期研究了拟南芥MATE转运蛋白ABS3亚家族中的五个基因,并发现该亚家族中基因的过表达会使植物产生真叶发育快、叶柄变短、衰老加快以及矮化丛生的表型,因此本研究中对ABS3亚家族中ABS3L4、ABS3L5以及ABS3L6基因的功能进行了研究,主要获得以下结果:(1)构建拟南芥MATE转运蛋白家族的系统进化树,并对ABS3L4、ABS3L5以及ABS3L6蛋白序列与ABS3进行同源序列比对分析,发现其有高度的序列相似性。(2)探究了ABS3L4、ABS3L5以及ABS3L6基因的时空表达特异性。一方面通过半定量RT-PCR检测到ABS3L4、ABS3L5以及ABS3L6基因在拟南芥不同发育阶段的各个组织中表达略有差异,但均在花器官中有表达。另一方面通过GUS组织染色证明了ABS3L6基因在拟南芥幼苗子叶及真叶的水孔部位,下胚轴及主根中,叶片托叶中,花柱处,花瓣中有不同程度的表达。(3)成功构建了ABS3L4、ABS3L5以及ABS3L6蛋白融合绿色荧光蛋白或红色荧光蛋白的瞬时表达载体,并借助拟南芥叶肉细胞原生质体瞬时表达体系对蛋白的亚细胞定位情况进行研究,发现这三个蛋白与ABS4的共定位情况略有差异,并且其在亚细胞结构上的定位情况也不尽相同。(4)成功构建并获得了ABS3L4、ABS3L5以及ABS3L6基因的过表达转基因系,且均观察到与ABS3基因过表达突变体相似的表型,同时在RNA水平上验证了过表达转基因系中基因的上调。(5)成功获得并验证了ABS3L4、ABS3L5以及ABS3L6基因的T-DNA插入功能缺失突变体。并在本实验室前期研究的基础上继续对该亚家族的八个基因进行构建八重功能缺失突变体,目前已经获得七纯一杂多重基因功能缺失突变体。
[Abstract]:MATE (Multidrug and Toxic Compound Extrusion) family transporter is a transporter widely existed in organisms, which can transport multiple drugs and toxin compounds. In plants, MATE transporters help plant efflux metabolites, antibiotics, cationic compounds and other substances, and are involved in regulating the distribution, accumulation and balance of many substances in plants, so as to maintain normal growth and development of plants. In our previous study, five genes of Arabidopsis thaliana MATE transporter subfamily ABS3, and found that the overexpression of the subfamily phenotype will enable the plant to produce the leaf petiole shorter, fast development and accelerated aging and dwarf tufted, therefore the ABS3 subfamily of ABS3L4, ABS3L5 and ABS3L6 genes of the in the study were studied. The main results are summarized as follows: (1) phylogenetic tree of MATE transporter family in Arabidopsis, and ABS3L4, ABS3L5 and ABS3L6 protein sequence and ABS3 sequence analysis, the sequence of a high degree of similarity. (2) the spatio-temporal expression specificity of ABS3L4, ABS3L5 and ABS3L6 genes was explored. On the one hand, semi quantitative RT-PCR showed that ABS3L4, ABS3L5 and ABS3L6 genes expressed slightly differently in all tissues of Arabidopsis thaliana at different developmental stages, but they were all expressed in floral organs. On the other hand through the GUS staining showed that the ABS3L6 gene in Arabidopsis hole parts of cotyledon and leaf, hypocotyl and root, leaf stipules, office style, there are different degrees of expression in petals. (3) ABS3L4 was constructed successfully, and the instantaneous ABS3L5 ABS3L6 fusion protein green fluorescent protein or red fluorescent protein expression vector, and with the help of Arabidopsis mesophyll protoplast transient expression studies on protein subcellular localization system, found that the co localization of slight differences between the three protein and ABS4, and its subcellular structure the situation is not the same. (4) successfully constructed and obtained transgenic lines with over expression of ABS3L4, ABS3L5 and ABS3L6 genes, and observed similar phenotypes with ABS3 gene overexpression mutants. Meanwhile, the gene up-regulated in transgenic lines was verified at RNA level. (5) the T-DNA insertion deletion mutants of ABS3L4, ABS3L5 and ABS3L6 genes were successfully obtained and verified. And continue to build the eight functions of deletion mutants of eight genes of the subfamily based on the previous study of our laboratory, has produced seven hybrid multiple gene deletion mutants single function.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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