本文關(guān)鍵詞：擬南芥三個(gè)ABS3亞家族MATE轉(zhuǎn)運(yùn)蛋白的基因功能研究　出處：《西北農(nóng)林科技大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

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【摘要】：MATE(Multidrug and Toxic Compound Extrusion)家族轉(zhuǎn)運(yùn)蛋白是生物體內(nèi)廣泛存在的一種可以轉(zhuǎn)運(yùn)多種藥物及毒素化合物的轉(zhuǎn)運(yùn)蛋白。在植物中,MATE轉(zhuǎn)運(yùn)蛋白幫助植物外排代謝產(chǎn)物、抗生素、陽離子化合物等物質(zhì),并參與調(diào)節(jié)植物體內(nèi)多種物質(zhì)的分布、積累和平衡,從而維持植物正常的生長發(fā)育過程。本實(shí)驗(yàn)室前期研究了擬南芥MATE轉(zhuǎn)運(yùn)蛋白ABS3亞家族中的五個(gè)基因,并發(fā)現(xiàn)該亞家族中基因的過表達(dá)會(huì)使植物產(chǎn)生真葉發(fā)育快、葉柄變短、衰老加快以及矮化叢生的表型,因此本研究中對(duì)ABS3亞家族中ABS3L4、ABS3L5以及ABS3L6基因的功能進(jìn)行了研究,主要獲得以下結(jié)果:(1)構(gòu)建擬南芥MATE轉(zhuǎn)運(yùn)蛋白家族的系統(tǒng)進(jìn)化樹,并對(duì)ABS3L4、ABS3L5以及ABS3L6蛋白序列與ABS3進(jìn)行同源序列比對(duì)分析,發(fā)現(xiàn)其有高度的序列相似性。(2)探究了ABS3L4、ABS3L5以及ABS3L6基因的時(shí)空表達(dá)特異性。一方面通過半定量RT-PCR檢測到ABS3L4、ABS3L5以及ABS3L6基因在擬南芥不同發(fā)育階段的各個(gè)組織中表達(dá)略有差異,但均在花器官中有表達(dá)。另一方面通過GUS組織染色證明了ABS3L6基因在擬南芥幼苗子葉及真葉的水孔部位,下胚軸及主根中,葉片托葉中,花柱處,花瓣中有不同程度的表達(dá)。(3)成功構(gòu)建了ABS3L4、ABS3L5以及ABS3L6蛋白融合綠色熒光蛋白或紅色熒光蛋白的瞬時(shí)表達(dá)載體,并借助擬南芥葉肉細(xì)胞原生質(zhì)體瞬時(shí)表達(dá)體系對(duì)蛋白的亞細(xì)胞定位情況進(jìn)行研究,發(fā)現(xiàn)這三個(gè)蛋白與ABS4的共定位情況略有差異,并且其在亞細(xì)胞結(jié)構(gòu)上的定位情況也不盡相同。(4)成功構(gòu)建并獲得了ABS3L4、ABS3L5以及ABS3L6基因的過表達(dá)轉(zhuǎn)基因系,且均觀察到與ABS3基因過表達(dá)突變體相似的表型,同時(shí)在RNA水平上驗(yàn)證了過表達(dá)轉(zhuǎn)基因系中基因的上調(diào)。(5)成功獲得并驗(yàn)證了ABS3L4、ABS3L5以及ABS3L6基因的T-DNA插入功能缺失突變體。并在本實(shí)驗(yàn)室前期研究的基礎(chǔ)上繼續(xù)對(duì)該亞家族的八個(gè)基因進(jìn)行構(gòu)建八重功能缺失突變體,目前已經(jīng)獲得七純一雜多重基因功能缺失突變體。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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本文編號(hào)：1339118