【摘要】：水稻不僅是主要的糧食作物,同時(shí)也是重要的模式植物,對(duì)水稻相關(guān)農(nóng)藝性狀功能基因的鑒定意義重大。本實(shí)驗(yàn)室前期對(duì)水稻育性控制相關(guān)信號(hào)通路開(kāi)展研究,通過(guò)酵母雙雜交篩選,從水稻雌雄蕊形成期幼穗cDNA文庫(kù)中分離到若干與Rho蛋白OsRacD相互作用蛋白的編碼基因,其中包括一種Rho GTP酶激活蛋白基因,命名為OsRhoGAP2(GenBank登錄號(hào):AY 364311)。本研究對(duì)該基因及其啟動(dòng)子開(kāi)展系統(tǒng)的功能鑒定和元件分析,以此評(píng)估該基因在分子設(shè)計(jì)育種上的應(yīng)用潛力。基于本實(shí)驗(yàn)室前期構(gòu)建的該基因啟動(dòng)子及5個(gè)5’端缺失片段載體,分別轉(zhuǎn)化篩選擬南芥,每個(gè)轉(zhuǎn)基因擬南芥均獲得3個(gè)以上的純合株系。通過(guò)GUS組織化學(xué)染色、酶活力測(cè)定結(jié)合缺失實(shí)驗(yàn)分析,研究不同啟動(dòng)子片段驅(qū)動(dòng)GUS報(bào)告基因在生長(zhǎng)發(fā)育不同階段、不同組織中的表達(dá),發(fā)現(xiàn)在OsRhoGAP2啟動(dòng)子5’端的-703bp~-289bp區(qū)域內(nèi)可能著存在1個(gè)重要的花藥特異性元件,驅(qū)動(dòng)了GUS基因在轉(zhuǎn)基因擬南芥的花藥中表達(dá),尤其是在小孢子發(fā)育的初期,而在成熟期時(shí),擬南芥的營(yíng)養(yǎng)器官(根、莖、葉)以及繁殖器官(果莢)中均沒(méi)有表達(dá),由此推測(cè),OsRhoGAP2基因可能在小孢子發(fā)育初期表達(dá),對(duì)維持花藥正常發(fā)育有重要作用。通過(guò)5種激素(IAA、6-BA、GA、SA、MeJA)、5種脅迫處理(高溫、低溫、干旱、高鹽、黑暗)對(duì)生長(zhǎng)到10d的轉(zhuǎn)基因擬南芥進(jìn)行噴施處理,結(jié)合GUS染色,研究不同啟動(dòng)子片段對(duì)激素、非生物脅迫的響應(yīng),發(fā)現(xiàn)該基因啟動(dòng)子可能是1個(gè)與IAA誘導(dǎo)相關(guān)及脅迫誘導(dǎo)相關(guān)的啟動(dòng)子,在IAA及高溫、干旱、高鹽誘導(dǎo)條件下能驅(qū)動(dòng)下游基因表達(dá),而對(duì)其他激素沒(méi)有響應(yīng)。進(jìn)一步推測(cè),OsRhoGAP2基因可能參與到了IAA誘導(dǎo)相關(guān)及逆境脅迫誘導(dǎo)相關(guān)的應(yīng)答過(guò)程。結(jié)合5’端的缺失實(shí)驗(yàn)表明,在該基因啟動(dòng)子的-1292 bp~-948 bp存在1個(gè)重要的MeJA響應(yīng)元件,推測(cè)該基因可能參與了MeJA信號(hào)通路。基于本實(shí)驗(yàn)室前期工作,本研究對(duì)OsRhoGAP2過(guò)表達(dá)轉(zhuǎn)基因水稻T_2代進(jìn)行了分子檢測(cè)和表型分析,旨在了解該基因在生產(chǎn)上的應(yīng)用潛力。通過(guò)提取轉(zhuǎn)基因水稻基因組DNA結(jié)合PCR擴(kuò)增,對(duì)T_2代轉(zhuǎn)基因水稻進(jìn)行檢測(cè),結(jié)果發(fā)現(xiàn)90%以上都是陽(yáng)性植株;通過(guò)qRT-PCR檢測(cè),發(fā)現(xiàn)在T_2代轉(zhuǎn)基因水稻中,OsRhoGAP2基因表達(dá)量均在14倍以上,其中最高的達(dá)到了229倍;對(duì)T_2代轉(zhuǎn)基因水稻農(nóng)藝學(xué)性狀進(jìn)行統(tǒng)計(jì)分析,結(jié)果發(fā)現(xiàn),與野生型相比,其有效分蘗數(shù)有了顯著提高,提高了32.35%-38.75%。提示在分子設(shè)計(jì)育種中,可以通過(guò)提高OsRhoGAP2基因的表達(dá)水平,增進(jìn)水稻的有效分蘗數(shù),進(jìn)一步來(lái)提高水稻的產(chǎn)量。本文對(duì)水稻Rho GTP酶激活蛋白基因OsRhoGAP2的研究顯示,該基因啟動(dòng)子及其編碼蛋白在分子設(shè)計(jì)育種上有重要的應(yīng)用潛力。本研究發(fā)現(xiàn)該基因的啟動(dòng)子主要驅(qū)動(dòng)報(bào)告基因在花藥中表達(dá),同時(shí),該基因啟動(dòng)子對(duì)IAA及高溫、干旱、高鹽等非生物脅迫具有響應(yīng),可以應(yīng)用于植物基因工程、植物品系改良、提高植物抗逆性等方面;同時(shí),本研究的結(jié)果提示,該基因可以作為提高水稻有效分蘗的候選基因。
[Abstract]:Rice is not only a major grain crop, but also an important model plant. It is of great significance for identification of rice-related agronomic traits and functional genes. OsRacD interacting protein encoding gene, including a Rho GTP enzyme-activated protein gene, named OsRho GAP2 (GenBank login number: AY 364311). This study carried out systematic functional identification and element analysis of the gene and its promoter, in order to evaluate the potential of the gene in molecular design breeding. More than three homozygous lines were obtained from each transgenic Arabidopsis thaliana. GUS histochemical staining, enzyme activity assay and deletion assay were used to study the different stages of GUS reporter gene driven by different promoter fragments. The expression of GUS gene in the anther of transgenic Arabidopsis thaliana was driven by the presence of an important anther-specific element in the 5'-703bp~-289bp region of OsRhoGAP2 promoter, especially in the early stage of microspore development, and in the mature stage, the vegetative organs (roots, stems, leaves) and reproduction of Arabidopsis thaliana. OsRhoGAP2 gene may be expressed at the early stage of microspore development and play an important role in maintaining normal anther development. Transgenic Arabidopsis thaliana was sprayed with 5 hormones (IAA, 6-BA, GA, SA, MeJA), 5 stress treatments (high temperature, low temperature, drought, high salinity, dark) for 10 days, combined with GUS infection. It was found that the promoter of OsRhoGAP2 gene may be a promoter related to IAA induction and stress induction, and it can drive the expression of downstream genes under IAA and high temperature, drought and salt induction, but not other hormones. In combination with the 5'-terminal deletion assay, an important MeJA response element was found in the promoter of the gene - 1292 BP ~ - 948 bp, suggesting that the gene may be involved in the MeJA signaling pathway. Molecular detection and phenotypic analysis of T_2 generation rice were carried out in order to understand the potential application of the gene in rice production.By extracting the genomic DNA of transgenic rice combined with PCR amplification, more than 90% of T_2 generation transgenic rice were found to be positive plants.By qRT-PCR detection, OsRhoGAP2 gene table was found in T_2 generation transgenic rice. The results showed that the number of effective tillers increased by 32.35% - 38.75% compared with the wild type, suggesting that the expression level of OsRhoGAP2 gene could be increased in molecular design breeding. The study of rice Rho GTP enzyme-activating protein gene OsRho GAP2 showed that the promoter and its coding protein had important application potential in molecular design and breeding. Because the promoter is responsive to IAA and abiotic stresses such as high temperature, drought and high salinity, it can be used in plant genetic engineering, plant strain improvement and stress tolerance improvement, etc. Meanwhile, the results of this study suggest that the promoter can be used as a candidate gene for improving effective tillering of rice.
【學(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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