水稻Rho GTP酶激活蛋白基因OsRhoGAP2及其啟動子在分子育種上的應用潛力評估
發(fā)布時間：2018-08-14 20:01
【摘要】：水稻不僅是主要的糧食作物,同時也是重要的模式植物,對水稻相關農(nóng)藝性狀功能基因的鑒定意義重大。本實驗室前期對水稻育性控制相關信號通路開展研究,通過酵母雙雜交篩選,從水稻雌雄蕊形成期幼穗cDNA文庫中分離到若干與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:AY 364311)。本研究對該基因及其啟動子開展系統(tǒng)的功能鑒定和元件分析,以此評估該基因在分子設計育種上的應用潛力�；诒緦嶒炇仪捌跇�(gòu)建的該基因啟動子及5個5’端缺失片段載體,分別轉(zhuǎn)化篩選擬南芥,每個轉(zhuǎn)基因擬南芥均獲得3個以上的純合株系。通過GUS組織化學染色、酶活力測定結(jié)合缺失實驗分析,研究不同啟動子片段驅(qū)動GUS報告基因在生長發(fā)育不同階段、不同組織中的表達,發(fā)現(xiàn)在OsRhoGAP2啟動子5’端的-703bp~-289bp區(qū)域內(nèi)可能著存在1個重要的花藥特異性元件,驅(qū)動了GUS基因在轉(zhuǎn)基因擬南芥的花藥中表達,尤其是在小孢子發(fā)育的初期,而在成熟期時,擬南芥的營養(yǎng)器官(根、莖、葉)以及繁殖器官(果莢)中均沒有表達,由此推測,OsRhoGAP2基因可能在小孢子發(fā)育初期表達,對維持花藥正常發(fā)育有重要作用。通過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)對生長到10d的轉(zhuǎn)基因擬南芥進行噴施處理,結(jié)合GUS染色,研究不同啟動子片段對激素、非生物脅迫的響應,發(fā)現(xiàn)該基因啟動子可能是1個與IAA誘導相關及脅迫誘導相關的啟動子,在IAA及高溫、干旱、高鹽誘導條件下能驅(qū)動下游基因表達,而對其他激素沒有響應。進一步推測,OsRhoGAP2基因可能參與到了IAA誘導相關及逆境脅迫誘導相關的應答過程。結(jié)合5’端的缺失實驗表明,在該基因啟動子的-1292 bp~-948 bp存在1個重要的MeJA響應元件,推測該基因可能參與了MeJA信號通路�；诒緦嶒炇仪捌诠ぷ�,本研究對OsRhoGAP2過表達轉(zhuǎn)基因水稻T_2代進行了分子檢測和表型分析,旨在了解該基因在生產(chǎn)上的應用潛力。通過提取轉(zhuǎn)基因水稻基因組DNA結(jié)合PCR擴增,對T_2代轉(zhuǎn)基因水稻進行檢測,結(jié)果發(fā)現(xiàn)90%以上都是陽性植株;通過qRT-PCR檢測,發(fā)現(xiàn)在T_2代轉(zhuǎn)基因水稻中,OsRhoGAP2基因表達量均在14倍以上,其中最高的達到了229倍;對T_2代轉(zhuǎn)基因水稻農(nóng)藝學性狀進行統(tǒng)計分析,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%。提示在分子設計育種中,可以通過提高OsRhoGAP2基因的表達水平,增進水稻的有效分蘗數(shù),進一步來提高水稻的產(chǎn)量。本文對水稻Rho GTP酶激活蛋白基因OsRhoGAP2的研究顯示,該基因啟動子及其編碼蛋白在分子設計育種上有重要的應用潛力。本研究發(fā)現(xiàn)該基因的啟動子主要驅(qū)動報告基因在花藥中表達,同時,該基因啟動子對IAA及高溫、干旱、高鹽等非生物脅迫具有響應,可以應用于植物基因工程、植物品系改良、提高植物抗逆性等方面;同時,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.
【學位授予單位】：河南師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2
[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.
【學位授予單位】：河南師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2
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