水稻Rho GTP酶激活蛋白基因OsRhoGAP2及其启动子在分子育种上的应用潜力评估

发布时间：2018-08-14 20:01

【摘要】：水稻不仅是主要的粮食作物,同时也是重要的模式植物,对水稻相关农艺性状功能基因的鉴定意义重大。本实验室前期对水稻育性控制相关信号通路开展研究,通过酵母双杂交筛选,从水稻雌雄蕊形成期幼穗cDNA文库中分离到若干与Rho蛋白OsRacD相互作用蛋白的编码基因,其中包括一种Rho GTP酶激活蛋白基因,命名为OsRhoGAP2(GenBank登录号:AY 364311)。本研究对该基因及其启动子开展系统的功能鉴定和元件分析,以此评估该基因在分子设计育种上的应用潜力。基于本实验室前期构建的该基因启动子及5个5’端缺失片段载体,分别转化筛选拟南芥,每个转基因拟南芥均获得3个以上的纯合株系。通过GUS组织化学染色、酶活力测定结合缺失实验分析,研究不同启动子片段驱动GUS报告基因在生长发育不同阶段、不同组织中的表达,发现在OsRhoGAP2启动子5’端的-703bp~-289bp区域内可能着存在1个重要的花药特异性元件,驱动了GUS基因在转基因拟南芥的花药中表达,尤其是在小孢子发育的初期,而在成熟期时,拟南芥的营养器官(根、茎、叶)以及繁殖器官(果荚)中均没有表达,由此推测,OsRhoGAP2基因可能在小孢子发育初期表达,对维持花药正常发育有重要作用。通过5种激素(IAA、6-BA、GA、SA、MeJA)、5种胁迫处理(高温、低温、干旱、高盐、黑暗)对生长到10d的转基因拟南芥进行喷施处理,结合GUS染色,研究不同启动子片段对激素、非生物胁迫的响应,发现该基因启动子可能是1个与IAA诱导相关及胁迫诱导相关的启动子,在IAA及高温、干旱、高盐诱导条件下能驱动下游基因表达,而对其他激素没有响应。进一步推测,OsRhoGAP2基因可能参与到了IAA诱导相关及逆境胁迫诱导相关的应答过程。结合5’端的缺失实验表明,在该基因启动子的-1292 bp~-948 bp存在1个重要的MeJA响应元件,推测该基因可能参与了MeJA信号通路。基于本实验室前期工作,本研究对OsRhoGAP2过表达转基因水稻T_2代进行了分子检测和表型分析,旨在了解该基因在生产上的应用潜力。通过提取转基因水稻基因组DNA结合PCR扩增,对T_2代转基因水稻进行检测,结果发现90%以上都是阳性植株;通过qRT-PCR检测,发现在T_2代转基因水稻中,OsRhoGAP2基因表达量均在14倍以上,其中最高的达到了229倍;对T_2代转基因水稻农艺学性状进行统计分析,结果发现,与野生型相比,其有效分蘖数有了显著提高,提高了32.35%-38.75%。提示在分子设计育种中,可以通过提高OsRhoGAP2基因的表达水平,增进水稻的有效分蘖数,进一步来提高水稻的产量。本文对水稻Rho GTP酶激活蛋白基因OsRhoGAP2的研究显示,该基因启动子及其编码蛋白在分子设计育种上有重要的应用潜力。本研究发现该基因的启动子主要驱动报告基因在花药中表达,同时,该基因启动子对IAA及高温、干旱、高盐等非生物胁迫具有响应,可以应用于植物基因工程、植物品系改良、提高植物抗逆性等方面;同时,本研究的结果提示,该基因可以作为提高水稻有效分蘖的候选基因。
[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.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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