目录
摘要Ⅱ
关键词Ⅱ
AbstractⅢ
引言
前言1
1材料与方法2
1.1材料 2
1.1.1供试材料与试剂2
1．1．2 培养基与试剂配方 2
1.2方法 3
1.2.1 GSTOsGAO原核表达载体的构建3
1.2.2 GSTOsGAO融合蛋白的表达与纯化4
1.2.3 免疫印迹检测4
2 结果与分析5
2.1 GSTOsGAO原核表达载体的构建5
2.2 GSTOsGAO原核表达载体的表达与纯化7
2.3 免疫印迹检测7
3 讨论 8
致谢 9
参考文献9
水稻OsGAO基因的原核表达
摘 要
在水稻的雄性生殖发育的过程中，涉及到了一系列的生物学事件，需要精确的转录调控才能完成这一生物学过程。即使截止至目前已经报道了涉及到水稻绒毡层调控的部分调控因子，但是关于水稻绒毡层调控基因和调控机理仍需不断的探索研究和数据积累。本实验在对宁粳4号野生型水稻一个无花粉雄性不育突变体进行表型鉴定和细胞学观察，确定其突变基因为水稻绒毡层调控基因基础上，通过图位克隆成功克隆了该不育基因，并将该基因进行原核表达，提取其表达蛋白。并对其表达蛋白的功能进行一定的预测，以期能够进一步揭示该基因在水稻花粉不育中所起的作用和作用机理，为不育系的创造提供理论和基因资源。本实验成功将雄性不育基因OsGAO基因在原核生物大肠杆菌中成功表达，并对其表达蛋白进行纯化，通过免疫印迹法鉴定该蛋白成功表达并提纯成功。在成功提取表达蛋白质的基础上，我们通过基因序列的比对预测该基因所表达的蛋白具有乙二醛氧化酶活性，但仍需进一步实验验证。
Prokaryotic expression of rice OsGAO gene
ABSTRACT
In the process of male reproductive development of rice, a series of biological events are involv *51今日免费论文网|www.51jrft.com +Q: ^351916072* 
ed, which requires precise transcriptional regulation to complete this biological process. Even though some of the regulatory factors involved in the regulation of rice tapetum have been reported so far, the genes and mechanism of regulation of rice tapetum still require continuous exploration and data accumulation. In this experiment, on the basis of phenotypic identification and cytological observation of a pollenfree male sterility mutant of Ningjing No. 4 wildtype rice, the mutant gene was determined to be a rice tapetum regulatory gene. Sterile gene, and prokaryotic expression of the gene, extracting its expressed protein. It also predicts the function of the protein expressed in order to further reveal the role and mechanism of the gene in rice pollen sterility, and to provide theory and genetic resources for the creation of sterile lines. In this experiment, the male sterility gene OsGAO gene was successfully expressed in prokaryotic E. coli, and the expressed protein was purified. The protein was successfully expressed and purified by immunoblotting. On the basis of successfully extracting the expressed protein, we predict that the protein expressed by the gene has glyoxal oxidase activity through the comparison of gene sequences, but further experimental verification is still required.
KEY WORDS： Male sterility; OsGAO gene; Prokaryotic expression; Protein purification;GLOX
水稻（Oryza sativa L.）是世界上最重要的粮食作物之一，为了有充足的粮食储备，大部分亚洲国家将提高水稻产量作为育种的主要目标。中国60%以上的居民以稻米为主食, 水稻生产事关国家口粮绝对安全。依靠科技创新推进水稻产业发展十分重要。【1】在杂交水稻的生产过程中，雄性不育这一性状被广泛的应用，所以研究水稻雄性不育的相关机理显得尤为重要。
本实验在对水稻一雄性不育植株进行表型鉴定和细胞学观察的基础上，发现该雄性不育基因为调控绒毡层的基因。在水稻中已报到的MYB转录因子 GAMYB【2】；bHLH(basic HelixLoopHelix)转录因子UDT(Undeveloped Tapetum)【3】、 TDR（Tapetum Degeneration Retardation）【4】、EAT1（Eternal Tapetum 1）【5】和TIP2（TDR Interacting Protein2)【67】，Phdfinger蛋白PTC1（Perisant Tapetal Cell1）【8】；细胞凋亡抑制因子OsAPI5【9】；bZIP转录因子 OsTGA10【10】等通过互作或者直接激活各自的靶基因参与精确调控绒毡层PCD(programmed cell death)和花粉外壁的形成。由于本实验所用的雄性不育突变株的基因也为调控绒毡层的基因，但对其调控机理并不清楚，所以我们对其表达的蛋白进行酶活测定，从而研究其调控绒毡层发育的机理。

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