【作者】 李勇；

【导师】 杨光圣；

【作者基本信息】 华中农业大学 ， 作物生物技术， 2008， 硕士

【摘要】 油菜是食用植物油和植物蛋白的最主要来源,也是潜在的仅次于豆粕的大宗饲用蛋白源,而且在现代工业上的用途也日益广泛。随着人们生活水平的不断提高,对油菜产量和品质的要求也相应提高,因此,开展油菜杂种优势利用研究,把品质育种与杂种优势利用结合起来,选育高产优质油菜品种已成为目前油菜育种的主攻方向。油菜雄性不育无疑是利用油菜杂种优势的一个有效途径,并且已经广泛地应用于生产实践。关于甘蓝型油菜的不育机制,一直以来是人们所研究的热点,吴建勇(2006)构建了甘蓝型油菜显性核不育材料Rs1046AB的抑制差减(SSH)文库,研究表明雄配子发育的各个时期有212个表达序列标签(EST)存在差异表达,并且就显性核不育的不育机制提出假设,即不育材料中Rad23基因表达很微弱,从而导致不能正常识别DNA的损伤,因此在花粉母细胞发育的早期就产生了各种各样的减数分裂不正常行为,并最终导致不育花粉的产生。研究表明,参与DNA修复的Rad23蛋白具有类似泛素(ubiquitin)的功能,并且与DNA损伤修复因子(XPC)结合,一起识别损伤的部位。如果Rad23基因失活,则不能有效地进行DNA修复,增加了细胞对紫外辐射的敏感度,引起细胞的损伤。本研究以甘蓝型油菜显性核不育纯合两型系Rs1046AB,隐性核不育9012AB,为研究对象,在Rs1046AB的SSH文库中选取可能与小孢子发育阶段DNA修复有关的的2个EST:1-B09,GenBank登录号为EE392255;2-I05,GenBank登录号为EE392341。希望通过RACE技术,得到全长cDNA,进一步获得基因组的信息,获得以下结果:1、采用RACE技术,从油菜Rs1046AB花粉中克隆得到候选EST:1-B09的全长cDNA,命名为Brassica napus DNA repair protein RAD23 gene,(Bnrad23 gene)GeneBank登录号为EU306600;并且克隆了部分启动子序列,与编码区一起命名为Brassica napus RAD23-like gene,promoter region,5′UTR,and complete sequence,GeneBank登录号为EU306601;另一个侯选EST:2-I05的部分cDNA序列,命名为Brassica napus RAD23-like protein cDNA,GeneBank登录号为EU306599。此外,本研究还对Brassica napus rad23蛋白的性质及结构进行了部分预测。2、通过半定量RT-PCR,分析了2种EST在Rs1046AB,9012AB中的表达情况,探讨了与DNA修复相关的基因与甘蓝型油菜雄配子败育的联系与可能的机制。3、通过染色体步移的方法,克隆了1-B09的部分启动子区域,通过生物信息学分析,发现包括2个TATAbox,3个CAAT框,5个GATAbox,多处在启动基因花粉特异性表达中起重要作用的顺式元件,以及多种与胁迫相关的顺式元件。并且在Rs1046AB的不育和可育株中,扩增出Bnrad23的基因组序列,通过比较,发现二者在基因组水平上并不存在差异。4、通过对获得的2-I05的部分序列与1-B09全长cDNA比较,发现2-I05已包含了完整的编码区域,并且通过分析,认为这两个基因可能属于同一个基因家族。更多还原

【Abstract】 Rapeseed is one of the most important resource of edible oil and vegetable protein, and also can be used in modern industry. With the improvement of people’s life, we need to improve the yield and quality of rapeseed. So, utilization of heterosis to breed the high yield and high quality rapeseed hybrids has become the main aim for rapeseed breeding.Genic male sterility of rapeseed has been widely applied and it is a efficient way to realize heterosis.The mechanism of genic male sterility in Brassica napus has been studied by some researchers. Wu(2006) established the SSH library in dominant genic male sterility Brassica napus L Rs1046AB. The research revealed that there were 212 ESTs expressed differently in the development of male gametogenesis.And there was a assumption that ,the expression of rad23 gene in the sterility materials is weak, so it can not recognize the damaged DNA,therefore,there are abnormal meiosis behaviors in the early development of pollen mother cells, which eventually led to the formation of sterile pollen.Some researches reveal that, rad23 protein has similar function to ubiquitin,and it can bind to the XPC factor, the protein complex can recognize the damaged DNA. If the rad23 gene was inactive, the sensitivity to UV of the cell maybe enhanced, and the procession of repairing DNA is inefficient, which eventually led to damage of the cell.In this paper, the homozygous DGMS two-type line Rs1046AB and two-type heterozygous RGMS 9012AB were used as materials. Two ESTs associated with the DNA repair: 1-B09 (GeneBank accession number: EE392255), 2-I05 (GeneBank accession number: EE392341) was selected in the experiment. In this article, cloning the full length cDNA of rad23 , and function analysis of it were carried out. Primary results are listed as follows:1、Full-length cDNA of this EST: 1-B09, named Brassica napus DNA repair protein RAD23 mRNA (Bnrad23 gene) was cloned (GeneBank accession number: EU306600) in pollen of Rs1046AB using rapid-amplification of cDNA ends(RACE). Promoter regions of Brassica napus DNA repair gene were cloned. Together with the coding sequence it was called Brassica napus RAD23-like gene, promoter region, 5’ UTR, and complete sequence(GenBank accession number: EU306601); The part cDNA of another EST 2-I05 was cloned(GeneBank accession number: EU306599). Characterization and structure of rad23 protein were partially predicted, too.2、Analysis of the expressing of the 2 ESTs were done in Rs1046AB, 9012AB by RT-PCR,the relationship of DNA repair and male sterility in Brassica napus was partially predicted, too.3、By using chromosome walking technique, promoter regions of Bnrad23 gene were cloned from Rs1046AB. Two TATA boxes, three CAAT boxes, five GAAT boxes, several cis elements which have significance for initiating gene pollen-specific expression, and a series of cis elements relevant to stresses were found in the promoter region. There are no difference between sterility and fertility on the genome level of Bnrad23.4、Analysis of the part cDNA of 2-I05, it maybe contain the whole coding sequence .The 2-I05 and the 1-B09 are possibly the same gene family.更多还原