【作者】 贺热情；

【导师】 刘选明；

【作者基本信息】 湖南大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 激活标记技术(activation tagging)是构建功能获得突变体为研究对象,在植物功能基因组学的研究中具有重要的作用。该技术在模式植物拟南芥中已经得到广泛的应用,但是在油菜中还未见报道。我们以优质高产的油菜品种1244W6为材料,采用浸花蕾方法,将含有激活标记和Basta筛选标记质粒pSKI015的农杆菌进行转化,成功构建了油菜激活标记突变体库。通过Basta筛选,结果共获得约30000个转基因株系(T1代)。转化效率约为1%。对突变体库的T1代转基因植株表型进行观察,共发现39个株系有明显的表型变化,约占转化植株总数的千分之五,包括叶色、分枝数、植株的育性以及开花期等方面的变异。这将为开展油菜分子遗传学研究提供丰富的资源。将突变体T2代种子播种在含不同浓度植物激素的营养液中,结果筛选出激素反应突变体63个。这些激素反应突变体的获得,为揭示油菜激素代谢调控及激素调控油菜生长发育、产量和含油量的分子机理提供了材料。采用TAIL-PCR技术,克隆了W1016赤霉素反应突变体T-DNA插入位点基因组旁邻序列。通过序列比对,发现该序列与拟南芥基因组中的细胞色素P450单加氧酶基因(At1g01190)序列相似。接着我们便以这旁邻序列作为出发点,采用Genome Walker的方法进一步克隆到了油菜中P450基因的全长序列,这为深入研究该基因在油菜激素代谢调控中的功能奠定了基础,同时也建立了克隆油菜突变体相关基因的新方法。更多还原

【Abstract】 Activation tagging is an important strategy in plant genomics by generating gain-of-function mutants. This method in arabidopsis has been widely applied, but not reported in napus.An activation tagging mutant library of Brassica napus 1244w6 was constructed by inflorescence dipping method, and pSKI015 plasmid with Basta screening marker was transformed by Agrobacterial tumefaciens containing 30000 independent T1 transformants were obtained through basta screening. The efficiency of transformation is about 1%. Library of mutant T1 generation of transgenic plants have observed phenotype, a total of 39 lines have significant phenotypic change about 5%o of transformed plants, changes in leaf color, branch number, plant fertility as well as the variation in flowering periods.This would be provided abundant resources for the development of molecular genetics of Brassica napus.About 63 phytohormone response mutants were obtained in the seeds of T2 transformants screened, which was sowed in nutrient solution with different phytohormone. So, these mutants could provide some materials to reveal and clarify the molecular mechanism of the phytohormone metabolism in Brassica napus growth and development, and regulation in yield and oil content of Brassica napus.The T-DNA flanking sequence of gibberellin response mutant W1016 was cloned by TAIL-PCR. which is quite similar to the cytochrome P450 monooxygenase gene (At1g01190) in arabidopsis thaliana genome. Then we take this known flanking sequence, to clone the full-length sequence of Brassica napus P450 genes by using Genome Walker. This research lays the foundation of further study on this gene in oilseed rape in the functional regulation of hormone metabolism, and also established a new approach of clone rape-related genes from mutant.更多还原