油菜BAC文库和水稻BAC克隆BIBAC亚克隆文库的构建及相关克隆筛选

发布时间：2019-01-30 08:42

【摘要】：本研究工作由两部分组成,第一部分是关于油菜BAC文库的构建以及相关克隆的筛选;第二部分是水稻BAC克隆BIBAC亚克隆的构建和相关克隆的筛选。细菌人工染色体(bacterial artificial chromosome,BAC)文库和人工双元染色体(binary bacterial artificial chromosome,BIBAC)文库在基因组的研究方面扮演了重要的角色。油菜是一种重要的农作物,具有很好的经济效益。通过油菜菜籽榨取的植物油不仅饱和脂肪酸的含量低而且还含有多种氨基酸、维生素,非常有益于人体的健康。本研究中,利用中国农科院油料作物研究所师家勤副研究员提供的油菜叶片,构建了一个油菜的BAC文库,用于相关基因的筛选和克隆。油菜BAC文库总计103,680个克隆,存放在270个384孔板中,文库平均插入片段大小为122.6kb,按油菜基因组1000M计算,覆盖油菜基因组12.5倍,空载率低于2%,无细胞器污染;利用连锁标记,对整个油菜BAC文库进行筛选,成功地筛选到4个阳性克隆,这些克隆携带了与油菜性状相关的基因。该部分工作对于通过遗传改良提高油菜的产量和品质具有特殊的意义。BAC载体不能用来直接转化植物,需借助BIBAC载体,通过BIBAC载体构建的亚克隆文库,能够直接转化植株。利用Sau3AI内切酶对水稻BAC文库中筛选出来的阳性克隆进行部分酶切,构建了一个水稻BIBAC亚克隆文库。该文库共768个克隆,平均插入片段大小为18kb,覆盖度为69倍。利用分子标记设计引物,对整个文库进行筛选,得到72个与水稻性状相关的阳性克隆基因,并进行了测序验证,这些克隆将直接用于水稻遗传改良。
[Abstract]:This research consists of two parts: the first part is about the construction of rape BAC library and the screening of related clones; the second part is about the construction of BIBAC subclone of rice BAC clone and the screening of related clone. Bacterial artificial chromosome (bacterial artificial chromosome,BAC library and artificial binary chromosome (binary bacterial artificial chromosome,BIBAC library play an important role in genome research. Rape is an important crop with good economic benefits. The vegetable oil extracted from rapeseed is not only low in saturated fatty acids but also contains a variety of amino acids and vitamins, which is very beneficial to human health. In this study, a rape BAC library was constructed by using the leaves of oilseed rape from Shijiaqin Research Institute of Chinese Academy of Agricultural Sciences, which was used to screen and clone related genes. The rape BAC library was composed of 103680 clones and was stored in 270 384-well plates. The average inserted fragment size of the library was 122.6 kb. According to the rape genome 1000M, the rape genome was covered with 12.5 times of the rape genome, the no-load rate was less than 2 parts, and no organelle contamination was found. The whole rape BAC library was screened by linkage markers, and four positive clones were successfully screened, which carried genes related to rape traits. This part of the work has special significance for improving the yield and quality of rape through genetic improvement. BAC vector can not be used to transform plants directly. It needs the help of BIBAC vector and the subclone library constructed by BIBAC vector, which can directly transform plants. A rice BIBAC subclone library was constructed by partial digestion of the positive clones from rice BAC library by Sau3AI endonuclease. There are 768 clones in this library. The average insertion fragment size is 18 kb and the coverage is 69 times. The whole library was screened with primers designed by molecular markers and 72 positive genes related to rice traits were obtained and sequenced. These clones will be directly used in rice genetic improvement.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4;S511

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