【作者】 薄存沛；

【导师】 徐有明； 魏文辉；

【作者基本信息】 华中农业大学 ， 植物学， 2010， 硕士

【摘要】 油菜是重要的油料作物之一。新疆野生油菜是野芥(Sinapis arvensis)分布在中国新疆的一个自然群体,它生长势强,抗病虫害、抗菌核病能力强,具有不育胞质,是油菜育种的优良野生种质资源。甘蓝型油菜-新疆野生油菜二体异附加系(B. napus-S. arvensis disomic alien addition line)是新疆野生油菜18号与甘蓝型油菜(Brassica napus)品种中双4号原生质体融合,连续4次回交后得到的新型油菜恢复系,它具有一对来源于新疆野生油菜的染色体,该染色体上携带育性恢复基因,克隆该恢复基因对探索油菜育性恢复机制、培育优良转基因油菜恢复系具有重要的科学意义和生产价值。本研究构建了新疆野生油菜基因组BAC文库,并以甘蓝型油菜-新疆野生油菜二体异附加系BAC文库为材料,对筛选新疆野生油菜染色体来源的BAC克隆进行了研究。研究结果如下：新疆野生油菜基因组BAC文库的构建：用低熔点琼脂糖包埋细胞核的方法制备高分子量DNA,HindⅢ酶切,电洗脱回收100-300kb大片段DNA,与pCC1BACHTM载体连接,用0.025μm的透析膜悬浮在10%的PEGS000中冰上脱盐浓缩,转化E.coilEP1300感受态细胞,得到新疆野生油菜基因组BAC文库,保存在38块384孔板中。随机挑取100个克隆,用Notl酶切,脉冲电泳检测插入片段大小,插入片段在50-250kb之间,平均插入片段105kb,该文库共有14592个克隆,覆盖了新疆野生油菜基因组4.3倍。野生油菜特定染色体来源BAC克隆的筛选：以新疆野生油菜基因组DNA为探针,直接对甘蓝型油菜-新疆野生油菜二体异附加系BAC文库的克隆进行菌落原位杂交,以期获得野生油菜特定染色体来源的BAC克隆,但未获得理想的结果,下一步需要以合适剂量的甘蓝型油菜基因组DNA进行封阻等方法继续开展相关研究。更多还原

【Abstract】 Rape is one of the important oil crops. Xinjiang wild rape is a wild mustard (Sinapis arvensis) found in a natural population of Xinjiang, China, it has strong growth potential, strong resistance to pests and diseases, and strong resistance to Sclerotinia sclerotiorum, as well as sterile cytoplasm, so it is a fine breeding of wild rapeseed germplasm. Brassica-Xinjiang wild rape disomic addition lines (B. napus-S. arvensis disomic alien addition line) is a new rapeseed restorer line which is obtained through protoplast fusion between Xinjiang wild rape 18 and ZhongShuang 4 (Brassica napus).and continuously backcross four times.The disomic addition lines have a pair of chromosomes from Xinjiang wild rape, which carrying the fertility restorer gene. Cloning of the restorer gene is to explore recovery mechanism, and cultivate good transgenic rapeseed restorer lines, It has important scientific significance and production value. The study has constructed wild rape genomic BAC library and studied BAC clones of Xinjiang wild rape chromosome from Brassica napus-Xinjiang wild rape disomic addition lines of BAC library. The results are as follows:Construction of Xinjiang wild rape genomic BAC library:With low melting point agarose embedded nuclei,high molecular weight DNA were prepared, then digested by HindⅢ, through electro elution,100-300kb large fragment DNA were recovered and linked with pCC1BACHTM vector, the connection was desalted and concentrated with 0.025μm dialysis membrane suspended in 10% PEG8000 on ice, then transfered into E.coil EP1300 competent cells. Xinjiang wild rape genomic BAC library had been constructed, and stored in 38 384 well plates. Randomly selected 100 clones, digested with NotI, tested insert size with PFGE, the library has 14,592 clones, with the insert size of 50-250kb, average insert of 105kb, covering the genome of Xinjiang wild rape 4.3 times.Screening specific BAC clones from wild rape chromosome:Xinjiang wild rape genomic DNA as a probe, directly hybridized colony in situ to the Brassica napus-Xinjiang wild rape disomic addition lines of clone BAC library to obtain a specific chromosome of wild rape sources of BAC clones, but get no desired results, the right dose of Brassica napus genome DNA for blocking will be carried on for the further correlation research.更多还原