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以我国抗病、优质、丰产的棉花优良品种中棉所12号为材料,对棉花细菌人工染色体(BAC)文库构建过程中的一些关键技术,如plug的制备、DNA的部分消化、酶切片段的选择、插入片段与载体的摩尔比值、连接产物的浓缩等进行了研究,建立了构建棉花BAC文库的适宜方法体系。依照该方法初步构建了含有38000个克隆的棉花BAC文库。经检测,插入片段平均大小约为120kb,蓝斑率小于0.5%,空载率小于1%。插入片段与载体的最适摩尔比为1∶15,一次转化可以获得约2000个克隆,cfu·μl 1高达4。该方法为进一步构建中棉所12号多倍基因组的BAC文库、从而进行棉花基因组有关研究奠定了基础。
In this paper, some key technologies in the process of BAC library construction, such as preparation of plug, partial digestion of DNA, restriction enzyme digestion The molar ratio of inserted fragment to the carrier and the concentration of the ligated product were studied. The suitable method system for constructing the cotton BAC library was established. According to this method, we initially constructed a cotton BAC library containing 38000 clones. After testing, the average insert size of about 120kb, blue spot rate of less than 0.5%, no-load rate of less than 1%. The optimal molar ratio of insert to vector was 1:15, about 2000 clones were obtained in a single transformation, and cfu · μl 1 was up to 4. This method lays the foundation for further research on cotton genomics to further construct BAC library of Cultivars 12 of China Cotton.