棉花线粒体基因组细菌人工染色体文库的构建  

Construction of BAC Library of Cotton Cytoplasmic Male Sterility(mt DNA)

作　　者：代培红[1] 孟志刚[1] 罗淑萍[2] 张锐[1] 程海刚[1] 郭三堆[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]新疆农业大学农学院,乌鲁木齐830052

出　　处：《新疆农业大学学报》2007年第4期57-62,共6页Journal of Xinjiang Agricultural University

基　　金：国家"863"计划项目(2002AA207005)

摘　　要：以P30A细胞质雄性不育系黄化苗为材料,采用蔗糖密度差速离心和不连续蔗糖密度梯度超速离心提取棉花线粒体,低熔点琼脂糖包埋和原位消化裂解的方法制备高分子量的线粒体DNA(mtDNA),经BamHⅠ,HindⅢ酶切和PCR扩增进行纯度鉴定,并对其进行部分酶切,酶切片段与载体连接,电击转入大肠杆菌DH10B中,成功的构建了棉花线粒体基因组BAC文库。该文库共挑取3 840个单克隆,平均插入片段大小为15.5 kb,覆盖率超过70倍,研究结果表明该基因文库具有较高的质量。A procedure to isolate mtDNA from cotton seedlings has been developed. The protocol allows cotton mitochondrion of high purity and yield to be isolated by using differential centrifugation,discontinuous sucrose gradient centrifugation of sucrose density. High molecular weight mtDNA was prepared by embedding low melt point agrose and instu digesting and lysis. Then the mtDNA of cytoplasmic male ste- rility material P30A was partly digested by restriction endonuclease, and was ligated with the BAC vector through T4 ligase. Electroporating into E. coli DH10B, the BAC library of cotton mtDNA was successful- ly constructed. The library consisted of about 3 840 clones with insert size 15.5 kb. It was calculated that the library contains approximate 70 times of the cotton mitochondrial genome. It was demonstrated this genome library has high quality.

关 键 词：棉花 细胞质雄性不育 线粒体基因组(mtDNA) 细菌人工染色体 文库 

分 类 号：Q785[生物学—分子生物学]