大豆细胞质雄性不育系配套保持系线粒体基因组BAC文库的构建

BAC Library Construction for Mitochondrial Genome of A Maintainer(JLCMS1) Associated with Cytoplasmic Male Sterility Soybean

以大豆细胞质雄性不育系配套保持系材料(JLCMS1)的黄化苗为材料,纯化得到线粒体。低熔点琼脂糖包埋线粒体成胶块,消化裂解后用HindⅢ部分酶切,通过脉冲场电泳回收40～60 kb酶切片段。将回收产物连接到载体pIndigo-BAC 5,通过电击转化大肠杆菌DH10B感受态细胞,获得的BAC文库包含2 000个单克隆,平均插入片段大小约为50 kb,约覆盖大豆线粒体基因组250倍以上。

A soybean maintainer of the coytoplasmic male sterility soybean（JLCMS1） was used for mitochondria isolation with high purity.Purified mitochondria was embedded into plugs by low melt point agarose,then the plugs was lysed and partially digested by HindⅢ.The DNA fragments between 40 and 60 kb size were selected by pulsed field gel electrophoresis.The size-selection product was ligated to BAC vector pIndigo-BAC5 and then transformed into competent cell of DH10B by electroperation.The constructed BAC library consist of 2 000 clones with an average 50 kb inserts and covered more than 250 times of the soybean mitochondrial genome used in this research.