中国水仙BAC文库构建的研究

Construction and Characteristics of a BAC Library of Narcissus tazetta var.chinensis

为更深入地挖掘中国水仙的基因组信息,筛选与中国水仙品质相关的功能基因,以中国水仙品种‘金盏银台’的黄化叶片为材料,采用改良Zhang法获得高分子量核DNA,经酶切、连接和转化,首次构建了中国水仙基因组BAC文库。结果表明,采用改良zhang法获取的核DNA分子量大于1 Mb,适合BAC文库的构建;优化了连接、转化体系,发现载体和插入片段的摩尔比为5：1时,连接、转化效率最高;经检测,该文库包括69120个克隆,插入片段的平均大小约87 kb,空载率小于1%,大约50%的克隆大小在80~90 kb之间;Southern blot结果表明该文库未受到细胞器DNA的污染。

To further study Narcissus genome and screen functional genes,a BAC library was constructed for the first time in the world.The high molecular weight DNA（HMW-DNA） was isolated from Narcissus etiolation leaves used improved Zhang＇s method.The HMW-DNA was cut by Hind III and ligated to BAC vectors;the ligations were transformed into Escherichia coli DH10B.The results showed that the DNA isolated by improved Zhang＇s method was higher than 1 Mb,which was suitable for BAC library construction.The ligation and transform methods were improved;the optimum mole ratio between vectors and DNA fragments was 5：1.The library consists of 69120 clones with an average insert size of 87 kb,about 50% clones contain 80 to 90 kb insert DNA,and less than 1% of the clones do not contain inserts.Screening of BAC library with chloroplast DNA probes by Southern blot indicated that less than 1% of the clones contained organelle-derived DNA.