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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (10): 194-197.doi: 10.11924/j.issn.1000-6850.2012-0287

• 林学 园艺 园林 • 上一篇    下一篇

中国水仙BAC文库构建的研究

王江波 潘东明 潘腾飞 郭志雄   

  • 收稿日期:2012-02-06 修回日期:2012-02-24 出版日期:2012-04-05 发布日期:2012-04-05
  • 基金资助:

    台湾果树新品种与品质控制新技术引进创新研究

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

  • Received:2012-02-06 Revised:2012-02-24 Online:2012-04-05 Published:2012-04-05

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摘要：

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。

关键词: 压条繁殖, 压条繁殖

Abstract:

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.

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