大白菜抗霜霉病同源基因的筛选与亚克隆文库的构建

Identification of downy mildew resistance analog gene and construction of sub-clone library on Chinese cabbage

根据植物抗霜霉病基因保守区设计简并引物,利用三维PCR方法对大白菜（Brassica campestris ssp.pekien-sis）‘85-1’BAC文库进行筛选,从19 200个BAC克隆中筛选到含有目的基因的10个阳性克隆。利用HindⅡ对其中的94-G-17克隆进行酶切,回收1~5 kb的DNA片段并连接到载体pUCHindⅡ？BAP上,构建了含有6 200个克隆的亚克隆文库。经电泳检测,插入片段在1~5 kb。该亚克隆文库的构建为克隆大白菜抗霜霉病同源基因全长奠定了基础。

The degenerated primers were designed according to the conservative domain of most disease resistant genes of downy mildew in plants.Screening the Chinese cabbage ＇85-1＇ BAC library through three steps of PCR,10 positive clones containing target gene from 19200 BAC clones were obtained.Digesting the BAC clone 94-G-17 with HindⅡ,1-5 kb fragments were collected.Then,these fragments were ligated on the pUCHindⅡ/BAP vectors,and a sub-clone library containing 6 200 clones were obtained.By electrophoresis detection,the insert fragments were from 1 kb to 5 kb.The sub-clone library could be used for obtaining the complete sequence of downy mildew resistance gene in Chinese cabbage.