苦荞BAC文库的构建及BAC末端序列分析

Construction of BAC Library of Fagopyrum tataricum and Analysis of BAC End Sequences

目的:为了深入挖掘苦荞的基因组信息,筛选与苦荞品质相关的功能基因。方法:以苦荞品种"晋荞麦2号"为材料,参考Peterson等方法通过提取高分子量核DNA,经酶切、连接和转化构建苦荞基因组BAC文库;随机挑选48个BAC克隆进行序列末端测序。结果:该文库包括30 000个克隆,插入片段平均大小约为123 kb,空载率小于1%,覆盖苦荞基因组6.9倍。共得到89条BAC末端序列,通过序列比对分析发现,7条(8%)有比对结果。其中,有末端序列与rpoTm2、Trrap、MDR1等已知基因相似,这些基因与DNA锚定,物质跨膜运输和磷酸转移酶活性等功能有关。分析还发现末端序列40G19-F可能是苦荞基因组的重复序列。结论:苦荞BAC文库构建为今后苦荞基因的克隆及全基因组测序奠定了基础。

Objective： To further study Fagopyrum tataricum genome and to screen the functional genes. Methods： The variety JINQIAO No. 2（ Fagopyrum tataricum） native to Shanxi,was used for BAC library construction. The high molecular weight DNA（ HMWDNA） was isolated from Fagopyrum tataricum leaves used the methods of Peterson. The HMW-DNA was cut by Hind Ⅲ and ligated to BAC vectors; the ligations were transformed into Escherichia coli DH10 B. Then 48 BAC clones were randomly selected and sequenced BAC end sequences. Results： The library consisted of 30 000 clones with an average insert size of about 123 kb and the empty clone ratio was less than 1%. The library represented an equivalent about 6. 9 fold size of Fagopyrum tataricum genome. Which obtained 89 BAC end sequences,among which 7 sequences（ 8%） had alignment results when comparing with NCBI Gen Bank database. The blast hits contained some genes with known function,such as rpo Tm2,Trrap and MDR1 genes,etc. Those genes were associated with DNA binding,transmembrane transport and phosphotransferase activity function. And it was also found that the sequence of 40G19-F was probably repetitive sequences in Fagopyrum tataricum genome. Conclusion： The BAC library will be helpful for the gene cloning and whole genome sequencing of Fagopyrum tataricum.