新疆甘草DNA片段的克隆、序列分析及其基因组DNA文库的构建

目的克隆新疆甘草DNA片段 ,构建新疆甘草基因组DNA文库。方法对新疆产 4种甘草进行随机扩增多态性DNA(RAPD)分析 ,回收甘草随机引物s12 1的PCR产物中 5 0 0bp左右的带 ,克隆到pMD18 T载体中 ,经电泳鉴定后测定序列 ,进行序列分析。用Sau3AI对所提新疆甘草基因组DNA进行酶切 ,回收 15～ 2 3kb之间的酶切产物片段 ,然后与LambdavectorBamHIArms进行连接、包装形成文库 ,计算文库重组噬菌体滴度、包装效率。结果获得了甘草 4 90bp的DNA片段 ,所建文库的重组噬菌体滴度为 3× 10 6pfu/ml、包装效率为 6× 10 6重组子 /μg载体DNA。结论成功克隆了新疆甘草DNA片段 ,并构建了基因组DNA文库 ,为进一步研究奠定了基础。

Clong and sequence analysis of the DNA fragment and construction of the genomic DNA library in G.uralensis fisch ex DC in Sinkiang

PurposeThe aim is to clone the characteristic DNA fragment and to construct the genomic DNA library in G. uralensis fisch ex DC.MethodsAccording to the result of RAPD analysis in Glycyrrhiza L in Sinkiang, about 750 bp segment was prepared from PCR product with the primer s121 of arbitrary sequence in G.uralensis fisch ex DC in Sinkiang,and cloned into vector pMD18-T and sequenced.The genomic DNA from G.uralensis fisch ex DC in Sinkiang was partially digested with Sau3AI.The large-scale digesting products which contain 15～23 kb size range of fragments were separated by electrophoresis. Suitable molecular ratio of the separated fragments to Lambda vector BamH I Arms was chosen to proceed the ligation by T4 DNA ligase. The ligated DNA was added to a Packagene Extract.and the packaged phage was formed. The titer of the phage and the packaging efficiency of arms were calculated.ResultsA 490 bp DNA fragment was obtained. The titer of the phage was 3×10 6 pfu/ml and the packaging efficiency of arms was 6×10 6 recombinants/μg DNA.ConclusionThe results demonstrate that DNA fragment has been successfully cloned and the genomic DNA library has been constructed in G.uralensis fisch ex DC in Sinkiang.