稀有海洋放线菌Salinispora arenicola大片段DNA基因组文库的构建  

Construct Library with Large Genomic DNA Fragments from Rare Marine Actinomycete Salinispora arenicola

作　　者：马艳玲[1] 邓海[1] 刘中来[1] 邓灵福[1] 刘艳丽[1] 祁超[1] 

机构地区：[1]华中师范大学生命科学学院,湖北武汉430079

出　　处：《生物技术》2010年第3期1-3,共3页Biotechnology

基　　金：国家自然科学基金项目(20772040)资助

摘　　要：目的:构建稀有海洋放线菌S.arenicola基因组文库。方法:以稀有海洋放线菌S.arenicola为实验材料,随机剪切提取的总DNA,5′-磷酸末端补平回收40kb左右的DNA片段,与pWEBTM载体连接,经包装蛋白包装成噬菌体后侵染宿主细胞E.coliEPI100,构建该菌株的基因组文库,并对该文库进行质量鉴定。结果:成功构建了稀有海洋放线菌S.arenicola的基因组文库,效价达6.5×104CFU/mL,得到3000个阳性克隆子,远远大于按覆盖率为99%计算至少所需的657个阳性克隆子数,且平均插入片段长度为36kb,重组率100%。阳性克隆子保存于96孔板中,-80℃保存。结论:所构建文库的各项指标均达到要求,为了进一步评估S.arenicola所能合成的所有潜在天然产物,还需要进一步检测该文库中包含有生物合成基因簇的大肠杆菌的表达情况。Objective： To construct a genomie library of rare marine aetinomycete S. arenicola. Method： Rare marine aetinomycete S. arenicola was taken as the material and its total genome was randomly cut. Fragments from 40 kb were ligated into the pWEBTM vector, packaged into phage particles with lambda packaging extracts, and transfected into E. coli EPI100. The genomie library was construted. Result： A genomie library of rare marine actinomycete S. arenicola was successfully constructed, and the titer of the library is 6.5 × 10^4 CFU/mL, obtained 3 000 clones. According coverage rate is 99% to calculate,it needed 657 clones, but finally obtained 3 000 clones, far greater than the minimum value of 657. The average length of the inserts is 36kb and recombination rate is 100%. The eolones were stored in 96 -well mierolite plates at -800C. Conclusion： The constructed eosmids library meet the requirements as a standard genomic library. In order to assess all the latent bioactive natnral products from S. arenicola, fitrther investigation will be carried out to heterogeneously express the biosynthetic gene clusters in E. coli EPI100 from this library.

关 键 词：海洋放线菌S.arenicola 基因组文库 噬菌体 

分 类 号：Q785[生物学—分子生物学]