海洋放线菌Streptomyces sp.大片段DNA基因组文库的构建

Library Construct with Large Genomic DNA Fragments from Marine Actinomycete Streptomyces sp.

目的:构建稀有海洋放线菌Streptomyces sp.基因组文库。方法:以稀有海洋放线菌Streptomyces sp.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Streptomyces sp.的基因组文库,效价达9.0×104CFU/mL,得到4000个阳性克隆子,远远大于按覆盖率为99%计算至少所需的837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Streptomyces sp.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 genomic library of rare marine actinomycete Streptomyces sp.Method：Rare marine actinomycete Streptomyces sp.was taken as the material and its total genome was cut randomly.Fragments from about 40 kb were ligated into the pWEBTM vector,packaged into phage particles with lambda packaging extracts,and transfected into E.coli EPI100.The genomic library was construted.Result：A genomic library of rare marine actinomycete Streptomyces sp.was successfully constructed,and the titer of the library is 9.0×104CFU/mL,obtained 4 000 clones.According to the coverage rate of 99% to calculate,it needed 837 clones,but finally obtained 4 000 clones,far greater than the minimum value of 837.The average length of the inserts is 36kb and the recombination rate is 100%.The colones were stored in 96-well microlite plates at-80℃.Conclusion：The constructed cosmids library meets the requirements as a standard genomic library.In order to assess all the latent natural bioactive products from Streptomyces sp.,further investigation will be carried out to express the biosynthetic gene clusters in E.coli EPI100 from this library heterogeneously.