Library Construct With Genomic DNA Fragments And Cloning Function Gene Of From Marine Actinomycete And Sequence Analysis

Rare marine actinomycete Salinispora arenicola and Streptomyces sp. were taken as the material and their total genome were 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 libraries were constructed. The titer of the library of Salinispora arenicola is 6.5×104 CFU/mL, obtained 3,000 clones. According to the coverage rate 99.9% to calculate, it needed 1380 clones, but finally obtained 65,000 clones, far greater than the minimum value of 1380. The average length of the inserts is 36Kb and the recombination rate is 100%. The positive clones were stored in 96-well microlite plates at-80℃. The titer of the library of Streptomyces sp. is 9.0×104CFU/mL, obtained 4000 clones. According to the coverage rate of 99% to calculate, it needed 837 clones, but finally obtained 4000 clones, far greater than the minimum value of 837. The average length of the inserts is 36kb and the recombination rate is 100%. The clones were stored in 96-well microlite plates at -80℃. The constructed cosmids library meets the requirements as a standard genomic library. In order to assess all the latent natural bioactive products from Salinispora arenicola and Streptomyces sp., further investigation will be carried out to express the biosynthetic gene clusters in E.coli EPI100 from this library heterogeneously.To obtain the fragments related to NRPS and Halogenase gene cluster, Degenerate primers were designed based on the conserved sequences of the NRPS and halide gene cluster from other microbes, the NRPS and Halogenase gene fragments were obtained by PCR, and molecular biological softwares were used to analyze homologies. The NRPS and Halogenase gene cluster fragments contain 660 bp and 555 bp nucleotide acids, encoding 220 and 185 amino acids respectively. Homology comparisons with NRPS and Halogenase gene sequences with Salinispora arenicola CNS-205 are 99% and 98% respectively. The 660 bp and 555 bp fragments related to NRPS and Halogenase gene cluster are obtained. It will help to isolate the full lengths related to NRPS and Halogenase gene clusters and to study their possible roles during biosynthesis further.