Livestock Streptomyces Regulate Gene Cloning And Biotechnology Neomycin Stable Genetic Engineering Bacteria Build

The dissertation is divided into two parts:1. In order to study the regulatory gene in thienamycin producing strain (S.cattleya A250) and on the basis of observation that a small molecular,diffusible compound which could restore a mutant the ability of producing thienamycin as well as sporulation was possibly excreted by the wild strain, cloning the regulatory gene from S.cattleya was attempted. Two pairs of primers were designed considering both the highly conserved regions of LuxI,one of the two component regulatory system of luxI/luxR, and the characteristics of biased codon usage of Streptomyces spp genes. An about 200bp fragment was amplified after PCR reaction with the total DNA of S.cattelaya as the template .Using the PCR product as a probe, enzyme digested total DNA of S.cattleya were hybridized, and EcoRI-BamHI 4.8kb,PstI-PstI 3.5kb, BglII-BglII 1.8kb highly positive signals were shown. The 4.5kb-5.5kb of EcoRI-BamHI digested total DNA fragment was recovered and cloned into the same enzymes treated pBluescript SK(-) vector ,two positive colonies were obtained after hybridization. A recombination plamid,pSN1,was obtained. The Southern hybridization pattern of pSN1 was the same as that of the total DNA of S.cattleya . Further Southern blotting experiment localized the positive signal area into a SmaI-XmnI 0.8kb fragment.Two intact ORFs,ORF1 and ORF2,were found after sequenced the total cloned fragment of pSN1 .ORF1 containing the PCR product consisted of 1,800bps with ATG as start codon and TGA as stop codon,and was named sctI, The Genbank accession number is AF111267. No persuasive homology of the amino acids coded by sctI was found of proteins in Genbank. The G+C% of ORF1 is 69.4% and the 1^st,2^nd,3^rd of sctI is 68.0%,45.5%,98.2% respectively, complying the characteristics of streptomyces. spp genes. An unusual phenomenon was noticed in Sma1-BstN1 249bp region upstream of the ORF1 of which the GC content is 59.1%,considering the GC content of the genome of common Streptomycs .spp is about 73%. This...