The Study On The Chitinase Gene And The Antibiotic Biosynthesis Gene Cluster Of Streptomyces Roseoflavus

Streptomyces spp., G+ bacteria in the genus, are important producers of antibiotics and other pharmacologically active compounds, such as chitinase. The microorganism chitinase is one of anti-fungi protein, and it has the important function in the mechanism of defending the phytopathogenic fungi though breaking down walls of plant cells. Totally 360 strains of Streptomyces were isolated from rhizosphere soil in this study, and were tested for their antagonistic against Verticillium dahliae V41. On the double-layer agar test, 161 isolates showed activity of antibiosis. Another 24 strains of Streptomyces spp. that were kept in our lab, were also tested and showed strong antibiosis activity against Verticillium dahliae. And 115 out of the 185 antagonistic strains showed chitinolytic activity by biological assay. A mothod of extracting DNA of Streptomyces strains for PCR molecular detection was developed though assessing the influence of four different DNA extraction methods , such as boiling methods, STE, SDS, Microwave methods. And DNAs were than amplified with the 16SrDNA primers of Streptomyes spp. the 1.6Kb sequence segments were amplified by all methods except STE method, and the boiling method was especially fit on DNA exaction for the PCR molecular detection.The 115 strains of antagonistic Streptomyces were detected for chitinase genes using designed PCR primers (Chi-1/Chi-2 and Chi-3/Chi-4) which derived from the conservative sequences of 26 cloned chitinase genes from Streptomyces spp. A specific DNA fragment was amplified by PCR from 112 out of 115 strains of'Streptomyces with chitinolytic activity. It was demonstrated that the PCR-mediated method to detect the chitinase-producing Streptomyces spp. from soil environment is possible and effective, and the primers designed were also qualified.The strains Men-myco-93-63 isolated from potato scab decline soil was identified as S.roseoflavus, it can produce antibiotics that had a strong antibiosis activity against Verticillium dahliae. In this study, the related biocontrol genes were cloned by different ways:1. After getting the chitinase catalytic domain of Men-myco-93-63 by PCR, the catalytic domain was then connected with S. lividans ChiC fragment which contains three domains (cellulose binding domain, chitin-binding domain, signal peptide domain) and was cloned into pET23b(+). The recombinant plasmid pLCH was transformed into E. coli JM109(DE3). The transformnt was induced expressing with IPTG and its chitinase activity was be found on the chitin culture medium.2. The genomic library of Men-myco-93-63 was constructed in E. Coli LE392 using the E.coli-Streptomyces cosmid shuttle vector pKC505. And then the recombinanted plasmids were isolated and transformed into the protoplasm of S.lividans TK54. The transformants showed an antibiosis activity against Verticillium dahliae had not been found.3. PCR detection of antibiotic biosynthesis related genes of Men-myco-93-63 was done using specific PCR primers, such as polyketide synthase gene (PKS)primers, glucose dehydratase(STR)primers, and nonribosomal peptide synthetas(NRPS) primers. We got the 500bp specific sequences with PKS primers and 750bp specific sequences with STR primers. But those sequences weren't identical with the prospective PKS or STR sequences. The relationship between the PCR sequences and the antibiotic biosynthesis genes would be definitized by gene disruption experiment.4. PCR detection of antibiotic biosynthesis gene of Men-myco-93-63 was done using PCR primers designed according several specific antibiotic resistant genes. The ï¿¡ -lactamas gene sequences were detected from Men-myco-93-63. The plasmid library of Men-myco-93-63 was constructed using pKC1139 plasmid and two clones including P -lactamas gene were screened from the library by the PCR primers. Both of the insert segments included 3 -lactamas ORF by sequencing. But the double end sequences of 3 -lactamas ORF of insert segments weren't identical with any cloned protein sequences inGenbank. The relationship between the insert segments and the antibiotic biosynthesis genes would be definitized by gene disruption experiment.