| Streptomyces roseoflavus Men-myco-93-63, which isolated from the potato scab decline field, was an excellent strain to control some plant diseases such as cucumber powdery mildew, cotton verticillium wilt and so on. The research was carried out to extract the active agent to inhibit the Streptomyces scabies from the fermentation and clone the genes related to antibiotic biosynthesis.S. roseoflavus Men-myco-93-63 was shake-flask culture in oatmeal medium and could produce 172.2 mg crude antibiotic in 1000 ml fermentation. The activity of the crude antibiotic was detected with oxford cup and the results showed that it could inhibit S. scabies S87 and Verticillium dahliaeV41 invariably. The quantitative assays also showed that 0.3mg/ml crude antibiotic was enough to inhibit S. scabies S87, but not to S. lividans. The crude antibiotic was analyzed by TLC and the results showed that it was the suitable developing solvent while the ratio of methyl alcohol and chloroform was 1:6 and 7 components could be separated on silica gel plate. The component Ⅲ, Ⅳ, Ⅶ had the antibacterial action to S. scabies S87 and the component Ⅲ was the most active agent. The component Ⅶ had the fungistatic action to V. dahliae V41 yet. Then the component III was prepared largely on preparative silica gel plate and the analysis of TLC and HPLC expressed it was very purity. The UV spectrum of the component Ⅲ showed that the maximum absorb wavelength (λmax) was 198.1nm and 274.5nm.The value of MIC (minimum inhibitory concentration) was determined and the results showed that S. lividans TK54 with pIJ702 could be inhibited when one milliliter MM medium had 80μl component Ⅲ. S. roseoflavus Men-myco-93-63 could be resistant higher concentration component III and the resistance gene was cloned by the shotgun method in S. lividans TK54 which located in the 1.7kb DNA fragment. This fragment had 4 ORFs, ORF1 encoded 386aa which was homologous to beta-galactosidase from S. grieus and S. colicolor. ORF2 which may be a new gene in streptomyces encoded a putative protein comprising 83aa. N-terminal of the putative protein was high homologous to RNA binding domain of ribosomal protein L2 from S. avermitilis MA-4680, S. collinus, S. coelicolor and C-terminal was very homologous to the endoglucanase from S. nanchangensis and S. avermitilis MA-4680. ORF3 encoded a putative proteinhomologous to aldehyde/ ketone reductase from Kimococcus radiotolerans SRS30216. ORF4 was located in the complementary strand corresponding to ORF3 and encoded 115aa which was homologous lowly to multidrug resistance protein (SpcT-like efflux protein, 450aa) from S. avermitilis MA-4680. The coding sequence should be subcloned and identify the function.The clone library to isolate the gene related to the antibiotic biosynthesis was constructed by cloning Sau3A I partial-digested genomic DNA fragments ofS. roseoflavus Men-myco-93-63 into the plasmid pIJ702 in the host S. lividans TK54. This library was screened by inhibition to the indicate strain S. scabies S87. It was found that 3 clones had the ability to antagonize to S. scabies S87 and SA-3 was the strongest antagonist. The plasmid isolated from SA-3 was named pSA3 and about 8.5kb extrinsic fragment was detected. The function of pSA3 was identified and it was proved that S. lividans TK54 transformed by pSA3 could inhibit S. scabies S87, but no active to V. dahliaeWAl. The fermentation was prunosus when the transformant was shake-flask culture in oatmeal medium and the extraction with ethyl acetate could inhibit S. scabies S87. The HPLC analysis of the extraction demonstrated that they were very different between S. lividans TK54 transformed by pSA3 and by pIJ702. The analysis of sequence with Frameplot2.3 showed that the fragment included 10 ORFs and 2 incomplete ORFs. The results of blastp showed 2 ORFs were homologous to whiB (transcriptional regulator) and RNA/DNA helicase which had DEXDc (DEAD-like helicases superfamily) conserved domain and another ORF had HTHXRE (Helix-turn-helix, xenobiotic respohse element family of transcriptional regulators) and COG 1426 conserved domain, which was homologous lowly to DNA-binding protein. The putative proteins encoded by other 7 ORFs which had not homologous sequence were unknown about the functions. The fragment was likely related to regulation of the antibiotic biosynthesis. The extrinsic fragment gene sequence and the HPLC analysis of the transformant fermentation showed that this fragment was involved in the biosynthesis of the antibiotic in S. roseoflavus Men-myco-93-63. |
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