| Zhongshengmycin,produced by Streptomyces lavendulae var. Hainanensis, is a N-glycoside agricultural antibiotic. It is a broad-spectrum and low-toxicity antibiotic without pollution to environment. It has good control effect against a variety of bacteria and fungal diseases. Now it has been widely used, and has made huge economic, social and ecological benefits.First, a cosmid library of Streptomyces lavendulae var. Hainanensis B-7 was successfully constructed with the genome DNA of B-7 and the cosmid vector pOJ446 as original material. The coverage rate of the library was 99.99%, which could cover the entire B-7 genome more than ten times. Second, after construction of the cosmid library, a PCR-based method for screening the library was developed. The library was screened with zpsA DNA sequence as PCR target. A positive clone was screened after 5 repeat PCR process which spent over 15 days, and completed more than 100 PCR reactions. This screening method was rapid, simple, convenient, economic, low false-positive rates and no-isotope in comparision with traditional methods.Third, the insert DNA sequence in the positive clone was sequenced, and its length was over 29719 bp which contained 75.5% G+C. The high G+C content is same as that of a typical streptomyces genome. At last, the DNA sequence was analyzed with several informatics software and online tools, such as DNAMAN 6.0, FramePlot 2.3.2, Blast and GeneMark.hmm for Prokaryotes (Version 2.4). It showed that there were 25 complete ORFs in this DNA segment. In these 25 ORFs, five ORFs were speculated to code ABC transport system, and 2 ORFs were speculated to code transcriptional regulators, and 15 ORFs were speculated to code zhongshengmycin synthetases, and 2 ORFs were speculated to code resistance proteins, and one was speculated to code an unknown protein. 5 ABC transport system genes, 2 transcription regulation genes and 2 structural genes were not similar with any cloned streptothricin biosynthesis genes, so they could be new zhongshengmycin biosynthesis genes.Five putative ABC transporter proteins include two ATP binding subunits, two transmembrane subunits, an substrate binding subunit which constitute a complete ABC type transporter system. It could be inferred that this system could have a function to transfer zhongshengmycin out of cells. orf5 was speculated to code GntR family transcription factors. It was speculated to be involved with the development of zhongshengmeycin-producing strain and the Zhongshengmycin resistance, and its mechanism may be related to interaction of ABC transporter proteins and GntR transcription factor. orf21 and orf24 may code resistance proteins of Zhongshengmycin. orf21 may code aminoglycoside 3'-phosphotransferase. It has been widely studied for streptothricin acetyltransferase, and orf24 is highly similar with these known genes, so it was definitively regarded as a zhongshengmycin resistance gene. In 15 hypothetic structural genes, two structural genes, orf 7 and orf25, were first cloned in the study of streptothricin. It was speculated that ORF7 is a carbamoyltransferase responsible for the carbamoylation of gulosamin, and ORF25 is a hydroxylase responsible for the hydroxylation modification of streptolidin. It was proposed that the function of other 12 structural genes resembled with known streptothricin biosynthesis genes except orf10. In GenBank, SttN was noted as amino-transferase responsible for the synthesis of gulosamin, however we thought that ORF10 and SttN should be responsible for the synthesis of streptolidin.The cloning of Zhongshengmycin biosynthetic gene cluster and the analysis of their function established a foundation for thd function identification of zhongshengmycin genes and genetic modification. Through molecular biology methods, we could advance the content of high-effective member in the zhonghshenymycin mixture, or obtain the strain to produce only a specific member, or obtain the strain to generate some zhongshengmycin derivatives, and so on.
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