| Streptomyces netropsis SD-07was isolated in2007from the mountain soil in the southern of Jinan, Shandong Province. It could produce polyene macrolide antibiotics with broad spectrum and high antifungal activity. The polyene macrolides produced by S.netropsis SD-07is a mixture. Among them, the percentage of pentaene macrolides is the maximum percentage, heptaene macrolide followed, the percentage of tetraene macrolide and hexaene macrolide are very low. Some of these polyene macrolide compisitions have new chemical formula, and their antifungal activity is far higher than amphotericin B, a widespread used polyene macrolide antifungal antibiotic at present. So that it is worth further researching for developing new antifungal antibiotics. In order to well study the functions of the biosynthesis genes of antibiotics and its regulation mechanisms in the future, it is necessary to know the squence information of biosynthesis gene clusters of antibiotics, and to develop genetic operating systems of S.netropsis SD-07. So, this thesis includes the following three parts:1. Scanning the complete genome sequence of S.netropsis SD-07and analyzing the antibiotic synthesis gene cluster.The genome of S.netropsis SD-07has about7685111bp, includes384scaffolds. Its G+C%is71.7%. Using genetic forecasting software and NCBI online sequence blast tool,60orfs relating to the antibiotic synthesis encoding genes have been identified, locating in37scaffolds. Using ASMPKS, PKS modular (polyketide synthases) analysis system database to forecast the possible modules and possible synthetic products of S.netropsis SD-07, it is found that these orfs contains32KS (ketosynthase) domains:27AT (acyl transferase) domains,42ACP (acyl carrier protein) domains,12DH (dehydratase) domains,24KR (ketoreductase) domains,4 ER (enoyl reductase) domains and2TE (thioesterase) domains. These informations lay good foundation for the further research focusing on the functions of antibiotic synthetic gene cluster and its regulation mechanisms, though they don’t give the complete sequence information of polyene macrolide antibiotic synthetic gene cluster.2. Establishing the antibiotics synthetic gene library of S.netropsis SD-07It is worth to establish antibiotics synthetic gene library of S.netropsis SD-07, because the gene library can not only be used to obtain the complete sequence of antibiotic synthetic gene cluster, but also be used to reveal the gene functions and the regulation mechanisms of gene expression.Firstly, genomic DNA library of S.netropsis SD-07is established using a fosmid plasmid pJTU2554, containing2304clones, every clone contains about34kb length of the exogenous DNA fragment on the vector plasmid. The coverage of the genomic DNA library is99.45%if we assume the genome of S.netropsis SD-07contains8×106bp.Secondly, two probes (KS-AT and DH-KR) have been obtained by PCR reaction using primes deaigned according to the consensus sequence of polyketone synthase (PKS). Total101clones have been selected after two rounds of nucleic acid hybridization (Southern blot) between genomic DNA of2304clonies and the two probes (KS-AT or DH-KR). All of these101clones have been proved containing KS sequence by PCR. So that, the antibiotic synthesis gene library has been constituted by these101clones, its coverage is99.9%. It lays good foundation for obtaining the complete sequence antibiotics synthetic gene cluster in the future.3. Successfully develping the genetic transfer systems of S.netropsis SD-07.Although genetic transfer systems in Streptomyces have been already established, the commenly used methods including conjugational transfer, protoplast transformation and electroporation. But until now there has been no any successful report in Streptomyces netropsis. In this thesis, we successfully achieve conjugational transfer from the methyl-deficient Escherichia coli strain ET12567, carrying an integrative plasmid pSET152and an assist plasmid pUZ8002, to S.netropsis SD-07and stably integration into the chromosome.The conjugation frequency has been greatly increased (near10,000times) after optimizing the influcing factors. The optical conditions of conjugation for Streptomyces netropsis SD-07are:the ratio of donor and receptor is1:1(108:108) conidia are heat shocked at45℃for10min or15min and then incubate at30℃for6h before they are used for conjugation; the conjugation medium is GSY with60niM Ca2+and60mM Mg2+; incubate at30℃for16-20h before spreading45ug/ml nalidixic acid and30ug/ml apramycin. Under such conditions, the highest conjugation frequency is about6×10-4.New factors affecting conjugational transfer frequency Found.The extra addition of Ca2+and its concentration is the most important factor influencing the efficiency of conjugation transfer. Ca2+is about10-100times better than Mg2+for improving conjugation transfer frequency.60mM CaC12+,60mM MgCl2is regarded as the optical combination for increasing the conjugational transfer frequency. Secondly, the organic nitrogen source and its concentration is another important factor influencing the efficiency of conjugation transfer. The effects of the above two factors on conjugation frequency are far more than the previously reported factors, such as the ratio of donor and receptor, heat shock condition and germination time of conidia, antibiotic overlapping time, etc..Electroporation is a commonly used genetic operating system. It is efficient and easy to do. For Streptomyces netropsis SD-07, the relationship between the electric shock conditions and the survival percentages of the receptor cells, the lysozyme pretreatment conditions of the receptor cells and the survival percentages of the receptor cells, have been investigated. Though electroporation has not completely finished, the experimental data and results give valuable enlightenment for later study. |
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