【作者】 张安娜；

【导师】 张雪洪；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 本实验室从土壤中分离出了一株能产灰藤黄菌素(griseolutein)的链霉菌——灰藤黄链霉菌(Streptomyces griseoluteus)。灰藤黄菌素是具有吩嗪核心结构的一种抗生素,具有很强的抗菌活性,对革兰氏阳性菌和阴性菌的抑制效果比吩嗪其它衍生物如绿脓菌素( pyocyanine )、绿针菌素( chlororaphin )和吩嗪-1-羧酸(phenazine-1-carboxylic acid)都要好。有研究证实,其较强的抗菌活性主要归因于吩嗪核心结构上C-6的取代基羟基乙酰甲基或1,2-二羟基乙氧基甲基。但是到目前为止还没有关于灰藤黄菌素生物合成基因的报道。为证实灰藤黄链霉菌中存在吩嗪核心结构的合成基因及后续转化基因,本研究构建了灰藤黄链霉菌的Fosmid基因组文库,为后续的实验奠定了基础。通过比对吩嗪核心结构的保守序列,设计了特异性引物,采用多轮PCR的方法筛选文库,最终筛选到7个阳性克隆。通过酶谱分析的方法,确定了最有可能包含完整吩嗪合成基因簇的克隆10D11,采用鸟枪法进行测序,并用PCR的方法补全缺口。最终,测得32973bp的碱基序列。采用ORF和同源性分析的方法,共得到27个ORF,其中6个为吩嗪生物合成的基因sghBCDEFG。ORF21-25为后续转化基因,最终的产物为灰藤黄菌素。更多还原

【Abstract】 Streptomyces griseoluteus, a strain which can produce griseolutein, is isolated from soil. It has been proved that griseolutein has strong antibacterial activities. It has very good inhibitory effects against both Gram-positive bacteria and Gram-negative bacteria. The structure of griseolutein is similar to phenazine nucleus. Compared with other phenazine derivatives, such as pyocyanine, chlororaphin and phenazine-1-carboxylic acid, griseolutein has the strongest bacteriostatic effect. These facts suggest that the strong bacteriostatic activity of griseolutein would be dependent on groups such as 1, 2-hydroxyethoxymethyl group or hydroxyacetyl- methyl group at 6-position of phenazine nucleus. But so far, no research on griseolutein biosynthetic genes has been reported.In order to confirm that there are phenazine nucleus synthesis genes and subsequent transformation genes on the chromosome of S. griseoluteus, we constructed Fosmid genomic library of S. griseoluteus. It also lays the foundation for following experiments. By blasting the conserved sequences of phenazine nucleus, we designed specific primers. Then we used several rounds of PCR to screen the Fosmid library, and finally got seven positive clones. Through analyzing the digest map of restriction enzyme, we selected the clone 10D11 which is most likely to contain a complete gene cluster of phenazine synthesis. With shotgun sequencing method, and PCR to complete gaps, we obtained the sequence of 32973bp bases in the end. Using ORF analysis and homology analysis methods, we identified a total of 27 ORFs in 10D11, of which 6 are for phenazine biosynthesis, which are called sghBCDEFG.. ORF21-25 is a gene cluster for following transformation. The final product is griseolutein.更多还原