【作者】 田云龙；

【导师】 朱昌雄；

【作者基本信息】 中国农业科学院 ， 农业微生物， 2010， 博士

【摘要】 中生菌素是一种由淡紫灰链霉菌海南变种(Streptomyces lavendulae var. hainanensis)产生的氨基糖苷类农用抗生素,具有广谱、高效、低毒、无污染等特点,对多种细菌及真菌病害具有良好的防治效果,目前已经得到了广泛的应用。为了利用分子生物学的手段构建高产工程菌,进一步提高中生菌素的生产水平,本研究计划克隆中生菌素生物合成基因簇并对其功能进行分析。首先以中生菌素产生菌的基因组DNA和粘粒载体pOJ446为材料,构建了基因组粘粒文库。获得的文库覆盖率超过99.99%,可以覆盖淡紫灰链霉菌海南变种B-7基因组10次左右,具有非常高的完整性和代表性。其次,在文库构建成功之后,确立了一种基于PCR的文库筛选方法。以之前克隆到的中生菌素生物合成基因zpsA片段为筛选目标,对构建的粘粒文库进行基于PCR的筛选。经过5轮共计100多个PCR反应筛选出含有zpsA基因片段的阳性克隆,所有操作耗时15天左右,效率较高。与传统文库筛选方法相比,该方法具有快速、简便、经济、假阳性低且不需要使用同位素等优点。第三,筛选到阳性克隆后,对筛选到的阳性克隆中DNA插入片段进行测序,最终得到了长为29719 bp的DNA序列,其G+C含量达75.5%,与典型的链霉菌基因组特征相符。最后,使用DNAMAN、FramePlot 2.3.2、Blast和GeneMark.hmm for Prokaryotes (Version 2.4)等多种生物信息学软件和在线工具对得到的DNA序列进行分析,表明其中含有25个完整的orf。推测其中5个属于ABC类型转运系统;2个编码转录调控因子;15个编码结构基因;2个编码抗性基因;1个为功能未知的基因。其中5个转运基因、2个转录调控因子基因和2个结构基因共9个orf与以往克隆到的链丝菌素生物合成基因没有同源性,属于新的基因。对25个orf的功能进行了分析。推测5个ABC转运系统orf编码2个ATP结合亚基、2个跨膜亚基、1个PBP2类型的底物结合亚基,从而构成一个完整的ABC转运系统。可推测这一ABC转运系统具有把中生菌素转运出产生菌胞外的功能;推测orf5编码的是GntR家族转录调控因子,ORF5可能与中生菌素产生菌的发育、中生菌素合成或抗性有关,其机理涉及到ABC转运系统与GntR转录调控因子的协同作用;推测orf21和orf24编码中生菌素抗性蛋白。orf21编码氨基糖苷3’-磷酸转移酶。orf24编码链丝菌素乙酰转移酶,对于这种抗性机制已经有了大量报道,并得到了实验验证,因此可以确定其具有中生菌素的抗性功能。15个结构基因orf中有2个是首次在链丝菌素的研究中被克隆到,即orf7和orf25。推测ORF7是参与古洛糖胺进行甲酰胺化的氨基甲酰胺转移酶,ORF25是对链里定进行羟基化修饰的羟化酶。另外,分析认为orf10编码的产物与中生菌素结构中链里定部分的合成有关,而并非以往研究中对于其同源基因sttN所注释的“与古洛糖胺部分的合成有关”。15个结构基因orf中剩余的12个orf与其相应的已经得到克隆的链丝菌素生物合成基因的同源基因的功能相同。中生菌素生物合成基因簇的成功克隆,以及基因功能的初步推测分析,为中生菌素生物合成基因的功能鉴定及遗传改造奠定了基础。通过对转录调控基因、抗性基因以及结构基因的改造,我们可以达到提高中生菌素产生菌发酵产物中高效组分在混合物中的含量、或是得到仅产某种成分的菌株、或是得到可以产生中生菌素衍生物的菌株等目的。更多还原

【Abstract】 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.更多还原