生防链霉菌Act12中沉默基因簇的激活与产物鉴定

发布时间：2018-10-11 14:41

【摘要】：生防链霉菌Act12是从自青藏高原分离获得的一株多效放线菌,前期的科学研究表明该菌株具有多种良好的生物活性,例如抑制病原真菌的生长,促进草莓、甜瓜和棉花等多种农作物的生长等。通过对该菌株进行全基因组测序并提交antiSMASH在线分析发现Act12基因组中可能含有丰富的次级代谢产物生物合成基因簇,然而,对实验室条件下Act12的发酵液进行检测发现该菌株含有的次级代谢产物的含量及种类都较少,这表明Act12中绝大多数基因簇是沉默的或低表达的。为了激活Act12中的沉默基因簇,挖掘该菌株中可能的活性产物资源,并进一步探究相关的活性机理,我们通过同源重组的方法构建了Act12的四株可稳定遗传的突变株,包括可能的LuxR家族调控因子SPA4754的缺失突变株Δspa4754、可能的TetR家族调控因子SPA0520的缺失突变株Δspa0520、全局性调控基因nsdA的缺失突变株ΔnsdA和全局性调控基因bldA的缺失突变株ΔbldA。通过病原微生物拮抗活性实验以及比较代谢图谱学分析后发现:缺失突变菌株Δspa0520对几类病原供试菌的抑制活性较野生型菌株Act12的活性显著增强。HPLC的代谢图谱学分析结果表明,突变株Δspa0520的发酵产物中较野生型Act12出现一显著差异峰。对突变株Δspa0520进行大量发酵,纯化显著差异峰所对应的化合物,通过高分辨质谱分析等手段对目的化合物进行初步的结构分析及鉴定,最终分析得出该化合物为寡霉素D。同时,对突变株Δspa0520进行遗传互补实验获得相应的遗传互补菌株Δspa0520C,并检测其对病原微生物拮抗活性以及比较代谢图谱学分析,结果发现:遗传互补菌株Δspa0520C对几类病原供试菌的抑制活性及其HPLC代谢图谱与野生型Act12保持一致。缺失突变菌株Δspa0520的相关实验结果表明spa0520(GenBank Accession No.KY368145)可能是寡霉素D生物合成途径中的负调控基因,也可能具有多效调控作用,这为研究有关链霉菌Act12的其它次级代谢产物的调控机理提供了帮助,同时采用敲除负调控基因的方法为激活相关的沉默基因簇提供了有益的借鉴。
[Abstract]:Biocontrol Streptomyces Act12 is a multifunctional actinomycetes isolated from the Qinghai-Tibet Plateau. Previous scientific studies have shown that the strain has many good biological activities, such as inhibiting the growth of pathogenic fungi and promoting strawberry. The growth of a variety of crops, such as melon and cotton. By sequencing the whole genome of the strain and submitting it to antiSMASH online analysis, it was found that the Act12 genome may contain abundant secondary metabolites biosynthesis gene clusters, however, The detection of the fermentation broth of Act12 in laboratory showed that there were few secondary metabolites in this strain, which indicated that the majority of gene clusters in Act12 were silent or low expressed. In order to activate the silencing gene cluster in Act12, excavate the possible active product resources in the strain, and further explore the related activity mechanism, we constructed four stable genetic mutants of Act12 by homologous recombination. The deletion mutant 螖 spa4754, of the possible LuxR family regulator SPA4754, the deletion mutant 螖 spa0520, of the TetR family regulator SPA0520, the deletion mutant 螖 nsdA of the global regulatory gene nsdA and the deletion mutant 螖 bldA. of the global regulatory gene bldA The antagonistic activity of pathogenic microorganisms and the comparative analysis of metabolic atlas showed that the inhibitory activity of deletion mutant 螖 spa0520 was significantly higher than that of wild-type strain Act12. The results of metabolic atlas of HPLC showed that the inhibitory activity of deletion mutant 螖 spa0520 was significantly higher than that of wild-type strain Act12. There was a significant difference peak in the fermentation products of the mutant 螖 spa0520 compared with the wild-type Act12. The mutant 螖 spa0520 was fermented in large quantities, and the compounds corresponding to the significant difference peak were purified. The target compounds were analyzed and identified by means of high resolution mass spectrometry. Finally, the compounds were identified as oligomycin D. At the same time, the genetic complementary strain 螖 spa0520C, was obtained by genetic complementation test on the mutant 螖 spa0520, and its antagonistic activity against pathogenic microorganisms was detected and the comparative metabolic map analysis was carried out. The results showed that the inhibitory activity of genetic complementary strain 螖 spa0520C and its HPLC metabolic map were consistent with those of wild type Act12. The experimental results of deletion mutant 螖 spa0520 suggest that spa0520 (GenBank Accession No.KY368145 may be a negative regulation gene in the biosynthesis pathway of oligomycin D, and may also have multiple effects. It is helpful to study the regulation mechanism of other secondary metabolites of Streptomyces Act12, and to use the method of knockout of negative regulatory genes to activate the related silencing gene clusters.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476.1;Q78

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