亮氨酰氨肽酶基因的阻断对刺糖多孢菌生长及次级代谢产物合成的影响

发布时间：2018-06-01 15:49

本文选题：刺糖多孢菌 + 亮氨酰氨肽酶基因　；参考：《微生物学报》2016年04期

【摘要】：【目的】构建亮氨酰氨肽酶基因(pep A)被阻断的刺糖多孢菌工程菌株,并鉴定该基因对刺糖多孢菌菌丝形态、生物量、菌体全蛋白表达水平及产多杀菌素能力的影响,探究该基因调控多杀菌素合成的可能机制。【方法】利用PCR扩增刺糖多孢菌中的pep A基因同源片段,经酶切连接技术构建敲除载体p OJ260-pep A;通过接合转移和单交换同源重组将该载体整合至刺糖多孢菌染色体中,获得工程菌株S.sp-△pep A;利用培养特征、形态学、高效液相色谱、SDS-PAGE等方法对菌株进行研究分析。【结果】工程菌株S.sp-△pep A菌丝片段化程度加剧,生长态势被延缓且生物量降低,但有效促进了多杀菌素的生物合成。阻断亮氨酰胺肽酶基因的表达使刺糖多孢菌菌体全蛋白表达情况发生明显改变,找到表达水平显著上调的差异蛋白核糖体蛋白亚基和醛基脱氢酶,核糖体蛋白亚基通过影响蛋白质代谢对菌体生长产生影响;醛基脱氢酶则可与乙醇脱氢酶、乙酰辅酶A的合成酶相互作用影响辅酶A合成,而辅酶A是合成多杀菌素的重要底物。【结论】在刺糖多孢菌合成多杀菌素的次级代谢过程中,pep A基因作为负调控因子发挥作用。
[Abstract]:[objective] to construct an engineering strain of Polysporium punctatus, which was blocked by Leucylaminopeptidase gene (Pep A), and to identify the effects of the gene on mycelium morphology, biomass, total protein expression and the ability of producing bacteriosporins. [methods] the homologous fragment of pep A gene was amplified by PCR. The knockout vector p OJ260-pep A was constructed by digesting ligation technique. The vector was integrated into the chromosome of polysporaria spp by conjugation transfer and mono-exchange homologous recombination, and the engineering strain S.sp- pep A was obtained. High performance liquid chromatography (HPLC) and SDS-PAGE were used to study and analyze the strain. [results] the hyphae fragmentation of S.sp- pep A was intensified, the growth trend was delayed and the biomass was decreased, but the biosynthesis of spsp- pep A was promoted effectively. Blocking the expression of leucine aminopeptidase gene changed the whole protein expression of polyspora spp, and found the differential protein ribosomal protein subunit and aldehyde dehydrogenase, which were upregulated significantly. Ribosomal protein subunits affect cell growth by affecting protein metabolism, while aldehyde dehydrogenase interacts with alcohol dehydrogenase and acetyl coenzyme A synthase. Coenzyme A is an important substrate for synthesizing bactericosporins. [conclusion] the pep A gene plays a role as a negative regulatory factor in the secondary metabolism of the biosynthesis of germicosporins by polyspora.
【作者单位】：湖南师范大学生命科学学院微生物分子生物学国家重点实验室培育基地;
【基金】：国家“863”计划(2011AA10A203) 国家“973”计划(2012CB722301) 国家自然科学基金(31070006) 湖南省2011协同创新中心项目(20134486) 湖南省教育厅项目(10CY013)~~
【分类号】：Q93;Q78

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