本文選題：刺糖多孢菌 + 亮氨酰氨肽酶基因�。� 參考：《微生物學(xué)報(bào)》2016年04期

【摘要】：【目的】構(gòu)建亮氨酰氨肽酶基因(pep A)被阻斷的刺糖多孢菌工程菌株,并鑒定該基因?qū)Υ烫嵌噫呔z形態(tài)、生物量、菌體全蛋白表達(dá)水平及產(chǎn)多殺菌素能力的影響,探究該基因調(diào)控多殺菌素合成的可能機(jī)制。【方法】利用PCR擴(kuò)增刺糖多孢菌中的pep A基因同源片段,經(jīng)酶切連接技術(shù)構(gòu)建敲除載體p OJ260-pep A;通過接合轉(zhuǎn)移和單交換同源重組將該載體整合至刺糖多孢菌染色體中,獲得工程菌株S.sp-△pep A;利用培養(yǎng)特征、形態(tài)學(xué)、高效液相色譜、SDS-PAGE等方法對(duì)菌株進(jìn)行研究分析�！窘Y(jié)果】工程菌株S.sp-△pep A菌絲片段化程度加劇,生長態(tài)勢(shì)被延緩且生物量降低,但有效促進(jìn)了多殺菌素的生物合成。阻斷亮氨酰胺肽酶基因的表達(dá)使刺糖多孢菌菌體全蛋白表達(dá)情況發(fā)生明顯改變,找到表達(dá)水平顯著上調(diào)的差異蛋白核糖體蛋白亞基和醛基脫氫酶,核糖體蛋白亞基通過影響蛋白質(zhì)代謝對(duì)菌體生長產(chǎn)生影響;醛基脫氫酶則可與乙醇脫氫酶、乙酰輔酶A的合成酶相互作用影響輔酶A合成,而輔酶A是合成多殺菌素的重要底物�！窘Y(jié)論】在刺糖多孢菌合成多殺菌素的次級(jí)代謝過程中,pep A基因作為負(fù)調(diào)控因子發(fā)揮作用。
[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.
【作者單位】： 湖南師范大學(xué)生命科學(xué)學(xué)院微生物分子生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室培育基地;
【基金】：國家“863”計(jì)劃(2011AA10A203) 國家“973”計(jì)劃(2012CB722301) 國家自然科學(xué)基金(31070006) 湖南省2011協(xié)同創(chuàng)新中心項(xiàng)目(20134486) 湖南省教育廳項(xiàng)目(10CY013)~~
【分類號(hào)】：Q93;Q78

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