本文選題：曲霉N-’ + PYC�。� 參考：《現(xiàn)代食品科技》2017年02期

【摘要】：為了解曲霉(Aspergillus sp.)N1-14’的高產(chǎn)L-蘋果酸(LMA)機(jī)制,提取其總DNA作模板,設(shè)計(jì)同源引物擴(kuò)增包含丙酮酸羧化酶基因(pyc)和蘋果酸脫氫酶基因(mdh)全長的片段并測(cè)序,再參考測(cè)序結(jié)果找到丙酮酸羧化酶(PYC)和蘋果酸脫氫酶(MDH)的編碼區(qū),設(shè)計(jì)引物從N1-14’總cDNA擴(kuò)增出其編碼序列并通過TA克隆測(cè)序。測(cè)序結(jié)果顯示pyc基因編碼區(qū)長3582 bp,編碼1193aa,分析結(jié)果顯示PYC氨基酸序列在曲霉屬內(nèi)相當(dāng)保守,同源性高達(dá)90%以上,其中N1-14’在兩個(gè)保守位點(diǎn)出現(xiàn)突變,833位的A位于一個(gè)環(huán)區(qū)和1022位的F位于α-螺旋中部,可能與其高產(chǎn)酸活性相關(guān);mdh編碼區(qū)全長1023 bp,編碼340aa。MDH氨基酸序列高度保守,突變株同樣有兩個(gè)保守區(qū)出現(xiàn)氨基酸點(diǎn)突變,且兩點(diǎn)均出現(xiàn)在α-螺旋區(qū)域。本試驗(yàn)主要克隆兩個(gè)曲霉N1-14’產(chǎn)L-蘋果酸通路關(guān)鍵酶基因,分析其種屬的特異性及預(yù)測(cè)特異氨基酸位點(diǎn)的功能,為繼續(xù)探究N1-14’的高產(chǎn)LMA機(jī)制及相應(yīng)的基因工程改造提升產(chǎn)酸水平奠定基礎(chǔ)。
[Abstract]:In order to understand the mechanism of high yield LMAs of Aspergillus sp. N1-14', the total DNA of Aspergillus sp. N1-14 'was extracted as template, and the fragments containing pyruvate carboxylase gene (PYC) and malate dehydrogenase gene (mdh) were amplified by homologous primers and sequenced. The coding regions of pyruvate carboxylase (Pyc) and malate dehydrogenase (MDH) were found by referring to the sequencing results. Primers were designed to amplify the coding sequence from N1-14'total cDNA and sequenced by TA cloning. The results of sequencing showed that the coding region of pyc gene was 3582 BP, encoding 1193A. The results showed that the amino acid sequence of PYC was conserved in Aspergillus, and the homology was over 90%. Among them, A of N1-14 'mutation at two conserved sites is located in a ring region and F at position 1022 is located in the middle of 偽 -helix, which may be related to its high acid activity. The coding region of mdh is 1023 BP in length. The amino acid sequence encoding 340aa.MDH is highly conserved, and the amino acid sequence of N1-14' is highly conserved. There were two amino acid point mutations in two conserved regions of the mutant, and both appeared in the 偽 -helix region. In this study, two key enzyme genes of L- malic acid pathway produced by Aspergillus N1-14 'were cloned, and their species-specificity and function of predicting specific amino acid sites were analyzed. The results laid a foundation for further study on the mechanism of high yield LMA of N1-14 'and the corresponding genetic engineering to improve acid production level.
【作者單位】： 中國科學(xué)院南海海洋研究所;廣東省微生物研究所省部共建華南應(yīng)用微生物國家重點(diǎn)實(shí)驗(yàn)室廣東省菌種保藏與應(yīng)用重點(diǎn)實(shí)驗(yàn)室廣東省微生物新技術(shù)公共實(shí)驗(yàn)室;中國科學(xué)院大學(xué);廣東環(huán)凱微生物科技有限公司;
【基金】：國家自然科學(xué)基金項(xiàng)目(31271940)
【分類號(hào)】：TS202.3
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本文編號(hào)：1885700