本文選題：酒酒球菌　切入點(diǎn)：環(huán)丙烷脂肪酸合酶基因　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：在葡萄酒釀造過程中,主要利用酒酒球菌(Oenococcus oeni)啟動(dòng)并進(jìn)行蘋果酸-乳酸發(fā)酵以改善葡萄酒的風(fēng)味,而這主要得益于酒酒球菌較強(qiáng)的耐脅迫能力。在酒酒球菌適應(yīng)脅迫環(huán)境過程中,膜成分的自我調(diào)節(jié)是一個(gè)重要機(jī)制。菌體主要改變細(xì)胞膜的流動(dòng)性和剛性,以應(yīng)對(duì)外界的脅迫條件,維持細(xì)胞正常的生理功能。其中,細(xì)胞膜中環(huán)丙烷脂肪酸(cyclopropane fatty acid,CFA)的合成在細(xì)菌適應(yīng)急劇變化的環(huán)境中非常重要,在酒酒球菌應(yīng)對(duì)多種脅迫環(huán)境機(jī)制中都發(fā)揮著作用。研究和解析環(huán)丙烷脂肪酸合酶基因(cyclopropane fatty acid synthase gene,cfa)的功能和菌株耐受性方面的相關(guān)性可以更好地探究酒酒球菌應(yīng)對(duì)脅迫環(huán)境進(jìn)行的膜組分變化機(jī)制;而植物乳桿菌作為啟動(dòng)蘋果酸-乳酸發(fā)酵的另一重要菌種,本研究為進(jìn)一步促進(jìn)其啟動(dòng)蘋果酸-乳酸發(fā)酵的廣泛實(shí)現(xiàn)提供思路和基礎(chǔ)。本文首先選擇pH作為菌株篩選的脅迫因素,篩選出野生耐酸、酸敏酒酒球菌,確定耐酸野生菌的生長極限。然后,對(duì)耐酸、酸敏的野生菌和突變菌進(jìn)行環(huán)丙烷脂肪酸合酶基因進(jìn)行測(cè)序比對(duì)。在此基礎(chǔ)上,將野生耐酸、酸敏菌株的cfa基因?qū)氪竽c桿菌中進(jìn)行誘導(dǎo)、表達(dá),并獲得純化產(chǎn)物;同時(shí)將相同的基因?qū)胫参锶闂U菌中進(jìn)行表達(dá),對(duì)重組植物乳桿菌的存活能力、降解蘋果酸能力及膜脂肪酸成分變化進(jìn)行檢測(cè)。主要完成的研究結(jié)果如下:(1)在pH 2.8條件下篩選實(shí)驗(yàn)室保藏的35株酒酒球菌,初步篩選出5株較耐酸菌株;在pH 2.6、2.8條件下培養(yǎng)5株菌,測(cè)定生存能力(即OD600值)。最終確定酒酒球菌CS-7b及ME-5b的耐酸能力最強(qiáng),生長極限為pH 2.6。(2)對(duì)酒酒球菌CS-7b、ME-5b及酸敏菌株SX-1b與實(shí)驗(yàn)室已有突變菌a3(耐酸)及b1(酸敏)進(jìn)行cfa基因進(jìn)行測(cè)序比對(duì),耐酸菌株的野生型和誘變型的序列相同,較Oenococcus oeni PSU-1發(fā)生了3處堿基突變,而酸敏菌野生型、突變型的序列則與Oenococcus oeni PSU-1一致。(3)在大腸桿菌中成功轉(zhuǎn)入SX-1b與CS-7b的cfa基因,構(gòu)建重組大腸桿菌R1、R2。確定誘導(dǎo)條件,16℃、150 rpm誘導(dǎo)19 h,并過Ni-NTA柱,獲得純化蛋白。(4)將SX-1b與CS-7b的cfa基因在植物乳桿菌ATCC33222中表達(dá),構(gòu)建重組載體pMG36e-cfa1和pMG36e-cfa2,并得到對(duì)應(yīng)重組菌L1和L2。對(duì)植物乳桿菌工程菌的耐酸能力進(jìn)行檢測(cè),并檢測(cè)脅迫條件下菌體環(huán)丙烷脂肪酸含量變化。結(jié)果顯示在pH 3.6培養(yǎng)基中培養(yǎng),重組菌L1和L2的生長情況明顯優(yōu)于含空載體的植物乳桿菌L0,且L2的生長優(yōu)于L1。而在p H 3.4培養(yǎng)基中培養(yǎng),L0沒有生長跡象,只有重組菌L1和L2正常生長,cfa基因的導(dǎo)入突破了植物乳桿菌耐酸的生長極限。在酸脅迫環(huán)境中培養(yǎng)重組植物乳桿菌發(fā)現(xiàn),環(huán)丙烷脂肪酸的含量急劇上升,且外源cfa基因的導(dǎo)入提升了增幅。L-蘋果酸降解試驗(yàn)顯示,所有植物乳桿菌工程菌能保持正常蘋果酸-乳酸發(fā)酵能力。
[Abstract]:In the process of wine brewing, malic acid-lactic acid fermentation was initiated by Oenococcus oeniensis in order to improve the wine flavor. This is mainly due to the strong stress tolerance of Bacillus cereus. The self-regulation of membrane component is an important mechanism in the process of adapting to stress environment. The bacteria mainly change the fluidity and rigidity of cell membrane. The synthesis of cyclopropane fatty acid (Cypropane fatty) on cell membrane is very important for bacteria to adapt to the rapidly changing environment. The study and analysis of the function of cyclopropane fatty acid synthase gene and the relationship between the strain tolerance and the function of cyclopropane fatty acid synthase gene can better explore the relationship between Bacillus cereus and Bacillus cereus. The change mechanism of membrane components in stress environment; Lactobacillus plantarum is another important strain of malolactic acid fermentation. This study provides a basis for further promoting the widespread realization of malic acid-lactic acid fermentation. Firstly, we select pH as the stress factor for screening strains, and select wild acid-resistant, acid-sensitive Bacillus cereus. The growth limit of acid-tolerant wild bacteria was determined. Then, the acid-tolerant, acid-sensitive wild bacteria and mutant bacteria were sequenced and compared with those of cyclopropane fatty acid synthase genes. The cfa gene of the acid-sensitive strain was induced, expressed and purified by E. coli, and the same gene was introduced into Lactobacillus plantarum to express the survival ability of the recombinant Lactobacillus plantarum. The main results were as follows: 1) 35 strains of Bacillus wine were screened under pH 2.8, and 5 strains of acid-resistant strains were screened. Five strains were cultured at pH 2.6 ~ 2.8.The survival ability (i.e. OD600 value) was determined. Finally, the acid-tolerant ability of CS-7b and ME-5b was the strongest. The cfa gene sequence of CS-7bME-5b and acid-sensitive strain SX-1b was compared with that of A3 (acid-tolerant) and b1 (acid-sensitive) strains. The sequence of wild and mutagenic strains was the same. Compared with Oenococcus oeni PSU-1, there were three base mutations, while the wild type of acid-sensitive bacteria, the sequence of which was consistent with that of Oenococcus oeni PSU-1, was successfully transferred into the cfa gene of SX-1b and CS-7b in E. coli. The recombinant Escherichia coli R1 / R2 was constructed. The induction conditions were determined for 19 h after induction at 16 鈩，

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