植物乳杆菌细菌素基因座遗传分析与基因plnEF外源表达研究
发布时间：2019-02-22 20:44
【摘要】：植物乳杆菌可以适应恶劣的葡萄酒环境,启动苹果酸-乳酸发酵,具有产生植物乳杆菌细菌素的能力,抑制或杀死葡萄酒中有害乳酸菌,防止葡萄酒的破败,部分替代二氧化硫的作用,从而降低葡萄酒中二氧化硫的使用。因此筛选产细菌素的植物乳杆菌进行苹果酸-乳酸发酵,十分必要。本试验首先通过共培养诱导培养产生细菌素,从14株植物乳杆菌中成功筛选获得两株产细菌素的植物乳杆菌XJ25和PC520。其次,利用两株菌基因组重测序手段,进一步比较分析其植物乳杆菌素(Plantaricin,pln)基因座间存在的差异,获得植物乳杆菌素pln基因座遗传图谱。最后构建基因plnE和plnF的外源表达载体,诱导产生大量重组蛋白,并对其进行初步的纯化。主要研究结果如下:1)14株植物乳杆菌中,菌株XJ25和PC520抑菌范围大,抑菌性较强,且对醋酸菌有一定的抑制作用;植物乳杆菌中共培养现象是普遍存在的,菌株XJ25和PC520与粪肠球菌XJ26M共培养诱导产生细菌素。通过对7株乳酸菌(XJA2,201,205,216,509,594,542)进行16S rRNA及recA基因的扩增分析,鉴定7株菌为植物乳杆菌,并且RAPD分析12株植物乳杆菌随机扩增片段的多态性,表明了其基因组DNA的多态性。2)菌株XJ25和PC520的pln基因座结构相似,均由6个操纵子(plnEFI、pln JKLR、plnGHSTUVW、plnABCD、plnMNOP、plnWXY)组成,其中plnABCD是三组分调节操纵子。并且菌株XJ25和PC520均存在一个新的未知功能的orf1。植物乳杆菌菌株PC520的部分基因(pln Y,pln S)等发生缺失,菌株XJ25的plnF基因编码蛋白在14位(A-S)和42位(V-I)有两个氨基酸突变。3)通过一步定向克隆技术,引物设计与线性质粒两端同源片段,成功将目的片段插入质粒中,转化的效率很高。优化重组蛋白诱导表达条件,选择在18℃,150 rpm,IPTG浓度0.6 mM,诱导时间12 h,进行扩大培养。4)重组蛋白PlnE,PlnF1,Pln F2经AKTA蛋白纯化分析,超滤脱盐等,去除部分杂蛋白,所测蛋白浓度为600-1000 ng/μl,仍需进一步纯化;纯化后蛋白进行抑菌活性检测表明,重组蛋白PlnE和Pln F具有抑菌活性,且蛋白PlnE和蛋白PlnF之间存在协同作用。
[Abstract]:Lactobacillus plantarum can adapt to bad wine environment, start malolactic acid fermentation, have the ability to produce lactobacillus plantarum bacteriocin, inhibit or kill harmful lactic acid bacteria in wine, and prevent wine decay. Partially replace the role of sulfur dioxide, thereby reducing the use of sulfur dioxide in wine. Therefore, it is necessary to screen Lactobacillus plantarum producing bacteriocin for malic acid-lactic fermentation. In this experiment, two bacteriocin producing strains of Lactobacillus plantarum (XJ25 and PC520.) were obtained from 14 strains of Lactobacillus plantarum. Secondly, the genetic map of pln locus of Lactobacillus plantarum (Plantaricin,pln) was obtained by comparing and analyzing the difference between the two strains by means of genome resequencing. Finally, the exogenous expression vectors of plnE and plnF were constructed, and a large number of recombinant proteins were induced and purified. The main results are as follows: 1) among the 14 strains of Lactobacillus plantarum, XJ25 and PC520 have a wide range of inhibition, strong bacteriostatic, and have a certain inhibitory effect on acetic acid bacteria; The co-culture of Lactobacillus plantarum was common. The co-culture of XJ25 and PC520 with Enterococcus faecalis XJ26M induced the production of bacteriocin. Seven strains of Lactobacillus plantarum (XJA2201205216509594542) were identified as Lactobacillus plantarum by 16s rRNA and recA gene amplification, and the polymorphism of random amplified fragments of 12 strains of Lactobacillus plantarum was analyzed by RAPD. The results showed that the pln loci of XJ25 and PC520 were similar, and they were composed of six plnEFI,pln JKLR,plnGHSTUVW,plnABCD,plnMNOP,plnWXY, in which plnABCD was a three-component regulatory operon. And both XJ25 and PC520 have a new orf1. with unknown function. Some genes of Lactobacillus plantarum PC520 (pln Yappln S) were deleted. The plnF gene encoding protein of XJ25 had two amino acid mutations at 14 (A-S) and 42 (V-I) sites. 3) one step directional cloning technique was used. The primers were designed to be homologous to the linear plasmids, and the target fragments were inserted into the plasmids successfully, and the transformation efficiency was very high. The expression conditions of recombinant protein were optimized. The recombinant protein PlnE,PlnF1,Pln F2 was cultured for 12 h at 18 鈩，
本文编号：2428609
[Abstract]:Lactobacillus plantarum can adapt to bad wine environment, start malolactic acid fermentation, have the ability to produce lactobacillus plantarum bacteriocin, inhibit or kill harmful lactic acid bacteria in wine, and prevent wine decay. Partially replace the role of sulfur dioxide, thereby reducing the use of sulfur dioxide in wine. Therefore, it is necessary to screen Lactobacillus plantarum producing bacteriocin for malic acid-lactic fermentation. In this experiment, two bacteriocin producing strains of Lactobacillus plantarum (XJ25 and PC520.) were obtained from 14 strains of Lactobacillus plantarum. Secondly, the genetic map of pln locus of Lactobacillus plantarum (Plantaricin,pln) was obtained by comparing and analyzing the difference between the two strains by means of genome resequencing. Finally, the exogenous expression vectors of plnE and plnF were constructed, and a large number of recombinant proteins were induced and purified. The main results are as follows: 1) among the 14 strains of Lactobacillus plantarum, XJ25 and PC520 have a wide range of inhibition, strong bacteriostatic, and have a certain inhibitory effect on acetic acid bacteria; The co-culture of Lactobacillus plantarum was common. The co-culture of XJ25 and PC520 with Enterococcus faecalis XJ26M induced the production of bacteriocin. Seven strains of Lactobacillus plantarum (XJA2201205216509594542) were identified as Lactobacillus plantarum by 16s rRNA and recA gene amplification, and the polymorphism of random amplified fragments of 12 strains of Lactobacillus plantarum was analyzed by RAPD. The results showed that the pln loci of XJ25 and PC520 were similar, and they were composed of six plnEFI,pln JKLR,plnGHSTUVW,plnABCD,plnMNOP,plnWXY, in which plnABCD was a three-component regulatory operon. And both XJ25 and PC520 have a new orf1. with unknown function. Some genes of Lactobacillus plantarum PC520 (pln Yappln S) were deleted. The plnF gene encoding protein of XJ25 had two amino acid mutations at 14 (A-S) and 42 (V-I) sites. 3) one step directional cloning technique was used. The primers were designed to be homologous to the linear plasmids, and the target fragments were inserted into the plasmids successfully, and the transformation efficiency was very high. The expression conditions of recombinant protein were optimized. The recombinant protein PlnE,PlnF1,Pln F2 was cultured for 12 h at 18 鈩，
本文编号：2428609
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