弧菌YeaZ基因表达、蛋白性质及其对环境微生物促生长作用研究
发布时间：2018-07-22 11:27
【摘要】：弧菌广泛分布于江河湖泊、海洋、高盐土壤等自然环境,跟人类生活密切相关,有些种是水产养殖动物的益生菌,有些能够引起人和动物疾病。弧菌在恶劣环境如低温、辐射、缺乏营养等情况下能进入休眠状态,即活的非可培养状态(VBNC),用一般方法检测不到VBNC细菌,但仍有一定代谢活力,条件适宜时,会重新生长。许多革兰氏阳性细菌存在一种细菌复苏促进因子蛋白,能促进不同状态细菌细胞的生长及VBNC状态细胞复苏,在部分革兰氏阴性细菌中发现了复苏促进因子活性的类似蛋白,具有明显的促细胞生长活性,但其作用机制有待阐明,通过研究VBNC细菌复苏,可以获得更多有应用价值新种,探索VBNC菌未被发现的新生物功能。本文从哈维弧菌(Vibrio harveyi)和溶藻胶弧菌(Vibrio alginolyticus)基因组扩增出一种类似于革兰氏阳性细菌细胞复苏促进因子的yeaZ基因,片段大小为702 bp,能够编码233氨基酸残基。yeaaZ基因与大肠杆菌糖蛋白酶、耶尔森菌M22肽酶YeaZ基因序列相似性分别为59%和55%,通过对不同种类弧菌复苏促进因子yeaZ基因的检测,进一步发现该类yeaaZ基因在弧菌类群中普遍存在,序列相似性在67%-94%。将yeaZ基因进行原核表达,SDS-PAGE电泳分析发现重组蛋白分子量为30 kDa。将YeaZ蛋白添加到VBNC状态的哈维弧菌细胞悬液中,能明显促进非可培养细胞的复苏,添加YeaZ蛋白的实验组在28℃培养8 h后细胞实现复苏,可培养细菌数量最大为2.88×15 cfu/ml,随着哈维弧菌在VBNC状态停留时间延长,能复苏的可培养细菌数量逐渐变小。当细菌在VBNC保持120 d后,添加YeaZ蛋白,可培养细菌数量为1.13×103 cfu/ml。其他对照组不能实现细胞复苏,检测不到可培养细菌。用人工合成底物 4-Methylumbelliferyl-β-D-N-N'-N"-t.riacetylchitotriose检测发现弧菌复苏促进因子YeaZ具有微弱溶菌酶活性。溶菌酶比活力为7.05 U/mg,最适pH为5.0,在pH 4.0-7.0和20-50℃温度下酶较稳定,当温度超过50℃时,酶活力丧失。Ca2+、Mg2+、Zn2+对酶有抑制作用,1 mmol/1的Co2+对溶菌酶活力有微弱促进作用。通过PCR定点突变技术对其溶菌酶活性中心位点氨基酸Thr-71和Asp-112进行了突变,发现突变的YeaZ溶菌酶活性发生部分降低,分别为4.75和2.50 U/mg,其突变体仍具有复苏促进作用。以偶氮酪蛋白及不同种类人工合成蛋白酶底物BAPNA、ATEE和BTEE为底物测定YeaZ的蛋白酶活性,发现YeaZ能不同程度的水解这些蛋白底物。其中以偶氮酪蛋白为底物的蛋白水解酶比活力为3 0 U/mg,以BAPNA为底物的酰胺酶比活力为1190 U/mg,以ATEE和BTEE为底物的酯酶比活力分别为365和197.5 U/mg。同时以体外定点突变技术对蛋白酶可能的活性位点氨基酸Asp88、Ser185和Trp169进行了定点突变,构建了不同突变体的表达载体,并在大肠杆菌BL21进行了表达。发现突变后的蛋白水解能力、蛋白酰胺酶活性以及酯酶活性都有不同程度下降或者完全丧失。其中突变体Asp88-Ala、Ser185-Leu、Trp169-Glu的蛋白水解能力分别下降了 66.67%、0.00%和 50.000%。突变体 Asp88-Ala、Trp169-Glu胰蛋白酶活力分别下降了 84.93%和32.88%,而同时带有Asp88-Ala和Ser185-Leu突变的突变体蛋白酶活力.完全丧失,说明这几个氨基酸是蛋白酶活性中心。采用平板计数和变性梯度凝胶电泳的方法研究了 YeaZ蛋白白对极端环境样品土壤可培养细菌的影响,发现添加YeaZ的实验组可培养细菌的数量和多样性都有一定程度的提高,其中可培养细菌数量分别从0.17×103和 2.03×103 cfu/g增 增加到 1.00×103 和 5.55×103 cfu/g,增加了 2-5 倍;从DGGE图谱可以看出添加YeaZ的实验组条带明显比对照组多。从添加YeaZ的实验组分离得到南极微球菌(Micrococcuss antarcticus)、玫瑰色库克菌(Kocuria rose)、新疆盐地杆菌(Salinibacterium xinjiangense)、南极游动球菌(Planococcusantarcticus)、Bacillusoceanisediminis、短短芽抱杆菌(Brevibacillusbrevis)、解木聚糖类芽抱杆菌(Paenibacillvs xylanilyticus)、Microbacterium maritypicum、枯草芽孢杆菌(Bacillus subtilis)、嗜盐芽孢杆菌(Bacillusalcalophilus)、Bacillusniabensis、杜氏大洋单胞菌(Oceanimonas doudroffii)和台湾佐氏菌(Zobellella taiwanensis)等更多细菌种类。论文还研究了西部荒漠草原土壤可培养细菌季节动态变化与土壤理化因子之间的相关性,发现荒漠草原可培养细菌季节动态变化显著,土壤细菌数量垂直分布明显,可培养细菌数量与全年降雨量和温度关联较大。1月、4月、7月和10月可培养细菌数量分别为0.13×107、4.09×107、5.33×107和1.80×l07cfu/g,全年不同季节共分离出不同的菌株72株,分别属于厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、变形菌门(Proteobacteria)和拟杆菌门(Bacteroidates)。芽抱杆菌属(Bacillus)作为优势菌属在不同月份所占比例分别5.56%、28.57%、50.000%和20.83%。从荒漠草原分离出的放线菌主要为链霉菌属(Atreptomyces)、小单孢菌属(Micromonspora)、链轮丝菌属(Streptoverticillium)、间抱囊菌属(Intrasporangium),其中链霉菌属是优势菌群,占放线菌属的50.000%,真菌类型单一,主要为交链孢霉属(Alternaria)和芽枝霉属(Cladosporium)。土壤温度和含水率是影响土壤微生物数量和多样性的主要因素,另外土壤营养元素(N,P,K)在不同程度上对土壤微生物数量也有一定影响。
[Abstract]:Vibrio is widely distributed in rivers, lakes, oceans, high salt soil and other natural environments, which are closely related to human life. Some species are probiotics of aquaculture animals, some can cause human and animal diseases. Vibrio can enter dormant conditions such as low temperature, radiation, and lack of nutrition, that is, the living non culturable state (VBNC). The general method does not detect VBNC bacteria, but still has a certain metabolic activity. When conditions are suitable, it will grow again. Many Gram-positive bacteria have a bacterial resuscitation promoting factor protein, which can promote the growth of bacterial cells in different states and the recovery of VBNC cells. In some Gram-negative bacteria, the activity of the resuscitation promoting factor is found. Similar proteins have obvious cell growth activity, but their mechanism of action remains to be elucidated. By studying the resuscitation of VBNC bacteria, more valuable new species can be obtained and new biological functions of VBNC bacteria have not been discovered. This article amplified a genomes from Vibrio Harvey (Vibrio harveyi) and Vibrio alginolyticus (Vibrio alginolyticus). The yeaZ gene, similar to gram positive bacterial cell resuscitation factor, is 702 BP, which can encode 233 amino acid residues.YeaaZ gene and Escherichia coli protease, and the similarity of the YeaZ gene sequence of Jerson M22 peptidase YeaZ gene is 59% and 55%, respectively, by detecting the yeaZ gene of different Vibrio resuscitation factor. It was found that the yeaaZ gene was ubiquitous in Vibrio groups. Sequence similarity was prokaryotic expression of yeaZ gene in 67%-94%.. SDS-PAGE electrophoresis analysis found that the molecular weight of recombinant protein was 30 kDa. and YeaZ protein was added to the Vibrio Harvey cell suspension of VBNC state. It could promote the resuscitation of non culturable cells and add the real YeaZ protein. After 8 h culture at 28 C, the cells recovered and the maximum number of cultured bacteria was 2.88 * 15 cfu/ml. With the prolongation of the residence time of Vibrio Harvey in VBNC state, the number of returnable bacteria became smaller and smaller. When the bacteria kept 120 d in VBNC, the number of cultured bacteria was 1.13 x 103 cfu/ml. other control groups. 4-Methylumbelliferyl- beta -D-N-N'-N "-t.riacetylchitotriose detection showed that Vibrio resuscitation factor YeaZ had weak lysozyme activity. The specific activity of the lysozyme was 7.05 U/mg, the optimum pH was 5, and the enzyme was stable at pH 4.0-7.0 and 20-50 C, when the temperature exceeded the temperature. At 50, the enzyme activity lost.Ca2+, Mg2+ and Zn2+ inhibited the enzyme, and the Co2+ of 1 mmol/1 had a weak promoting effect on the activity of lysozyme. The mutation of the amino acid Thr-71 and Asp-112 of the lysozyme activity site was carried out by PCR fixed-point mutation. It was found that the mutant YeaZ lysozyme activity was partly reduced, 4.75 and 2.50 U/mg respectively. BAPNA, ATEE and BTEE were used to determine the protease activity of YeaZ by using azo casein and different kinds of synthetic proteinase substrates. It was found that YeaZ could hydrolyze these protein substrates at different degrees. The specific activity of the protein hydrolysase with azo casein as the substrate was 30 U/mg, and BAPNA was used as the substrate. The specific activity of amidase was 1190 U/mg, and the esterase specific activity of ATEE and BTEE was 365 and 197.5 U/mg., respectively. At the same time, fixed-point mutation was carried out on the possible active site amino acid Asp88, Ser185 and Trp169 in vitro. The expression vector of different mutants was constructed and expressed in Escherichia coli BL21. The proteolysis, protease activity and esterase activity of the mutant Asp88-Ala, Ser185-Leu and Trp169-Glu decreased by 66.67%, 0% and 50.000%. mutants Asp88-Ala, and Trp169-Glu trypsin activity decreased by 84.93% and 32., respectively. 88%, the mutant protease activity with Asp88-Ala and Ser185-Leu mutations at the same time was completely lost, indicating that these amino acids were the protease activity centers. The effect of YeaZ white on the cultivated bacteria in the extreme environmental samples was studied by the method of plate counting and denaturing gradient gel electrophoresis. The experimental group adding YeaZ was found. The number and diversity of the cultured bacteria increased to a certain extent, in which the number of cultured bacteria increased from 0.17 x 103 and 2.03 x 103 cfu/g to 1 x 103 and 5.55 * 103 cfu/g, and increased by 2-5 times. From the DGGE map, the experimental group adding YeaZ was obviously more than the control group. Isolated from the experimental group adding YeaZ was obtained. Micrococcus Antarctica (Micrococcuss antarcticus), Kocuria Rose (Kocuria Rose), Bacillus salinis (Salinibacterium xinjiangense) in Xinjiang, Planococcusantarcticus, Bacillusoceanisediminis, Bacillus sprout (Brevibacillusbrevis), Bacillus sprout (Paenibacillvs xylanilyticus), Microbacter, and Microbacter Ium maritypicum, Bacillus subtilis (Bacillus subtilis), Bacillus halophila (Bacillusalcalophilus), Bacillusniabensis, oceanic monomonas Duchenne (Oceanimonas doudroffii) and zoella Taiwan (Zobellella taiwanensis), and so on. This paper also studied the seasonal dynamic changes of cultivated bacteria in the soil of western desert steppe. The correlation between soil physicochemical factors and soil physical and chemical factors showed that the seasonal dynamic changes of the cultivated bacteria in the desert steppe were significant, the vertical distribution of soil bacteria was obvious. The number of cultivated bacteria was associated with the annual rainfall and temperature.1 months. In April, July and October, the number of cultured bacteria were 0.13 * 107,4.09 * 107,5.33 * 107 and 1.80 x l07cfu/g, respectively. 72 strains of strains were isolated in different seasons, which were Firmicutes, Actinobacteria, Proteobacteria, Bacteroidates and Bacillus as the dominant bacteria in 5.56%, 28.57%, 50% and 20.83%. from the desert steppe. The isolated actinomycetes were mainly Streptomyces (Atreptomyces), small monospora (Micromonspora), Streptomyces (Streptoverticillium) and inter cysts (Intrasporangium), among which Streptomyces were dominant, 50% of actinomycetes, and single fungi, mainly Streptomyces (Alternaria) and sprout genus (Cladosporium). Soil temperature and water content are the main factors affecting the number and diversity of soil microbes. In addition, the soil nutrient elements (N, P, K) also have some influence on the soil microbial number in varying degrees.
【学位授予单位】：兰州理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q93
[Abstract]:Vibrio is widely distributed in rivers, lakes, oceans, high salt soil and other natural environments, which are closely related to human life. Some species are probiotics of aquaculture animals, some can cause human and animal diseases. Vibrio can enter dormant conditions such as low temperature, radiation, and lack of nutrition, that is, the living non culturable state (VBNC). The general method does not detect VBNC bacteria, but still has a certain metabolic activity. When conditions are suitable, it will grow again. Many Gram-positive bacteria have a bacterial resuscitation promoting factor protein, which can promote the growth of bacterial cells in different states and the recovery of VBNC cells. In some Gram-negative bacteria, the activity of the resuscitation promoting factor is found. Similar proteins have obvious cell growth activity, but their mechanism of action remains to be elucidated. By studying the resuscitation of VBNC bacteria, more valuable new species can be obtained and new biological functions of VBNC bacteria have not been discovered. This article amplified a genomes from Vibrio Harvey (Vibrio harveyi) and Vibrio alginolyticus (Vibrio alginolyticus). The yeaZ gene, similar to gram positive bacterial cell resuscitation factor, is 702 BP, which can encode 233 amino acid residues.YeaaZ gene and Escherichia coli protease, and the similarity of the YeaZ gene sequence of Jerson M22 peptidase YeaZ gene is 59% and 55%, respectively, by detecting the yeaZ gene of different Vibrio resuscitation factor. It was found that the yeaaZ gene was ubiquitous in Vibrio groups. Sequence similarity was prokaryotic expression of yeaZ gene in 67%-94%.. SDS-PAGE electrophoresis analysis found that the molecular weight of recombinant protein was 30 kDa. and YeaZ protein was added to the Vibrio Harvey cell suspension of VBNC state. It could promote the resuscitation of non culturable cells and add the real YeaZ protein. After 8 h culture at 28 C, the cells recovered and the maximum number of cultured bacteria was 2.88 * 15 cfu/ml. With the prolongation of the residence time of Vibrio Harvey in VBNC state, the number of returnable bacteria became smaller and smaller. When the bacteria kept 120 d in VBNC, the number of cultured bacteria was 1.13 x 103 cfu/ml. other control groups. 4-Methylumbelliferyl- beta -D-N-N'-N "-t.riacetylchitotriose detection showed that Vibrio resuscitation factor YeaZ had weak lysozyme activity. The specific activity of the lysozyme was 7.05 U/mg, the optimum pH was 5, and the enzyme was stable at pH 4.0-7.0 and 20-50 C, when the temperature exceeded the temperature. At 50, the enzyme activity lost.Ca2+, Mg2+ and Zn2+ inhibited the enzyme, and the Co2+ of 1 mmol/1 had a weak promoting effect on the activity of lysozyme. The mutation of the amino acid Thr-71 and Asp-112 of the lysozyme activity site was carried out by PCR fixed-point mutation. It was found that the mutant YeaZ lysozyme activity was partly reduced, 4.75 and 2.50 U/mg respectively. BAPNA, ATEE and BTEE were used to determine the protease activity of YeaZ by using azo casein and different kinds of synthetic proteinase substrates. It was found that YeaZ could hydrolyze these protein substrates at different degrees. The specific activity of the protein hydrolysase with azo casein as the substrate was 30 U/mg, and BAPNA was used as the substrate. The specific activity of amidase was 1190 U/mg, and the esterase specific activity of ATEE and BTEE was 365 and 197.5 U/mg., respectively. At the same time, fixed-point mutation was carried out on the possible active site amino acid Asp88, Ser185 and Trp169 in vitro. The expression vector of different mutants was constructed and expressed in Escherichia coli BL21. The proteolysis, protease activity and esterase activity of the mutant Asp88-Ala, Ser185-Leu and Trp169-Glu decreased by 66.67%, 0% and 50.000%. mutants Asp88-Ala, and Trp169-Glu trypsin activity decreased by 84.93% and 32., respectively. 88%, the mutant protease activity with Asp88-Ala and Ser185-Leu mutations at the same time was completely lost, indicating that these amino acids were the protease activity centers. The effect of YeaZ white on the cultivated bacteria in the extreme environmental samples was studied by the method of plate counting and denaturing gradient gel electrophoresis. The experimental group adding YeaZ was found. The number and diversity of the cultured bacteria increased to a certain extent, in which the number of cultured bacteria increased from 0.17 x 103 and 2.03 x 103 cfu/g to 1 x 103 and 5.55 * 103 cfu/g, and increased by 2-5 times. From the DGGE map, the experimental group adding YeaZ was obviously more than the control group. Isolated from the experimental group adding YeaZ was obtained. Micrococcus Antarctica (Micrococcuss antarcticus), Kocuria Rose (Kocuria Rose), Bacillus salinis (Salinibacterium xinjiangense) in Xinjiang, Planococcusantarcticus, Bacillusoceanisediminis, Bacillus sprout (Brevibacillusbrevis), Bacillus sprout (Paenibacillvs xylanilyticus), Microbacter, and Microbacter Ium maritypicum, Bacillus subtilis (Bacillus subtilis), Bacillus halophila (Bacillusalcalophilus), Bacillusniabensis, oceanic monomonas Duchenne (Oceanimonas doudroffii) and zoella Taiwan (Zobellella taiwanensis), and so on. This paper also studied the seasonal dynamic changes of cultivated bacteria in the soil of western desert steppe. The correlation between soil physicochemical factors and soil physical and chemical factors showed that the seasonal dynamic changes of the cultivated bacteria in the desert steppe were significant, the vertical distribution of soil bacteria was obvious. The number of cultivated bacteria was associated with the annual rainfall and temperature.1 months. In April, July and October, the number of cultured bacteria were 0.13 * 107,4.09 * 107,5.33 * 107 and 1.80 x l07cfu/g, respectively. 72 strains of strains were isolated in different seasons, which were Firmicutes, Actinobacteria, Proteobacteria, Bacteroidates and Bacillus as the dominant bacteria in 5.56%, 28.57%, 50% and 20.83%. from the desert steppe. The isolated actinomycetes were mainly Streptomyces (Atreptomyces), small monospora (Micromonspora), Streptomyces (Streptoverticillium) and inter cysts (Intrasporangium), among which Streptomyces were dominant, 50% of actinomycetes, and single fungi, mainly Streptomyces (Alternaria) and sprout genus (Cladosporium). Soil temperature and water content are the main factors affecting the number and diversity of soil microbes. In addition, the soil nutrient elements (N, P, K) also have some influence on the soil microbial number in varying degrees.
【学位授予单位】：兰州理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q93
【参考文献】
相关期刊论文 前10条
1 赵爱花;杜晓军;臧婧;张守仁;焦志华;;宝天曼落叶阔叶林土壤细菌多样性[J];生物多样性;2015年05期
2 孙嘉兴;;土壤全量氮磷钾测定方法的研究[J];中国农业信息;2015年07期
3 丛立新;马玉杰;梁俊超;周会敏;;结核分枝杆菌休眠复苏促进因子的研究进展[J];中国兽医杂志;2014年05期
4 刘秉儒;张秀珍;胡天华;李文金;;贺兰山不同海拔典型植被带土壤微生物多样性[J];生态学报;2013年22期
5 陈晓娟;吴小红;刘守龙;袁红朝;李苗苗;朱捍华;葛体达;童成立;吴金水;;不同耕地利用方式下土壤微生物活性及群落结构特性分析:基于PLFA和MicroResp~(TM)方法[J];环境科学;2013年06期
6 焦迎春;卢素锦;;青海大通周边地区不同土壤结构微生物种群研究[J];北方园艺;2013年08期
7 赵明君;贾俊涛;陈吉祥;;副溶血弧菌一种糖蛋白酶(复苏促进因子)在大肠杆菌中的表达及性质[J];中国海洋大学学报(自然科学版);2012年03期
8 慈龙骏;;极端干旱荒漠的“荒漠化”[J];科学通报;2011年31期
9 代金霞;赵辉;;宁夏荒漠草原固沙植物群落土壤微生物数量及土壤酶活性研究[J];江苏农业科学;2011年04期
10 王洪涛;金研铭;马丽华;李世荣;;不同复层次群落下土壤理化性质分析[J];安徽农业科学;2011年13期
相关会议论文 前1条
1 刘忠泉;张宗德;邢爱英;陈曦;李自慧;古淑香;贾红艳;杜博平;马s，
本文编号：2137288
本文链接：https://www.wllwen.com/shoufeilunwen/jckxbs/2137288.html
