弧菌YeaZ基因表達(dá)、蛋白性質(zhì)及其對(duì)環(huán)境微生物促生長作用研究
發(fā)布時(shí)間：2018-07-22 11:27
【摘要】：弧菌廣泛分布于江河湖泊、海洋、高鹽土壤等自然環(huán)境,跟人類生活密切相關(guān),有些種是水產(chǎn)養(yǎng)殖動(dòng)物的益生菌,有些能夠引起人和動(dòng)物疾病�；【趷毫迎h(huán)境如低溫、輻射、缺乏營養(yǎng)等情況下能進(jìn)入休眠狀態(tài),即活的非可培養(yǎng)狀態(tài)(VBNC),用一般方法檢測不到VBNC細(xì)菌,但仍有一定代謝活力,條件適宜時(shí),會(huì)重新生長。許多革蘭氏陽性細(xì)菌存在一種細(xì)菌復(fù)蘇促進(jìn)因子蛋白,能促進(jìn)不同狀態(tài)細(xì)菌細(xì)胞的生長及VBNC狀態(tài)細(xì)胞復(fù)蘇,在部分革蘭氏陰性細(xì)菌中發(fā)現(xiàn)了復(fù)蘇促進(jìn)因子活性的類似蛋白,具有明顯的促細(xì)胞生長活性,但其作用機(jī)制有待闡明,通過研究VBNC細(xì)菌復(fù)蘇,可以獲得更多有應(yīng)用價(jià)值新種,探索VBNC菌未被發(fā)現(xiàn)的新生物功能。本文從哈維弧菌(Vibrio harveyi)和溶藻膠弧菌(Vibrio alginolyticus)基因組擴(kuò)增出一種類似于革蘭氏陽性細(xì)菌細(xì)胞復(fù)蘇促進(jìn)因子的yeaZ基因,片段大小為702 bp,能夠編碼233氨基酸殘基。yeaaZ基因與大腸桿菌糖蛋白酶、耶爾森菌M22肽酶YeaZ基因序列相似性分別為59%和55%,通過對(duì)不同種類弧菌復(fù)蘇促進(jìn)因子yeaZ基因的檢測,進(jìn)一步發(fā)現(xiàn)該類yeaaZ基因在弧菌類群中普遍存在,序列相似性在67%-94%。將yeaZ基因進(jìn)行原核表達(dá),SDS-PAGE電泳分析發(fā)現(xiàn)重組蛋白分子量為30 kDa。將YeaZ蛋白添加到VBNC狀態(tài)的哈維弧菌細(xì)胞懸液中,能明顯促進(jìn)非可培養(yǎng)細(xì)胞的復(fù)蘇,添加YeaZ蛋白的實(shí)驗(yàn)組在28℃培養(yǎng)8 h后細(xì)胞實(shí)現(xiàn)復(fù)蘇,可培養(yǎng)細(xì)菌數(shù)量最大為2.88×15 cfu/ml,隨著哈維弧菌在VBNC狀態(tài)停留時(shí)間延長,能復(fù)蘇的可培養(yǎng)細(xì)菌數(shù)量逐漸變小。當(dāng)細(xì)菌在VBNC保持120 d后,添加YeaZ蛋白,可培養(yǎng)細(xì)菌數(shù)量為1.13×103 cfu/ml。其他對(duì)照組不能實(shí)現(xiàn)細(xì)胞復(fù)蘇,檢測不到可培養(yǎng)細(xì)菌。用人工合成底物 4-Methylumbelliferyl-β-D-N-N'-N"-t.riacetylchitotriose檢測發(fā)現(xiàn)弧菌復(fù)蘇促進(jìn)因子YeaZ具有微弱溶菌酶活性。溶菌酶比活力為7.05 U/mg,最適pH為5.0,在pH 4.0-7.0和20-50℃溫度下酶較穩(wěn)定,當(dāng)溫度超過50℃時(shí),酶活力喪失。Ca2+、Mg2+、Zn2+對(duì)酶有抑制作用,1 mmol/1的Co2+對(duì)溶菌酶活力有微弱促進(jìn)作用。通過PCR定點(diǎn)突變技術(shù)對(duì)其溶菌酶活性中心位點(diǎn)氨基酸Thr-71和Asp-112進(jìn)行了突變,發(fā)現(xiàn)突變的YeaZ溶菌酶活性發(fā)生部分降低,分別為4.75和2.50 U/mg,其突變體仍具有復(fù)蘇促進(jìn)作用。以偶氮酪蛋白及不同種類人工合成蛋白酶底物BAPNA、ATEE和BTEE為底物測定YeaZ的蛋白酶活性,發(fā)現(xiàn)YeaZ能不同程度的水解這些蛋白底物。其中以偶氮酪蛋白為底物的蛋白水解酶比活力為3 0 U/mg,以BAPNA為底物的酰胺酶比活力為1190 U/mg,以ATEE和BTEE為底物的酯酶比活力分別為365和197.5 U/mg。同時(shí)以體外定點(diǎn)突變技術(shù)對(duì)蛋白酶可能的活性位點(diǎn)氨基酸Asp88、Ser185和Trp169進(jìn)行了定點(diǎn)突變,構(gòu)建了不同突變體的表達(dá)載體,并在大腸桿菌BL21進(jìn)行了表達(dá)。發(fā)現(xiàn)突變后的蛋白水解能力、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喪失。其中突變體Asp88-Ala、Ser185-Leu、Trp169-Glu的蛋白水解能力分別下降了 66.67%、0.00%和 50.000%。突變體 Asp88-Ala、Trp169-Glu胰蛋白酶活力分別下降了 84.93%和32.88%,而同時(shí)帶有Asp88-Ala和Ser185-Leu突變的突變體蛋白酶活力.完全喪失,說明這幾個(gè)氨基酸是蛋白酶活性中心。采用平板計(jì)數(shù)和變性梯度凝膠電泳的方法研究了 YeaZ蛋白白對(duì)極端環(huán)境樣品土壤可培養(yǎng)細(xì)菌的影響,發(fā)現(xiàn)添加YeaZ的實(shí)驗(yàn)組可培養(yǎng)細(xì)菌的數(shù)量和多樣性都有一定程度的提高,其中可培養(yǎng)細(xì)菌數(shù)量分別從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;從DGGE圖譜可以看出添加YeaZ的實(shí)驗(yàn)組條帶明顯比對(duì)照組多。從添加YeaZ的實(shí)驗(yàn)組分離得到南極微球菌(Micrococcuss antarcticus)、玫瑰色庫克菌(Kocuria rose)、新疆鹽地桿菌(Salinibacterium xinjiangense)、南極游動(dòng)球菌(Planococcusantarcticus)、Bacillusoceanisediminis、短短芽抱桿菌(Brevibacillusbrevis)、解木聚糖類芽抱桿菌(Paenibacillvs xylanilyticus)、Microbacterium maritypicum、枯草芽孢桿菌(Bacillus subtilis)、嗜鹽芽孢桿菌(Bacillusalcalophilus)、Bacillusniabensis、杜氏大洋單胞菌(Oceanimonas doudroffii)和臺(tái)灣佐氏菌(Zobellella taiwanensis)等更多細(xì)菌種類。論文還研究了西部荒漠草原土壤可培養(yǎng)細(xì)菌季節(jié)動(dòng)態(tài)變化與土壤理化因子之間的相關(guān)性,發(fā)現(xiàn)荒漠草原可培養(yǎng)細(xì)菌季節(jié)動(dòng)態(tài)變化顯著,土壤細(xì)菌數(shù)量垂直分布明顯,可培養(yǎng)細(xì)菌數(shù)量與全年降雨量和溫度關(guān)聯(lián)較大。1月、4月、7月和10月可培養(yǎng)細(xì)菌數(shù)量分別為0.13×107、4.09×107、5.33×107和1.80×l07cfu/g,全年不同季節(jié)共分離出不同的菌株72株,分別屬于厚壁菌門(Firmicutes)、放線菌門(Actinobacteria)、變形菌門(Proteobacteria)和擬桿菌門(Bacteroidates)。芽抱桿菌屬(Bacillus)作為優(yōu)勢菌屬在不同月份所占比例分別5.56%、28.57%、50.000%和20.83%。從荒漠草原分離出的放線菌主要為鏈霉菌屬(Atreptomyces)、小單孢菌屬(Micromonspora)、鏈輪絲菌屬(Streptoverticillium)、間抱囊菌屬(Intrasporangium),其中鏈霉菌屬是優(yōu)勢菌群,占放線菌屬的50.000%,真菌類型單一,主要為交鏈孢霉屬(Alternaria)和芽枝霉屬(Cladosporium)。土壤溫度和含水率是影響土壤微生物數(shù)量和多樣性的主要因素,另外土壤營養(yǎng)元素(N,P,K)在不同程度上對(duì)土壤微生物數(shù)量也有一定影響。
[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.
【學(xué)位授予單位】：蘭州理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：蘭州理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q93
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 趙愛花;杜曉軍;臧婧;張守仁;焦志華;;寶天曼落葉闊葉林土壤細(xì)菌多樣性[J];生物多樣性;2015年05期
2 孫嘉興;;土壤全量氮磷鉀測定方法的研究[J];中國農(nóng)業(yè)信息;2015年07期
3 叢立新;馬玉杰;梁俊超;周會(huì)敏;;結(jié)核分枝桿菌休眠復(fù)蘇促進(jìn)因子的研究進(jìn)展[J];中國獸醫(yī)雜志;2014年05期
4 劉秉儒;張秀珍;胡天華;李文金;;賀蘭山不同海拔典型植被帶土壤微生物多樣性[J];生態(tài)學(xué)報(bào);2013年22期
5 陳曉娟;吳小紅;劉守龍;袁紅朝;李苗苗;朱捍華;葛體達(dá);童成立;吳金水;;不同耕地利用方式下土壤微生物活性及群落結(jié)構(gòu)特性分析:基于PLFA和MicroResp~(TM)方法[J];環(huán)境科學(xué);2013年06期
6 焦迎春;盧素錦;;青海大通周邊地區(qū)不同土壤結(jié)構(gòu)微生物種群研究[J];北方園藝;2013年08期
7 趙明君;賈俊濤;陳吉祥;;副溶血弧菌一種糖蛋白酶(復(fù)蘇促進(jìn)因子)在大腸桿菌中的表達(dá)及性質(zhì)[J];中國海洋大學(xué)學(xué)報(bào)(自然科學(xué)版);2012年03期
8 慈龍駿;;極端干旱荒漠的“荒漠化”[J];科學(xué)通報(bào);2011年31期
9 代金霞;趙輝;;寧夏荒漠草原固沙植物群落土壤微生物數(shù)量及土壤酶活性研究[J];江蘇農(nóng)業(yè)科學(xué);2011年04期
10 王洪濤;金研銘;馬麗華;李世榮;;不同復(fù)層次群落下土壤理化性質(zhì)分析[J];安徽農(nóng)業(yè)科學(xué);2011年13期
相關(guān)會(huì)議論文 前1條
1 劉忠泉;張宗德;邢愛英;陳曦;李自慧;古淑香;賈紅艷;杜博平;馬s，
本文編號(hào)：2137288
本文鏈接：http://sikaile.net/shoufeilunwen/jckxbs/2137288.html
最近更新
教材專著
