DnaK基因调控酸土脂环酸芽孢杆菌嗜酸耐热生理特性研究
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摘要
酸土脂环酸芽孢杆菌具有嗜酸、耐热的双重生理特性，能经受酸性条件下的巴氏杀菌过程而存活，研究认为生物抵御不良环境的能力与其体内的热休克蛋白（HeatShock Protein，HSP）有关，该蛋白广泛分布于微生物、动物、植物等各种生物体内，其中对HSP70（DnaK）的研究最深入。DnaK生物学功能广泛，不仅在细胞生长、发育、分化过程中的基因转录、蛋白质合成、折叠、跨膜运输、分解和立体构象的维持等方面发挥重要作用，而且在生物体内广泛地参与了多种复杂的功能，与生物在高温、酸、碱、氧化应激等逆境下的生存关系密切。
本论文利用同源克隆和基因组步移技术克隆得到酸土脂环酸芽孢杆菌DSM3922TDnaK基因，利用Real Time RT-PCR技术分析酸土脂环酸芽孢杆菌DnaK基因在酸、热、冷胁迫下的表达差异，从mRNA水平探索DnaK蛋白在该菌对酸、热逆境应答中的功能，并在大肠杆菌中异源表达酸土脂环酸芽孢杆菌DnaK基因，研究该基因对宿主菌耐高温、酸、碱、氧化胁迫抗性的影响，主要研究结果如下：
1.成功获取酸土脂环酸芽孢杆菌DSM3922TDnaK基因全序列，其ORF全长1,854bp，编码617个氨基酸，其推测氨基酸序列与来源于A. acidocaldarius LAA1的分子伴侣蛋白DnaK同源性最高，相似性达86%。
2. Real Time RT-PCR试验结果表明：该基因在正常生长条件下组成性表达，在热、酸胁迫下表达量在短时间内迅速上调，70℃、80℃、90℃热胁迫5min时，DnaK基因的相对表达量分别为对照组的1.4倍、1.7倍和2.5倍；酸胁迫1h表达量升至最高，为对照组的4.3倍，随后表达量下调，胁迫3h时降至最低，只有对照组的0.08倍；冷胁迫条件下，DnaK基因的表达量迅速下调，冷胁迫处理1h，其表达量仅为对照组的0.48倍，随着处理时间延长，表达量继续降低，处理6h时，表达量为对照组的0.25倍。
3.构建重组质粒pET28a-DnaK，转化大肠杆菌BL21，诱导其表达酸土脂环酸芽孢杆菌DnaK基因，目标蛋白主要以胞内可溶形式在大肠杆菌中成功表达，重组DnaK经一步亲和层析，得到电泳级纯蛋白，为酸土脂环酸芽孢杆菌DnaK蛋白的结构、功能及与其他热休克蛋白的相互作用机制等的研究提供材料支持。
4.利用IPTG诱导重组大肠杆菌表达DnaK基因，研究了DnaK基因表达对重组大肠杆菌热、酸、碱及氧化胁迫抗性的影响，结果表明，经过热、酸、氧化胁迫后，重组大肠杆菌的存活率明显高于对照菌株，而两种菌株的存活率在碱胁迫条件下相差不大，说明酸土脂环酸芽孢杆菌DnaK基因在大肠杆菌中异源表达能显著提高宿主菌对高温、酸及氧化胁迫的抗性。
本论文克隆了酸土脂环酸芽孢杆菌DnaK基因，利用Real Time RT-PCR技术分析了Alicyclobacillus acidoterrestris DnaK基因在热、酸等胁迫条件下的表达差异，从分子水平揭示酸土脂环酸芽孢杆菌耐酸、热逆境的生理适应机制，并进一步研究了其DnaK基因异源表达对宿主耐热、酸、氧化冲击等胁迫抗性的影响，不仅为揭示微生物的环境适应化机制奠定理论基础，也为发酵工业生产中利用转基因技术提高发酵微生物的胁迫抗性，开发对发酵工业生产中主要胁迫因素有较强耐受性的生产菌株具有重要的应用价值。
Alicyclobacillus acidoterrestris is a genus of aerobic, acidophilic, thermophilic,soil-dwelling bacteria, with the ability to survive traditional pasteurization process under theacid condition. This capacity of resisting adverse environment is related to its heat shockproteins(HSPS). HSPS are widely distributed in organisms like microorganism, animal and plant,among which, HSP70(DnaK) was analyzed extensively. DnaK is of wide biological functions,for it not only exerts important influence on gene transcription, protein synthesis, folding andmembrane transport, catabolism and maintenance of stereo conformation in the process ofcellular growth, development and differentiation, but also widely involves in various complexfunctions of organisms. DnaK play a key role in protecting organisms from hostile environments,such as high temperture, acid, alkali, oxidative stress and other adversities.
This paper has obtained DSM3922TDnaK gene from Alicyclobacillus acidoterrestris byapplying homologous cloning and genome-walking cloning, analyzed expression differences ofDnaK gene in Alicyclobacillus acidoterrestris under acid, heat and cold stress by Real TimeRT-PCR, and explored the functions of DnaK protein of A.acidoterrestris under heat and acidstresses from the mRNA level. Furthermore, the heterologous expression of DnaK gene inE.coli will be performed to investigate the effect on high temperature, acid, alkaline andoxidation stress resistance of host becteria. The main research results were as follows:
1. It had succeed in obtaining complete sequence of DSM3922TDnaK gene fromAlicyclobacillus acidoterrestris. The DnaK gene contains a complete ORF of1,854bp,encoding617aa, which deduced amino acid sequence shared the highest homology with DnaKfrom A. acidocaldarius LAA1(86%).
2. RT-PCR experimental results showed that: DnaK was constitutively expressed undernormal growth condition, with expression rapidly rises under heat and acid stress within shorttime. When heat shock took place at70℃,80°C and90°C, the relative expression of DnaK wasrapidly increased and reached1.4times,1.7times and2.5times of control group respectively;when acid stress lasts for1h, expression rises to the highest value as4.3times of control group,and then expression of DnaK gene rapidly decreases; when stress lasts for3h, it decreases to the lowest value as0.08time of control group; under cold stress condition, expression of DnaKgene rapidly decreases, and when cold stress lasts for1h, the expression becomes0.48time ofcontrol group, and with extending of treatment time, expression goes on decreasing. Whentreatment time lasts for6h, the expression becomes0.25time of control group. So we canconclude that DnaK has made significant influence on survival of alicyclobacillusacidoterrestris under acid and heat stress, which may have close relation with specialphysiological function involving heat and acid endurance of Alicyclobacillus acidoterrestris.
3. It has constructed recombinant plasmids pET28a-DnaK, transformed colon bacillusBL21and induced it to express DnaK of Alicyclobacillus acidoterrestris, and made targetprotein mainly in the method of intracellular soluble form and successfully expressed in colonbacillus, as well as made reconstructed DnaK got further affinity chromatography and obtainedpure electrophoresis protein, and accordingly set material support for exploring the structure,function and interaction mechanism with other heat shock protein of A. acidoterrestris DnaK.
4. It has applied IPTG to induce recombinant Escherichia coli to express DnaK gene, andthen researched the influence of DnaK gene expression on heat, acid, alkaline and oxidizationstress resistance of recombinant Escherichia coli. The results indicated that survival rate ofrecombinant E. coli after heat, acid and oxidization stress is obviously higher than controlgroup, while survival rate of both groups is of little difference under alkaline stress, andindicates that heterologous expression of DnaK gene from Alicyclobacillus acidoterrestris incolon bacillus can significantly improve endurance for high temperature, oxidization impactand acid stress of host strain.
DnaK gene was cloned from Alicyclobacillus acidoterrestris and analyzed expressiondifferences of DnaK gene in Alicyclobacillus acidoterrestris under heat and acid stress by RealTime RT-PCR in this paper, as well as revealed physiological adaptation mechanism ofAlicyclobacillus acidoterrestris under acid and heat stress from the molecule level, and furtherresearched the influence of heterologous expression of DnaK gene on heat and acid enduranceof host bacteria. It has not only set theoretical basis for elucidating environmental adaptation ofmicroorganism and acid and heat endurance mechanism but also has important applicationvalue for applying transgenic technology to improve stress resistance of fermentedmicroorganism in the production of fermentation industry and develop the newmicroorganisms with high production and preferable stress tolerant properties for main expressin the production of fermentation industry.
本论文利用同源克隆和基因组步移技术克隆得到酸土脂环酸芽孢杆菌DSM3922TDnaK基因，利用Real Time RT-PCR技术分析酸土脂环酸芽孢杆菌DnaK基因在酸、热、冷胁迫下的表达差异，从mRNA水平探索DnaK蛋白在该菌对酸、热逆境应答中的功能，并在大肠杆菌中异源表达酸土脂环酸芽孢杆菌DnaK基因，研究该基因对宿主菌耐高温、酸、碱、氧化胁迫抗性的影响，主要研究结果如下：
1.成功获取酸土脂环酸芽孢杆菌DSM3922TDnaK基因全序列，其ORF全长1,854bp，编码617个氨基酸，其推测氨基酸序列与来源于A. acidocaldarius LAA1的分子伴侣蛋白DnaK同源性最高，相似性达86%。
2. Real Time RT-PCR试验结果表明：该基因在正常生长条件下组成性表达，在热、酸胁迫下表达量在短时间内迅速上调，70℃、80℃、90℃热胁迫5min时，DnaK基因的相对表达量分别为对照组的1.4倍、1.7倍和2.5倍；酸胁迫1h表达量升至最高，为对照组的4.3倍，随后表达量下调，胁迫3h时降至最低，只有对照组的0.08倍；冷胁迫条件下，DnaK基因的表达量迅速下调，冷胁迫处理1h，其表达量仅为对照组的0.48倍，随着处理时间延长，表达量继续降低，处理6h时，表达量为对照组的0.25倍。
3.构建重组质粒pET28a-DnaK，转化大肠杆菌BL21，诱导其表达酸土脂环酸芽孢杆菌DnaK基因，目标蛋白主要以胞内可溶形式在大肠杆菌中成功表达，重组DnaK经一步亲和层析，得到电泳级纯蛋白，为酸土脂环酸芽孢杆菌DnaK蛋白的结构、功能及与其他热休克蛋白的相互作用机制等的研究提供材料支持。
4.利用IPTG诱导重组大肠杆菌表达DnaK基因，研究了DnaK基因表达对重组大肠杆菌热、酸、碱及氧化胁迫抗性的影响，结果表明，经过热、酸、氧化胁迫后，重组大肠杆菌的存活率明显高于对照菌株，而两种菌株的存活率在碱胁迫条件下相差不大，说明酸土脂环酸芽孢杆菌DnaK基因在大肠杆菌中异源表达能显著提高宿主菌对高温、酸及氧化胁迫的抗性。
本论文克隆了酸土脂环酸芽孢杆菌DnaK基因，利用Real Time RT-PCR技术分析了Alicyclobacillus acidoterrestris DnaK基因在热、酸等胁迫条件下的表达差异，从分子水平揭示酸土脂环酸芽孢杆菌耐酸、热逆境的生理适应机制，并进一步研究了其DnaK基因异源表达对宿主耐热、酸、氧化冲击等胁迫抗性的影响，不仅为揭示微生物的环境适应化机制奠定理论基础，也为发酵工业生产中利用转基因技术提高发酵微生物的胁迫抗性，开发对发酵工业生产中主要胁迫因素有较强耐受性的生产菌株具有重要的应用价值。
Alicyclobacillus acidoterrestris is a genus of aerobic, acidophilic, thermophilic,soil-dwelling bacteria, with the ability to survive traditional pasteurization process under theacid condition. This capacity of resisting adverse environment is related to its heat shockproteins(HSPS). HSPS are widely distributed in organisms like microorganism, animal and plant,among which, HSP70(DnaK) was analyzed extensively. DnaK is of wide biological functions,for it not only exerts important influence on gene transcription, protein synthesis, folding andmembrane transport, catabolism and maintenance of stereo conformation in the process ofcellular growth, development and differentiation, but also widely involves in various complexfunctions of organisms. DnaK play a key role in protecting organisms from hostile environments,such as high temperture, acid, alkali, oxidative stress and other adversities.
This paper has obtained DSM3922TDnaK gene from Alicyclobacillus acidoterrestris byapplying homologous cloning and genome-walking cloning, analyzed expression differences ofDnaK gene in Alicyclobacillus acidoterrestris under acid, heat and cold stress by Real TimeRT-PCR, and explored the functions of DnaK protein of A.acidoterrestris under heat and acidstresses from the mRNA level. Furthermore, the heterologous expression of DnaK gene inE.coli will be performed to investigate the effect on high temperature, acid, alkaline andoxidation stress resistance of host becteria. The main research results were as follows:
1. It had succeed in obtaining complete sequence of DSM3922TDnaK gene fromAlicyclobacillus acidoterrestris. The DnaK gene contains a complete ORF of1,854bp,encoding617aa, which deduced amino acid sequence shared the highest homology with DnaKfrom A. acidocaldarius LAA1(86%).
2. RT-PCR experimental results showed that: DnaK was constitutively expressed undernormal growth condition, with expression rapidly rises under heat and acid stress within shorttime. When heat shock took place at70℃,80°C and90°C, the relative expression of DnaK wasrapidly increased and reached1.4times,1.7times and2.5times of control group respectively;when acid stress lasts for1h, expression rises to the highest value as4.3times of control group,and then expression of DnaK gene rapidly decreases; when stress lasts for3h, it decreases to the lowest value as0.08time of control group; under cold stress condition, expression of DnaKgene rapidly decreases, and when cold stress lasts for1h, the expression becomes0.48time ofcontrol group, and with extending of treatment time, expression goes on decreasing. Whentreatment time lasts for6h, the expression becomes0.25time of control group. So we canconclude that DnaK has made significant influence on survival of alicyclobacillusacidoterrestris under acid and heat stress, which may have close relation with specialphysiological function involving heat and acid endurance of Alicyclobacillus acidoterrestris.
3. It has constructed recombinant plasmids pET28a-DnaK, transformed colon bacillusBL21and induced it to express DnaK of Alicyclobacillus acidoterrestris, and made targetprotein mainly in the method of intracellular soluble form and successfully expressed in colonbacillus, as well as made reconstructed DnaK got further affinity chromatography and obtainedpure electrophoresis protein, and accordingly set material support for exploring the structure,function and interaction mechanism with other heat shock protein of A. acidoterrestris DnaK.
4. It has applied IPTG to induce recombinant Escherichia coli to express DnaK gene, andthen researched the influence of DnaK gene expression on heat, acid, alkaline and oxidizationstress resistance of recombinant Escherichia coli. The results indicated that survival rate ofrecombinant E. coli after heat, acid and oxidization stress is obviously higher than controlgroup, while survival rate of both groups is of little difference under alkaline stress, andindicates that heterologous expression of DnaK gene from Alicyclobacillus acidoterrestris incolon bacillus can significantly improve endurance for high temperature, oxidization impactand acid stress of host strain.
DnaK gene was cloned from Alicyclobacillus acidoterrestris and analyzed expressiondifferences of DnaK gene in Alicyclobacillus acidoterrestris under heat and acid stress by RealTime RT-PCR in this paper, as well as revealed physiological adaptation mechanism ofAlicyclobacillus acidoterrestris under acid and heat stress from the molecule level, and furtherresearched the influence of heterologous expression of DnaK gene on heat and acid enduranceof host bacteria. It has not only set theoretical basis for elucidating environmental adaptation ofmicroorganism and acid and heat endurance mechanism but also has important applicationvalue for applying transgenic technology to improve stress resistance of fermentedmicroorganism in the production of fermentation industry and develop the newmicroorganisms with high production and preferable stress tolerant properties for main expressin the production of fermentation industry.
引文
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