| 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. |
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