Screening Of Major Cold Shock Proteins Of Bacillus Cereus D2

Bacillus cereus D2 is a kind of cold tolerant bacteria which can survive at low temperatures and has the function of repairing heavy metal soil.However,at present,national and international scientists mainly focus on the remediation ability of heavy metal contaminated soil in this strain,but the mechanism of cold shock of Bacillus cereus has not been reported.Cold shock protein(Csp)is a kind of protein expressed by microorganisms in low temperature environment to help the body recover normal life activities,and it is an indispensable adaptation of microorganisms to low temperature environment.In this article,Bacillus cereus D2 was chosen as research object and its growth curve at different temperatures was measured,and based on this,the Arrhenius curve was constructed in order to set the subsequent cold induction temperature.The Csp genes were searched via NCBI and Universal Protein database,and identified by PCR and sequencing.Tricine SDS-PAGE was used to analyze the protein expression changes before and after cold induction,and qRT-PCR was used to compare the mRNA expression levels of the cold shock protein genes after cold induction,so as to find out the high expression cold shock proteins after cold induction.The high expression cold shock proteins were expressed in E.coli.The growth curves of these E.coli were measured in order to define major cold shock proteins.we compared the cold shock proteins in various bacteria in NCBI and,based on it,the Phylogenetic tree of cold shock proteins in Bacillus cereus D2 was built with MEGA7.0.Then,by comparing the number and proportion of hydrophobic amino acids,primary structure,secondary structure and spatial structure,the structural differences between key cold shock proteins in Bacillus cereus D2 and other cold shock proteins were analyzed.The growth curve of Bacillus cereus D2 at different temperatures showed that the growth rate of the strain decreased rapidly with the decrease of temperature,and it grew slowly at 8?.The Arrhenius curve showed that the growth rate from 8? to 10? is slower than that from 10? to 15?,which means that the strain is most sensitive to temperature change at 10?,so 10? is selected as the cold shock temperature.Six cold shock proteins were screened from the strain according to the database comparison,and their existence was confirmed by cloning and sequencing.After 2 hours of cold induction,7 kD differential protein was expressed in Bacillus cereus D2.Real time fluorescent quantitative PCR showed that the mRNA expression levels of Csp1,Csp2,Csp3 and Csp6 increased significantly after cold induction.The corresponding Csp-pet-28 a vector was constructed and overexpressed in E.coli.The comparison of amino acid sequence and structure shows that csp2 and csp6 belong to CspB and CspC families,respectively.And the alanine content in the key cold shock proteins is much higher.It is suggested that the formation of ? helix on leucine may be the main difference between the key cold shock proteins and other cold shock proteins.The above research provides a theoretical basis for the utilization and research of cold resistant microorganisms and cold shock proteins in practical application.