The Relatinship Between Chaperonin And Parasporal Crystal Proteins Of Bacillus Thuringensis

Bacillus thuringiensis?Bt?has been used as a biopesticide in agriculture,which is because of its advantages of efficient and broad insecticidal spectrum.The distinguished insecticidal characteristics of this bacterium are due to its ability for producing insecticidal crystal proteins?ICPs?during sporulation.The expression and regulation of B.thuringiensis cry gene by P19?P20?ORF1 and ORF2 were also studied as well.However the effect of groel?groes on the transcription and expression of cry gene is still not clear.In this paper,the expression of groel?groes of Bacillus thuringiensis strain 4.0718 at four phases-middle vegetative phase?T1?,early sporulation phase?T2?,middle sporulation phase?T3?and late sporulation phase?T4?was identified by immunoblotting,to find the relationship between GroEL?GroES and parasporal crystal protein.We constructed the prokaryotic expression vector pET28a-GroEL and pET28a-GroES,GroEL and GroES fusion protein successfully expressed in E.coli BL21?DE3?.The anti-GroEL and anti-GroES antibody acquired by immunizing rabbit with purified recombinant-GroEL and recombinant-GroES.Western blotting result revealed that both GroEL?60Kda?and GroES?10Kda?were all detected throughout the entire fermentation process,the expression of groel and groes were significantly upregulated in the T2 phase.therefore,the expression abundance of GroEL and GroES protein were significantly lower than the 30s ribosomal protein.In this study,the mutant strain named ?groel which knockout the groel gene of KCTF12 strain of Bacillus thuringiensis species has been constructed by the method of homologous recombination technology.On the side of phenotype,the cracking time appears delayed longly owing to lacking of groel gene,likewise the size of insecticidal crystal proteins reduce seriously rather than the original strain.In this work,we used the engineering strain XBU001?1Ac-GFP?as the object,observed GroEL,GroES and Cry proteins in samples collected at in consequent time point with confocal laser scanning microscopy.The results showed that green fluorescence of Cry proteins and red fluorescence of GroEL and GroES overlapped,during 10-25h of fermentation process,intensity of red fluorescence was enhanced with green fluorescence.Then intensity of green fluorescent reached a peak,and red fluorescent reduced during 25-35h.when red fluorescent could not detected,green fluorescent remained stable during 35-55h,These results above indicated that GroEL and GroES protein may be directly or indirectly interacted to insecticidal crystal proteins at early sporulation phase.GroEL and GroES protein were analyzed by bioinformatics tools-String 9.1,we found out GroEL could interact to GroS?DnaK?Tu?BT9727 2551?BT9727 4829?HrcA?FtsH?GrpE and SecA1 proteins,and GroES could interact to GroL?DnaK?FtsH?AcaP?BT9727₀237?RpsM?GrpE?BT9727₄313?GuaA and NrdR proteins.This is the first use of immunofluorescence technology to research relationship between GroEL?GroES and crystal protein of Bacillus thuringiensis from different periods,laying an important theoretical foundation for the study of formation of its insecticidal crystal proteins and providing valuable experiment date to the interactions between the various metabolites.