Bt Transcription Factor HD73₁186 And HD73₁919 Functional Analysis

Bacillus thuringiensis?Bt?is a very important microbial insecticide,but there are some drawbacks,such as slow insecticidal effect,crystal protein instability after the cleavage of mother cells.Increasing the expression of protein crystals and preventing the cleavage of mother cells can not release crystal proteins and become a solution to effectively increase the insecticidal activity.In the previous study,the key hydrolase CwlC,which affects the cleavage of the mother cells,is controlled by the?^K factor and is positively regulated by GerE.Whether there are other transcription factors regulate the initiation and transcription of cwlC.The purpose of this study was to find the transcription mechanism of the key hydrolytic enzymes of the mother cell lysis.According to the laboratory data analysis,four transcription factors were selected:HD73₁186,HD73₁919,HD73₀806,and HD73₂081.Early use of bioinformatics analysis found that HD73₁186,HD73₁919,HD73₀806,and HD73₂081 are transcription factors.The mutant vectors pRN5101₁186,pRN5101₁919,pRN5101₀806,and pRN5101₂081 were successfully constructed by overlapping PCR methods.HD73₁186,HD73₁919 and HD73₂081 genes in HD73 were successfully knocked out by high-temperature screening of homologous recombination,and the insertion mutants HD??1186?,HD??1919?and HD??2081?were screened out..Determination of?-galactosidase found that transcription factors HD73₁186 and HD73₁919 do not regulate transcription of the cwlC gene Using light microscopy,the mutants HD??1186?,HD??1919?,and HD??2081?were found to be indistinguishable from wild-type HD73,indicating that deletion of the HD73₁186,HD73₁919,and HD73₂081 genes did not affect cleavage of the mother cells.Using bioinformatics analysis,it was found that HD73₁186 may be involved in the regulation of glycerol metabolism.The transcription unit of HD73₁186 gene cluster was found by RT-PCR:HD73₁186 is co-transcribed with HD73₁187,HD73₁188 is co-transcribed with HD73₁189,and HD73₁190 is transcribed separately.The pHT304-18Z expression vector pHTP₁₁₈₆ was successfully constructed and was transformed into wild-type HD73 and mutant HD??1186?,and the?-galactosidase was determined.The results showed that the transcription of HD73₁186 gene was not controlled by the transcription factor HD73₁186.After bioinformatics analysis of the upstream and downstream genes of HD73₁919,it was found that HD73₁919 may be related to the utilization of Fe³⁺.After measuring the growth curve,it was found that the absence of the HD73₁919 gene did not affect the growth of the strain In the previous study,SpoIIID,a binding protein associated with blastocyst cleavage.Thus,the transcriptional regulation of the key hydrolase gene cwlC,which affects the cleavage of mother cells,was thought.The P_cwlC promoter was constructed and ligated into pHT304-18Z expression vector pHTP_cwlC,which was then electroporated into wild-type HD73 and mutant HD??spoIIID?.The activity of the P_cwlC promoter in the HD(pHTP_cwlC)strain was lower than that in the?(pHTP_cwlC)strain by?-galactosidase analysis.The results indicate that deletion of the spoIIID gene can affect the transcription activity of the P_cwlCwlC promoter,cwlC gene Transcription is positively regulated by SpoIIID.At the same time,we built HD73₀806 and HD73₂081.Four insertional mutation vectors were successfully constructed in this study.Among them,the mutant strains HD??1186??HD??1919?and HD??2081?were successfully screened,which laid a good molecular basis.It provides an important theoretical basis for the genetic improvement of Bacillus thuringiensis in the future,and it is expected to construct a new Bt engineering strain.