The Study On Expression And Transcriptional Regulation Of Sip1Ab1 Gene From Bacillus Thuringiensis

Bacillus thuringiensis?Bt?is widely used as microbial pesticides.The main feature of Bt is the formation of spores and parasporal crystals during the stable growth period.The parasporal crystals are mainly composed by insecticidal proteins Cry and Cyt.The insecticidal protein also includes vegetative insecticidal protein Vip and the secreted insecticidal protein Sip.There are two sip genes?sip1Aa1 and sip1Ab1?encoding Sip proteins have been found so far.There are some reports show that Sip proteins have specific insecticidal activities against Coleoptera pests,but the transcriptional regulation mechanisms of sip genes are unknown.This subject is focused on the research of transcription mechanism of sip1Ab1 gene.1.It was determined that the insecticidal proteins in QZL38 strain encoded by cry8 genes and sip1Ab1 gene through mass spectrometry analysis of crystals and genome sequencing.The supernatant of QZL38 strain cultured in LB medium was concentrated and analyzed by SDS-PAGE.The results showed that the secretion of Sip1Ab1 protein reached the peak in the transition period.sip1Ab1 gene and its promoter were constructed on the p HT304 expression vector and transferred into HD73 strain.It was proved that the Sip1Ab1 protein was also expressed in the transition period in the HD73 strain and there was no difference in the expression level with the QZL38 strain.2.The 5?-RACE experiment determined that the transcription start site of the sip1Ab1 gene is G which 35 bases upstream from the start codon?ATG?and the-10?TTATAA?and-35?TAATAT?region of sip1Ab1 promoter are determined.Bioinformatics analysis of the sip1Ab1 promoter revealed that there are two AbrB putative binding sites in the promoter of sip1Ab1 gene.It is speculated that transcription factor AbrB regulates sip1Ab1 gene.HD?abr B?abr B gene mutant strain of HD73 strain?was successfully screened.The truncated promoter P_sip1Ab1-S?-201 to+36?and the full-length promoter P_sip1Ab1-L?-531 to+36?of sip1Ab1 gene were constructed in the p HT304 vectors fusioning lac Z gene.The two vectors were transferred into HD73 and HD?abr B strain respectively.The transcriptional activities of P_sip1Ab1-L and P_sip1Ab1-S were tested in HD73strain and HD73?abr B respectively.It showed that the transcriptional activities of P_sip1Ab1-L and P_sip1Ab1-S were lost in the HD73?abr B strain and the transcriptional activities of P_sip1Ab1-S is two-thirds of P_sip1Ab1-L in HD73 strain.These indicated that the truncated sequence also vital for the transcription of sip1Ab1 gene and AbrB regulates the transcription of sip1Ab1 gene.3.In order to verify AbrB directly regulates the sip1Ab1 gene promoter,the AbrB protein was first purified by nickel column affinity chromatography and the sip1Ab1 gene promoter was also divided into two parts P_sip1Ab1-F?-531 to-202?and P_sip1Ab1-S?-201 to+36?.The results of electrophoretic mobility shift assay?EMSA?proved that the AbrB protein directly binds to Psip1Ab1-F and Psip1Ab1-S?4.The sip1Ab1 promoter was transferred into HD?spo0A,HD?cod Y and HD?sig H strain.It was found that the spore initiation transcription factors Spo0A an d Sig H don't regulate sip1Ab1.The transcription activity of sip1Ab1 promoter in HD?cod Y is lower than in the HD73 wild strain.It indicates that transcriptional factor Cod Y positively regulates sip1Ab1 gene.