Effect Of Sporulation Genes On The Expression Of Cry Genes From Bacillus Thuringiensis G03

Bacillus thuringiensis is the most widely used as insecticidal micriobe for its high toxin, safety and the other merits. Comparing with the other Bacillus species, Bt could also procude crystal composing of insecticidal proteins. Sporulation genes of B.subtilis, as a kind of mode strain of bacillus, is clearer than the other Bacillus species. The study on Insecticidal Crystal Proteins(ICPs) is mainly focused on the expression and regulation of cry gene encoding ICPs. But the research about the relation between production of ICPs and sporulation genes is less. We have primarily studied the relationship of sporulation genes and the regulation of ICPs genes' expression.We had constructed and screened a B. thuringiensis G03 Mini-Tn10 insertion mutant library for loss of spore and crystal. One clone named G03(spoâ…¢AE)::Mini-Tn10 was isolated, which locus inserted is in the front of spoâ…¢AE. The Mini-Tn10 has only one insertion locus on the chrosome DNA confirmed by southern blotting. The production of ICPs was severely reduced owing to the inactivation of spoâ…¢AE gene.The transcription of cry genes encoding ICPs is regulated by sigmaE and sigmaK, in which the sigmaE plays a primary role during this process. So the reduction of sigmaE activity may result from the inactivaton of spoâ…¢AE gene. In the network of sporulation, the transcription of spoâ…¢AE gene is controlled by the sigmaE which is at the upstream of spoâ…¢AE gene. And the Spoâ…¢AE protein is essential for the activation of sigmaK which is at the downstream of network. There have no reports about the Spoâ…¢AE affecting the activity of sigmaE. In order to explore the relationship between the sporulation and the production of ICPs, more relative work had been done below.The plasmid pRAE was firstly constrcuted for knock-out of spoâ…¢AE gene, then introduced into G03 strain. The relative mutant strain G03(spoâ…¢AEâ–³) could not form spore and crystal. Besides, a little insecticidal protein was produced by SDS-PAGE analysis. The promoter of cry1Aa was cloned from Bt G03 and the plasmid pHTcry1Aa containing cry1Aa'-lacZ was constructed. The analysis of transcription suggested that the activity of cry1Aa gene was severely reduced both in G03(spoâ…¢AEâ–³) and in G03(spoâ…¢AE)::Mini-Tn10 strains. Then the promoter of spoâ…¡D gene was cloned which was controlled by sigmaE directedly. The plasmid pHTspoâ…¡D was constructed contianning spoâ…¡D'-'lacZ.The transcription suggested that the activity of spoâ…¡D gene also severely decreased in G03(spoâ…¢AEâ–³) and G03(spoâ…¢AE)::Mini-Tn10 strains, and this also suggested that the reduction of sigmaE activity was resulted from the inactivation of spoâ…¢AE gene. In a word, the spoâ…¢AE gene mutant strain lost the ability of production of spore and ICPs due to the inactivation of spoâ…¢AE gene that is essential to keep high activity of the sigmaE. During the sporulation, the spoâ…¢AE gene regulated the activity of sigmaE through positive feedback. The deletion of spoâ…¢AE gene resluted in reduction of sigmaE activity and then inhibited the activity of cry1Aa gene.In order to know the effect of upstream gene of spoâ…¢AE gene on the activity of sigmaE, we studied the funtion of the spoâ…£F operon, which activates the sigmaK directly, in production of spore and ICPs. The plasmid pRIVF was constructed and introduced into G03 strain. The spoâ…£F operon was deleted by homologous recombination and the mutant strain G03(spoâ…£Fâ–³) was obtained. This muant strain also lost the ability of sporulation and production of crystal. At the same time, the production of ICPs is severely decreased by SDS-PAGE analysis. The complementary strain was constructed to identify the function of spoâ…£F operon, and the ability of production spore and ICPs was partially complemented. Then plasmids pHTcry1Aa and pHTspoâ…¡D were introduced into muant strain G03(spoâ…£Fâ–³) respectively. Results indicated that the activity of cry1Aa and spoâ…¡D genes was also decreased. So the activity of sigmaE was decreased due to deletion of spoâ…£F operon and resulted in the reduction of expression of cry1Aa gene. Furthemore, the analysis of SDS-PAGE suggested that the spoâ…£F operon has less effect on the activity of sigmaE than spoâ…¢AE gene does.