| Bacillus thuringiensis is the most widely used as insecticidal microbe for its high toxin, safety and the other merits. Comparing with the other Bacillus species, Bt could also procude crystal composed of insecticidal proteins. The study on insecticidal crystal proteins (ICPs) is mainly focused on the expression and regulation of cry gene encoding ICPs. Production of ICPs is ralatetd to sporulating-specific genes.SigE and SigK, encoded by sigE and sigK gene, respectively, are important sigma factors in the sporulation process. Many known genes and operons are controlled by these sporulation-specific factors, as well as most of cry genes in Bacillus thuringiensis.The transcription of spoIIIAE gene is regulated by SigE, whose product, SpoⅢAE, is necessary for activating SigK factor. Research showed that the disruption of spoIIIAE gene disables Bt to sporulate nor crystallize.In this study, part of sigE and sigK gene is based on commercial strain Bacillus thuringiensis subsp. kurstaki HD-73. Plasmids pMAD and pRNK were firstly constrcucted for deleting sigE and sigK gene, introduced into HD-73. Then sigE and sigK deletion mutants HD-73 (ΔsigE) and HD-73 (ΔsigK) were constructed by homologous recombination. The results showed that the expression of insecticidal crystal proteins was severely reduced in these mutants, and growth speed of the mutants was affected intensively. The lacZ gene was fused with the promoter of the cry1Aa gene, and expressed in HD-73 (ΔsigE), HD-73 (ΔsigK) and HD-73 acrystalliferous mutant respectively. The activity ofβ-galactosidase expressed in HD-73 (ΔsigE) and HD-73 (ΔsigK) mutant was much lower than that in the HD-73 acrystalliferous mutant. Gene spoⅡD was controlled by sigE directedly. The plasmid pHTspoⅡD-lacZ, containing spoⅡD-lacZ were expressed in HD-73 (ΔsigE), HD-73 (ΔsigK) and HD-73 wild strains. The transcription suggested that the activity of spoⅡD gene also severely decreased in HD-73 (ΔsigK), and this suggested that the reduction of spoⅡD activity was resulted from the inactivation of sigK gene. Then the promoter of spoIIG, was cloned together with lacZ, named pHTsigE-lacZ. The transcription suggested that the activity of sigE gene also severely decreased in HD-73 (ΔsigK), and this also suggested that the reduction of SigE activity was resulted from the inactivation of sigK gene. For mutant HD-73 (ΔsigE), the ability of sporulation and crystallization of Cry protein was normally complemented by the expression of spoIIG operon, which contained the sigE gene after transformation with recombinant plasmid, and growth speed of the complement strain also recovered. Up to now, we have not got the sigK recovered stains.All the results indicated that for the B. thuringiensis subsp. kurstaki strain, sigE and sigK gene are essential to sporulate and crystallize. And there must be complex regulation system between sigE and sigK. Part of research on spoⅢAE gene is based on strain Bacillus thuringiensis G03. Inactivation of spoⅢAE gene reduced the transcription of sigE.β-galactosidase activity analysis indicated that the transcription of sigE gene in G03 (△spoⅢA E) mutant decreased seriousely. In Bacillus subtilis 168, deletion of spoⅢAE gene did not influence either transcription nor activity of SigE. The influence of spoⅢAE gene on activity of SigE differs in Bt and Bs.sigE,sigK and spoⅢAE genes play important roles during sporulation process. sigE and sigK encode two important sigma factors, SigE and SigK, which regulate handreds of genes. Acquirement of HD-73 (ΔsigE) and HD-73 (ΔsigK) mutants will redound to understanding mechanism of Cry protein expression and regulation, crystallization and its relationship with sporulation. It is important to explore different fuctions of genes in Bt and Bs.
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