RESUMO
The spoIVF operon exists in Bacillus universally. Two proteins encoded by the spoIVF operon are essential for the sporulation of Bacillus subtilis. In this study, a spoIVF operon disruption mutant G03 (spoIVF-), in which the spoIVF operon was deleted, was constructed by homologous recombination. The result showed that the mutant strain lost the ability of sporulation. At the same time, the expression of Insecticidal Crystal Protein (ICP) was severely reduced in G03 (spoIVF-) mutant strain and resulted in no crystals. The lacZ gene was fused with the promoter of the cry1Aa gene and expressed in mutant strain G03 (spoIVF-) and G03 wild strain. The activity of beta-galactosidase much lower in mutant G03 (spoIVF-) strain than in the wild-type strain. This further suggested that the activity of sigmaE and sigmaK factors was affected in mutant G03 (spoIVF-) strain. The ability of sporulation and production of Insecticide Crystal Protein was complemented by the expression of spoIVF operon through the vector of pSTK in the mutant strain. In all, The spoIVF operon is essential for the sporulation and the expression of cry gene controlled by sigmaE and sigmaK factors.
Assuntos
Bacillus thuringiensis/fisiologia , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Deleção de Genes , Proteínas Hemolisinas/metabolismo , Óperon , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas/genética , Esporos Bacterianos/genética , Esporos Bacterianos/fisiologiaRESUMO
The current status of production and application of biopesticides for pest control in China is briefly reviewed, with a focus on research advances in microbial control with Bacillus thuringiensis (Bt). These have led to improvements in Bt production, exploitation of Bt gene resources, and development of engineered Bt insecticides and transgenic Bt crops that have expanded host ranges and increased efficacy against target pests. Both conventional and biotechnology approaches need to be employed to achieve further progress in discovery, production technology, formulation processing, development of quality standards and recommended use patterns.
Assuntos
Bacillus thuringiensis/fisiologia , Pesquisa Biomédica , Biotecnologia , Insetos/microbiologia , Controle Biológico de Vetores/tendências , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Toxinas Bacterianas , China , Endotoxinas , Alimentos Geneticamente Modificados , Proteínas Hemolisinas , Inseticidas , Controle Biológico de Vetores/estatística & dados numéricos , Plantas Geneticamente ModificadasRESUMO
The full length cry2Ab gene was cloned by PCR-RFLP method from Bt strain B-Pr-88, which was isolated in China with high toxicity to the Lepidopteran insect pests. Nucleic acid sequence analysis showed that this gene was 1902 base pairs encoding 633 amino acids. This cry gene was named cry2Ab4 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The full open reading frame sequence of the cry2Ab4 gene was amplified with a pair of PCR primers L2ab5/L2ab3 designed according to its DNA sequence,and inserted into the BamH I /EcoR I sites of E. coli expression vector pET21b to obtain the recombinant plasmid pET-2Ab4. The result of SDS-PAGE proved that Cry2Ab4 could be expressed as a 60 kD protein in E. coli BL21 (DE3)strain induced by IPTG. Bioassay of the expressed product of the cry2Ab4 gene showed that Cry2Ab4 was highly toxic to the larvae of Helicoverpa armigera and Leguminivora glycinivorella, moderately active to the larvae of Plutella xylostella and Chilo suppressalis, but not insecticidal to the larvae of Spodotera exigua and Ostrinia furnacalis. Our result indicated that cry2Ab4 gene could be used as a novel gene for generation of transgenic plants and engineered microorganism.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/biossíntese , Endotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Proteínas Recombinantes/biossíntese , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Clonagem Molecular , Endotoxinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Proteínas Hemolisinas/genética , Controle Biológico de Vetores , Proteínas Recombinantes/genética , Análise de Sequência de DNARESUMO
The full length sequence of the promoter and gfp gene were obtained respectively by PCR with two pairs unique primers PxyF/R and primers gfpF/R, which were designed according to the gfp gene and promoter sequence of xylase operon from Bacillus subtilis 168, and the DNA template plasmids pHT315-xyIR and pGFPuv. Furthermore, the fused translational expression cassette PxylR-gfp was constructed using overlapping PCR technique with the primers pair PxyF/gfpR and the mixture of above PCR production. After being digested by Kpn I and Sph I , PxylR-gfp expression cassette was inserted into E. coli-B. thuringiensis shuttle vecter pHT315 and E. coli-B. subtilis shuttle vecter pRP22, and the resulted recombinant plasmids were named as pGFP315 and pGFP22 respectively. Both recombinant plasmids were transferred into B. subtilis lab strain 168 and the resulted transformants are bright green performance under 365 nm UV light. However, only pGFP22 can be introduced into the natural strain B916. The transformants containing pGFP22 have bright green performance under 365 nm UV light and was named B916-gfp. Antifungal activities testing results proved that there is no obvious difference between B916 and the engineered strains B916-gfp. Research results also showed that the stability of B916-gfp was 94% after growth about 175 generations at 37 degrees C, and the losing rate of plasmid was less than 3.5 x 10(-4) per generation.