Insecticidal protein genes from Bacillus thuringiensis are currently the most widely used insect-resistant genes. They have been transferred to many crops for breeding and production. Among them, cotton, maize, potato and other insect-resistant crops are commercialized, creating considerable economic benefit. In this review, we summarized advances in identifying functional genes and transgenic crops for insect resistance, compared different strategies for enhancing vigor of insecticidal protein and utilizing gene stacking as well as listing valuable groups of stacked genes. In addition, the methods for multiple gene transformation was discussed.
Animals ; Bacterial Proteins ; genetics ; Crops, Agricultural ; genetics ; Endotoxins ; genetics ; Hemolysin Proteins ; genetics ; Insecta ; Plants, Genetically Modified

Actinobacillus pleuropneumoniae ; drug effects ; genetics ; Animals ; Bacterial Proteins ; genetics ; Blotting, Western ; Drug Resistance, Bacterial ; genetics ; Green Fluorescent Proteins ; genetics ; Hemolysin Proteins ; genetics ; Hemolysis ; Mice ; Mutation ; Polymerase Chain Reaction

Sorghum (Sorghum bicolor L.) was one of the most important crops in the world next to wheat, rice, maize, soybean and barley. Using the callus derived from immature inflorescence as the recipients, we efficiently transformed sorghum varieties 115, ICS21B and 5-27 with the insecticidal Bacillus thuringiensis (Bt) cry1Ab gene carried in the T-DNA of binary vectors which contained hygromycin resistance gene and gus gene via Agrobacterium tumefaciens. After gradient selection with hygromycin, a total of 21 independent transgenic plant lines, 52 transgenic plants were regenerated, and the average stably transformation efficiency was 1.9%. The integration and transcription of cry1Ab gene in transgenic sorghum was confirmed by PCR analysis, Southern blotting and RT-PCR analysis. The Bt proteins were expressed in most transgenic plants with different level from plant to plant by Western blotting and ELISA assay. According to insect bioassay in laboratory, the transgenic plants with a relatively high level of Bt gene expression displayed insect-resistance to pink rice borer (Sesamina inferens).
Agrobacterium tumefaciens ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Endotoxins ; genetics ; metabolism ; Hemolysin Proteins ; genetics ; metabolism ; Pest Control, Biological ; Plants, Genetically Modified ; genetics ; Sorghum ; genetics ; Transformation, Genetic

The alpha-hemolysin protein of Staphylococcus aureus, which was expressed in Escherichia coli BL21 (DE3) with recombinant pET32a(+)-alpha-HL plasmid, was purified with gel filtration chromatography (GFC) and Ni-NTA spin columns. The quality and biological characteristic were compared. First, the purified products were analyzed with SDS-PAGE, and the expected protein band was with a molecular mass of 53 kD. Second, protein concentration was determined by the method of Bradford, and the median hemolytic dose potency (HD50) was finally analyzed with rabbit erythrocyte. The protein purified with GFC was 0.337 mg/mL, its hemolysis activity was 1519 HU/mg, and hemolysin yield was 14.04%. Meanwhile, the protein purified with the Ni-NTA Spin Columns was 0.35 mg/mL, its hemolysis activity was 1463 HU/mg, and hemolysin yield was 17.5%, respectively. The results showed that there is no significant difference in the quality, hemolysis activity and yield of the recombinant proteins purified with Ni-NTA spin columns and GFC.
Chromatography, Gel ; methods ; Escherichia coli ; genetics ; metabolism ; Hemolysin Proteins ; genetics ; isolation & purification ; Nitrilotriacetic Acid ; analogs & derivatives ; Organometallic Compounds ; Recombinant Proteins ; genetics ; isolation & purification ; Staphylococcus aureus ; genetics ; metabolism

OBJECTIVETo analyze factors related to the virulence associated genes of Leptospires.

METHODSTwelve putative virulence associated genes were detected by polymerase chain reaction (PCR) method in 38 reference strains, 81 field strains of Leptospira interrogans isolated from patients or animals, and 12 avirulent strains of Leptospira biflexa.

RESULTSThese putative virulent genes were widely distributed among the strains of Leptospira interrogans, but only few of them were detected in Leptospira biflexa. Gene lipL32 was detected in all strains of Leptospira interrogans. Distribution of gene lipL36 was varied significantly with detected rates from 0 to 90.91%. Gene la1608 had a positive rate of 87.50% for strains of serogroup Icterohaemorrhagiae, but was only detected in few strains of other serogroups with a range from 0 to 25.00%. Rate of detection on gene sphA was 17.65% in Leptospira interrogans, and was absent in serovar hardjo reference strain.

CONCLUSIONResults indicated that these genes might be of importance for the virulence and pathogenicity of Leptospira interrogans, while gene lipL32 might be one of the common antigens. Gene lipL36 might be involved in serogroup specificity with genetic diversity, but gene la1608 was as one of the genes with specificity for serogroup Icterohaemorrhagiae. However, serovar hadjo might hold quite different genetic characteristics when compared with the other serovars of Leptospires.

Bacterial Outer Membrane Proteins ; genetics ; Bacterial Proteins ; genetics ; Carbohydrate Dehydrogenases ; genetics ; Flagellin ; genetics ; Genes, Bacterial ; genetics ; Hemolysin Proteins ; genetics ; Leptospira ; genetics ; pathogenicity ; Lipoproteins ; genetics ; Polymerase Chain Reaction ; Virulence ; genetics ; Virulence Factors ; genetics

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.
Animals ; Bacillus thuringiensis/genetics* ; Bacillus thuringiensis Toxins ; Bacterial Proteins/metabolism* ; Endotoxins/metabolism* ; Hemolysin Proteins/metabolism* ; Insecta/metabolism* ; Insecticide Resistance/genetics* ; Insecticides/pharmacology* ; Pest Control, Biological

cry1Ah1, one of holo-type cry genes, cloned in this laboratory from Bacillus thuringiensis strain has been patented in China, and it encoded a protein with strong insecticidal activity against certain lepidopteran insect pests, such as Chilo suppressalis. cry1Ah1 gene is exhibiting good application prospects. In order to improve the expression level of cry1Ah1 gene in rice, and investigate the effect of codon usage preference of gene expression, we designed five different optimized schemes for cry1Ah1 insecticidal critical fragment in accordance with bias of rice codon, to improve G+C content, removed the shear signal and unstable factors. Optimized cry1Ah1 genes were transformed into Escherichia coli Rosetta (DE3) respectively, and 65 kDa polypeptides was expressed normally in inclusion body separately. All of these expressed polypeptides showed insecticidal activity against 2nd-instar larvae of Plutella xylostella and neonate of Chilo suppressalis. After transformation with modified cry1Ah1 genes into Var nippobare, the transgenic rice seedlings were detected by PCR, the positive rate containing target gene was more than 87%. Afterwards, the results of real-time RT-PCR and ELISA assay indicated that the highest expression level of five modified cry1Ah1 genes was that using the highest frequent codons. Average expression amount of Cry1Ah1 polypeptides was 0.104% of total soluble proteins from the positive transgenic rice.
Animals ; Bacillus thuringiensis ; genetics ; metabolism ; Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; genetics ; Endotoxins ; biosynthesis ; genetics ; Hemolysin Proteins ; biosynthesis ; genetics ; Insecticides ; Lepidoptera ; Oryza ; genetics ; Pest Control, Biological ; methods ; Plants, Genetically Modified ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

N-acyl-homoserine lactones (AHLs), are widely conserved signal molecules present in quorum-sensing systems of many Gram-negative bacteria. AHLs molecules mediate the expression of virulence genes of a range of bacterial pathogens. Recently, it has been reported that AiiA protein, which widely exists in Bacillus species, can inactivate the AHLs by hydrolyzing the lactone bond of AHLs, thus attenuate the diseases caused by the expression of virulence genes of bacterial pathogens. Bacillus thuringiensis, a type of Gram-positive bacteria, has been used extensively as a microbial insecticide in the last few decades. However, most of important insecticidal B. thuringiensis strains have not been exploited for bacterial disease control because they usually do not produce antibiotics that are effective against bacteria and fungi. The discovery of AiiA protein in B. thuringiensis shows the application potential of B. thuringiensis on biocontrol against bacterial diseases. In this study, in order to construct the B. thuringiensis recombinant strain that has high expression of AiiA protein, the promoter of insecticidal crystal protein coding gene cry3Aa of B. thuringiensis was selected. The promoter of gene cry3Aa is a non-sporulation promoter, it promotes the transcription earlier and longer than the promoters of other cry genes. The promoter of AiiA protein coding gene aiiA was replaced with the promoter of gene cry3Aa by overlapping PCR, resulting fusion gene pro3A-aiiA. The gene pro3A-aiiA was inserted into shuttle vector pHT304 at site BamH I / Sph I , resulting recombinant plasmid pBMB686. The plasmid pBMB686 was introduced into B. thuringiensis acrystalliferous strain BMB171, the resulting strain BMB686 had a higher and more stable expression level of protein AiiA comparing with the parental strain BMB171. Furthermore, the strain BMB686 exhibited stronger ability of AHLs inactivation and much more effective restraint to the potato's soft rot disease caused by Erwinia carotovora than those of the parental strain BMB171. From these results, it was concluded that the B. thuringiensis strain harvesting the fusion gene pro3A-aiiA may be utilized in the future to control bacterial diseases which are mediated by the AHL quorum-sensing signals.
Acyl-Butyrolactones ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Daucus carota ; microbiology ; Endotoxins ; genetics ; metabolism ; Hemolysin Proteins ; genetics ; metabolism ; Metalloendopeptidases ; genetics ; metabolism ; Models, Genetic ; Pectobacterium carotovorum ; pathogenicity ; Plant Diseases ; microbiology ; prevention & control ; Promoter Regions, Genetic ; genetics ; Recombinant Proteins ; genetics ; metabolism

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.
Bacillus thuringiensis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Endotoxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genes, Bacterial ; Hemolysin Proteins ; biosynthesis ; genetics ; Pest Control, Biological ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.
Insecticides/metabolism* ; Bacillus thuringiensis ; Endotoxins/pharmacology* ; Bacillus thuringiensis Toxins/metabolism* ; Hemolysin Proteins/pharmacology* ; Bacterial Proteins/chemistry* ; Plants, Genetically Modified/genetics* ; Pest Control, Biological