Bacillus thuringiensis subspecies morrisoni strain HD12, whose genome harbors multiple insecticidal protein-encoding genes, includes eight cry genes, as indicated by genome sequencing. This strain produces crystals th...Bacillus thuringiensis subspecies morrisoni strain HD12, whose genome harbors multiple insecticidal protein-encoding genes, includes eight cry genes, as indicated by genome sequencing. This strain produces crystals that are toxic to lepidopteran species. These crystal inclusions were purified by sucrose gradients and sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE）, followed by liquid chromatography-mass spectrometry, and found to contain five proteins （Cry1Da, Cry1Ae, Cry1Bb, Cry1Fb, and CrylJa）. The transcriptional activities of the cry1Da, cry1Ae, cry1Bb, cry1Fb, and cry11Ja promoters indicated that transcription of crylDa is controlled by SigE; however, the other four cry genes were found to be controlled by both SigE and SigK. The activities of the crylJa and crylFb promoters were the strongest among the five genes studied. These promoters were therefore used to direct the expression of the Cry1Ac- and Cry2Ab-encoding genes concurrently in a single strain. Our findings indicate that promoters with the same transcriptional profile may be used to direct the expression of different cry genes in one strain. Our results are expected to be valuable for the construction of strains with efficient expression of multiple cry genes in order to overcome current limitations associated with the application of B. thuringiensis-based insecticides.展开更多
Bacillus thuringiensis (Bt) strain C002 contains crylAa, cry2Ab, crylCa insecticidal crystal genes and an unkown gene cryX, among which crylCa is located in a 6 -9 kb EcoR I fragment of the chromosomal DNA. The total ...Bacillus thuringiensis (Bt) strain C002 contains crylAa, cry2Ab, crylCa insecticidal crystal genes and an unkown gene cryX, among which crylCa is located in a 6 -9 kb EcoR I fragment of the chromosomal DNA. The total DNA and the plasmids DNA libraries of C002 were constructed in Bt-E. coli shuttle plasmid pHT315 by inserting 6 - 9 kb chromosomal and plasmid DNA fragments prepared respectively with EcoR I complete and 5au3A I partial digestion. On the basis of every 50 transformants pooled together from 5-10 tubes, the pools containing about 2 000 transformants from the plasmids DNA library and 400 transformants from the total DNA library were rapidly screened by PCR-RFLP. Clones containing crylAa, cryX, crylCa , and cry2Ab were isolated and named as pHT-1Aa, pHT-X, pHT-1Ca and pHT-2Ab respectively. Restriction analysis indicated that pHT-1Aa, pHT-1Ca and pHT-2Ab had the typical physical map of the homologous cry genes. Furthermore, each plasmid was transferred into Bt acrystalliferous strain cryB- by eletropora-tion. SDS-PAGE result showed that transformant of pHT-1Ca expressed 130 kDa protein and bioassay result proved its high toxicity against Spodotera exigua 1st instar larvae with 100% corrected motality.展开更多
The present study aims to isolate the unknown and known serotypes of Bacilllus thuringiensis (Bt) from natural objects in Kyrgyzstan. A total of 83 Bt strains were isolated from natural substrates, of which 30% were t...The present study aims to isolate the unknown and known serotypes of Bacilllus thuringiensis (Bt) from natural objects in Kyrgyzstan. A total of 83 Bt strains were isolated from natural substrates, of which 30% were taken from the soil and litter samples, 69.7% from dead insects and about 0.3% from slugs. Serological examination revealed that such subspecies as var. thuringiensis (H-1), var. alesti (H-3), var. sotto (H-4a4b) and var. entomocidus (H-6) predominated in the upper horizon of soils in all climatic zones. In the dead insects such species as subsp. thuringiensis, subsp. galleria, subsp. sotto, subsp. kurstaki, subsp. Aizawai and subsp. Entomocidus dominated. A set of Bt strains isolated from insects and soil samples, selected from different ecosystems in Kyrgyzstan was molecular taxonomically characterized using the pycA gene as marker for phylogenetic reconstruction. Within the Bacillus cereus sensu lato species complex, all Kyrgyz isolates were shown to belong to the B. cereus subspecies thuringiensis. Most isolates were assigned to the lineage Bt tolworthi, with two isolates each belonging to the lineages Bt kurstaki and Bt sotto. A high degree of cry gene diversity was demonstrated in the set of Bt isolates, with several gene copies simultaneously present in a single strain;a particularly conspicuous trait was the frequent combination of Lepidopteran-specific cryI with Dipteran-specific cryIV genes in the same Bt isolate.展开更多
基金supported by a grant from the National Natural Science Foundation of China(31530095 and 31300085)
文摘Bacillus thuringiensis subspecies morrisoni strain HD12, whose genome harbors multiple insecticidal protein-encoding genes, includes eight cry genes, as indicated by genome sequencing. This strain produces crystals that are toxic to lepidopteran species. These crystal inclusions were purified by sucrose gradients and sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE）, followed by liquid chromatography-mass spectrometry, and found to contain five proteins （Cry1Da, Cry1Ae, Cry1Bb, Cry1Fb, and CrylJa）. The transcriptional activities of the cry1Da, cry1Ae, cry1Bb, cry1Fb, and cry11Ja promoters indicated that transcription of crylDa is controlled by SigE; however, the other four cry genes were found to be controlled by both SigE and SigK. The activities of the crylJa and crylFb promoters were the strongest among the five genes studied. These promoters were therefore used to direct the expression of the Cry1Ac- and Cry2Ab-encoding genes concurrently in a single strain. Our findings indicate that promoters with the same transcriptional profile may be used to direct the expression of different cry genes in one strain. Our results are expected to be valuable for the construction of strains with efficient expression of multiple cry genes in order to overcome current limitations associated with the application of B. thuringiensis-based insecticides.
文摘Bacillus thuringiensis (Bt) strain C002 contains crylAa, cry2Ab, crylCa insecticidal crystal genes and an unkown gene cryX, among which crylCa is located in a 6 -9 kb EcoR I fragment of the chromosomal DNA. The total DNA and the plasmids DNA libraries of C002 were constructed in Bt-E. coli shuttle plasmid pHT315 by inserting 6 - 9 kb chromosomal and plasmid DNA fragments prepared respectively with EcoR I complete and 5au3A I partial digestion. On the basis of every 50 transformants pooled together from 5-10 tubes, the pools containing about 2 000 transformants from the plasmids DNA library and 400 transformants from the total DNA library were rapidly screened by PCR-RFLP. Clones containing crylAa, cryX, crylCa , and cry2Ab were isolated and named as pHT-1Aa, pHT-X, pHT-1Ca and pHT-2Ab respectively. Restriction analysis indicated that pHT-1Aa, pHT-1Ca and pHT-2Ab had the typical physical map of the homologous cry genes. Furthermore, each plasmid was transferred into Bt acrystalliferous strain cryB- by eletropora-tion. SDS-PAGE result showed that transformant of pHT-1Ca expressed 130 kDa protein and bioassay result proved its high toxicity against Spodotera exigua 1st instar larvae with 100% corrected motality.
文摘The present study aims to isolate the unknown and known serotypes of Bacilllus thuringiensis (Bt) from natural objects in Kyrgyzstan. A total of 83 Bt strains were isolated from natural substrates, of which 30% were taken from the soil and litter samples, 69.7% from dead insects and about 0.3% from slugs. Serological examination revealed that such subspecies as var. thuringiensis (H-1), var. alesti (H-3), var. sotto (H-4a4b) and var. entomocidus (H-6) predominated in the upper horizon of soils in all climatic zones. In the dead insects such species as subsp. thuringiensis, subsp. galleria, subsp. sotto, subsp. kurstaki, subsp. Aizawai and subsp. Entomocidus dominated. A set of Bt strains isolated from insects and soil samples, selected from different ecosystems in Kyrgyzstan was molecular taxonomically characterized using the pycA gene as marker for phylogenetic reconstruction. Within the Bacillus cereus sensu lato species complex, all Kyrgyz isolates were shown to belong to the B. cereus subspecies thuringiensis. Most isolates were assigned to the lineage Bt tolworthi, with two isolates each belonging to the lineages Bt kurstaki and Bt sotto. A high degree of cry gene diversity was demonstrated in the set of Bt isolates, with several gene copies simultaneously present in a single strain;a particularly conspicuous trait was the frequent combination of Lepidopteran-specific cryI with Dipteran-specific cryIV genes in the same Bt isolate.
