| Bacillus thuringiensis (Bt) strain MC28was isolated from Mu Chuan virgin forest in Sichuan Province of China. It can form spherical parasporal crystals during the sporulation stage and exhibit a broad insecticidal activity against Dipteran and Lepidopteran pests. In previous study,5insecticidal crystal protein genes were identified and cloned using PCR-RFLP (restricted fragment length polymorphisms, RFLP) method. Among these genes, Cry54Aal protein has insecticidal activity against Helicoverpa armigera.(Lepidoptera), Laphygma exigua (Lepidoptera) and Aedes aegypti (Diptera); Cry30Fal protein was toxic to Nilaparvata lugens (Homoptera).For studying genomic structure and evolutionary relationship of Bt strain MC28, whole-genome sequencing of MC28was performed using Illumina GA II sequencing technology. A total of18,324,961filtered paired-end reads were obtained and451-fold coverage of the genome was achieved using an Illumina Solexa GA II. About97.13%of the reads were assembled into113scaffolds using the SOAPdenovo alignment tool. Gaps within and between the scaffolds were confirmed and closed using primer walks, long-distance PCR amplification, and the construction of a fosmid library using an ABI3730capillary sequencer. Finally, the6.68Mb whole-genome sequence of MC28was obtained.The gene prediction of the whole-genome of Bt MC28was performed using the Glimmer3.02software. Then, the blast software was used to compare the predicted protein with the Nr (non-redundant protein sequences) databases. The best alignment results were as the annotation of the corresponding proteins. Synteny of Bt MC28, Bt CT-43, and Bt YBT-020was performed using the perl+SVG tool. The results indicated that Bt MC28, Bt CT-43, and Bt YBT-020had similar genomic structure and significant genomic synteny. Both the MEGA and the Splits Tree softwares were used to structure phylogenetic tree of Bt MC28and other relatived strains. These results all showed that there was further evolutionary distance between Bt MC28and other Bt strains. But Bt MC28, Be Rock3-28, Be Rock4-18, Be Rock1-3, and Be Rock3-29were in a in a separate branch.The6.68Mb genome of MC28is found to contain8replicons:a circular chromosome (5,414,461bp) encoding5,279predicted open reading frames (ORFs), and7circular plasmids: pMC8, pMC54, pMC95, pMC183, pMC189, pMC319and pMC429, These plasmids contain a total of1,278predicted ORFs (Table1). The G+C content of the chromosome is35.41%, and those of the plasmids range from32.11%to34.78%(Table1). The MC28genome encodes74tRNA and45rRNA operons. Based on Bt MC28genome, we found three plasmids which all contain insecticidal crystal protein genes from Bt strain MC28, and other six novle insecticidal crystal protein genes (cry54Ab1, cry68Aa1, cry69Aa1, cry69Aa2, cry70Bal, and cytlDa1) also were found from these plasmids. So far, eleven insecticidal crystal protein genes have been found from Bt strain MC28. Plasmid pMC189is found to harbor seven insecticidal crystal genes:cry30Fal, cry53Ab1, cry54Aa1, cry54Ab1, cry68Aa1, cyt1Da1, and cyt2Aa3. Plasmid pMC95is found to harbor three cry genes:cry4Cc1, cry69Aa1, and cry70Ba1. Plasmid pMC183is found to contain only one cry gene, cry69Aa2whose encoding protein is96.2%identical with Cry69Aal protein.Length of cry68Aa1, cry70Ba1, and cytlDal gene is2,511bp,2,427bp and1,527bp, respectively. In order to study insecticidal activity of these genes, these genes were amplified by PCR and inserted into prokaryotic expression vector pET-28a, respectively. Then, recombinant plasmids were transferred into E. coli BL21(DE3) pLysS and expressed by induction of IPTG. The molecular weight of expressed products of cry68Aa1, cry70Ba1, and cytlDal were95kDa,90kDa, and60kDa, respectively. These were the same as deduced molecular mass of Cry68Aal, Cry70Ba1, and CytlDal, respectively. Results of insect toxicity assay indicated that both Cry68Aal and Cry70Bal were toxic to L. exigua (Lepidoptera) with LC50as20.16μg/ml (95%confidence limit16.34-35.72μ/ml) and30.22μg/ml (95%confidence limit27.15-36.53μg/ml), but were no toxic to H. armigera (Lepidoptera) and A. aegypti (Diptera). The CytlDal protein had no any insecticidal activity against these insects.The cry69Aa1gene (3,651bp) encoded1,216amino acids. It had a deduced molecular mass of137.1kDa and39%identity with Cry4Ba protein as indicated by amino acid homology. Sequence analysis indicated that Cry69Aal and other known large Cry proteins contain eight conserved blocks. So Cry69Aal is a novel member of large Cry protein. For studying insecticidal activity of Cry69Aa1protein, the full-length sequence of cry69Aa1gene was inserted into a shuttle vector, pSTK, and expressed in a crystalliferous mutant Bt subsp. kurstaki HD73-. The transformant was examined with scanning electron microscope (SEM). The result showed that when cry69Aa1gene was expressed in the crystalliferous mutant Bt HD73-, it could form a large number of spherical parasporal crystals. The bioassay results inicated that spore-crystal complex of the transformants was highly toxic to the larvae of Culex quinquefasciatus (Diptera)(LCso=23.7μug/ml,95%confidence:20.5-26.4μg/ml).The cry54Ab1operon is composed of two ORFs oriented in the same direction. The first ORF, a cry54A-like gene, encoded743amino acids and it had a deduced molecular mass of84.5kDa. Based on encoded amino acid sequence homology, this cry gene was classified as cry54Ab1. The second ORF, located downstream of cry54Ab1, was separated from the cry54Ab1gene by a67bp untranslated gap. It is here called orf2. The orf2gene corresponded to a polypeptide of503amino acids and it had a deduced molecular mass of58.2kDa. In order to study insecticidal activity of cry54Ab1operon, the cry54Ab1, orf2, and the whole cry54Ab1operon were used to construct recombinant plasmid using shuttle vector pSTK, respectively. These recombinant plasmids were then expressed in the crystalliferous mutant Bt HD73-. The transformant was examined with SEM. The result showed that when the whole cry54Ab1operon were expressed in the crystalliferous mutant Bt HD73-, it could form a large number of spherical parasporal crystals. However, expression of cry54Abl or orf2alone in the crystalliferous mutant Bt HD73-did not produce any parasporal crystals. SDS-PAGE analysis indicated that when the whole cry54Ab1operon were expressed in the crystalliferous mutant Bt HD73-, it could produce a protein of approximately80kDa Cry54Ab1and another protein of approximately60kDa ORF2. However, expression of cry54Ab1or orf2alone in the crystalliferous mutant Bt HD73-, less expressed products were observed. The orf2gene even was not expressed in the crystalliferous mutant Bt HD73-. The bioassay results showed that the transformants expressing either the whole cry54Ab1operon or the cry54Abl gene alone showed larvicidal activity against C. quinquefasciatus (Diptera). However, transformants contained recombinant plasmid pSTK-orf2showed no larvicidal activity against C. quinquefasciatus. Transformants expressing the cry54Ab1gene alone had less than one-tenth the insecticidal activity of transformants expressing the whole cry54Ab1operon. Thus, ORF2protein can not only contribute to formation of parasporal crystals of Cry54Abl protein but also enhance the insecticidal activity of Cry54Ab1. |
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