Characterization of a New Highly Toxic Isolate of Bacillus thuringiensis from the Diapausing Larvae of Silkworm and Identification of Cry1A 22 Gene
1. College of Life and Technology Science, Guangxi University, Nanning, 530004, China
2. Hainan Provincial Key Lab for Crop Molecular Breeding, Sanya, 572025, China
3. Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, China
4. Jiaxing Silkworm Farm, Jiaxing, 314000, China
* Those authors contributed equally
Bt Research, 2010, Vol. 1, No. 1 doi: 10.5376/bt.2010.01.0002
Received: 15 Aug., 2010 Accepted: 19 Sep., 2010 Published: 28 Dec., 2010
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Preferred citation for this article:
Xie et al., 2010, Characterization of a New Highly Toxic Isolate of Bacillus thuringiensis from the Diapausing Larvae of Silkworm and Identification of cry1A 22 Gene, Bt Research (online), Vol.1, No.2 (DOI: 10.5376/bt.2010.01.0002)
We have isolated 218 Bacillus isolates from the dissected guts of 100 diapausing larvae of the silkworm, Bombyx mori, collected from silkworm farmers in the Hangjiahu area of Zhejiang Province. Six isolates were identified as Bacillus thuringiensis strains. The strain named as W015-1 is highly toxic to the lepidopteran Plutella xylostella and is deposited in the HITAR Bacillus Collections with Accession No 20050509W015.
Strain W015-1 can synthesize bipyramidal crystals during sporulation as under light and scanning electron microscopes. SDS-PAGE analysis showed that the dominant protein has a molecular mass of about 130 kD. The plasmid profile was revealed based on the technology of pulsed field gel electrophoresis (PFGE). W015-1 has a similar in size, number and banding pattern to the reference strain Btk HD73 and is completely different to Btk HD1 and Bti AND508. When we employed RFLP-PCR to identify the genotype of the strains, the results indicated that Bt strain W015-1 has a cry 1A genotype with different enzyme cutting sites compared to the reference strain HD73.
The full coding sequence of the crystal toxin was cloned (GenBank Accession number EU282379) by combining the techniques of PCR-RFLP and inverted PCR and was designated as Cry1Aa22 according to the nomenclature system proposed by Crickmore et al. Sequence analysis revealed that this gene contained an open reading frame of 3 534 nucleotides encoding a protein of 1 178 amino acids residues containing three typical toxin domains, and is highly homologous with then Cry 1Ac family. There are three existing differences with the sequence of known cry 1Ac1, at sites, 233(T/R), 448(M/I) and 1158(K/E).
We ligated the cry1Ac22 into E. coli expression vector pQE30 to construct pQE30-Cry1Ac22 and then the recombinant plasmids were transformed into E. coli M15 to express an inclusion protein
of 133 kD. The inclusion of cry1Ac22 can be hydrolysed to a trypsin-activated form with a molecular weight of about 80 kD with the trypsin treatment. Larvicidal assays of the trypsin-activated form of cry1Ac22 were carried out and demonstrated high insecticidal activity against larvae of Plutella xylostella (LC50: 4.135 ×108 cfu/mL; 95% FL: 3.368~5.122 ×108 cfu/mL), which was much higher than that of model strain HD 73.
W015-1 and the reference strains are dissimilar in plasmid profiles, cry genotypes and crystal proteins. Thus, it is believed that Bt W015-1 could be a potential biopesticide alternative to Btk.
Silkworm; Bacillus thuringiensis; Parasporal crystal toxin; cry genotype; Cry1Ac22; Plasmid profile; Insecticidal activity