Identification, cloning and expression of the insecticidal protein genes from Bacillus thuringiensis isolate Rpp39

Abstract:

[Objective] We characterized strain Rpp39 isolated from the soil of Sichuan, China. A type of cry2Aa gene was cloned and expressed in E. coli. [Methods] We characterized strain Rpp39 by scanning electron microscope, PCR-RFLP identification and SDS-PAGE analysis. We cloned cry2Aa gene by PCR clone. We constructed the recombinant plasmid pET-2Aa, and transformed E.coli.BL21(DE3) to express by IPTG. We determined the toxicity of expressed products to the larvae of Plutella xylostella and Chilo supperssalis through bioassay. [Results] The strain Rpp39 contained three types parasporal crystals, diamond, squareness and round, observed under the scanning electron microscope. SDS-PAGE analysis showed that two kinds of molecular mass of insecticidal crystal proteins, one was about 130kD and the other 60 kDa, were expressed in isolate Rpp39. The strain contained cry1Aa, cry1Ab, cry1Ac, cry1Ia and cry2Aa gene, analyzed by the PCR-RFLP method. One cry2Aa-type gene of Rpp39 was cloned and designated as cry2Aa12 by Bacillus thuringiensis Insecticidal Crystal Proteins Nomenclature Committee. Sequence analysis revealed that this gene contained an open reading frame of 1902 nucleotides encoding a protein of 634 amino acids. Compared with Cry2Aa1 protein, Cry2Aa12 protein has shown as high as 99.7% amino acid homology. We amplified the full open reading frame sequence of the cry2Aa12 gene by use of a pair of PCR primers P3/P4 designed according to its DNA sequence, and the PCR product was inserted into the Nde I / BamH I site of E. coli expression vector pET-30a to obtain the recombinant plasmid pET-2Aa. The result of SDS-PAGE proved that Cry2Aa12 could be expressed as 65 kDa protein in E. coli BL21(DE3) strain induced by IPTG. We found that Cry2Aa12 was highly toxic to the larvae of Plutella xylostella and Chilo supperssalis through bioassay, with LC50 as 5.4 mg/mL and 22.3 mg/mL, respectively. [Conclusion] Strain Rpp39 and cry2Aa12 gene cloned from strain Rpp39 were came from Sichuan ecological condition in China. They could be affluent in the resources of strain and gene. It has important significance to accumulate the resource.

Key words:Bacillus thuringiensis; indentification; cry2Aa12 gene; cloning and expression; insecticidal activity