Insecticidal Activities Of Recombinant CryIAc Protein From Bacillus Thuringiensis And Solid Fermentation Products From Aspergillus Terreus PNR2

In this study, we ascertained the existence of cryⅠAc gene in one Bacillus thuringiensis strain HD-73 by PCR-RFLP. According to sequences of crylAc category in GenBank, two pairs of primers (ORF-1, ORF-2 and COM-1, COM-2) were designed to amplify the ORF and full-length (including promoter, ORF and terminator) of crylAc gene. Sequencing results showed a total length of 4202 bp for full-length gene, and 3537 bp for ORF encoding 1178 amino acids. By analysis using Antheprot 4.3 Software, we preliminarily forecast that the molecular weight of the protein was 133.35 kDa, and the isoelectric (pⅠ) point was 4.825. The result of comparison using Blast showed that the nucleic acid sequence of this gene has homology of 99% with several other published crylAc genes, as well as the amino acid sequence homology of 99.7%.The ORF of cryⅠAc gene was cloned into pMD19-T vector, digested with Bam HI and Sal I, and then ligated to pET-30a (+) vector digested with the same enzymes. The resulting recombinant expression plasmid pETcO was transferred into E. coli BL21 (DE3) for inducible expression using IPTG. Expression products were detected by SDS-PAGE and found to express CryⅠAc protein with the molecular weight of about 130 kDa.We also extract CrylAc protoxin from Bacillus thuringiensis HD-73 by the method of isoelectric precipitation, after seed culturing in NB liquid medium together with fermentation in in the 1/2 NB liquid medium for 72 h. Detected by SDS-PAGE, the protoxin has identical molecular weight to the recombinant protein.Bioassays of recombinant CrylAc were carried out against Spodoptera litura and Plutella xylostella using leaf-soaking method. Results showed high insecticidal activities against both insects. LC50 were 2.334μg/ml and 4.444μg/ml, respectively. Dihydricitrinone and cytochalasin D, two kinds of secondary metabolites, were separated and purified from one Aspergillus tereus strain PNR2 by solid-state fermentation. Bioassays of these two compounds were carried out against Spodoptera litura, Plutella xylostella and Bemisia tabaci using leaf-soaking method. The result showed that corrected morality rates of dihydrocitrinone (200μg/ml) were merely 5.7%(S. litura),3.2%(P. xylostella) and 7.4% (B. tabaci), respectively; Cytochalasin N (200μg/ml) resulted in a morality of 12.5% against B. tabaci, as well as relatively higher toxicities against S. litura and P. xylostella with LC50 of 856.556μg/ml and 98.026μg/ml.