| Bacillus thuringiensis is currently the most widely used microbe in biocontrol. Because of the long-time use of Bt formulation, it causes a series of problems, such as resistance and narrow-spectrum of insecticide. The clone of novel Bt gene could broaden the types of Bt insecticidal genes and expand of the target pesticides. Especially, the genes that had insecticidal activity to the more difficult-controlled pests could be genic resources for cultivating insect-resistant transgenic plants. In this paper, 285 Bt strains which had preserved in the laborory were identifided by use of PCR-RFLP. The results show that those strains contain cry1, cry2, cry3, cry4/10, cry8,cry9, cry11, cry21, cry22, cry30, cry32, cry40 and vip3A. Fifty-one isolates harbored cry1 genes, 45 isolates harbored cry2 genes, 97 isolates harbored cry8 genes, 13 isolates harbored cry32 genes, 10 isolates harbored cry4/10 genes, 9 isolates harbored cry30 genes, 5 isolates harbored cry22 genes, 2 isolates harbored cry11 genes and cry3 genes, 1 isolates harbored cry9 genes, 1 isolates harbored cry21 genes, 1 isolates harbored cry40 genes,39 isolates harbored vip3A genes and 106 strains were not identified genotypes. Twelve isolates harbored both cry8C genes and cry8F genes, 5 isolates harbored both cry8E genes and cry8F genes, 5 isolates harbored cry1Aa genes,cry1Ea genes,cry1Be genes,cry1Ah genes,cry1Ai genes,cry2Ab genes and vip3Aa1 genes.SDS-PAGE of parasporal crystal in 285 wild strains assay indicated that isolates containing cry1 genes and cry2 genes mostly expressed 130kDa and 60kDa protein bands and formed bipyramidal crystals, isolates containing cry22 genes expressed 80 kDa protein bands and formed spherical crystals which are bigger than spores, isolates containing cry32 genes expressed 130kDa and 40kDa protein bands and formed bipyramidal crystals, isolates containing cry8 genes mostly expressed 130kDa bands and formed spherical crystals. Strains of which genetypes were not identified expressed various protein bands and formed various crystals.Full-length of cry22 gene were cloned from strain FZ-4. One novel cry22 gene, first discovery inland, cloned from strain FZ-4, had been nominated as cry22Aa3 by International Nomenclature Committee of Bt as GenBank accession numbers EU715020.Four novel genes were found by cloning partial sequence of cry4 genes and cry32 genes. Strain FBG-133 and Strain FZ-2 all contained a new cry32 genes, the two partial sequences of a novel cry32 gene from strain FBG-133 and strain FZ-2 shared 86% and 91% high-comparability with cry32Ca1 gene (AB075461.1) and cry32Aa1 gene(AY008143.1), respectively. The fragment (1563 bp) cloned from strain FBG-25 had 97% sequence high-comparability with the cry54Aa1 genes(EU339367). The fragment (1418 bp) cloned from strain FPT-3 shared no comparability with Bt genes which were known in GenBank. One unknown gene might exist and needed further study.97 Bt strains which contained cry8 genes carried on bioassay to larvae of Holotrichia oblira. 3 strains of them (Hw-11, FBG-74 and FBG-17) had high toxicity towards larvae of Holotrichia oblira with an LC50 of 1.0716 108spores/mL, 3.6109 108spores/mL and 11.5792 108spores/mL, respectively. 44 strains of them had high toxicity to larvae of Anomala corpulenta, and the FPT-38 and Hw-2 had the highest activity which the corrected mortality were 100%. 106 strains which couldn't be identified any cry gene types had low or no activity to Holotrichia oblira, Holotrichia parallela, Anomala corpulenta, Heliothis armigera and Plutella xylostella. We should expand the scope of pests to find their insecticidal activity in the future. |
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