Identification Of Cry Genes From Bacillus Thuringiensis In Mengding Mountain Of Sichuan, And The Cloning And Expression Of Novel Holetype Cry Gene

Bacillus thuringiensis (Bt), as a safeties to human and animals, pollution-free microbial insecticides, plays a very important role in pest prevention and cure of agriculture, forestry and sanitary insect pest. It becomes a most successful development and apply of microbial pesticide at abroad and home. So to futher dig abundant resource of Bt in our country, screen high effective and broad-spectrum insecticidal activity of Bt strain, analyse their cry gene types, isolate and clone novel pesticidal genes will have important meanings in theories and practices for developping microbial insecticides, constructing high toxicity and broadspectrum engineering strains and breeding insect resistant transgenic plants. This study described a systematic study of Bt resources from the soil in MengDing mountain of Yaan city in Sichuan province. A novel pesticidal protein genes was cloned and expressed. The concrete results are as follows:1. According to different altitude and environment, we collected 285 soil samples and isolated 78 Bt strains from these soil samples by using sodium acetate-antibiotics method. The average, rate was 19%. Observed by electron microscope, These Bt strains produced different patterns of parasporal crystals, such as long bipyramid, short bipyramid, cuboidal, round and abnormity, which showed the diversity of Bt resources in Mengding mountain. The cry gene-types of 78 Bt isolates were identified by using PCR-RFLP system.68 isolates harbored cry1 genes,28 isolates harbored cry2 genes,2 isolates harbored cry3 genes,2 isolates harbored cry9 genes,3 isolates harbored cry4/10 genes and 3 isolates harbored cry3 genes. Using SDS-PAGE method to analyse insecticidal crystal proteins of these Bt strains, the result showed that four different kinds of insecticidal crystal proteins were expressed, whose molecular weight were between 40 kDa and 130 kDa. Some Bt strains didn't produce any PCR products in PCR-RFLP analysis. But SDS-PAGE assay indicated that these isolates produced crystal proteins, which suggested that they may contain potentially novel Cry toxin genes.2.28 Bt strains which contained cry2 genes were identified by using general primers of cry2 gene. Then restriction enzyme DdeⅠwas used to digest these PCR productions for identification of cry gene-types. The results showed that the size of of the Bt strain JF19-2 amplified fragment was different from enzyme cut pieces of known cry2 genes, which indicated that JF19-2 contained a novel cry 2 holetype gene.3. According to amplified fragment of JF19-2 by using general primers of cry2 genes, a pair of specific primers SP1 and SP2 was designed, in upstream and downstream of amplified fragment. Unknown sequence of the novel cry2 gene was amplified by Tail-PCR method. Then amplified sequences was cloned and sequenced. Sequencing result was matched with known sequence of the novel cry2 gene. A 3,285 bp nucleotide sequence was obtained. There was a 1,905 bp ORF in the obtained sequence, which encoded a protein of 635 amino acids with a predicted molecular mass of 70.8 kDa and isoelectric point of 4.952.This protein was an alkalescent protein, which mainly contained four kinds of Amino acids:Ile, Asn, Ser and Thr, accounted for 11.2%,10.57%,9.94% and 8.36%, respectively. Compared with other known Cry2 proteins, Cry2Ag1 and Cry2Ab1 had shown as high as 92% Amino acids sequence homology. This, novel gene was registered in the GeneBank databases under accession number ACH91610. This gene was designed as cry2Agl in the Bacillus thuringiensis Toxin Nomenclature Committee.4. The full open reading frame sequence of the cry2Ag1 gene was amplified with a pair of PCR primers P1/P2 designed according to cry2Agl gene sequence, and inserted into the NcoⅠ/XhoⅠsite of E. coli expression vector pET-22b(+) to obtain the recombinant plasmid pET-2Ag1. The pET-2Agl was transduced into E. coli BL21 (DE3) pLysS. The analysis of SDS-PAGE showed that the cry2Agl gene produced 60kDa protein in E.coli by IPTG induction, which smaller than calculated Cry2Ag1 protein (70kDa) according to cry2Agl gene. Bioassay of the expressed product of the cry2Agl gene showed that Cry2Agl was highly toxic to both Lepidoptera P.xylostella, H.armigera and Dipteral A.aegypti with LC50 as 23.47μg/mL,9.74μg/mL and 2.54μg/mL, respectively.