Characterization Of Vip3Genes In Bacillus Thuringiensis From Four Sites In Ya’an And Clone And Expression Of Novel Vip3A Gene

Vegetative insecticidal protein, expressed and excreted into the medium during vegetative period by Bacillus thuringiensis, was first discovered by Estruch in1996. Vip3A protein has a wide spectrum and high insecticidal activity on Lepidopera. The amino acids of vip3A greatly differed from the known amino acids of insecticidal crystal protein while the mechanism of insecticidal activity is different from the crystal protein as well. Therefore, Vip3A was proposed as novel insecticides. Studies on Vip3A will assist in broadening the pesticidal spectrum, retarding resistance of insect and modifying insecticidal protein by gene engineering.Ya’an, with a complicated geomorphology environment, is located in the western region of Sichuan basin. Novel vegetative insecticidal protein is probable to be found from the soil samples. This study described a systematic study on Bt resourcees screening and vip3gene cloning from soil samples of JinFengshi, MengDing mountain, ZhouGong mountain and BiFengxia. A novel vip3A gene was cloned and expressed. The concrete results are as follows:1. A total of465soil samples were collected from four sampling sites in Ya’an and765Bt strains were isolated using sodium acetate-antibiotics method. The average rate was164%. Through observations by electron microscope, these Bt strains produced different patterns of parasporal crystals, such as long bipyramid, short bipyramid, round and abnormity, which indicated that diversity of Bt resources was resided in Ya’an. These Bt strains with unique parasporal crystals patterns mainly showed proteins with40kDa and1lOkDa in molecular weight by SDS-PAGE.2.745Bt strain were identified to contain vip3genes by PCR using general primers of Vip3s-Vip3a and V32s-V32a, which accounted for97.4%in our collection of isolates. The vip3A genotype of the745Bt isolates were identified by using PCR-RFLP with special designed primers of vip3N2-5-vip3N2-3. Products of1.486kb in length were obtained from624isolates that accounted for81.6%of isolates with vip3A genes. Then restriction enzyme TaqI was used to digest these PCR productions for identification of vip3A genotypes. The results showed that restriction patterns of603Bt strains were similar to that of vip3Aal with DNA framents of828bp,386bp and273bp in length. The result showed that vip3Aa gene were the most abundant genotype in our collection and further confirmed that vip3A gene was extremely conserved. PCR-RFLP pattern of21Bt strains were different from that of vip3Aa. Bt strain ZG151-20amplified frament was cut into1100bp and300bp in length.The characterization PCR products of strain ZG151-20was inserted into the vector of pEASY-T1.Blast result showed that it was as high as97%Amino acids sequence homology.It demonstrated that it was a novel vip3Aa gene. A novel vip3Aa gene from ZG151-20was cloned by full-length PCR method. This novel gene was registered in the GeneBank databases under accession number JQ731616(Vip3Aa48). vip3Aa48was inserted into a pET28expression vector and transformed into E.coliBL21(DE3)pLysS. The analysis of SDS-PAGE showed that the vip3Aa48gene produced88.6kDa protein in E.coli by IPTG induction. The bioassay results indicated that vip3Aa48was highly toxic to Spodoptera exigua with the LC50values of18.2μg/mL. However,Vip3Aa48only caused severe stuning of neonate larvae of Helicocerpa armigera.