Localization Of Vip3A And Chitinase Gene Of Bacillus Thuringiensis Strain WB50

The leading biological pesticide, Bacillus thuringiensis (Bt), is a ubiquitous bacterium that forms several crystal proteins during the stationary phase of its growth cycle. Since S. Ishiwata first isolated B. thuringiensis, there are currently an estimated 60,000 isolates of Bt with different insect host spectra throughout the world. Most of them display toxicity against over 3,000 species from more than 16 orders of insects. However, a number of insect populations of various species with different levels of resistance to Bt are reported since the mid-1980s. The development of resistance against Cry proteins in susceptible larvae as well as the insensitivity insects renders it imperative to search for novel insecticidal proteins.Novel vegetative insecticidal proteins (VIPs), produced by Bt during its vegetative stages of growth, have been identified since 1996. These proteins comprise three classes including VIP1, VIP2 and VIP3. Chitinases playing an important role during the biocontrol against a number of phytopathogenic insects, fungi and wireworm etc. have also been investigated in recent years. The efficacy of VIP3A and chitinases in controlling some of the agronomically important pests has made up for the deficiency of Cry proteins, broaden the spectrum and provided the possibility of efficacious usage of Cry proteins.After localizing the gene, a physical map of plasmids or chromosome whichharbors the gene can be constructed and furthermore the whole gene sequence also would be determined. It will help to thoroughly study on the gene structure, function and regulation, discover novel genes, and search for the correlation among genes.In order to gain a better result of Southern blotting, a modified procedure for isolation of Bt plasmids was presented in this paper. Compared with standard alkaline lysis, BGSC or boiling protocol, the modified protocol gave reproducible, complete plasmid profiles for Bt. The DNA preparations were usually sufficiently pure to be analyzed without further treatment.In current research, vip3A gene which codes for VIP3A was cloned from the plasmids isolation of Bt strain WB50. The sequence of 2,460 bp was deduced and deposited in GenBank under the accession number AY295778. The nucleotide sequence analysis indicated that it had 99% homology with the known vip3A genes with on-line Blast software.Preliminary localization of the gene was performed through PCR using plasmids and chromosomal DNA as template to amplify vip3A gene. The presence of vip3A gene on the plasmids was preliminarily confirmed basing on no purposed fragment obtained from chromosomal DNA. To further investigate the localization of the gene in WB50, we used 468 bp fragment derived entirely from an interior portion of the cloned gene as probe in hybridization. Southern hybridization analysis of this strain showed that vip3A gene was located on a plasmid of 31.8 kb in size.Localization of chi gene coding for chininase was carried out through PCR, using plasmids, total DNA and chromosomal DNA of WB50 as template to amplify chi gene, respectively. In contrast to vip3A gene, chi gene wasprimarily suggested to localize on chromosomal DNA from the absence of PCR product from plasmids DNA. Accurate localization of this gene in WB50 was investigated by Southern hybridization analysis showing that chi gene was localized on chromosomal DNA.