MSc

The study entitled ‘Cloning of genes encoding insecticidal proteins (cry/vip genes) of Bacillus thuringiensis from Western Ghats of Kerala’ was carried out in the Molecular Biology Laboratory of the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2005- 2007. The crystal protein genes (cry/vip) of B. thuringiensis possess insecticidal activity against larvae of insect orders Lepidoptera, Diptera and Coleoptera. In the present study, an attempt was made to isolate and clone cry genes of B. thuringiensis from the Western Ghats of Kerala.

Bacillus thuringiensis strains were isolated from soil samples collected from different locations of the Western Ghats of Kerala. The pure colonies obtained were stab inoculated and stored under refrigerated conditions. Variability among the isolates were studied by various cultural, morphological and biochemical tests. The insecticidal activity of the isolates was determined by bioassay against the major lepidopteran pest of cucurbitaceous vegetables, the pumpkin caterpillar.

The information on cry1A gene sequences of different species of Bacillus thuringiensis available in the public domain NCBI was collected and subjected to multiple sequence alignment to detect conserved boxes of the gene among species. Based on the data, one pair of gene specific primer was designed for amplification of partial cry1A gene fragment of about 800bp in B. thuringiensis isolates.

Total DNA was isolated from the B. thuringiensis strains of Western Ghats of Kerala. Profiling of cry1 and cry4 genes of bacterial isolates were done using universal primers for cry1 and cry4. Amplification was obtained with cry1 gene for seven isolates and with cry4 gene primer for two isolates. The amplicons obtained with universal cry1 primer from two isolates and with cry4 primer from one isolate were used for cloning.
The amplicons obtained with cry1 and cry4 primers were eluted, cloned in pGEMT vector and transformed into competent cells. High level of recombination was observed on blue-white screening. Recombination of the insert was confirmed by PCR of the plasmid isolated from white colonies. The cloned fragments were sequenced. The amplicon obtained with cry4 primer in the second isolate was sequenced after purifying the PCR product.

The cry1ky5 and cry1em11 sequences when subjected to Blast search revealed significant levels of homology with cry1 genes reported from other B. thuringiensis strains deposited in the public domain. The cry4em10 sequence when subjected to Blast search, showed high level of similarity with cry4 genes from B. thuringiensis. The cry4ky1 sequence showed similarity with cry genes of different species of B. thuringiensis. The sequences were also subjected to various sequence analysis using bioinformatics tools which include ORF finder, SOPMA, GENSCAN, AASTAT and TCAG tools of Biology Workbench and Interproscan.

PCR of all the isolates found positive in cry1 gene profiling, was done with cry1A primer designed during this study. Amplification was obtained with cry1A primer in two isolates. The amplicon obtained in one isolate was subjected to sequencing after purifying the PCR product. The cry1Aky3 sequence showed similarity with other cry genes of Bacillus thuringiensis present in the NCBI databank. 1500 bp long variable region of cry1 was amplified in two isolates using specific primers.
Future research works should be focused on the isolation of B. thuringiensis from completely undisturbed ecological niches. Novelty of cry genes can be detected by restriction digestion of the genes. Characterization of novel full-length cry genes and its expression in transgenic crops will help to develop resistant varieties thereby reducing insecticide applications and resistance development in insect pest populations.