MSc

The study entitled “Molecular cloning of cry genes specific to dipterans from native Bacillus thuringiensis Berliner” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2007- 2009.
The crystal proteins encoded by various cry genes present in B. thuringiensis exhibit insecticidal activity against members of the orders Lepidoptera, Diptera and Coleoptera. In the present study, an attempt was made to screen and clone dipteran specific cry genes from native isolates of B. thuringiensis.
Twenty Bacillus thuringiensis isolates obtained from different locations of the Western Ghats of Kerala and available in the repository at CPBMB were characterized by routine cultural, morphological and biochemical tests (Cappuccino and Sherman, 1992). Abundance of crystal protein produced during sporulation was assessed by staining. The specificity of Bt strain to a target insect depends on its cry gene content and the insecticidal property can be predicted by molecular tools like polymerase chain reaction (PCR). Profiling of eight dipteran-specific cry genes (cry 2, 4, 10, 11, 16, 17, 19 and 21) was carried out in twenty native Bt isolates using PCR with specific primers. B. thuringiensis subsp. israelensis strain 4Q1 served as positive control. Eleven native isolates contained cry4 gene and four isolates contained two different dipteran-specific genes. Based on the abundance of crystal protein and also the cry gene profile, four isolates were selected for further bioassay and molecular studies.
The insecticidal activity of the native Bt isolates was determined by bioassay against the dipteran insect Drosophila melanogaster (Vinegar fly). Crystal protein-spore suspension of the selected isolates (Bt-23, Bt-133, Bt-190 and Bt-242) and reference strain 4Q1 at five different spore concentrations were mixed in the semi-synthetic diet for third instar larvae. The percent mortality of larvae was directly proportional to the concentration of spores. 4Q1 was found to be highly toxic, with 100% mortality in 24h. Bt-23 recorded lowest LC50 value among the native isolates, indicating high toxicity of crystal protein.
The dipteran-specific cry4 gene was amplified by PCR in four selected isolates. The amplified PCR product 800bp of isolate Bt-23 was eluted and ligated into pGEMT vector further cloned in E. coli JM 109 strain and sequenced. Remaining three isolates of 400bp amplified fragment were eluted and purified product directly sequenced without cloning.
The gene sequences were analyzed by using various bioinformatics tools like clustalW, Blastn and ORF finder. All the sequenced gene fragments showed 100 per cent homology with cry4A gene. Forward and reverse primers were designed for cry4A full length gene and PCR amplification was done with the selected Bt isolates. But none of the isolates gave amplification for cry4A full length gene primer.
The present study was helpful in predicting the insecticidal activity of twenty native isolates of B. thuringiensis. Twelve of these isolates harboured at least one dipteran-specific gene. Bioassay of four selected isolates on Drosophila melanogaster revealed that Bt-23 was the best with the lowest LC50 value. Sequence analysis of the cry4 genes amplified through PCR revealed homology with cry4A accessions in the database. The isolate Bt-23 could be further exploited as a bioagent for controlling dipteran pests of crop plants.