H. M. Mahadeva Swamy

Screening, Diversity and Partial Sequence Comparison of Vegetative Insecticidal Protein (vip3A) Genes in the Local Isolates of Bacillus thuringiensis Berliner

Characterization, direct sequencing of the PCR amplicon and phylogenetic relationship was done to discover a novel Vip protein genes of the Bt isolates, to improve the prospects for insect control, more Vip proteins should be sought out and researched to predict their insecticidal activity. Characterization was based on direct sequencing of PCR amplicon using primers specific to vip3A gene was presented here. 12 out of 18 isolates screened were positive for vip gene-specific primers. Homology search for the partial sequences using BLAST showed that 11 isolates had high similarity to vip3Aa gene and only one fragment with vip3Ae gene (25–100% at nucleotide and amino acid level). Phylogenetic analysis showed that the gene sequences were responsible for geographic separation for divergence within vip genes, consistent with the evaluation of distinct bacterial population. Despite the geographical distances, strains harbouring vip genes have originated from common ancestors may significantly contribute to control resistant insect pests. Some strains have evolved to be quite distinct and others remain as members of closely related groups. The reported method is a powerful tool to find novel Vip3A proteins from large-scale Bt strains which is effective in terms of time and cost. Further the Vip proteins produced by different strains of B. thuringiensis are unique in terms of the sequence divergence and hence may also differ in their insecticidal activities.

Analysis of opportunities and challenges in patenting of Bacillus thuringiensis insecticidal crystal protein genes.

Bacillus thuringiensis (Bt) is the most widely used microbial control agent. The broad spectrum of susceptible hosts, production on artificial media and ease of application has caused the widespread use of this bacterium against several pests in agriculture, forest and vectors of human diseases. B.thuringiensis toxins are highly species specific which provide economic, environmental benefits, potential for future control and spread of the technology worldwide. This makes the B. thuringiensis crystal proteins an interesting tool for the implementation in integrated pest management programs. It has gained importance over the last 100 years for its biocontrol properties which is used in this review as a case study and analysis of the patents granted on B. thuringiensis was carried out. This study categorizes a number of patents related to B.thuringiensis insecticidal crystal proteins, application of B.thuringiensis insecticidal crystal proteins and the development of patentable technologies. The analyses were done using various criteria like patenting trends over the years, assignees playing a major role, comparison of the technology used in different patents and the patenting activity across the insect orders. Patent documents related to bacterium B.thuringiensis contain a trove of technical and commercial information and thus, patent analysis is considered as a useful tool for R management and techno economical development. Patent analysis also helps identifying and evaluating new and alternate technologies, keeping abreast with latest technologies for business interests, finding solutions to technical problems and ideas for new innovative trends.

Expression of cry3A gene and its toxicity against Asian Gray Weevil Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae).

Coleopterans are the most damaging pests of many agricultural and forestry crops; there is an urgent need to develop effective biopesticides against these insects. Enhancers of Bt toxicity typify an opportunity to improve currently available commercial products into more effective control agents against diverse pests. A 1.9 kb DNA fragment, PCR amplified from native isolates of Bt using cry3A gene specific primers was cloned in expression vector pQE‐80L and then used for transformation of Escherichia coli M15 cells. The sequence of the cloned crystal protein gene showed almost complete homology with a Coleopteran active Cry3A toxin gene with 117 mutations scattered in different domain regions encoding a protein of 645 amino acid residues in length, with a predicted molecular mass of 77.4 kDa. Phylogenetic analysis could be compulsive for new/novel Bacillus thuringiensis strains, allowing them to be grouped with related Cry proteins. The toxicity of Bt protein was determined against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) LC50 152 ng cm−2. Genes coding for Coleopteran active Cry3A proteins have been isolated and their efficient expression will provide the tools necessary to increase the efficacy of Cry‐based biopesticide against economically important beetles.

Isolation, Molecular Characterization and Expression of a New Cry3a Gene from Native Isolates of Bacillus thuringiensis and Its Toxicity Against Asian Grey Weevil Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae)

The genetic diversity and distribution of cry genes in Bacillus thuringiensis strains vary based on geographical location. Each habitat may contain novel B. thuringiensis isolates that have more toxic effects on target spectra of insects. To obtain novel B. thuringiensis strains for the production of Cry proteins, isolation of numerous new B. thuringiensis strains is becoming a routine activity in many industries. The B. thuringiensis Cry3A gene is regulated by a different mechanism from that of most of the other cry genes. 1.935-kb DNA fragment of Cry3A gene was PCR amplified using gene-specific primers, cloned in expression vector pQE-80 L and then used for transformation of E. coli M15 cells. The optimum expression was obtained with 1 mM IPTG at 37°C for 3 h. Nucleotide sequencing of the Cry3A gene revealed an open reading frame of 1,935 bp, encoding a protein of 645 amino acid residues in length, with a predicted molecular mass of 77.4 kDa. These full-length gene sequences were deposited at NCBI GenBank with accession number JQ038134 and JQ038135. Present work is now being directed toward the isolation and expression of the new Cry3A gene to access its effect against Asian grey weevil Myllocerus undecimpustulatus undatus (Coleoptera: Curculionidae). The availability of the recombinant protein will also allow the isolation and identification of its receptor in the insect midgut. Additionally, novel activities/specificities based on the sequence of the new Cry3A gene can be explored by the use of techniques such as DNA shuffling or point mutations. This system offers an additional method for potentially improving the efficacy of Bt insecticidal proteins efficiently, stably, and safely particularly against anti-lepidopteran insect pests. In other words, Bt technology will be a key to future growth of agriculture.