| Popis: |
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. |
