PCR-aided Synthesis Of The Bt CRY3Bb Gene Against Coleoptera Pests, Expression In E.coli And Transformation Of Tabacco Plants

Coleoptera are also named beetle. There are more known species of Coleoptcra than any other group of organisms, with over 330,000 described species. They live throughout the world (except Antarctica), but are most speciose in the tropics. The loss of the crops because of Coleoptera pests become more and more serious. Traditional chemicial pesticide which pollute environment seriously cannot work well because of its beetles and high progenitive rate. Planting resistant varieties has been considered as the most effective measure for protection against this pest. The practice of regular breeding has not significantly solving the problem of insect-resistance due to lack of gemiplast resource. The application of Coleoptera -specific Bt toxin proteins by means of the technique of genetic engineering in transgenic plants provides a new way of controlling this pests.Poor expression in plants is a well-reported characteristic of wild type Bt insect control proteins results in unsuccessfully commercial use. To increase expression of Bt genes and obtain insect resistance against Coleoptera -insect in plants, codon usage of wild type Bt genes is optimized and modified according to plant preferred codons and instability sequence elements is regulated or removed purposefully for efficient plant expression. The main results are summarized as follows:1. GFMcry3Bb insecticidal protein gene was designed and synthesized. Modifications and optimization in nucleotide sequence of cry3Bb gene did not alter the amino acid sequence of the Cry3Bb protein. 44 of potential polyadneylylation signal sequence, one of intron cleavage sequence and 4 of ATTTA sequence were removed entirely. A+T rich regions (>4 of consecutive A/T) were decreased from 66 to 16 during GFMcry3Bb designed. In comparison with the wild type cry3Bb gene, the ratio of GC content was increased from 32.96% of the wild type to 49.43% of the synthetic gene, a ratio more close to that of most plant genes. There are 65.8% of codons in wild type gene not suitable for expression in plant were modified to plant favorable condons without amino acid change. The synthesized GFMcry3Bb Bt gene using successive PCR was cloned into pBluescript IISK vector. Results of sequencing for recombinant plasmid were correct completely.2. Prokaryotic fusion gene expression vector, pGEX~GFMcry3Bb, was constructed. A high level of expression of fusion protein in E.coli was detected after IPTG induction and purified proteins were obtained by affinity chromatography with Glutathione Sepharose 4B.3. The purified GFMCry3Bb proteins were mixed with Freund's complete or incomplete adjuvant as antigen to immune rabbits. ELISA assay revealed that the tiler of the prepared antiserum against GFMCry3Bb protein was as high as 1: 10000. Purified IgG labeled with Alkaline Phosphatase by glutaraldehyde one-step method was used as enzyme-linked immunosorbent assay to detect content and level of GFMcry3Bb gene expression in transgenic plants.4. Synthesized GFMcry3Bb gene was cloned into plant expression vector. Recombinant plant expression vector was transferred into tobacco {Nicotiami labacum L.) plant via Agrobacterium-mediated transformation of tobacco leaf disks.5. Integration and expression of foreign gene were detected in a number of transgenic tobacco plants by southern blot analysis and ELISA analysis. The results indicated that synthesized GFMcry3Bb gene was integrated into tobacco genome, expression level of the modified Bt gene reached 0.001%~0.l% of overall soluble proteins.