Activity Analysis Of Bacillus Thuringiensis Cry8Ca2 Gene And Its Expression In Transgenic Nicotiana Tabacum L.

The cry8-type genes from Bacillus thuringiensis strains were toxic to a number of Coleopteran pests such as leaf beetles, scarabs and so on. The cry8C gene from Bt strain HBF-1 was cloned and sequenced. There was one amino acid difference with cry8Cal gene from Bt strain Buibui and it was designed as cry8Ca2 gene (AY518201) by International Bt Insecticidal Protein Gene Nomenclatural Committee. The cry8Ca2 gene's insecticidal activity was further researched in this report.With random mutation using PCR amplification more than 35 individual clones were gotten and transferred into Bt strain HD-73^-. The expressed proteins were detected their insecticidal activity against Anomala corpulenta larvae. The result showed that the two mutants had increased their toxicity to A. corpulenta larvae compared with wild type. The sequence analysis indicated the mutation sites were Q₄₃₉ to P₄₃₉ and E₆₄₂ to G₆₄₂.Nine truncated Cry8Ca2 proteins were induced in E. coli BL21(DE3) containing nine deleted fragments from cry8Ca2 gene by PCR method. The results from the activities of nine truncated proteins against A. corpulenta larvae showed the activity fragment of cry8Ca2 gene toxic to A. corpulenta larvae was between 70-659 amino acids.To improve the Cry8Ca2 insecticidal protein expression in transgenic plants, the cry8Ca2 gene was modified with plant preference codon usage and mutated at two sites, Q₄₃₉ to P₄₃₉ and E₆₄₂ to G₆₄₂. The modified cry8Ca2 gene (mcry8Ca) has 86.88% similarity with cry8Ca2 gene in nucleiode acid sequence and the content of G+C increased from 37% to 46%.The root-specific promoter TobRB7 was cloned from Nicotiana tabacum L. NC89 using PCR amplification. The new promoter TobRB7 had 2 nt difference with the sequence which was reported by Yamamoto et al. The promoter-reporter gene fusion assay showed that the TobRB7 promoter drove the GUS gene specific-expression in root tissue. The four plant expression constructs, pBTmCN (TobRB7 promoter, mcrySCa2 gene),pBSmCN (CaMV35S promoter: mcrySCa 2gene),pBI121 (CaMV35 promoter: GUS gene)and pBTGN (TobRB7 promoter: GUS gene), were transformed into Nicotiana tabacum by Agrobacterium tumefaciens. PCR results verified the integration of the mcry8Ca2 gene into the host genome of tobacco transgenic plants. Western blot indicated that the foreign CrySCa2 protein was correctly expressed in these transgenic tobacco plants.Bioassay results showed that these transgenic tobacco plants are highly toxic to A. corpulenta and A. exoleta larvae and lessen the damage caused by their feeding. These results demonstrate the feasibility of using transgenic plant technology to control crop underground pests such as scarabaeoidae larvae.