RESUMO
cry1Ah1, one of holo-type cry genes, cloned in this laboratory from Bacillus thuringiensis strain has been patented in China, and it encoded a protein with strong insecticidal activity against certain lepidopteran insect pests, such as Chilo suppressalis. cry1Ah1 gene is exhibiting good application prospects. In order to improve the expression level of cry1Ah1 gene in rice, and investigate the effect of codon usage preference of gene expression, we designed five different optimized schemes for cry1Ah1 insecticidal critical fragment in accordance with bias of rice codon, to improve G+C content, removed the shear signal and unstable factors. Optimized cry1Ah1 genes were transformed into Escherichia coli Rosetta (DE3) respectively, and 65 kDa polypeptides was expressed normally in inclusion body separately. All of these expressed polypeptides showed insecticidal activity against 2nd-instar larvae of Plutella xylostella and neonate of Chilo suppressalis. After transformation with modified cry1Ah1 genes into Var nippobare, the transgenic rice seedlings were detected by PCR, the positive rate containing target gene was more than 87%. Afterwards, the results of real-time RT-PCR and ELISA assay indicated that the highest expression level of five modified cry1Ah1 genes was that using the highest frequent codons. Average expression amount of Cry1Ah1 polypeptides was 0.104% of total soluble proteins from the positive transgenic rice.
