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Abstract Cry2Aa gene from prokaryotic organism Bacillus thuringiensis consists of low GC content and some elements that affect gene expression in eukaryotic organisms, and the introduction of the unmodified gene into rice resulted in low levels of protein expression and insect resistance. In this paper, the proportion of rice (Oryza sativa L.) preferred codons and GC content in optimized Cry2Aa gene were increased, the potential Poly(A) addition signal sequences and the intron-exon boundary sequences were eliminated by synonymous codon substitution; then the optimized Cry2Aa gene was added with PR1a signal peptide sequence at 5' end and KDEL sequence at 3' end, and the secondary structure of its mRNA was optimized by using Mfold software. As a result, the GC content of the optimized gene Cry2Aa# was 57.29%，25.87% of the total nucleotides and 67.51% of the total codons were replaced in the optimized Cry2Aa# gene; the sequences which had been shown to destabilize mRNA in eukaryotic organisms were eliminated, codon usage frequency in the optimized Cry2Aa# gene kept consistent with the rice genome; In addition, nine stable mRNA stem-loop structures were eliminated, and the free energy of mRNA of the optimized Cry2Aa# gene decreased by 23.9 kcal/mol comparing with the free energy before optimization. The plant expression vector containing the optimized Cry2Aa# gene was constructed, and the optimized Cry2Aa# gene was transformed into rice via Agrobacterium-mediated method; the results of ELISA showed that the Cry2Aa# protein concentration ranged from 8.62 to 18.88 μg/g fresh weight (FW) of leaf at tillering stage in different transgenic lines; the results of bioassay showed that the transgenic lines were well resistant to rice leaf roller (Cnaphalocrocis medinalis); that indicated the approaches for optimization of Cry2Aa# gene were viable. This study not only validats the methods for gene optimization, but also provides an excellent insect-resistant genetic resource for rice breeding.
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Received: 16 May 2013
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