Research Article

A novel synthetic Cry1Ab gene resists rice insect pests

Published: April 03, 2014
Genet. Mol. Res. 13 (2) : 2394-2408 DOI: 10.4238/2014.April.3.12

Abstract

A few insect control genes of Bacillus thuringiensis have been modified successfully to increase the expression in plants by replacing rare codons, increasing GC content, and avoiding the DNA elements that could cause premature transcription termination, mRNA instability, and potential methylation. However, the modification process was intricate and often confused researchers. In this study, we adopted a simple method to modify Cry1Ab only by individually replacing its amino acid sequence with corresponding rice-preferred codons based on analysis of 92,188 coding DNA sequences. Unexpectedly, all elements of A+T richness, which terminate or destabilize transcription in plants, were avoided in the newly designed mCry1Ab. However, mCry1Ab had 2 notable features: less synonymous codons and high GC content. mCry1Ab only employed 22 of the 61 codons to encode protein and had an enhanced GC content of 65%. The increase in GC content caused abundant potential methylation signals to emerge in mCry1Ab. To test whether mCry1Ab could be expressed in rice, we transferred it into Oryza japonica variety Wanjing97. Insect bioassays revealed that transgenic plants harboring this gene driven by 2 promoters, CaMV35S and OsTSP I, were highly resistant to rice leaffolder (Cnaphalocrocis medinalis). Analysis of R0 to R2 generation plants indicated that the mCry1Ab was inherited stably by the progeny. Our study provided a simple modified method for expressing exogenous genes in rice and confirmed that less synonymous codons and high GC content do not affect transgene expression in rice.

A few insect control genes of Bacillus thuringiensis have been modified successfully to increase the expression in plants by replacing rare codons, increasing GC content, and avoiding the DNA elements that could cause premature transcription termination, mRNA instability, and potential methylation. However, the modification process was intricate and often confused researchers. In this study, we adopted a simple method to modify Cry1Ab only by individually replacing its amino acid sequence with corresponding rice-preferred codons based on analysis of 92,188 coding DNA sequences. Unexpectedly, all elements of A+T richness, which terminate or destabilize transcription in plants, were avoided in the newly designed mCry1Ab. However, mCry1Ab had 2 notable features: less synonymous codons and high GC content. mCry1Ab only employed 22 of the 61 codons to encode protein and had an enhanced GC content of 65%. The increase in GC content caused abundant potential methylation signals to emerge in mCry1Ab. To test whether mCry1Ab could be expressed in rice, we transferred it into Oryza japonica variety Wanjing97. Insect bioassays revealed that transgenic plants harboring this gene driven by 2 promoters, CaMV35S and OsTSP I, were highly resistant to rice leaffolder (Cnaphalocrocis medinalis). Analysis of R0 to R2 generation plants indicated that the mCry1Ab was inherited stably by the progeny. Our study provided a simple modified method for expressing exogenous genes in rice and confirmed that less synonymous codons and high GC content do not affect transgene expression in rice.