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In this study, an optimized Cry2Aa gene was obtained after codon optimization based on the preferred codons in rice. The novel fusion gene Cry2Aa #  was designed by adding the sequence coding the signal peptide of PR1a at the 5' end and the endoplasmic reticulum retention signal peptide KDEL at the 3' end of the optimized Cry2Aa gene, respectively. The Cry2Aa #  and Bar genes were transformed into 4008S, a photoperiod-sensitive genic male sterile (PGMS) line in rice, using the Agrobacterium transfer method. A total of 65 regenerated plantlets confirmed by PCR were produced, in which eight transgenic lines had single-copy insertions as confirmed by Southern blot analysis. The high variability of Cry2Aa #  gene expression was observed among independent transgenic lines with single-copy insertion, and the spatiotemporal difference of Cry2Aa protein expression was discovered in each transgenic line. The results showed that the transgenic lines were highly resistant to glufosinate, rice leaf roller and striped stem borer, which not only confirmed the effective optimization of the Cry2Aa gene but also produced a useful germplasm for breeding insect-resistant and herbicide-tolerant hybrid rice varieties.

Optimization of the Cry2Aa gene and development of insect-resistant and herbicide-tolerant photoperiod-sensitive genic male sterile rice