Identification of Herbicide-tolerant Gene aroAA1501 Split Site
SONG Ya-Ya, YANG Jiang-Tao, WANG Zhi-Xing*, WANG Xu-Jing*
Key Laboratory of Safety Evaluation (Molecular) of Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Sciences, Institute of Biotechnology, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Abstract：Gene splitting technology can effectively avoid the escape of target traits in genetically modified crops to the environment. In order to cultivate a new glyphosate-resistant aroAA1501 transgenic rice (Oryza sativa) using gene resolution technology, 13 possible resolution sites were screened out based on the advanced structure of aroAA1501 protein in this study. aroAA1501 was split into N-terminal (An) and C-terminal (Ac) by PCR, and is combined with N-terminal (In) and C-terminal (Ic) of SspDnaE intein to form fusion genes An-In and Ic-Ac. Based on pETDuet-1, a total of 40 prokaryotic expression vectors containing An-In, Ic-Ac and An-In / Ic-Ac were constructed. The constructed prokaryotic expression vector was introduced into the auxotrophic Escherichia coli strain ER2799. It was proved by functional complementation tests that aroAA1501 was split at the positions 141/142, 224/225, and 230/231. The protein was reassembled into a functional intact protein mediated by the protein intein. The glyphosate tolerance test proved that 230/231 was the most suitable resolving site. The reassembled protein after resolving was 3 times more tolerant to glyphosate than the complete aroAA1501 protein. This study provides basic data for the later cultivation of glyphosate-resistant transgenic rice with aroAA1501 gene.
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