耐除草剂基因aroAA1501可拆分位点的鉴定  

Identification of Herbicide-tolerant Gene aroAA1501 Split Site

作　　者：宋亚亚 杨江涛[1] 王志兴[1] 王旭静[1] 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)

机构地区：[1]中国农业科学院生物技术研究所,农业农村部农业转基因生物安全评价(分子)重点实验室,北京100081

出　　处：《农业生物技术学报》2020年第4期743-753,共11页Journal of Agricultural Biotechnology

基　　金：转基因新品种培育重大专项(2016zx08010-003)。

摘　　要：基因拆分技术(gene splitting)能有效避免转基因作物中目标性状向环境中的逃逸。为了利用基因拆分技术培育新型耐草甘膦(glyphosate)的转aroAA1501基因水稻(Oryza sativa),本研究依据aroAA1501蛋白的高级结构筛选出13个可能的拆分位点,PCR的方法将aroAA1501拆分成N端(An)和C端(Ac) 2部分,并分别与Ssp DnaE intein的N端(In)和C端(Ic)结合形成融合基因An-In和Ic-Ac。以pETDuet-1为基础载体,构建了单独含有An-In、Ic-Ac和同时含有An-In/Ic-Ac的原核表达载体共40个。将构建好的原核表达载体导入营养缺陷型大肠杆菌(Escherichia coli)菌株ER2799中,通过功能互补试验证明aroAA1501在140/141、224/225和230/231位点处拆分后,拆分开的蛋白在蛋白内含肽intein介导下重新组装成有功能的完整蛋白。草甘膦耐受性试验证明230/231是最适宜的拆分位点,拆分后重新组装的蛋白对草甘膦的耐受性是完整aroAA1501蛋白的3倍。对含有aroAA1501、224N-In/Ic-225C、230N-In/Ic-231C和140N-In/Ic-141C的ER2799菌株中外源基因以及表达的重组蛋白分别进行qRT-PCR以及ELISA酶活分析,结果表明基因拆分后重组菌株的抗性差异是由于蛋白水平差异造成的而不是转录水平的差异造成的。此研究为后期培育耐草甘膦的转aroAA1501基因水稻提供了基础资料。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 Ssp DnaE intein to form fusion genes An-In and IcAc. 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 140/141, 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. The expression of exogenous genes and recombinant proteins in ER2799 strains containing aroAA1501, 224 N-In/Ic-225 C, 230 N-In/Ic-231 C and 140 N-In/Ic-141 C were analyzed by qRT-PCR and ELISA respectively, and it was proved that the difference of resistance of the recombinant strains after gene splitting was caused by the difference of protein level rather than the difference of transcription level. This study provides basic data for the later cultivation of glyphosate-resistant transgenic rice with aroAA1501 gene.

关 键 词：aroAA1501基因 基因拆分 可拆分位点 原核表达系统 

分 类 号：S33[农业科学—作物遗传育种]