Fu Jianmei, Liu Biao
Nanjing Institute of Environmental Sciences, Ministry of Ecology and Environment, Nanjing, China.
Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
Front Bioeng Biotechnol. 2020 Jun 30;8:685. doi: 10.3389/fbioe.2020.00685. eCollection 2020.
Current risk assessments of transgenic crops do not take into consideration whether exogenous proteins interact with endogenous proteins and thereby induce unintended effects in the crops. Therefore, the unintended effects through protein interactions in insect-resistant transgenic rice merit investigation. Here, a yeast two-hybrid assay was used to evaluate interactions between (Bt) protein-derived Cry1Ab/c insect resistance rice Huahui-1 and the endogenous proteins of its parental rice Minghui-63. The authenticity of the strongest interactions of Cry1Ab/c and 14 endogenous proteins involved in photosynthesis and stress resistance, which may be primarily responsible for the significant phenotypic differences between transgenic Huahui-1 and parental Minghui-63, were then analyzed and validated by subcellular co-localization, bimolecular fluorescence complementation and co-immunoprecipitation. As the exogenous full-length Cry1Ab/c protein was found to have self-activating activity, we cleaved it - into three segments based on its three domains, and these were screened for interaction with host proteins using the yeast two-hybrid assay. Sixty endogenous proteins related to the regulation of photosynthesis, stress tolerance, and substance metabolism were found to interact with the Cry1Ab/c protein. The results of bimolecular fluorescence complementation and co-immunoprecipitation verified the interactions between the full-length Cry1Ab/c protein and 12 endogenous proteins involved in photosynthesis 23KD, G, PSBP, Rubisco, Trx, THF1 and stress resistance CAMTAs, DAHP, E3s, HKMTs, KIN13A, FREE1. We used a combination of yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation to identify Cry1Ab/c interacting with rice proteins that seem to be associated with the observed unintended effects on photosynthesis and stress resistance between Huahui-1 and Minghui-63 rice plants, and analyze the possible interaction mechanisms by comparing differences in cell localization and interaction sites between these interactions. The results herein provide a molecular analytical system to qualify and quantify the interactions between exogenous proteins and the endogenous proteins of the recipient crop. It could help elucidate both the positive and negative effects of creating transgenic plants and predict their potential risks as well as net crop quality and yield.
目前对转基因作物的风险评估并未考虑外源蛋白是否与内源蛋白相互作用，从而在作物中引发非预期效应。因此，抗虫转基因水稻中通过蛋白质相互作用产生的非预期效应值得研究。在此，利用酵母双杂交试验评估了苏云金芽孢杆菌（Bt）蛋白Cry1Ab/c抗虫水稻华恢1号与其亲本水稻明恢63的内源蛋白之间的相互作用。随后，通过亚细胞共定位、双分子荧光互补和免疫共沉淀分析并验证了Cry1Ab/c与14种参与光合作用和抗逆性的内源蛋白之间最强相互作用的真实性，这些蛋白可能是转基因华恢1号与其亲本明恢63之间显著表型差异的主要原因。由于发现外源全长Cry1Ab/c蛋白具有自激活活性，我们根据其三个结构域将其切割成三个片段，并利用酵母双杂交试验筛选它们与宿主蛋白的相互作用。发现60种与光合作用调控、胁迫耐受性和物质代谢相关的内源蛋白与Cry1Ab/c蛋白相互作用。双分子荧光互补和免疫共沉淀结果验证了全长Cry1Ab/c蛋白与12种参与光合作用（23KD、G、PSBP、Rubisco、Trx、THF1）和抗逆性（钙调蛋白、3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶、泛素连接酶、组蛋白赖氨酸甲基转移酶、类受体蛋白激酶13A、自噬相关蛋白1）的内源蛋白之间的相互作用。我们结合酵母双杂交、双分子荧光互补和免疫共沉淀来鉴定与水稻蛋白相互作用的Cry1Ab/c，这些水稻蛋白似乎与华恢1号和明恢63水稻植株之间观察到的对光合作用和抗逆性的非预期效应有关，并通过比较这些相互作用在细胞定位和相互作用位点上的差异来分析可能的相互作用机制。本文的结果提供了一个分子分析系统，用于鉴定和量化外源蛋白与受体作物内源蛋白之间的相互作用。它有助于阐明创建转基因植物的正负效应，并预测其潜在风险以及作物的净质量和产量。
