Abstract
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 Bacillus thuringiensis (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.
Highlights
Transgenic crops, known as genetically modified crops, refer to crops and their descendants produced by DNA recombination technology to integrate exogenous genes (DNA sequences are introduced into target cells through genetic engineering or viral infection, etc.) into the genomes of recipient crops, in a way that is changing their genetic composition (Herdt, 2005)
It has been reported that exogenous genes in transgenic crops might induce unintended effects by disrupting host genes or rearranging DNA sequences at the insertion site (Forsbach et al, 2003), which causes somatic variation during the transgenic crop cultivation process (Labra et al, 2001; Noro et al, 2007), consuming excess host material and energy for their own expression (Zeller et al, 2010) as exogenous proteins interact with endogenous proteins in the recipient crops
Our results showed that the Cry1Ab/c-bihybrid vector pGBKT7 (BD) yeast strain Y2HGold bearing the full-length Cry1Ab/c protein developed normally on nutrient-deficient SD/-Trp medium, but turned blue on SD/-Trp/X medium supplemented with x-alphagal (X)
Summary
Transgenic crops, known as genetically modified crops, refer to crops and their descendants produced by DNA recombination technology to integrate exogenous genes (DNA sequences are introduced into target cells through genetic engineering or viral infection, etc.) into the genomes of recipient crops, in a way that is changing their genetic composition (Herdt, 2005). Transgenic crops have considerable potential economic, environmental, and social benefits. Uncertainty regarding their unintended effects has been a major constraint in their commercial development and exploitation. The crop cannot express naturally the exogenous proteins, the transgenic crops could express these proteins through integrating exogenous genes into the crop genome in all tissues throughout the growth stages (Fu et al, 2018), so these exogenous proteins might potentially interact with endogenous proteins to produce unintended biological effects in the transgenic crop.
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