转基因稻米的非靶向代谢组变异分析及真菌毒素风险预警

Analysis of Non-targeted Metabolomic Variation in Transgenic Rice and Warning of Mycotoxin Risk

采用液相色谱-质谱联用技术(HPLC-MS)研究粳稻品种日本晴(FP1)和PJ574(FP2)及其相应的转基因品种(FT16、FT23)的种子的代谢组学,旨在评价转基因稻米与其亲本代谢物组成的实质等同性,从代谢组变异和真菌毒素含量两方面评价稻米中的风险因素。结果:利用主成分分析和偏最小二乘法-判别分析等多元统计方法从稻米样本检出448种代谢物。筛选差异代谢物发现L-乳酸、玉米赤霉烯酮、10E,12Z-十八碳二烯酸和精胺代谢物浓度变异幅度受品种差异的影响较大。FP1-FP2组、FP1-FT16组、FP2-FT23组分别有9,23,13个特有差异代谢物,FP1-FP2组特有差异代谢物主要包括肌醇、硬脂酸酰胺等;FP1-FT16组特有差异代谢物主要包括脱氧鸟苷酸、咖啡盐和葡萄糖酸;FP2-FT23组特有差异代谢物主要包括前列腺素B2、L-胱氨酸、16-羟基棕榈酸,FP1-FT16组变异幅度最大。转基因育种和品种差异的效应类似,都在一定程度上影响精氨酸生物合成及多条氨基酸代谢通路,而不同品种的转基因稻米中代谢通路的变异不同。经酶联免疫法(ELISA)实测验证,稻米样本中的玉米赤霉烯酮含量在4.2~6μg/kg之间,两亲本品种间差异显著(P<0.01),而转基因稻米的玉米赤霉烯酮含量与亲本实质等同。ITS高通量测序结果表明镰刀菌属为稻米内生真菌的常见菌属。结论:转基因育种引发的稻米代谢组分和代谢通路变异虽可能影响稻米的营养品质,但幅度不大于品种差异,位于安全范围内。在4个货架期内的稻米样本中检出低剂量(小于6μg/kg)的镰刀菌毒素玉米赤霉烯酮,虽低于国家标准(60μg/kg),但其可能由稻米内生镰刀真菌合成,在特定条件下存在真菌爆发生长并大量产毒的风险。

The main objectives were to evaluate the substantial equivalence of metabolite composition in transgenic rice and its parents,and assess the risk factors in rice from two aspects of metabolomic variation and mycotoxin content.High performance liquid chromatography-mass spectrometry(HPLC-MS)was used to analyze Japonica rice varieties Nipponbare(FP1)and PJ574(FP2)and their corresponding transgenic lines(FT16 and FT23).The results were as follows:multivariate statistical methods such as principal component analysis and partial least squares discriminant analysis were used to detect 448 metabolites from rice samples.Different metabolites were screened and it was found that the concentration variations of zearalenone,L-lactate,10E,12Z-octadecanodienoic acid and spermine were significantly affected by gene modification or variety differences.There were 9,23 and 13 unique differential metabolites in group FP1-FP2,FP1-FT16 and FP2-FT23,respectively,with the highest variation in FP1-FT16.The unique differential metabolites of group FP1-FT16 mainly included deoxyguanolate,coffee salt and gluconic acid.The distinct metabolites of FP2-FT23 were mainly prostaglandin B2,L-cystine,16-hydroxypalmitic acid.The unique differential metabolites of FP1-FP2 consisted of inositol and stearate amides.The effects of transgenic breeding and variety differences were similar,both of which affect arginine biosynthesis and multiple amino acid metabolic pathways to a certain extent,but the variation of metabolic pathways in different varieties of transgenic rice was different.Enzyme-linked immunoassay(ELISA)showed that the zearalenone content in rice samples ranged from 4.2 to 6μg/kg,with a significant difference between the two parent varieties(P<0.01),but the content of zearalenone in transgenic rices was substantial equivalence to that in the parents.Fusarium was found to be a common endophytic fungus in rice by high-throughput sequencing.Conclusions:Although the variation of metabolic components and metabolic pathways induced by transgenic breeding may affect the nutritional quality of rice,the extent of variation is not greater than that of rice varieties,and it is within the safe range.A low dose(less than 6μg/kg)of Fusarium toxin zearalenone was detected in rice samples from four rice samples by untargeted metabolomic analysis technique.Although lower than the national standard(60μg/kg),Fusarium toxin may be synthesized by endophytic Fusarium fungi in rice,and there is a risk of fungal outbreaks under certain conditions with significant accumulation of toxicity.