基于微滴式数字PCR定量检测转基因玉米'CC-2'外源基因和内参基因在土壤中的存留  

Quantitative Detection of Soil Persistence of Exogenous and Reference Gene from Genetically Modified Maize(Zea mays)'CC-2'by Droplet Digital PCR

作　　者：董姗姗 肖泽华 章嫡妮 刘燕 DONG Shan-Shan;XIAO Ze-Hua;ZHANG Di-Ni;LIU Yan(Key Laboratory for Biosafety of Environmental Protection,Nanjing Institute of Environmental Science,Ministry of Ecology and Environment,Nanjing 210042,China;Institute of Biodiversity Science,Fudan University,Shanghai 200438,China)

机构地区：[1]生态环境部南京环境科学研究所环境保护生物安全重点实验室,南京210042 [2]复旦大学生物多样性科学研究所,上海200438

出　　处：《农业生物技术学报》2022年第5期1014-1022,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31901231);国家重点研发计划(2020YFC1806305-02)。

摘　　要：监测转基因作物外源基因在土壤中的持留水平及其动态变化,是评估转基因作物对土壤生态系统潜在风险的重要内容。本研究以耐除草剂转基因玉米(Zea mays)'CC-2'和非转基因对照玉米'郑58'为实验材料,采用微滴式数字PCR(droplet digital PCR,ddPCR)技术,定量检测不同生长时期玉米根系土壤中耐除草剂基因CP4-EPSPS(Agrobacterium tumefaciens strain CP45-enolpyruvyl shikimate-3-phosphate synthase)和玉米内参基因zSSIIb(Zea mays starch synthase isoform zSTSII-2)的拷贝数。结果显示,ddPCR对CP4-EPSPS和zSSIIb基因的定量限(limit of quantitation,LOQ)分别为(0.48±0.07)和(0.22±0.04)copies/µL;转基因玉米'CC-2'各生育期根系土壤中CP4-EPSPS的拷贝数均低于定量限,随着生长时期的推移,其阳性检出率呈下降趋势;'CC-2'根系土壤中CP4-EPSPS与zSSIIb基因拷贝数无显著差异;在玉米不同生育期,'CC-2'与'郑58'根系土壤中zSSIIb基因的阳性检出率和拷贝数的变化趋势相似,但是在拔节期'郑58'根系土壤中zSSIIb拷贝数显著高于'CC-2'(P<0.05)。实验结果表明转基因玉米中的外源基因可以通过根系分泌物进入土壤环境,但浓度很低,且随生育期推移呈迅速下降趋势,说明ddPCR技术能够灵敏、精准地对土壤中存留的植物DNA进行定量分析。本研究为转基因作物外源基因在土壤中的定量检测提供新的方法和借鉴。Monitoring the persistence and dynamic changes of transgenes in the soil from genetically modified(GM)crop is an important issue in assessing the potential risks of GM crops.The present study applied a novel droplet digital polymerase chain reaction(ddPCR)method to quantitatively detect the copy number concentrations of exogenous gene CP4-EPSPS(Agrobacterium tumefaciens strain CP45-enolpyruvyl shikimate-3-phosphate synthase)and reference gene zSSIIb(Zea mays starch synthase isoform zSTSII-2)fragments in the rhizosphere soil of herbicide-resistant transgenic maize(Zea mays)'CC-2'and its non-transgenic control line'Zheng-58',at different growth stages of maize(seedling stage,jointing stage,silking stage,milk-ripe stage and full-ripe stage).The limit of quantitation(LOQ)of ddPCR for the CP4-EPSPS and zSSIIb system was(0.48±0.07)and(0.22±0.04)copies/µL,respectively.The copy number concentrations of CP4-EPSPS were lower than the limit of quantitation in the rhizosphere soil of GM maize'CC-2'at all growth stages,and the positive detection rate of CP4-EPSPS obviously declined with increasing growth stage.There was no significant difference between CP4-EPSPS and zSSIIb concentrations in rhizosphere soil of'CC-2'.At different growth stage,there was a similar change tendency in the positive detection rate and concentration of zSSIIb gene fragments in rhizosphere soil of'CC-2'and'Zheng-58',but the copy number concentration of zSSIIb in rhizosphere soil of'Zheng-58'was significantly higher than that of'CC-2'at the jointing stage(P<0.05).Test results showed that the transgene fragments of GM maize could enter into the soil environment through root exudates,but the concentration was very low and showed a decreasing trend with the growth stage,which indicates that the ddPCR method is suitable for sensitive and precise quantitative analysis of the persistent plant DNA fragments in soil environment.This study provided a novel method and reference for quantitative detection of transgene from GM crops in soil.

关 键 词：转基因玉米'CC-2' 微滴式数字PCR(ddPCR) CP4-EPSPS(Agrobacterium tumefaciens strain CP45-enolpyruvyl shikimate-3-phosphate synthase) 基因水平转移 土壤生态系统 

分 类 号：S181[农业科学—农业基础科学]