摘要
【目的】耐除草剂大豆SHZD3201、DBN9004、中黄6106(ZH10-6)相继获得生产应用安全证书,针对这3个转化体建立二重实时荧光PCR检测方法,应用于大豆转基因转化体种子纯度的精准、快速鉴定,为大豆转基因品种种子质量安全监管提供技术支撑。【方法】搜集耐除草剂大豆SHZD3201、DBN9004和中黄6106 3个转化体以及4个大豆内标准基因Lectin的实时荧光PCR方法,通过比较ΔCt值,遴选出二重实时荧光PCR的最佳大豆内标准基因检测方法;设置转化体和Lectin不同引物/探针浓度,优化二重实时荧光PCR的反应体系;根据不同类型的特异性测试样品测试二重实时荧光PCR方法的特异性;设置2 000、200、20、10、5和1拷贝的梯度样品,测试方法的检出限。利用一步提取液快速研磨单粒种子,以稀释后的粗提液直接作为模板进行二重实时荧光PCR扩增,进而开展耐除草剂大豆SHZD3201、DBN9004和中黄6106单粒种子纯度的检测。【结果】经测试计算最小ΔCt值,在SHZD3201、DBN9004和中黄6106转化体二重实时荧光PCR中,遴选出最佳大豆内标准基因Lectin检测方法。经优化,SHZD3201/Lectin二重实时荧光PCR反应体系中,SHZD3201转化体的引物/探针浓度为0.3μmol·L^(-1)/0.15μmol·L^(-1),Lectin的引物/探针浓度为0.3μmol·L^(-1)/0.15μmol·L^(-1);DBN9004/Lectin二重实时荧光PCR反应体系中,DBN9004转化体的引物/探针浓度为0.4μmol·L^(-1)/0.2μmol·L^(-1),Lectin的引物/探针浓度为0.4μmol·L^(-1)/0.2μmol·L^(-1);中黄6106/Lectin二重实时荧光PCR反应体系中,中黄6106转化体的引物/探针浓度为0.4μmol·L^(-1)/0.2μmol·L^(-1),Lectin的引物/探针浓度为0.5μmol·L^(-1)/0.25μmol·L^(-1)。3种方法特异性良好,检出限均达10个拷贝。利用100粒模拟单粒种子样本,快速研磨,模板稀释5倍后,采用二重实时荧光PCR快速扩增,成功地检测了SHZD3201、DBN9004和中黄6106种子转化体纯度。【结论】成功建立了耐除草剂大豆SHZD3201/Lectin、DBN9004/Lectin和中黄6106/Lectin的二重实时荧光PCR方法,结合一步提取液的DNA快速提取方法,实现了大豆转基因种子转化体纯度精准快速检测。
【Objective】Production and application safety certificates have been successively granted to herbicide-tolerant soybean,including SHZD3201,DBN9004,and ZH10-6.The objective of this study is to establish duplex real-time fluorescence PCR detection methods for these three events,which are applied to the accurate and rapid identification of the purity of soybean transgenic event seeds,and to provide technical support for the quality and safety supervision of soybean transgenic varieties seeds.【Method】Three event-specific real-time PCR methods for herbicide-tolerant soybean:SHZD3201,DBN9004,ZH10-6,and four soybean-specific real-time PCR methods targeting the Lectin were collected.Through the comparison of ΔCt values,the most effective soybean reference gene detection approach for duplex real-time fluorescence PCR was chosen.The study further adjusted the concentration of primers and probes for both events and Lectin,optimizing the reaction system of duplex real-time fluorescence PCR.The duplex real-time fluorescence PCR methods underwent specificity testing using diverse types of samples.The limit of detection(LOD) was assessed using range of gradient samples containing 2 000,200,20,10,5,and 1copies.Using one-step extraction solution for rapid grinding of individual seeds,the diluted crude extract was directly used for duplex real-time fluorescence PCR amplification,thereby conducting event purity testing of individual seeds for herbicide-tolerant soybeans SHZD3201,DBN9004,and ZH10-6.【Result】Following the determination of the minimum ΔCt value through testing and calculation,the optimal soybean reference gene for detecting Lectin was selected in duplex real-time fluorescence PCR of SHZD3201,DBN9004,and ZH10-6 events.After optimization,the SHZD3201/Lectin duplex real-time fluorescence PCR reaction system exhibited optimal performance with the primer/probe concentration of 0.3 μmol·L^(-1)/0.15μmol·L^(-1) for SHZD3201 event and the primer/probe concentration of 0.3 μmol·L^(-1)/0.15 μmol·L^(-1) for Lectin.In the DBN9004/Lectin duplex real-time fluorescence PCR reaction system,the primer/probe concentrations for both the DBN9004event and Lectin were identical,setting at 0.4 μmol·L^(-1) for the prime and 0.2 μmol·L^(-1) for the probe.In the duplex real-time fluorescence PCR reaction system of ZH10-6/Lectin,the primer/probe concentration of ZH10-6 event was 0.4 μmol·L^(-1)/0.2μmol·L^(-1),the primer/probe concentration for the Lectin was 0.5 μmol·L^(-1)/0.25 μmol·L^(-1).The three methods had good specificity with the LOD of 10 copies each.Using 100 simulated single-seed samples,after rapid grinding and a 5-fold dilution of the template,the direct duplex real-time fluorescence PCR was used for rapid amplification,and the event purity of transgenic seeds of SHZD3201,DBN9004,and ZH10-6 was successfully detected.【Conclusion】This study successfully established three duplex real-time fluorescence PCR methods for herbicide-tolerant soybean SHZD3201,DBN9004,and ZH10-6.By combining a rapid DNA extraction method using one-step extraction solution,the research achieved precise and rapid detection of event purity in transgenic soybean seeds.
作者
李允静
任雪贞
肖芳
晋芳
高鸿飞
景琦
武玉花
孙全
李俊
王培
翟杉杉
金石桥
吴刚
LI YunJing;REN XueZhen;XIAO Fang;JIN Fang;GAO HongFei;JING Qi;WU YuHua;SUN Quan;LI Jun;WANG Pei;ZHAI ShanShan;JIN ShiQiao;WU Gang(Oil Crops Research Institute of Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture and Rural Affairs/Inspection and Testing Center(Wuhan)for Plant Ecological Environment Safety,Ministry of Agriculture and Rural Affairs/Key Laboratory of Agricultural Genetically Modified Organisms Traceability,Ministry of Agriculture and Rural Affairs,Wuhan 430062;National Agro-Tech Extension and Service Center,Beijing 100125)
出处
《中国农业科学》
CAS
CSCD
北大核心
2024年第23期4698-4711,共14页
Scientia Agricultura Sinica
基金
科技创新2030—重大项目(2022ZD04019)
中国农业科学院科技创新工程(CAAS-ASYIP-2021-OCRI)。
