摘要
抗逆大豆IND-ØØ41Ø-5已经批准获得我国进口用作加工原料的安全证书,含有外源HaHB4基因和bar标记基因。本研究根据抗逆大豆IND-ØØ41Ø-5转化体5′端和3′端插入位点旁侧序列信息,设计19对引物,结合罗萨里奥农业生物技术学院公司的1对引物,针对20对引物组合利用定性PCR技术进行引物特异性筛选,结果显示5′端的14号引物特异性良好,优化后获得最佳反应体系和反应程序;经测试,该引物特异性好、稳定性高,检出限达0.1%,扩增片段大小248 bp。经8家有资质实验室对本方法的特异性、检出限、重复性和再现性进行验证,各项参数均达到国家标准要求。本研究成功建立了抗逆大豆IND-ØØ41Ø-5转化体定性PCR检测方法,为IND-ØØ41Ø-5转化体在国内的安全监管提供技术支撑。
Stress-resistant soybean IND-ØØ41Ø-5,which contains HaHB4 gene and bar marker gene,has been granted a safety certificate for importation as a processing material into China.In this study,We designed 19 primer pairs based on the 5´and 3´flanking sequences of the stress-resistant soybean IND-ØØ41Ø-5 event,in combination with one pair of primer providing by Instituto de Agrobiotecnologia Rosario(INDEAR)S.A.The specif-ic screening for the 20 primer pairs was performed using qualitative PCR technology.The results indicated that the primer pair of 14 at the 5´flanking sequence had good specificity.After optimization,the best reaction system and reaction procedure were obtained.The primer pair was tested for their specificity and stability,with limit of detec-tion(LOD)of 0.1%and an amplified fragment size of 248 bp.The specificity,LOD,repeatability and reproducibility of this method were verified by eight qualified laboratories,all of which met the requirements of national standards.This study successfully established a qualitative PCR method to detect the stress-resistant soybean IND-ØØ41Ø-5 event,which can provide technical support for the safety regulation of IND-ØØ41Ø-5 event in China.
作者
肖芳
李允静
武玉花
李俊
高鸿飞
翟杉杉
吴刚
梁晋刚
XIAO Fang;LI Yun-jing;WU Yuhua;LI Jun;GAO Hong-fei;ZHAI Shan-shan;WU Gang;LIANG Jin-gang(Oil Crops Research Institute of Chinese Academy of Agricultural Science/Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture and Rural Affairs/Supervision and Test 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,China;Development Center of Science and Technology,Ministry of Agriculture and Rural Affairs,Beijing 100176,China)
出处
《中国油料作物学报》
CAS
CSCD
北大核心
2024年第5期1148-1156,共9页
Chinese Journal of Oil Crop Sciences
基金
农业生物育种重大项目(2022ZD04019)
中国农业科学院科技创新工程项目(CAAS-ASYIP-2021-OCRI)。
