转基因耐除草剂大豆及产品中外源成分检测方法的建立

分别从DNA和蛋白质2个水平上对转基因耐除草剂大豆及其产品的检测方法进行了研究．设计合成引物检测大豆内参照基因Lectin（大豆凝集素）及转基因抗除草剂Roundup Ready大豆外源基因，包括来自土壤细菌Agrobacterium tumefaciens株系CP4的5-烯醇丙酮酸莽草酸-3-磷酸合酶（5-enolpyruvylshikimate-3phosphate synthase，EPSPS）基因、花椰菜花叶病毒（Cauliflower mosaicvirus，CaMV）35S启动子、胭脂碱合酶3’端的转录终止子（nopaline synthase，NOS）．应用优化的常规PCR方法，对大豆及其加工产品进行了检测，检测灵敏度可达0．1％．通过PCR方法从转基因大豆中扩增出CP4-EPSPS基因，在大肠杆菌中表达，利用表达的外源蛋白质作为抗原免疫家免，得到该蛋白质的多克隆抗体，并建立了一套基于蛋白质印迹杂交（western hybridization）的检测转基因大豆及其粗加工品的方法，其检测极限达到1％以下．此两方法互相配合，互相印证，有助于规范化转基因检测方法的建立．

Methods for detection of extraneous gene and its protein product in transgenic herbicide-tolerance soybeans and their processed products

A PCR-based method and a immunological-based method were developed to detect extraneous genes in transgenic herbicide-tolerance soybeans and their processed products in this study.The primers for the gene of endogenous Lectin and foreign DNAs in Roundup Ready(RR) soybean,i.e.35S promoter(from cauliflower mosaic virus),NOS(nopaline synthase) terminator and CP4-EPSPS(5-enolpyruvylshikimate-3-phosphate from CP4) have been designed and synthesized.The detection limit based on qualitative PCR reaches 0.1% of transgenic content.Also,the CP4-EPSPSgene was amplified from RR soybean by PCR and expressed in E.coli.The anti-CP4-EPSPS antibodies were produced by immunized rabbits with the purified recombinant CP4-EPSPS protein, and used to develop western hybridization-based method in detecting the RR soybeans and their crude processed products.The detection limitation of this method is able to reach as low as 1% of transgenic components.These two can be cooperated to set up the standard methods for detecting genetically modified crops and their processed products.