摘要
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 tum efaciens株系CP4的5-烯醇丙酮酸莽草酸-3-磷酸合酶(5-enolpyruvylsh ik im ate-3-phosphate synthase,EPSPS)基因、花椰菜花叶病毒(Cau liflowermosaic virus,CaMV)35S启动子、胭脂碱合酶3′端的转录终止子(nopalinesynthase,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兔,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 hybrid ization)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.
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 hybridizationbased 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.
出处
《华南农业大学学报》
CAS
CSCD
北大核心
2005年第4期67-72,共6页
Journal of South China Agricultural University
基金
广东省科技计划重大专项项目(20034049)
广东省农业科技项目
关键词
转基因耐除草剂大豆
大豆加工产品
定性PCR
外源蛋白质检测
transgenic herbicide-tolerance soybean
processed soybean products
qualitative PCR
extraneous protein detection
