【作者】 余春燕；

【导师】 励建荣；

【作者基本信息】 浙江工商大学 ， 食品科学， 2007， 硕士

【摘要】 转基因大豆及其制品日益增多，已经直接或间接地影响到人们的生活。也正因如此，转基因大豆及其制品对人体健康及生态环境的影响引起人们的广泛关注。为满足消费者选择权和知情权以及出于国际贸易的需要，转基因食品的检测手段也越来越引起各国政府和有关食品监督机构的重视。随着各国有关转基因标签法的建立和不断完善，对食品中的转基因成分含量的下限已有所规定，而且阈值越来越低。这就要求各国不仅能使定性检测技术日趋简捷和准确，更要求能探索出更加适合检测需要的定量手段。基于目前国际上对转基因食品还没有建立统一完善的定性和定量检测体系，为了探索建立转基因检测体系的策略，本研究以抗草甘膦转基因大豆及其加工产品为材料，试图建立经济灵敏、准确可靠的转基因大豆及其制品的内外源基因检测体系。本研究主要取得了以下结果：1、建立了以CTAB法为基础的转基因大豆及其粗加工和深加工产品抽提基因组DNA的技术。其结果显示：利用传统CTAB法对大豆种子抽提的基因组DNA基本完整，能完全满足PCR扩增要求；对粗加工产品（豆粕，豆奶粉）而言，延长CTAB法中抽提缓冲液的抽提时间为40～60min能得到基本完整的DNA；而对于深加工制品色拉油而言，在此基础上还需增加前处理阶段，即将样品适量溶解于TE缓冲液并充分混匀搅拌；此外对于酱油而言，抽提前用100mmoL/L的Tris-HCl洗2～3次可在DNA溶出前去掉水溶性和部分脂溶性色素以及部分多糖。2、设计合成检测转基因大豆内源基因Lectin、外源基因CaMV35S启动子、NOS终止子及抗除草剂基因EPSPS基因的特异性引物。应用优化的常规PCR方法，对大豆及其加工产品豆粕、豆奶粉、酱油、色拉油等进行了检测。所有样品均检测到内标基因Lectin，其中在大豆和豆粕、豆奶粉中用此法可检测到外源基因CaMV35S、NOS和EPSPS基因片段。3、采用常规PCR技术在深加工产品色拉油、酱油样品中未检测出外源基因CaMV35S启动子和NOS终止子，故利用巢式PCR技术，设计两对引物，成功扩增出CaMV35S启动子的大小两段基因片段，分别为195bp和117bp。本研究建立的常规PCR方法检测的相对灵敏度可达到0.1％，完全满足国际检测的需求。4、利用TaqMan^?探针法荧光定量PCR技术对转基因大豆及豆粕进行荧光定量PCR检测技术的研究，从内源Lectin基因的扩增情况表明TaqMan^?探针法荧光定量PCR技术重现性和灵敏度良好。在此基础上本文对大豆和豆粕的CaMV35S启动子和EPSPS基因分别进行了定量检测。结果表明在对转基因大豆的检测中，由CaMV35S启动子和目的基因EPSPS标准曲线计算得出样品中转基因百分含量分别为3.02％和3.00％，两者相差0.02％；在转基因豆粕的检测中，由CaMV35S启动子和目的基因EPSPS标准曲线计算得出样品中转基因百分含量分别为3.48％和3.49％，两者相差0.01％；且R²值都达到了0.99以上，可作为大豆和豆粕中转基因成分定量检测的供选方法。更多还原

【Abstract】 Recently transgenic soybean and products have been increasing day by day and regarded as one of the main foods of human consumption directly or indirectly. People pay more and more attention to the effects of transgenic soybeans and the products on human’s health and ecological environment. In order to meet consumer’s right to choose and the right of being in know and to meet the needs of international business, the detection methods of the genetically modified food causes more and more attention of the food supervisory organization and the governments in all countries. With the labeling administration on genetically modified food in different countries and territories, the lowest limit of content in food have been already regulated, and threshold value becomes lower and lower. Quantitative detection is becoming more important. This requires all the countries not only to develope the qualitative methods with high facility and precision, but also to explore the quantitative way to meet the detection needs.However, the unified detection system has not been established internationally at present. For exploring the strategy for detection of introduced DNA, transgenic herbicide tolerance soybean （Roundup Ready, RR） and processed products have been used as materials to establishing the economic and reliable detection method in this study.The main results obtained are as follows: 1、To establish the DNA extracted methods from RR soybean and other processed products. Results indicate: DNAs in RR soybean have been extracted easily by CTAB methods and amplified clearly by conventional PCR. It’s feasible for soy flour and defatted soy meal to extend the DNAs extracted time with 40min up to 60min. As for soybean oil, it’s absolutely necessary to dissolve samples in TE liquid firstly. And as for soy sauce, wash samples at least twice or thrice with Tris-HCl （100mmoL/L） at first, it conduce to throw off lipochrome and amylose.2、The primers of the endogenous Lectin and introduced DNAs in RR soybean, i.e.CaMV35S promoter, NOS terminator and EPSPS have been designed and synthesized. Conventional polymerase chain reaction （PCR） were successfully used to amplify the endogenous Lectin and RR soybean specific genes in soybean, defatted soy meal, soy flour, oil and sauce. At last, Lectin gene was amplified and observed in all samples, other introduced genes were detected successfully in soybean, defatted soy meal and soy flour.3、CaMV35S promoter and NOS terminator were not detected in soy oil and sauce samples by conventional PCR method. Double pair of primers were designed and Nested-PCR was adoptted to amplify the CaMV35S promoter including 195bp and 117bp successfully. With the Conventional PCR, relative detection limits of transgenic content reached 0.1% and would be adequate for meeting needs of international detection.4、Amplifying Lectin gene as criterion to evaluate the sensitivity and accuracy of real-time fluorescence quantitative PCR.TaqMan^? probe was utilized in quantitative detecting of CaMV35S promoter and EPSPS in soybean and soy defatted meal. Results as follows: CaMV35S promoter and EPSPS standard curves were eatablished by standard transgenic RR soybean. On the basis of these two standard curves, the contents of trasgenetic factor in soybean samples were calculated with 3.02% and 3.00% expectively. In the same way, defatted soy meal were 3.48% and 3.49% expectively. Because of its good repeatability and reliability, TaqMan^? probe method is absolutely adequate for the quantitative detection of trasgenetic factor in RR soybean and defatted soy meal.更多还原