导　　师： 刘阳

学科专业： 083201

授予学位： 硕士

作　　者： ;

机构地区： 中国农业科学院

摘　　要： 自转基因作物问世以来，其食用安全性一直受到广泛关注。本研究以转Cry1Ab基因大米为原料，通过六种DNA提取方法的比较，选择最佳的DNA提取方法进行样品DNA的提取。然后利用定性PCR和实时荧光定量PCR方法，研究米饭、米粉、米果和米酒的加工过程对大米内源（SPS）、外源（Pubi、Cry1Ab、NOS和Hpt）基因的片段大小及外源基因（Pubi和Cry1Ab）含量变化的影响。为食品中转基因成分的检测、转基因食品加工工艺参数的设置以及转基因食品食用安全性评价提供一定的科学依据。 （1）转Cry1Ab基因大米及其加工食品DNA提取方法的确定。分别采用三种CTAB（Cetyltrithylammonium Bromide）法、一种碱裂解法和两种试剂盒法提取转Cry1Ab基因大米及其米果加工产品的DNA，探讨适用于转基因大米加工食品的DNA提取方法。结果表明：虽然天恩泽试剂盒法所提取的DNA产率最低，但其DNA的完整性、纯度和PCR扩增效果最好，故选择该法进行下面的研究。 （2）转Cry1Ab基因大米内、外源基因在食品加工过程中降解规律的研究。将转Cry1Ab基因大米分别制成米饭、米粉、米果和米酒产品，采用定性PCR方法对四种食品加工过程中各操作单元样品的内、外源基因进行检测，从而分析各加工操作对内、外源基因的降解影响。结果显示：在米饭和米粉的加工过程中各基因的降解并不显著，仅大于1500bp的片段发生了降解，且NOS终止子和Hpt标记基因并未观察到任何降解。米果加工过程中，油炸对各基因的降解最为严重，然后依次是微波、焙烤、煮制和两次干燥过程。米酒的发酵过程对内、外源基因有显著的降解作用，其作用效果高于其他三种米制品的加工过程。然而，四种产品的加工过程都没有将内、外源基因降解到200bp以下（除NOS基因以外）。比较各基因的稳定性发现：在米饭和米粉的加工过程中Cry1Ab、Pubi、Hpt和SPS基因的稳定性相似；而在米果和米酒的制备过程中，SPS基因最稳定，然后依次为Cry1Ab基因、Pubi启动子、Hpt基因和NOS终止子。 （3）转Cry1Ab基因大米外源基因含量在食品加工过程中变化规律的研究。利用实时荧光定量PCR技术对四种米制品加工过程中各操作单元样品进行外源基因含量的检测，研究不同加工工艺对食品中转基因成分含量的影响。结果表明：米饭和米粉制备过程中，Cry1Ab基因的百分含量不低于100/%，Pubi启动子含量不高于83/%（除磨浆样品外）。在米果加工过程中，油炸样品的外源基因含量最低，约为30/%，微波和焙烤样品的外源基因含量均低于61/%，这表明油炸导致外源基因的降解最严重，显著高于微波和焙烤过程。米酒发酵过程对外源基因含量降低的影响大于其他加工过程（除油炸和焙烤后Cry1Ab基因含量外），除煮制样品外其他各样品的外源基因含量都低于52/%。因此，在米饭和米粉的制备过程中Cry1Ab基因的稳定性最高，其次是SPS基因和Pubi启动子；而在米果和米酒的加工过程中SPS基因最稳定，然后依次为Cry1Ab和Pubi基因。此外，在检测食品中转基因成分含量时，选择不同的外源基因作为转基因成分的代表基因会得到不同的检测结果，这与所选择的外源基因对内源基因的相对稳定性有关。 Since the advent of the transgenic crops, their food security has attracted widespread attention. Inthis study, genetically modified rice with Cry1Ab was used as raw material, by comparing six DNAextraction methods we selected the most suitable DNA extraction method to extract DNAs of samples.Then the qualitative PCR and real-time fluorescent quantitative PCR methods were used to study theimpact of the processing procedures of steamed rice, rice noodles, rice crackers and sweet rice wine onthe degradation of endogenous /(SPS/) and exogenous /(Pub, Cry1Ab, NOS and Hpt/) genes and thecontent changes of exogenous /(Pubi and Cry1Ab/) genes. The aim was to provide some scientific basisfor the detection of exogenous genes in foods, setting parameters during genetically modified foodprocessing and safety assessment of GM foods. /(1/) Establishment of DNA extraction method for GM rice and GM rice-derived food products.Three CTAB /(Cetyltrithylammonium Bromide/)-based methods, an alkaline lysis method and twocommercial kits from Tiangen and Tiandz were used to extract DNA from genetically modified ricewith Cry1Ab and its rice crackers products. The results showed that although the yield of the DNAextracted by Tiandz kit was the lowest, Tiandz kit method gained highest quality of DNA integrity,purity and PCR amplifiability. So Tiandz kit was selected to extract DNA of samples for thedownstream study. /(2/) Degradation of endogenous and exogenous genes of genetically modified rice with Cry1Abduring Food Processing. Genetically modified rice with Cry1Ab was used to produce four processedfoods /(steamed rice, rice noodles, rice crackers and sweet rice wine/), and then the qualitative PCR wasused to detect the degradation of endogenous and exogenous genes during food processing. The resultssuggested that during the processing of steamed rice and rice noodles, the procedures were so mild thatonly genes larger than1500bp were degraded, and no degradation of NOS terminator and Hpt gene wasdetected. For the processing rice crackers, frying was the most severe procedure, followed bymicrowaving and baking, boiling, and drying. For the sweet rice wine, fermentation had more impact onthe degradation of genes than the other processing procedures. However, all procedures we studied didnot lead to degradation of genes to below200bp, except for NOS terminator. In the case of stability ofthe genes studied, we found during the processing of steamed rice and rice noodles, the stabilities ofCry1Ab, Pubi, Hpt and SPS gene were similar; while during the preparation of rice crackers and sweetrice wine, SPS gene was the most, followed by the Cry1Ab target gene, Hpt marker gene, Pubi promoterand NOS terminater. /(3/) Effect of processing procedures on the content changes of exogenous genes during foodprocessing of genetically modified rice with Cry1Ab. Real-time fluorescence quantitative PCRtechnology was used to detect the content changes of exogenous genes during the processing of fourrice products. The results indicated during the preparation of rice and rice flour, the content of Cry1Abgene was not less than100/%, and the content of Pubi promoter was not more than85/%, except for samples of grinding. For rice crackers, the minimum content of exogenous genes was the fried sample,only about30/%. While the contents of the exogenous genes of both microwaved and baked sampleswere below61/%. This result indicated the processing of frying caused the most serious degradation ofexogenous genes, which was significantly severer than the processings of baking and microwaving. Theprocessing procedures of sweet rice wine were the most severe on the reduction of the content ofexogenous genes /(except for the content of Cry1Ab gene after frying and baking/), and the content ofexogenous genes of each sample was below52/%/(except for boiled sample/). As to the stability of thegenes, we found during the preparation of the steamed rice and rice noodles, Cry1Ab gene was the moststable，followed by SPS gene and Pubi promoter. While during the processing of rice crackers and sweetrice wine, SPS gene was the most, followed by Cry1Ab gene and Pubi promoter. In addition, whendetect the content of genetically modified components in foods, selecting different exogenous genes as arepresentative of genetically modified material will gain different results, which depended on therelative stabilities of exogenous genes to endogenous genes.

关 键 词： 转 基因大米 食品加工 内源基因 外源基因 降解

分 类 号： [TS205]

领　　域： [轻工技术与工程] [轻工技术与工程]