【作者】 田庆常；

【导师】 陈集双；

【作者基本信息】 浙江理工大学 ， 生化与分子生物学， 2011， 硕士

【摘要】 随着转基因产品的日益丰富,人们对转基因产品安全的关注越来越多。关于转基因产品是否安全至今还不能够给出确切的答案。为了转基因产品安全管理的需要,应尽快发展和完善转基因检测方法。目前转基因检测的方法各种各样,各有自己的特点。探索一种实用、简便、有效的检测方法对加强对转基因技术的研究和对转基因产品的管理都有重要的意义。本研究根据转基因植物中所转入外源基因的特征,结合微管杂交的方法,建立了一套简便而且有效的转基因检测方法。大多数的转基因植物中以CaMV35S启动子和NOS终止子作为外源基因的启动子和终止子。本研究首先设计针对CaMV35S启动子和NOS终止子的引物,然后对携带CaMV35S启动子和NOS终止子的质粒pBI121进行PCR扩增,扩增产物标记后作为探针。其次将尼龙膜剪切成大小合适的膜条,将多个样品DNA固定于膜条上,然后膜条与探针在2ml离心管中与探针杂交、检测获得杂交信号。这个方法可以实现一两条探针检测多个样品。最后利用PCR方法对杂交所得结果进行验证,结果发现PCR检测结果与膜条杂交结果相一致,说明微管杂交法检测多个植物样品中外源基因的方法是可行的。对于单一的转基因植物,转入的外源基因中不仅含有启动子和终止子,而且还含有目的基因和标记基因。本研究以转基因棉花为样品,根据转入其中的外源基因可能包含基因的种类设计了一组寡核苷酸探针,然后将氨基化的寡核苷酸探针固定到EDC处理的膜条上。样品DNA标记后与固定在膜条上的寡核苷酸探针在2ml离心管中杂交、检测获得杂交信号。结果发现这种方法可以检测到一个样品中的多个外源基因。最后利用上述基因的引物对样品DNA进行PCR检测,验证结果发现PCR检测结果与膜条杂交结果相一致,说明微管杂交法检测植物样品中多个外源基因的方法是可行的。新霉素磷酸转移酶(neomycin phosphotransferaseⅡ,NPTⅡ)基因是转基因植物中常用的标记基因,能够翻译出RNA,表达蛋白质。本研究以NPTⅡ基因靶标,检测植物样品外源基因表达的RNA产物。利用PCR方法扩增质粒pBI121中的NPTⅡ基因,然后将扩增产物标记成探针。RNA样品被固定到膜条上之后,在2ml离心管中与探针杂交,然后检测杂交信号。结果显示一条探针能够检测多个样品中外源基因的RNA产物。为了验证杂交结果的可靠性,利用NPTⅡ基因的引物对样品RNA进行RT-PCR检测,验证结果发现PCR检测结果与膜条杂交结果相一致,说明微管杂交法检测多个植物样品中外源基因RNA产物的方法是可行的。更多还原

【Abstract】 Fallowing the abundance of the genetically modified product (GMP), people pay more attention to the safety of GMP. Currently, we haven’t rendered reliable answer to the safety of GMP. It’s necessary to develop and improve the detection methods for the safety control of GMP.At present, there are kinds of distinguishing detection methods. It’s significant to develop a kind of functional, handy, effective detection method for the advancement of transgenic biotechnology and the safety control of GMP.According to the feature of exogenous genes in transgenic plants and combining the microtube hybridization, we established a group of functional, handy, effective detection method.Most of exogenous genes in genetically modified plants apply CaMV35S and NOS as their promoter and terminator. In this study, firstly we design pairs of primers according to the CaMV35S and NOS and amplify the plasmid pBI121 that contains CaMV35S and NOS by PCR and label the PCR products as probes. After clipping the nylon membrane into strips, the DNA of multi-samples is fixed on the strips, hybridized with probes and the hybridization signal is detected in 2ml microtubes. The result is that this method can detect multi-samples by one or two probes. At last it shows that the result of hybridization and PCR are unanimous after the hybridization signal is verified by PCR, and it shows that to detect exogenous genes in multi-samples is available by microtube hybridization.For a single transgenic plant, it contains not only the promoter and terminator, but also the goal-gene and mark-gene. In this study we use genetically modified cotton as the sample, and design a group of oligonucleotide probes according to the exogenous genes in genetically modified tobacco. Then the amination probes are fixed on nylon strips processed by EDC. The DNA of sample is hybridized with the oligonucleotide probes after being marked and the hybridization signal is detected in 2ml microtube. The result is that this method can detect multi-exogenous gene in one sample. At last the sample is detected by PCR with the primers of exogenous genes in genetically modified tobacco, and it finds that the result of hybridization and PCR are unanimous, and it shows that to detect multi-exogenous genes in one sample is available by microtube hybridization.Neomycin phosphotransferaseⅡ(NPTⅡ)gene usually used as labeled gene can be translated into RNA and be transcribed into protein. In this study we detect the RNA translated by the exogenous gene in multi-samples with NPTⅡprobe. Firstly we amplify the pBI121 that contains NPTⅡwith PCR and labeled the product of PCR to be probe. RNA samples are fixed onto the nylon strips, and then hybridized with the probe and the hybridization signal is detected in the 2ml microtube. It found that one probe can detect RNA translated by exogenous gene in multi-samples. To verify the hybridization signal, RNA samples of multi-samples are detected by RT-PCR with NPTⅡprimers. Then it finds that the result of hybridization and PCR are unanimous after the hybridization signal is verified by RT-PCR, and it shows that to detect RNA translated by exogenous genes in multi-samples is available by microtube hybridization.更多还原