【作者】 刘建越；

【导师】 邓欣； 高必达； 康林；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2006， 硕士

【摘要】 自1983年世界首例转基因烟草作物问世以来，转基因技术在世界范围内对农作物的生产和遗传改良产生深刻的影响，但基因工程可能导致我们无法预测的植物性状改变，有时这种改变在短时期内甚至难以觉察，但却可能给环境、生态系统、人身健康带来巨大的风险。为了加强对转基因作物及产品的管理，各国都出台了各种管理办法和政策。因此，开展转基因检测方法研究具有重大的社会意义和实际意义。本研究针对转基因番木瓜和番茄，分别建立了常规定性PCR和实时荧光PCR检测技术。主要研究内容如下： 转基因番木瓜和番茄常规PCR检测技术 1．选用核酮糖1，5-二磷酸羧化酶／加氧酶大亚基基因(ribulose bisphosphate carboxylase／oxygenase large subunit)作为植物通用的内源对照基因，设计通用引物，采用番木瓜，番茄，草莓进行试验研究。结果表明，RBCL基因可以作为转基因番木瓜和番茄的内源基因。 2．针对转基因番木瓜和番茄中常见的外源基因序列CaMV35S启动子序列，NOS终止子序列，NPTII基因，GUS基因，CH5B基因，OSMOTIN基因，PRSV-CP基因，PRSV-RP基因分别设计特异引物，进行了普通定性PCR检测研究。并优化了PCR反应体系。 3．设计了35S／NOS引物，35S／CP引物，实现了一对引物定性PCR可以检测到2个或以上外源基因片断。 4．建立了双重和三重定性PCR的检测方法，实现了“一管多检”。并优化了PCR反应体系。 5．同时对PRSV-CP基因，PRSV-RP基因，CH5B基因，OSMOTIN基因，GUS基因的全序列进行克隆测序，提取其阳性质粒作为转基因检测过程中的阳性对照物质；测定出不同转PRSV-CP基因番木瓜品系中35S和CP基因之间的不同边界连接序列。 转基因番木瓜和番茄实时荧光PCR检测技术 6．首次利用SYBR Green实时荧光PCR进行转基因检测，并建立了SYBR Green实时荧光PCR对转基因水果通用内源基因RBCL基因，外源CaMV35S基因，NOS基因，NPTII基因，PRSV-CP基因，PRSV-RP基因，CH5B基因，OSMOTIN基因，GUS基因的检测。 7．建立Taqman探针实时荧光PCR方法对转基因水果通用内源基因RBCL基因，外源CaMV35S基因，NOS基因，NPTII基因，PRSV-CP基因，PRSV-RP基因，CH5B基因，OSMOTIN基因，GUS基因的检测；同时也建立了双重Taqman实时荧光PCR检测转基因番木瓜Rainbow，并优化了双重实时荧光-PCR反应体系。 通过以上研究，建立了多套适合于各进出境口岸检测转基因番木瓜和番茄的定性PCR技术。更多还原

【Abstract】 Since the first genetically modified crop tobacco came into being in the world in 1983, the genetically modified technology has had a profound influence on the yield and quality of the crops and created enormous economic and social benefits. However, genetic engineering may lead to unpredictable plant property changes that even difficult to be aware in short-term, may bring the enormous risks for environment, ecosystem, personal health and so on. In order to strengthen the control of transgenic crops and products, many countries have issued their own managements and policies. Thus, the detection for genetically modified crops is urgently needed. Based on that, this research has established the methods of common PCR and Real-time fluorescence PCR respectively of genetically modified papaya and tomato, The main contents of the paper are as follows: Genetically modified papaya and tomato common qualitative PCR detection technology1. Selected ribulose bisphosphate carboxylase/oxygenase large subunit gene (RBCL gene) as endogenesis reference gene and design universal primes, choosed papayas, tomatoes and strawberrys as the experiment materials . The result indicated: RBCL gene can be regarded as endogenous gene in the genetically modified papaya and tomato.2. Aimed at the common extraneous sequence of genetically modified papaya and tomato: CaMV35S promoter sequence, the NOS termination sequence, the NPTII gene, the GUS gene, the CH5B gene, the OSMOTIN gene, the PRSV-CP gene and the PRSV-RP gene, then designed separately specially primes, acted as the ordinary qualitative PCR examination research, and optimized the PCR system.3. Designed primes of 35S/NOS and 35S/CP, carried out a pair primes to check 2 or more of the extraneous fragment, and optimized the PCR system.4.Established dual and trial qualitative PCR detection methods, and optimized the PCR system.5.Cloned and sequenced the PRSV-CP gene, the PRSV-RP gene, the CH5B gene, the OSMOTIN gene, the GUS gene, extracted their plasmids as the positive template; and also confirmed different boundary connection sequence in 35S and the PRSV-CP gene between genetically modified papaya of the PRSV-CP strains.更多还原