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关于转基因技术或者转基因农作物的

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genetically modified organism (GMO);oligonucleotide microarray;GMO screening;multiplex-PCR;event-specific;integration junction sequence
随着转基因农作物检测技术的不断发展，PCR技术已经成为最主要的检测方法之一。但是常规的PCR每次只能扩增一个基因，而单独的多重PCR和常规电泳相结合的技术由于受多重PCR技术自身限制，每次扩增的基因数目也很有限，因此不能满足当前转基因农作物检测的要求。 寡核苷酸芯片是一种在基片上点有若干寡核苷酸探针的基因芯片，每个点都含有序列特异的探针以和感兴趣的基因相互补结合。因此，寡核苷酸芯片可以和多重PCR技术相结合用来同时检测多种目标基因。 在这篇论文中，我们开发了多重PCR技术和DNA芯片技术相结合的方法，在同一个反应中同时检测多个目标基因，以达到检测转基因农作物的目的。 我们设计了两大类芯片，其中一类用来检测样本是否是转基因农作物，另一类用来检测样本来自于哪一种转基因品系。 对于第一类芯片，我们使用了20种探针用以检测转基因农作物，按照具体功能可以分为三种：第一种，根据一些普通元件例如启动子、终止子、报告基因等来检测是否为转基因农作物；第二种，根据目标基因例如抗除草剂或是抗虫害基因来做特定基因的检测确认；第三种，根据物种特异性基因来检测该样本来自于哪种作物。为保证方法的有效性，设立不同的阳性对照和阴性对照来鉴定该实验方法，并且通过常规PCR反应和测序来进一步验证。结果表明这种方法可以快速识别样本是否为转基因农作物，并且花费少，效率提高。这种方法可以检测目前95％以上的转基因作物，并且对于大豆的最低检出率是0.5％，玉米是1％。 对于第二类芯片，根据唯一的、品系特异的宿主与插入片断间连接区域的基因序列，通过多重PCR和寡核苷酸芯片相结合的技术，以达到检测出样本来自于哪种转基因品系的目的。转基因农作物商品大豆(GTS 40-3-2)和六种转基因玉米(MON810，MON863，Bt176，Bt11，GA21和T25)通过这种方法进行检测。结果表明对于这些转基因大豆和玉米，该方法均可以适用。 以上两种芯片被证明是一种新的转基因农作物检测的常规方法。
With the increasing development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection. And an oligonucleotide microarray is a glass chip to the surface of which an array of oligonucleotides was fixed as spots, each containing numerous copies of a sequence-specific probe that is complementary to a gene of interest. So it is used to detected tens or more targets synchronously. In this research, we developed a multiplex polymerase chain reaction (multiplex-PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a consecutive reaction to detect a GMO. We designed two types DNA microarray, one to detect whether the sample was from GMO, the other to detect which event the sample was from. For the first type, there are a total of 20 probes for detecting a GMO in a DNA microarray which can be classified into three categories according to their purpose: the first for screening GMO from un-transgenic plants based on the common elements such as promoter, reporter and terminator genes; the second for specific gene confirmation based on the target gene sequences such as herbicide-resistance or insect-resistance genes; the third for species-specific genes which the sequences are unique for different plant species. To ensure the reliability of this method, different kinds of positive and negative controls were used in DNA microarray. Commercial GM soybean, maize, rapeseed and cotton were identified by means of this method and further confirmed by PCR analysis and sequencing. The results indicate that this method discriminates between the GMOs very quickly and in a cost-saving and more time efficient way. It can detect more than 95％of currently commercial GMO plants and the limits of detection are 0.5％for soybean and 1％for maize. For the second type, an event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity using multiplex-PCR together with oligonucleotide microarray. Commercial GM soybean (GTS 40-3-2), six GM maize events (MON810, MON863, Bt176, Bt11, GA21, and T25) were detected by this method. The results indicate that it is a suitable method for the identification of these GM soybean and maizes. This method is proved to be a new method for routine analysis of GMOs.

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转基因技术与传统技术的关系 自从人类耕种作物以来，我们的祖先就从未停止过作物的遗传改良。过去的几千年里农作物改良的方式主要是对自然突变产生的优良基因和重组体的选择和利用，通过随机和自然的方式来积累优良基因。遗传学创立后近百年的动植物育种则是采用人工杂交的方法，进行优良基因的重组和外源基因的导入而实现遗传改良。 因此，转基因技术与传统技术是一脉相承的，其本质都是通过获得优良基因进行遗传改良。但在基因转移的范围和效率上，转基因技术与传统育种技术有两点重要区别。第一，传统技术一般只能在生物种内个体间实现基因转移，而转基因技术所转移的基因则不受生物体间亲缘关系的限制