转基因生物检测技术研究进展

转基因技术的发展,促进了转基因生物（genetically modified organisms,GMO）的数量、多样性不断增加,给GMO检测工作提出了新的挑战,同时也促进了GMO检测方法的发展。GMO检测新方法突破了传统方法中一次只能分析一个靶目标的限制,新方法一次可以检测多个靶目标,向高通量检测的方向发展。作者对最新报道的GMO检测方法作一概述,主要包括实时荧光PCR多靶分析方法,锁式探针－基因芯片方法、液相芯片方法,多重PCR－毛细管电泳方法,生物发光检测方法,以及为GMO检测提供方法决策的支持系统（DSS、GMO示踪）等。     

多重PCR结合变性高效液相色谱技术转基因小麦检测方法的建立

为了满足对小麦转基因成分高通量快速检测的需要,建立了一种新的转基因小麦检测方法。通过对不同稀释倍数的转基因小麦品系B7361中的内源基因Wx012、外源基因ubiquitin、bar、nos、uidA进行单一PCR和多重PCR扩增,应用琼脂糖凝胶和变性高效液相色谱（DHPLC,denaturing high performance liquid chromatography）技术分离PCR 产物,进行灵敏度分析,并用非转基因小麦京花1号籽粒、大豆籽粒、麦片、转基因小麦B7361加工成的油炸制品为样本,对建立的检测体系进行检测。结果表明,样品经多重PCR扩增和DHPLC分离后,能够得到准确可靠的转基因图谱。与凝胶电泳方法比较,本研究所建立的多重PCRDHPLC具有更高的检测通量和灵敏度(能够同时检测5个基因,灵敏度达到1 ng·μL^-1),能够满足小麦转基因成分的高通量快速检测的要求,同时也为转基因产品检测提供了一种新的技术手段。     

单倍体玉米育种方法在转基因玉米育种中的应用

用单倍体育种方法获取转基因玉米纯合自交系,减少花粉在环境中释放量,避免基因漂移影响生态环境,绝大部分过程不受区域限制,不受限制区域无花粉散失。     

Prediction of the combined effect of various GM contamination sources of seed: A case study of oilseed rape under Danish conditions

Abstract

The potential GM contamination at each step in seed production can be predicted for conditions that correspond to a worst-case scenario. When the combined effect of the various GM contamination stages is calculated, the worst-case predictions of the different stages are normally added. This additive procedure to estimate the combined effect of GM contamination may be relevant to estimate the combined effect where a single farm is concerned, but may result in estimation errors when harvested seeds from a large number of farms are mixed. In this study, the consequences of treating the different stages of GM contamination as independent stochastic effects on the combined GM contamination have been estimated. A case study of GM contamination in organic and conventional oilseed rape crops under Danish conditions indicated that the greater part of GM contamination occurred at farm level and much less during transport and storage. Generally, it may be concluded that it is important to consider this uncertainty in the estimates of adventitious presence at each seed production step and also whether the different steps are independent.     

Centres of Crop Diversity and/or Origin, Genetically Modified Crops and Implications for Plant Genetic Resources Conservation

The concept of centres of crop diversity and/or origin of agriculture is briefly reviewed. The conservation status of crop genetic resources, either ex situ or in situ, cultivated or wild, has been assessed for species of the Central American and Mexican centre, demonstrating that that region is indeed one of the important centres of crop diversity for human kind. Furthermore, biotechnological developments with regard to the creation and spread of genetically modified crops have been analyzed. The likelihood of unintentional introgression of genetically modified traits into conventional seed lots, crops as well as into germplasm collections have been assessed. Related biosafety measures as well as the possible implications of intellectual property rights on transgenic crops and/or genes are being discussed vis-a-vis the possible implications they might have for germplasm management. The Central American crop genetic resources situation has been used as a “case study” to illustrate the potential impact of the spreading of GM varieties on the genetic diversity in genebanks and farmers’ fields and the need for effective and efficient conservation efforts. Conservation management strategies and practices are being proposed of mitigate the potential negative impact of GM crops on the conservation efforts.     

食用大豆油中DNA的快速提取和转基因成分定性检测

转基因生物（GMO）制品中转基因成分检测的难点在于深加工产品中DNA的提取。本试验以食用大豆油为研究对象,研究操作简单、费用低廉的大豆深加工产品DNA提取的新方法。利用大豆内源基因Lectin设计引物,对所获得的DNA进行PCR检测,结果证实通过本方法提取的DNA纯度足以进行PCR反应。利用转基因大豆转化载体序列设计引物,进一步对其转基因成分进行检测,结果表明,用本方法从转基因大豆油中提取的DNA含有转基因成分。     

35S启动子定量PCR非同源竞争模板的构建

利用35S启动子特异引物,以大肠杆菌基因组DNA为异源模板,经低严谨度PCR扩增并回收克隆适宜DNA片段,构建了35S启动子的非同源竞争对照。测序结果表明,该片段与35S启动子相应序列即靶序列除两端引物序列完全相同外,其他部分没有同源性。利用该非同源竞争对照,建立了转基因玉米35S启动子的QC-PCR技术体系,并进行了校正。     

深加工转基因产品检测技术的研究进展

对国内外深加工产品转基因成分的检测技术进行了综述,重点介绍了目前各类转基因检测方法的发展情况,特别是深加工产品转基因检测技术的研究现状和发展趋势、制约因素、需要解决的关键技术。提出了今后在深加工产品转基因检测技术上的重点研究方向。     

美国转基因生物监管机制探究

美国是世界上转基因生物技术和商业化最为成熟的国家,其代表的以产品为导向的监管政策在全球独树一帜.美国坚持“实质等同性”和“个案分析”作为转基因生物的监管原则,以《生物技术的监管协调框架》为基本纲领,建立了以农业部、食品与药品管理局、环保局三大管理机构为中心的转基因生物监管体系,并制定了一系列相关法律和监管流程.介绍了美国转基因生物监管的基本理念和原则,综述了转基因生物监管机构及其职责,在此基因上总结了转基因生物监管的特点.     

转基因油菜筛查检测策略研究

根据收集到的国内外已商业化转基因油菜品种的基因元件信息,构建了油菜转基因检测分子特征矩阵,在分析基因元件使用频率的基础上,建立了转基因油菜的筛查检测策略。研究发现,利用P-CaMV35S、P-FMV35S和bar这三个元件组合,对现有转基因油菜品种的筛查检出率可达100%。将P-CaMV35S、P-FMV35S、bar、CP4-¬EPSPS、PAT和T-NOS组合,每个转化事件可检出至少两次,增加了检测的可靠性。同时,分析了不同转化事件中同名基因元件的序列差异,进一步验证了分子特征矩阵筛查方法的可行性。