转基因猪外源生长激素基因的定位研究——非同位素标记染色体原位杂交技术

近几年来发展起来的染色体原位杂交技术是进行基因定位研究的重要手段。它为进一步进行基因定点整合研究提供依据。本文采用非同位素标记染色体原位杂交技术对经显微注射得到的转基因猪进行了内源和外源猪生长激素(PGH)基因在染色体上定位的研究。 用BhlI对pSMTPGH注射基因(本室制备)进行单酶切,选取3.5kb片段以Dig进行标记。为了提高灵敏度,我们采用了胶体金标记抗体技术并结合使用银增加试剂。利用染色体的组型分析,对杂交点的分布进行了统计学分析,发现内源PGH基因位于12p~(11),与国外Thomsen,P.D.等报道的一致。插入的外源PGH基因呈随机分布,但对某一转基因猪来说,外源基因的插入是集中在某一染色体上。这一结果的发现为进行转基因猪外源基因定点整合提供了依据,也为研究外源基因整合位点和转基因猪表观性状的关系准备了条件。     

PCR技术定性检测转基因大豆方法的建立与研究

以转基因大豆(RRS品系)为材料,采用PCR技术,通过特异性引物和探针,对转基因大豆中的内源性基因Lectin和外源基因35S启动子、NOS终止子及CP4-EPSPS进行定性检测,旨在建立一种简单、快速、高效的检测方法,并将该方法用于我国进口自美国大豆实施检测,以评判是否为转基因大豆,为我国的商业谈判提供依据,同时也可满足百姓的知情权。     

家蝇介导的水稻基因逃逸风险评估(英文)

【目的】对转基因作物进行生态风险评估是大面积种植前的一个必要步骤,水稻Oryza sativa访花昆虫有上百种,包括家蝇Musca domestica。本研究旨在明确访花昆虫家蝇介导转基因水稻外源基因逃逸的风险。【方法】2010年,我们使用转基因水稻B1, B6和G8-7作为父本(花粉供体),用同源非转基因水稻Jiazao 935和Wuyunjing 7作为母本（花粉受体）,并用家蝇作为授粉昆虫,在浙江大学华家池和长兴试验基地开展了田间种植试验,对收割的后代水稻种子进行室内种植培养,对种苗用潮霉素B和草甘膦处理进行转基因杂交种检测,对存活植株进行潮霉素和草甘膦抗性基因PCR检测,测试家蝇介导的转基因水稻外源基因逃逸频率。【结果】对浙江两个测试基地3个转基因水稻品种共计超过216 500粒后代水稻种子进行的检测及结果表明,在毗邻区域杂交种少,家蝇授粉区和无家蝇授粉区转基因水稻外源基因向非转基因水稻逃逸频率均较低(0~0.64%)。【结论】家蝇介导的转基因水稻外源基因逃逸频率较低,家蝇没有增加转基因水稻外源基因逃逸的风险。     

根癌农杆菌介导的转新城疫病毒融合蛋白基因水稻植株的获得

利用转基因植物作为生物反应器可以表达重组蛋白、生产外源蛋白质,也可以成为动物疫苗的廉价生产系统。以编码新城疫病毒融合蛋白(NDV-F)的基因为外源基因,以玉米泛素蛋白(Ubi)启动子为启动子,以潮霉素磷酸转移酶(HPT)基因作为选择标记基因,β-半乳糖苷酸酶(GUS)基因作为报告基因构建了适宜于农杆菌介导转化水稻的表达质粒pUNDV,并通过农杆菌介导转化水稻,获得了多株转基因植株。通过PCR分析和GUS活性检测,证实含有NDV-F基因的T-DNA已整合到水稻核基因组中,为研制廉价安全的转基因水稻新城疫基因工程疫苗奠定了基础。     

抗凝血酶Ⅲ转基因山羊外源基因拷贝数及其蛋白表达量的测定

目的:检测抗凝血酶Ⅲ(ATⅢ)转基因山羊外源基因的拷贝数及ATⅢ蛋白的表达量,分析拷贝数与蛋白表达量的相关性。方法:用Primer 5.0软件设计外源基因ATⅢ及山羊管家基因GAPDH的引物,以倍比稀释的标准品进行实时荧光定量PCR,制作标准曲线,通过标准曲线计算外源基因拷贝数;通过酶显色底物法检测ATⅢ蛋白的含量。结果:依据ATⅢ和GAPDH基因标准曲线方程计算得到的山羊个体间外源基因的拷贝数相差很大,最少的为5,最多为25,且不同转基因山羊间ATⅢ表达水平的差别达10倍以上。结论:外源基因表达水平在受到拷贝数的影响外,也受到整合位点等其他因素的重要影响。     

应用荧光原位杂交技术检测人β^E珠蛋白基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态。检测结果表明,外源人β^E珠蛋白基因已稳定地整合于小鼠染色体上,FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合反进行染色体定位检测。     

转基因定量检测的不确定度研究

目前,欧盟、日本对转基因产品都实行基于转基因含量(阈值)强制标识制度。世界各国都采用实时荧光PCR方法来开展食品成分的相对定量检测工作,以样品的内、外源基因的拷贝数之比来近似代表样品中的转基因质量分数。为了便于用户正确理解检验结果,在转基因定量检测结果报告中必须报结果的不确定度,分析了转基因定量的不确定度来源,参照化学分析中的有关方法,给出了转基因定量检测中外源基因和内源基因的标准曲线的不确定度测算公式,并以转基因大豆为试材,利用方法的室内验证数据进行不确定度计算,可供相关实验室参考。     

转基因小鼠中外源基因部分内含子序列的缺失及其对转录的影响

利用PCR扩增及PCR测序在显微注射法产生的转基因小鼠中发现,整合在小鼠染色体上的肌球蛋白轻链2启动子(myosinlightchain2promoter,MLC2)-糜酶(chymase)外源融合基因存在两种形式,一种为全长的融合基因,另一种在糜酶结构基因的第一内含子中缺失了213bp的序列。RT-PCR结果表明,缺失了部分内含子序列的外源融合基因不能在转基因小鼠心脏中表达.而全长的外源融合基因则能较高水平地表达,竞争性PCR定量实验表明在200ng心脏总RNA反转录产物中约含5.05(±1.38)×106个糜酶cDNA分子。上述结果表明,糜酶结构基因的第一内含子可能对MLC2-糜酶基因的表达具有调控作用。     

毕赤酵母基因工程菌胞内AOX酶的检测方法

巴斯德毕赤酵母(Pichia pastoris)作为外源基因的表达宿主,已成功表达出一系列胞内和胞外蛋白[1～6],并已建立起了一套较成熟的发酵工艺.巴斯德毕赤酵母基因工程菌的外源基因,由胞内AOX酶(乙醇氧化酶)基因启动子调控.在非甲醇碳源条件下(如甘油或葡萄糖),AOX酶基因表达被抑制,外源基因也处于不表达状态.而以甲醇为唯一碳源时,AOX酶在胞内大量合成,同时外源基因被调控表达.在一般情况下,AOX酶的变化直接反映了外源基因的表达状况,因此通过分析检测胞内AOX酶的含量和变化速率,就可以确定外源基因所处的状态.     

多重PCR技术快速检测烤后烟叶转基因成分

为提高烟叶转基因成分检测效率,建立了烤后烟叶中3种外源基因成分的多重PCR检测技术体系,通过优化该反应体系中的Mg2+、d NTP、r Taq酶、引物浓度及退火温度等参数,实验最适条件为将4对相等摩尔浓度引物预先按1∶1∶1∶1(V/V)混合,在Mg2+为1.5 mmol/L、d NTP为125μmol/L、r Taq酶1 U、混合引物1μmol/L,DNA 100 ng,退火温度65℃,循环数35个的反应条件下,在一次PCR反应中便可同时检测烟草内源基因NR,外源基因35S启动子、NOS终止子、NPT II筛选标记基因。优化后的反应体系能够有效地检测出转基因烤后烟叶百分比含量为0.9%(V/V)的转基因成分。     

转基因生物及其产品检测技术和标准化

随着转基因生物及其产品的大规模商业化以及消费者对其安全性的担心,很多国家和地区纷纷出台包括转基因产品标签制度在内的转基因生物安全管理的法律法规,为保障转基因产品标签制度的实施以及消费者知情权和选择权,转基因产品检测分析方法及其标准化研究受到人们广泛重视。目前,国内外常用的转基因产品检测方法主要包括针对转基因产品中的目的核酸DNA分子或者其编码的蛋白质分子,本文将对转基因生物及其产品检测方法及其标准化的进展及发展趋势做一概述。     

利用决策树方法建立转基因植物环境生物安全评价诊断平台

转基因技术及其产品是解决世界粮食问题的重要途径之一, 但是包括食品和环境安全在内的转基因生物安全评价是转基因技术及其产品商品化应用的前提和保证。现有的人为生物安全评价方法存在着一定的不足, 难以应对数量日益增加和内容日趋复杂的转基因产品的安全评价需求, 因此找寻一种客观和高效的评价方法势在必行。决策树(decision tree)方法是现今广泛使用的数据挖掘和分析的决策方法之一, 通过将需要解答问题的层层分解并分别解决, 最终得到理想的决策结果, 在处理复杂问题方面具有独特的优势。本文旨在通过介绍决策树的概念、特性、种类及其构建方法, 探索将决策树方法应用于建立转基因植物环境生物安全评价诊断平台的可行性, 并分析构建的诊断平台在高效、准确和客观地进行转基因植物环境生物安全评价, 以及对新一代转基因产品环境安全性的预测和普及环境安全知识等方面的优势, 为进一步推动转基因技术的发展和转基因产品的安全利用奠定基础。     

Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize

Detection of nonauthorized genetically modified organisms (GMOs) has always presented an analytical challenge because the complete sequence data needed to detect them are generally unavailable although sequence similarity to known GMOs can be expected. A new approach, differential quantitative polymerase chain reaction (PCR), for detection of nonauthorized GMOs is presented here. This method is based on the presence of several common elements (e.g., promoter, genes of interest) in different GMOs. A statistical model was developed to study the difference between the number of molecules of such a common sequence and the number of molecules identifying the approved GMO (as determined by border-fragment-based PCR) and the donor organism of the common sequence. When this difference differs statistically from zero, the presence of a nonauthorized GMO can be inferred. The interest and scope of such an approach were tested on a case study of different proportions of genetically modified maize events, with the P35S promoter as the Cauliflower Mosaic Virus common sequence. The presence of a nonauthorized GMO was successfully detected in the mixtures analyzed and in the presence of (donor organism of P35S promoter). This method could be easily transposed to other common GMO sequences and other species and is applicable to other detection areas such as microbiology.     

Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection

Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.     

GMDD: a database of GMO detection methods

Background  

Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed.     

A semi-quantitative approach to GMO risk-benefit analysis

In many countries there are increasing calls for the benefits of genetically modified organisms (GMOs) to be considered as well as the risks, and for a risk-benefit analysis to form an integral part of GMO regulatory frameworks. This trend represents a shift away from the strict emphasis on risks, which is encapsulated in the Precautionary Principle that forms the basis for the Cartagena Protocol on Biosafety, and which is reflected in the national legislation of many countries. The introduction of risk-benefit analysis of GMOs would be facilitated if clear methodologies were available to support the analysis. Up to now, methodologies for risk-benefit analysis that would be applicable to the introduction of GMOs have not been well defined. This paper describes a relatively simple semi-quantitative methodology that could be easily applied as a decision support tool, giving particular consideration to the needs of regulators in developing countries where there are limited resources and experience. The application of the methodology is demonstrated using the release of an insect resistant maize variety in South Africa as a case study. The applicability of the method in the South African regulatory system is also discussed, as an example of what might be involved in introducing changes into an existing regulatory process.     

The development and standardization of testing methods for genetically modified organisms and their derived products

As the worldwide commercialization of genetically modified organisms (GMOs) increases and consumers concern the safety of GMOs, many countries and regions are issuing labeling regulations on GMOs and their products. Analytical methods and their standardization for GM ingredients in foods and feed are essential for the implementation of labeling regulations. To date, the GMO testing methods are mainly based on the inserted DNA sequences and newly produced proteins in GMOs. This paper presents an overview of GMO testing methods as well as their standardization.     

国际上主要国家和地区农业转基因产品的标识制度

转基因标识是表明产品含有转基因成分或者由转基因生物生产、加工而成的一种标识。随着全球转基因技术研发和应用的不断推进,国际上对农业转基因产品的标识管理更加关注与重视。通过阐述农业转基因产品标识制度的形成与发展过程,研究欧盟、美国、加拿大、日本、韩国等主要国家和地区的转基因产品标识管理制度,总结出成分关注标识、过程关注标识、自愿标识、强制性标识、定性标识、定量标识、全面标识、目录标识等不同标识类别的特点与利弊,并分析了国际上关于标识豁免及阴性标识等方面的政策规定,为我国的农业转基因产品标识管理工作提供了启示与参考。     

Implications of natural propagule flow for containment of genetically modified forest trees

Propagule flow in populations of virtually all organisms has importance for both the genetic cohesion of the species and for its interaction with natural selection. It’s relevance` for the deployment of genetically modified organisms (GMOs) is that propagules can be expected to move, under a wide range of circumstances, and will carry transgenic elements with them. Any consideration of the potential risks of deploying GMOs in the wild must include an assessment of how far and how fast introduced elements are transferred to surrounding conspecific (and sometimes congeneric) populations. In practice, we need estimates of the rates/distances of both pollen and seed movement. There are analytical methods to characterize seed (maternity), pollen (paternity), and established offspring (parent-pair) data, but spatial limitations restrict the area that one can study, and these approaches require modification for application to propagule flow in GMOs. We can apply indirect methods to estimate male gamete dispersal based on pollen pool analysis for single mothers, when some degree of precision can be sacrificed in return for compensating gains in the spatial coverage, but the loss of precision is problematic for GMO tracking. Special methods have been developed for GMO tracking, and we shall show how to assess spatial movement of both transgene-carrying seeds and pollen and will illustrate with an example from Brassica napus, a well-studied crop species. Institute of Forest Biotechnology conference on Ecological Risks Associated with the Products of Forest Biotechnology     

Testing the interaction between analytical modules: an example with Roundup Ready<Superscript>®</Superscript>soybean line GTS 40-3-2

Background  

The modular approach to analysis of genetically modified organisms (GMOs) relies on the independence of the modules combined (i.e. DNA extraction and GM quantification). The validity of this assumption has to be proved on the basis of specific performance criteria.