转基因组学分析技术研究进展

转基因技术备受世人关注,且转基因作物关乎人体健康和生态环境,因此对转基因作物的安全评价地位极其重要,各种评价方法也在不断前进与发展。组学分析技术成为安全评价工作的新思路。本文主要论述了转基因组学分析技术的必要性,组学分析技术的发展,世界主要转基因作物组学评价发展情况及未来转基因组学分析技术的发展趋势,以期对转基因安全评价工作提供新的思路和方向。     

Bt抗虫基因在作物中的应用

基因工程是现代生物技术在生物农药研制方面的重要手段之一,其中以苏云金芽芽孢菌（Bt）杀虫蛋白为基础的转基因作物是该类研究的热点。由于转Bt基因作物在抗虫、减少化学污染方面的良好性能,玉米、棉花等转基因作物已在世界范围内商业化。我国转Bt基因作物研究起步晚,但发展迅速。目前已先后批准了甜椒、番茄以及番木瓜3种作物作为商业化生产。与此同时,Bt转基因作物在基因漂移、抗虫性以及生物安全性方面存在争论,需要更多的实验支持和更深入细致的研究。     

Detection of genetically modified organisms in foreign-made processed foods containing corn and potato

Investigations of the validity of labeling regarding genetically modified (GM) products were conducted using polymerase chain reaction (PCR) methods for foreign-made processed foods made from corn and potato purchased in the Tokyo area and in the USA. Several kinds of GM crops were detected in 12 of 32 samples of processed corn samples. More than two GM events for which safety reviews have been completed in Japan were simultaneously detected in 10 samples. GM events MON810 and Bt11 were most frequently detected in the samples by qualitative PCR methods. MON810 was detected in 11 of the 12 samples, and Bt11 was detected in 6 of the 12 samples. In addition, Roundup Ready soy was detected in one of the 12 samples. On the other hand, CBH351, for which the safety assessment was withdrawn in Japan, was not detected in any of the 12 samples. A trial quantitative analysis was performed on six of the GM maize qualitatively positive samples. The estimated amounts of GM maize in these samples ranged from 0.2 to 2.8%, except for one sample, which contained 24.1%. For this sample, the total amount found by event-specific quantitative analysis was 23.8%. Additionally, Roundup Ready soy was detected in one sample of 21 potato-processed foods, although GM potatoes were not detected in any sample.     

转基因食品安全性评价研究进展

自1996年以来,转基因作物的大规模商业化生产为人们带来了巨大的社会经济效益,但是转基因技术存在一定的风险性,因此加强转基因食品的安全性评价和标准化管理显得尤为迫切和重要。本文从营养学、毒理学、过敏性等方面综述了转基因食品的食用安全性评价,并多角度探讨了转基因食品安全性评价的关键问题,包括用不同动物实验评价转基因食品的食用安全性,新型转基因植物的安全性评价,以及转基因食品的食用安全标准化等,以期使读者对转基因食品的食用安全性有更加系统、全面的了解。     

Regulatory policy on genetically modified breeding stack in key countries and the current status in Korea

With an increasing interest and demand for biotechnology crops in agriculture worldwide, genetically modified (GM) breeding stacks produced by conventional breeding of previously approved GM single events remain popular for farmers in GM crop cultivation countries. However, regulations on stacks vary in each country. Currently, Korea requires approval for all breeding stacks intended for cultivation. To determine whether the stack is subject to a full safety assessment as a new GM crop, molecular characterization, protein expression, composition analysis, and agronomic characterization data are required. Korea’s regulatory policy on stacks has not adopted the high-covers-low concept; therefore, subcombinations of already approved higher combination events are subject to breeding stack review if any subcombination was purposefully bred for cultivation use. This review will help promote the efficient management of GM breeding stacks in Korea in the future.

     

Molecular Identification of Four Genetically Modified Maize (Bt11, Bt176, Mon810 and T25) by Duplex Quantitative Real-Time PCR

In response to the increasing number of genetically modified (GM) events released on the market, control laboratories explore various strategies to simplify and reduce the number of tests needed to characterise the content in genetically modified organism (GMO) of a given sample. Lastly, multiplexing is considered as one of the possible ways to decrease the time and cost of analysis. Here, we report the development of four duplex polymerase chain reaction (PCR) tests for the identification and the quantification of four maize transformation events from which commercial lines have been authorised in Europe namely, Bt11 and Bt176 (Syngenta, DE, USA), Mon810 MaisGard? (Monsanto, MO, USA) and T25 Liberty Link? (Bayer CropScience, Monheim, Germany). The duplex PCR tests combine a maize-specific PCR test hybridising in the Adh1 locus with an event-specific detection system designed on a junction fragment for each of these four GM maize. Real-time PCR tests, suitable to comply with the European regulation, were designed by using Taqman® chemistry.     

Horizontal Transfer of DNA From GM Crops to Bacteria and to Mammalian Cells

ABSTRACT: The evidence for horizontal gene transfer from genetically modified (GM) crops to bacteria and mammalian cells is reviewed. The conclusion is that while horizontal gene transfer can and has occurred, such events are rare. However, even rare events may have an ecological impact, and thus the genes encoded by DNA introduced into a GM plant should be the focus of biosafety considerations. In the case of antibiotic resistance markers, the chances of increasing the fitness of any bacteria acquiring the genes from a GM plant is remote. There is also no known risk associated with the remote possibility that mammalian cells could be transformed with these genes and express the proteins.     

Adaptation of the ToxRTool to Assess the Reliability of Toxicology Studies Conducted with Genetically Modified Crops and Implications for Future Safety Testing

To determine the reliability of food safety studies carried out in rodents with genetically modified (GM) crops, a Food Safety Study Reliability Tool (FSSRTool) was adapted from the European Centre for the Validation of Alternative Methods’ (ECVAM) ToxRTool. Reliability was defined as the inherent quality of the study with regard to use of standardized testing methodology, full documentation of experimental procedures and results, and the plausibility of the findings. Codex guidelines for GM crop safety evaluations indicate toxicology studies are not needed when comparability of the GM crop to its conventional counterpart has been demonstrated. This guidance notwithstanding, animal feeding studies have routinely been conducted with GM crops, but their conclusions on safety are not always consistent. To accurately evaluate potential risks from GM crops, risk assessors need clearly interpretable results from reliable studies. The development of the FSSRTool, which provides the user with a means of assessing the reliability of a toxicology study to inform risk assessment, is discussed. Its application to the body of literature on GM crop food safety studies demonstrates that reliable studies report no toxicologically relevant differences between rodents fed GM crops or their non-GM comparators.     

Monitoring of Bt11 and Bt176 genetically modified maize in food sold commercially in Brazil from 2005 to 2007

BACKGROUND: The first genetically modified (GM) maize lines were approved for trading in Brazil after December 2007 and they were T25, MON810, Bt11, NK603 and GA21. The polymerase chain reaction (PCR) method was employed to monitor the presence of Bt11 and nested PCR was used to detect the presence of Bt176 in 81 maize‐derived products (maize flour, corn meal, maize flour flakes and polenta) that were sold in Brazilian market from 2005 to 2007, before the release of GM maize in Brazil. RESULTS: The PCR detection limit for Bt11 was 10 g kg^?1 and for nested PCR of Bt176 it was 1 g kg^?1. All Brazilian samples analyzed showed no positive signal for these GM maize events. CONCLUSION: Bt11 and Bt176 GM maize lines were not detected by specific PCR in 81 maize‐derived food samples sold in Brazil from 2005 to 2007, before the commercial release of GM maize in Brazil. These Brazilian food industries were in compliance with the rules stipulated by the current legislation with respect to consumer requirements about GMO labeling. Copyright © 2010 Society of Chemical Industry     

Monitoring of genetically modified food and feed in the Tunisian market using qualitative and quantitative real-time PCR

Genetically modified organisms (GMO) invade more and more the agricultural production in the world. Although there are no legislations on GM labeling and cultivation of GM crops in Tunisia, the present study aims to check the status of GMO in Tunisian market using qualitative and quantitative real time-PCR (QRT-PCR). Three-hundred-sixty five samples were collected and different DNA extraction methods were adapted and optimized. Specific primers targeting 35S promoter from Cauliflower mosaic virus (CaMV) and nopaline synthase terminator from Agrobacterium tumefaciens (At) were used for the detection of the GMO insert and Taxon specific primers for the detection of plant species. Validated Taqman® probes (EU-RL) targeting event specific regions of the maize events MON810, Bt11, and the soybean event RRS were used for the quantification studies. Seven food and feed products showed different amounts of RRS (1.9%), MON810 (2.1%), and Bt11 (1.6%). The results demonstrate for the first time the presence of GMO in Tunisian markets reinforcing the need for the development of accurate quantitative methods in routine analyses.     

Comparative analysis of genetically modified brown rice with conventional rice varieties for the safety assessment

Compositional analysis of genetically modified (GM) rice (OsCK1), non‐GM comparator and reference rice as commercial rice was conducted for the safety assessment. The tolerance intervals (TI) of commercial rice were also set to illustrate compositional variability. GM rice, non‐GM rice and reference rice were compared using principal component analysis (PCA) and partial least squares‐discriminant analysis (PLS‐DA) to assess the impact of genetic modification vs. environmental influence on the rice component. Seventeen components in forty‐six analysed components were significantly different between GM rice and non‐GM rice. But, the mean ranges of the seventeen components were all within those of the reference TIs. These results demonstrate that the nutrition components of GM rice (OsCK1) fell within the range of natural variability and were biologically equivalent to non‐GM rice. In addition, multivariate analysis by PCA and PLS‐DA revealed that environmental factors such as growing locations were important due to the variability of the rice metabolite profiles. These comparisons lead to the conclusion that disease‐resistant transgenic rice (OsCK1) is compositionally equivalent to and as safe and nutritious as conventional rice varieties grown commercially.     

The integrated risk assessment of transgenic rice Oryza sativa: A comparative proteomics approach

The identification of unintended effects resulting from genetic modification processes is an important but difficult aspect of evaluations of the biosafety of genetically modified organisms (GMOs). Non-targeted techniques offer considerable potential for improving the detection of unintended effects of genetic modification in crop plants. In this study, total seed protein expression patterns of two strains of transgenic rice (Bt rice and PEPC rice) were examined using a comparative proteomics approach with two-dimensional electrophoresis (2-DE), and differences determined comparing to each line’s non GM counterpart. The results indicated that some of the seed proteins from the two transgenic rice lines differed in their relative intensities. Twenty eight proteins were successfully identified with MALDI-TOF-MS, five of which were well-characterized and these were discussed. In summary, transgenic rice were found to differ in their protein contents from their non-GM counterparts, which might raise concerns regarding their potential risks for human health and ecology.     

环介导等温扩增技术在转基因成分检测中的应用

随着全球转基因作物商业化种植面积持续增长,转基因成分检测作为转基因作物安全管理的重要技术支撑受到越来越多的关注,世界各国与地区不断致力于转基因成分检测技术的开发。环介导等温扩增（loop-mediatedisothermal amplification,LAMP）技术由于简单快速等特点,近年来在转基因检测领域备受青睐。本文对LAMP技术在转基因成分检测中的最新研究与应用及其发展前景加以综述。     

食品中转基因表达蛋白的危险性评估

随着转基因技术的商业化发展,直接或间接来源于转基因作物的食品日益增多。对食品中外源性基因表达蛋白质的研究是转基因食品安全性评价工作中的重要部分。本文从安全食用历史、生物信息学分析、体外稳定性研究、作用方式研究、毒理学试验等方面,对目前常见转基因表达蛋白的危险性评估进行了综述。     

Real-time multiplex PCR: An accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize-containing food

A very sensitive and new real-time multiplex PCR method for the quantification of genetically modified (GM) maize crops in food materials was developed and validated for an ABI Prism 7700 Sequence Detection System. In the assay described, fluorescence-labelled TaqMan probes were chosen to detect the amplified DNA fragments during PCR. In this multiplex approach, maize-specific DNA (zein) and 35S-CaMV promoter-specific DNA fragments are amplified in the same tube. The method was tested for the detection and quantification of the four maize events that are approved in Europe and contain the 35S-CaMV promoter: Bt11, Bt176, Mon810 and T25 maize. Quantification was based on a standard curve prepared from certified maize flour reference material prepared by the Institute for Reference Materials and Measurements. Quantification within the range of the standard curve (0.05-1% GM maize) and up to 100% was possible. Repeatability of the method for each GM maize event was determined; coefficients of variations ranged from 28-40%. In addition, three internal Nestlé laboratories successfully applied this method and comparable results were obtained.     

Multivariate analysis for the safety assessment of genetically modified rices in the anti‐nutrients and phenolic compounds

A safety assessment of genetically modified (GM) rice Agb0102 (resveratrol synthesis) and Agb0103 (drought‐tolerant) were conducted by comparing with their non‐GM comparators. Phytic acid, trypsin inhibitors and phenolic acids of the rices were analysed to identify the biological equivalences and the impacts of the environment. The analytical tools were principal component analysis (PCA), Pearson's correlation analysis and hierarchical clustering analysis (HCA). The PCA results of phytic acid and trypsin inhibitors revealed no clear separation among rices due to breeding conditions, environmental conditions or among various cultivars. The total, bound, free and ester forms of phenolic acids were not separated in the environmental conditions and different cultivars. The HCA analysis showed strong relationship between GM rice and non‐GM rice. The concentrations of anti‐nutrient and phenolic compounds of the GM rices were not different from those of non‐GM comparators and that various chemometric tools were useful to describe the separation of GM and non‐GM groups.     

Combinatory SYBR® Green Real-Time PCR Screening Approach for Tracing Materials Derived from Genetically Modified Rice

Two real-time PCR approaches for the detection of genetically modified (GM) rice were tested: (1) a combination of SYBR® Green real-time PCR methods detecting the 35S promoter (P-35S) of Cauliflower Mosaic Virus, the nopaline synthase terminator (T-nos) of Agrobacterium tumefaciens and the Bacillus thuringiensis (Bt) CryIAb/Ac toxins and (2) a P-35S/T-nos duplex TaqMan® real-time PCR system. Both systems correctly recognized their respective targets in control samples of Bt63, Kefeng6 and KMD1 insect-resistant and LLRice62 and LLRice601 herbicide-resistant rice. Due to its lesser specificity but broader genetically modified organism (GMO) coverage capacity, the SYBR® Green real-time PCR approach was further tested in more detail. Melting curve, capillary and agarose gel electrophoresis analyses indicated that the P-35S, T-nos and CryIAb/Ac targets in the GM rice events are similar to the corresponding targets present in many known GMOs. High-resolution melting analysis showed that the CryIAb/Ac targets of the GM rice events Bt63 and Kefeng6 matched best the corresponding Bt11 CryIAb sequence. Digital PCR analysis on genomic DNA from the GM rice Bt63 and Kefeng6 events indicated that both GMO contained multiple inserts. Sensitivity tests showed that all SYBR® Green real-time PCR methods could detect their targets at less than an estimated five copies per reaction. Finally, it was shown that these SYBR® Green real-time PCR methods could detect low levels of their targets in rice consignments originating from China. Together, these results demonstrated that a ‘P-35S and T-nos and CryIAb/Ac’ combinatory SYBR® Green real-time PCR screening is highly suited to trace the respective targets including the possible presence of Bt63, Kefeng6 and KMD1 GM rice materials in food products.     

转Bt基因水稻种子中Bt蛋白含量测定方法的研究

采用ELISA技术检测转Bt基因水稻中Bt蛋白的含量,判断水稻样品中是否含有转基因成分。利用研磨、酶标、孵育等技术对样品进行前处理。采用阳性质控物浓度等倍稀释方法,建立标准曲线,相关系数为0.997 4。用一系列不同转基因含量的标准基体材料,分析方法的最低检测限,灵敏度达0.1%。通过对我国进入生产性试验的转Bt基因水稻品系TT51-1和科丰6号的测试,表明该方法与普通PCR方法真实性和灵敏度一致,可以广泛应用于转Bt基因水稻及其粗加工产品的转基因成分检测。为转基因生物安全监管和安全性评价提供技术支撑。     

Screening food products for the presence of CaMV 35S promoter and NOS 3′ terminator

Biotechnology has enabled the modification of agricultural materials in a very precise way, thereby improving productivity and yields of economically important crops. There are a number of methods available for detecting genetically modified organisms (GMOs). In the present investigation, a qualitative PCR technique has been adopted in order to discriminate between genetically modified and non‐modified food products. The qualitative PCR assay employs primers specific for genetic elements that are used to generate genetically engineered agricultural crops. Two of the most common primers used for the detection of GMOs, 35S promoter and NOS 3′ terminator, have been tested over a panel of 24 food products purchased from the local market. The results indicated that, out of the 24 food products tested, three products gave positive results with the 35S promoter. The NOS 3′ primers gave negative results with all tested samples. Copyright © 2005 Society of Chemical Industry     

药用/工业用转基因植物数据库的构建

药用/工业用转基因植物已经成为转基因生物新的增长点。本研究收集了以植物作为生物反应器生产药用/工业用产品的数据信息300多份,转化事件近200条,并完成信息上传108条。每个转化事件收集了包括外源蛋白表达量、毒性、致敏性、临床数据、安全等级在内的20多种信息。利用Linux Apache Mysql PHP （LAMP）架构首次设计了药用/工业用转基因植物数据库,面向用户不同需求设计了初级检索和高级检索两种检索方式。该数据库具有综合性、专业性、实用性和首创性的特点,为药用/工业用转基因植物新品种的研发提供信息帮助,为政府机构审批和监管以及制定其安全评价标准提供技术支撑。