转基因植物环境安全评价策略

构建完善的转基因植物环境安全评价技术体系是保障转基因生物产业健康发展的重要组成部分。本文综述了转基因植物环境安全评价技术发展历程与趋势,归纳了转基因植物环境安全评价的思路与内容。转基因植物环境安全评价应分为潜在风险分析、风险假设验证、风险特征描述等3个步骤,并采用逐层评价模式;安全评价应贯穿转基因植物新品种研发与产业化全程,包括应用前预测、研发中筛选、推广前评价、推广后监测。此外,基于科学性和个案分析原则,本文对复合性状、非生物胁迫抗性等新型转基因植物环境安全评价策略进行了探讨。     

复合性状转基因植物安全性评价的研究进展

随着转基因技术的不断发展,转基因作物实现了产业化。目前,单性状的转基因作物无法满足农业发展的需求,而复合性状转基因作物拓宽了转基因作物的功能,提高了资源的利用率,满足了农民多元化需求,具有广阔的应用前景,是转基因植物发展的新方向。由于转基因技术有一定的风险,因此对转基因作物的安全性监管较为重要,对于复合性状转基因作物,不同国家的安全性评价制度却不尽相同。综述了不同国家对复合性状转基因作物的安全性评价体制,旨为我国复合性状作物提供监管依据。     

安全标记基因在转基因植物中的应用

转基因植物的抗性标记一直是转基因生物安全性争论的焦点,是限制转基因植物应用的瓶颈之一。筛选安全标记基因替代抗生素标记基因已成为解决转基因植物安全性和促进转基因植物应用的重要策略。综述了生物安全标记基因的产生背景、系统分类、筛选原理及不同起源的标记基因在植物基因工程中的应用和存在问题。选用植物内源标记基因已成为转基因植物安全标记基因研究的重要方向。     

转基因抗虫烟草研究进展

烟草为模式植物,也是外源杀虫基因最早转化成功的植物。文章从转Bt内毒素基因,植物凝集素GNA,Plec,AHA基因,蛋白酶抑制剂PIⅠ,PIⅡ,MTI,SKTI基因,昆虫特异性神经毒素基因,几丁质酶基因,畸形细胞分泌蛋白基因以及双抗虫基因等方面综述了转基因抗虫烟草的抗虫性、转基因抗虫烟草的经济性状等,展望了转基因抗虫烟草的研究和应用前景,以期对烟草害虫的治理尤其是对其他转基因抗虫作物的培育和研究有借鉴作用。     

转Bt基因植物中外源基因时空动态表达的研究现状

在转Bt基因植株中 ,外源基因的时空动态表达对于害虫的防治和转基因安全评价管理具有重要意义。利用生物测定法和酶联免疫吸附测定法 (ELISA) ,对植物不同组织在同一发育阶段、同一组织在不同的发育阶段以及不同转基因植株的外源基因的时空动态表达进行研究。本文综述了转基因植物中外源基因时空动态表达的研究进展和现状。     

抗虫抗除草剂转基因玉米HiII-NGc-1的遗传稳定性分析

目的基因和目标性状遗传稳定性是转基因植物新品种选育和产业化的重要前提。以进入农业部环境释放的抗虫抗除草剂双价转基因玉米Hi II-NGc-1为试验材料,采用PCR、RT-PCR和免疫试纸条技术检测转基因玉米连续3代目的基因cry NGc和bar整合及表达的遗传稳定性,采用心叶期除草剂草铵膦抗性鉴定和心叶期/穗期亚洲玉米螟抗性鉴定实验详细分析了目标性状的遗传稳定性。研究结果表明:Cry NGc和PAT蛋白在Hi II-NGc-1中连续3代遗传稳定,高世代稳定耐受正常5倍大田除草剂草铵膦使用浓度,心叶期和穗期高抗亚洲玉米螟性状稳定,具有潜在的产业化应用价值。转基因玉米Hi II-NGc-1目的基因和目标性状遗传稳定性的明确为开展下一步生物安全评价奠定了基础,对复合性状转基因抗虫抗除草剂玉米新品种的选育和产业化具有重要意义。     

融合基因在转基因植物中的应用

目前双基因和多基因转基因植物已经商品化,并展现了广泛的应用前景。但在转基因植物研究中,使多个基因同时在植物体中表达调控依然很难实现,是植物基因工程和生物技术发展中的难点。融合基因表达载体作为一种新型的方法,弥补了获得双价或多价转基因植物传统方法的缺点,具有更高的应用价值。本文对目前构建融合基因的方法作了评述,并对比较新颖的连接肽2A和LP4做了详细介绍。     

转基因植物中标记基因去除方法的研究进展

随着越来越多的转基因植物品种的出现,转基因植物的环境安全及食用安全越来越受到人们的广泛关注。目前存在的问题之一是转基因植物中抗生素或除草剂抗性的选择标记基因的存留。为了提高转基因植物的安全性,将转基因植物中选择性标记基因去除,不仅利于同一转基因植物的多次操作,而且更容易被人们所接受。就目前转基因植物中选择性标记基因去除的方法及其优缺点进行了横纵向的比较,希望对相关研究领域方法的选择方向具有指导意义。     

基因枪法在水稻工程育种中的应用

至今,基因枪法以其实用性和有效性应用于水稻工程育种已有十几年。本文阐述了其在水稻工程育种的六个主要领域的应用现状,同时评价了外源基因在转基因水稻中的稳定性和对转基因水稻主要农艺性状的影响。当然,基因枪法的转化效率还有待提高,随着转不同基因的工程水稻的日益增多,进一步的安全性试验显得十分必要,它是转基因水稻商品化的前提。     

转基因技术与大豆品质改良

大豆是重要的食用油、食用蛋白和饲用蛋白原料。应用转基因技术对调节大豆品质形成的关键基因进行定向操作,可大大加速优质大豆品种的选育进程,培育出适合不同消费需求的多样化优质大豆品种。文章主要综述了近年来转基因技术在大豆品质性状改良方面的应用进展,同时也简要介绍了目前已经发展起来的安全转基因技术。     

转基因植物的基因漂流风险

讨论了近10年有关转基因植物漂流方面的5个主要研究领域,转基因进入相关植物野生种或近缘种中的实证性研究,以花粉为煤体的基因漂流特性研究,基因漂流实验和风险评价方法研究,基因漂流风险安全性评价标准的争论和基因漂流的长期生态效应研究,提出了目前应进一步开展的研究课题。     

抗病毒基因工程与转基因植物释放的环境风险评估

全面介绍了抗病毒转基因植物释放的风险性，包括重组（ｒｅｃｏｍｂｉｎａｔｉｏｎ）、衣壳转移（ｔｒａｎｓｃａｐ－ｓｉｄａｔｉｏｎ）和协生作用（ｓｙｎｅｒｇｉｓｍ）等，并指出，我国应对抗病毒转基因植物释放产生的风险进行系统评估，在缺乏对可能产生的风险的认识之前，应控制进行大规模的田间试验。     

Evaluation of a non-targeted "omic" approach in the safety assessment of genetically modified plants

Genetically modified plants must be approved before release in the European Union, and the approval is generally based upon a comparison of various characteristics between the transgenic plant and a conventional counterpart. As a case study, focusing on safety assessment of genetically modified plants, we here report the development and characterisation of six independently transformed ARABIDOPSIS THALIANA lines modified in the flavonoid biosynthesis. Analyses of integration events and comparative analysis for characterisation of the intended effects were performed by PCR, quantitative Real-time PCR, and High Performance Liquid Chromatography. Analysis by cDNA microarray was used as a non-targeted approach for the identification of potential unintended effects caused by the transformation. The results revealed that, although the transgenic lines possessed different types of integration events, no unintended effects were identified. However, we found that the majority of genes showing differential expression were identified as stress-related genes and that environmental conditions had a large impact on the expression of several genes, proteins, and metabolites. We suggest that the microarray approach has the potential to become a useful tool for screening of unintended effects, but state that it is crucial to have substantial information on the natural variation in traditional crops in order to be able to interpret "omics" data correctly within the framework of food safety assessment strategies of novel plant varieties, including genetically modified plant varieties.     

Changes in fitness-associated traits due to the stacking of transgenic glyphosate resistance and insect resistance in Brassica napus L

Increasingly, genetically modified crops are being developed to express multiple 'stacked' traits for different types of transgenes, for example, herbicide resistance, insect resistance, crop quality and tolerance to environmental stresses. The release of crops that express multiple traits could result in ecological changes in weedy environments if feral crop plants or hybrids formed with compatible weeds results in more competitive plants outside of agriculture. To examine the effects of combining transgenes, we developed a stacked line of canola (Brassica napus L.) from a segregating F(2) population that expresses both transgenic glyphosate resistance (CP4 EPSPS) and lepidopteran insect resistance (Cry1Ac). Fitness-associated traits were evaluated between this stacked genotype and five other Brassica genotypes in constructed mesocosm plant communities exposed to insect herbivores (Plutella xylostella L.) or glyphosate-drift. Vegetative biomass, seed production and relative fecundity were all reduced in stacked trait plants when compared with non-transgenic plants in control treatments, indicating potential costs of expressing multiple transgenes without selection pressure. Although costs of the transgenes were offset by selective treatment, the stacked genotype continued to produce fewer seeds than either single transgenic line. However, the increase in fitness of the stacked genotype under selective pressure contributed to an increased number of seeds within the mesocosm community carrying unselected, hitchhiking transgenes. These results demonstrate that the stacking of these transgenes in canola results in fitness costs and benefits that are dependent on the type and strength of selection pressure, and could also contribute to changes in plant communities through hitchhiking of unselected traits.     

基因工程植物的安全性问题

转基因植物的研究进展很迅速,但基因工程植物是否安全一直争论不休,主要表现在转基因食品的安全性及生态安全性问题上.转基因食品的安全性涉及这些食品的过敏性、毒性以及抗生素标记基因的安全性几个方面.转基因植物的生态安全性包括基因漂流、是否能诱发昆虫产生Bt抗性和对生物多样性的影响等.本文针对这些问题,对转基因植物潜在危害以及国际上现有的评价作简要综述.     

转Bt基因作物Bt毒蛋白在土壤中的安全性研究

商业化的转Bt基因作物获准在田间大面积种植,使其释放的Bt毒蛋白对土壤生态系统的生态风险性问题成为人们关注的焦点,本文综述了转Bt抗虫作物以植株残体、根系分泌物、花粉等形式释放的Bt毒蛋白通过田间耕作等方式进入土壤后的一些安全性问题,包括土壤活性颗粒对Bt毒蛋白的吸附作用。Bt毒蛋白在土壤中的杀虫活性、存留,土壤微生物对Bt毒蛋白的降解作用以及Bt毒蛋白对土壤生物的影响等。     

Transgenic plants: performance,release and containment

This review focuses on transgenic plants, from the initial stages of the genetic modification process in the laboratory to their release stage in the field and indicates possible areas of concern and strategies for dealing with them. The classes of marker genes and issues about their safety, the gene flow and strategies that are used to isolate transgenic plants genetically are specifically examined. In addition, an assessment is provided of the phenomena which affect the performance of transgenic plants, such as gene disruption, the pleiotropic effect on plant phenotype and genetic variation. Finally, strategies are suggested for preventing unexpected consequences of transgenic plant production.The author is with the Department of Genetics, University of Leeds, Leeds LS2 9JT, UK     

Expression of endogenous proteins in maize hybrids in a multi-location field trial in India

Genetically modified (GM) crops undergo large scale multi-location field trials to characterize agronomics, composition, and the concentration of newly expressed protein(s) [herein referred to as transgenic protein(s)]. The concentration of transgenic proteins in different plant tissues and across the developmental stages of the plant is considered in the safety assessment of GM crops. Reference or housekeeping proteins are expected to maintain a relatively stable expression pattern in healthy plants given their role in cellular functions. Understanding the effects of genotype, growth stage and location on the concentration of endogenous housekeeping proteins may provide insight into the contribution these factors could have on transgenic protein concentrations in GM crops. The concentrations of three endogenous proteins (actin, elongation factor 1-alpha, and glyceraldehyde 3-phosphate dehydrogenase) were measured in several different maize hybrids grown across multiple field locations over 2 years. Leaf samples were collected from healthy plants at three developmental stages across the growing seasons, and protein concentrations were quantified by indirect enzyme-linked immunosorbent assay (ELISA) for each protein. In general, the concentrations of these three endogenous proteins were relatively consistent across hybrid backgrounds, when compared within one growth stage and location (2–26%CV), whereas the concentrations of proteins in the same hybrid and growth stage across different locations were more variable (12–64%CV). In general, the protein concentrations in 2013 and 2014 show similar trends in variability. Some degree of variability in protein concentrations should be expected for both transgenic and endogenous plant-expressed proteins. In the case of GM crops, the potential variation in protein concentrations due to location effects is captured in the current model of multi-location field testing.     

Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.