转基因植物中标记基因的安全性新策略

转基因植物中的除草剂和抗生素抗性标记基因潜在的生态环境和食用安全性令人担忧。解决转基因植物中抗性标记基因安全性问题有两种途径：一是转化时仍使用抗性标记基因,转基因植物再生成功后,在释放大田前将标记基因剔除;二是发展安全性标记基因用于植物遗传转化。本文综述了三种标记基因剔除系统和几种安全性标记基因在植物转化中的应用进展。     

遗传修饰工程体的生态安全性

遗传修饰工程体的生态安全性引起了人们的广泛关注。本文对转基因植物外源基因逃逸、对非靶标生物的影响、抗生素抗性基因的安全性以及生防工程菌的生态安全性等问题作了讨论。     

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。     

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。     

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.     

培育无选择标记的转基因植物

目前,几乎所有的植物遗传转化都要通过使用选择标记基因,如抗生素或除草剂抗性基因等来筛选转化子,虽然没有研究结果表明选择标记基因影响人类健康或环境安全,但近年来也引发了人们对转基因产品安全性的担心。为了消除公众对转基因食品的安全性顾虑,无选择标记的转基因植物应运而生。本文综述了共转化系统、位点特异性重组系统(包括FLP/FRT、Cre/lox、R/RS及Gin/gix系统)和转座子系统(Ac/Ds转座子系统)在培育无选择标记转基因植物中的应用。     

新一代转基因植物研究进展

转基因植物具有抗病、抗虫、抗逆、高产、营养成分改善等优良性状,但其安全性引起了人们的关注。新一代植物转基因技术,如叶绿体基因工程、基因约束、多基因共转、去除抗性标记基因、对外源基因进行实时监控、抗性管理策略、最小程度地改变基因等技术的发展,将使未来的转基因植物更好地适应人们的需求,更有利于消费者食用安全和生态环境的可持续发展 。     

卡那霉素在植物转基因中的应用及其抗性基因的生物安全性评价

介绍了卡那霉素在转基因植物筛选中的作用机理及其在植物转化过程和转化后代中的应用现状。卡那霉素不仅在植物转化过程中可起到筛选作用,而且在转化后代中可通过其对后代进行遗传分析和测定种子纯度,同时还可用于后代田间成株的筛选。随着卡那霉素的广泛使用,卡那霉素抗性基因的安全性问题日益受到重视。概述了转基因植物的杂草化、卡那霉素抗性基因的水平扩散、抗生素医疗安全性和食用安全性等方面的研究进展 。     

转基因食品的安全性探讨

转基因食品近几年成了大众关注的一个热点问题,但普遍存在一些错误观点。从转基因食品常用的基因,如抗生素抗性基因、Bt基因、抗除草剂抗性基因、人或动物基因等,分析其食用安全性, 排除对转基因食品的疑惑。     

转基因植物中的卡那霉素抗性

在转基因植物中一个经常使用的标记基因就是卡那霉素抗性基因。对转基因植物的安全性估价将对于现代植物育种的普及和接受十分有利。对于这个标记基因的生物安全性进行估测认为,没有必要对含这个基因的转基因植物进行注册的限制。     

Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics

Selectable marker gene systems are vital for the development of transgenic crops. Since the creation of the first transgenic plants in the early 1980s and their subsequent commercialization worldwide over almost an entire decade, antibiotic and herbicide resistance selectable marker gene systems have been an integral feature of plant genetic modification. Without them, creating transgenic crops is not feasible on purely economic and practical terms. These systems allow the relatively straightforward identification and selection of plants that have stably incorporated not only the marker genes but also genes of interest, for example herbicide tolerance and pest resistance. Bacterial antibiotic resistance genes are also crucial in molecular biology manipulations in the laboratory. An unprecedented debate has accompanied the development and commercialization of transgenic crops. Divergent policies and their implementation in the European Union on one hand and the rest of the world on the other (industrialized and developing countries alike), have resulted in disputes with serious consequences on agricultural policy, world trade and food security. A lot of research effort has been directed towards the development of marker-free transformation or systems to remove selectable markers. Such research has been in a large part motivated by perceived problems with antibiotic resistance selectable markers; however, it is not justified from a safety point of view. The aim of this review is to discuss in some detail the currently available scientific evidence that overwhelmingly argues for the safety of these marker gene systems. Our conclusion, supported by numerous studies, most of which are commissioned by some of the very parties that have taken a position against the use of antibiotic selectable marker gene systems, is that there is no scientific basis to argue against the use and presence of selectable marker genes as a class in transgenic plants.     

Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants

Selectable markers of bacterial origin such as the neomycin phosphotransferase type II gene, which can confer kanamycin resistance to transgenic plants, represent an invaluable tool for plant engineering. However, since all currently used antibiotic-resistance genes are of bacterial origin, there have been concerns about horizontal gene transfer from transgenic plants back to bacteria, which may result in antibiotic resistance. Here we characterize a plant gene, Atwbc19, the gene that encodes an Arabidopsis thaliana ATP binding cassette (ABC) transporter and confers antibiotic resistance to transgenic plants. The mechanism of resistance is novel, and the levels of resistance achieved are comparable to those attained through expression of bacterial antibiotic-resistance genes in transgenic tobacco using the CaMV 35S promoter. Because ABC transporters are endogenous to plants, the use of Atwbc19 as a selectable marker in transgenic plants may provide a practical alternative to current bacterial marker genes in terms of the risk for horizontal transfer of resistance genes.     

无标记（Marker—Free）：转基因植物研究的新趋势

目前 ,几乎所有的植物遗传转化中都要使用选择性标记基因诸如抗生素或除草剂抗性基因等来筛选转化子。为了消除由此而引起的公众的安全性顾虑 ,一种全新的发展策略即获取无选择标记的转基因植物应运而生。无选择标记的转基因植物具有许多独特的优势 ,如消除大众对转基因植物中含有选择标记基因而引起的恐惧及可以反复地向已转化的植物中叠加外源基因等 ,因此 ,这种新策略 (无标记 )有着巨大的应用潜力。本文对获得无标记转基因植物的一些途径做一综述。     

无标记(Marker-Free):转基因植物研究的新趋势

目前,几乎所有的植物遗传转化中都要使用选择性标记基因诸如抗生素或除草剂抗性基因等来筛选转化子.为了消除由此而引起的公众的安全性顾虑,一种全新的发展策略即获取无选择标记的转基因植物应运而生.无选择标记的转基因植物具有许多独特的优势,如消除大众对转基因植物中含有选择标记基因而引起的恐惧及可以反复地向已转化的植物中叠加外源基因等,因此,这种新策略(无标记)有着巨大的应用潜力.本文对获得无标记转基因植物的一些途径做一综述.     

Marker Gene Controversy in Transgenic Plants

Biosafety implications of selectable marker genes that are integrated into the transgenic plants are discussed. In the laboratory, selectable marker genes are used at two stages to distinguish transformed cells out of a large population of nontransformed cells: 1) initial assembly of gene cassettes is generally done in E. coli on easily manipulatable plasmid vectors that contain the selectable marker genes which often code for antibiotic inactivating enzymes, and 2) Then the gene cassettes are inserted into the plant genome by various transformation methods. For selection of transformed plant cells, antibiotic and herbicide resistance genes are widely used. Consequently, transgenic plants can end up with DNA sequences of selectable markers that are functional in E. coli and plants. The potential for horizontal gene transfer of selectable markers from transgenic plants to other organisms both in the environment and in the intestine of humans and animals is evaluated. Mechanisms and consequences of the transfer of marker genes from plants to other organisms is examined. Strategies to avoid marker genes in plants are discussed. It is possible to avoid the use of controversial selectable markers in the construction of transgenic plants.     

Environmentally friendly approaches to genetic engineering

Summary Several environmental problems related to plant genetic engineering may prohibit advancement of this technology and prevent realization of its full potential. One such common concern is the demonstrated escape of foreign genes through pollen dispersal from transgenic crop plants to their weedy relatives, creating super weeds or causing gene pollution among other crops or toxicity of transgenic pollen to nontarget insects. The high rates of gene flow from crops to wild relatives (as high as 38% in sunflower and 50% in strawberries) are certainly a serious concern. Maternal inheritance of the herbicide resistance gene via chloroplast genetic engineering has been shown to be a practical solution to these problems. Another common concern is the suboptimal production of Bacillus thuringiensis (Bt) insecticidal protein or reliance on a single (or similar) B.t. protein in commercial transgenic crops, resulting in B.t. resistance among target pests. Clearly, different insecticidal proteins should be produced in lethal quantities to decrease the development of resistance. Such hyperexpression of a novel B.t. protein in chloroplasts has resulted in 100% mortality of insects that are up to 40 000-fold resistant to other B.t. proteins. Yet another concern is the presence of antibiotic resistance genes in transgenic plants that could inactivate oral doses of the antibiotic or be transferred to pathogenic microbes in the GI tract or in soil, rendering them resistant to treatment with such antibiotics. Cotransformation and elimination of antibiotic resistant genes from transgenic plants using transposable elements via breeding are promising new approaches. Genetic engineering efforts have also addressed yet another concern, i.e., the accumulation and persistence of plastics in our environment by production of biodegradable plastics. Recent approaches and accomplishments in addressing these environmental concerns via chloroplast genetic engineering are discussed in this review.     

Strategies for developing marker-free transgenic plants

The development of marker-free transgenic plants has responded to public concerns over the safety of biotechnology crops. It seems that continued work in this area will soon remove the question of unwanted marker genes from the debate concerning the public acceptability of transgenic crop plants. Selectable marker genes are co-introduced with genes of interest to identify those cells that have integrated the DNA into their genome. Despite the large number of different selection systems, marker genes that confer resistance to the antibiotics, hygromycin (hpt) and kanamycin (nptII) or herbicide phosphinothricin (bar), have been used in most transgenic research and crop development techniques. The techniques that remove marker gene are under development and will eventually facilitate more precise and subtle engineering of the plant genome, with widespread applications in both fundamental research and biotechnology. In addition to allaying public concerns, the absence of resistance genes in transgenic plants could reduce the costs of developing biotechnology crops and lessen the need for time-consuming safety evaluations, thereby speeding up the commercial production of biotechnology crops. Many research results and various techniques have been developed to produce marker-free transgenic plants. This review describes the strategies for eliminating selectable marker genes to generate marker-free transgenic plants, focusing on the three significant marker-free technologies, co-transformation, site-specific recombinase-mediated excision, and non-selected transformation.     

Marker-free transformation: increasing transformation frequency by the use of regeneration-promoting genes

The generation of transgenic plants free of antibiotic resistance markers is a major challenge to plant biologists and plant breeders. Currently, there are two main strategies to achieve this goal: one approach is to excise or segregate marker genes from the host genome after regeneration of transgenic plants, and the second is based on so-called 'marker-free' transformation. Marker-free transformation has been successfully demonstrated by the use of several plant and non-plant genes that are capable of promoting explant regeneration. This approach appears not only to be effective for the generation of marker-free transgenic plants, but also has great potential to improve the transformation frequency of recalcitrant species.     

A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time‐consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co‐introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic‐resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high‐throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant‐specific DNA herbicides.