转基因植物中标记基因的剔除

在目前的植物转化系统中,要求在关注基因或目的基因转入细胞时,同时有标记基因存在.标记基因主要是抗生素或除草剂的抗性基因.借标记基因的表达可以将转化细胞从大量的未转化细胞中筛选出来,但标记基因的继续存在,特别是在转基因食品中,是人们广泛关注的问题.培育无标记基因的转基因植株已成为植物生物工程研究中的新课题.该文介绍了剔除标记基因的两种方法:分离剔除和重组剔除,并对近年来这两种方法在培育无标记基因的转基因植物中的应用和进展作了介绍.     

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.     

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.     

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。     

转基因植物中的标记基因研究新进展

文章综述了转基因植物中标记基因研究的新进展,主要包括以下3个方面：第一是采用共转化、位点特异性重组和转座子等技术对传统抗性标记基因进行消除,以利于对同一作物进行多次转基因操作;第二是完善各种已应用的以糖类代谢酶基因、耐胁迫酶类基因和绿色荧光蛋白基因等为安全标记基因的转化体系,并大力研究、开发潜在的汞离子还原酶基因、叶绿体合成关键酶基因等作为安全标记基因;第三是着力发展无标记基因、无载体骨架的简单高效转化体系。此外,还展望了安全标记的应用前景。     

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。     

A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop

It is generally thought that transformation of plant cells using Agrobacterium tumefaciens occurs at a very low frequency. Therefore, selection marker genes are used to identify the rare plants that have taken up foreign DNA. Genes encoding antibiotic and herbicide resistance are widely used for this purpose in plant transformation. Over the past several years, consumer and environmental groups have expressed concern about the use of antibiotic- and herbicide-resistance genes from an ecological and food safety perspective. Although no scientific basis has been determined for these concerns, generating marker-free plants would certainly contribute to the public acceptance of transgenic crops. Several methods have been reported to create marker gene-free transformed plants, for example co-transformation, transposable elements, site-specific recombination, or intrachromosomal recombination. Not only are most of these systems time-consuming and inefficient, but they are also employed on the assumption that isolation of transformants without a selective marker gene is not feasible. Here we present a method that permits the identification of transgenic plants without the use of selectable markers. This strategy relies on the transformation of tissue explants or cells with a virulent A. tumefaciens strain and selection of transformed cells or shoots after PCR analysis. Incubation of potato explants with A. tumefaciens strain AGL0 resulted in transformed shoots at an efficiency of 1-5% of the harvested shoots, depending on the potato genotype used. Because this system does not require genetic segregation or site-specific DNA-deletion systems to remove marker genes, it may provide a reliable and efficient tool for generating transgenic plants for commercial use, especially in vegetatively propagated species like potato and cassava.     

An efficient and novel plant selectable marker based on organomercurial resistance

A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments. So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance. However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation. The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve as an excellent selectable marker for plant transformation.     

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.     

正向选择的生物安全标记基因

标记基因的产生方便了植物的转化,随着转基因植物的迅速发展及商品化,人类更关注抗性标记基因的安全性。目前解决的有效途径是发展正向选择系统,使用非抗性的生物安全标记基因,主要包括糖类代谢酶基因（pmi和xylA）、干扰氨基酸代谢酶基因（ak和dapA）、绿色荧光蛋白基因（gfp）、β-葡萄糖苷酸酶基因（gus）、核糖醇操纵子（rtl）和叶绿素生物合成基因（hemL）等。     

Selectable marker genes in transgenic plants: applications, alternatives and biosafety

Approximately fifty marker genes used for transgenic and transplastomic plant research or crop development have been assessed for efficiency, biosafety, scientific applications and commercialization. Selectable marker genes can be divided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or non-conditional on the presence of external substrates. Positive selectable marker genes are defined as those that promote the growth of transformed tissue whereas negative selectable marker genes result in the death of the transformed tissue. The positive selectable marker genes that are conditional on the use of toxic agents, such as antibiotics, herbicides or drugs were the first to be developed and exploited. More recent developments include positive selectable marker genes that are conditional on non-toxic agents that may be substrates for growth or that induce growth and differentiation of the transformed tissues. Newer strategies include positive selectable marker genes which are not conditional on external substrates but which alter the physiological processes that govern plant development. A valuable companion to the selectable marker genes are the reporter genes, which do not provide a cell with a selective advantage, but which can be used to monitor transgenic events and manually separate transgenic material from non-transformed material. They fall into two categories depending on whether they are conditional or non-conditional on the presence of external substrates. Some reporter genes can be adapted to function as selectable marker genes through the development of novel substrates. Despite the large number of marker genes that exist for plants, only a few marker genes are used for most plant research and crop development. As the production of transgenic plants is labor intensive, expensive and difficult for most species, practical issues govern the choice of selectable marker genes that are used. Many of the genes have specific limitations or have not been sufficiently tested to merit their widespread use. For research, a variety of selection systems are essential as no single selectable marker gene was found to be sufficient for all circumstances. Although, no adverse biosafety effects have been reported for the marker genes that have been adopted for widespread use, biosafety concerns should help direct which markers will be chosen for future crop development. Common sense dictates that marker genes conferring resistance to significant therapeutic antibiotics should not be used. An area of research that is growing rapidly but is still in its infancy is the development of strategies for eliminating selectable marker genes to generate marker-free plants. Among the several technologies described, two have emerged with significant potential. The simplest is the co-transformation of genes of interest with selectable marker genes followed by the segregation of the separate genes through conventional genetics. The more complicated strategy is the use of site-specific recombinases, under the control of inducible promoters, to excise the marker genes and excision machinery from the transgenic plant after selection has been achieved. In this review each of the genes and processes will be examined to assess the alternatives that exist for producing 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.     

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T₁ soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.     

Marker-free transgenic plants

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, selection is based on antibiotic or herbicide resistance. Due mainly to consumer concerns, a suite of strategies (site-specific recombination, homologous recombination, transposition and co-transformation) have been developed to eliminate the marker gene from the nuclear or chloroplast genome after selection. Current efforts concentrate on systems where marker genes are eliminated efficiently soon after transformation. Alternatively, transgenic plants are produced by the use of marker genes that do not rely on antibiotic or herbicide resistance but instead promote regeneration after transformation. Here, the merits and shortcomings of different approaches and possible directions for their future development are discussed.     

<Emphasis Type="Italic">ptxD</Emphasis> gene in combination with phosphite serves as a highly effective selection system to generate transgenic cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Key message

This report demonstrates the usefulness of ptxD/phosphite as a selection system that not only provides a highly efficient and simple means to generate transgenic cotton plants, but also helps address many of the concerns related to the use of antibiotic and herbicide resistance genes in the production of transgenic crops.

Abstract

Two of the most popular dominant selectable marker systems for plant transformation are based on either antibiotic or herbicide resistance genes. Due to concerns regarding their safety and in order to stack multiple traits in a single plant, there is a need for alternative selectable marker genes. The ptxD gene, derived from Pseudomonas stutzeri WM88, that confers to cells the ability to convert phosphite (Phi) into orthophosphate (Pi) offers an alternative selectable marker gene as demonstrated for tobacco and maize. Here, we show that the ptxD gene in combination with a protocol based on selection medium containing Phi, as the sole source of phosphorus (P), can serve as an effective and efficient system to select for transformed cells and generate transgenic cotton plants. Fluorescence microscopy examination of the cultures under selection and molecular analyses on the regenerated plants demonstrate the efficacy of the system in recovering cotton transformants following Agrobacterium-mediated transformation. Under the ptxD/Phi selection, an average of 3.43 transgenic events per 100 infected explants were recovered as opposed to only 0.41% recovery when bar/phosphinothricin (PPT) selection was used. The event recovery rates for nptII/kanamycin and hpt/hygromycin systems were 2.88 and 2.47%, respectively. Molecular analysis on regenerated events showed a selection efficiency of ~?97% under the ptxD/Phi system. Thus, ptxD/Phi has proven to be a very efficient, positive selection system for the generation of transgenic cotton plants with equal or higher transformation efficiencies compared to the commonly used, negative selection systems.

     

Recent advances in development of marker-free transgenic plants: Regulation and biosafety concern

During the efficient genetic transformation of plants with the gene of interest, some selectable marker genes are also used in order to identify the transgenic plant cells or tissues. Usually, antibiotic- or herbicide-selective agents and their corresponding resistance genes are used to introduce economically valuable genes into crop plants. From the biosafety authority and consumer viewpoints, the presence of selectable marker genes in released transgenic crops may be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with herbicides or antibiotics, respectively. Sexual crossing also raises the problem of transgene expression because redundancy of transgenes in the genome may trigger homology-dependent gene silencing. The future potential of transgenic technologies for crop improvement depends greatly on our abilities to engineer stable expression of multiple transgenic traits in a predictable fashion and to prevent the transfer of undesirable transgenic material to non-transgenic crops and related species. Therefore, it is now essential to develop an efficient marker-free transgenic system. These considerations underline the development of various approaches designed to facilitate timely elimination of transgenes when their function is no longer needed. Due to the limiting number of available selectable marker genes, in future the stacking of transgenes will be increasingly desirable. The production of marker-free transgenic plants is now a critical requisite for their commercial deployment and also for engineering multiple and complex trait. Here we describe the current technologies to eliminate the selectable marker genes (SMG) in order to develop marker-free transgenic plants and also discuss the regulation and biosafety concern of genetically modified (GM) crops.     

Genetic transformation of apple (<Emphasis Type="Italic">Malus</Emphasis> x <Emphasis Type="Italic">domestica</Emphasis>) without use of a selectable marker gene

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, antibiotic or herbicide resistance marker genes are preferred because they tend to be most efficient. Due mainly to consumer and grower concerns, considerable effort is being put into developing strategies (site-specific recombination, homologous recombination, transposition, and cotransformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. For the commercialization of genetically transformed plants, use of a completely marker-free technology would be desirable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations for the public. With this goal in mind, a technique for apple transformation was developed without use of any selectable marker. Transformation of the apple genotype “M.26” with the constructs pPin2Att35SGUSintron and pPin2MpNPR1 was achieved. In different experiments, 22.0–25.4% of regenerants showed integration of the gene of interest. Southern analysis in some transformed lines confirmed the integration of one copy of the gene. Some of these transformed lines have been propagated and used to determine the uniformity of transformed tissues in the plantlets. The majority of the lines are uniformly transformed plants, although some lines are chimeric, as also occurs with the conventional transformation procedure using a selectable marker gene. A second genotype of apple, “Galaxy,” was also transformed with the same constructs, with a transformation efficiency of 13%.     

质体基因工程中选择标记基因研究进展

与细胞核基因工程相比,质体基因工程能更安全、精确和高效地对外源基因进行表达,作为下一代转基因技术已广泛用于基础研究和生物技术应用领域。与细胞核基因工程一样,质体基因工程中也需要合适的选择标记基因用于转化子的筛选和同质化,但基于质体基因组的多拷贝性和母系遗传特点,转化子的同质化需要一个长期的筛选过程,这就决定了质体基因工程中选择标记基因的选择标准将不同于细胞核基因工程中广泛使用的现行标准。目前,质体基因工程的遗传转化操作中使用较多的是抗生素选择标记基因,出于安全性考虑,需要找到可替换、安全的选择标记基因或有效的标记基因删除方法。本文在对质体基因工程研究的相关文献分析基础之上,对主要使用的选择标记基因及其删除体系进行了综述,并对比了其优缺点,同时探讨了质体基因工程中所使用的报告基因,以期为现有选择标记基因及其删除体系的改进和开发提供一定参考,进一步推动质体基因工程,尤其是单子叶植物质体基因工程的发展。     

Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene

Public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers. We describe the development of an effective transformation system for generating such marker-free transgenic plants, without the need for repeated transformation or sexual crossing. This system combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. The described system can be generally applied to existing transformation protocols, and was tested in strawberry using a model vector in which site-specific recombination leads to a functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding sequence, thereby enabling the histochemical monitoring of recombination events. Fully marker-free transgenic strawberry plants were obtained following two different selection/regeneration strategies.