转基因植物中标记基因研究概况

标记基因在植物基因工程中具有重要的作用。它在遗传转化中的关键作用是区分转化和非转化的细胞,以筛选并鉴定出转化的细胞、组织和转基因植株。目前,已报道的标记基因种类很多,划分标准也各不相同。出于对生态环境和转基因食品的生物安全性考虑,从传统的选择标记基因、与激素代谢相关的基因、与氨基酸代谢相关的基因、与糖类代谢相关的基因、能解除化合物毒性（或胁迫）的基因、编码能产生特定荧光物质的蛋白酶类的基因、利用颜色差异性筛选转化体的相关基因及抗性标记基因的敲除技术八个不同的方面,综述了标记基因的种类、作用原理、应用价值及存在的问题。在标记基因的综合应用方面,详细总结了标记基因与组织（或器官）特异性启动子和MAT载体系统的结合应用,以及P．葡萄糖苷酸酶作为多功能标记基因的综合应用。最后,对标记基因的发展前景进行了探讨分析。     

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。     

植物转化中的安全标记基因

转基因植物中的除草剂或抗生素抗性标记基因的生态环境和食用安全性一直颇有争议.糖类分解代谢酶基因作为安全标记基因,近年来在植物转化中显示了巨大应用潜力.这类标记基因编码产物是筛选剂糖类的分解代谢酶,使转化细胞能利用筛选剂糖类作为主要碳源从而获得优势生长,非转化细胞则因饥饿生长被抑制但不被杀死,故称为正筛选系统(positive selection system).目前木糖异构酶基因（xylose isomerase,xylA）和磷酸甘露糖异构酶基因（phosphomannose isomerase,pmi)等安全标记基因已成功应用于植物转化.     

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。     

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。     

转基因植物中卡那霉素抗体（Ｋan^r）标记基因的生物安全性

卡那霉素抗性（Kan^r)基因是转基因植物中广泛使用的一类标记基因,其生物安全性受到普遍关注,本文详细讲座了转基因植物中Kan^r基因的漂流及其对自然生态环境的影响,并对Kan^r基因及编码蛋白ＡＰＨ（３′）－Ⅱ的人畜食用安全性进行了综述分析。     

植物功能性靶向基因标记的研究进展

随着功能基因组学的发展,分子标记技术正朝着功能性靶向基因标记的方向发展。因功能标记是根据与表型紧密相关的功能基因内部特定区域多态性基序开发而来的,所以此类标记不需要进一步的验证就可在不同的遗传背景下确定等位基因的有无。从靶向基因标记和功能标记、保守DNA和基因家族标记、转座子标记、抗性基因标记、RNA标记和靶向指纹标记几方面综述了植物功能性靶向基因标记的研究进展,旨在为分子标记的开发与应用提供理论基础。     

代谢关键酶基因作为转基因植物选择标记的研究进展

在植物基因工程中,需要用到选择标记基因以便筛选转化子。随着转基因植物的商品化,人们越来越关注其安全性问题,其中主要涉及选择标记的安全性。为了提高转基因植物的安全性,生物学家们开始寻找生物安全的选择标记。本文分别从糖代谢、氨基酸代谢、激素代谢、核苷酸代谢、蛋白质代谢等方面综述了当前以代谢关键酶基因作为转基因植物选择标记的研究进展,此类选择标记为解决转基因植物的安全性问题提供了一条有效途径。     

利用FLP/frt重组系统产生无选择标记的转基因烟草植株

在植物转基因植株产生过程中,对转化细胞进行抗性筛选是通用程序,转化细胞的抗性一般是抗生素抗性或除草剂抗性,将赋予转化细胞抗性的选择标记基因删除是提高转基因植物生物安全性的重要措施。来自于啤酒酵母的FLP/frt位点特异性重组系统可有效删除同向定点重组位点frt之间的基因。通过多步骤重组,建立了可在植物中广泛应用的FLP/frt位点特异性重组系统。该系统包括含有frt位点的植物表达载体pCAMBIA1300-betA-frt-als-frt和含有由热诱导启动子hsp启动的FLP重组酶基因的植物表达载体pCAMBIA1300-hsp-FLP-hpt。利用二次转化的方式将二者先后转入烟草植株,热激处理后,热诱导型启动子hsp调控的重组酶FLP基因的表达催化位于选择标记基因als两侧同向frt位点间的重组反应,有效地删除了选择标记基因als。41%的经热激处理的二次转化植株发生了选择标记基因的删除,表明该系统在获得无选择标记基因的转基因植株中有很好的应用价值。     

开发利用土壤杆菌的无标记植物性状转化法

日本烟草产业（ＪＴ）公司成功地开发了可从性状转化植物高效地去除选择性标记的利用土壤杆菌的无标记性状转化法。不仅限于双子叶植物,也能进行单子叶植物水稻的基因导入。ＪＴ公司在川崎市召开的日本育种学会上发表了该项成果。在植物的性状转化中使用了卡那霉素等基因,但是,如果能排除标记,就能反复使用单一标记,所以希望此法能在需要导入代谢系基因群等多种基因时使用。因此,选择标记的去除法是植物重组育种必需的技术。ＪＴ公司开发的载体是在其１９９４年开发的、土壤杆菌（不仅能向双子叶植物中导入基因而且也能向水稻和玉米等…     

用绿色荧光蛋白监测转基因植物中选择标记基因的消除

绿色荧光蛋白(GFP)可直接进行活体观察,它的这个优点可被用于监测转基因植物中选择标记基因的消除。为此,构建了植物表达载体pGNG,将绿色荧光蛋白基因(gfp)和卡那霉素抗性基因表达盒(NosP-nptll-NosT)一起克隆在两个同向的lox位点间,在第一个lox位点上游置有CaMV 35S启动子以驱动GFP表达,第二个lox位点下游置有不含启动子的大肠杆菌β-葡萄糖醛酸酶(GUS)基因。首先在含卡那霉素(Kan)的培养基上筛选出转pGNG的烟草,借助绿色荧光可容易地检出表达GFP的转化体。然后用另一转化载体pCambia1300Cre二次转化表达GFP的转基因植物,利用另一选择标记基因潮霉素抗性基因(hpt)进行筛选,在获得的再生植株中,Cre重组酶的表达消除了转化体中两lox位点间的gfp和nptll。实验结果表明可借助GFP荧光的消失,快速选出nptII被消除的二次转化体,同时GUS(作为目的蛋白) 在CaMV 35S启动子驱动下获得表达。最后利用后代的分离将hpt和cre除去。     

Production of Marker-free Transgenic Tobacco Plants by FLP/frt Recombination System

Selectable marker genes that usually encode antibiotic or herbicide resistances are widely used for the selection of transgenic plants, but they become unnecessary and undesirable after transformation selection. An important strategy to improve the transgenic plants' biosafety is to eliminate the marker genes after successful selection. In the FLP/frt site-specific system of 2-μm plasmid from Saccharomyces cerevisiae, the FLP enzyme efficiently catalyzes recombination between two directly repeated FLP recombination target (frt) sites, eliminating the sequence between them. By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs, the system can be applied to eliminate the marker genes after selection. Through a series of procedures, the plant FLP/frt site-specific recombination system was constructed, which included the frt-containing vector pCAMBIA1300-betA-frt-als-frt and the FLP expression vector pCAMBIA1300-hsp-FLP-hpt. The FLP recombinase gene was introduced into transgenic (betA-frt-als-frt) tobacco plants by re-transformation. In re-transgenic plants, after heat-shock treatment, the marker gene als flanked by two identical orientation frt sites could be excised by the inducible expression of FLP recombinase under the control of hsp promoter. Excision of the als gene was found in 41 % re-transgenic tobacco plants, which indicated that this system could make a great contribution obtaining the marker-free 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.     

Development of visible markers for transgenic plants and their availability for environmental risk assessment

Monitoring of transgenic plants in the field is important, but risk assessment has entailed laborious use of invisible marker genes. Here, we assessed three easily visible marker transgenes--green fluorescent protein (GFP), R, and Nicotiana tabacum homeobox (NTH) 15 genes--for their potential use as marker genes for monitoring genetically modified plants. Transgenic Arabidopsis thaliana plants for each of these genes were visibly distinguished from wild-type plants. We determined the germination rate, 3-week fresh weight, time to first flowering, and seed weight of the transgenic plants to evaluate whether the expression of these marker genes affected the growth of the host. Introduction of GFP gene had no effect on the evaluated parameters, and we then used the GFP gene as a marker to assess the outcrossing frequency between transgenic and two Arabidopsis species. Our results showed that the hybridization frequency between transgenic plants and Arabidopsis thaliana was 0.24%, and between transformants and Arabidopsis lyrata it was 2.6% under experimental condition. Out-crossing frequency was decreased by extending the distance between two kinds of plants. Thus, the GFP gene is a useful marker for assessing the whereabouts of transgenes/transformants in the field. We also demonstrated that the GFP gene is possibly applicable as a selection marker in the process of generation of transgenic plants.     

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     

转基因植物生物安全标记基因

在大量转基因植物被推向市场的同时,人们对转基因植物对环境及人类健康等许多方面可能存在的风险感到担扰。标记基因的生物安全性成为人们普遍关注的问题之一。新的标记基因不仅要求能够对转基因植物进行筛选和鉴定,而且必须对环境和生物都是安全的。概述并评价了绿色荧光蛋白基因、核糖醇操纵子、6磷酸甘露糖异构酶基因、木糖异构酶基因以及谷氨酸1半醛转氨酶基因等生物安全标记基因及其最新研究进展 。     

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.