植物的遗传转化及其应用(英文)

近年来,植物遗传转化研究有了长足的发展。已经达到能够通过简单的遗传控制手段研究具有新表现型的植物,甚至达到进入商业化的程度。这些手段包括植物生物学的主要研究技术以及植物组织培养和树种改良的一些实用方法。尽管采用农瘤杆菌和鸟枪法等技术的植物遗传转化系统已经得到了广泛的应用,但是在如何开发具有能够得到控制表达的转基因高产植物方面,在如何使所得到的转基因植物远离遗传危害等方面,目前的转化系统遇到了极大的技术挑战。已经提出了各种各样的方法用于将新基因稳定地导入120多种不同植物的核基因组。本文将讨论这些遗传转化系统所需的生物学要求和实际应用方面的需求、基因转化和转基因表达的研究策略、遗传转化植物的鉴定以及转基因植物与大众的认可。本文将分为七个部分加以讨论:一、导言;二 、基因转化到细胞里的方法;三、植物遗传转化策略;四、植物遗传转化的鉴定;五、植物遗传转化的实际应用;六、转基因植物与环境;七、未来植物遗传转化的需求与发展方向。     

转基因植物中标记基因的消除

随着转基因植物的商业化,植物遗传转化技术将为农业生产带来一场新的革命,新的基因转化程序要求转基因为单拷贝,不带有标记基因,并在不同的转化体中表达一致,稳定遗传,本文讨论了转基因植物中有关标记基因及其安全性和标记基因消除的方法等问题。     

农杆菌介导的植物遗传转化研究进展

农杆菌介导的转基因方法是目前植物遗传转化的重要方法之一。本文从农杆菌转化原理、菌株比较及载体发展入手,系统讨论了植物转化受体对转化效率的影响,同时分别综述了农杆菌介导转化技术在双子叶和单子叶植物转化应用中的最新进展。     

Advances in Pollen Mediated Genetic Transformation

植物遗传转化技术是植物科学基础理论与应用研究的有力武器,已成为植物遗传改良的重要途径之一。但是、目前遗传转化所采用的受体系统,大都需要体外培养和植株再生过程,才能获得转基因植株。其中、基因型限制和遗传变异是该技术不可逾越的两大障碍。花粉管通道法可省去转化体的离体培养,不过、多数植物受花器结构的限制而难以经花柱注射ＤＮＡ,只能向子房注射,并不是真正的“花粉管通道”。又由于此法外源基因的导入发生在授粉之后,因此该方法亦不属于花粉遗传转化。利用小孢子胚胎发生体系进行遗传转化与利用花粉作为外源ＤＮＡ的媒介,继而、通过授粉受精获得转基因种子,是目前花粉遗传转化的两个重要方面和活跃的研究方向。前者仍需要离体再生系统,后者则可以利用植物自身的再生机制,本文称之为花粉介导法（ｐｏｌｅｎ－ｍｅｄｉａｔｅｄｔｒａｎｓｆｏｒｍａｔｉｏｎ）。该方法通过自然的胚胎发育过程获得转基因子代,避免了组织培养过程中的遗传变异和转基因植株的嵌合现象。可望成为简便快速的植物遗传转化体系。目前对花粉介导的遗传转化进行专门评述的文献较少,本文对该领域的研究分三个层次进行了综述。一、外源基因转化方法小孢子或由小孢子形成的胚状体是很有潜力的遗传转化受体     

安全转基因植物培育技术研究进展

由于关系到转基因植物的产业化前景,安全型转基因植物培育越来越受到公众的关注。在植物遗传转化体系中,绝大多数选择标记基因来源于细菌,对人类健康和环境安全存在潜在风险,因此无选择标记转基因植物培育受到科研工作者的高度重视。本文综述了安全型转基因植物的培育途径,包括共转化系统、位点特异性重组系统、转座子系统、同源重组系统、不依赖于组织培养的简易转化技术及再生相关基因利用等技术,探讨了各种途径的优缺点,以期推动安全型转基因植物培育和转基因植物产业化进程。     

叶绿体转化及其用于疫苗表达研究的最新进展

随着植物转基因研究的不断深入,核基因组转化的转基因沉默现象严重影响了基因工程的应用效果。植物叶绿体遗传转化以叶绿体基因组为平台对植物进行遗传操作,外源基因定点整合及母性遗传特性能较好地解决"顺式失活"和"位置效应"等类的基因沉默问题和转基因逃逸等安全问题,成为植物基因工程发展的新方向,在工业、农业及医药生物领域发挥了重要作用,也为生产廉价、安全的植物疫苗提供了新思路。本文在简要介绍叶绿体转化的原理、转化方法与优势的基础上,重点综述了近年来通过该技术表达的一些重要的病毒抗原和细菌抗原。最后,对叶绿体转化技术在表达外源基因方面存在的问题进行分析。未来随着叶绿体基因表达、调控机制研究的逐渐深入及相关技术体系的日臻完善,叶绿体转化有望成为疫苗生产的生力军。     

安全型转基因植物培育技术研究进展

由于关系到转基因植物的产业化前景,安全型转基因植物培育越来越受到公众的关注。在植物遗传转化体系中,绝大多数选择标记基因来源于细菌,对人类健康和环境安全存在潜在风险,因此无选择标记转基因植物培育受到科研工作者的高度重视。本文综述了安全型转基因植物的培育途径,包括共转化系统、位点特异性重组系统、转座子系统、同源重组系统、不依赖于组织培养的简易转化技术及再生相关基因利用等技术,探讨了各种途径的优缺点,以期推动安全型转基因植物培育和转基因植物产业化进程。     

转基因植物中外源基因的遗传学行为

１９８４年,首次利用农杆菌Ｔｉ质粒将外源基因导人烟草获得成功［１」,随后,转基因禾本科作物在水稻上也获得了成功［２,３］。随着各种遗传转化技术的创立与改进,近十几年来,在许多作物上都获得了转基因植株。植物遗传转化技术在基础研究和应用研究中的价值得到了很大体现。尤其是在应用研究上,植物遗传转化技术与常规的育种技术相比,它以超越种间隔离的特点吸引了广大的分子育种家投入到这方面的研究。迄今为止,许多有应用前景的基因已导入到双子叶植物和单子叶植物,并有少量转基因植物已释放到大田应用［４,５］。我们实验室…     

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.     

基于纳米基因载体的动植物遗传转化研究进展

转基因技术在动植物优良新品种的培育中发挥着重要作用,而随着纳米生物技术的发展,基于纳米材料构建基因载体的动植物转基因技术,对于发展动植物转基因新方法以及加速转基因种质材料的大规模制备、优良新品种的培育进程具有更为重要的意义。综述了纳米基因载体的种类与性质,并结合动植物遗传育种的研究进展,分析了纳米基因载体相比于其他载体的特点及优势,同时,重点阐述了基于纳米基因载体的基因转染技术的基本原理和操作过程,及其在动植物遗传转化中的应用,以期为动植物基因工程改造提供新思路。     

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.     

茎尖法转基因棉花植株真实性鉴定方法探究

棉花茎尖转化法具备不受基因型限制、转化周期短的优点,是理想的棉花转化体系,但据报道其所获得的转基因植株普遍存在遗传不稳定、高代植株基因丢失的现象。以陆地棉TM?1品种为受体材料,利用茎尖转化法将DsRed2载体转入棉花,经卡那霉素筛选获得16株抗性植株,进一步PCR扩增靶基因,获得6株DsRed2基因片段PCR检测为阳性的植株,初步判断该6株为茎尖转化法获得的转基因植株,但经紫外照射,6个转基因植株均未检测到红色荧光。对其进行靶基因RT?PCR,发现DsRed2基因在6个转基因植株中仅有极低量的表达或无表达。进一步对DsRed2载体的非T?DNA片段,即载体骨架部分进行PCR以及植株内生菌培养检测,结果表明,6个转基因植株均含有完整的DsRed2载体,植株可培养出含有完整载体的内生菌,且内生菌经农杆菌16S核糖体RNA（16S rRNA）片段PCR检测结果为阳性,推测由于茎尖侵染形成农杆菌与植株共生关系,造成假阳性株的现象,进而导致高代转基因植株基因丢失、遗传不稳定的现象。旨在建立一套完整的茎尖法转基因棉花植株真实性的鉴定方法,为进一步深入研究提供参考依据。     

Genetic transformation technology: Status and problems

Summary Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.     

Agrobacterium-mediated genetic transformation of plants: biology and biotechnology

Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at understanding and improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains, plasmids and technologies currently used for the successful transformation of numerous plant species. Unlike the role of bacterial proteins, the role of host factors in the transformation process has remained obscure for nearly a century of Agrobacterium research, and only recently have we begun to understand how Agrobacterium hijacks host factors and cellular processes during the transformation process. The identification of such factors and studies of these processes hold great promise for the future of plant biotechnology and plant genetic engineering, as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants.     

Cytokinin vectors mediate marker-free and backbone-free plant transformation

Conventional Agrobacterium-mediated transformation methods rely on complex and genotype-specific tissue culture media for selection, proliferation, and regeneration of genetically modified cells. Resulting transgenic plants may not only contain selectable marker genes but also carry fragments of the vector backbone. Here, we describe a new method for the production of transgenic plants that lack such foreign DNA. This method employs vectors containing the bacterial isopentenyltransferase (ipt) gene as backbone integration marker. Agrobacterium strains carrying the resulting ipt gene-containing "cytokinin" vectors were used to infect explants of various Solanaceous plant species as well as canola (Brassica napus). Upon transfer to hormone-free media, 1.8% to 9.9% of the infected explants produced shoots that contained a marker-free T-DNA while lacking the backbone integration marker. These frequencies often equal or exceed those for backbone-free conventional transformation.     

Genetic Transformation of Major Wine Grape Cultivars of Vitis Vinifera L.

We have developed an Agrobacterium-mediated transformation system for a number of important grapevine cultivars used in wine production. Transgenic plants were obtained for the seven cultivars: Cabernet Sauvignon, Shiraz, Chardonnay, Riesling, Sauvignon Blanc, Chenin Blanc and Muscat Gordo Blanco. Embryogenic callus was initiated from anther filaments and genotypic differences were observed for initiation and subsequent proliferation with Chardonnay responding most favourably to culture conditions. The transformation system allowed the recovery of germinating transgenic embryos 10–12 weeks after Agrobacterium inoculation and plants within 18 weeks. Examination of the expression patterns of the green fluorescent protein gene under the control of the CAMV35S promoter in leaf tissue of transgenic plants showed that for up to 35% of plants the pattern was not uniform. The successful transformation of a genetically diverse group of wine grape cultivars indicates that the transformation system may have general application to an even wider range of Vitis vinifera cultivars.     

Gene Manipulation of a Heavy Metal Hyperaccumulator Species Thlaspi caerulescens L. via Agrobacterium-mediated Transformation

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species.     

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。