Tumor DNA structure in plant cells transformed by A. tumefaciens

Crown gall tumors are induced in plants by infection with the soil bacterium Agrobacterium tumefaciens. Because the tumor induction involves transfer of a portion of the tumor-inducing (Ti) plasmid DNA from the bacterium to the plant cells, this system is of interest for the study of genetic exchange as well as tumor induction. The boundaries of the transferred DNA (T-DNA) have been cloned from transformed plant cells of tobacco. Detailed mapping with restriction enzymes and nucleotide sequence analysis of two independent clones were used to study the molecular structure of the ends of the T-DNA. One clone contains the two ends of the T-DNA joined together; the other contains one end of the T-DNA joined to repetitive plant DNA sequences. These studies provide direct evidence that the T-DNA can be integrated into the plant genome. In addition, the data suggest that in the plant, T-DNA can be tandemly repeated. Sequence analysis of the junction of crown gall clone 1 reveals several direct repeats as well as an inverted repeat; these structures may be involved in the transfer of the DNA from Agrobacterium to plant cells.     

Single-Stranded DNA Binding Protein Encoded by the virE Locus of Agrobacterium tumefaciens

The transfer process of T (transfer)-DNA of Agrobacterium tumefaciens is activated after the induction of the expression of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. The vir gene products then act to generate a free transferable single-stranded copy of the T-DNA, designated the T-strand. Although some vir proteins are responsible for the synthesis of the T-strand, others may mediate T-strand transfer to plant cells as part of a DNA-protein complex. Here, a novel 69-kilodalton vir-specific single-stranded DNA binding protein is identified in Agrobacterium harboring a nopaline-type Ti plasmid. This protein binds single-stranded but not double-stranded DNA regardless of nucleotide sequence composition. The molecular size of the vir-specific single-stranded DNA binding protein and its relative abundance in acetosyringone-induced Agrobacterium suggested that it might be the product of the virE locus; molecular cloning and expression of the virE region in Escherichia coli confirmed this prediction.     

Transfer of Agrobacterium DNA to Plants Requires a T-DNA Border But Not the virE Locus

Agrobacterium tumefaciens induces tumors in plants by transferring and integrating oncogenes (T-DNA) into the chromosomes of host plant cells. Agrobacterium strains were used to transfer complementary DNA copies of a potato spindle tuber viroid (PSTV) to plant cells at a wound site on tomato plant stems. Subsequently, infectious viroid RNA was found in the leaves of these plants, indicating systemic PSTV infection. This process utilized the T-DNA transfer mechanisms of Agrobacterium since PSTV infection required most virulence genes (vir) as well as one of the DNA sequences that flank either side of the Agrobacterium T-DNA. However, transfer still occurred from virE mutants of Agrobacterium, strains that fail to induce tumors even though a completely functional T-DNA is present. The virE gene seems to be directly involved in the integration of foreign DNA into plant chromosomes.     

Introduction of genetic material into plant cells

The tumor-inducing (Ti) plasmid of the soil microorganism Agrobacterium tumefaciens is the agent of crown gall disease in dicotyledonous plants. The Ti plasmid contains two regions that are essential for the production of transformed cells. One of these regions, termed transfer DNA, induces tumor formation and is found in all established plant tumor lines; the other, termed the virulence region, is essential for the formation but not the maintenance of tumors. Transfer DNA, which transfers to the plant genomes in a somewhat predictable manner, can be increased in size by the insertion of foreign DNA without its transferring ability being affected. The tumor-causing genes can be removed so that they no longer interfere with normal plant growth and differentiation. This modified Ti plasmid can thus be used as a vector for the transfer of foreign genes into plants.     

参与农杆菌侵染及T-DNA转运过程植物蛋白的研究进展和思考

农杆菌是一种革兰氏阴性土壤病原细菌,携带具有天然转基因功能的Ti质粒或Ri质粒,能将一部分遗传物质插入到寄主植物的染色体上,使其稳定遗传和表达,赋予植物新的性状。所以农杆菌作为最有效的转化媒介,已被广泛应用于多数双子叶植物和部分单子叶植物转基因研究。虽然农杆菌介导的转化技术具有操作简单、成本低廉,转基因沉默几率小,插入基因拷贝数少等优点,但农杆菌介导植物遗传转化是一个复杂的生物学过程,需要一系列农杆菌蛋白和植物蛋白相互作用,共同完成外源基因的转入和整合。植物相关蛋白在转化过程中起着重要作用。其中,阿拉伯半乳聚糖蛋白(AGP)、植物根钙粘附蛋白和类玻连蛋白等参与农杆菌附着于植物细胞表面的过程;鸟苷三磷酸腺苷酶（GTPase）和BTI蛋白协助T-DNA和Vir效应蛋白进入植物细胞;actin、GIP、VIP等蛋白参与T-DNA复合体在细胞质中的运输的过程;与Vir效应蛋白互作的VIP1、VIP2、KAPa、PP2C、Roc等蛋白协助T-DNA定位于植物细胞核;组蛋白、VIP1和VIP2等引导T-DNA在植物基因组上的整合。由于植物种类间存在巨大差异,上述一些植物蛋白基因的过表达虽能提高农杆菌转化某些植物的转化效率,但不能提高另一些植物的转化效率。在容易被农杆菌遗传转化的植物如拟南芥、水稻中的研究表明,VIP1、VIP2、AGP、H2A等蛋白与农杆菌转化关系密切,但这些蛋白在利用农杆菌转化较难的作物如小麦、玉米中的功能还不明确,因而需要在不同植物中继续筛选和鉴定与T-DNA转化相关重要蛋白的编码基因。目前,农杆菌介导的植物遗传转化有2个显著特点,一是农杆菌介导转化烟草、拟南芥、水稻等模式植物的技术日渐成熟,二是农杆菌介导转化小麦、玉米、大豆等重要作物的技术仍然没有本质突破,植物相关蛋白在T-DNA转运、整合等过程中的作用还需要深入研究和进一步明确。文章主要对参与农杆菌介导遗传转化植物整个过程中相关植物蛋白的研究进展进行了综述,以期为提高农杆菌转化顽拗型作物的转化效率提供参考。     

Molecular basis for the auxin-independent phenotype of crown gall tumor tissues

The transfer of specific Ti (tumor-inducing) plasmid sequences, the T-DNA, from Agrobacterium tumefaciens to a wide range of plants results in the formation of crown gall tumors. These tissues differ from most plant cells in that they can be grown in vitro in the absence of added phytohormones. Here, data are presented that offer an explanation for the auxin-independent phenotype of crown gall tissues. It is shown that crude cell-free extracts prepared from three bacterial species harboring pTiA6 gene 1 could convert L-tryptophan to indole-3-acetamide; control extracts lacking gene 1 could not carry out the reaction. Other reports indicate that the pTiA6 gene 2 product can convert indole-3-acetamide to indole-3-acetic acid, a naturally occurring auxin of plants. It is concluded that the auxin-independent phenotype of crown gall tissue involves the introduction of Ti plasmid sequences encoding a two-step pathway for auxin synthesis.     

Site-Specific Nick in the T-DNA Border Sequence as a Result of Agrobacterium vir Gene Expression

The T-DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the expression of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. The vir gene products act in trans to mobilize the T-DNA element from the bacterial Ti plasmid. The T-DNA is bounded by 25-base pair direct repeat sequences, which are the only sequences on the element essential for transfer. Thus, specific reactions must occur at the border sites to generate a transferable T-DNA copy. The T-DNA border sequences were shown in this study to be specifically nicked after vir gene activation. Border nicks were detected on the bottom strand just after the third or fourth base (+/- one or two nucleotides) of the 25-base pair transferpromoting sequence. Naturally occurring and base-substituted derivatives of the 25-base pair sequences are effective substrates for acetosyringone-induced border cleavage, whereas derivatives carrying only the first 15 or last 19 base pairs of the 25-base pair sequence are not. Site-specific border cleavages occur within 12 hours after acetosyringone induction and probably represent an early step in the T-DNA transfer process.     

Definition of a bacterial type IV secretion pathway for a DNA substrate

Bacteria use conjugation systems, a subfamily of the type IV secretion systems, to transfer DNA to recipient cells. Despite 50 years of research, the architecture and mechanism of action of the channel mediating DNA transfer across the bacterial cell envelope remains obscure. By use of a sensitive, quantifiable assay termed transfer DNA immunoprecipitation (TrIP), we identify contacts between a DNA substrate (T-DNA) and 6 of 12 components of the VirB/D4 conjugation system of the phytopathogen Agrobacterium tumefaciens. Our results define the translocation pathway for a DNA substrate through a bacterial conjugation machine, specifying the contributions of each subunit of the secretory apparatus to substrate passage.     

质粒拯救型T-DNA标签质粒的构建

[目的]为了高效率地获得转基因植物的旁邻序列。[方法]以pUC18及pWM101为出发质粒,通过用限制性内切酶EcoRⅠ和HindⅢ双酶切质粒pUC18,得到含大肠杆菌复制起点及氨苄青霉素抗性基因的pUC18大片断,并将这段DNA整合到pWM101的T-DNA之中构建重组质粒。[结果]该质粒利用根癌农杆菌转化植物后,可利用潮霉素筛选转化细胞,拯救质粒则可转化大肠杆菌后,以氨苄青霉素进行筛选。[结论]该研究为以后的用T-DNA标签研究植物功能奠定了基础。     

Gene transfer in cereals

Until recently, gene transfer in plants was achieved only by sexual hybridization. Now, in addition, plant genetic manipulation, with the use of both recombinant DNA and protoplast fusion technology, is being applied to an increasing range of plants. The soil bacterium Agrobacterium tumefaciens, with its associated plasmid, is used as a vector for introducing DNA into the genomes of dicotyledonous plants, but it has not proved suitable for cereals. Instead, the direct uptake of plasmid DNA into cereal protoplasts is being used for the transformation of cells in rice, wheat, and maize. Transformation efficiencies, in some cases, are becoming comparable to those obtained in dicotyledons with Agrobacterium. In rice it is now possible to regenerate efficiently whole plants from protoplasts, and this capability may soon be extended to the other cereals. By means of direct interaction of cereal protoplasts with plasmids, coupled with improved procedures for the regeneration of plants from their protoplasts, gene transfer in the cereals is becoming established at the frontiers of recombinant DNA technology.     

A simple and general method for transferring genes into plants

Transformed petunia, tobacco, and tomato plants have been produced by means of a novel leaf disk transformation-regeneration method. Surface-sterilized leaf disks were inoculated with an Agrobacterium tumefaciens strain containing a modified tumor-inducing plasmid (in which the phytohormone biosynthetic genes from transferred DNA had been deleted and replaced with a chimeric gene for kanamycin resistance) and cultured for 2 days. The leaf disks were then transferred to selective medium containing kanamycin. Shoot regeneration occurred within 2 to 4 weeks, and transformants were confirmed by their ability to form roots in medium containing kanamycin. This method for producing transformed plants combines gene transfer, plant regeneration, and effective selection for transformants into a single process and should be applicable to plant species that can be infected by Agrobacterium and regenerated from leaf explants.     

Crown gall disease and prospects for genetic manipulation of plants

Agrobacterium tumefaciens incites crown gall tumors when bacterial DNA integrates into plant nuclear DNA. Plant cells can express these integrated bacterial genes. Following insertion of desired genes into bacterial DNA using recombinant DNA techniques, this system permits introduction of these new genes into plant DNA. We discuss the potential for genetic manipulation of plants using Agrobacterium tumefaciens and the related organism Agrobacterium rhizogenes.     

番茄外源基因转化系统的研究

本文结合反义ＡＣＣ基因的番茄中的导入,就番茄外源基因转化系统建立中的几个问题做了研究。结果表明,转基因番茄的组织培养比普通番茄更复杂和困难,需要对其培养基的激素配比做系统研究,以选择更适合的培养基,抗生素的使用要掌握好剂量,在番茄的子叶上以５０ｍｇ／Ｌ为宜;叶盘法转化番茄子叶时采用的菌液浓度及处理时间对转化也有很大的影响,以农杆菌过夜培养液用ＭＳ液体培养基稀释１０倍,浸泡时间为５ｍｉｎ为宜;转基因     

Transgenic plants as tools to study the molecular organization of plant genes

Transgenic plants are generated in nature by Agrobacterium tumefaciens, a pathogen that produces disease through the transfer of some of its own DNA into susceptible plants. The genes are carried on a plasmid. Much has been learned about how the plasmid is transferred, how the plasmid-borne genes are organized, regulated, and expressed, and how the bacteria's pathogenic effects are produced. The A. tumefaciens plasmid has been manipulated for use as a general vector for the transfer of specific segments of foreign DNA of interest (from plants and other sources) into plants; the activities of various genes and their regulation by enhancer and silencer sequences have been assessed. Future uses of the vector (or others like it that have different host ranges) by the agriculture industry are expected to aid in moving into vulnerable plants specific genes that will protect them from such killers as nonselective herbicides, insects, and viruses.     

根癌农杆菌介导玉米遗传转化的研究进展

对根癌农杆菌介导的玉米基因转化中包括外植体、基本培养基及其附加物、共培养温度、信号分子与vir基因的活化的几个关键因素研究进展作了论述。近年来的研究发现,大小为1~2mm的幼胚是较为理想的转化外植体,在外植体培养基本培养基一般多选用MS和N6,在基本培养基上附加有机氮和氨基酸、金属离子均有利于转化效率的提高。转化中的共培养温度一般在200C~250C之间。研究认为根癌农杆菌转化禾本科植物的困难在于禾本科植物缺乏酚类物质,难以诱导Vir基因活化表达,因此在转化中多采用加入外源的酚类物质如乙酰丁香酮来提高玉米转化的成功率。     

Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens

Transformed Arabidopsis thaliana plants have been produced by a modified leaf disk transformation-regeneration method. Leaf pieces from sterilely grown plants were precultured for 2 days and inoculated with an Agrobacterium tumefaciens strain containing an avirulent Ti (tumor-inducing) plasmid with a chimeric gene encoding hygromycin resistance. After cocultivation for 2 days, the leaf pieces were placed on a medium that selects for hygromycin resistance. Shoots regenerated within 3 months and were excised, rooted, and transferred to soil. Transformation was confirmed by opine production, hygromycin resistance, and DNA blot hybridization of both primary transformants and progeny. This process for producing transgenic Arabidopsis plants should enhance the usefulness of the species for experimental biology.     

Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene

A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.     

根瘤农杆菌二组分体VirA/VirG介导的侵染性

根瘤农杆菌（Agrobacterium tumefaciens）是存在于土壤中的革兰氏阴性菌，可以侵染很多种双子叶植物、单子叶植物以及裸子植物的受伤部位，用位于其Ti质粒上vir基因产物来诱导植物产生冠瘿瘤，因此，根瘤农杆菌广泛用于植物基因工程研究，是个农业工程菌。研究表明vir基因的表达受VirA/VirG二组分体的介导。回顾和总结了根瘤农杆菌的VirA/VirG二组分体的结构、作用机理、以及所感知的环境因子，和这些环境因子通过VirA/VirG二组分体对vir基因的表达调控分子机制。     

Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants

The luciferase gene from the firefly, Photinus pyralis, was used as a reporter of gene expression by light production in transfected plant cells and transgenic plants. A complementary DNA clone of the firefly luciferase gene under the control of a plant virus promoter (cauliflower mosaic virus 35S RNA promoter) was introduced into plant protoplast cells (Daucus carota) by electroporation and into plants (Nicotiana tabacum) by use of the Agrobacterium tumefaciens tumor-inducing plasmid. Extracts from electroporated cells (24 hours after the introduction of DNA) and from transgenic plants produce light when mixed with the substrates luciferin and adenosine triphosphate. Light produced by the action of luciferase was also detected in undisrupted leaves or cells in culture from transgenic plants incubated in luciferin and in whole transgenic plants "watered" with luciferin. Although light was detected in most organs in intact, transgenic plants (leaves, stems, and roots), the pattern of luminescence appeared to reflect both the organ-specific distribution of luciferase and the pathway for uptake of luciferin through the vasculature of the plant.