发根农杆菌Ri质粒rol基因研究进展及在林木改良上的应用

Ri质粒上携带的诱根基因的表达不仅能导致植物被感染部位形成大量的毛根,而且由毛根容易获得再生的转化植株,这些植株可表现出许多能稳定遗传的表型变异,在植物遗传改良中有着广阔的应用前景。20世纪80年代以来,国内外学者对发根农杆菌Ri质粒及其rol基因进行了广泛深入的研究。本文重点综述了农杆碱型发根农杆菌Ri质粒的结构与功能,rol基因的位点与特征,Ri质粒的转化与rol基因的表达对植物生长发育的影响及在林木遗传改良上应用等方面的研究现状,并讨论了Ri质粒rol基因在林木遗传改良应用上存在的问题与应用前景。     

农杆菌介导的单子叶植物遗传转化研究进展

根癌农杆菌(Agrobacterium tumefaciens)Ti质粒转化系统的建立使植物遗传工程进入了一个飞速发展的时期。近年来,发根农杆菌(A.rhizogenes)Ri质粒毛根转化系统的研究十分迅速,展示了美好的前景,农杆菌介导的植物遗传转化已成为目前研究和应用最广泛的系统。但是,农杆菌的宿主范围一般仅限于双子叶植物和一些裸子植物,这就直接防碍着这种比较完善的基因转移技术在单子叶植物,尤其是禾谷类作物转化的应用。本文介绍了农杆菌介导的单子叶植物遗传转化的进展;对扩大农杆菌宿主范围、实现对单子叶植物转化的途径进行了探讨。 (一)农杆菌介导的单子叶植物转化的方法 目前建立的单子叶植物基因转移系统有:(1)农杆菌载体系统;(2)外源DNA     

Ri质粒介导的毛状根体系建立及其在植物次生代谢产物合成中的研究进展

发根农杆菌Ri质粒可诱导植物产生毛状根体系,该体系具有遗传性状稳定且增殖速度快的特点,可用于药用植物次生代谢产物的生产研究,为利用生物反应器技术进行药用植物有效成分工业化水平的发酵培养开辟了新途径。本文主要综述了发根农杆菌Ri质粒介导的植物毛状根体系遗传转化机理,并对毛状根体系在药用植物次生代谢产物生产中的研究现状进行了深入分析,为从基因水平上调控植物次生代谢产物的合成提供新思路。     

NaCl胁迫对转基因杨树外源基因表达的影响

以具高抗虫性的转抗虫基因‘741杨’及在此基础上转入了发根农杆菌Ri质粒T-DNA株系的组培苗为材料,研究了转基因株系BtCrylAc抗虫基因和发根基因的表达及其对NaCl胁迫的反应。结果表明,转入Ri质粒T- DNA上的rol基因后,导致苗木根系数目增加,根系长度减小,IAA和GA含量显著提高,抗虫BtCrylAc基因编码的毒蛋白的表达量降低;随着NaCl胁迫强度的增加,苗高、根系数量、叶绿素含量及IAA、GA含量逐步降低,而根系的长度加大,Bt毒蛋白含量显著提高,表明NaCl胁迫使转基因杨外源Bt毒蛋白基因的表达增强,而发根农杆菌Ri质粒T-DNA的表达下降。     

紫花苜蓿外源基因共转化植株的再生

高含硫氨基酸蛋白(HNP)基因和发根(rol)基因由发根农杆菌介导转入紫花苜蓿, 成功地从子叶毛根组织诱导转基因植株再生. 子叶是较理想的转化受体, 毛根年龄与紫花苜蓿体细胞胚分化频率呈负相关. 共转化植株高产、优质性状的产生对紫花苜蓿新品种的培育有重要意义.     

Ri质粒介导的DNA转移及诱发甘草毛根再生植株

本文报道了通过三亲交配将PB1121双元中间载体从大肠杆菌C600转移到发根农杆菌R1000(PRiA4b)中,在含Km、Sm的MinA选择平板上获得了转移子,上述转移子菌液用注射法感染甘草胚轴、茎等外植体,在无激素含1mg/mlcb,0.1mg/mlkm的MS培养基上培养,两周后诱发出毛根,切下毛根在同上培养基上6周长出不定芽,继续培养长成再生植株,经检测转化株毛根中存在甘露碱和GUS基因产物,具有Km抗性和激素自主性生长特性,证明Ri质粒不仅可介导GUS基因转移还可诱发毛根再生植株,提供了简化、高效的植物遗传转化系统。     

根癌农杆菌介导法在禾本科植物遗传转化中的应用

在植物基因工程中，农杆菌质粒介导的遗传转化是比较完善与有效的基因转移方法，但在单子叶植物中应用较少。文章介绍了农杆菌介导遗传转化技术在禾本科植物改良中的研究进展，并对此法的应用前景作了分析。     

快速克隆DNA大片段的方法

文章采用菌落PCR的技术,快速扩增发根农杆菌Ri质粒rol（包括rolA、rolB、rolC）基因的DNA片段并用于测序分析。克服了由于Ri质粒过大,提取困难等问题。     

Ri质粒诱导的植物发根培养系及其应用

阐述了具Ri质粒的发根农杆菌的生物学特性,介绍了毛状根的诱导、筛选及增殖培养的具体方法,提出了农杆菌转化植物细胞的影响因素,对国内外利用植物发根培养系进行次生代谢物质生产方面的研究进行了综述,并对Ri质粒诱导的发根培养系的应用作了展望。     

高等植物在发根农杆菌介导下的遗传转化

本文介绍了发根农杆菌的生物学特性、遗传转化的操作技术、RiT-DNA转化体的形态特征以及发根农杆菌转化的利用。与根癌农杆菌不同,农杆碱型发根农杆菌的RiT-DNA含有生长素合成基因、农杆碱、甘露碱合成基因,不含有细胞分裂素合成基因,它的转化体首先是转化根。发根农杆菌介导的植物遗传转化,在次生代谢产物生产,植物抗逆性育种以及细菌与植物进化关系的研究等方面具有广泛的应用前景。     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

Use of ri-mediated transformation for production of transgenic plants

Summary Agrobacterium rhizogenes-mediated transformation has been used to obtain transgenic plants in 89 different taxa, representing 79 species from 55 genera and 27 families. A diverse range of dicotyledonous plant families is represented, including one Gymnosperm family. In addition to the Ri plasmid, over half these plants have been transformed with foreign genes, including agronomically useful traits. Plants regenerated from hairy roots often show altered plant morphology such as dwarfing, increased rooting, altered flowering, wrinkled leaves and/or increased branching due to rol gene expression. These altered phenotypic features can have potential applications for plant improvement especially in the horticultural industry where such morphological alterations may be desirable. Use of A. rhizogenes and rol gene transformation has tremendous potential for genetic manipulation of plants and has been of particular benefit for improvement of ornamental and woody plants.     

A critical review on use of Agrobacterium rhizogenes and their associated binary vectors for plant transformation

Agrobacterium rhizogenes, along with A. tumefaciens, has been used to affect genetic transformation in plants for many years. Detailed studies conducted in the past have uncovered the basic mechanism of foreign gene transfer and the implication of Ri/Ti plasmids in this process. A number of reviews exist describing the usage of binary vectors with A. tumefaciens, but no comprehensive account of the numerous binary vectors employed with A. rhizogenes and their successful applications has been published till date. In this review, we recollect a brief history of development of Ri-plasmid/Ri-T-DNA based binary vectors systems and their successful implementation with A. rhizogenes for different applications. The modification of native Ri plasmid to introduce foreign genes followed by development of binary vector using Ri plasmid and how it facilitated rapid and feasible genetic manipulation, earlier impossible with native Ri plasmid, have been discussed. An important milestone was the development of inducible plant expressing promoter systems which made expression of toxic genes in plant systems possible. The successful application of binary vectors in conjunction with A. rhizogenes in gene silencing and genome editing studies which are relatively newer developments, demonstrating the amenability and adaptability of hairy roots systems to make possible studying previously intractable research areas have been summarized in the present review.     

Prospects for applications ofrol genes for crop improvement

Prospects for the applications ofrol genes for crop improvement are discussed. As suggested in many reports, rol genes are suitable tools to modify plant developmental processes, such as formation of adventitious roots and release of axillary buds from apical dominance. Practical applications, however, might be hampered by the many pleiotropic side effects that are observed in plants transformed withrol genes. Alternative approaches need to be developed, therefore, to overcome these undesired effects. We offer a novel approach for application that is clearly different from earlier strategies, and that is based on the application ofrol genes incombination plants; i.e., plants consisting of an untransformed scion grafted on a rootstock transformed with arol gene. In rose it was demonstrated for the first time that expression ofrol genes in rootstocks led to an accelerated release of axillary buds of the untransformed scion, but without the transmission of many undesired pleiotropic effects. We expect that this stimulation will result in a changed plant architecture leading to a more efficient production of roses. Alternatively, the pleiotropic effects may be overcome by employingrol genes that are driven by organ- or tissue-specific promoters, leading to a more defined expression of these genes.     

Transgenic hairy roots. recent trends and applications

Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic roots produced by A. rhizogenes infection is characterized by high growth rate and genetic stability. These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants. Hairy root cultures offer promise for production of valuable secondary metabolites in many plants. The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel. Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots. Development of bioreactor models for hairy root cultures is still a recent phenomenon. It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium. Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants. In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest. In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement. Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest. This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A. rhizogenes.     

Structure and function of root-inducing (Ri) plasmids and their relation to tumor-inducing (Ti) plasmids

The abilities of Agrobacterium tumefaciens and A. rhizogenes to transform dicotyle-dons and cause crown gall and hairy root disease are caused by the presence of tumor inducing (Ti) and root inducing (Ri) plasmids. During transformation plasmid T-DNA (transferred DNA) is inserted into the plant genome. The T-region is flanked by 25 bp direct repeats, which are essential for transfer. The T-regions contain oncogenes that are expressed in the plants. Some of these code for enzymes that synthesize auxin or cytokinin. Another type, present in Ri plasmids only, appears to impose a high hormone sensitivity on the infected tissue. The T-DNA also contains genes for enzymes synthesizing opines, which the bacteria catabolize. The T-DNA transfer is initiated by the induction of genes in the virulence (vir) region of the plasmid by phenolic compounds secreted by wounded tissue. The products of the vir -genes and of chromosomal genes mediate transfer of T-DNA to the plant cells. Crown gall disease is caused by production of auxin and cytokinin by the transferred T-DNA. The T-DNA of Ri plasmids codes for at least three genes that each can induce root formation, and that together cause hairy root formation from plant tissue. Current results indicate that the products of these genes induce a potential for increased auxin sensitivity that is expressed when the transformed cells are subjected to a certain level of auxin. After this stage the transformed roots can be grown in culture without exogenous supply of hormones.     

香石竹毛状根诱导、离体培养及其植株再生

为了探讨利用发根农杆菌遗传转化所产生的毛状根来创新香石竹种质的可能性,本文采用叶盘法,建立了发根农杆菌Agrobacterium rhizogenes对香石竹Dianthus caryophyllus L.叶片外植体的遗传转化及其植株再生体系。结果表明,发根农杆菌ATCC15834感染香石竹幼嫩叶片外植体12 d后,从叶片外植体切口中脉处产生白色毛状根,21 d后约90%的叶片外植体产生毛状根。所获得的无菌毛状根能在无外源激素的MS固体和液体培养基中快速自主生长。PCR扩增和硅胶薄层层析结果显示发根农杆菌Ri质粒的rol B和rol C基因以及冠瘿碱合成酶基因已在香石竹毛状根基因组中整合并得到表达。将毛状根置于MS+6-BA 1.0-3.0 mg/L+NAA 0.1-0.2 mg/L中培养15 d后产生淡黄绿色的疏松愈伤组织。愈伤组织不定芽分化的最适培养基为MS+6-BA 2.0 mg/L+NAA 0.02 mg/L,培养6周后不定芽分化率为100%;平均每个愈伤组织产生30-40个不定芽;将不定芽转至1/2 MS或1/2 MS+0.5 mg/L NAA的培养基中10 d后产生不定根,发育成再生植株。再生植株移植于栽培基质中20 d后,成活率达95%以上。     

发根农杆菌Ri质粒及其在植物科学中的应用

阐述了发根农杆菌的性质及Ri质粒的结构和功能,介绍了毛状根的诱导及其鉴定方法,并对Ri质粒在植物基因工程、植物次生代谢产物生产、植物品种改良和植物栽培等方面的应用进行了综述。     

Genetically Transformed Plant Roots as a Model for Studying Specific Metabolism and Symbiotic Contacts of the Root System

Genetic transformation of plants mediated by Ri plasmid ofAgrobacterium rhizogenes occupies a special place in plant cell engineering, since this technique based on a natural phenomenon allows cultivation of isolated growing plant roots on hormone-free media. Application of wild-type unmodified agrobacterial strains allows us to obtain root cultures capable of long-term growth in vitro due to an increased sensitivity of the cells to auxins while other biochemical properties remain unaltered. A collection of pRi T-DNA transformed roots of certain dicotyledons was made; some strains in it are used to study synthesis of secondary metabolites in root cells. Thein vitro cultivated roots could synthesize root-specific metabolites, which makes possible their application for large-scale biotechnological production of ecologically pure crude drugs. Cocultivation of pRi T-DNA transformed roots with arbuscular mycorrhizal fungi makes possible vital study of all stages of obligate symbiont development and interaction with plant roots. Dual axenic culture of AM fungi and pRi T-DNA transformed plants can be used to make a collection of the most valuable endomycorrhizal fungal species and to produce considerable quantities of homogeneous fungal inoculums.