发根农杆菌附着植物细胞的离子效应

本文将光学显微镜、电镜技术和附着定量分析方法结合起来,研究了发根农杆菌各菌株附着烟草叶肉原生质体的离子效应。实验结果表明：发根农杆菌不同菌株对共培养基离子强度的敏感性有差异。菌株Ｒ１０００附着植物细胞及其引起民植物细胞的聚集成对离子强度不敏感,而菌株Ａ４和１５８３４则非常敏感;农杆对材料细胞附着的多与其引起植物细胞的聚集程度有关性;共培养基中Ｍｇ＾２＋,Ｍｎ＾２＋等二价阳离子（不包括Ｃａ＾２＋）是     

发根农杆菌及其应用

农杆菌是一类侵染性非常广泛的Ｇ-土壤杆菌,农杆菌质粒介导的基因转移系统是植物基因工程中比较完善与有效的基因转移方法。在众多的转基因植物中有80%是由农杆菌介导转化的,但其中大部分是有根癌农杆菌Ｔｉ质粒介导法获得的。根癌农杆菌含有的Ｔｉ质粒,能诱导被侵染的植物细胞形成肿瘤,即诱发冠瘿瘤。发根农杆菌含有Ｒｉ质粒,侵染植物后能诱发植物细胞产生毛发状根,即发根瘤。近年来的研究发现发根农杆菌Ｒｉ质粒介导法比Ｔｉ质粒法具有一定优越性而被广泛重视,而且由其转化获得转基因植物和生物有效成分的报道愈来愈多,本文主要讨论发根农…     

农杆菌与植物细胞的相互作用

近年来,植物遗传工程的研究进入了一个飞速发展的时期。T_i质粒和R_i质粒是应用最普遍的两个基因工程的载体。它们是分别存在于根癌农杆菌(Agrobaeterium tumofaciens)和发根农杆菌(A.rhizogenes)细胞核外的一种双链环状DNA。人们将控制优良性状(如高产、抗病虫害、抗旱等)的基因通过一定方法整合到T_i或R_i质粒上的T-DNA区,然后借助于农杆菌对植物的感染,将外源基因引入植物细胞并整合     

发根农杆菌菌株的综合鉴定及活力比较

利用3-酮乳糖产物法、差异酸生成实验和游动性实验鉴定发根农杆菌菌株A4、R1205、R1000、R1601、R1022和15834的菌株类型和活力。结果表明,R1205、R1601、R1000、A4为Ⅱ型农杆菌,其活力从大到小依次是R1000、R1205、A4、R1601。利用PCR方法鉴定表明,A4、R1205、R1000和R1601为发根农杆菌,而R1022和15834未出现阳性结果。黄瓜遗传转化力鉴定结果表明,R1000的遗传转化力最大,达到79.16%,其它菌株依次是R1205、R1601、A4。根据上述三方面综合鉴定,R1000菌株活力最大。     

农杆菌T—DNA介导的植物转基因的分子机制

前言 根瘤农杆菌(Agrobacterium tumefaciens)与发根农根菌(A.rhizogenes)同属根瘤菌科,是革兰氏阴性植物病原菌。前者感染植物引起冠瘿病(Crop gall tumour),冠瘿病用含有抗生素的培养基培养与除菌。可无限增殖;后者感染植物诱发出许多不定根,把不定根培养在上述培养基上也可迅速生长,多次分枝成毛状称毛状根(hairy root)。农杆菌感染机理很复杂。其感染作用起始于受伤的植物细胞分泌的大量化学物质时期。受伤植物细胞分泌的酚分子诱导农杆菌怀有的Ti质粒或Ri质粒上的基因活化,尔后农杆菌紧附植物细胞并把Ti质粒或Ri质粒上一部分DNA(称作T—DNA,tranfer DNA)转移到植物染色体上。T.DNA共价整合后编译合成的新的低分量的代谢物,称为冠瘿碱。     

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     

发根农杆菌Ri质粒在药用植物生物工程中的应用

发根农杆菌(Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓ)与根癌农杆菌(Ａ.ｔｕｍｅｆａｃｉｅｎｓ)属于根瘤菌科,均为革兰氏阴性菌[1]。它们在侵染植物后引起的症状不同,含有Ｔｉ质粒的根癌农杆菌在侵染植物后形成冠瘿瘤(ｃｒｏｗｎｇａｌｌ),而含有Ｒｉ质粒的发根农杆菌表现与其不同,它在感染植物后在植物的伤口部位诱发产生毛状根(ｈａｉｒｙｒｏｏｔ)。由发根农杆菌侵染植物诱导产生的毛状根具有生长快、分枝多、根毛多等特点。发根农杆菌Ｒｉ质粒与根癌农杆菌Ｔｉ质粒结构相似,都具有高效率转移的Ｔ-ＤＮＡ区和致病的Ｖｉｒ区…     

纤维植物罗布麻发根的诱导及植株再生

利用3种发根农杆菌(LBA9402.R601,和R1000)转化纤维植物罗布麻无菌种子苗的根茎叶不同外植体部位,首次诱导其生成发根并实现了直接由发根途径的植株再生.罗布麻发根诱导与所用的发根农杆菌菌株,外植体部位及光周期密切相关.发根农杆菌LBA9402感染罗布麻的根外植体,实现了最高转化率达100%.与LBA9402及R601相比,被发根农杆菌R1000感染的根外植体适合在黑暗环境下培养.其诱导生成的发根密度可达平均每个外植体22条.在不加激素的1/2 MS培养基上,LBA9402和R601诱导产生的发根可以诱导生成不定芽,不定芽诱导率达20%.不定芽切下后,在不加激素的1/2 MS培养基上2周内可以诱导生根.通过聚合酶链式反应(PCR)对发根及再生植株进行了鉴定,证明发根农杆菌的T-DNA插入了植物的基因组.为罗布麻的分子育种建立了稳定的转化及再生体系,为下一步通过转入外源基因改善其农艺性状奠定了基础.     

4种发根农杆菌对朱砂根组培无菌叶片毛状根诱导的影响

以朱砂根(Ardisia crenata Sims)组培无菌叶片为材料,用4种发根农杆菌菌株(A4、ATCC15834、LBA9402和R1601)分别侵染进行毛状根诱导,比较朱砂根叶片毛状根诱导的最适培养基种类、预培养时间、侵染方式、共培养时间以及不同发根农杆菌的致根能力。研究表明:(1)朱砂根无菌叶片毛状根诱导最适培养基为1/2MS培养基,预培养2d、共培养2d,毛状根诱导率最高(31.87%)。(2)最佳侵染方式以剪好的幼叶和活化好的菌液(100mg/L AS)一起在28℃、180r/min黑暗条件下共振荡8~15min。(3)4种发根农杆菌均能诱导朱砂根叶片毛状根产生,但A4、ATCC15834效果最好,其致根能力大小顺序依次为ATCC15834A4LBA9402R1601。(4)PCR分子鉴定表明,发根农杆菌Ri质粒T-DNA已成功整合到宿主细胞核基因组中。     

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。     

Observation by SEM of the attachment ofAgrobacterium tumefaciens to the surface of vinca,asparagus and rice cells

Transformation of vinca cells was performed by the co-cultivation of cell-wall regenerated vinca protoplasts withAgrobacterium tumefaciens. Using thisin vitro and single cell system, attachment of the bacteria to the surface of vinca cells was observed by scanning electron microscopy (SEM). Figures of the bacteria polarly binding to the plant cell wall were often observed. AsEscherichia coli does not attach to the plant cells at all, the observed attachment ofA. tumefaciens is suggested as a characteristic feature in crown gall induction. Even though no evidence of transformation was obtained by the co-cultivation methods, a similar attachment was observed in the cell-wall regenerated protoplasts of rice. The bacteria also attached to the surface of isolated mesophyll cells of asparagus and root hairs of rice. From these observation, we concluded that the attachment is not the limiting step of crown gall induction byA. tumefaciens in monocotyledonous plants. Extracellular fibrils like pili were observed with a few strains of A.tumefaciens for the first time. These fibrils were observed regardless of their ability of attachment and infectivity.     

Polyethylene glycol-assisted transfection of Streptomyces protoplasts

In the presence of polyethylene glycol (concentration optimum 20%), protoplasts of appropriate Streptomyces strains could be transfected by deoxyribonucleic acid (DNA) of five temperate phages (phi C31, VP5, R4, phi 448, and S14) belonging to four different immunity groups. Quantitation of transfection was made possible by plating the transfection mixture with excess uninfected protoplasts in soft agar overlays on protoplast regeneration medium so that plaques were easily detected. Optimum frequencies of transfection in the ranges of 10(-6)/DNA molecule and 10(-5)/viable protoplast were invariably obtained. It appeared that single DNA molecules initiated transfection events, and that the conformation of the DNA (i.e., circular or linear) was not important. Inhibition of transfection by ethylenediaminetetraacetic acid suggested that divalent cations were also observed. A minor subpopulation of protoplasts appeared to be particularly sensitive to transfection (i.e., "competent") in some DNA-host combinations. In such cases the size of this subpopulation was the major limiting factor in obtaining high transfection frequencies. The same protoplast     

Agrobacterium rhizogenes mutants that fail to bind to plant cells.

Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria to bind to carrot cells. The parent strain bound to carrot cells in greatest numbers in low-ionic-strength media such as 4% sucrose but still showed significant binding in Murashige-Skoog tissue culture medium. All of the mutants showed reduced binding in 4% sucrose after 2 h of incubation with carrot cells. One mutant was delayed in binding in 4% sucrose. This mutant and one other mutant also showed reduced binding to carrot cells in Murashige-Skoog medium. To determine whether the Tn5 insertion was responsible for the mutant phenotype, DNA containing the Tn5 insertion was cloned from the mutant bacteria and used to introduce Tn5 into the parent strain in the same location as in the original mutant by marker exchange. The resulting transconjugants had the same avirulent, nonattaching phenotype as the original mutants, suggesting that the mutant phenotype was due to the Tn5 insertion. The cloned DNA containing the Tn5 insertion was also tested for homology to DNA of known genes that affect attachment of Agrobacterium tumefaciens to plant cells by DNA hybridization. No homology to chv, att, or pscA clones was observed. In addition, cloned chv, att, and pscA genes from A. tumefaciens were unable to complement the attachment-minus A. rhizogenes mutants. Thus, the A. rhizogenes nonattaching mutants appear to be different from the previously described A. tumefaciens mutants.     

中药植物黄山药发根基因的遗传转化

以发根农杆菌菌株A4转染已预培养1d的黄山药茎段后,共感染3d,其转化效果最佳;转化毛状根在无生长调节物质的MS培养基上培养可获得丛状芽,并发育成植株。     

Colonization of arbuscular-mycorrhizal fungi on Ri T-DNA transformed roots in synthetic medium

Hairy root culture of tomato (Lycopersicon esculantum L.) was induced with three strains of Agrobacterium rhizogenes namely A4, ATCC 15834 and LBA 9402. The best response in terms of growth of hairy root was observed with A. rhizogenes strain A4 and LBA 9402 followed by ATCC 15834. Hairy roots were maintained on Murashige and Skoog (MS) medium but it could also grow on minimal (M) medium. Spores of Gigaspora margarita were isolated by wet sieving and decanting method and further recovered by sucrose density gradient method. A new method for surface sterilization of spores has been described which is simpler than the methods described earlier. Surface sterilized spores of G. margarita were used for inoculation of transformed roots grown on M medium as it was found more favourable for germination and growth of spores. During co-cultivation, mycorrhizal spore germination and its penetration into root cortex were observed. Inter and intracellular mycelial spread and formation of arbuscules were also observed in the cortical region of transformed roots of this plant.     

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca²⁺concentration was lowered by ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.Abbreviations ConA concanavalin A - CW calcofluor white - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - -Man -methyl-d-mannoside     

Spontaneous protoplast formation in Methanobacterium bryantii.

Methanobacterium bryantii was found to undergo rapid lysis when grown in a prereduced chemically defined medium under H2-CO2 (4:1, vol/vol). The addition of 20 mM MgCl2 to the medium gave, rather than rapid lysis, a gradual formation of phase-dark spherical bodies which in thin section appeared as true protoplasts. In general, the protoplasts were stabilized by divalent but not monovalent cations and, unlike whole cells, were sensitive to lysis by Triton X-100. Electron microscopic examination revealed that protoplast formation was preceded by a general breakdown of the cell wall with an apparent squeezing out of the protoplast through the degraded wall. The growth of cells was greatly increased and not accompanied by detectable lysis in a medium modified by elevating the levels of nickel and ammonium.     

Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli

Inductively coupled plasma emission spectroscopy was used to quantitate the metal cations bound to outer and cytoplasmic membranes and to extracted lipopolysaccharide from several Escherichia coli K12 strains. The outer membrane was found to be enriched in both calcium and magnesium relative to the cytoplasmic membrane. Both membranes contained significant levels of iron, aluminum, and zinc. The multivalent cation content of the lipopolysaccharide resembled that of the intact outer membrane. Lipopolysaccharide extracted from wild-type k12 strains contained higher levels of Mg than Ca regardless of the growth medium, but the medium used for growth did affect the relative amounts of bound Mg as well as the levels of the minor cations iron, aluminum, and zinc. In contrast, lipopolysaccharide isolated from a deep rough mutant strain, D21f2, contained more Ca than Mg. Electrodialysis of lipopolysaccharide from wild-type k12 strains removed 1 mol of Mg per mol of lipopolysaccharide but did not significantly affect the level of other bound metal ions. Dialysis of lipopolysaccharide against sodium (ethylenedinitrilo)tetraacetate removed most of the Mg and Ca, resulting in a sodium salt. The equimolar replacement of divalent cations with sodium in the sodium salt resulted in a net loss of counterion change. The sodium salt was dialyzed against either tris(hydroxymethyl)aminomethane hydrochloride, CaCl2, MgCl2, or TbCl3, and the resulting lipopolysaccharide salts were analyzed for their ionic composition. It was shown that tris(hydroxymethyl)aminomethane and Ca can replace some but not all of the Na bound to the sodium salt, but all of the other multivalent cations tested replaced Na, resulting in uniform lipopolysaccharide salts. Lipopolysaccharide isolated from the deep rough mutant strain D21f2 was also converted into a sodium salt. Relative to the wild-type lipopolysaccharide, Na was able to neutralize the anionic charge to a greater extent in the mutant lipopolysaccharide. Our results suggest that the loss of specific groups in the core region of the lipopolysaccharide from the mutant strain results in a more open structure that allows the binding of larger cations and of more monovalent cations.     

CATION UPTAKE BY INTACT NUCLEI FROM RAT CEREBRAL CORTEX

A method is described for determining the uptake of cations by intact nuclei from rat cerebral cortex. The divalent cations of magnesium, manganese and calcium were found to be concentrated in the nuclear pellet to levels well above the initial concentration of the medium, whereas little uptake of either potassium or sodium was observed. The binding of divalent cations to the nuclei will produce a cationic environment quite different from that of the external medium. Such localized differences may play a role in the control of nuclear activities.     

Yeast Frataxin Is Stabilized by Low Salt Concentrations: Cold Denaturation Disentangles Ionic Strength Effects from Specific Interactions

Frataxins are a family of metal binding proteins associated with the human Friedreich''s ataxia disease. Here, we have addressed the effect of non-specifically binding salts on the stability of the yeast ortholog Yfh1. This protein is a sensitive model since its stability is strongly dependent on the environment, in particular on ionic strength. Yfh1 also offers the unique advantage that its cold denaturation can be observed above the freezing point of water, thus allowing the facile construction of the whole protein stability curve and hence the measurement of accurate thermodynamic parameters for unfolding. We systematically measured the effect of several cations and, as a control, of different anions. We show that, while strongly susceptible to ionic strength, as it would be in the cellular environment, Yfh1 stability is sensitive not only to divalent cations, which bind specifically, but also to monovalent cations. We pinpoint the structural bases of the stability and hypothesize that the destabilization induced by an unusual cluster of negatively charged residues favours the entrance of water molecules into the hydrophobic core, consistent with the generally accepted mechanism of cold denaturation.