棉花与番茄抗棉花黄萎病不依赖于Ve1

黄萎病是我国棉花的主要病害之一,发掘抗病基因和阐明抗病机制是开展棉花抗病分子育种的基础.本研究将目前唯一的植物抗黄萎病主效基因Ve1分别在本氏烟和陆地棉中超量表达,以探讨其在防控棉花黄萎病中的价值.研究发现,Ve1基因在本氏烟中超量表达后并未对番茄大丽轮枝菌2个生理小种和棉花黄萎病菌产生明显抗性.RT-PCR分析表明,Ve1并不能激活烟草抗病相关基因的表达,推测本氏烟中可能不存在完整的Ve1介导的抗黄萎病信号路径.Ve1超量表达的转基因棉花接种棉花黄萎病菌"V991"后表现出与本氏烟类似的结果,同时发现,陆地棉对番茄大丽轮枝菌1号生理小种表现出高抗性.利用番茄抗/感黄萎病近等基因系"Craigella GCR218"/"Craigella GCR26"进一步研究发现,2个番茄材料均对棉花落叶型强致病力黄萎病菌"V991"免疫,这暗示番茄对棉花黄萎病菌的抗性不依赖于Ve1.分子鉴定表明,棉花黄萎病菌与番茄大丽轮枝菌1号和2号生理小种存在明显区别,番茄大丽轮枝菌1号和2号生理小种均属于非落叶型黄萎病菌.本研究中鉴定的棉花黄萎病菌均不含有ave1基因,这可能是在棉花中超量表达Ve1并不能增强其棉花黄萎病菌抗性的直接原因.通过病毒介导的基因沉默,在抑制GbSERK1的表达后能显著削弱海岛棉对黄萎病菌的抗性,证实"海7124"中存在类似于Ve1的下游抗病信号路径.本研究还对棉花类受体蛋白的进化以及Ve1抗病信号路径在棉花抗黄萎病机制研究中的价值进行了探讨.     

VIGS技术高通量筛选棉花抗黄萎病相关基因

黄萎病是严重影响棉花生产的一种土传性维管束真菌病害,长期以来,因缺乏行之有效的手段,有关棉花对黄萎病菌抗性分子机制的研究难度很大,进展缓慢.病毒诱导基因沉默(VIGS)这一反向遗传学技术的建立和应用,为研究和阐明棉花基因的功能提供了新的契机.本研究挑选可能与棉花抗黄萎病相关的基因,通过VIGS方法进一步明确和验证这些基因在棉花抗黄萎病菌中的作用.共成功构建了62个基因的VIGS载体,通过重复筛选实验,最终筛选出一些与棉花抗黄萎病相关的基因,其中乙烯信号途径正调控因子EIN2,植物病原应答诱导蛋白基因DIR,木葡聚糖内转糖苷酶/水解酶(XTH)被沉默后,沉默棉花植株对黄萎病菌的抗性明显减弱,表明这些基因可能通过不同的信号途径参与棉花对黄萎病菌的抗性.     

病毒基因沉默抑制子及其作用机制

基因沉默或RNA 沉默是植物、真菌以及无脊椎动物抵御病毒侵染的重要机制, 为了应对这种机制, 病毒编码RNA 沉默抑制子抑制宿主的基因沉默, 抵抗由基因沉默介导的宿主对病毒的抗性. 病毒编码的RNA 沉默抑制子, 又被称为病毒基因沉默抑制子, 广泛存在于各种植物RNA 病毒和DNA 病毒以及部分动物病毒中. 近年来, 针对病毒基因沉默抑制子作用机制的研究表明, 病毒基因沉默抑制子通过与宿主基因沉默通路中的RNA 或者关键蛋白分子相互作用, 发挥抑制宿主对病毒的抗性以及干扰宿主正常的基因表达调控的功能. 由于植物基因沉默通路的复杂性, 病毒基因沉默抑制子的作用机制也是复杂而多样的.     

寄主诱导的基因沉默在增强植物真菌病害抗性方面的研究进展

寄主诱导的基因沉默(host-induced gene silencing,HIGS)技术以RNAi技术为基础,可从反向遗传学角度研究基因功能,同时可创制具有持久抗性且稳定遗传的抗病品种,为增强植物抗病性提供了新的视角.本文阐明了HIGS技术的原理,总结了近年来该技术在增强植物真菌病害抗性方面最新的研究进展,分析了HI...     

植物抗病毒分子机制

在与植物病毒的长期斗争中,植物进化出多种抗病毒机制,其中RNA沉默和R基因介导的病毒抗性是最受人们关注的两种机制.一方面,RNA沉默是植物抵抗病毒侵染的重要手段.植物在病毒侵染过程中可形成病毒来源的双链RNA,经过DCL蛋白的切割、加工形成sRNA,与AGO蛋白结合形成RISC指导病毒RNA的沉默,用于清除病毒.相应地,病毒在与植物的竞争中进化出RNA沉默抑制子,抑制宿主RNA沉默系统以逃避宿主RNA沉默抗病毒反应,增强致病能力.另一方面,植物也进化出R基因介导植物对包括病毒在内的多类病原的抗性.R蛋白直接或间接识别病毒因子,通过一系列的信号转导途径激活植物防御反应,限制病毒的进一步侵染.对植物抗病毒的研究有助于人们对植物抗病分子基础的理解,有重要的科学意义和潜在应用价值.本文综述了植物抗病毒分子机制的重要进展.     

植物RNA沉默抗病机制与应用研究进展

RNA沉默是真核生物基因表达调控的保守机制,在植物生长发育以及响应生物和非生物胁迫过程中发挥着非常重要的作用。跨界RNA沉默与种间RNA沉默为开发基于RNA沉默的作物病害防控体系提供了理论基础。本文概括了植物RNA沉默保守途径,归纳了RNA沉默在植物-病原互作研究中的代表性研究,阐述了基于RNA沉默开发的宿主诱导基因沉默、喷施诱导基因沉默和微生物诱导基因沉默技术的原理,以及应用研究现状,以期为开发基于RNA沉默的新型作物病害防控技术提供参考。     

棉花黄萎病病原菌诱导侵染对转基因抗病棉花生理性状的影响

通过研究棉花黄萎病病原菌诱导侵染对转基因抗病棉花生理性状的影响,旨在为提高转基因抗病棉花抗病性提供理论依据。Byd、28p两品种棉花为供试材料,应用花粉管通道法将几丁质酶与β-1,3-葡聚糖双价抗病基因导入棉花株系中,多年筛选获得不同抗性水平的棉花株系;棉种经棉花黄萎病病菌侵染后,苗期测定棉株内的生理生化变化。结果显示,转基因抗病棉株体内的酶活性已达对照水平,而感病及耐病棉株内的酶活性均有不同程度的变化。所测定的棉株内的酶活性及脯氨酸含量的变化与棉株的抗病性相关。     

云南野生茄资源黄萎病苗期人工接种抗性鉴定分析

黄萎病是目前茄子生产中的主要病害之一,广泛收集、鉴定、筛选抗性资源,尤其是从野生近缘种中发掘抗性基因并培育抗病品种,是解决茄子黄萎病危害的优选途径。云南省拥有丰富的野生茄子资源但尚未有效利用。本研究针对云南省茄子主产区收集到的3种黄萎病菌株,通过形态学鉴定和真菌18S r DNA/ITS鉴定,均属于大丽轮枝菌,并通过致病力鉴定,筛选出一株强致病力菌株(QZ-S);应用菌株QZ-S,通过苗期人工接种的方法对45份云南野生茄子资源开展黄萎病抗性鉴定,最终筛选出2份高抗材料(蒜芥茄和喀西茄)、2份抗病材料(水茄和多裂水茄)、6份中抗材料(1份刺天茄和5份红茄);此外,还筛选到了1份黄萎病高感材料239-3-2。本研究筛选出的材料可应用于茄子黄萎病抗病育种,为茄子及其他作物黄萎病抗病育种提供抗源。     

植物中病毒来源的小RNA介导的RNA沉默

植物RNA 沉默机制的主要功能之一是具有抗病毒作用. 在被病毒侵染的宿主细胞中发现的病毒来源的小RNA表明, 宿主的RNA沉默机制可以靶向病毒RNA. 随着vsiRNAs 高通量测序技术的发展, 近年来的遗传学研究揭示了vsiRNAs 的起源和组成以及它们调控基因表达的潜在功能. 本文简述了vsiRNAs 的起源和生物合成过程, 并着重围绕在抗病毒过程中vsiRNAs 介导的对病毒基因组和宿主转录本的RNA 沉默现象进行综述, 以更好地理解植物中vsiRNAs 在病毒致病性以及和宿主互作中的作用机制.     

陆地棉品种和骨干品系黄萎病抗性鉴定

选育和推广抗病品种是防治陆地棉黄萎病的主要措施,为了早日实现多类型、多区域大面积抗病品种的应用,本研究选取107份遗传背景差异较大的种质,利用河北省农林科学院棉花研究所小安舍试验站黄萎病病圃进行了3年黄萎病抗性重复鉴定。鉴定得到抗病品系8个,占7.5%;耐病品种(系)20个,占18.7%。本研究表明,当前被作为育种亲本的抗病品系还太少,需要深入开展抗病遗传机制,以及与其他经济性状协同改良的关系,为陆地棉抗病育种提供理论指导;达到抗病或接近抗病水平的大部分品种(系)来自于海陆野远缘后代,具有外源基因血统,证明了远缘杂交是陆地棉黄萎病抗性改良的有效手段。     

Overexpression of GhPFN2 enhances protection against Verticillium dahliae invasion in cotton

Growing evidence indicates that actin cytoskeleton is involved in plant innate immune responses,but the functional mechanism remains largely unknown.Here,we investigated the behavior of a cotton profilin gene(GhPFN2) in response to Verticillium dahliae invasion,and evaluated its contribution to plant defense against this soil-borne fungal pathogen.GhPFN2 expression was up-regulated when cotton root was inoculated with V.dahliae,and the actin architecture was reorganized in the infected root cells,with a clear increase in the density of filamentous actin and the extent of actin bundling.Compared to the wild type,GhPFN2-overexpressing cotton plants showed enhanced protection against V.dahliae infection and the actin cytoskeleton organization in root epidermal cells was clearly altered,which phenocopied that of the wild-type(WT) root cells challenged with V.dahliae.These results provide a solid line of evidence showing that actin cytoskeleton reorganization involving GhPFN2 is important for defense against V.dahliae infection.     

The Arabidopsis thaliana DNA-binding protein AHL19 mediates verticillium wilt resistance

Verticillium spp. are destructive soilborne fungal pathogens that cause vascular wilt diseases in a wide range of plant species. Verticillium wilts are particularly notorious, and genetic resistance in crop plants is the most favorable means of disease control. In a gain-of-function screen using an activation-tagged Arabidopsis mutant collection, we identified four mutants, A1 to A4, which displayed enhanced resistance toward the vascular wilt species Verticillium dahliae, V. albo-atrum and V. longisporum but not to Fusarium oxysporum f. sp. raphani. Further testing revealed that mutant A2 displayed enhanced Ralstonia solanacearum resistance, while mutants A1 and A3 were more susceptible toward Pseudomonas syringae pv. tomato. Identification of the activation tag insertion site in the A1 mutant revealed an insertion in close proximity to the gene encoding AHL19, which was constitutively expressed in the mutant. AHL19 knock-out alleles were found to display enhanced Verticillium susceptibility whereas overexpression of AHL19 resulted in enhanced Verticillium resistance, showing that AHL19 acts as a positive regulator of plant defense.     

Evaluation of Compost Amendments for Control of Vascular Wilt Diseases

Vascular wilt fungal pathogens cause heavy economic losses to a wide range of crops; amongst them are Fusarium oxysporum f. sp. melonis (FOM) and Verticillium dahliae Kleb. Several strategies for controlling these pathogens have been introduced, such as soil solarization, resistant rootstocks and biological control. In this study, the suppressive ability of seven different compost amendments and the plant growth-promoting rhizobacterium Paenibacillus alvei K165 (with proven activity against V. dahliae ) were tested against FOM in melon and V. dahliae in eggplant. It was shown that K165 had a suppressive effect against the pathogens in all experiments. On the contrary, the composts exhibited a narrow spectrum of effectiveness against the pathogens. Two composts were effective against V. dahliae and one against FOM. Moreover, we investigated the potential of the various compost samples and K165 to induce resistance in an Arabidopsis thaliana – V. dahliae or FOM model system. It was demonstrated that three composts and K165 were effective against V. dahliae ; whereas, one compost and K165 were effective against FOM. In a naturally V.dahliae infested field, the ability of K165 to enhance the suppressive effect of one of the compost amendments, was evaluated. It was demonstrated that fortification of the compost with strain K165 significantly reduced disease severity, whereas the single application of the compost was not sufficient to significantly protect the plants.     

高效大丽轮枝菌(Verticillium dahliae) 基因敲除体系的构建

[目的]为了深入研究大丽轮枝菌(Verticillium dahliae)致病基因的功能,构建高效大丽轮枝菌基因敲除体系.[方法]融合PCR构建基因敲除载体;利用农杆菌介导法转化大丽轮枝菌;使用在T-DNA之间加入致死基因的双元载体,使T-DNA随机插入转化子在添加5-氟脱氧尿苷的培养基上不能存活,实现对随机插入转化子的"反向筛选".[结果]对大丽轮枝菌腺嘌呤合成酶基因和几丁质合成酶基因进行基因敲除验证,基因敲除转化子在总转化子中的比例分别达到87%和44%.[结论]成功构建大丽轮枝菌高效基因敲除体系,为大丽轮枝菌致病基因的功能验证提供了技术平台.     

Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens

Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens.     

Proteomic analysis of the sea-island cotton roots infected by wilt pathogen Verticillium dahliae

Verticillium wilt of cotton is a vascular disease mainly caused by the soil-born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt-resistant sea-island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock-inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2-DE combined with local EST database-assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease.