内生真菌印度梨形孢诱导小麦抗根腐病作用研究

【目的】明确印度梨形孢(Piriformospora indica)诱导小麦对根腐病产生抗性的作用机制。【方法】用印度梨形孢悬液浸种,以无菌培养液为对照,用病原菌禾谷镰孢菌(Fusarium graminearum)侵染小麦,对其相关生理生化指标及转录组变化进行分析。【结果】禾谷镰孢菌能诱导小麦产生过氧化氢,降低细胞内水含量,破坏细胞膜的稳定性;根部定殖印度梨形孢的小麦细胞内抗氧化酶活性增强,活性氧自由基含量降低,胞内水含量提高,细胞膜稳定性增强;印度梨形孢定殖能改变由于病原菌引起的mRNA转录组变化,抗性相关基因的表达增强。综合表明印度梨形孢定殖能有效地提高小麦对禾谷镰孢菌的抗性。【结论】研究结果为深入理解植物与微生物互作、开发新型高效环保抗根腐病生物制剂提供理论和实验依据。     

内生真菌印度梨形孢诱导小麦抗根腐病作用研究

【目的】明确印度梨形孢(Piriformospora indica)诱导小麦对根腐病产生抗性的作用机制。【方法】用印度梨形孢悬液浸种,以无菌培养液为对照,用病原菌禾谷镰孢菌(Fusarium graminearum)侵染小麦,对其相关生理生化指标及转录组变化进行分析。【结果】禾谷镰孢菌能诱导小麦产生过氧化氢,降低细胞内水含量,破坏细胞膜的稳定性;根部定殖印度梨形孢的小麦细胞内抗氧化酶活性增强,活性氧自由基含量降低,胞内水含量提高,细胞膜稳定性增强;印度梨形孢定殖能改变由于病原菌引起的mRNA转录组变化,抗性相关基因的表达增强。综合表明印度梨形孢定殖能有效地提高小麦对禾谷镰孢菌的抗性。【结论】研究结果为深入理解植物与微生物互作、开发新型高效环保抗根腐病生物制剂提供理论和实验依据。     

印度梨形孢对铁皮石斛种子萌发和原球茎生长的影响

为探究印度梨形孢(Piriformospora indica)对铁皮石斛(Dendrobium officinale)种子萌发和原球茎生长的影响,在铁皮石斛种子离体培养和原球茎生长阶段分别接种印度梨形孢,对其形态发育特征和生理特性进行研究.结果表明,接种印度梨形孢的铁皮石斛种子的起始萌发时间提前,接种印度梨形孢的铁皮石...     

印度梨形孢-植物共生关系的建立及共生体对重金属胁迫的响应

重金属污染土壤的治理一直是全球热点问题,植物-微生物联合修复技术因其高效、经济和减少二次污染而被广泛关注.印度梨形孢是一种能够与植物共生的真菌,它能够通过与植物建立共生关系的方式来提高宿主植物的生长和耐胁迫能力.本文系统总结了印度梨形孢-植物体共生关系的建立过程,以及重金属胁迫下共生体的生长、光合作用、抗性生理指标、防...     

miRNA响应印度梨形孢定殖调控大麦生长发育的作用机制

【背景】内生真菌印度梨形孢(Piriformospora indica)定殖植物可以显著促进植物生长发育。miRNA已被证实在植物体的生长发育中具有调控作用。【目的】揭示印度梨形孢定殖大麦促进大麦生长发育过程中miRNA对印度梨形孢定殖的响应及对大麦生长发育的调控作用。【方法】提取大麦总RNA,实施转录组测序并进行序列比对与数据挖掘;使用高效液相色谱检测大麦生长素等激素水平变化。【结果】印度梨形孢对大麦有显著促生作用;全转录组测序结果显示：印度梨形孢侵染3 d较空白对照有18个差异表达的miRNA,其中11个miRNA上调、7个miRNA下调;侵染7 d与空白对照相比24个差异表达的miRNA,其中11个miRNA上调、13个miRNA下调;侵染3 d与侵染7 d相比有3个miRNA上调、6个miRNA下调。GO功能富集分析与KEGG通路分析显示,差异表达miRNA的靶基因主要参与转录、细胞分裂、生长素信号的感知和转导、光合作用和激素刺激响应。靶基因所参与的途径与大麦生长发育密切相关,暗示miRNA对印度梨形孢定殖过程做出了积极响应。代谢产物分析表明miRNA参与的调控路径的代谢产物发生改变。【结论】本研究以miRNA为入手点,探究了miRNA对大麦生长发育的调控机制,为揭示印度梨形孢的促生机制提供了新的研究方向。     

印度梨形孢(Piriformospora indica)增强植物抗逆境胁迫能力的研究进展

印度梨形孢是一种可在多种植物根部定殖的内生真菌,能与多种植物形成共生体,提高植物对外界营养的吸收能力,促进次生代谢产物的积累,提高植物对生物及非生物胁迫的抵抗能力,同时可增加植物的生物产量,对宿主植物产生许多有益影响。因此,印度梨形孢作为优良的生物防治和土壤改良因子,在农业生产方面显示出巨大的应用前景。本文结合本课题组近年研究结果及近10年间相关科学工作者的研究,系统总结了印度梨形孢在增强植物抗生物胁迫与非生物胁迫方面的研究进展,旨在为更好地发挥其潜在价值提供参考。     

蒺藜苜蓿MtROP10基因的克隆及其功能初探

目的:克隆并研究蒺藜苜蓿ROP基因的功能,为研究该基因家族在共生途径中的作用提供依据.方法:采用RACE方法,从蒺藜苜蓿中克隆MtROP基因,利用生物信息软件比对同源性及ROP蛋白特征结构分析,利用RT-PCR方法分析该基因的组织特异性表达,构建该基因的过表达载体并转化拟南芥.结果:获得了蒺藜苜蓿ROP家族中与拟南芥ROP10高度同源的MtROP10全长序列.氨基酸编码序列具有明显的ROP家族蛋白的结构域特征.该基因在花中高表达,根中低表达.拟南芥中过表达MtROP10,可导致根毛变粗、变短、分叉.结论:MtROP10属于植物ROP家族蛋白,可能在根毛的极性生长方面具有较为重要的功能.     

蒺藜内生真菌发酵液抗菌及抗肿瘤活性初步研究

【目的】筛选出具有抗菌和抗肿瘤活性的蒺藜内生真菌。【方法】采用牛津杯法、稻瘟菌模型及肿瘤细胞模型评价蒺藜内生真菌PDB和察氏培养基发酵产物的抗菌活性和肿瘤细胞毒性。【结果】PDB培养基发酵液和察氏培养基发酵液抑菌圈直径大于10 mm的菌株分别占总菌株数的19.05%和23.81%。PDB培养基发酵液和察氏培养基发酵液对稻瘟菌的最小抑制浓度(MIC)低于10%的菌株分别占总菌株数的19.05%和47.61%。对肿瘤细胞抑制率高于50%的PDB发酵产物占PDB发酵产物总数的52.38%, 而对肿瘤细胞抑制率高于50%的察氏发酵产物占察氏发酵产物总数的28.57%。【结论】部分蒺藜内生真菌的发酵产物具有抗菌和抗肿瘤活性。     

蒺藜苜蓿MtbHLH148转录因子的克隆与转化及其功能分析

bHLH(Basic helix loop helix, bHLH)转录因子家族是植物最大的转录因子家族之一,广泛参与植物生长发育和盐胁迫应答机制。该研究利用同源克隆的方法克隆蒺藜苜蓿(Medicago truncatula)的MtbHLH148基因,采用qRT PCR方法分析MtbHLH148基因在蒺藜苜蓿中的表达特性,构建超表达载体并通过农杆菌侵染法转化拟南芥(Arabidopsis thaliana),对转基因拟南芥的耐盐性相关功能进行分析研究。结果显示：(1)从蒺藜苜蓿中获得MtbHLH148基因,该基因cDNA全长1 343 bp,包含开放阅读框为603 bp,编码 201 个氨基酸,蛋白分子量22.7 kD,等电点为11.76;蛋白结构分析显示,该蛋白无跨膜结构域,无信号肽,为亲水性蛋白;含有精氨酸／赖氨酸残基的保守结构域和典型的bHLH结构域;二级结构以α 螺旋和无规则卷曲为主。(2)亚细胞定位表明,MtbHLH148蛋白定位在细胞核。(3)进化树分析表明,MtbHLH148与大豆(Glycine max)的亲缘性最近;启动子分析发现,该基因启动子区域含有光响应元件、MYB结合位点以及ABA应答元件ABRE,可能参与非生物胁迫。(4)qRT PCR分析发现,MtbHLH148基因在蒺藜苜蓿的茎中表达量最高,叶中表达量最低,且MtbHLH148基因受ABA(100 μmol/L)诱导并在盐胁迫(200 mmol/L NaCl)处理8 h内表达量上调,而在低温(4 ℃)处理时表达量明显下调。(5)成功构建超表达载体pCAMBIA3301 MtbHLH148并转化拟南芥获得16个抗性株系,经鉴定有12个过表达株系,其中表达量最高的转基因株系为OE8;对OE8株系耐盐性功能分析发现,转基因拟南芥植株的发芽率明显高于野生型,盐胁迫下转基因拟南芥的根长是野生型的1.5倍,表明其耐盐性得到了增强。研究表明,MtbHLH148基因可能在盐胁迫调节机制中具有一定的调控作用。     

DNA transfer by highly asymmetric somatic hybridisation in Medicago truncatula (+) Medicago rugosaand Medicago truncatula(+) Medicago scutellata

A regenerable line of Medicago truncatula (Jemalong 2HA) as a recipient species, was fused with the sexually incompatible species Medicago scutellata or Medicago rugosa. The treatments described maintain the chromosome number of the recipient but enable the transfer of small amounts of DNA of the donor species, probably by intergenomic recombination. Without a chromosome number-change fusion products can readily regenerate to produce fertile plants; and potentially a library with a diverse array of new genetic material. The selection of fused cells is based on treatment of the recipient cells with iodoacetamide (IOA), a non-regenerable donor, γ-irradiation of the donor, and regeneration on a medium favouring the recipient. DNA transfer was demonstrated by amplified fragment length polymorphism (AFLP), Southern hybridisation and changed morphology. Received: 21 December 2000 / Accepted: 5 April 2001     

Repetitive somatic embryogenesis in Medicago truncatula ssp. Narbonensis and M. truncatula Gaertn cv. Jemalong

Medicago truncatula ssp Narbonensis and four genotypes of M. truncatula Gaertn cv. Jemalong were tested for their somatic embryogenesis potential using a two-step protocol. In the first step, embryogenic callus was induced in folioles isolated from shoots grown in vitro and cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid and zeatin. In the second step, somatic embryos were allowed to develop from the induced callus in MS growth-regulator-free medium. Individual somatic embryos were then isolated and transferred again to growth regulator free medium where they formed secondary somatic embryos in repetitive cycles. Conversion of somatic embryos into plantlets was achieved by isolating late-torpedo-phase somatic embryos with distinct cotyledons and reculturing them onto MS growth regulator free medium. The system of repetitive somatic embryogenesis in M. truncatula described here represents a permanent source of embryogenic material that can be used for the genetic modification of this species. Received: 7 August 1997 / Revision received: 22 December 1997 / Accepted: 20 January 1998     

Influence of genes determining supernodulation on root colonization by the mycorrhizal fungus Glomus mosseae in Pisum sativum and Medicago truncatula mutants

Pisum sativum (pea) mutants of the wild type cv. Frisson and six supernodulating Medicago truncatula mutants of the wild-type cv. Jemalong line J5 for their ability to form endomycorrhizas. The six mutants of M. truncatula were shown to be allelic mutants of the same gene Mtsym12, whereas distinct genes (sym28 and sym29) are known to determine the supernodulation character of the P64 and P88 pea mutants, respectively. Mutant P88 of pea and the majority of the M. truncatula mutants were significantly more colonized by the mycorrhizal fungus Glomus mosseae than their corresponding wild types, 4 weeks and 30 days after inoculation, respectively. These differences were expressed essentially in transversal intensity rather than in length intensity of root colonization and appeared to correspond to an increase in arbuscule formation. Results are discussed in relation to the mutated genes and, in particular, whether the observed effects are due indirectly to plant physiological modifications or are a direct result of possible common factors of regulation of nodulation and mycorrhizal development. Accepted: 9 February 2000     

Piriformospora indica,a plant-root-colonising fungus enhances growth and tolerance of wheat to seedlings damping-off caused by Fusarium oxysporum

In greenhouse experiments, we investigated the potentials of Piriformospora indica (Pi) to penetrate and colonise roots of wheat and to induce beneficial effects on growth as well as to reduce seedlings damping-off disease of wheat caused by the soil-borne fungus Fusarium oxysporum (Fo). By microscopy we observed the intracellular hyphae within root cortex cells of 7–14 day-old plants and chlamydospores within root cortex cells of 14–28 day-old plants and within root hair cells of 21–28 day-old plants. Moreover, diagnostic PCR based on the β-tubulin gene marker confirmed the presence of Pi in roots of inoculated plants. Also, we found that plants inoculated with Pi exhibited a better growth of roots and shoots as well as early flowering as compared with non-treated plants. Moreover, Pi conferred tolerance of wheat to seedlings damping-off i.e. by reducing the harmful effects of Fo on infected plants.     

Growth and senescence of Medicago truncatula cultured cells are associated with characteristic mitochondrial morphology

Here mitochondrial morphology and dynamics were investigated in Medicago truncatula cell-suspension cultures during growth and senescence. Cell biology techniques were used to measure cell growth and death in culture. Mitochondrial morphology was investigated in vivo using a membrane potential sensor probe coupled with confocal microscopy. Expression of a senescence-associated gene (MtSAG) was evaluated in different cell-growth phases. Mitochondria appeared as numerous, punctuate organelles in cells at the beginning of the subculture cycle, while interconnected networks were observed in actively growing cells. In senescent cells, giant mitochondria were associated with dying cells. The release of cytochrome c from mitochondria was detected in different growth phases of cultured cells. Studies on plant cell cultures allowed us to identify physiological and molecular markers of senescence and cell death, and to associate distinct mitochondrial morphology with cells under different physiological conditions.     

Nitrogen fixation is synchronized with carbon metabolism in Lotus japonicus and Medicago truncatula nodules under salt stress

Abstract

In the present work, the response to NaCl applied at the vegetative stage to Medicago truncatula and Lotus japonicus has been evaluated in order to ascertain whether the effect of salt stress on nitrogen fixation is due to a limitation on nodular carbon metabolism. Results show maximum sucrose synthase (SS) and alkaline invertase (AI) activities were obtained at the vegetative stage, when maximum nitrogenase activity was detected in both species. SS activity decreased with the salt treatment, providing evidence of the regulatory role of this enzyme for the carbon supply to the bacteroids. Phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activities could account for higher nitrogen fixation efficiency detected in L. japonicus nodules and isocitrate dehydrogenase (ICDH) activity compensated for the carbon limitations that occur under salt stress. These results support that nitrogenase inhibition in nodules experiencing salt stress is doubt to a carbon flux shortage, as result of carbon metabolism enzymes activities down-regulation.     

Repeated leaf wounding alters the colonization of Medicago truncatula roots by beneficial and pathogenic microorganisms

In nature, plants are subject to various stresses that are often accompanied by wounding of the aboveground tissues. As wounding affects plants locally and systemically, we investigated the impact of leaf wounding on interactions of Medicago truncatula with root-colonizing microorganisms, such as the arbuscular mycorrhizal (AM) fungus Glomus intraradices, the pathogenic oomycete Aphanomyces euteiches and the nitrogen-fixing bacterium Sinorhizobium meliloti. To obtain a long-lasting wound response, repeated wounding was performed and resulted in locally and systemically increased jasmonic acid (JA) levels accompanied by the expression of jasmonate-induced genes, among them the genes encoding allene oxide cyclase 1 (MtAOC1) and a putative cell wall-bound invertase (cwINV). After repeated wounding, colonization with the AM fungus was increased, suggesting a role of jasmonates as positive regulators of mycorrhization, whereas the interaction with the rhizobacterium was not affected. In contrast, wounded plants appeared to be less susceptible to pathogens which might be caused by JA-induced defence mechanisms. The effects of wounding on mycorrhization and pathogen infection could be partially mimicked by foliar application of JA. In addition to JA itself, the positive effect on mycorrhization might be mediated by systemically induced cwINV, which was previously shown to exhibit a regulatory function on interaction with AM fungi.     

Nitrogen fixation persists under conditions of salt stress in transgenic Medicago truncatula plants expressing a cyanobacterial flavodoxin

Several recent studies have demonstrated that the expression of a cyanobacterial flavodoxin in plants can provide tolerance to a wide range of environmental stresses. Indeed, this strategy has been proposed as a potentially powerful biotechnological tool to generate multiple‐tolerant crops. To determine whether flavodoxin expression specifically increased tolerance to salt stress and whether it might also preserve legume nitrogen fixation under saline conditions, the flavodoxin gene was introduced into the model legume Medicago truncatula. Expression of flavodoxin did not confer saline tolerance to the whole plant, although the sensitive nitrogen‐fixing activity was maintained under salt stress in flavodoxin‐expressing plants. Our results indicate that flavodoxin induced small but significant changes in the enzymatic activities involved in the nodule redox balance that might be responsible for the positive effect on nitrogen fixation. Expression of flavodoxin can be regarded as a potential tool to improve legume symbiotic performance under salt stress, and possibly other environmental stresses.     

Transformation of floral organs with GFP in Medicago truncatula

 A high frequency of embryogenesis and transformation from all parts of flowers of two lines of Medicago truncatula R-108–1 and Jemalong J5 were obtained. Using this flower system, we obtained transgenic plants expressing promoter-uidA gene fusions as well as the gfp living cell color reporter gene. Moreover, this method allows us to save time and to use a smaller greenhouse surface for the culture of donor plants. Southern hybridization showed that the internal gfp fragment had the expected size and the number of T-DNA copies integrated in the plant genome varied between one and three. These data suggest that the presence of the GFP protein has no toxic effects, since no rearrangement of the gfp reporter gene was detected in the regenerated plants. Received: 25 May 1999 / Revision received: 2 August 1999 / Accepted: 2 August 1999