拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象,该文通过建立拟南芥．大豆疫霉菌（Arabidopsis thaliana—Phytophthora sojae）非寄主互作系统,筛选对大豆疫霉菌感病的拟南芥突变体,为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T—DNA插入突变体离体叶片,从代表12000个独立转化株系的40000株T3代T。DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定,离体叶片接菌后3天内出现明显的水渍状病斑,4—5天后产生大量卵孢子和／或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明,581—51突变体含有4个T-DNA插入事件,其感病性状可能由隐性单基因控制。     

尖镰孢古巴专化型Focr4-1701突变体的生物学表型研究

由尖镰孢古巴专化型(Fusarium oxysporum f.sp.cubense,Foc)引起的香蕉枯萎病是香蕉生产中的毁灭性病害之一。Foc有4个小种,其中4号小种(Focr4)因其寄主范围广且无高抗病性的香蕉品种,对香蕉产业的危害最大,其致病机制至今尚不清楚。为研究其致病机制,本文对Focr4野生型菌株(Focr4-193-6)、因其T-DNA插入导致致病性严重减弱的突变体Focr4-1701、T-DNA插入标签基因敲除子△Focr4-1701及敲除基因互补子△Focr4-1701-cp-1为材料,从致病性测定、玻璃纸穿透试验、孢子形态观察、产孢量与孢子萌发率测定、粗毒素测定等方面对其与致病性相关的生物学表型进行了测定。结果表明:T-DNA插入突变体Focr4-1701和基因敲除突变体△Focr4-1701接种后的香蕉植株叶片没有表现发病症状,敲除基因互补菌株与野生型菌株一样表现出发病症状;T-DNA插入突变体和基因敲除突变体不能穿透玻璃纸生长,T-DNA插入突变体及基因敲除突变体在产孢量、孢子萌发率和产毒素能力上均极显著低于野生型菌株及互补菌株。这些表型性状差异说明Focr4-1701致病性严重减弱可能是T-DNA插入位点标签基因失活后导致了菌丝体侵染能力下降及产毒素能力降低造成的。     

银杏叶片三种酶活性变化与抗疫霉菌关系的研究

将疫霉菌离体接种于银杏叶片,发现不同龄期银杏叶片对疫霉菌的抗病性有差异,叶龄为100d的抗病性较强,叶龄为20d的抗病性较弱。不同银杏品种叶片对疫霉菌的抗性也有差异,潮田1号和大佛手2号较抗病,大佛手1号和桂G86-1较感病。不同龄期叶片接种后,其过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)的酶活性变化与抗病性成正相关,而多酚氧化酶(PPOD)的酶活性变化与抗病性没有相关性;不同银杏品种叶片接种后,品种间的抗病性与PPOD酶活性变化成正相关,而POD和PAL的酶活性变化与品种间的抗病性没有相关性。可见,POD、PPOD和PAL的酶活性变化没有明显规律性,不能作为衡量银杏叶片抗病性的生化指标。     

大豆疫霉菌的EMS化学诱变

以甲基磺酸乙酯(ethylmethane sulfonate,EMS)为诱变剂,通过其对大豆疫霉菌Phytophthora sojae休止孢萌发的影响,确定化学诱变条件。通过收集单卵孢子,建立了包含640个单卵孢子系的突变体库,其中约有50%的诱变菌系在培养性状和菌落形态方面发生了明显变化,菌落形态多样,表现出较紧密或松散,近圆形或不规则;气生菌丝减少,生长速度较慢或快;在卵孢子产量方面,8.13%的菌系有增加,20.41%的菌系减少,27.82%的菌系极少或者没有卵孢子产生,43.64%的菌系卵孢子产量类似野生型。以质膜氢离子泵蛋白基因PsPMA1(plasma membrane H+-ATPase1)为对象,通过TILLING技术,从320个大豆疫霉菌突变体中获得9个突变体,进一步确认了EMS对大豆疫霉菌的诱变效果,并且估算EMS对大豆疫霉菌的诱变频率至多每115kb发生一个核苷酸变异。新构建的突变体库为开展大豆疫霉病菌的功能基因组研究奠定了遗传材料基础。     

拟南芥T-DNA插入突变体atsuc3的PCR鉴定

应用两种植物T-DNA插入突变体PCR鉴定方法,即“三引物法”和“双引物法”对拟南芥T-DNA插入突变体atsuc3（目的基因两条染色体均发生T-DNA插入的纯合突变体）进行鉴定和比较的结果表明,“三引物法”由于易产生非特异性扩增而无法得到PCR鉴定结果,从而导致鉴定失败;“双引物法”则可避免此种现象,得到可靠、有效的鉴定结果。     

快速筛选拟南芥受精和早期胚胎发生相关基因的方法

阐明拟南芥受精和早期胚胎发生过程对理解被子植物生殖发育有着重要的指导意义,而利用正向遗传学方法研究拟南芥突变体的表型及其分子机理是探究植物基因功能最常用的一种方法。基于常规的插入突变(包括T-DNA和转座子)、化学诱变(如ethylmethane sulfonate,EMS)和高能射线方法构建的突变体库中假阳性突变体多,难以高效筛选到受精和早期胚胎发生相关基因的突变体。为解决这一难题,本研究建立了一种构建T-DNA插入突变体文库的新方法。即在载体p CAMBIA1302的T-DNA元件上增加花粉特异荧光标记基因(p LAT52∷EGFP),并遗传转化具有四分体花粉的Columbia野生型拟南芥突变体qrt1-2;对获得的突变体库可利用花粉荧光快速排除假阳性突变体,并采用反向PCR(inverse-PCR)扩增技术确定突变位点。此方法在筛选拟南芥受精和早期胚胎发生相关基因突变体上的成功应用表明,其是一种效率高、针对性强、操作相对快捷方便的拟南芥突变体筛选方法。     

大豆疫霉对寄主大豆和非寄主菜豆根及根分泌物的响应与其对寄主选择性的关系

【背景】大豆疫霉根腐病作为大豆生产上的一种毁灭性病害已被美国、加拿大等多国报道,其病原菌大豆疫霉(Phtophthora sojae Kaufmann and Gerdemann)为典型的土传病原菌。近年来,土传病原菌与植物根系的互作成为研究土传病原菌寄主选择机制的主要方向。【目的】探究寄主大豆和非寄主菜豆根及根分泌物对大豆疫霉的不同影响,阐明这种影响与大豆疫霉对寄主选择的关系。【方法】应用原位土培法种植大豆疫霉感病品种Sloan、抗病品种Williams82和非寄主菜豆一点红,测定了单个大豆疫霉游动孢子对寄主大豆和非寄主菜豆幼根的侵染行为,收集了寄主及非寄主根分泌物,测定了根分泌物对大豆疫霉游动孢子的趋化作用,包括吸引游动孢子的能力以及对游动孢子成囊、孢囊萌发和芽管生长的影响。【结果】大豆疫霉单个游动孢子对寄主大豆幼根表现强烈趋向性,沿着根面进行多次试探性接触后在根尖伸长区快速成囊并萌发,产生的芽管顶端贴附在幼根表面,在感病大豆品种根面上的芽管比抗病大豆品种上的短且粗,而对非寄主菜豆幼根则不具有趋向性,接触一次后即远离,最终在距离幼根75μm的位置成囊萌发,且芽管生长不具有方向性。此外,大豆疫霉游动孢子对抗病、感病大豆和非寄主菜豆幼根的侵染行为差异完全在根分泌物试验中重现,即寄主大豆根分泌物对大豆疫霉游动孢子具有较强的趋向作用,能够有效吸引游动孢子,促进游动孢子快速成囊及萌发,抑制芽管的伸长,而非寄主菜豆根分泌物不具有上述作用。【结论】大豆疫霉对寄主的选择性与根分泌物有关,为进一步了解大豆疫霉的寄主选择机制提供理论依据。     

水稻剑叶突变体的表型及T-DNA旁邻基因分析

从已构建的水稻（Oryza sativa L.）T-DNA插入突变体中鉴定获得一株穗部额外发育出叶片的突变体,并根据该叶片的形态学位置将其命名为剑叶突变体（J4）。研究表明这种额外发育的叶片呈现明显的缺陷,主要表现为叶片短小、表皮细胞变小、叶片中维管束数目减少等。进一步通过TAIL-PCR和inverse-PCR的方法克隆该突变体中T-DNA插入位置的旁邻序列,从而准确地将T-DNA定位到2号染色体上。基因表达分析显示,T-DNA插入位置附近的AK100376基因在J4突变体以及表型类似突变体neck leaf 1中的表达均被明显下调,可初步将其确定为与剑叶突变体表型相关的候选基因。     

农杆菌介导的灰葡萄孢T-DNA插入突变体库构建及插入位点分析

【目的】利用农杆菌(Agrobacterium tumefaciens)介导法对灰葡萄孢(Botrytis cinerea)进行转化,构建T-DNA插入突变体库,为从分子水平上认识灰葡萄孢的致病机制打下基础。【方法】以含有pCAMBIA 1390双元载体的农杆菌对灰葡萄孢进行转化,利用潮霉素进行筛选。对抗性稳定的转化子进行生物学和形态学观察,采用离体番茄叶片进行致病性测定。利用TAIL-PCR技术对突变体中T-DNA的旁侧序列进行克隆。【结果】得到了一些突变体,表现为生长速率减缓、产孢能力下降、致病力减弱等。克隆并分析了其中一个突变体中T-DNA插入的位置和旁侧序列。【结论】本实验建立了农杆菌介导的灰葡萄孢转化体系,构建了T-DNA插入的灰葡萄孢突变体库。用TAIL-PCR进行突变体中T-DNA旁侧序列的分析是可行的。     

梨黑星菌粗毒素对抗病和感病梨离体叶片生理特性的影响

以不同抗病性梨品种为材料,研究了梨黑星菌粗毒素对梨离体叶片的过氧化物酶(POD)活性、苯丙氨酸解氨酶(PAL)活性、丙二醛(MDA)含量、细胞膜相对透性以及叶绿素含量变化的影响.研究表明,毒素接种后抗病和感病品种叶片的POD和PAL活性、MDA含量和相对电导率均呈上升趋势,其间会有1个或2个峰值出现,且抗病品种叶片的POD和PAL活性高于感病品种,而细胞内MDA含量和相对电导率增加比率低于感病品种;同时,毒素使梨离体叶片的叶绿素、类胡萝卜素含量下降,且感病品种的下降幅度大于抗病品种.总之,梨离体叶片POD和PAL的活性变化与梨品种抗病性呈正相关,叶片MDA含量和相对电导率变化与梨品种抗病性呈负相关,抗病品种离体叶片对毒素的毒害有更强的抵抗力.     

农杆菌介导巨桉Eg5高效遗传转化

以巨桉(Eucalyptus grandis)无性系Eg5叶片为外植体, 探讨了农杆菌(Agrobacterium tumefaciens)侵染时间、共培养pH值和共培养时间对瞬时转化效率的影响, 分析了不同筛选策略对遗传转化植株筛选效果的影响。结果表明, 外植体侵染45分钟, 共培养pH值为5.8, 共培养3天所得到的瞬时转化效率最高; 逐步提高卡那霉素(Km)浓度筛选转基因植株有效, 筛选率达到15%, 转化率达到0.26%。经过GUS染色分析和PCR检测, 证实为转基因植株。     

Developmental anatomy of cotyledons and leaves in has mutant of Arabidopsis thaliana

Summary. In this work, we analyzed the developmental anatomy of cotyledons and leaves in the has mutant of Arabidopsis thaliana. It is a recessive T-DNA insertion mutation that causes changes in the size, shape, and tissue organization of the cotyledons and leaves of has plants. Analysis of has cotyledons revealed a prominent decrease in the cell number and an increase in the area of cotyledon cells and intercellular spaces of has plants. At early stages of development, has leaves are fingerlike structures, but later they develop small, lobed blades with rare trichomes. An important characteristic of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. In addition, both cotyledons and leaves display a disrupted pattern of vascular bundles. Furthermore, mutant plants are defective in root and shoot morphology, indicating that the has mutation affects a number of aspects in plant development. Correspondence and reprints: Institute of Botany and “Jevremovac” Botanical Garden, Faculty of Biology, Belgrade University, Takovska 43, 11 000 Belgrade, Serbia.     

树鼩细菌感染模型的建立及抗菌药物的治疗效果评价(英文)

在抗微生物感染药物开发过程中, 动物模型是必不可少的。虽然目前已经用啮齿类动物建立了一些细菌感染动物模型, 但在小型灵长类动物中还很少见。这里首次报道两个树鼩细菌感染动物模型。第一种是在三度烫伤后的皮肤表面接种 5×106 CFU 的金黄色葡萄球菌构建的皮肤烫伤感染模型。这个数量的金黄色葡萄球菌可以造成 7 d 持续性感染, 并且在第 4天可以看到明显的炎症反应。第二种是用绿脓杆菌构建的涤纶补片感染模型, 接种 2×106 CFU 的绿脓杆菌同样可以引起持续 6 d 感染, 并在第三天在伤口处观察到大量的脓液。进一步用这两种模型评价头孢哌酮钠和左氧氟沙星的治疗效果。左氧氟沙星和头孢哌酮钠在皮肤烫伤感染模型中能分别将 100 mg 皮肤组织中的细菌降低到 4log10 和 5log10 CFU, 并且在涤纶补片植入感染模型中这两种抗生素都能显著地将感染的细菌降低了 4log10 CFU (P<0.05)。结果表明用金黄色葡萄球菌和绿脓杆菌成功构建了两个细菌感染的树鼩模型。此外, 树鼩对金黄色葡萄球菌和绿脓杆菌很敏感, 适合用于构建细菌感染动物模型和评价新的抗细菌感染药物的效果。     

椒样薄荷、薄荷和苏格兰留兰香精油与抗生素的协同抑菌功能

以开花期的椒样薄荷(Mentha × piperita)、薄荷(M. haplocalyx)和苏格兰留兰香(M. × gentilis)叶片部位提取的精油为研究对象, 通过GC-MS分析, 并采用纸片扩散法研究了3种精油单独使用及与抗生素联合使用时对金黄色葡萄球菌、蜡状芽孢杆菌、大肠杆菌、绿脓杆菌和肺炎克雷伯氏菌的抑制情况。结果表明, (1) 椒样薄荷与薄荷精油中含量最高的成分为薄荷醇、薄荷酮和异薄荷酮, 苏格兰留兰香精油的主要成分为香芹酮和柠檬烯。薄荷和苏格兰留兰香精油符合欧洲药典与ISO标准, 椒样薄荷需要继续改良以提高其精油品质与抑菌功能。(2) 精油单独使用时, Pseudomonas aeruginosa ATCC 15442对椒样薄荷精油和薄荷精油敏感; P. aeruginosa ATCC 27853对薄荷精油和苏格兰留兰香精油敏感。精油与抗生素联合使用时抑菌范围和强度均有所改变: 绿脓杆菌的2个菌株对精油与抗生素的组合最为敏感, 其中, 椒样薄荷精油与头孢他啶的组合对P. aeruginosa ATCC 15442显示出最强的增效作用, 薄荷精油与头孢他啶混合之后对P. aeruginosa ATCC 27853出现拮抗作用。Staphylococcus aureus ATCC 25923对所有精油以及精油与抗生素混合物均有抗性。(3) 椒样薄荷、薄荷和苏格兰留兰香精油的不同成分及其含量差异不仅对精油品质有影响, 而且影响精油对测试菌种的抑制作用, 可考虑将其作为薄荷属植物品质育种的参考指标。     

UV-B辐射对拟南芥种子萌发和幼苗生长的影响

以哥伦比亚生态型(Columbia-0)拟南芥(Arabidopsis thaliana)为实验材料, 人工模拟UV-B辐照处理拟南芥种子, 统计其发芽势和发芽率, 测定根长、株高、叶绿素、可溶性糖、可溶性蛋白以及丙二醛(MDA)含量, 研究UV-B辐照处理对拟南芥种子萌发和幼苗生长的影响。研究结果表明, 低剂量的UV-B辐照可以促进拟南芥种子萌发和幼苗生长, 并且最佳辐照剂量为1.0 kJ·m^–2(P<0.05), 此时的发芽势、发芽率、根长、株高、叶绿素、可溶性糖及可溶性蛋白的含量均达到最大, 而丙二醛的含量变化则不明显(P>0.05)。当辐照剂量大于1.0 kJ·m^–2时, 促进作用逐渐变小, 并且随着辐照剂量的增加, 表现出了抑制作用。实验结果表明, 适当剂量的UV-B辐照在一定程度上可以促进拟南芥种子萌发和幼苗生长, 而过高剂量的辐照则对其产生抑制或损伤作用。     

气候变化对6种荒漠植物分布的潜在影响

分析气候变化对植物分布的影响,对保护生物多样性具有重要意义。利用CART(分类和回归树)模型及A2和B2情景,分析了气候变化对短叶假木贼(Anabasis brevifolia)、裸果木(Gymnocarpos przewalskii)、梭梭(Haloxylon ammoden-dron)、膜果麻黄(Ephedra przewalskii)、驼绒藜(Ceratoides latens)和喀什膜果麻黄(Ephedra przewalskii var.kaschgarica)分布范围及空间格局的影响。结果表明:气候变化下,这些植物目前适宜分布范围减小;从新适宜及总适宜分布范围而言,短叶假木贼和梭梭从1991-2020年到2051-2080年时段增加,之后减小,其它植物从1991-2020年到2081-2100年时段减小;喀什膜果麻黄和驼绒藜适宜分布范围减小并破碎化,其它植物向目前适宜分布的西部、西北部(或青海西南部)、昆仑山、阿尔金山和祁连山区扩展;除驼绒藜和喀什膜果麻黄与年均气温变化具显著相关性外,其它植物分布范围与年均气温和降水量变化的相关性较弱(P0.05),除驼绒藜、喀什膜果麻黄和裸果木目前分布范围与年均气温和降水量变化的回归关系较强外,其它植物分布范围与年均气温和年降水量变化多元线性回归关系较弱。上述研究结果表明,气候变化下,这些植物空间分布格局改变,目前分布范围减少,新适宜及总适宜分布范围近期增加,随着气候变化程度的增强,又逐渐减小。     

Arabidopsis nonhost resistance gene PSS1 confers immunity against an oomycete and a fungal pathogen but not a bacterial pathogen that cause diseases in soybean

ABSTRACT: BACKGROUND: Nonhost resistance (NHR) provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Three Arabidopsis thaliana PEN (penetration deficient) genes, PEN1, 2 and 3 have been shown to provide NHR against the barley pathogen Blumeria graminis f. sp. hordei at the prehaustorial level. Arabidopsis pen1-1 mutant lacking the PEN1 gene is penetrated by the hemibiotrophic oomycete pathogen Phytophthora sojae, the causal organism of the root and stem rot disease in soybean. We investigated if there is any novel nonhost resistance mechanism in Arabidopsis against the soybean pathogen, P. sojae. RESULTS: The P. sojae susceptible (pss) 1 mutant was identified by screening a mutant population created in the Arabidopsis pen1-1 mutant that lacks penetration resistance against the non adapted barley biotrophic fungal pathogen, Blumeria graminis f. sp. hordei. Segregation data suggested that PEN1 is not epistatic to PSS1. Responses of pss1 and pen1-1 to P. sojae invasion were distinct and suggest that PSS1 may act at both pre- and post-haustorial levels, while PEN1 acts at the pre-haustorial level against this soybean pathogen. Therefore, PSS1 encodes a new form of nonhost resistance. The pss1 mutant is also infected by the necrotrophic fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome in soybean. Thus, a common NHR mechanism is operative in Arabidopsis against both hemibiotrophic oomycetes and necrotrophic fungal pathogens that are pathogenic to soybean. However, PSS1 does not play any role in immunity against the bacterial pathogen, Pseudomonas syringae pv. glycinea, that causes bacterial blight in soybean. We mapped PSS1 to a region very close to the southern telomere of chromosome 3 that carries no known disease resistance genes. CONCLUSIONS: The study revealed that Arabidopsis PSS1 is a novel nonhost resistance gene that confers a new form of nonhost resistance against both a hemibiotrophic oomycete pathogen, P. sojae and a necrotrophic fungal pathogen, F. virguliforme that cause diseases in soybean. However, this gene does not play any role in the immunity of Arabidopsis to the bacterial pathogen, P. syringae pv. glycinea, which causes bacterial blight in soybean. Identification and further characterization of the PSS1 gene would provide further insights into a new form of nonhost resistance in Arabidopsis, which could be utilized in improving resistance of soybean to two serious pathogens.     

The elicitor-induced oxidative processes in leaves of Solanum species with differential polygenic resistance to Phytophthora infestans

Previous studies have shown that suspension-cultured cells of Solanum genotypes with various polygenic resistances to Phytophthora infestans differed in activities of early oxidative processes in response to culture filtrate (CF) from this pathogen. These studies have now been extended by analysing production of reactive oxygen species (ROS), lipid peroxidation and Lipoxygenase (LOX, E.C.1.13.11.12) activity induced by CF in detached leaves of S. tuberosum cv Bzura and clone H-8105, polygenically resistant and susceptible, respectively, as well as S. nigrum, nonhost, completely resistant. The relative increase in the ROS production was higher in the susceptible clone H-8105 than in both resistant genotypes. Lipid peroxidation increased only in the nonhost S. nigrum. An increase in lipid peroxidation in S. nigrum leaves coincided with enhanced LOX activity. In both S. tuberosum genotypes, significant increases in LOX activity were delayed and unaccompanied by changes in the level of lipid peroxidation. LOX activity attained a higher level in both of the resistant genotypes than in the susceptible one. The present results suggest that the involvement of both ROS production and LOX activity in the defense strategy in Solanum species/P. infestans interactions.     

Integration of XAS techniques and genetic methodologies to explore Cs-tolerance in Arabidopsis

We have characterised genetically and phenotypically a T-DNA insertion mutant line of A. thaliana (L.) Heynh. selected for Cs resistance when germinating and growing on Cs concentrations up to 600microM, lethal for the wild type. Measures of concentration and localisation of Cs, K, and Ca have been conducted on plants grown in vivo also utilising synchrotron light-based techniques as micro-SRXF (synchrotron radiation induced X-ray micro-fluorescence) and micro-XANES (micro X-ray absorption near edge structure) spectroscopy. We report here an attempt to apply micro-XANES to investigate Cs speciation and to measure the Cs content of living plants. The results obtained with micro-SRXF and micro-XANES spectroscopy complemented the genetic and physiological analyses: a comparison between wild type and mutant plants led to the conclusion that in our case a single gene mutation impairs Cs uptake and translocation, K and Ca homeostasis and plant biomass production.