苹果树腐烂病菌细胞色素P450基因Vmcyp5的功能

【目的】研究表明,细胞色素P450(CYP)在死体营养型真菌的毒素合成代谢中发挥重要作用,预测可能与病原菌致病相关。论文对苹果树腐烂病菌(Valsa mali)毒素合成基因簇中的1个上调表达的CYP基因Vmcyp5进行生物学功能研究,明确CYP基因对病原菌致病力影响,为细胞色素P450基因家族对苹果树腐烂病菌致病机理的进一步研究提供依据。【方法】通过Double-joint PCR和PEG介导的原生质体转化技术获得具有G418抗性的突变体,并对突变体进行PCR检测及Southern blotting验证得到单拷贝敲除突变体。将目的基因片段重新导入敲除突变体,筛选获得互补突变体。最终对野生型菌株及敲除突变体、互补突变体进行菌落、产孢及致病力观察,利用SPSS软件对数据进行差异显著性分析,并利用q RT-PCR技术分析突变体黑色素基因簇的表达水平。【结果】通过基因敲除技术获得1个Vmcyp5基因的敲除突变体。与野生型菌株相比,Vmcyp5基因的敲除突变体菌落呈白色,产孢量减少51.3%。q RT-PCR分析发现敲除突变体黑色素基因簇基因表达量降低。重要的是,敲除突变体致病力较野生型菌株降低24.5%。互补突变体菌落颜色、产孢及致病力近似恢复至野生型菌株水平。【结论】Vmcyp5基因与病原菌黑色素合成、子实体的产生和致病力相关。     

真菌细胞色素P450在大肠杆菌中的表达

【背景】真菌细胞色素P450蛋白在大肠杆菌中表达水平低甚至不表达,近期研究发现通过对该类蛋白氨基端(N端)氨基酸序列的修饰可优化其表达水平。【目的】在大肠杆菌系统中表达预测功能为P450酶的焦曲霉094102菌株的Au8002蛋白,为真菌P450蛋白在大肠杆菌表达系统中的N端氨基酸序列修饰策略提供有效依据。【方法】对野生型P450蛋白Au8002的氨基酸序列进行分析,对其N端序列进行了3种序列修饰,并在诱导蛋白表达时添加P450生物合成前体5-氨基乙酰丙酸(5-ALA),研究N端氨基酸序列修饰策略及前体添加对真菌P450在大肠杆菌中蛋白表达的影响。【结果】SDS-PAGE和Westernblot检测结果显示,对目的蛋白进行的3种氨基酸序列修饰均使Au8002蛋白获得了表达,前体5-ALA的添加提高了目的蛋白表达量。其中对目的蛋白进行N端全长截短时可部分增加其可溶性,同时也验证了其特征性的CO结合能力。【结论】对预测为P450酶的菌株094102蛋白Au8002氨基端(N端)氨基酸序列的修饰有效解决了其在大肠杆菌内不表达的难题,实现了其可溶性表达;另一方面P450生物合成前体5-ALA的添加也能有效提高该类蛋白的表达水平,上述策略对改善其它该类蛋白在大肠杆菌内的表达水平具有借鉴意义。     

微小杆菌属(Exiguobacterium)细菌的能量代谢途径分析

【目的】微小杆菌属(Exiguobacterium)细菌广泛分布于海洋及非海洋环境中,具有多种代谢途径以适应复杂多样的生境。本研究从能量代谢途径角度出发,探究该属菌株对不同生境的适应能力。【方法】从美国国家生物科技数据中心(National Center for Biotechnology Information, NCBI)数据库中获取146个Exiguobacterium属菌株的基因组,查找并统计光营养、厌氧呼吸和底物代谢等多种能量代谢途径的关键蛋白或关键酶基因在各菌株基因组中的分布,包括光营养型的视紫红质基因、厌氧呼吸营养型的钼辅因子合成蛋白基因,以及底物代谢营养型中乙醛酸分流途径的异柠檬酸裂解酶及苹果酸合酶基因等。根据对应的氨基酸序列构建视紫红质、MoaC和异柠檬酸裂解酶的系统发育树,分析不同能量代谢途径在该属菌株进化过程中的保守性,推测其对于该属菌株的重要性。【结果】Exiguobacterium属中50%的种具有视紫红质基因,其中分离自非海洋生境的菌株更趋向于含有视紫红质基因。本研究所统计的全部非海洋生境菌株中,含有视紫红质基因的菌株占比约为70%,而在海洋生境菌株中该比例...     

出芽短梗霉菌株PA-2全基因组测序及分析

【背景】出芽短梗霉菌株PA-2是一株分离自青海省海东市平安区患病杨树叶片上的真菌,前期研究表明该菌株具有除草和抑菌能力,说明其在生物农药方面具有潜在的应用前景。【目的】了解菌株PA-2的基因组序列信息,挖掘其生防相关功能基因簇,为进一步研究解析该菌株生防机理及生防功能改造提供遗传背景信息。【方法】利用IlluminaHiSeq高通量测序平台对生防菌株PA-2进行全基因组测序,用生物信息学的方法对测序数据进行基因组组装、基因预测及功能注释、碳水化合物活性酶预测、次级代谢产物合成基因簇预测,利用刚果红染色等方法对水解酶活性进行衡量。【结果】菌株PA-2基因组序列全长28 932 793 bp,平均GC含量为50%,共编码10 839个基因,预测到该菌株具有4个已知的次级代谢产物合成基因簇,编码Melanin、Burnettramic Acid A、ACR-Toxin I、Abscisic Acid,该菌株能水解纤维素和果胶。【结论】有助于在基因组层面上解析菌株PA-2生防机制的内在原因,为深入了解出芽短梗霉菌次级代谢物合成途径提供参考,对菌株PA-2的下一步相关研究具有重要意义。     

膜泡运输蛋白VdSec22参与大丽轮枝菌胞外蛋白的分泌和致病性

摘要:【目的】验证大丽轮枝菌分泌途径中一个膜泡运输蛋白VdSec22的功能,为防治棉花黄萎病提供潜在的生物靶点。【方法】利用“正负双向筛选法”的方法,构建大丽轮枝菌VdSec22蛋白编码基因缺失的突变体菌株ΔQF。通过农杆菌介导的转化,将其编码基因VdSec22重新导入ΔQF构建了功能回补菌株CΔQF。以野生型菌株为对照,检测上述菌株分泌胞外蛋白(果胶酶、纤维素酶和毒素蛋白)的能力;采用蘸根接种的方法,检测上述菌株对棉花致病性的差异。同时通过定量PCR检测内质网分子伴侣表达量的方法,推断突变体菌株ΔQF中是否发生了内质网应激反应。【结果】成功构建了基因敲除突变体ΔQF和功能回补菌株CΔQF。突变体菌株ΔQF的果胶酶、纤维素酶、毒素蛋白的分泌能力和对棉花的致病性均较野生型减弱,并且产生了内质网应激反应。重新导入VdSec22基因可弥补突变体菌株ΔQF的上述缺陷。【结论】VdSec22是大丽轮枝菌的一个重要分泌途径蛋白,在大丽轮枝菌诸多胞外致病蛋白的分泌和棉花致病性中起重要作用。VdSec22可作为防治棉花黄萎病的潜在生物靶点。     

白假丝酵母Vps74蛋白的鉴定及其功能

【背景】Vps74/GOLPH3是参与高尔基体蛋白糖基化修饰的关键蛋白,并且是重要的磷酸磷脂酰肌醇效应因子,在胞内参与多种信号通路。【目的】鉴定白假丝酵母Vps74蛋白,并探索其在该病原菌压力应答、蛋白分泌、形态发生及致病过程中的功能。【方法】采用在线序列比对方法,初步鉴定白假丝酵母Vps74蛋白;采用两步PCR介导的同源重组方法,构建白假丝酵母vps74基因缺失菌株vps74Δ/Δ及回补菌株VPS74c;采用反向遗传学方法,探究Vps74在白假丝酵母的压力应答、蛋白分泌、形态发生及致病过程中的功能。【结果】白假丝酵母中存在典型的Vps74/GOLPH3同源蛋白,Vps74参与蛋白糖基化修饰过程,vps74基因缺失导致白假丝酵母蛋白分泌能力、形态发生能力、黏附能力以及侵染宿主能力的显著降低。【结论】Vps74通过影响蛋白分泌、形态发生、黏附、嵌入式生长等过程,在白假丝酵母致病过程中发挥重要作用。     

广西南宁市柑橘木虱虫生真菌种类调查

自柑橘木虱Diaphorina citri体上分离虫生真菌,通过形态特征和ITS序列分析,鉴定虫生真菌的种类;通过致病性测定,明确昆虫病原真菌种类。结果发现,自死体柑橘木虱上分离到18个虫生真菌菌株,隶属4种真菌;自活体上分离到985个虫生真菌菌株,隶属25种真菌。致病性测定结果发现,仅4种真菌对柑橘木虱成虫有致病性,包括刀孢蜡蚧菌Lecanicillium psalliotae、球孢白僵菌Beauveria bassiana、爪哇虫草Cordyceps javanica和淡紫紫孢菌Purpureocillium lilacinum,该4种真菌的分生孢子悬浮液（浓度5×10^7个孢子/mL）接种柑橘木虱成虫后10 d的累计死亡率分别为100%、100%、98.89%和43.33%。其中爪哇虫草仅自死体柑橘木虱上分离到,刀孢蜡蚧菌仅在活体上分离到,球孢白僵菌和淡紫紫孢菌在死体和活体上均被分离到。可见,柑橘木虱活体上的虫生真菌种类丰富。生产上应加强保护和利用昆虫病原真菌,提高其对柑橘木虱的自然抑制力。     

利用非经典分泌蛋白质实现脂肪酶A的分泌表达

【目的】以枯草芽孢杆菌脂肪酶A(Lipase A)为报告蛋白,尝试利用4种非经典分泌蛋白质及其前50个氨基酸作为分泌信号以实现其分泌表达。【方法】我们扩增了脂肪酶A的编码基因和非经典分泌蛋白质的编码序列,构建了8种针对脂肪酶A的分泌表达载体,并转化至枯草芽孢杆菌WB800菌株,通过测定重组菌株的酶活、利用蛋白质电泳和免疫印迹等技术检测脂肪酶A的分泌情况【结果】以Pdh A的氨基酸序列和Sod A、Eno的前50氨基酸序列作为分泌信号的重组菌株较好的实现了脂肪酶A的分泌表达。【结论】部分非经分泌蛋白质的编码基因或其前50个氨基酸序列能够引导脂肪酶A分泌至细胞外。     

内蒙古鄂尔多斯一古盐湖真菌多样性及其功能酶的初步筛选

【目的】探索内蒙古鄂尔多斯一古盐湖土壤沉积物和湖水样品中的真菌多样性,并初步筛选出产功能酶的活性菌株。【方法】采用含有2.5%、5%、10%及15%NaCl的3种分离培养基,利用稀释平板法分离可培养真菌,基于形态学和ITS序列分析对获得菌株进行系统分类学分析。利用6种筛选培养基定性检测盐湖真菌的产酶活性。【结果】共分离到2 121株真菌,共45个形态种,根据形态学研究和ITS序列的系统分类学分析将其归类为27个属,优势属为曲霉属(Aspergillus)。功能酶筛选结果表明,45个形态种中有22个形态种具有产酶活性,其中6个形态种只产蛋白酶,6个只产纤维素酶,2个只产复合酶,1个只产淀粉酶。有4个形态种可同时产3种酶,有3个可同时产2种酶。【结论】内蒙古鄂尔多斯地区盐湖中真菌多样性丰富,产酶特性良好,研究工作为耐(嗜)盐真菌资源的进一步开发和利用提供依据。     

禾本科植物内生真菌研究20：基于NRPS5基因的我国东部早熟禾亚科植物内生真菌系统发育学分析

【背景】冷季型禾草-Epichloae真菌内生共生体在我国广泛存在,Epichloae内生真菌具有遗传多样性。【目的】选取来自江苏、山东和安徽等6省共8地的早熟禾亚科植物中的21个Epichlo?sp.菌株,检测菌株的NRPS5基因;分析不同宿主内Epichlo?属内生真菌NRPS5基因多样性。【方法】提取Epichlo?spp.菌株DNA,利用特异性引物对菌株中NRPS5基因进行PCR检测和序列分析,并基于NRPS5基因构建最大简约法系统发育树。【结果】NRPS5基因可能广泛分布于我国东部冷季型禾本科植物Epichloae内生真菌中。在同种内生真菌和同一宿主(除鹅观草)中的不同种内生真菌中NRPS5基因相对保守,但不同宿主的内生真菌其碱基序列差异大,与宿主植物采集地点、采集时间和子座的有无均无相关性,NRPS5基因的差异主要与宿主植物有关。这说明NRPS5基因序列比较保守,推测属于NRPS蛋白的功能核心区。【结论】NRPS5基因序列相对保守,可作为真菌遗传多样性分析的一种辅助手段。     

The biotrophy-associated secreted protein 4 (BAS4) participates in the transition of Magnaporthe oryzae from the biotrophic to the necrotrophic phase

The physiological and metabolic processes of host plants are manipulated and remodeled by phytopathogenic fungi during infection, revealed obvious signs of biotrophy of the hemibiotrophic pathogen. As we known that effector proteins play key roles in interaction of hemibiotrophic fungi and their host plants. BAS4 (biotrophy-associated secreted protein 4) is an EIHM (extrainvasive hyphal membrane) matrix protein that was highly expressed in infectious hyphae. In order to study whether BAS4 is involved in the transition of rice blast fungus from biotrophic to necrotrophic phase, The susceptible rice cultivar Lijiangxintuanheigu (LTH) that were pre-treated with prokaryotic expression product of BAS4 and then followed with inoculation of the blast strain, more serious blast disease symptom, more biomass such as sporulation and fungal relative growth, and lower expression level of pathogenicity-related genes appeared in lesion of the rice leaves than those of the PBS-pretreated-leaves followed with inoculation of the same blast strain, which demonstrating that BAS4 invitro changed rice defense system to facilitate infection of rice blast strain. And the susceptible rice cultivar (LTH) were inoculated withBAS4-overexpressed blast strain, we also found more serious blast disease symptom and more biomass also appeared in lesion of leaves inoculated with BAS4-overexpressed strain than those of leaves inoculated with the wild-type strain, and expression level of pathogenicity-related genes appeared lower in biotrophic phase and higher in necrotrophic phase of infection, indicating BAS4 maybe in vivo regulate defense system of rice to facilitate transition of biotrophic to necrotrophic phase. Our data demonstrates that BAS4 in vitro and in vivo participates in transition from the biotrophic to the necrotrophic phase of Magnaporthe oryzae.     

真菌中G蛋白信号调控因子蛋白类型与其理化性质的关系

[目的]G蛋白信号调控因子(RGS)作为G蛋白信号转导途径的负调控因子,在植物病原菌的致病性和有性生殖调控方面发挥着重要作用.研究真菌中RGS蛋白类型与其理化性质及特征的关系,为今后深入开展不同真菌中具有不同类别RGS的功能解析打下坚实的理论基础.[方法]前期对模式生物、病原菌、非致病菌等49个真菌中229个RGS蛋白...     

Plant resistance signalling hijacked by a necrotrophic fungal pathogen

The strategies used by necrotrophic fungal pathogens to infect plants are often perceived as lacking the sophistication of their haustorium producing, host defence suppressing, biotrophic counterparts. There is also a relative paucity of knowledge regarding how effective gene-for-gene based resistance reactions might function against necrotrophic plant pathogens. However, recent data has emerged from a number of systems which has highlighted that particular species of necrotrophic (and/or hemibiotrophic) fungi, have evolved very sophisticated strategies for plant infection which appear, in fact, to hijack the host resistance responses that are commonly deployed against biotrophs. Both disease resistance (R) protein homologues and mitogen-activated protein kinase (MAPK) cascades commonly associated with incompatible disease resistance responses; appear to be targeted by necrotrophic fungi during compatible disease interactions. These findings highlight an emerging sophistication in the strategies deployed by necrotrophic fungi to infect plants.Key words: Mycosphaerella graminicola, Septoria tritici, Triticum aestivum, mitogen-activated protein kinase, programmed cell death, fungal pathogen, disease resistance, disease susceptibility, toxin     

The hemibiotrophic lifestyle of Colletotrichum species

Colletotrichum species infect several economically important crop plants. To establish a compatible parasitic interaction, a specialized infection cell, the melanized appressorium, is differentiated on the cuticle of the host. After penetration, an infection vesicle and primary hyphae are formed. These structures do not kill the host cell and show some similarities with haustoria formed by powdery mildews and rust fungi. Therefore, this stage of infection is called biotrophic. Later in the infection process, necrotrophic secondary hyphae spread within and kill the host tissue. The lifestyle of Colletotrichum species is called hemibiotrophic, as biotrophic and necrotrophic developmental stages are sequentially established. As most Colletotrichum species are accessible to molecular techniques, genes can be identified and functionally characterized. Here we demonstrate that Agrobacterium tumefaciens-mediated transformation is a well-suited method for tagging of genes mediating compatibility in the Colletotrichum graminicola–maize interaction.     

Characterization of a Heterobasidion irregulare endo‐rhamnogalacturonase that mediate growth on pectin

Heterobasidion irregulare is one of five Heterobasidion annosum sensu lato (s.l.) species, which are destructive pathogens in boreal and temperate forests of the northern hemisphere that causes root and butt rot in conifer. A gene encoding endo‐rhamnogalacturonase (HIRHG), which belongs to the glycoside hydrolase family 28 (GH28), was found in a quantitative trait loci (QTL) region for virulence in Heterobasidion. In this study, we showed that HIRHG is highly upregulated during necrotrophic infection of Norway spruce compared with growth in liquid culture and that the HIRHG encoded protein is produced during fungal growth on complex carbon sources. Phylogenetic analysis of endo‐rhamnogalacturonases revealed that rhamnogalacturonase genes have been lost in most of the biotrophic and hemibiotrophic plant pathogens investigated but were common in necrotrophic pathogens and saprophytic fungi. Heterologous expression of the HIRHG gene in the hemibiotrophic fungus Magnaporthe oryzae increased its capacity to grow on pectin; however, the transformed M. oryzae isolates showed significant less infection of rice leaves compared to the wild type.     

拮抗植物病原真菌链霉菌菌株ZH-356的鉴定及其生防评价

[目的]对实验室分离到的菌株ZH-356进行鉴定并评价其对植物病原真菌的生物防治效果,为研发针对植物真菌病害的生防菌剂提供理论指导。[方法]通过平板对峙法确定菌株ZH-356抗菌谱,并通过16S rRNA基因序列分析确定其种属,利用离体枝条的苹果树腐烂病菌感染预防试验和患腐烂病苹果树的防治试验评价其生防效果。[结果]菌株ZH-356鉴定为链霉菌属,与直丝紫链霉菌(Streptomyces rectiviolaceus)相似性最高,为99.71%。抗菌谱试验表明,菌株ZH-356对苹果树腐烂病菌、小麦赤霉病菌、小麦根腐病菌和番茄早疫病菌等多种植物病原真菌均具有较强的抑制作用,这种抑制作用可导致苹果树腐烂病菌菌丝变粗、交叉扭曲、分支变少且容易断裂。此外,ZH-356产生的抑菌活性物质对温度和酸碱度具有高度稳定性,并且该活性物质只存在于其胞内,只有当ZH-356遇到植物病原真菌时才会被分泌出来以抑制它们的生长。在离体枝条的苹果树腐烂病菌感染预防试验中,ZH-356对苹果树腐烂病防效可达94%以上,而在患腐烂病苹果树的防治试验中,ZH-356菌制剂对苹果树腐烂病的防效高达100%。[结论]链霉菌ZH-356抑菌谱广,对多种植物病原真菌均具有良好的拮抗活性,可作为防治植物真菌病害的生防菌株,为基于ZH-356菌株的生防菌剂的开发和防治苹果树腐烂病等植物真菌病害奠定了基础。