Plant amphipathic proteins delay the hypersensitive response caused by harpinPssandPseudomonas syringaepv.syringae

Harpin_Pssfrom the plant pathogenPseudomonas syringaepv.syringaeis a proteinaceous elicitor that induces a hypersensitive response (HR) in non-host plants. The plant products which recognize harpin_Pssin the triggering of the HR are not yet known. According to the elicitor-receptor model, we hypothesize that an exogenous cell membrane receptor infiltrated into the intercellular space will interfere with the interaction between harpin_Pssand the putative receptor. We demonstrate a plant amphipathic protein (AP1) which can postpone the HR induced by harpin_Pssas well asP. syringaepv.syringae.AP1 was extracted by solubilizing proteins from healthy leaves in the non-polar n-octanol buffer followed by a polar Tris buffer. The amphipathic extracts were then further separated by gel filtration and anion exchange chromatography to obtain highly purified AP1. Similar proteins can be extracted from cotton, tomato, and sweet pepper. The N-terminal amino acid sequence of AP1 is conserved among cotton, tomato, and sweet pepper. The postponement of the harpin_Pss-mediated HR was characterized as a competitive dosage-dependent pattern of AP1. An analysis of the bacterial population development indicates that the effect of AP1 on the postponement of bacteria-mediated HR was attributed to the suppression of bacterial growth during the early stages of the HR. The time course analysis of the infiltration indicates that the postponement of HR resulted from the co-interaction between AP1 and the bacteria. Based on these results, we suggest that the postponement of bacteria-mediated HR is due to the interference of the interaction between harpin_Pssand the putative receptor in the plant. Our research provides a new approach to elucidating the role that plants may play in the nonhost response caused by pathogens.     

Prochloraz,a potent inducer of the microsomal cytochrome P-450 system

Prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-imidazole-1-carboxamide), a recently developed agricultural fungicide, is a potent inducer of microsomal enzymes. Rats fed 7 days with a prochloraz-contaminated diet (2500 ppm) showed an increase in hepatic cytochrome P-450, cytochrome b₅, and microsomal protein level; aniline hydroxylase, 7-ethoxycoumarin dealkylase, 7-ethoxyresorufin dealkylase, NADPH-cytochrome c reductase, and epoxide hydrolase were significantly induced. At a lower dose (100 ppm), only an increase in cytochrome P-450 and 7-ethoxyresorufin dealkylase was noticed. As shown with aniline hydroxylase and 7-ethoxycoumarin dealkylase, prochloraz is also a potent inhibitor of drug-metabolizing enzymes. The interaction of prochloraz with hepatic microsomal fraction from rat liver was also studied. Prochloraz binds to oxidized cytochrome P-450 to produce a type II spectral change; the compound also binds to reduced cytochrome P-450. The binding of some ligands (7-ethoxycoumarin, n-octylamine, aniline, and imidazole) to oxidized cytochrome P-450 was determined after induction by prochloraz. Japanese quails (Coturnix coturnix) fed 7 days with a prochloraz-contaminated diet (2000 ppm) showed a dramatic increase in liver weight (2.5-fold) and both hepatic and duodenal cytochrome P-450 (9- and 12-fold, respectively).     

Ozone for control of post-harvest decay of table grapes caused byRhizopus stolonifer

The effect of ozone on post-harvest decay of table grapes was studied both with regard to its effectiveness and its possible mode of action. Ozone concentrations fell rapidly upon contact with organic matter and the amount which reacted with grape berries and the microflora on their surface was about 0.1mgg^-1, when supplied at a rate of 8mgmin^-1for 20min. The dose applied could be increased by longer periods of exposure, but symptoms of toxicity were observed on certain cultivars. The number of colony forming units (cfu) of fungi, yeasts and bacteria naturally present on the berry surface was considerably reduced by a 20min exposure to ozone. Ozone treatments significantly reduced the extent of berry decay caused by fungi following cold storage and increased shelf-life. A significant decrease in decay was observed in berries that were treated with ozone either before or after being inoculated withRhizopus stolonifer.This finding indicates that, in addition to its sterilizing effect, ozone also induced resistance to post-harvest decay development. The phytoalexins resveratrol and pterostilbene were elicited by ozone treatments, at levels similar to those produced by uv-c irradiation. Resveratrol accumulated in greater quantities than pterostilbene. Inoculation withR. stoloniferin addition to ozone treatment, raised the levels of both stilbenes even more. Exposing berries to ozone was almost as effective as SO₂fumigation for the control of storage decay caused byR. stoloniferand no deleterious effects were observed on the appearance of the grape cluster. Ozone treatments can therefore be considered as a possible substitute for SO₂fumigation for the control of post-harvest fungal decay.     

The relative tolerances of wild and cultivated oats to infection byErysiphe graminisf.sp.avenae: I. The effects of infection on vegetative growth and yield

The effects of infection by the powdery mildew fungusErysiphe graminisf.spavenaewere studied in one line of wild oat (Avena fatua), and two cultivars, Lustre and Peniarth, of cultivated oat (A. sativa) to determine if the wild oat was more tolerant of infection than the two cultivated oats. Seven weeks after inoculation, when the plants were 10-weeks-old with fully expanded flag leaves, the fungus had colonized approx. 40% of the leaf surfaces of wild oat and cv. Lustre but only about 30% of the leaf surfaces of cv. Peniarth. The lower leaves of cv. Peniarth were at least as susceptible, if not more so, than those of the other two lines but the upper leaves, including the flag leaf, were much more resistant. Although cv. Peniarth supported the production of about half the number of mildew conidia as the wild oat and cv. Lustre its total dry weight and grain yield were reduced to the greatest extent. The wild oat was clearly much more tolerant of mildew infection than cv. Peniarth and slightly more tolerant than cv. Lustre. The greater tolerances of the wild oat and cv. Lustre compared to cv. Peniarth appeared to be due to the lower sensitivities of their metabolism to the activities of the mildew fungus.     

大豆RSC4抗病候选基因Glyma.14G204700的克隆及其生物信息学分析

为明确大豆抗大豆花叶病毒（soybean mosaic virus，SMV）SC4株系的基因位点R_SC4（resistance to SMV strain SC4，R_SC4）的候选基因Glyma.14G204700在大豆不同抗性品种中的序列结构特征和保守结构域，以齐黄1号、科丰1号、大白麻和南农1138-2共4个大豆品种为材料，通过基因克隆获得大豆Glyma.14G204700基因的cDNA全长序列，并采用生物信息学方法分析其序列特征和编码蛋白的理化特性及结构特征。结果表明，大豆Glyma.14G204700基因在4个大豆品种中的cDNA全长为4 719~4 776 bp，编码1 295~1 307个氨基酸，预测蛋白的分子量为148.38~149.33 kD，等电点为5.53~5.61，均为具较强亲水性的非分泌蛋白。Glyma.14G204700蛋白含有植物抗病基因家族蛋白的保守功能域——核苷酸结合域和富含亮氨酸重复结构域。该蛋白的二级结构主要由α-螺旋、无规则卷曲、延伸链和β-折叠组成，所占比例分别为59.45%~61.08%、28.65%~30.15%、8.11%~8.88%和1.61%~2.16%。启动子序列分析发现该基因含有脱落酸、低温及干旱等多种逆境胁迫响应元件。表明大豆R_SC4抗病候选基因Glyma.14G204700在大豆不同抗性品种中具有不同的等位变异，优异等位变异的鉴定和开发可为抗SMV大豆育种提供基础材料。     

PCR cloning, DNA sequencing and phylogenetic analysis of a xylanase gene from the phytopathogenic fungusAscochyta pisiLib.

A gene encoding a xylanase,xyl1, was isolated from the phytopathogenic fungusAscochyta pisiLib. by PCR cloning using degenerate primers. DNA sequence analysis revealed an open reading frame of 736 bp interrupted by an intron of 55 bp. The ORF encodes a predicted protein of 227 amino acids. The precise splicing site of the intron was identified from the sequence of a PCR product obtained using the same degenerated primers on a cDNA template. The cDNA product and a northern blot demonstrated that the gene is transcribed into mRNA when the fungus is cultured in media containing xylan as sole carbon source. The Neighbour-Joining method using the Clustal W(1.5) program demonstrated that theA. pisixylanase is a member of the family 11 glycosyl hydrolases, and that this family represents at least five phylogenetically consistent groups. The family 11 glycosyl hydrolases can be linked with family 10 glycosyl hydrolyses through bifunctional enzymes fromRuminococcus flavefaciensand, to a lesser extentNeocallimastix patriciarum.     

Analysis of two acetylcholinesterase genes in Bombyx mori

Two acetylcholinesterases (AChE, EC 3.1.1.7) cDNAs were identified and cloned from silkworm, Bombyx mori. One of those, BmAChE-o cDNA, is comprised of 3197 nucleotides which encode 638 amino acids, having an amino acid sequence homology of 72% with Drosophila melanogaster Ace-orthologous AChE (AO-AChE). In some species, another AChE group based on the sequence, Drosophila Ace-paralogous AChE (AP-AChE) has been recognized in relation to organophosphate- or carbamate-resistance, but there have been few reports of AP-AChE among lepidopteran species. However, we isolated the AP-AChE from lepidopteran silkworm, and cloned full ORF as BmAChE-p, which cDNA consisted of 2465 nucleotides that encode 683 amino acids. The homologies with other AP-AChEs were over 60% when compared. Although silkworm is not a target of pesticides, the genomic information obtained in this study will contribute to insecticide-resistance study on lepidopteran pest species.     

十字花科黑腐病菌hrp基因致病功能分析

通过对Xcc 8004菌株的hrp基因进行逐一敲除,系统研究单个hrp基因对Xcc致病性的贡献。结果表明,9个hrc(hypersensitive response and conserved)基因单独突变后在满身红萝卜上的致病性和在辣椒ECW-10R上激发HR的能力完全丧失;9个hrp基因中,hrpW突变后在辣椒ECW-10R上仍能产生HR,满身红萝卜上致病性减弱,其余hrp基因突变后致病性和过敏反应均丧失;4个hpa(hrp associated protein)基因突变后,hpaA和hpaB突变体完全丧失在满身红萝卜上的毒性也不能在辣椒ECW-10R上引起HR,hpa1和hpaP突变后致病性和HR显著减弱。另外,通过RT-PCR对Xcc 8004 hrp基因簇的每个基因受hrpX和hrpG的调控情况进行分析,结果表明所有hrp基因在不同程度上都受到hrpG、hrpX的正向调控。     

Pest control by pathogens — fungi, viruses and bacteria Control of chrysanthemum pests with verticillium lecanii

Integrated pest control on chrysanthemum is once again becoming a possibility.Verti-cillium lecanii is one of the key components as, under the right conditions, it will provide good control of several pest species. High humidity for a number of nights per week is critical for reliable pest control with this fungal pathogen. These conditions can be obtained easily and safely by fogging water over the crop at night. Four consecutive nights of high humidity per week or a cycling two nights of high humidity and two nights ambient have given excellent pest control with no adverse effect to the crop.     

利用BSMV Agro/LIC进行小麦条锈菌耐高温 相关基因功能分析的互作体系构建

为利用HIGS技术研究条锈菌耐高温胁迫相关基因的功能,需要构建小麦品种与条锈菌及大麦条纹花叶病毒Barley stripe mosaic virus(BSMV)亲和互作体系。本研究选用20个常温下(10℃)高感条锈病的小麦品种和22个耐高温条锈菌菌株,进行高温(21℃)条件下接种亲和性分析,发现11个品种高感条锈病、3个菌株耐高温性稳定且毒性谱较宽。在此基础上,利用含有小麦TaPDS基因片段的重组BSMV侵染上述候选小麦品种,确定‘Local Red'等6个品种与BSMV亲和程度符合试验要求。筛选出小麦品种‘Local Red'与菌株15335-2、15323-4等组成的最佳互作组合,适合利用BSMV Agro/LIC方法开展小麦条锈菌耐高温相关基因功能的研究。     

荔枝霜疫霉自噬相关基因的鉴定与表达分析

 细胞自噬(Autophagy)是一种真核生物中高度保守的胞内物质降解和循环再利用的生理过程,其调控细胞动态平衡、压力适应和细胞程序性死亡,与植物病原真菌生长发育、孢子形成、侵染等一系列过程密切相关。荔枝霜疫霉(Peronophythora litchii)侵染引起的荔枝霜疫病严重危害荔枝生产及采后贮藏。本研究利用模式物种中已知的自噬相关蛋白,采用同源比对的方法在荔枝霜疫霉基因组数据库中鉴定到19个同源蛋白,分属16种蛋白类型。分析基因结构和蛋白保守功能域,发现荔枝霜疫霉自噬相关基因均具有内含子,且数目差异明显;所鉴定候选蛋白都具有相应分组的保守功能域,而PlATG1a在C端具有2个额外的ATG11功能域,PlATG11则在其N端包含1个APG17功能域。进一步对具有核心功能且多成员的自噬相关蛋白ATG1、ATG6、ATG8和ATG18进行系统进化和序列模块分析发现,这些蛋白在不同物种之间十分保守,但是其同源蛋白间存在分化。通过转录组数据分析和qRT-PCR验证结果显示,荔枝霜疫霉中自噬相关基因在生长发育和侵染阶段均有表达,与菌丝阶段相比,PlATG1a、 PlATG2、PlATG3、PlATG6a、PlATG8、PlATG18a基因在游动孢子阶段特异上调表达,其中PlATG8最为显著;PlVPS34在孢子囊阶段特异诱导表达,PlATG1b、PlATG12、PlATG17在孢子囊与游动孢子阶段上调表达但是在侵染阶段下调表达;PlATG6b、PlATG9、PlATG10、PlATG13、PlATG14及PlVPS15基因在生长发育和侵染阶段均表现出不同程度的上调表达,PlATG7、PlATG11、PlATG18b 则在侵染阶段显著下调表达。结果表明,细胞自噬可能参与调控荔枝霜疫霉的生长发育及致病过程。     

壳二孢叶枯病病原菌Ascochyta rabiei胁迫下鹰嘴豆防御相关基因的表达分析

为挖掘获得新的抗性基因,培育鹰嘴豆Cicer arietinum抗壳二孢叶枯病(病原菌为Ascochyta rabiei)新品种,以项目组前期获得的102个差异表达的新基因为基础,随机选取29个基因进行同源性分析,以鹰嘴豆Actin(EU529707)和Ef-1α(AJ004960)作为参考基因,利用实时荧光定量PCR技术检测这29个基因在宿主植物鹰嘴豆抗性品种系选03中的表达规律。结果显示,基于同源性分析结果可将29个基因大致分成4类,涉及信号传导机制、细胞运输、转录和细胞拯救、防御、毒性;功能分析结果显示,功能未知的基因数目最多,达到了11个,其中多数为鹰嘴豆未定性基因。这29个基因在A. rabiei胁迫下都发生了不同程度的差异表达,表达差异时间点集中在胁迫后72 h,并在96 h恢复至正常表达水平。其中解毒相关基因474在72 h时相对表达水平最高,是对照处理0 h的19.773倍,抗氧化修复相关基因1137的相对表达水平最低,约为对照处理0 h的1/3。筛选获得的差异表达基因中,表达上调的基因有10个,表达下调的基因有16个,其余3个基因的表达差异不明显。上调表达基因可能与鹰嘴豆应对A. rabiei侵染的应答机制有关,其中与免疫应激相关的蛋白基因如谷胱甘肽S-转移酶、咖啡酰辅酶A、泛素蛋白基因等可能直接参与了鹰嘴豆应对A. rabiei的免疫识别和防御。     

西瓜噬酸菌Aac-5AAA ATPases基因moxR和ruvB功能研究

AAA ATPases(ATPase associated with diverse cellular activities)是一种与细胞活性相关的酶,广泛存在于所有生物中,同时具有蛋白酶和分子伴侣活性。为了研究AAA ATPases相关基因缺失对西瓜噬酸菌(Acidovorax citrulli)的影响,本研究通过同源重组双交换的方法构建西瓜噬酸菌moxR基因以及ruv B基因全缺失突变菌株及互补菌株,测定了突变菌株的致病性、烟草过敏反应、运动性、生长能力、生物膜形成等;并利用实时荧光定量PCR方法测定了AAA ATPases与Ⅲ型分泌系统相关基因(hrc N和hrp E)、毒性蛋白相关基因(trb C和vir B)、鞭毛相关基因(fliR和fli M)、菌毛相关基因(pin C-1和pin C-2)以及AAA ATPases亚家族Ruv B相关基因(ruv A和ruv C)的关系。结果表明,moxR基因以及ruv B基因全缺失突变菌株的致病力降低、运动性减弱、生长能力下降、生物膜形成能力增强、烟草过敏性反应减弱、突变菌株中重要的致病基因hrc N和hrp E表达量显著下调,表明AAA ATPases相关基因moxR、ruv B在西瓜噬酸菌的致病力上发挥重要作用。     

水稻白叶枯病菌毒性基因调控hrp基因表达的分析

水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)侵染寄主水稻,引起水稻白叶枯病(bacterial leaf blight,BLB)。病原菌主要依赖hrp基因簇编码的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)将效应蛋白(T3SS effectors,T3SEs)注入水稻细胞中,激发水稻的抗(感)病性。同源性搜索结果显示,植物病原黄单胞菌中已鉴定的一些毒性基因在Xoo的代表菌株PXO99A中保守存在。为了明确这些毒性基因对hrp基因表达调控的影响,本研究利用pK18mob-W介导的定点突变方法,成功获得了14个毒性基因的突变体;在突变体中,利用hrp∶∶gusA融合表达体系,通过GUS活性定量测定检测了hrpG、hrpX和hrpB1的启动子活性;通过荧光定量PCR技术,检测了这3个基因的mRNA水平。结果显示,双组分调控系统ColR/ColS、RpfC/RpfG和转录调控子Clp负调控hrpG和hrpB1的表达;Trh和Xrv A通过HrpG-HrpX途径正调控hrpB1的表达;HpaR1和Fur不依赖于HrpG仅通过HrpX正调控hrpB1的表达。这些调控关系的鉴定为解析水稻黄单胞菌hrp调控网络提供了新的线索。     

玉米BTB家族基因的鉴定与表达规律分析

本研究利用生物信息学方法对玉米BTB家族基因进行系统发育、保守结构域、组织特异性以及在玉米抵抗生物和非生物胁迫过程中的表达规律进行分析.发现玉米70个BTB家族蛋白除AC206223.3外均含有保守的BTB结构域,可分为11个亚家族.玉米BTB家族基因具有明显的组织表达特性,在高温、低温、紫外、高盐和干旱胁迫下以及拟轮...     

大豆疫霉MAPK基因的鉴定与转录分析

 疫霉菌包括大豆疫霉等重要植物病原物,属于茸鞭生物界卵菌门,在进化上与真菌相差甚远;由于分离培养与遗传转化等相对困难,目前对其生长发育和致病机理的研究相对滞后。本研究综合运用基因组学和转录组学方法,首先对植物病原卵菌与真菌的蛋白激酶特别是MAPK进行了鉴定和比较,然后对大豆疫霉MAPK的基因结构、蛋白功能域以及转录模式等进行深入分析。结果表明,植物病原卵菌比真菌含有更多的蛋白激酶(包括MAPK),且卵菌及其近缘物种硅藻的MAPK与真菌在进化上相对独立;大豆疫霉的14个MAPK中,4个具有非典型的磷酸化唇序列,7个含有PH、C2、WW、PAS等与细胞信号转导相关的其它功能域;转录分析表明,大部分MAPK可能在大豆疫霉生长发育与致病的整个过程或特定过程中发挥重要作用。本文通过对蛋白激酶特别是MAPK的分析揭示了植物病原卵菌(与真菌相比)在细胞信号转导网络与机制上的独特性与复杂性,可为进一步研究疫霉菌MAPK的生物学功能及其信号调控机制提供参考。     

利用枯草芽胞杆菌表达系统进行马铃薯抗细菌基因的筛选

 抗菌肽是植物先天免疫系统的重要组成部分,具有良好的广谱抗菌活性,因此作为抗生素的替代品有较好的应用前景。马铃薯野生种(Solanum demissum)作为重要的种质资源,含有丰富的抗性基因。本研究利用指示菌内置文库抗性基因筛选技术,将马铃薯野生种cDNA文库转入指示菌枯草芽胞杆菌内,筛选出两种具有广谱抗菌活性的抗菌肽SdR2332和SdR2770。课题组分别测定了它们的热稳定性、酶稳定性、紫外线稳定性、最低抑菌浓度(minimum inhibitory concentration, MIC)和溶血性,旨在明确这两种新抗菌肽的实际可应用性;同时通过测定抗菌肽处理后病原菌活性氧(reactive oxygen species, ROS)迸发量、病原菌细胞形态观察和基因表达分析来探究抗菌肽的抑菌机制及防效。结果表明,抗菌肽SdR2332和SdR2770均具有良好的广谱抗细菌活性,且在低浓度下即可致死病原菌;它们还具有较为稳定的理化性质和较低的溶血性。经抗菌肽SdR2332和SdR2770处理后的青枯菌(Ralstonia solancearum)出现了明显的活性氧迸发现象,电镜下观察发现其细胞有一定程度的皱缩和破损,这表明抗菌肽对病原菌有杀伤作用。在qRT-PCR分析中,接种马铃薯的植株中SdR2770表达量明显上调,推测可能与植物抗病相关。本研究结果为新抗菌肽的研发提供了一定的理论基础和参考价值。     

玉米NF-YC亚基基因的鉴定与表达规律分析

0 引言 NF-Y(nuclear factor-Y,NF-Y)是一类与CCAAT基序结合的转录因子,由NF-YA、NF-YB和NF-YC 3个亚基组成[1-2].NF-YC参与并调控植物生长发育的进程以及在生物和非生物胁迫响应过程中起作用[3].AtNF-YC1通过与AtXTH21基因启动子区的CCAAT-box结合...     

哈茨木霉对黄瓜尖孢镰刀菌的抑制作用和抗性相关基因表达

采用室内筛选与田间防效相结合的方法,对哈茨木霉抑制黄瓜尖孢镰刀菌的拮抗机制进行了研究。对峙培养结果显示,木霉菌和病原菌间均形成了较明显的抑菌圈,对病原菌的抑菌率达66.7%~85.8%,其中菌株TG、TM对枯萎病菌抑制作用较强。木霉菌可有效提高根系对病原菌的抗性,黄瓜植株接种枯萎病菌后,根系细胞大量死亡,而先接种木霉菌再接种病原菌后则减小了对根系的伤害。田间防效试验结果表明,TG和TM孢子悬浮液浓度在108个/mL时防效最好,分别为54.9%和49.4%。接种木霉菌植株根系抗性基因的表达量均高于对照植株,呈双峰趋势。在第6天时抗性基因表达量最高,WRKY6、MYB、PR-1、PAL、GST和GLU的表达量分别为对照的5.15、5.22、6.07、6.00、3.16、和16.15倍。表明木霉菌通过激活与胁迫相关的基因表达提高了对病原菌的抗性。