辣椒多聚半乳糖醛酸酶基因CaPG的克隆与序列分析

以耐贮辣椒品系P98为材料,采用RACE方法,首次获得辣椒果实多聚半乳糖醛酶(PG)基因的全长cDNA,命名为CaPG,登录号为FJ596175.序列分析结果表明,该基因cDNA长1 668 bp,5′非编码区为119 bp,3′非编码区为442 bp,CDS长1 107 bp,编码368个氨基酸.Blast比对发现,该基因核苷酸序列与已报道的番茄和番木瓜PG基因具有84%和85%的相似性.聚类分析表明,该基因与番茄和番木瓜的亲缘关系较近,与拟南芥PG基因的亲缘关系较远.     

桃多聚半乳糖醛酸酶基因的cDNA克隆及序列分析

以白粉桃成熟果实为材料,用Trizol法提取桃总RNA,根据已发表的多聚半乳糖醛酸酶(PG)基因的序列设计合成一对特异引物,经 RT-PCR 扩增出一条1 188 bp的目的片段,包括一个393个氨基酸组成的开放阅读框.通过TA克隆,把它连接到pGEM-T vector上.PCR、酶切鉴定后进行基因测序,结果表明所克隆的PG基因与GenBank中肥城桃PG基因序列(AF095577)同源性为98.65%,相应的氨基酸的同源性为97.46%,说明此片段为桃PG基因片段.     

果实多聚半乳糖醛酸酶分子生物学研究进展

多聚半乳糖醛酸酶（PG酶）是一种在植物细胞壁降解中起重要的作用的酶。作者介绍了PG酶在果实成熟软化中的作用。概述了PG酶基因及其表达调控,评述了乙烯对PG酶合成的影响。     

植物多聚半乳糖醛酸酶功能研究进展

综述了近年来多聚半乳糖醛酸酶 (PG)在果实软化、器官脱落、花粉授粉、种子发育、胁迫反应等中作用的研究进展 ,结合实验结果讨论了PG与乙烯的关系 ,以及PG在植物病害发生和逆境反应中的作用。分析了PG的多基因家族所表现出来的功能多样性     

真菌多聚半乳糖醛酸酶研究进展

真菌多聚半乳糖醛酸酶是降解植物细胞壁果胶的主要降解酶之一,是植物病原真菌的致病因子之一。文中对真菌多聚半乳糖醛酸酶及其序列特征、多聚半乳糖醛酸酶的基因及其序列特征、多聚半乳糖醛酸酶的表达调控以及与病原真菌致病力之间的关系等方面进行了综述。     

胡萝卜抗冻蛋白失去PGIP家族抑制多聚半乳糖醛酸酶的活性

含有LRR基序的胡萝卜抗冻蛋白虽然具有抗冻活性,但却属于植物PGIP家族。胡萝卜抗冻蛋白虽然在氨基酸序列上属于PGIP家族,但却失去了抑制外源真菌的PGase活性,并且获得了一个重要的活性——抑制冰晶的生长和重结晶。胡萝卜抗冻蛋白的这种活性的变化一直被认为是由于植物自身长期进化的结果,并认为最初的DcAFP也应当具有抑制PGase的活性。采用酵母双杂交来分析DcAFP是否还拥有PGIP的活性。通过RT-PCR克隆了真菌互格链格孢（Alternaria alternata）的PGase的cDNA,然后分别将PGase与DcAFP的完整编码框构建成酵母双杂交的捕获质粒和诱饵质粒,经过预实验表明两者都不能产生自激活作用,酵母双杂交实验表明两者不能产生相互作用,说明DcAFP完全失去了抑制PGase的活性,这种活性的丢失是由于位于6-螺旋上凹面的LRR基序中非保守的氨基酸残基发生了大量的碱性氨基酸的取代,导致结合的凹面从负电荷富集区变成了正电荷表面,从而不能通过静电作用与PGase的正电荷表面相结合。     

加工番茄多聚半乳糖醛酸酶（PG）基因的cDNA克隆的全序列测定

以加工番茄“87－5”为材料,采用反转录PCR（RT－PCR）技术将加工番茄G基因的cDNA序列克隆到pGEM-T载体上,并进行了全序列测定分析,结果表明,加工番茄的PG基因的cDNA与国内外报道的番茄PG基因的cDNA,在碱基序列及氨基酸序列上均有差异,说明番茄的PG基因具有多态性。     

多种抑制剂对灰葡萄孢菌多聚半乳糖醛酸酶和漆酶活性的影响

目的:研究EDTA、CaCl2、红酵母酸和肠菌素对Botrytis cinerea多聚半乳糖醛酸酶(PG)和漆酶(LC)的抑制作用。方法:分离得到2株具有致病性、能抵抗杀菌剂以及分泌PG和LC的灰葡萄孢菌。观察四种抑制剂对B.Cinerea感染苹果的防治效果。将菌种活化后分别给予四种抑制剂(EDTA、CaCl2、红酵母酸和肠菌素)处理7d后,测定不同组别菌体干重和总蛋白含量。利用酶动力学的方法比较四种抑制剂EDTA、CaCl2、红酵母酸和肠菌素对两株灰葡萄孢菌中PG和LC活性的影响。结果:EDTA和红酵母酸能显著提高健康苹果对B.Cinerea感染的抵抗能力,而氯化钙和肠菌素则对已感染了B.Cinerea的苹果感染面积的扩大有较好的控制作用。肠菌素对两菌株LC酶活性的抑制作用最强、抑制率达70-80%,氯化钙对PG活性的抑制作用最强、抑制率达45%。结论:EDTA和红酵母酸可预防B.Cinerea的感染,肠菌素和氯化钙对B.Cinerea的感染有治疗作用。其防治作用可能与抑制B.Cinerea中PG和LC的活性有关。     

立枯丝核菌对高粱幼苗活性氧及抗氧化酶活性的影响北大核心CSCD

以立枯丝核菌AG1-IA接种‘龙杂19号’高粱幼苗,通过盆栽试验在不同侵染时间观测分析高粱幼苗生长及生理代谢相关指标,揭示立枯丝核菌侵染对高粱生长、渗透调节物质及其抗氧化酶活性的影响机理。结果表明,(1)高粱幼苗株高、根长、地上(茎和叶片)鲜质量和干质量、地下(根)鲜质量和干质量均随接种时间延长呈下降趋势,且在接菌72 h时较对照依次分别显著下降了41.0%、29.2%、50.0%、50.0%、53.3%和50.0%。(2)幼苗叶片叶绿素a含量在接菌72 h时较对照显著下降45.3%,叶片PSⅡ最大光化学效率(Fv/Fm)值随接菌时间增加而逐渐下降。(3)幼苗叶片内MDA、O^(-·)_(2)和H_(2)O_(2)含量随接菌时间增加均呈逐渐上升趋势,在接菌72 h时较对照分别显著上升244.6%、140.4%和137.0%;叶片SOD、POD、APX和CAT的活性在接菌后变化趋势不尽相同,但在接菌72 h时较对照分别显著上升了16.5%、60.3%、50.0%和36.5%。(4)幼苗叶片可溶性蛋白和可溶性糖含量随接菌时间增加均呈逐渐上升趋势,并在接菌24~72 h增幅均达到显著水平。研究发现,接种立枯丝核菌可致高粱幼苗植株产生病斑,体内活性氧过量积累,膜质发生显著氧化损伤;侵染前期高粱植株主要以积累更多的渗透调节物质来抵御立枯丝核菌带来的伤害,侵染后期随植株受到伤害加重,同时诱导植株抗氧化酶类活性显著增强,以维持高粱体内活性氧代谢稳态平衡,减少植物膜进一步氧化损伤。     

Control of blast and sheath blight diseases of rice using antifungal metabolites produced by Streptomyces sp. PM5

Two antifungal aliphatic compounds, SPM5C-1 and SPM5C-2 with a lactone and ketone carbonyl unit, respectively obtained from Streptomyces sp. PM5 were evaluated under in vitro and in vivo conditions against major rice pathogens, Pyricularia oryzae and Rhizoctonia solani. These compounds were dissolved in distilled water/medium to get the required concentrations. The well diffusion bioassay indicated that the of SPM5C-1 remarkably inhibited the mycelial growth of P. oryzae and R. solani in comparison to SPM5C-2. Though SPM5C-2 showed low antifungal activity against P. oryzae, it was not active against R. solani. Further, SPM5C-1 completely inhibited the growth of P. oryzae and R. solani at concentrations of 25, 50, 75 and 100 μg/ml. Greenhouse experiments revealed that spraying of SPM5C-1 at 500 μg/ml on rice significantly decreased blast and sheath blight development by 76.1% and 82.3%, respectively, as compared to the control with a corresponding increase in rice grain yield.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)^– RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)^– RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.     

AsSlt2基因对茄链格孢细胞壁完整性的调控

【背景】由茄链格孢(Alternaria solani)引起的马铃薯早疫病被普遍认为是马铃薯生产上的第二大叶部病害,在马铃薯各产区普遍发生,给马铃薯生产造成了巨大的经济损失。【目的】明确AsSlt2基因对茄链格孢细胞壁完整性的影响。【方法】在含有刚果红、细胞壁降解酶和十二烷基硫酸钠(sodiumdodecylsulfate,SDS)等细胞壁胁迫的培养基上观察ΔAsSlt2缺失突变株的生长情况,计算相对生长抑制率;通过实时荧光定量PCR (RT-qPCR)方法检测ΔAsSlt2菌株中细胞壁合成相关基因的表达情况;进一步检测ΔAsSlt2细胞壁中几丁质的含量及胞外酶活性。【结果】ΔAsSlt2缺失突变株对SDS、刚果红、细胞壁降解酶等细胞壁胁迫的敏感性增强,在加入细胞壁降解酶后突变株原生质体释放量显著增多;ΔAsSlt2对外源氧胁迫更敏感,突变株胞外过氧化物酶和漆酶活性均显著降低;进一步研究发现,ΔAsSlt2细胞壁中几丁质含量减少,几丁质合成相关基因与漆酶合成相关基因的表达量均明显降低。【结论】AsSlt2基因在茄链格孢细胞壁的完整性及抵御外界胁迫方面发挥重要作用。     

Linear plasmid DNAs of the plant pathogenic fungus Rhizoctonia solani with unique terminal structures

Summary Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3-exonuclease and 5-exonuclease even after treatment with proteinase K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of the plasmids is discussed.     

水稻OsMBF1c基因的克隆及表达分析

多蛋白桥联因子1(multi protein bridging factor 1, MBF1)在植物应对逆境胁迫中起着重要的作用,而对于水稻中MBF1是否参与重金属胁迫响应机制目前尚未见相关报道。为了揭示水稻MBF1家族与重金属胁迫的相关性及其潜在作用机制,该研究利用PCR技术克隆水稻OsMBF1c基因的全长编码序列,通过生物信息学对基因功能进行分析和预测,并通过实时荧光定量PCR(RT-qPCR)分析其在镉(Cd)胁迫下的表达特征。结果表明:(1)OsMBF1c的全长编码序列为468 bp,共编码155个氨基酸,相对分子量为16.154 kDa。(2)OsMBF1c与大麦TdMBF1a.1亲缘关系最近,具有光、厌氧等环境因子诱导相关的顺式调节元件。(3)重金属Cd可诱导OsMBF1c表达且在时间上和组织中的表达水平具有特异性,100 μmol·L^-1 Cd 处理1 h 后,地上部分OsMBF1c表达量明显上调,为对照组的7倍; 100 μmol·L^-1 Cd 胁迫处理6 h后,根部OsMBF1c表达量上调为对照组的3倍。该研究结果进一步完善了非生物胁迫下MBF1家族的生物学功能研究。     

夏枯草PvDXS基因的克隆和表达分析

该研究在夏枯草转录组测序的基础上设计特异引物,采用逆转录PCR技术获得该基因的全长核苷酸序列,并进行生物信息学分析,采用qRT-PCR法分析PvDXS在夏枯草不同组织及不同外源性物质诱导下的表达量。结果表明:克隆得到的PvDXS基因开放阅读框2 181 bp,编码726个氨基酸,理论分子量为78 040.47 D,等电点为6.75,PvDXS蛋白具有Transketolase_C结构域和Transket_pyr结构域,系统进化树结果表明,PvDXS蛋白与丹参、长春花的DXS(SmDXS2、CrDXS2)亲缘关系较近,推测PvDXS属于第Ⅱ类DXS蛋白。qRT-PCR分析表明,PvDXS基因在叶中表达量高于果穗及茎。对果穗施加7种外源性物质处理24 h后,GA₃处理组该基因表达量升高,其他6种外源性物质处理后表达量均降低,其中CaCl₂、SNP、SA处理后该基因的表达量显著降低。PvDXS基因在不同组织中表达量差异较大,且受外源物质诱导表达。这为进一步研究PvDXS基因对夏枯草萜类成分合成途径中的功能及表达调控奠定基础。     

Functional validation of pathogenicity genes in rice sheath blight pathogen Rhizoctonia solani by a novel host-induced gene silencing system

Rice sheath blight, caused by the soilborne fungus Rhizoctonia solani, causes severe yield losses worldwide. Elucidation of the pathogenic mechanism of R. solani is highly desired. However, the lack of a stable genetic transformation system has made it challenging to examine genes' functions in this fungus. Here, we present functional validation of pathogenicity genes in the rice sheath blight pathogen R. solani by a newly established tobacco rattle virus (TRV)–host-induced gene silencing (HIGS) system using the virulent R. solani AG-1 IA strain GD-118. RNA interference constructs of 33 candidate pathogenicity genes were infiltrated into Nicotiana benthamiana leaves with the TRV-HIGS system. Of these constructs, 29 resulted in a significant reduction in necrosis caused by GD-118 infection. For further validation of one of the positive genes, trehalose-6-phosphate phosphatase (Rstps2), stable rice transformants harbouring the double-stranded RNA (dsRNA) construct for Rstps2 were created. The transformants exhibited reduced gene expression of Rstps2, virulence, and trehalose accumulation in GD-118. We showed that the dsRNA for Rstps2 was taken up by GD-118 mycelia and sclerotial differentiation of GD-118 was inhibited. These findings offer gene identification opportunities for the rice sheath blight pathogen and a theoretical basis for controlling this disease by spray-induced gene silencing.