儿茶素在棉苗对枯萎病抗性中的作用

 分析了枯萎病菌接种后棉苗组织内儿茶素含量的变化以及儿茶素对枯萎病菌及其多聚半乳糖醛酸酶活性的影响。健康棉苗组织中儿茶素含量随着棉苗的生长而增加。枯萎病菌接种后,病苗组织中儿茶素含量显著高于健康对照,而且抗病品种棉苗儿茶素含量的增加明显高于感病品种。经氟乐灵预处理的棉苗接种枯萎病菌后,儿茶素含量增加更快,增幅更大。说明儿茶素是品种抗性和氟乐灵诱发棉苗对枯萎病诱导抗性的机理之一。儿茶素可抑制枯萎病菌的菌丝生长、产孢及孢子萌发,对培养液中病菌的多聚半乳糖醛酸酶(PG)有抑制作用。儿茶素化学制品及棉苗组织提取液经TLC分离后获得的含儿茶素组分提取液对初步纯化的PG活性有明显的抑制作用。这些结果表明:儿茶素可能通过抑制病菌的菌丝生长、产孢以及其PG活性而在棉花对枯萎病的抗病性中起到作用。     

日本梨黑星病菌与欧洲梨黑星病菌多聚半乳糖醛酸酶研究

为判断日本梨黑星病菌与欧洲梨黑星病菌多聚半乳糖醛酸酶蛋白分子量是否相同,本研究对2种梨黑星病菌多聚半乳糖醛酸酶进行基因扩增、测序与比对,相似度为96%。为进一步判断两者多聚半乳糖醛酸酶的蛋白在后续加工过程中是否有所差异,进一步利用延伸法获得多聚半乳糖醛酸酶部分cDNA序列,并利用质粒pET-24a(+)对获得的cDNA序列表达、Ni-NTA柱纯化重组蛋白、用蛋白免疫小鼠等方法获得梨黑星病菌多聚半乳糖醛酸酶抗体,通过Western blotting方法进行了日本梨黑星病菌与欧洲梨黑星病菌多聚半乳糖醛酸酶分子量比对,结果证明2种梨黑星病菌的多聚半乳糖醛酸酶分子量没有区别。     

凸脐蠕孢菌玉米、高粱专化型的细胞壁降解酶活性及基因表达分析

为探明玉米专化型和高粱专化型凸脐蠕孢菌的细胞壁降解酶在致病过程中的作用,采用酶活性检测方法测定了2种专化型的细胞壁降解酶活性,并检测了相关基因的表达。结果表明:高粱专化型凸脐蠕孢菌的聚甲基半乳糖醛酸酶(PMG)活性为115.84 U/mg,略高于玉米专化型;玉米专化型的多聚半乳糖醛酸酶(PG)和纤维素酶(Cx)的活性分别为151.76 U/mg和168.53 U/mg,略高于高粱专化型;且同一种专化型菌株的细胞壁降解酶活性存在差异。2种专化型的细胞壁降解酶基因表达量存在差异,Cx基因在2种专化型互作过程中均随病程的延长而大幅度上调表达;高粱专化型的PG基因随病程的延长大幅度上调表达,而玉米专化型的PG基因随病程的延长上调表达量有所下降;高粱专化型的PMG基因随病程的延长大幅度上调表达,而玉米专化型的PMG基因随病程的延长下调表达。推测产酶能力、基因表达和基因时间表达的差异可能是引起凸脐蠕孢菌专化型致病专化性的诱因之一。     

禾谷镰孢漆酶样多铜氧化酶的鉴定及其表达模式

 子囊菌禾谷镰孢(Fusarium graminearum)可侵染玉米茎部造成严重的玉米茎腐病。漆酶样多铜氧化酶(Laccase-like multicopper oxidase)具有广泛的作用底物且在植物病原真菌中参与病菌侵染,促进病菌定殖。利用已知真菌漆酶的蛋白序列在禾谷镰孢中鉴定得到14个漆酶样多铜氧化酶,分属5种亚家族。通过对其在侵染玉米茎部不同时间后的芯片数据分析表明FGSG_02142、FGSG_05159在菌丝、孢子及侵染各阶段表达量均较高,FGSG_02328、FGSG_13185和FGSG_00142在侵染阶段表达量明显增加,其他基因表达量相对较低;试验进而利用qPCR检测了部分差异基因在接种玉米感病种质资源B73和抗病种质资源Mo17中的相对表达量,结果显示,禾谷镰孢FGSG_00142基因在B73中的表达量明显高于在抗病材料Mo17中的表达量,而毒素相关基因FGSG_02328在接种Mo17时表达也出现延迟,推测这2个基因均参与了禾谷镰孢的侵染过程。     

禾谷镰孢漆酶样多铜氧化酶的鉴定及其表达模式

 子囊菌禾谷镰孢(Fusarium graminearum)可侵染玉米茎部造成严重的玉米茎腐病。漆酶样多铜氧化酶(Laccase-like multicopper oxidase)具有广泛的作用底物且在植物病原真菌中参与病菌侵染,促进病菌定殖。利用已知真菌漆酶的蛋白序列在禾谷镰孢中鉴定得到14个漆酶样多铜氧化酶,分属5种亚家族。通过对其在侵染玉米茎部不同时间后的芯片数据分析表明FGSG_02142、FGSG_05159在菌丝、孢子及侵染各阶段表达量均较高,FGSG_02328、FGSG_13185和FGSG_00142在侵染阶段表达量明显增加,其他基因表达量相对较低;试验进而利用qPCR检测了部分差异基因在接种玉米感病种质资源B73和抗病种质资源Mo17中的相对表达量,结果显示,禾谷镰孢FGSG_00142基因在B73中的表达量明显高于在抗病材料Mo17中的表达量,而毒素相关基因FGSG_02328在接种Mo17时表达也出现延迟,推测这2个基因均参与了禾谷镰孢的侵染过程。     

甘蔗NBS-LRR类抗梢腐病基因的筛选及定量表达分析

本文旨在探讨核苷酸结合位点和富含亮氨酸重复(NBS-LRR)类基因参与甘蔗抗梢腐病菌侵染应答机制,为后续克隆关键抗病基因以及研究抗病机理提供理论依据。试验利用Illumina高通量转录组测序技术检测高抗梢腐病品种‘粤糖94-128’和高感梢腐病品种‘桂糖37号’接种前后NBS-LRR类抗梢腐病基因的表达情况,然后设计引物对显著差异表达基因进行不同接种时期下的荧光定量PCR验证。结果表明,16个NBS-LRR类基因在甘蔗叶片受到梢腐病菌侵染后持续上调表达,但表达趋势存在两种情况,13个基因在诱导后7 d显著或极显著上调表达,3个基因在诱导7 d后上调表达不显著,在诱导14 d后才显著上调表达。据此可将其分为瞬时基础防御基因(0~7 d)和滞后特异性防御基因(14~21 d),确定NBS-LRR类基因参与甘蔗防御梢腐病菌的侵染。     

菜豆普通花叶病毒RT-PCR承检测及3''末端序列分析

根据哈尔滨地区豇豆感病植株的症状,初步鉴定感染豇豆的病毒为菜豆普通花叶病毒(Bean common mosaic virus,BCMV).利用马铃薯Y病毒属通用引物扩增出病毒基因组3'末端序列,经BLAST检索表明该病毒为BCMV,将该序列与GenBank上的21个BCMV分离物的3'末端序列进行比较,显示其核苷酸序列与其他分离物的序列同源性为91.7%～97.3%.系统进化分析显示不同分离物可聚为5个类群,并显示出一定的寄主相关性.哈尔滨分离物BCMV-X与2个浙江分离物、1个澳大利亚分离物聚为一支,且该4株分离物的寄主均为豇豆.RNA二级结构分析显示BCMV基因组3'末端非编码区形成4个茎环结构,不同分离物的序列变化并未引起茎环结构的明显变化.     

3种防御酶在水稻抗稻曲病中的活性变化

不同水稻品种对稻曲病抗性存在明显差异。本研究选用12个不同抗、感病水稻品种,在抽穗前7~10 d,用注射法接种稻曲病菌分生孢子悬浮液。结果表明,在抗病品种中苯丙氨酸解氨酶（PAL）、过氧化物酶（POD）和多酚氧化酶（PPO）3种酶活性接种后迅速提高,变化幅度较大;而感病品种的酶活性在接种后缓慢增加,变化幅度小。由此说明,这3种防御酶与水稻抗稻曲病有一定的关系。     

枯萎病菌对西瓜不同抗感品种丙二醛含量及某些保护酶活性的影响

 本文以西瓜为材料,研究了西瓜苗期感染枯萎病菌(Fusarium oxysporum Schlecht.)后根部细胞内丙二醛含量、超氧化物歧化酶和过氧化氢酶活性的动态变化。结果表明,受枯萎病菌侵染的幼苗细胞内MDA含量的增加比率,抗病品种克伦生低于感病品种早花;SOD酶活性的下降比率,抗病品种克伦生低于感病品种早花;CAT酶活性增减的幅度,抗病品种克伦生低于感病品种早花。抗病品种克伦生在染病后的短时间内(48h左右)能使细胞内MDA、SOD酶和CAT酶的代谢基本恢复到正常状态,其自我调节和恢复正常状态的能力显著大于感病品种早花。     

茎基腐亚洲镰孢菌侵染抗感小麦品种的组织病理学观察

小麦茎基腐是由多种镰孢菌侵染的世界性土传病害,亚洲镰孢菌(Fusarium asiaticum)是我国冬小麦主产区茎基腐镰孢菌的优势种群,对小麦生产造成巨大损失。本研究利用绿色荧光蛋白报告基因标记亚洲镰孢菌,研究其侵染抗感小麦的病理组织学过程,建立了茎基腐病菌与寄主互作的直观性的研究体系,对病害防治及抗病育种具有重要意义。基于PEG-CaCl_2介导原生质体转化法将gfp导入亚洲镰孢菌株CF0915,对转化子进行荧光表达、PCR验证、遗传稳定性、生长特性及致病力分析,选取与野生型表现相近的转化子进行侵染分析。结果表明,绿色荧光蛋白基因(gfp)与潮霉素基因(hyg)PCR扩增表明gfp已整合入真菌基因组中,转化子菌丝与分生孢子表现强烈绿色荧光信号,gfp能够在转化子中稳定遗传,菌落形态、生长速度及致病力与野生型菌株无显著差异;将gfp标记病菌分生孢子接种感病品种1 d后,大量孢子附着于根毛及根表皮细胞开始萌发,接种2 d后观察到抗性品种分生孢子萌发;感病品种接种3 d后,菌丝直接侵入表皮细胞或沿表皮细胞间层定殖生长,扩展至皮层组织,8 d后菌丝从根部迅速扩展至茎基部,至第10 d大量菌丝充塞根皮层细胞,叶鞘维管束也被菌丝侵染,并产生大量大型分生孢子,植株表现褐色病斑,14 d后根部及茎维管束被大量菌丝体填充,而后产生大量厚垣孢子,至25 d大部分感病品种幼苗萎蔫死亡;与感病品种相比,抗性品种在整个侵染过程中表现时间滞后。本研究对引起茎基腐病的亚洲镰孢菌侵染小麦的组织学过程观察,为病菌致病机理的阐释及抗病资源的利用提供了重要理论依据。     

Polygalacturonase-inhibiting protein (PGIP) from Japanese pear: possible involvement in resistance against scab

Venturia nashicola is the causal agent of scab, a fungal disease affecting Asian pears. The Japanese pear cv. ‘Kousui’ is highly susceptible to the race 1 of this fungus whereas the cv. ‘Kinchaku’ and the non-host European pear cv. ‘Flemish Beauty’ are resistant. The aim of this work is to investigate the role of polygalacturonase-inhibiting proteins (PGIPs) of pear during the interactions with V. nashicola leading to susceptibility or resistance. PGIP protein was detected from immature fruit of Kousui and Kinchaku. It showed a molecular mass of 42 kDa that shifted to 35 kDa after chemical deglycosylation. The gene pgip was amplified by PCR using genomic DNA and/or cDNA from young leaves of Kousui, Kinchaku, and European pear cvs. Flemish Beauty, ‘Bartlett’, and an Asian wild pear strain ‘Mamenashi 12’, then sequenced after sub-cloning. Some conserved variations were identified in the sequence indicating that gene family also exists in pgip of Japanese pear and confirmed by Southern blot analysis. The expression of PGIP was studied in scab-inoculated leaves of the susceptible Kousui and the resistant Kinchaku and Flemish Beauty. pgip Gene and its encoding protein were highly and rapidly activated in these resistant plants. In addition, PGIP extracts derived from Kinchaku and Flemish Beauty partially inhibited the activity of polygalacturonase (PG) from V. nashicola suggesting a possible role of PGIP in limiting fungal growth frequently observed in these resistant cultivars.     

核基质结合区对马铃薯Y病毒全长非翻译CP基因介导抗病性的影响

 为探索核基质结合区(matrix attachment regions,MARs)对RNA介导的病毒抗性的影响,我们将从烟草中克隆到的核基质结合区TM2构建在包含马铃薯Y病毒全长非翻译CP基因的植物表达载体pRPVYCPN的表达盒的两侧,构建了植物表达载体pRTM2CPNTM2。采用农杆菌介导基因转化法,将表达载体pRPVYCPN和pRTM2CPNTM2转入烟草品种NC89中,分别获得了144株和344株转基因烟草。抗病性检测发现,核基质结合区的存在能明显提高RNA介导抗性的产生效率。在含MARs转基因植株中,抗病植株的比率为15.1%,而不含核基质结合区的转基因植株的抗病比率则为8.3%。这一研究结果对抗病毒植物的分子育种和转基因表达调控有指导意义。     

Fungal polygalacturonase activity reflects susceptibility of carnation cultivars to fusarium wilt

Carnation cultivars with different levels of partial resistance were inoculated with race 2 of Fusarium oxysporum f.sp. dianthi and monitored for accumulation of host phytoalexins, fungal escape from compartmentalization, production of fungal pectin-degrading enzymes and development of external disease symptoms. Accumulation of phytoalexins, assessed after 10 days in the first 5 cm above the inoculation site, was weakly (methoxydianthramide S) or not (hydroxydianthalexin B) correlated with resistance levels after 12 weeks. Fungal escape from compartmentalization, assessed after 3 weeks as percentages colonized plants at 8 cm above the inoculation site, was highly correlated with expression of susceptibility after 12 weeks. Polygalacturonase (PG) activity, assessed after 4 weeks in the first 5 cm above the inoculation site, was highly correlated to final disease development. Linear increases in disease severity were accompanied by quadratic increases in PG activity. In contrast to water-treated plants, that lacked any PG activity, inoculated plants contained two main groups of fungal PGs, the dominant forms of which had estimated pI values of 7.0 and minimally 9.5, respectively. Compared to those of the first group, enzymes of the second group were produced only in trace amounts in liquid media containing pectin or polygalacturonate as sole source of carbon. On these media, the fungus also produced a pectin methyl esterase (PME) with an estimated pI of 9.3. Besides PMEs of host origin, inoculated plants of susceptible cultivars contained the fungal PME while no more than traces were found in resistant ones.Assessment of phytoalexin production by the host during defense responses cannot replace monitoring of external symptoms as a resistance test. Assessment of fungal growth, whether by reisolations above the compartmentalization area or by measurement of PG activity, provides a both rapid and reliable prediction of disease development.     

Functional Characterization of Citrus Polygalacturonase-inhibiting Protein

A cDNA encoding a polygalacturonase-inhibiting protein gene (SaiPGIPA) was identified from the citrus cultivar Sainumphung (Citrus sp.), one of the most popular cultivars in northern Thailand. SaiPGIPA was expressed in Escherichia coli cells, and the functional properties of citrus PGIP were analyzed. The PGIP fusion protein inhibited by a maximum of about 60% of the endopolygalacturonase activity, and a mixture of the PGIP and fungal endopoly-galacturonase released oligogalacturonides from polygalacturonic acid. The mixture containing the oligogalactur-onides, endopolygalacturonase and PGIP induced expression of the PGIP gene and a chalcone synthase gene in citrus leaves. The mixture also induced resistance in cucumber leaves against Colletotrichum lagenarium. Received 5 September 2001/ Accepted in revised form 20 November 2001     

乙烯信号参与调控水稻纹枯病抗性的研究

 乙烯(ET)信号在植物抵抗各种逆境反应中具有重要作用,但目前关于乙烯信号在调控水稻纹枯病抗性中的作用仍缺乏系统研究。本研究以感纹枯病水稻品种Lemont和抗纹枯病水稻品种YSBR1为材料,分析了纹枯病菌接种后乙烯合成关键基因及信号传导途径中标志基因的表达水平,结果显示,纹枯病菌侵染可显著诱导相关基因的表达、激活乙烯信号。进一步采用乙烯合成抑制剂和信号激活剂处理水稻并进行接种鉴定,结果显示,两种化学剂分别可抑制或激活乙烯信号;无论是Lemont还是YSBR1,激活其乙烯信号均可显著增强抗病性,而抑制该信号均显著降低抗病性。对3个乙烯受体突变体(ethylene response2, etr2; ethylene response3, etr3; ethylene response sensor2, ers2)进行温室接种鉴定,发现突变体的病斑长度均极显著高于野生型对照。以上研究表明乙烯信号在水稻对纹枯病菌的防卫反应或基础抗性中具有十分重要的作用,结果将为进一步解析“水稻-纹枯病菌”间的互作机制、制定合适的病害综合防控策略提供理论基础。     

beta-1,3-Glucanase Activity in Peanut Seed (Arachis hypogaea) is Induced by Inoculation with Aspergillus flavus and Copurifies with a Conglutin-Like Protein

ABSTRACT Infection of peanut (Arachis hypogaea) seed by Aspergillus flavus and A. parasiticus is a serious problem that can result in aflatoxin contamination in the seed. Breeding resistant cultivars would be an effective approach to reduce aflatoxin accumulation. The objective of this study was to investigate the expression of the pathogenesis-related (PR) protein beta-1,3-glucanase and the isoform patterns in peanut seed inoculated with A. flavus. Peanut genotypes GT-YY9 and GT-YY20 (both resistant to A. flavus infection) and Georgia Green and A100 (both susceptible to A. flavus infection) were used in this study. The activities of beta-1,3-glucanase were similar in the uninfected seed of all genotypes, but increased significantly in the resistant genotypes after inoculation in comparison with the susceptible genotypes. An in-gel (native polyacrylamide gel electrophoresis [PAGE]) enzymatic activity assay of beta-1,3-glucanase revealed that there were more protein bands corresponding to beta-1,3-glucanase isoforms in the infected seed of resistant genotypes than in the infected seed of susceptible genotypes. Both acidic and basic beta-1,3-glucanase isoforms were detected in the isoelectric focusing gels. Thin-layer chromatography analysis of the hydrolytic products from the reaction mixtures of the substrate with the total protein extract or individual band of native PAGE revealed the presence of enzymatic hydrolytic oligomer products. The individual bands corresponding to the bands of beta-1,3-glucanase isoforms Glu 1 to 5 were separated on the sodium dodecyl sulfate-PAGE, resulting in two bands of 10 and 13 kDa, respectively. The sequences of fragments of the 13-kDa major protein band showed a high degree of homology to conglutin, a storage protein in peanut seed. Conglutin is reported as a peanut allergen, Ara h2. Our data provide the first evidences for peanut having beta-1,3-glucanase activities and the association with the resistance to A. flavus colonization in peanut seed. We have not directly demonstrated that conglutin has beta-1,3-glucanase activity.     

豇豆与锈菌互作中的活性氧代谢研究

 本文分析了不同抗性水平的豇豆(Vigna sesquipdalis Wight)品种与锈菌(Uromyces vignae Barcl)互作中活性氧(ac-tive oxygen species,AOS)的发生、防御酶活性及膜脂过氧化水平的变化规律及其与豇豆抗锈病性的关系。结果表明,在接种后,免疫品种(益农)和感病品种(揭上和金迪)的超氧物歧化酶(superoxide dismutase,SOD)比活性均升高,且升高幅度都在12 h出现高峰;抗病品种(金山和特青)开始时降低,至12h时回升,在24 h(高抗)和48 h(中抗)出现高峰;在24 h时,免疫和抗病品种的SOD比活性高于感病品种。在接种后12 h内,免疫和抗病品种的过氧化氢酶(catalase,CAT)比活性都下降;而感病品种均上升,并在12 h出现第1个高峰。受锈菌侵染后,豇豆各品种都产生超氧阴离子(superoxide anion,O₂^·),在多数测试时段中,免疫和抗病品种的O₂^·产率净变化值低于感病品种。丙二醛(malondialdehyde,MDA)含量变化幅度与品种抗性呈负相关,与O₂^·产率的变化趋势基本相吻合。这些结果表明AOS代谢在豇豆与锈菌互作中起重要作用。     

棉株体内几种生化物质与抗枯萎病之间关系的初步研究

 棉花抗枯萎病品种86-1、陕1155,感病品种岱15、徐州142。在棉幼苗三叶期用采自河南王屯的棉枯萎菌7号小种接种,接种后12小时,各抗病品种的葡萄糖、核糖含量显著增加,而感病品种则相反。接种后抗病品种的苯丙氨酸解氨酶(PAL)活性峰出现早于感病品种,相对酶活值也高于感病品种。接种后抗病品种蛋白质含量出现先低后高趋势,感病品种则始终保持较高水平。木质素含量的增加与棉花品种抗病性存在正相关性。伴随着PAL活性的增加,木质素含量也迅速增加。结果表明,棉花品种对枯萎病的抗性与PAL活性有密切关系,并通过代谢最终产物之一的木质素的增加得到表达。