黄萎病菌胁迫下野生茄子托鲁巴姆防卫反应的生理生化分析

以野生茄子托鲁巴姆(Solanum torvum Swartz)和苏崎茄(Solanum melongena L.)为主要材料,通过比较两种茄子在大丽轮枝菌(Verticillium dahliae Kleb)侵染过程中体内的生理生化指标变化,分析托鲁巴姆对黄萎病的抗性响应机制。结果表明:(1)与苏崎茄植株相比,托鲁巴姆植株表现出很强的自我防御和自我修复能力。(2)托鲁巴姆体内存在的活性氧清除系统(如SOD、POD、CAT等酶的活性)高于苏崎茄;在侵染后,托鲁巴姆体内各种酶的活性快速增加,其幅度高于苏崎茄;MDA的变化则恰恰相反。这个结果提示,黄萎病菌胁迫可能激活了托鲁巴姆体内活性氧清除系统,从而加快了某些防御物质(如木质素和抗菌物质绿原酸等)的形成,同时减缓或降低MDA等有害物质在植株体内的积累。(3)黄萎病菌侵染后各生理指标的响应时间表现出差异。托鲁巴姆中POD、PAL的活性和可溶性蛋白含量在侵染后的12h内就迅速作出响应(POD增加、PAL和可溶性蛋白减少);而SOD、PPO、CAT的活性和MDA的含量则在处理后初始阶段(至少12h)进行了一些调整,随后才进入持续性的增加或减少阶段。由此可见,托鲁巴姆对黄萎病病菌侵染的响应具有时序性,其体内POD酶、PAL酶和可溶性蛋白首先参与植物的防卫反应以应对黄萎病的胁迫,其它酶随后参与响应,它们的共同作用形成对黄萎病菌的有效防卫。     

云南野生茄资源黄萎病苗期人工接种抗性鉴定分析

黄萎病是目前茄子生产中的主要病害之一,广泛收集、鉴定、筛选抗性资源,尤其是从野生近缘种中发掘抗性基因并培育抗病品种,是解决茄子黄萎病危害的优选途径。云南省拥有丰富的野生茄子资源但尚未有效利用。本研究针对云南省茄子主产区收集到的3种黄萎病菌株,通过形态学鉴定和真菌18S r DNA/ITS鉴定,均属于大丽轮枝菌,并通过致病力鉴定,筛选出一株强致病力菌株(QZ-S);应用菌株QZ-S,通过苗期人工接种的方法对45份云南野生茄子资源开展黄萎病抗性鉴定,最终筛选出2份高抗材料(蒜芥茄和喀西茄)、2份抗病材料(水茄和多裂水茄)、6份中抗材料(1份刺天茄和5份红茄);此外,还筛选到了1份黄萎病高感材料239-3-2。本研究筛选出的材料可应用于茄子黄萎病抗病育种,为茄子及其他作物黄萎病抗病育种提供抗源。     

青枯菌诱导的花生基因表达谱SSH分析

以抗青枯病花生种质‘J4’和‘中花6号’、感青枯病花生品种‘中花12号’为材料,用强产青枯菌毒菌株(Ralstonia solanacearum)对其根系分别接种,采用抑制差减杂交(SSH)技术检测花生根系应答侵染的基因表达谱变化,并对文库中差异基因进行Real-time PCR分析。结果表明:经菌液PCR检测对挑选出的1 036阳性克隆片段进行测序及片段整合分析,获得162条花生基因,有功能注释的基因58条,其中44条基因参与了细胞结构(6%)、信号转导(12%)、抗病防御(5%)、转录调控(12%)等生理过程。用Real-time PCR技术对7个基因在‘中花6号’和‘中花12号’中的表达模式分析结果表明,6个基因在青枯菌侵染早期在抗病材料‘中花6号’中呈上调表达,可能与青枯病抗性直接相关。     

多菌灵胁迫下嫁接对茄子生长和抗氧化酶活性的影响

以野生茄托鲁巴姆(Solanum torvum)为砧木,以栽培品种西安绿茄(S.melongena)为接穗进行嫁接,测定不同施入频次多菌灵条件下嫁接茄子和自根茄子果实中的农药残留、生长发育状况和抗逆生理指标。结果表明,经过多菌灵施药1次、2次、3次处理后,嫁接茄果实中农药残留量比自根茄分别降低了12.7%～49.6%、11.4%～63.1%、13.5%～44.1%;嫁接茄株高、茎粗、植株鲜重和鲜果重相对于自根茄明显增加;嫁接茄根系活力比自根茄增加了6.8%～29.3%、8.7%～24.5%、11.1%～28.6%;嫁接茄叶片过氧化氢酶(CAT)活性、过氧化物酶(POD)活性、谷胱甘肽过氧化物酶(GSH-PX)活性相对于自根茄明显增加,而相对电导率比自根茄降低了16.3%～27.3%,19.7%～31.9%、20.5%～36.1%;嫁接茄丙二醛含量比自根茄降低了21.4%～31.6%、24.6%～37.9%、23.7%～39.0%。试验结果显示,施药2次和3次,各指标变化相对于施药1次明显。因此,在多菌灵胁迫下,嫁接茄子果实中的农药残留降低,植株长势明显增强,有关酶的代谢活性显著提高,从而缓解了多菌灵胁迫对茄子造成的危害。     

嫁接茄的黄萎病抗性与根际土壤生物学活性的关系

以野生茄托鲁巴姆(Solanum torvum)和刺茄(S.surattense)为砧木,西安绿茄(smelongena)为接穗,研究了嫁接茄的抗黄萎病效果及其根际土壤微生物种群数量和酶活性变化,探讨了嫁接茄的抗病性与根际土壤生物学活性的关系.结果表明:嫁接茄的抗病效果明显,其根际微生物种群数量及所占比例、根际土壤酶活性均发生了改变,且不同生育时期存在一定的动态变化.总体看,嫁接茄的抗病性增强,与其根际放线菌数量增加、放线菌数量与真菌数量的比值提高,根际土壤脱氢酶、多酚氧化酶活性提高有关.     

野生茄子(Solanum torvum)抗黄萎病相关基因StoVe1的克隆与分析

采用RT-PCR和RACE技术从野生茄子中扩增克隆到一个抗黄萎病相关基因,命名为StoVe1,其cDNA全长3 400bp,含有3 153 bp的完整开放阅读框,编码1 051个氨基酸,该基因编码的蛋白序列与刚果野茄、类番茄和番茄Ve1编码的氨基酸序列同源性分别为82%、81%和80%,且有很高的功能区段保守性.将该cDNA全长序列提交GenBank,登陆号为DQ020574.半定量PCR表明该基因为组成型表达,在根中表达最多,叶中最少.     

茄子砧木Na^＋、K^＋含量、Sk,Na运输与耐盐性关系研究

为考察不同茄子砧木在茄子耐盐性的作用,以茄子嫁接生产中常用的托鲁巴姆（Solanum torvum）、赤茄（Solanum integgrifolium）、刺茄（Solanum texanum）和刚果茄（Solanum sisymbriifl ium）为试材,研究了盐害指数、根系和地上部Na^＋、K^＋含量、Na^＋/K^＋比、SK,Na运输及其与耐盐性的关系。结果表明,各种砧木的盐害指数均随着盐浓度的增大呈上升趋势,同一盐浓度下,盐害指数由大到小依次为刚果茄〉赤茄〉刺茄〉托鲁巴姆。根系Na^＋含量、地上部K^＋含量、根系Na^＋/K^＋比及SK,Na运输在各盐浓度下均表现为托鲁巴姆〉刺茄〉赤茄〉刚果茄。地上部Na^＋含量、根中K^＋含量及地上部Na^＋/K^＋比在各盐浓度下均表现为托鲁巴姆〈刺茄〈赤茄〈刚果茄。茄子砧木耐盐性与根中Na^＋含量、根中Na^＋/K^＋比、SK,Na运输呈正相关,与地上部Na^＋含量、根中K^＋含量、地上部Na^＋/K^＋比呈负相关。这些结果表明,Na Cl处理下耐盐性强的砧木通过限制Na^＋向叶片中运输,增加了叶片中K^＋含量,从而降低Na^＋/K^＋比来提高植株耐盐性。     

野生茄子（Solanum torvum）抗黄萎病相关基因Sto Vel的克隆与分析

采用RT—PCR和RAcE技术从野生茄子中扩增克隆到一个抗黄萎病相关基因,命名为StoVel,其cDNA全长3400bp,含有3153bp的完整开放阅读框,编码1051个氨基酸,该基因编码的蛋白序列与刚果野茄、类番茄和番茄Vel编码的氨基酸序列同源性分别为82％、81％和80％,且有很高的功能区段保守性。将该cDNA全长序列提交Gen Bank,登陆号为DQ020574。半定量PCR表明该基因为组成型表达,在根中表达最多,叶中最少。     

葡萄抗白粉病相关基因γVPE的克隆与表达分析

该研究采用RT-PCR技术,从抗病的中国野生华东葡萄‘白河-35-1’和感病的欧洲葡萄‘佳丽酿’中克隆了液泡加工酶基因(γVPE),分别命名为VpγVPE和VvCγVPE。克隆的2个γVPE基因cDNA长度均为1 624bp,ORF为1 482bp,编码493个氨基酸。氨基酸多序列对比分析发现,‘白河-35-1’、‘佳丽酿’、‘无核白’和‘黑比诺’葡萄中的γVPE基因底物结合口袋域的3个关键氨基酸之一的丝氨酸(Ser395)均变为丙氨酸(Ala),与其他植物的VPE基因底物口袋结合域有所不同。实时荧光定量PCR表明,在白粉菌诱导后的不同时期内,γVPE基因在感病葡萄和抗病葡萄中的表达模式不同,抗病株系中VpγVPE基因的表达量在诱导后的前期(4h和48h)和后期(168h)均有所增加,而感病株系中VvCγVPE基因在诱导后4h表达量最高,随后降低。γVPE基因在白粉菌诱导后不同时期内表达量的变化,表明γVPE基因在一定程度上与葡萄的抗性相关。研究结果为进一步揭示γVPE基因在抗病过程中的分子机理奠定了基础。     

陆地棉GhSUMO基因的克隆与黄萎病菌诱导表达分析

SUMO化修饰与植物抗病防御、信号转导和耐旱等有着直接的关系.本文以抑制差减杂交(suppression subtractive hybridization,SSH)技术获得SUMO的EST为信息探针,对棉花EST数据库进行同源搜索和电子克隆,获得了全长为396 bp的SUMO基因编码区cDNA全长,我们将该基因命名为GhSUMO,推测该基因编码95个氨基酸.分别以抗黄萎病陆地棉品种豫棉21号的cDNA和DNA为模板,对该基因进行了PCR扩增验证,测序结果表明,GhSUMO基因序列与电子克隆序列一致,且没有内含子.蛋白序列分析表明,该蛋白具有保守泛素结构域和C端双Gly的断裂/连接位点,以及保守的疏水表面和Ulp1-Smt3互作位点.系统进化分析表明,该蛋白与蓖麻的同源序列表现了最高的相似性,与其它双子叶植物同源序列次之,而与单子叶植物的相似性较低.棉苗接菌后实时定量PCR结果显示,该基因表达量在接黄萎病菌的量48 h后明显上调,96 h达到未接菌对照的5倍以上.GhSUMO基因可受黄萎病菌诱导表达,表明该基因在陆地棉抗黄萎病的机制中可能发挥重要作用.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.     

水椰八角铁甲和椰心叶甲对椰子和银海枣的寄主选择性

【目的】水椰八角铁甲和椰心叶甲均为棕榈科植物的重要入侵害虫,两者的外部形态、取食部位和危害特征相似。研究它们的寄主选择性有助于了解这2种害虫的扩散和成灾机制。【方法】在室内用椰子和银海枣2种寄主植物分别饲养水椰八角铁甲和椰心叶甲,研究在不同寄主植物上水椰八角铁甲和椰心叶甲的存活率、产卵率、发育历期等以及这2种害虫对不同寄主植物的选择性。【结果】水椰八角铁甲在2种寄主上的存活率差异显著,除了卵期和蛹期之外,幼虫期各虫态在银海枣上的存活率明显比在椰子上的存活率高;椰心叶甲在椰子上的存活率高于银海枣,各虫态平均存活率分别为95%和86%。取食银海枣的水椰八角铁甲达标准卵量概率为0.23,取食椰子不产卵,无法完成整个世代;取食椰子的椰心叶甲达标准卵量概率为0.86,取食银海枣不产卵,也无法完成整个世代;水椰八角铁甲取食银海枣完成世代的实验种群趋势指数为12.55,椰心叶甲取食椰子完成世代的实验种群趋势指数为66.55。【结论】水椰八角铁甲和椰心叶甲分别对银海枣和椰子这2种寄主植物具有明显的选择性。在海南椰子树的数量远远超过银海枣,该实验结果在一定程度上解释了椰心叶甲在海南岛广泛分布而水椰八角铁甲只是零星发生的原因。     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。