马铃薯萜类合酶基因StHcS的功能鉴定

为了研究马铃薯萜类代谢物的生物合成机制,从马铃薯基因组数据中筛选到一个萜类合酶基因。通过RT-PCR方法,从致病疫霉侵染后的马铃薯品种‘费乌瑞它’中成功克隆到该基因,命名为StHcS,并对其进行生物信息学分析、生化功能鉴定及表达模式分析。结果表明:(1)序列分析显示StHcS编码区序列长1 497 bp,编码498个氨基酸,分子质量为74.78 kD。(2)StHcS基因编码的蛋白序列含有DDXXD催化功能域,与短柱茶(Camellia maliflora)中的四甲基环癸二烯甲醇合酶相似度最高。(3)蛋白体外催化实验和大肠杆菌代谢工程分析表明,StHcS可以催化生成倍半萜化合物四甲基环癸二烯甲醇(hedycaryol)。(4)基因表达分析显示,StHcS可以被致病疫霉侵染诱导表达反式法尼烯焦磷酸(FPP),且在侵染后72 h表达最高;GC-MS分析显示,在受侵染的马铃薯块茎中检测到四甲基环癸二烯甲醇。StHcS生化功能的鉴定为倍半萜合酶的研究提供了多样性,也是首次在马铃薯中发现的四甲基环癸二烯甲醇合酶,为马铃薯萜类代谢途径解析提供了参考。     

辣椒疫霉RXLR型效应子PcAvh2的序列多态性、基因转录特征及功能

【目的】分析辣椒疫霉中RXLR型效应子PcAvh2的序列多态性,研究该效应子在辣椒疫霉生长发育和侵染阶段的转录特征及其生物学功能。【方法】本研究通过高保真扩增,分析2个烟草疫霉、1个恶疫霉和31个辣椒疫霉菌株的PcAvh2序列;提取辣椒疫霉菌丝、游动孢子囊、游动孢子、萌发休止孢和7个侵染时间点(1.5、3、6、12、24、36、72 h)的本氏烟根部总RNA,利用RT-qPCR分析PcAvh2的转录表达水平;利用PVX瞬时表达系统,分析PcAvh2是否抑制6种效应子(BAX、INF1、PsojNIP、PsCRN63、PsAvh241、R3a/Avr3a)激发的植物免疫反应;利用CaCl_2-PEG介导的原生质体稳定转化技术,沉默PcAvh2基因,分析辣椒疫霉致病力的变化。【结果】PcAvh2为典型的RXLR效应子,在辣椒疫霉群体中该效应子具有10个等位基因,而且烟草疫霉和恶疫霉中也存在该效应子。该基因在辣椒疫霉的侵染阶段上调表达,它能够抑制6种效应子激发的植物免疫反应,进一步研究发现基因沉默导致辣椒疫霉的致病力显著下降。【结论】RXLR型效应子PcAvh2是辣椒疫霉中一个重要的侵染致病因子。     

致病疫霉PiSAK1的生物学功能分析

【背景】致病疫霉是引起世界范围内马铃薯晚疫病的重要病原菌。Stress-activated protein kinases (SAPKs)是一类胁迫激活的mitogen-activated protein kinases (MAPKs)，研究表明真菌SAPKs在调控细胞应答外界胁迫等方面有重要作用。致病疫霉中存在一个SAPK，即PiSAK1，其生物学功能并不明确。【目的】探究PiSAK1在致病疫霉生长发育、抵抗外界胁迫及侵染马铃薯过程中发挥的生物学功能。【方法】利用生物信息学手段分析PiSAK1的特性，通过RT-qPCR分析明确致病疫霉PiSAK1在不同发育阶段及侵染马铃薯不同时期的表达量，最后构建PiSAK1沉默、过表达菌株并测定其各项生物学表型。【结果】PiSAK1具有丝裂原活化蛋白激酶典型的Ser/Thr蛋白激酶催化结构域，并且与其他卵菌的SAPKs同属一个进化分支。致病疫霉PiSAK1分别在休止孢阶段、侵染马铃薯48 h时表达量最高，且0.3 mol/L NaCl及3 mmol/L H2O2胁迫刺激0.5 h后PiSAK1的表达量均显著升高。构建PiSAK1沉默、过表达菌株并测...     

对增强辣椒疫霉菌抗性的研究

病原物诱导型启动子能精确控制抗病基因在侵染位点的表达,是抗病基因工程的有效工具。prp1-1是来自马铃薯谷胱甘肽巯基转移酶基因启动子的一个273bp的片段,能够快速准确地启动被侵染位点抗病基因的表达;Rs-AFP2是具有对致病性丝状真菌的广谱抗性。该研究构建prp1-1调控Rs-AFP2基因表达的载体,经农杆菌介导转化法导入辣椒。逆转录PCR检测发现,转基因辣椒只在受到疫霉菌孢子侵染时,才由prp1-1启动Rs-AFP2基因的转录。用疫霉菌孢子灌根接种转基因辣椒T1代植株,35株T1代辣椒中有29株表现出明显的疫霉菌抗性。另将23株T1代辣椒种于人工气候箱,发现其形态和发育特征与相同条件下的非转基因植株无明显区别。研究表明,prp1-1调控Rs-AFP2的诱导表达达到了增强辣椒疫霉菌抗性的目的,而且避免了负面效应的发生。     

中国部分地区马铃薯寄主上致病疫霉SSR基因型分析

利用两对SSR引物对两个基因座Pi4B和Pi4G进行了PCR扩增,测定了中国部分地区66个致病疫霉Phyophthora infestans(马铃薯晚疫病菌)菌株和2个参考菌株的SSR基因型,并对菌株的基因型进行了鉴定和命名.在被测定的66个致病疫霉菌株中,共产生了7种SSR基因型D-03,D-05,D-06,G-02,H-01,F-01和F-06,其中F-06为本研究新命名的基因型.F-01基因型菌株53个,占总菌株数目的80.3%,该基因型为中国致病疫霉的优势基因型.在对两个基因座Pi4B和Pi4G产生的等位基因统计分析发现基因座Pi4B产生的多样性比Pi4G高.对SSR数据揭示的河北、黑龙江和云南3个不同省份致病疫霉遗传多样性的比较发现,河北省和黑龙江省致病疫霉遗传多样性几乎相同,然而与云南省致病疫霉有较大的遗传差异.此外,发现致病疫霉SSR基因型与其对甲霜灵抗性无相关性.     

一个马铃薯种质资源圃致病疫霉群体的分析

由致病疫霉Phytophthora infestans引起的晚疫病是马铃薯生产上最严重的病害之一,认识其群体结构特征,可为晚疫病防控策略的制定以及抗病品种的合理布局提供指导。对2009年采自宁夏一个种植有93个品种(品系)的马铃薯种质资源圃的致病疫霉进行了交配型、致病型和线粒体DNA单倍型分析,结果表明,116个致病疫霉菌株中存在A1、A2和自育型3种交配型,发生频率分别为24.1%、57.8%和18.1%,A2交配型为优势类型;对其中43个菌株的致病型进行测试,检测到两种致病类型:1.2.3.4.5.6.7.8.9.10.11和3.4.10,发生频率分别为95.3%和4.7%,可克服所有11个抗病基因的1.2.3.4.5.6.7.8.9.10.11类型占绝对优势;对62个菌株的线粒体DNA单倍型进行分析,检测到Ia和IIa两种类型,发生频率分别为74.2%和25.8%。综合表型和基因型数据分析发现,该马铃薯种质资源圃中致病疫霉群体致病型单一,但致病型毒力因子高度复合;线粒体DNA分析表明,该马铃薯种质资源圃引入了遗传背景较为复杂的致病疫霉"新"群体。     

马铃薯致病疫霉研究进展

马铃薯致病疫霉(Phytophthora infestans)属卵菌纲(Oomycetes)霜霉目(Peronosporales)腐霉科(Pythiaceae)疫霉属(Phytophthora),是马铃薯和番茄晚疫病病原菌。由于晚疫病对马铃薯生产的毁灭性和严重性,对致病疫霉的研究一直是关注的重点。本文首先对病害引起的症状、发生特点及流行规律进行阐述,对有性生殖发生的遗传规律和多种交配型共存的大环境下病原菌群体结构变异特点进行归纳总结。随着2009年致病疫霉基因组测序的完成,本文比对了疫霉属目前已完成测序各个种的基因组学特点,介绍了致病疫霉在效应子克隆方面的研究进展及线粒体基因组研究现状,阐述了功能基因组学的两个重要技术:高密度遗传连锁图谱(high density linkage mapping)和全基因组关联分析(genome-wide association study,GWAS),及其在挖掘致病疫霉重要功能基因上的应用。本文有助于了解致病疫霉研究热点及后续突破方向,可为深入解析致病疫霉的功能基因及致病机制提供参考,对开发马铃薯晚疫病菌药物靶标及预测病害的大规模流行趋势也具有重要意义。     

马铃薯StNPR4基因的克隆与功能分析

为探究StNPR4基因在马铃薯（Solanum tuberosum）中应对生物胁迫和非生物胁迫的功能,本研究通过克隆StNPR4的CDS序列和启动子序列,进行生物信息学分析;利用qRT-PCR进行组织表达特异性分析;同时构建了由其自身启动子驱动的StNPR4双元表达载体,转化马铃薯获得了转基因马铃薯,研究转基因马铃薯对水杨酸、致病疫霉和高盐胁迫的响应。结果显示：StNPR4具有典型的NPR1家族的功能结构域,启动子上具有响应于生物胁迫和非生物胁迫的顺式作用元件。StNPR4在叶中的表达量最高;StNPR4受SA诱导表达,且在转基因植株中的诱导表达程度高于对照;转基因马铃薯增强了对致病疫霉的抗性,在高盐胁迫下生根率更高。说明StNPR4不仅在马铃薯生物胁迫中发挥重要作用,而且在非生物胁迫中也扮演着重要角色。     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交(suppression subtractive hybridization,SSH)的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。     

致病疫霉RXLR效应蛋白相关研究进展

由致病疫霉(Phytophthora infestans(Mont.)de Bary)引起的晚疫病是马铃薯生产中最具毁灭性的病害。为了成功入侵和在寄主植物中繁衍,致病疫霉会向寄主细胞分泌一类RXLR效应蛋白以干扰植物免疫系统。自2005年克隆第一个晚疫病菌RXLR类无毒基因AVR3a以来,国内外学者从RXLR效应蛋白的结构、功能,以及与寄主靶标作用机理等多个方面展开了大量研究。随着高通量测序技术与效应子组学技术的发展,RXLR效应蛋白抑制植物免疫分子机制也取得了显著进展。RXLR效应蛋白的研究有助于揭示致病疫霉与马铃薯互作分子机制,并进一步为马铃薯抗病育种工作提供新思路。主要概述了致病疫霉RXLR效应蛋白的相关研究进展,重点介绍了致病疫霉AVR基因的克隆、定位、变异及功能等方面的最新进展,同时对未来值得关注的研究方向进行了探讨。     

Comparative analysis of Phytophthora infestans induced gene expression in potato cultivars with different levels of resistance

Differential gene expression was analyzed after infection with Phytophthora infestans in six potato cultivars with different levels of resistance to late blight. To verify the infection of the potato leaflets, the amount of phytopathogen mRNA within the plant material was quantified by real-time quantitative PCR. The expression of 182 genes selected from two subtracted cDNA libraries was studied with cDNA array hybridization using RNA from non-infected and infected potato leaflets. Gene up- and down-regulation were clearly detectable in all cultivars 72 h post inoculation. Gene expression patterns in susceptible cultivars differed from those in potato varieties with a higher level of resistance. In general, a stronger gene induction was observed in the susceptible cultivars compared to the moderately to highly resistant potato varieties. Five genes with the highest homology to stress and/or defence-related genes were induced specifically in the susceptible cultivars. Four genes responded to pathogen attack independently of the level of resistance of the cultivar used, and three genes were repressed in infected tissue of most cultivars. Even in the absence of P. infestans infection, six genes showed higher expression levels in the somewhat resistant cultivars Bettina and Matilda. Possible reasons for the different levels of gene expression are discussed.     

Purification of an isoform of patatin with antimicrobial activity against Phytophthora infestans.

Phytophthora infestans (Mont.) de Bary is infamous as the causal agent of the late blight epidemic contributing to the Irish potato famine of the mid 19th century and remains agriculture's most destructive disease as new mutations and migrations confound control measures. In efforts to develop resistant varieties, a somatic hybrid (the Wisconsin J series) between potato (Solanum tuberosum) and a wild relative (Solanum bulbocastanum) has been found to convey durable resistance against the pathogen. We screened the total protein (100 microg ml(-1)) of somatic hybrid varieties J138, J138A12, J101K12, J103K12, and J101K9 for in vitro spore germination inhibition of P. infestans. Since J138 exhibited maximum inhibition at 150 microg ml(-1) in comparison to other varieties, we purified a 40 kD protein from J138 tubers by assaying its ability to inhibit spore germination in P. infestans spores. The highly purified protein was able to inhibit P. infestans spore germination by 70% at the 2.5 microg ml(-1) concentration. The N-terminal sequence of this protein was found to have exact amino acid homology to patatin, the major storage protein of potato tubers. The inhibitory protein has the same molecular weight as patatin and cross-reacts with patatin antibodies. The infection of J138 plants with spores of P. infestans under greenhouse conditions showed that patatin is expressed in stem tissue 72 h after the plant is inoculated with field isolates of P. infestans (US8). In this communication, we report the purification, characterization and antifungal activity against spores of P. infestans of patatin-J from potato tubers.     

Race nonspecific resistance for potato late blight

The late blight fungus (Phytophthora infestans) rots susceptible species of potato plants. None of the major varieties of potato (Solanum tuberosum) grown in the USA is resistant to US-8, the most prevalent genotype of the fungus. Now, Junqi Song, James Bradeen and colleagues have cloned the RB gene from the wild diploid potato species, Solanum bulbocastanum, using a map-based approach in combination with long-range PCR. Transgenic plants containing the gene, normally fully susceptible, displayed broad-spectrum late blight resistance.     

Induction of a proteinase K inhibitor in a potato cultivar with a high degree of field resistance against Phytophthora infestans

A proteinase K inhibitor (PLPKI) was isolated from a potato cultivar with a high level of field resistance ( Solanum tuberosum L. cv. Pampeana INTA), after 24 h of infection with Phytophthora infestans , when inhibitory activity was markedly increased. Purification was performed by heat treatment, gel filtration chromatography and affinity chromatography. A size of 60 kDa was estimated by SDS-PAGE in partially denaturing conditions and by gel filtration. It is multimeric and the monomer has a molecular mass of 8.5–9.0 kDa. PLPKI is highly active against proteinase K (EC 3.4.21.14) but poorly inhibits two serine proteinases of animal origin, trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). A differential expression (determined by activity and immunoblotting assays) of PLPKI was observed between two potato cultivars with different degrees of field resistance to P. infestans . In the resistant cultivar (cv. Pampeana INTA) PLPKI induction (19-fold with respect to healthy leaves) occurred 24 h after infection and remained over basal levels after 48 h infection. By contrast, in the susceptible cultivar (cv. Bintje), no induction was observed.     

Inhibition of fungal and bacterial plant pathogens by synthetic peptides: in vitro growth inhibition, interaction between peptides and inhibition of disease progression

Four synthetic cationic peptides, pep6, pep7, pep11 and pep20, were tested alone and in combinations for their antimicrobial activities against economically important plant pathogenic fungi (Phytophthora infestans and Alternaria solani) and bacteria (Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica). In in vitro studies, P. infestans and A. solani were inhibited by all four peptides, while E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica were inhibited only by pep11 and pep20. All peptides completely inhibited P. infestans and A. solani on potato leaves and P. infestans on tubers at concentrations comparable to the in vitro IC50 (effective concentration for 50% growth inhibition) values, suggesting that these peptides are more potent in preventing infection than in inhibiting hyphal growth in vitro. Microscopic observations of P. infestans and A. solani when treated with these peptides revealed hyphal anomalies. In tuber-infectivity assays, pep11 and pep20 reduced bacterial softrot symptoms by 50% at 2.0 to 2.30 microM and by 100% at 20 microM. In assays involving two-way combinations of these peptides, growth inhibitions of fungi and bacteria by the combinations were no more than the sum of growth inhibitions by each peptide when used alone, indicating that they act additively. pep11 and pep20 are not phytotoxic to potato plants at 200 microM. With strong and broad-spectrum antimicrobial activities of pep11 and pep20 against fungi and bacteria, and with no antagonistic activities, the expression of these peptides in transgenic potato plants could lead to enhanced disease resistance against these pathogens.     

A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potato

Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans-membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans.