大丽轮枝菌(Verticillium dahliae VdLs.17)分泌组预测及分析

【目的】预测并分析大丽轮枝菌基因组范围内的分泌蛋白,为大丽轮枝菌分泌蛋白致病机理的研究奠定基础。【方法】利用已公布的大丽轮枝菌全基因组序列,组合使用生物信息学软件SignalP、TargetP、TMHMM、Big-pi和PROSITE,预测大丽轮枝菌基因组范围内所有分泌蛋白,定义为分泌组。统计分析分泌组中蛋白N-端信号肽特点;应用碳水化合物活性酶类数据库和病原菌-寄主互作蛋白数据库对分泌组蛋白进行注释,预测分泌组中潜在果胶酶、纤维素酶和病原菌寄主互作蛋白;利用真菌激发子的保守结构域,预测分泌组中潜在的激发子蛋白集;应用BLASTP程序比较分析大丽轮枝菌和黑白轮枝菌分泌组,获得大丽轮枝菌相对于黑白轮枝菌特异的分泌蛋白。【结果】大丽轮枝菌分泌组共有922个蛋白。信号肽分析表明,以19个氨基酸为信号肽的蛋白数目最多,非极性氨基酸丙氨酸的出现频率最高,而有带电侧链的氨基酸天冬氨酸和谷氨酸的出现频率最低,信号肽的-3和-1位置上的氨基酸相对保守。大丽轮枝菌分泌组含有158个潜在的碳水化合物活性酶类,其中,包括10个果胶水解酶和14个果胶裂解酶;190个潜在的病原菌-寄主互作蛋白、97个含有RxLx[EDQ]模体的蛋白和52个富含半胱氨酸的小分子量分泌蛋白;58个相对于黑白轮枝菌分泌组特异的蛋白。【结论】本文建立了预测大丽轮枝菌分泌组蛋白的方法。分泌组蛋白信号肽长度具有高度的变异性,氨基酸组成多为脂肪族氨基酸,序列在C-端结构域较为保守。分泌组中包含大量潜在的果胶降解酶、病原菌-寄主互作蛋白、RxLx[EDQ]模体蛋白和富含半胱氨酸的小分子量蛋白等致病相关蛋白。

黄萎病菌诱导下陆地棉酵母双杂交文库的构建及GhMAPKKK2互作蛋白的筛选

为了探究棉花响应黄萎病菌侵染的分子机制并筛选GhMAPKKK2的互作蛋白,以陆地棉XLZ35为材料,基于Gateway克隆技术构建了棉花黄萎病菌诱导的陆地棉酵母双杂交文库,其中初级文库和次级文库的库容分别为1.2×10^(7)和2.0×10^(7)CFU,插入片段重组率均为100%且平均长度大于1000 bp。次级文库质粒浓度为953 ng·μL^(-1),符合构建酵母文库的标准,可用于酵母双杂交筛选互作蛋白。构建了pGBKT7-GhMAPKKK2表达载体,并验证了该载体对酵母细胞无毒性且不存在自激活活性。以GhMAPKKK2为诱饵蛋白,利用酵母文库初步筛选,获得81个阳性互作蛋白,经测序对比选取6个候选蛋白,通过酵母双杂交及双分子荧光互补试验(BiFC)验证,明确了候选蛋白GhFLA与GhMAPKKK2存在互作关系。本研究结果为解析GhMAPKKK2调控棉花抗黄萎病反应分子机制及调控网络提供了重要参考。

棉花大丽轮枝菌(Verticillium dahliae)血清学检测——抗原处理的比较

分别以棉花黄萎病菌V20的菌丝研磨物、菌丝蛋白、超氧化物歧化酶酶带Ⅰ、超氧化物歧化酶酶带Ⅱ为抗原免疫家兔,制备了抗血清As1、As2、As3、As4。琼脂双扩散法(ADD)和间接ELISA方法测定其特异性,发现这4种抗血清既可与同种的抗原发生阳性反应,也可与不同种的抗原发生交叉反应。用异种抗原黑白轮枝菌和尖孢镰刀菌吸附这4种抗血清,排除其非特异性成分后,抗血清专化性明显提高,消除了交叉反应。特异性测定表明:经吸附后的4种抗血清可以准确地将大丽轮枝菌鉴定到种的水平,但仍不能区分其生理型和致病类型。另外,当分别使用菌丝研磨物或菌丝蛋白为特异性测定抗原时,ADD检测的结果虽然一致,但前者阳性反应沉淀线粗糙,轮廓不清晰,后者的沉淀线较细,且清晰、明显。因此适宜选择菌丝蛋白为抗原进行抗血清的特异性测定试验。

有关棉花黄萎病（Verticillium dahliae Kleb．）抗性遗传研究的几个问题

棉花黄萎病抗性遗传问题迄今尚未获得肯定的结论。据作者等估计，这可能和寄主（棉株）和寄生物（黄萎病镰刀菌）之间的关系、黄萎病菌不同菌系间的互作以及影响抗性鉴定的正确性等问题有关。为此，必须创设有利条件，使寄主和病原菌间的关系得以充分表现；必须了解病原菌菌系间的互作，寄主必须有较大的群体。这一切都是为了寄主和病原物问的关系得以充分体现，为抗性遗传研究奠定基础。抗性遗传研究宜用单菌系接种，并且宜按数量性状和质量性状遗传方式同时进行研究和分析，以便得到比较确切可靠的结果。

棉花黄萎病真菌Verticillium dahliae木聚糖酶基因的克隆、表达和酶学性质分析

【目的】从棉花黄萎病真菌Verticillium dahliae中克隆木聚糖酶基因,并在毕赤酵母中进行异源表达,研究酶学性质。【方法】通过多序列比对设计简并引物,扩增出真菌V.dahliae木聚糖酶基因片段,再采用基因组步行PCR技术获得全长木聚糖酶基因序列。经BLAST比对并结合GT-AG原则分析,该基因含有一个大小为63bp的内含子,利用DpnI介导的缺失方法对含内含子的全长木聚糖酶基因进行剪接,获得该基因的全长cDNA。将克隆到的cDNA在毕赤酵母GS115进行了表达,重组酶经纯化后进行酶学性质分析。【结果】BLAST比对显示,该cDNA推测的氨基酸序列和已知木聚糖酶的最高一致性为72%。测得该酶最适反应温度为45℃,最适反应pH值为6,在pH5-9维持50%以上的活性,对山毛榉材木聚糖具有最好的水解效果。Mg2+和Ca2+对酶有激活作用,分别提高了33.7%和16.6%,EDTA,β-巯基乙醇和NaN3对酶的活性基本没有影响,Tween-80和DMSO使酶活性提高了28.4%和12.8%。【结论】本文从引起棉花黄萎病的真菌V.dahliae中克隆到的木聚糖酶基因是在GenBank上登录的第一个来自棉花黄萎病真菌的木聚糖酶基因序列。本文所用的克隆方法可以高效的从植物病原真菌和白腐真菌克隆只含一个内含子的11家族的新木聚糖酶基因,避免了摸索原始菌株酶表达诱导条件,检测酶的活性等繁琐的操作。酶学性质分析显示该酶在低聚木糖的制备,面包改良上有潜在的应用价值。

轮枝菌(Verticillium dahliae和V.albo-atrum)的形态、致病力变异及变异机制

轮枝菌是重要的植物病原菌，该菌培养形态容易发生变异，对不同寄主植物的致病力变化也很大，这些特性与病害的防治密切相关。本文介绍了国内外对这些问题的近期研究与进展。

我国棉花大丽轮枝菌(Verticillium dahliae)电泳图谱分析(英文)

对24株来自不同地区、作物的不同致病类型的大丽轮枝菌,进行了同工酶和可溶性蛋白电泳图谱分析。结果表明,24株大丽轮枝菌苹果酸脱氢酶酶谱具有种的特异性,酯酶酶谱具有属的特异性。谷氨酸脱氢酶、过氧化物酶、超氧化物歧化酶和可溶性蛋白电泳图谱与病菌的致病类型有关,可将24株大丽轮枝菌分为2群,群Ⅰ包括20个菌株,均属于中等致病力或弱致病力的非落叶型菌系;群Ⅱ包括4株菌,均属于强致病力的落叶型菌系。

大丽轮枝菌(Verticillium dahliae)微菌核缺陷突变体的筛选及其侧端序列分析

为了获得大丽轮枝菌(Verticillium dahliae)微菌核缺陷突变体,利用农杆菌介导转化大丽轮枝菌,共获得270个T-DNA随机插入突变体,从中筛选出了4个气生菌丝发达、不产微菌核的突变体,利用高效TAIL-PCR技术,获得了其侧端序列,结合生物信息学方法,获得4个突变体T-DNA在大丽轮枝菌基因组插入位点的基因信息。

大丽轮枝菌(Verticillium dahliae)酯酶同工酶的测定

对棉花黄萎病菌不同致病力及致病类型菌系，不同寄主上的大力轮枝菌（Verticilliumdahliae）共14个菌系的酯酶，进行了聚丙烯酰胺凝胶平板电泳。结果表明棉花黄萎病不同致病类型菌系有其独特的酯酶同工酶谱。E6酶带的有无与致病力类型有关，E3酶带活性与病菌致病力有关，E3酶带活性与棉花发病病指关联度为0．85。不同寄主大丽轮枝菌酯酶同工酶总酶带数在7～12条之间，主酶带数在2～4条之间，按E3酶带活性强、中、弱划分为3个类型。E3酶带活性与病菌致病力有关，其活性大小与棉花、番茄、茄子病指的关联度均为0．774以上。说明酯酶同工酶技术可和于鉴定大丽轮枝菌同一种内不同致病类型致病力菌系。

The cotton WRKY transcription factor GhWRKY70 negatively regulates the defense response against Verticillium dahliae

WRKY transcription factors (TFs) play important roles in the regulation of biotic and abiotic stresses. However, the functions of most WRKY TFs in upland cotton (Gossypium hirsutum) are still unknown. In this study, we functionally identified a group Ⅲ WRKY transcription factor, GhWRKY70, in upland cotton. Reverse transcription-quantitative PCR analysis showed that GhWRKY70 expression was induced by Verticillium dahliae, salicylic acid (SA) and methyl jasmonate. Virus-induced gene silencing of GhWRKY70 increased the resistance of cotton to V. dahliae. Specifically, jasmonic acid (JA) response-associated genes were upregulated and SA-related genes were downregulated in GhWRKY70-silenced cotton plants. Overexpression of GhWRKY70 reduced tolerance to V. dahliae in Arabidopsis thaliana. Transgenic Arabidopsis plants showed increased expression of SA-associated genes and reduced expression of JA response-associated genes. These results suggest that GhWRKY70 negatively regulates tolerance to V. dahliae in at least two ways: (ⅰ) by upregulating the expression of SA-associated genes and (ⅱ) by reducing the expression of JA-associated genes.

The Influence of the Verticillium dahliae Kleb Infection on the Anti-Enzyme Inside the Body of the Cotton with Different Root Injured Degree

This study was to explore the influence of the Verticillium dahliae Kleb inflection on the anti-enzyme inside the body of the cotton with a different root injured degree. When the cotton seedling was long, with four leaves, it was flushed with water carefully, and then the following were obtained： （1） complete root seedling; （2） cut root seedling - by cutting off the lower part, 3-5 cm of the root, with a disinfected knife; （3） injured root seedling - by cutting off most of the side roots, but keeping the main root. Three kinds of cotton seedlings with different roots were immersed separately in different concentrations of the germ liquid （V. dahliae） of 20 mL each. Through 0- 48 h, the wilt degree of the seedling was recorded, and the related anti-enzyme of the variety was measured. After being immersed in the germ liquid, there was a significant difference in the wilt degree of the three kinds of injured root. When the germ liquid was in the ratio of 1：10, the complete root seedling was the lightest with no wilt; the injured root seedling was the second with a 2-degree wilt; but the cut root seedling was the most serious with a 3- degree wilt. At the same time, the changes in the peroxidase and malondialdehvde activities were determined. Peroxidase （POD） activities in the cut root seedling were 38.2 U mg^-1 min^-1, in the injured root seedling were 42.96 U mg^-1 min^-1, and in the complete root seedling were the highest at 49.2 U mg^-1 min^-1. The malondialdenvde （MDA） content in cut root seedling was 39.483 mmol g^-1, injured root seedling was 27.12 mmol g^-1, and the complete root seedling was only 3.845 mmol g^-1 The activity of the related anti-enzymes, such as POD was high or low, the quantity of the MDA was more or less, which they met the order of the harm of the seedlings. The change of SOD activities in cut root seedling was the most obvious as well. After injuring and inflecting the young roots, the exterior pathological reaction of the seedling and the dynamic state biochemical reaction of the related enzymes inside the plant body were studied. It showed that the plant exterior pathology responded to the test, with the internal biochemical reaction fitting together mutually.

大丽轮枝菌(Verticillim dahliae)突变体库的构建与分析

为了更加快捷地筛选大丽轮枝菌致病关键基因,利用聚乙二醇介导的原生质体转化法构建病原菌随机插入突变体库,对部分阳性转化子的生长表型指标和致病力进行分析,筛选生长发育与致病缺陷突变体并进行靶标基因定位。结果表明,本研究共获得13030多个阳性转化子,随机挑选5个转化子与V991野生型菌株进行比较分析,发现其中一个突变体在PDA和不同碳源培养基上的菌落直径、产孢量以及致病力均显著降低。侧翼序列测序结果结合Blast比对分析表明,该缺陷突变体中的潮霉素抗性基因表达盒定位于大丽轮枝菌3号染色体1528782 bp处,属于内切葡聚糖酶1基因(VDAG_04017)。综上所述,本研究通过大丽轮枝菌插入突变体库构建、突变体生长表型指标和致病力鉴定及靶标基因定位等一系列分子生物学手段,可初步鉴定病原菌致病相关基因,为从全基因组层面深入研究大丽轮枝菌致病机制奠定了一定基础。

RAPD Patterns Characteristic for VCGs of Cotton Verticillium Wilt Pathogen,Verticillium dahliae

Nine primers were employed to detect molecular polymorphisms in 103 Verticillium dahliae isolates that represent diverse groups of Vegetative Compatibility (VC). Our results showed that these isolates confer two distinctive RAPD groups (RPGs). RPG1, consists of isolates belonging to vegetative compatibility group I (VCG I ), while RPG2 encompasses VCGⅢ and VCGⅣ . The genetic diversity associated with VCGⅢ was greater than that associated with VCG1. Five RAPD fragments, with frequencies more than 0.96 in VCG I but less than 0.1 in VCGⅢ, produced characteristic fragments for VCG I (defoliating type), suggesting strong correlation between RPGs and VCGs.

Effects of Paclobutrazol on Physiological Parameters of Dahlia under Heat Stress

[Objectives] This study was conducted to investigate the effect of paclobutrazol （PBZ） on heat tolerance of dahlia.[Methods] A dahlia variety Danbanhuang was selected as the experimental material in this study. After 100, 200, 300 or 400 mg/L paclobutrazol was sprayed evenly to the leaves of dahlia seedlings, they were cultured in an incubator at high temperature （35 ℃/30 ℃, day/night） for two days, and then transferred to an incubator at normal temperature （25 ℃/20 ℃, day/night）. The physiological and biochemical parameters of the dahlia seedlings were measured before exposure to high temperature （D0）, after exposure to high temperature for two days （D2） and exposure to normal temperature for one day （R1）.[Results] Compared to the negative control, foliar application of paclobutrazol decreased the content of MDA, increased the contents of chlorophyll, proline, soluble protein, and the activity of SOD, POD and CAT in dahlia plants under heat stress. The contents of MDA, soluble protein and proline in PBZ treated dahlia plants increased when they were subject to high temperature stress （35 ℃/30 ℃, day/night）, and then decreased when the temperature returned to normal （25 ℃/20 ℃, day/night）, and CAT activity decreased at high temperature, and then increased at normal temperature, while the activity of SOD and POD kept rising during the entire experimental period.[Conclusions] Foliar application of paclobutrazol can alleviate the adverse effect caused by high temperature to dahlia plants, and the experimental data provide a theoretical basis for the application of paclobutrazol in dahlia cultivation in South China.

Tolerance of New Introgressive Hybrid and Backcross Forms Pathogenic Micromitisms (<i>Verticillium dahliae</i>Kleb and Fusarium oxysporum f.sp. vasinfectum)

The article is based on the use of experimental polyploidy method, with the introduction of new introgressive hybrid forms combining several species genotype with pathogenic Verticillium dahliae Kleb. and the effects of mycotoxins separated from the Fusarium oxysporum f.sp. vasinfectum micromicette on the yield of plant seeds. New artificial complex hypertension forms based on experimental polyploidy Verticillium dahliae Kleb. and Fusarium oxysporum f.sp. vasinfectum combine the potential of resistance to mycotoxins separated from microcrystals, making a tremendous contribution to the selection of new varieties and to the effectiveness of selection as a result of the use of genetic selective research as genetic-selective genetic-selector studies.

Down regulation of cotton GbTRP1 leads to accumulation of anthranilates and confers resistance to Verticillium dahliae

Background:Verticillium wilt,caused by Verticillium dahliae,is called a "cancer" disease of cotton.The discovery and identification of defense-related genes is essential for the breeding of Verticillium wilt-resistant varieties.In previous research we identified some possible broad-spectrum resistance genes.Here,we report a tryptophan synthesis-related gene GbTRP1 and its functional analysis in relation to the resistance of cotton to V.dahliae.Results:Expression analysis shows that GbTRP1 is suppressed at 1 h and 6 h post V.dahliae infection,but activated at 12 h and 24 h,and the expression of GbTRP1 is highly induced by treatment with salicylic acid and jasmonic acid.Sub-cellular localization studies show that GbTRP1 is localized in the chloroplast.Suppression of GbTRP1 expression leads to lesion-mimic phenotypes and activates the immune response in cotton by showing enhanced resistance to V.dahliae and B.cinerea.Metabolomic analysis shows that anthranilic compounds significantly accumulated in GbTRP1-silenced plants,and these metabolites can inhibit the growth of V.dahliae and B.cinerea in vitro.Conclusions:Our results show that suppression of GbTRPI expression dramatically activates the immune response and increases resistance of cotton to V.dahliae and B.cinerea,possibly due to the accumulation of anthranilate compounds.This study not only provides genetic resources for disease resistance breeding,but also may provide a basis for new chemical control methods for combatting of fungal disease in cotton.

Effects of Exogenous Ethylene and 1-Methylcyclopropene on of DpXTH Gene Expression in Dahlia(Dahlia pinnata Cav.) Flowers

Objectives] This study was conducted to investigate the regulatory mechanism of ethylene on DpXTH gene expression in dahlia （ Dahlia pinnata Cav.） flower petals.[Methods] Dahlia flower petals were treated with ethephon （ETH） and 1-methylcyclopropene （1-MCP） at different flower development stages and for different durations in vivo . Then, the relative expression of DpXTH1 and DpXTH2 in flower samples in different treatments was quantified with real-time PCR, using β-actin as a reference gene.[Results] The expression of DpXTH1 and DpXTH2 in ETH- or 1-MCP-treated dahlia flower petals increased at first, and decreased subsequently with increasing duration of treatment. The relative expression of both DpXTH1 and DpXTH2 in ETH-treated samples reached the maximum level at 12 h. Moreover, the relative expression of both DpXTH1 and DpXTH2 in dahlia flower petals treated with ETH at early stage of flowering was higher than that of the samples treated at other flowering stages; in 1-MCP-treated samples, it was higher at full-flowering stage than at other stages. The maximum relative expression levels of DpXTH1 and DpXTH2 in ETH- and 1-MCP-treated samples were all higher than those of control.[Conclusions] Exogenous ethylene can accelerate the opening of dahlia flowers, and 1-MCP can inhibit this process, so they can be used to regulate development of dahlia flowers.

Comparative Study of Inulin Extracts from Dahlia, Yam, and Gembili Tubers as Prebiotic

The addition of prebiotic in food today is mostly done. It is based on the ability of prebiotic to support the growth of probiotic. Inulin is a carbohydrate that serves as an effective prebiotic that cannot be digested by digestive enzymes. The purpose of this research is to assess utilization of inulin from several types of tubers include dahlia tubers, yam tu- bers, and gembili tubers as a source of prebiotic that tested in probiotic bacteria (L. casei and L. plantarum). The study used a Randomized Block Design method with two factors, the type of probiotic isolate and inulin extract from tubers. The result shows that the best treatment is isolates of L. casei obtained in the fermentation medium with the addition of inulin from gembili tubers. The best treatment has the following characteristics: an increase in total LAB 2.71 × 1010 cfu/ml, 1.50% total acid, pH 2.05 and the total sugars are 3.11%. Whereas in isolates of L. plantarum, the best treatment in the fermentation medium is with the addition of inulin from dahlia tubers. The LAB reaches 2.80 × 1010 cfu/ml, 1.29% total acid, pH 2.24 and 2.05% total sugars.

Test on Wilt-Resistance of Simple Hybrides F7 and Beckross Hybrid Lines F6 B1 of <i>G. hirsutum</i>L. of Cotton to New Virulent Isolates of Fungus <i>Fusarium verticillioides</i>and <i>Verticillium dahliae</i>

The article presents the results of studies on the resistance of hybrid cotton lines to a new virulent isolate (strain) of the fungus Fusarium verticillioides upon inoculation of the host plant. Based on the studies, it was found that the complex genotypic resistance of the studied lines, when the host plants are inoculated with isolates of -100 V. dahliae Kleb fungus and 103 Fusarium verticillioides fungi, depends on the degree of resistance of the parental forms and their combination ability.

外源添加L-脯氨酸对棉花黄萎病发生及其根际土壤微生物群落的影响

【目的】根系分泌物是植物与土壤微生物进行互作的信号媒介,对于植物病害发生和植株生长均具有重要调控功能。论文旨在明确棉花根系分泌物L-脯氨酸抵御棉花黄萎病发生的微生态机制,揭示L-脯氨酸介导的根际微生物与棉花黄萎病发生的互作关系,为构建生物防治土传病害的有益菌群提供新视角。【方法】通过温室盆栽试验,以外源添加不同浓度的L-脯氨酸(0、50、100、200和400 mmol·L^(-1))为试验处理,采用实时荧光定量PCR(real-time qPCR)和宏基因组测序技术分别测定不同处理的土壤中大丽轮枝菌(Verticillium dahliae)DNA拷贝数量和土壤微生物群落结构及功能,采用主成分分析比较不同处理的根际土壤微生物群落结构,利用冗余分析研究土壤养分因子与微生物群落结构的相关性,利用斯皮尔曼相关分析研究微生物群落结构功能代谢途径的相关性。【结果】与空白对照相比,低浓度(50 mmol·L^(-1))L-脯氨酸处理不能够减轻棉花黄萎病的发生,高浓度(100、200和400 mmol·L^(-1))L-脯氨酸处理的病情指数分别下降22.51%、60.23%和64.23%。qPCR结果表明,L-脯氨酸处理不能够显著降低土壤中大丽轮枝菌拷贝数量。宏基因组测序分析表明,L-脯氨酸处理后细菌多样性Shannon指数显著增加,真菌多样性Shannon指数呈下降趋势。在属水平上,L-脯氨酸处理后类诺卡氏菌属(Nocardioides)、溶杆菌属(Lysobacter)、节杆菌属(Arthrobacter)、Phycicoccus、假单胞菌属(Pseudomonas)和毛霉菌属(Mucor)的相对丰度呈上升趋势。线性判别分析表明,除浓度为100 mmol·L^(-1)的L-脯氨酸外,外源添加L-脯氨酸处理后根际土壤微生物KEGG通路的富集情况发生改变。冗余分析表明,细菌群落组成受pH、电导率、硝态氮、铵态氮和有机质显著影响,而真菌群落组成与铵态氮存在显著相关性。Spearman相关分析表明,细菌的KEGG代谢通路与pH、有机质、铵态氮含量呈负相关关系,而与电导率和硝态氮呈正相关关系;真菌的大部分KEGG代谢通路与土壤养分的相关性较差。【结论】外源添加适量L-脯氨酸通过改变土壤细菌群落结构和功能,增加有益微生物的相对丰度,从而影响棉花黄萎病的发生,但其不能够改变病原菌数量。同时,根际细菌群落组成和功能均与土壤养分存在相关性。