Cotton ethylene response factor Gh ERF91 is involved in the defense against Verticillium dahliae

Verticillium dahliae causes significant losses in cotton production.To reveal the mechanism of the defense response to V.dahliae in cotton,transcriptomic analyses were performed using cotton cultivars M138(V.dahliae-resistant)and P2(V.dahliae-susceptible).The results revealed 11,076 and 6,640 differentially expressed genes(DEGs)in response to V.dahliae,respectively.The weighted gene co-expression network analysis of 4,633 transcription factors(TFs)indicated a“MEblue”module containing 654 TFs that strongly correlate with resistance to V.dahliae.Among these TFs,the ethylene response factor Ghi_A05G10166(GhERF91)was identified as a putative hub gene with a defense response against V.dahliae.A virus-induced gene silencing assay and exogenous application of ethephon showed that GhERF91 is activated by ethylene and positively regulates the response to V.dahliae exposure in cotton.This study provides fundamental transcriptome data and a putative causal gene(GhERF91)associated with resistance to V.dahliae,as well as genetic resources for breeding V.dahliae-resistant cotton.

GhWRKY75 positively regulates GhPR6-5b via binding to a W-box TTGAC(C/T)to orchestrate cotton resistance to Verticillium dahliae

Verticillium dahliae is an important fungal pathogen affecting cotton yield and quality.Therefore,the mining of V.dahlia-resistance genes is urgently needed.Proteases and protease inhibitors play crucial roles in plant defense responses.However,the functions and regulatory mechanisms of the protease inhibitor PR6 gene family remain largely unknown.This study provides a comprehensive analysis of the PR6 gene family in the cotton genome.We performed genome-wide identification and functional characterization of the cotton GhPR6 gene family,which belongs to the potato protease inhibitor I family of inhibitors.Thirty-nine PR6s were identified in Gossypium arboreum,G.raimondii,G.barbadense,and G.hirsutum,and they were clustered into four groups.Based on the analysis of pathogen-induced and Ghlmm transcriptome data,Gh PR6-5b was identified as the key gene for V.dahliae resistance.Virus-induced gene silencing experiments revealed that cotton was more sensitive to V.dahliae V991after PR6-5b silencing.The present study established that GhWRKY75 plays an important role in resistance to Verticillium wilt in cotton by positively regulating GhPR6-5b expression by directly binding to the W-box TTGAC(T/C).Our findings established that GhWRKY75 is a potential candidate for improving cotton resistance to V.dahliae,and provide primary information for further investigations and the development of specific strategies to bolster the defense mechanisms of cotton against V.dahliae.

Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene(VdThit)confers resistance to Verticillium wilt in cotton

Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.In this study,we successfully generated two stable transgenic lines of cotton(Gossypium hirsutum L.),VdThitRNAi-1 and VdThit-RNAi-2,using host-induced gene silencing(HIGS)technology to introduce double-stranded RNA(dsRNA)targeting the thiamine transporter protein gene(VdThit).Southern blot analysis confirmed the presence of a single-copy insertion in each line.Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type(WT).The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.Real-time quantitative PCR(qRT-PCR)analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.Small RNA sequencing(sRNA-Seq)analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.Additionally,the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.Under field conditions,VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.In summary,our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton,thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.

Identification and virulence test of a new pathogen that causes verticillium striping on rapeseed in northwestern China

Five stems of rapeseed with abundant black microsclerotia were collected from Huangyuan County of Qinghai Province,China,and fungal isolates were obtained from the stems.They were identified based on morphology,molecular features and specific PCR detection.The results showed that the 10 fungal isolates belonged to Verticillium longisporum lineage A1/D3.One of the 10 isolates(HW7-1)was tested for virulence on three species of rapeseed,including B.napus Zhongshuang 9,B.rapa Qingyou 9 and B.juncea Tayou 2 by conidia inoculation of HW7-1 on roots of young seedlings.Control seedlings were inoculated with V.dahliae conidia or water alone.The seedlings of these treatments were transplanted in culture mix and incubated in a growth chamber(20℃).Results suggested that the control seedlings of three cultivars appeared quite healthy,while the seedlings inoculated with HW7-1 turned yellowing leaves,seedling stunting or even death after 22 days post-inoculation.V.longisporum was re-isolated from he yellow leaves,thus fulfilling Koch's postulates.Moreover,compared to the control treatments,inoculation with HW7-1 caused flowering delay and seed yield reduction on Tayou 2 with production of microsclerotia on the stems.To our knowledge,this is the first report of V.longisporum lineage A1/D3 on rapeseed in northwestern China.

A glycine-rich nuclear effector VdCE51 of Verticillium dahliae suppresses plant immune responses by inhibiting the accumulation of GhTRXH2

Verticillium dahliae is an important soil-borne fungal pathogen that causes great yield losses in many cash crops.Effectors of this fungus are known to regulate plant immunity but the mechanism much remains unclear.A glycine-rich nuclear effector,VdCE51,was able to suppress immune responses in tobacco against Botrytis cinerea and Sclerotinia sclerotiorum.This effector was a required factor for full virulence of V.dahliae,and its nuclear localization was a requisite for suppressing plant immunity.The thioredoxin GhTRXH2,identified as a positive regulator of plant immunity,was a host target of VdCE51.Our findings show a virulence regulating mechanism whereby the secreted nuclear effector VdCE51 interferes with the transcription of PR genes,and the SA signaling pathway by inhibiting the accumulation of GhTRXH2,thus suppressing plant immunity.

Expression analysis of the R2R3-MYB gene family in upland cotton and functional study of GhMYB3D5 in regulating Verticillium wilt resistance

Improving plant resistance to Verticillium wilt(VW),which causes massive losses in Gossypium hirsutum,is a global challenge.Crop plants need to efficiently allocate their limited energy resources to maintain a balance between growth and defense.However,few transcriptional regulators specifically respond to Verticillium dahliae and the underlying mechanism has not been identified in cotton.In this study,we found that the that expression of most R2R3-MYB members in cotton is significantly changed by V.dahliae infection relative to the other MYB types.One novel R2R3-MYB transcription factor(TF)that specifically responds to V.dahliae,GhMYB3D5,was identified.GhMYB3D5 was not expressed in 15 cotton tissues under normal conditions,but it was dramatically induced by V.dahliae stress.We functionally characterized its positive role and underlying mechanism in VW resistance.Upon V.dahliae infection,the up-regulated GhMYB3D5 bound to the GhADH1 promoter and activated GhADH1expression.In addition,GhMYB3D5 physically interacted with GhADH1 and further enhanced the transcriptional activation of GhADH1.Consequently,the transcriptional regulatory module GhMYB3D5-GhADH1 then promoted lignin accumulation by improving the transcriptional levels of genes related to lignin biosynthesis(GhPAL,GhC4H,Gh4CL,and GhPOD/GhLAC)in cotton,thereby enhancing cotton VW resistance.Our results demonstrated that the GhMYB3D5 promotes defense-induced lignin accumulation,which can be regarded as an effective way to orchestrate plant immunity and growth.

Knock-out of BnHva22c reduces the susceptibility of Brassica napus to infection with the fungal pathogen Verticillium longisporum

Verticillium longisporum(Vl43)is a soilborne hemibiotrophic fungal pathogen causing stem striping on oilseed rape(OSR)and severe yield losses.Breeding for resistant varieties is the most promising approach to control this disease.Here,we report the identification of Hva22c as a novel susceptibility factor and its potential for improving OSR resistance.Hva22c is a member of the Hva22 gene family,originally described for barley(Hordeum vulgare).Several Hva22 members have been located at the endoplasmic reticulum.Hva22c is up-regulated in response to Vl43 in both Arabidopsis and OSR.We demonstrate that knock-out of Hva22c in OSR by CRISPR/Cas9 and its homolog in Arabidopsis by T-DNA insertion reduced plants’susceptibility to Vl43 infection and impaired the development of disease symptoms.To understand the underlying mechanism,we analysed transcriptomic data from infected and non-infected roots of hva22c knock-out and wild type plants.We identified a homozygous mutant with frame-shifts in all four BnHva22c loci displaying a vastly altered transcriptional landscape at 6 dpi.Significantly,a large set of genes was suppressed under mock conditions including genes related to the endomembrane systems.Among the up-regulated genes we found several defense-related and phytohormone-responsive genes when comparing mutant to the wild type.These results demonstrate that Hva22c is functionally required for a fully compatible plant-fungus interaction.Its loss of function reduces plant susceptibility,most likely due to endoplasmatic reticulum and Golgi dysfunction accompanied by additionally activated defense responses.These findings can help improve OSR resistance to V.longisporum infection.

Screening and Identification of Cotton Verticillium Wilt Antagonistic Bacteria Strain 7-30

[Objective]The aim of this study was to screen the antagonistic spore-forming bacteria of Verticillium dahliae and identify its physiological and biochemical characteristics.[Method]Taking the cotton verticillium wilt pathogen Verticillium dahliae V-190 as the test microorganism,the antagonistic spore-forming bacteria were screened.Through the preliminary screening and secondary screening,an antagonistic bacteria strain 7-30 with stronger antibacterial activity was obtained,and its morphological characteristics,physiological and biochemical characteristics were also identified.[Result]84 antagonistic bacteria strains were isolated from soil in various places by the preliminary screening.Especially,18 strains with better antagonistic ability were screened again,so an antagonistic bacteria strain 7-30 with the diameter of inhibition zone 18.9 mm and stronger antibacterial activity was obtained.According to its morphological characteristics,physiological and biochemical characteristics,the antagonistic bacteria strain 7-30 was identified as Bacillus subtilis primarily.[Conclusion]The strain 7-30 was obtained as the antagonistic spore-forming bacteria of Verticillium dahliae.

不同花色牡丹品种亲缘关系的RAPD-PCR分析

用RAPD PCR技术对 7个花色 35个牡丹品种进行基因组DNA多态性分析 ,34个随机引物扩增出 4 18个DNA片段 ,其中 337条谱带表现多态性 ,多态性检测率为 80 .6 % ,表明受试牡丹栽培品种之间富含遗传多态性 ,其中18个受试牡丹品种产生特异性遗传标记 ,这些特异标记对各个牡丹品种具有一定的鉴别价值。将扩增图谱利用UP GMA方法构建遗传聚类树状图 ,分析品种间遗传关系。相似系数 1.5将 35个牡丹品种划分为 2个遗传聚类组 ,相似系数 1.0将其划分为 4个遗传聚类组 ,不同相似系数的遗传聚类划分与花色之间并非完全未有相关性。来源相同花色一致的牡丹品种之间亲缘关系相对较近 ,但产地来源对牡丹品种的亲缘关系的影响比花色更为突出。

RAPD-PCR对梨属植物品种鉴定的研究

用RAPD-PCR技术分析鉴定了梨属的18份品种材料(包括不同品种、杂交种和芽变种材料)。从OP系列8组共160个10碱基随机引物中,筛选出12种能很好地用于梨品种鉴定的引物。

利用RAPD-PCR技术区分4种仓虫幼虫的初步研究

以花斑皮蠹、谷斑皮蠹、黑毛皮蠹和印度谷螟等４种仓储害虫的幼虫为材料，借用小麦ＤＮＡ提取方法提取了虫体的ＤＮＡ，用Ｅ１２、Ｅ１５、Ｅ１８、Ｇ１０和Ｇ１４等５种引物采用ＲＡＰＤＩ程序进行扩增。电泳检测结果显示，５种引物均产生可用以区分这４种仓虫的扩增谱带。从ＤＮＡ提取到获得结果，只需４８ｈ。

猕猴桃模板DNA的提取及RAPD-PCR最佳反应体系的建立

以改良CTAB法从猕猴桃叶片中制备模板DNA ,优化了PCR热循环参数 ,建立了RAPD PCR扩增的最佳反应体系。实验结果表明 ,CTAB提取液中EDTA组分的浓度对模板提取影响很大 ,其最适浓度为 80mmol/L ;用异丙醇沉淀后不经乙醇洗涤纯化的DNA不会影响扩增效果。PCR热循环参数为 :94℃预变性 5min ;94℃变性 1min ,37℃退火 1min ,72℃延伸 2min ,循环 4 0次 ;最后在 72℃延伸 6min。

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。

11种兰属植物DNA的提取及RAPD-PCR实验体系的建立与优化

采用改进的CTAB-DNA提取方法，从11种兰属植物的嫩叶中提取总DNA。所得的DNA样品的A260／A280值在1．7～1．9之间，琼脂糖凝胶上主带清晰，较少降解，样品纯度高，DNA量大。另外，对影响RAPD-PCR的Mg^2＋、dNTPs、Taq酶、引物浓度等因素进行了优化。确定优化的反应体系为：75ng模板DNA，1×Buffer，2．5mmol／LMg^2＋，0．15mmol／LdNTPs，0．75UTaq酶，引物浓度0．4μmol／L，反应总体积为25山。该体系在20个供试兰属实验材料中获得较好的扩增结果。

利用正交设计优化兴安落叶松RAPD-PCR反应体系

以兴安落叶松针叶DNA为模板,对影响落叶松RAPD-PCR扩增的重要参数进行了优化试验,以期建立兴安落叶松RAPD-PCR反应的最佳体系。通过采用正交设计L16(45)对兴安落叶松RAPD-PCR反应的5因素(Taq酶、Mg2+、dNTP、模板DNA、引物)在4个水平上进行优化试验,结果表明兴安落叶松最佳的RAPD-PCR的反应体系(20μL)中含有模板90ng,0.5μmol·L-1的引物,1×反应缓冲液,DNTP各为0.25mmol·L-1,1U的TaqDNA聚合酶,Mg2+2.5mmol·L-1。在此基础上筛选出20个扩增稳定、多态性丰富的RAPD引物,并通过梯度PCR试验,确定了引物最佳退火温度。

松口蘑菌丝体的分离和RAPD-PCR分析

针对松口蘑 [Tricholomamatsutake(S .ItoetImai)Sing .]菌丝体分离培养困难和各种相关分离物目前难以用出菇试验鉴定的现实 ,采用 8种培养基配方 ,对 9个不同来源的松口蘑子实体的不同部位及菌根、菌土进行组织分离 ,计接种试管 81 0多支 ,结果从菌褶部位获得 94支慢生型的菌丝体分离菌株 ,从菌柄部位仅获得 1支快生型的菌丝体分离菌株。以马铃薯葡萄糖土壤滤液培养基 (PDAS)、马铃薯葡萄糖麦麸滤液培养基 (PDAW )、BM培养基、马铃薯葡萄糖琼脂培养基 (PDA)对菌褶进行组织分离 ,获慢生型菌丝体的成功率依次为 74 4%、35 5%、1 5 6%和 8 9%。以各分离菌株的来源松口蘑子实体和中日两国松口蘑研究者提供的分离菌株作为DNA参照样品 ,对从供试子实体、菌根、菌土进行组织分离获得的各种相关纯培养物进行亲菌鉴定。采用筛选的 1 7个随机引物介导 2 5个供试松口蘑子实体及其分离菌体的RAPD(RandomAmplifiedPolymorphicDNA) PCR反应 ,全部获得了清晰而稳定的DNA指纹图谱 ,结果一致表明 :每个松口蘑子实体的菌盖 (含菌褶 )、菌柄与从自身菌褶分离的慢生型纯培养菌丝体都具有相同的DNA指纹图谱 ,其相似系数为 1 0 0 0 ,明确揭示松口蘑个体的DNA同质性 ,而不同来源的供试松口蘑之间的相似系数在 0 934～ 0

天牛干标本DNA的提取及RAPD-PCR扩增反应

对标本馆保存多年的天牛标本进行基因组DNA的提取,并成功用于PAPD-PCR的扩增。结果显示基因组DNA的提取与标本的保存时间无直接的联系,而与标本保存的质量关系密切。用同一引物扩增,不同种天牛的扩增片段呈现多态性。

洋葱(Alliumcepa L.)RAPD-PCR反应体系及扩增程序的优化

为建立多态性高、稳定性好的洋葱RAPD-PCR反应体系,采用正交设计,研究了Taq酶、Mg2+、引物和dNTP 4种RAPD-PCR反应组分浓度变化对扩增结果的影响,在此基础上对模板DNA用量、扩增程序中退火温度和反应循环次数进行了筛选。试验结果表明,洋葱20μl RAPD-PCR优化反应体系为1×Buffer、2.0 mmo1/L Mg2+、1.0 UTaqDNA聚合酶、200μmo1/L dNTP、0.6μmo1/L引物、2%甘油和15 ng DNA模板;PCR扩增程序为94℃预变性4 m in;94℃变性30 s,35℃退火40 s,72℃延长1.5 m in,45个循环;72℃保温延伸7 m in。

黄瓜DNA提取及其RAPD-PCR反应体系的优化

利用改良的 SDS方法提取黄瓜干种子与一步法提取的黄花苗总 DNA进行 RAPD扩增 ,其结果与传统的 CTAB和 SDS的结果一样。这两种方法不仅节约成本 ,且简单快速 ,大大降低了 DNA的提取难度 ,适宜黄瓜杂交种子纯度鉴定。不同组织材料对 RAPD无影响。研究了 RAPD的影响因素 ,改进及优化了 RAPD反应程序。结果表明 :Mg2 +浓度的范围为 1.5～ 3.0 mmol· L- 1 ,d NTPs浓度范围为 0 .15～ 0 .2 5 mmol· L- 1 ,Taq聚合酶浓度范围为 0 .5～ 1.5 U,模板DNA浓度为 2 0～ 10 0 ng,反应以及引物浓度为 0 .2～ 0 .4μmol· L- 1 。引物的碱基组成以及不同存放时间的引物对 RAPD扩增也有影响。 DNA预变性 94℃进行 4 min,94℃变性 30 s,37℃退火 30 s,72℃延伸 1min,循环 35次后