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MAP kinase gene STK1 is required for hyphal, conidial, and appressorial development, toxin biosynthesis, pathogenicity, and hypertonic stress response in the plant pathogenic fungus Setosphaeria turcica 被引量:7
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作者 LI Po GONG Xiao-dong +7 位作者 JIA Hui FAN Yong-shan ZHANG Yun-feng CAO Zhi-yan HAO Zhi-min HAN Jian-min GU Shou-qin DONG Jin-gao 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2016年第12期2786-2794,共9页
The mitogen-activated protein kinase (MAPK), a key signal transduction component in the MAPK cascade pathway, regulates a variety of physiological activities in eukaryotes. However, little is known of the role MAPK ... The mitogen-activated protein kinase (MAPK), a key signal transduction component in the MAPK cascade pathway, regulates a variety of physiological activities in eukaryotes. However, little is known of the role MAPK plays in phytopathogenic fungi. In this research, we cloned the MAPK gene STK1 from the northern corn leaf blight pathogen Setosphaeria turcica and found that the gene shared high homology with the high osmolality glycerol (HOG) MAPK gene HOG1 of Saccharomy- ces cerevisiae. In addition, gene knockout technology was employed to investigate the function of STKI. Gene knockout mutants (KOs) were found to have altered hyphae morphology and no conidiogenesis, though they did show similar radial growth rate compared to the wild-type strain (WT). Furthermore, microscope observations indicated that STK1 KOs did not form normal appressoria at 48 h post-inoculation on a hydrophobic surface. STK1 KOs had reduced virulence, a significantly altered Helminthosporium turcicum (HT)-toxin composition, and diminished pathogenicity on the leaves of susceptible inbred corn OH43. Mycelium morphology appeared to be significantly swollen and the radial growth rates of STK1 KOs declined in comparison with WT under high osmotic stress. These results suggested that STK1 affects the hyphae development, conidiogenesis, and pathogenicity of S. turcica by regulating appressorium development and HT-toxin biosynthesis. Moreover, the gene appears to be involved in the hypertonic stress response in S. turcica. 展开更多
关键词 setosphaeria turcica MAPK CONIDIOGENESIS HT-toxin PATHOGENICITY
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Effects of MEK-Specific Inhibitor U0126 on the Conidial Germination,Appressorium Production,and Pathogenicity of Setosphaeria turcica 被引量:4
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作者 FAN Yong-shan GU Shou-qin +1 位作者 DONG Jin-gao DONG Bing-fang 《Agricultural Sciences in China》 CAS CSCD 2007年第1期78-85,共8页
Systemic studies on the effects of mitogen-activated protein kinase (MAPK) signal transduction pathway on the growth and development of Setosphaeria turcica is helpful not only in understanding the molecular mechani... Systemic studies on the effects of mitogen-activated protein kinase (MAPK) signal transduction pathway on the growth and development of Setosphaeria turcica is helpful not only in understanding the molecular mechanism of pathogenhost interaction but also in the effective control of the diseases caused by S. turcica. U0126, the specific MEK inhibitor, is used to treat S. turcica before the observation of the conidial germination, appressorium production, and pathogenicity of the pathogen. There is no significant effect of U0126 on the colony morphology and mycelium growth of the pathogen. After treatment with U0126, the growth of mycelium and conidia are normal, but the conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are significantly inhibited. Under the definite concentration scope, an increase in U0126 concentration increases the inhibition degree of conidial germination and appressorium production, but the inhibition degree decreases with elongation of treatment time. The conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are regulated by the MAPK pathway inhibited by U0126. 展开更多
关键词 setosphaeria turcica MAPK U0126 conidial germination appressorium production PATHOGENICITY
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Stk2,a Mitogen-Activated Protein Kinase from Setosphaeria turcica,Specifically Complements the Functions of the Fus3 and Kss1 of Saccharomyces cerevisiae in Filamentation,Invasive Growth,and Mating Behavior 被引量:3
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作者 GU Shou-qin YANG Yang +10 位作者 LI Po ZHANG Chang-zhi FAN Yu ZHANG Xiao-yu TIAN Lan HAO Zhi-min CAO Zhi-yan GONG Xiao-dong FAN Yong-shan HAN Jian-min DONG Jin-gao 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2013年第12期2209-2216,共8页
Setosphaeria turcica,an essential phytopathogenic fungus,is the primary cause of serious yield losses in corn; however,its pathogenic mechanism is poorly understood.We cloned STK2,a newly discovered mitogen-activated ... Setosphaeria turcica,an essential phytopathogenic fungus,is the primary cause of serious yield losses in corn; however,its pathogenic mechanism is poorly understood.We cloned STK2,a newly discovered mitogen-activated protein kinase gene with a deduced amino acid sequence that is 96% identical to MAK2 from Phaeosphaeria nodorum,56% identical to KSS1 and 57% identical to FUS3 from Saccharomyces cerevisiae.To deduce Stk2 function in S.turcica and to identify the genetic relationship between STK2 and KSS1/FUS3 from S.cerevisiae,a restructured vector containing the open reading frame of STK2 was transformed into a fus3/kss1 double deletion mutant of S.cerevisiae.The results show that the STK2 complementary strain clearly formed pseudohyphae and ascospores,and the strain grew on the surface of the medium after rinsing with sterile water and the characteristics of the complementary strain was the same as the wild-type strain.Moreover,STK2 complemented the function of KSS1 in filamentation and invasive growth,as well as the mating behavior of FUS3 in S.cerevisiae,however,its exact functions in S.turcica will be studied in the future research. 展开更多
关键词 setosphaeria turcica MAPK FILAMENTATION invasive growth mating behavior
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Purification and Structural Analysis of a Selective Toxin Fraction Produced by the Plant Pathogen Setosphaeria turcica 被引量:4
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作者 ZHANG Li-hui DONG Jin-gao +1 位作者 WANG Chao-hua LI Zheng-ping 《Agricultural Sciences in China》 CAS CSCD 2007年第4期452-457,共6页
Thirteen fractions from the pathogenic plant fungus Setosphaeria turcica race 1 were separated and collected using high performance liquid chromatography (HPLC). Their toxic activities were assayed through leaf punc... Thirteen fractions from the pathogenic plant fungus Setosphaeria turcica race 1 were separated and collected using high performance liquid chromatography (HPLC). Their toxic activities were assayed through leaf puncturing on corn differentials (OH43, OH43Ht1, OH43Ht2, and OH43HtN), and the results revealed that eight fractions were toxic and fraction 6 was specifically toxic to OH43Ht1, which could be taken as a gene-selective toxin fraction. Fraction 6 was finely purified via HPLC and condensed by freeze desiccation. Its chemical structure was analyzed with EI-MS, IR, HMBC, ^1H-NMR, and two-dimensional NMR. The results suggested that fraction 6 contained an unsaturated double bond, carbonyl and methylene groups with molecular weight of 142. 展开更多
关键词 setosphaeria turcica gene-specific toxin fraction structure analysis high performance liquid chromatography (HPLC)
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Melanin,DNA replication,and autophagy affect appressorium development in Setosphaeria turcica by regulating glycerol accumulation and metabolism 被引量:3
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作者 GUO Xiao-yue LIU Ning +4 位作者 LIU Bing-hui ZHOU Li-hong CAO Zhi-yan HAN Jian-min DONG Jin-gao 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2022年第3期762-773,共12页
Setosphaeria turcica(syn.Exserohilum turcicum)is the pathogenic fungus of maize(Zea mays)that causes northern leaf blight,which is a major maize disease worldwide.Melanized appressoria are highly specialized infection... Setosphaeria turcica(syn.Exserohilum turcicum)is the pathogenic fungus of maize(Zea mays)that causes northern leaf blight,which is a major maize disease worldwide.Melanized appressoria are highly specialized infection structures formed by germinated conidia of S.turcica that infect maize leaves.The appressorium penetrates the plant cuticle by generating turgor,and glycerol is known to be the main source of the turgor.Here,the infection position penetrated by the appressorium on maize leaves was investigated,most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells,and the appressorium structure was necessary for the infection,whether it occurred through epidermal cells or stomata.Then,to investigate the effects of key factors in the development of the appressorium,we studied the effects of three inhibitors,including a melanin inhibitor(tricyclazole,TCZ),a DNA replication inhibitor(hydroxyurea,HU),and an autophagy inhibitor(3-methyladenine,3-MA),on appressorium turgor and glycerol content.As results,appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments.The three inhibitors had the greatest effects on appressorium turgor pressure at this stage.Glycogen and liposomes are the main substances producing glycerol.It was also found inhibitors affected the distribution of glycogen and liposomes,which were detected in the conidia,the germ tube,and the appressorium during appressorium development.This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content,which was affected by the synthesis of melanin,DNA replication,and autophagy in the developing appressorium during a S.turcica infection. 展开更多
关键词 MELANIN DNA replication AUTOPHAGY APPRESSORIUM DEVELOPMENT setosphaeria turcica GLYCEROL
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Proteomics Identification of Differentially Expressed Leaf Proteins in Response to Setosphaeria turcica Infection in Resistant Maize 被引量:2
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作者 ZHANG Xiao-li SI Bing-wen +2 位作者 FAN Cheng-ming LI Hong-jie WANG Xiao-ming 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2014年第4期789-803,共15页
Northern corn leaf blight (NCLB), caused by the heterothallic ascomycete fungus Setosphaeria turcica, is a destructive foliar disease of maize and represents a serious threat to maize production worldwide. A compara... Northern corn leaf blight (NCLB), caused by the heterothallic ascomycete fungus Setosphaeria turcica, is a destructive foliar disease of maize and represents a serious threat to maize production worldwide. A comparative proteomic study was conducted to explore the molecular mechanisms underlying the defense responses of the maize resistant line A619 Ht2 to S. turcica race 13. Leaf proteins were extracted from mock and S. turcica-infected leaves after inoculated for 72 h and analyzed for differentially expressed proteins using two-dimensional electrophoresis and mass spectrometry identification. 137 proteins showed reproducible differences in abundance by more than 2-fold at least, including 50 up-regulated proteins and 87 down-regulated proteins. 48 protein spots were successfully identified by MS analysis, which included 10 unique, 6 up-regulated, 20 down-regulated and 12 disappeared protein spots. These identified proteins were classified into 9 functional groups and involved in multiple functions, particularly in energy metabolism (46%), protein destination and storage (12%), and disease defense (18%). Some defense-related proteins were upregulated such as 13-glucosidase, SOD, polyamines oxidase, HSC 70 and PPIases; while the expressions of photosynthesis- and metabolism-related proteins were down-regulated, by inoculation with S. turcica. The results indicated that a complex regulatory network was functioned in interaction between the resistant line A619 Ht2 and S. turcica. The resistance processes of A619 Ht2 mainly resided on directly releasing defense proteins, modulation of primary metabolism, affecting photosyntesis and carbohydrate metabolism. 展开更多
关键词 MAIZE setosphaeria turcica Ht2 gene differential proteomics
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Bioinformatic analysis and functional characterization of CFEM proteins in Setosphaeria turcica 被引量:2
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作者 WANG Jian-xia LONG Feng +5 位作者 ZHU Hang ZHANG Yan WU Jian-ying SHEN Shen DONG Jin-gao HAO Zhi-min 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2021年第9期2438-2449,共12页
Common in Fungal Extracellular Membrane(CFEM)domains are uniquely found in fungal extracellular membrane proteins which are important for pathogens.This study identified 13 StCFEM proteins in the genome of Setosphaeri... Common in Fungal Extracellular Membrane(CFEM)domains are uniquely found in fungal extracellular membrane proteins which are important for pathogens.This study identified 13 StCFEM proteins in the genome of Setosphaeria turcica,the hemibiotrophic fungus that causes northern corn leaf blight.Sequence alignment and WebLogo analysis of their CFEM domains indicated that the amino acids were highly conserved and that,with the exception of StCFEM1,2,3,and 6,they contained eight cysteines.Phylogenic analysis suggested that these 13 proteins(StCFEM1–13)could be divided into 2 clades based on the presence of the trans-membrane domain.Six StCFEM proteins with a signal peptide and without a trans-membrane domain were considered as candidate effector proteins.The CFEM domain in the candidate effector proteins could form a helical-basket structure homologous to Csa2 in Candida albicans.Transcriptome analysis suggested that the 13 genes were expressed during fungal infection and a yeast secretion assay revealed that these candidate effectors were secreted proteins.It was also found that StCFEM3,4,and 5 couldn't affect BAX/INF1-induced programmed cell death(PCD)in Nicotiana benthamiana and while StCFEM12 could suppress INF1-induced PCD,it showed no effect on BAXinduced PCD.This study found that there were 13 members of the S.turcica CFEM protein family and that StCFEM12 was a candidate effector.This study laid the foundation for illustrating the roles of CFEM proteins during the pathogenic processes of phytopathogens. 展开更多
关键词 CFEM domain EFFECTOR setosphaeria turcica signal peptide PCD
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Characterization of laccase gene StLAC6 and its involvement in the pathogenicity and peroxisome function in Setosphaeria turcica 被引量:1
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作者 LIU Ning ZHANG Qian-qian +4 位作者 JIA Hui ZHAO Bin ZHU Zi-ping CAO Zhi-yan DONG Jin-gao 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2022年第7期2019-2030,共12页
Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper ... Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall. The function of another major laccase gene, StLAC6, was studied here. The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. turcica, but the morphology and function of peroxisomes of knockout mutants were abnormal. The knockout of the StLAC6 gene resulted in increased contents of phenolic compounds and melanin and the sensitivity to fungicides increased compared with wild type strains. In the mutants of StLAC6, there is a significant change of the expression levels of other laccase genes. This study provides a new insight into laccase functions and the relationship of the laccase gene family in plant pathogenic fungi. 展开更多
关键词 setosphaeria turcica LACCASE StLAC6 PEROXISOME fungicides
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Transcriptional regulation of secondary metabolism and autophagy genes in response to DNA replication stress in Setosphaeria turcica
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作者 MENG Ya-nan ZHANG Xin-jie +8 位作者 ZENG Fan-li ZHAI Wei-bo LI Pan HU Jing-jing XIAO Sheng-lin HAO Zhi-min CAO Zhi-yan CHEN Chuan DONG Jin-gao 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2023年第4期1068-1081,共14页
The fungal pathogen Setosphaeria turcica causes northern corn leaf blight(NCLB),which leads to considerable crop losses.Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize ... The fungal pathogen Setosphaeria turcica causes northern corn leaf blight(NCLB),which leads to considerable crop losses.Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection.Previously,we demonstrated that the S.turcica triggers an S-phase checkpoint and ATR(Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection.However,how the regulatory mechanism works was still largely unknown.Here,we report a genome wide transcriptional profile analysis during appressorium formation in the present of DNA replication stress.We performed RNA-Seq analysis to identify S.tuicica genes responsive to DNA replication stress.In the current work,we found that appressorium-mediated maize infection by S.turcica is significantly blocked by S-phase checkpoint.A large serial of secondary metabolite and melanin biosynthesis genes were blocked in appressorium formation of S.turcica during the replication stress.The secondary metabolite biosynthesis genes including alcohol dehydrogenase GroES-like domain,multicopper oxidase,ABCtransporter families,cytochrome P450 and FAD-containing monooxygenase were related to plant pathogen infection.In addition,we demonstrated that autophagy in S.turcica is up-regulated by ATR as a defense response to stress.We identified StATG3,StATG4,StATG5,StATG7 and StATG16 genes for autophagy were induced by ATR-mediated S-phase checkpoint.We therefore propose that in response to genotoxic stress,S.turcica utilizes ATR-dependent pathway to turn off transcription of genes governing appressorium-mediated infection,and meanwhile inducing transcription of autophagy genes likely as a mechanism of self-protection,aside from the more conservative responses in eukaryotes. 展开更多
关键词 setosphaeria turcica APPRESSORIUM secondary metabolite AUTOPHAGY DNA replication stress
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Changes of SOD Activity in Corn Leaves Infected by Setosphaeria turcica with Different Pathogenicity
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作者 Yajun BAI Haiying LIU +1 位作者 Yongshan FAN Jiaojiao LI 《Plant Diseases and Pests》 CAS 2012年第2期51-53,共3页
[ Objective] The paper was to explore the relationship between SOD activity and resistance of corn against Setosphaeria turcica. [ Method] Strain YC and 01-23T of S. turcica with different pathogenicity were inoculate... [ Objective] The paper was to explore the relationship between SOD activity and resistance of corn against Setosphaeria turcica. [ Method] Strain YC and 01-23T of S. turcica with different pathogenicity were inoculated into corn leaves by fungal disc inoculation method, and the dynamic changes of SOD activity in infected corn leaves was measured. [Result] After inoculation of higly virulent strain YC and weakly virulent strain 01-23T of S. turcica, SOD activity in corn leaves within 1 -5 d showed obvious stages, which gradually decreased within 1 -3 d, then gradually increased within 3 -4 d, and decreased again within 4 -5 d. The SOD activity of corn leaves in inoculation treatment was higher than that in the treatment without inoculation within 1 - 4 d, which significantly decreased at fifth day. The pathogen with different pathogenicity would lead to different changes in SOD activity of corn leaves. SOD activity of corn leaves infected by weakly virulent strain 01-23T for the first day was extremely higher than that infected by strong virulent strain YC, but had no significant difference with highly virulent strain YC at 2 -3 d. [ Conclusion] SOD activity played an important role in initial resistant ability of corn against pathogen infection, and had no significant effect on resistance against pathogen extension after incidence. 展开更多
关键词 CORN setosphaeria turcica SOD activity PATHOGENICITY China
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玉米大斑病菌cDNA文库的构建及转录因子StMR1互作蛋白的筛选 被引量:1
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作者 王秋月 段鹏亮 +3 位作者 李海笑 刘宁 曹志艳 董金皋 《生物技术通报》 CAS CSCD 北大核心 2024年第6期281-289,共9页
【目的】筛选玉米大斑病菌(Setosphaeria turcica)转录因子的互作蛋白,解析黑色素调控转录因子StMR1调控玉米大斑病菌致病性的分子机制。为解析玉米大斑病菌侵染过程中转录因子的调控网络,阐明病菌的致病机理提供参考。【方法】收集玉... 【目的】筛选玉米大斑病菌(Setosphaeria turcica)转录因子的互作蛋白,解析黑色素调控转录因子StMR1调控玉米大斑病菌致病性的分子机制。为解析玉米大斑病菌侵染过程中转录因子的调控网络,阐明病菌的致病机理提供参考。【方法】收集玉米大斑病菌菌丝和孢子不同萌发阶段作为试验材料,采用Gateway方法构建玉米大斑病菌cDNA文库,使用同源重组的方法构建转录因子StMR1的诱饵载体,采用酵母双杂交技术筛选其互作蛋白并进行一对一验证。【结果】构建的玉米大斑病菌文库插入的平均片段长度大于1000 bp,初级文库及次级文库的库容量为1.2×107和1.04×107CFU,重组率为100%,可以用于酵母双杂交筛选。成功构建可以用于筛库的诱饵载体pGBKT7-StMR1,经初筛与复筛得到3个互作蛋白,一对一验证短链脱氢酶、糖基转移酶、富含亮氨酸重复序列蛋白均与转录因子StMR1存在互作。【结论】成功构建了丰富度高且质量好的玉米大斑病菌cDNA文库并筛选到了与转录因子StMR1互作的蛋白。 展开更多
关键词 玉米大斑病菌 CDNA文库 转录因子 酵母双杂交 互作蛋白
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玉米ACO基因家族生物信息学及表达模式分析
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作者 王程泽 张燕 +4 位作者 付伟 贾京哲 董金皋 申珅 郝志敏 《中国农业科学》 CAS CSCD 北大核心 2024年第7期1308-1318,共11页
【目的】对玉米1-氨基环丙烷-1-羧酸氧化酶ACO基因家族进行全基因组鉴定,分析其在玉米不同器官和不同发育时期以及响应外源激素和病菌侵染中的表达模式,为明确玉米ACO基因家族功能打下基础。【方法】利用生物信息学方法,在玉米B73自交... 【目的】对玉米1-氨基环丙烷-1-羧酸氧化酶ACO基因家族进行全基因组鉴定,分析其在玉米不同器官和不同发育时期以及响应外源激素和病菌侵染中的表达模式,为明确玉米ACO基因家族功能打下基础。【方法】利用生物信息学方法,在玉米B73自交系基因组中鉴定ACO,对其基因结构、蛋白质理化性质、家族成员间的亲缘关系以及保守基序进行分析,利用实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)技术分析ZmACO基因家族的表达模式。【结果】除ZmACO11外,ZmACO家族成员均具有Fe2+离子结合位点和底物抗坏血酸结合位点。系统发育分析显示,ZmACO_(2)与ScACO在同一分支,亲缘关系较近,Bootstrap值达98。基因表达分析表明,ZmACO_(2)、5、9、15、20、35在各发育时期均活跃表达,且在叶片中呈优势表达,因此选择上述6个基因进行下一步检测。喷施乙烯利后,上述6个基因的表达均有所波动,其中ZmACO_(2)的表达量受影响较大,变化幅度在8倍左右。在乙烯利处理的0—24 h内这6个基因的表达量存在波动,但在处理后24 h,6个基因的表达量均接近0。水杨酸处理后,ZmACO5的表达量受影响较大,变化倍数在2倍左右。其他基因的表达量在处理后24 h均接近0。ZmACO9、35在3—12 h的表达量存在波动,ZmACO_(2)、15、20表达量呈下调趋势。在响应生物胁迫方面,接种玉米大斑病菌(Setosphaeria turcica)后,ZmACO5、9的表达量变化幅度最大,在接种后第10天,这两个基因的表达量分别升至对照组的50和60倍。接种玉米小斑病菌(Cochlibolus heterostrophus)后,ZmACO5的表达量变化幅度较大,变化倍数在40—90倍。接种立枯丝核菌(Rhizoctonia solani)后,ZmACO5、35表达量变化幅度最大,在病菌接种的第3天达到200倍。【结论】ZmACO_(2)、5、20、35在玉米生长发育过程中表达变化最活跃;施加外源乙烯利和水杨酸可以对ZmACO的表达水平造成显著影响。病菌侵染玉米后ZmACO的表达水平产生显著变化,与生物胁迫应答关系密切。 展开更多
关键词 玉米ACO基因家族 实时荧光定量PCR 基因表达 乙烯利 水杨酸 玉米大斑病菌 玉米小斑病菌 立枯丝核菌
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玉米大斑病菌效应因子StSse19基因的克隆、表达模式分析及在原核系统中的表达
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作者 杨俊芳 尹贵波 +3 位作者 周启慧 刘玉卫 巩校东 谷守芹 《河北农业大学学报》 CAS CSCD 北大核心 2024年第3期66-71,104,共7页
玉米大斑病是由玉米大斑病菌引起的玉米叶部真菌性病害。本课题组前期通过对病菌侵染玉米的转录组分析发现,效应因子StSse19为重要的致病因子,但对其结构及功能尚不清晰。本研究以野生型菌株01-23的cDNA为模版,克隆StSse19并对其结构进... 玉米大斑病是由玉米大斑病菌引起的玉米叶部真菌性病害。本课题组前期通过对病菌侵染玉米的转录组分析发现,效应因子StSse19为重要的致病因子,但对其结构及功能尚不清晰。本研究以野生型菌株01-23的cDNA为模版,克隆StSse19并对其结构进行生物信息学预测;结合对病菌侵染玉米的RNA-Seq数据分析明确该基因的表达模式,进而利用qRT-PCR技术证实其表达模式;构建StSse19的融合表达载体pET28a-StSse19,在大肠杆菌BL21(DE3)中以IPTG诱导表达该目的基因,通过SDS-PAGE技术检测其表达情况。结果表明,StSse19的CDS序列由330个核苷酸组成,编码109个氨基酸残基,序列比对发现该蛋白无同源蛋白,表明其为大斑病菌的特异效应蛋白;对StSse19的表达模式分析发现,该基因在侵染玉米24 h的表达量显著提高,72 h表达量下降,推测该基因参与病菌的侵染过程;将StSse19在原核系统中进行表达,SDS-PAGE分析结果显示,该蛋白大小为28 kD,用Ni柱层析法结合Western blot检测显示获得了纯化的目的蛋白。该研究明确了StSse19基因的结构特征及表达模式、获得了StSse19在原核体系中的表达蛋白,为深入揭示其功能及作用机制奠定了基础。 展开更多
关键词 玉米大斑病菌 效应因子 StSse19 原核表达 QRT-PCR
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玉米大斑病菌细胞周期蛋白依赖性激酶结构亚基StCks1鉴定及其基因功能分析
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作者 张博文 赵丽雯 +4 位作者 徐璐 李盼 曾凡力 孟亚南 董金皋 《中国农业科学》 CAS CSCD 北大核心 2024年第5期900-908,共9页
【目的】Cks1(cyclin-dependent kinase subunit 1)是细胞周期蛋白依赖性激酶复合体CDK(cyclin-dependent kinase)的结构亚基,是细胞周期调控过程中的关键基因。论文旨在鉴定玉米大斑病菌(Setosphaeria turcica)StCks1,分析玉米大斑病... 【目的】Cks1(cyclin-dependent kinase subunit 1)是细胞周期蛋白依赖性激酶复合体CDK(cyclin-dependent kinase)的结构亚基,是细胞周期调控过程中的关键基因。论文旨在鉴定玉米大斑病菌(Setosphaeria turcica)StCks1,分析玉米大斑病菌附着胞发育过程中StCks1表达量差异及其互作蛋白,为进一步研究StCks1在附着胞发育中的作用打下基础。【方法】通过对玉米大斑病菌野生型菌株01-23全基因组数据分析,获取StCks1蛋白序列;利用软件MEGA 5.0和在线数据库Pfam、SMART对酿酒酵母、裂殖酵母和拟南芥等物种Cks1蛋白进行系统发育树构建和保守结构域分析;收集玉米大斑病菌附着胞发育不同时期样品进行转录组测序以及表达量分析。利用原核诱导表达系统,构建原核表达载体pGEX-6p-3-StCks1,并转化大肠杆菌表达菌株BL21,对重组蛋白GST-StCks1诱导表达纯化;通过GST pull-down、Western blot技术和酵母双杂交试验,鉴定StCks1的互作蛋白。【结果】玉米大斑病菌全基因组中鉴定到唯一StCks1蛋白编码基因,该蛋白由130个氨基酸组成;其三级结构主要由4股β折叠和3个α螺旋构成,含有HxPEPH(His-anyPro-Glu-Pro-His)保守位点和Cks保守结构域;通过转录组测序和表达量分析发现,StCks1在附着胞发育不同时期表达量显著上调;重组蛋白GST-StCks1在1 mmol·L^(-1) IPTG,30℃诱导2 h可获得大量可溶性蛋白;通过GST pull-down技术获取大量互作蛋白并进行质谱鉴定,通过Western blot和酵母双杂交试验,验证StCks1与细胞周期蛋白依赖性激酶CDK1存在互作。【结论】玉米大斑病菌中含有唯一的StCks1,通过与细胞周期蛋白依赖性激酶CDK1互作调控附着胞的发育。 展开更多
关键词 玉米大斑病菌 细胞周期调控 StCks1 原核诱导表达纯化 GST pull-down
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不同诱导因素对玉米大斑病菌附着胞产生的影响 被引量:17
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作者 范永山 曹志艳 +1 位作者 谷守芹 董金皋 《中国农业科学》 CAS CSCD 北大核心 2004年第5期769-772,共4页
附着胞是许多叶部病原真菌的重要侵染机构。在不同温度、光照条件、基质和培养时间下,对玉米大斑病菌1号小种和2号小种分别在玻璃平板和玉米叶片上进行附着胞的室内诱导,发现分生孢子的水悬浮液在玻璃平板和玉米叶片的上表面及下表面都... 附着胞是许多叶部病原真菌的重要侵染机构。在不同温度、光照条件、基质和培养时间下,对玉米大斑病菌1号小种和2号小种分别在玻璃平板和玉米叶片上进行附着胞的室内诱导,发现分生孢子的水悬浮液在玻璃平板和玉米叶片的上表面及下表面都可产生附着胞,附着胞都能正常萌发并产生侵入丝,但在玉米叶片上附着胞和侵入丝的产生时间要晚于玻璃平板,产生数量也少。玉米大斑病菌分生孢子在PD培养基中产生的附着胞数目少于清水处理。影响玉米大斑病菌附着胞生长和发育的决定因素是温度,在玻璃平板上10~36℃培养24h后就可以产生附着胞,但最适宜的温度是18~25℃,低于15℃和高于30℃都不利于附着胞的产生。不同菌株在同一玉米品种的叶片上产生的附着胞数量不同,但是同一菌株在抗病品种和感病品种间产生的附着胞数量差异不显著。 展开更多
关键词 诱导因素 玉米 大斑病菌 附着胞 温度 丝状真菌 寄主植物 互作
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玉米大斑病菌MAPK超家族的全基因组鉴定及途径模型建立 被引量:15
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作者 巩校东 张晓玉 +8 位作者 田兰 王星懿 李坡 张盼 王玥 范永山 韩建民 谷守芹 董金皋 《中国农业科学》 CAS CSCD 北大核心 2014年第9期1715-1724,共10页
【目的】从全基因组水平鉴定玉米大斑病菌(Setosphaeria turcica)的MAPK超家族基因,并对其进行系统进化、基因结构、多重序列比对及保守位点分析,构建玉米大斑病菌中的MAPK级联途径模型,为深入研究该植物病原菌中MAPK级联途径的功能奠... 【目的】从全基因组水平鉴定玉米大斑病菌(Setosphaeria turcica)的MAPK超家族基因,并对其进行系统进化、基因结构、多重序列比对及保守位点分析,构建玉米大斑病菌中的MAPK级联途径模型,为深入研究该植物病原菌中MAPK级联途径的功能奠定基础。【方法】利用玉米大斑病菌基因组数据库,通过基于隐马尔科夫模型的HMMER 3.0软件搜索基因组,鉴定并获得MAPK超家族成员序列、基因组定位信息;采用MEGA 5.0软件进行系统进化分析;通过GSDS工具进行基因结构分析;利用ClustalX、MEME工具分析MAPK蛋白激酶区的保守性及保守位点。【结果】在玉米大斑病菌基因组中发现了4个MAPK基因、3个MAPKK基因和3个MAPKKK基因。系统进化分析将MAPK分为Kss1/Fus3、Slt2、Hog1及Ime2 4类;MAPKK分为Pbs2、Ste7及Mkk1 3类;MAPKKK分为Ste11、Bck1及Ssk2 3类。基因组定位及基因结构分析表明,MAPK超家族散布在基因组中,且MAPK基因的结构最为复杂多样,MAPKK次之,MAPKKK结构最简单。多重序列比对与保守位点分析表明,玉米大斑病菌MAPK超家族的激酶结构域均高度保守,其中MAPK具有保守的"-TxY-"磷酸化位点,MAPKK含有保守的"-SD[V/I]WS-"磷酸化位点,MAPKKK含有"-G[S/T][V/P][F/M][W/Y]M[A/S]PEV-"特异性保守位点。【结论】全基因组分析表明,玉米大斑病菌中包括4个MAPK基因、3个MAPKK基因和3个MAPKKK基因。通过系统进化、基因结构及多重序列比对和保守位点分析,在玉米大斑病菌中构建了Fus3/Kss1-homolog、Slt2-homolog、Hog1-homolog和Ime2-homolog4条MAPK级联途径,其中Ime2 homolog为一条新发现的MAPK级联途径。该信息为深入解析植物病原真菌MAPK超家族的功能奠定了基础。 展开更多
关键词 玉米大斑病菌 MAPK基因超家族 系统进化 保守基序
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我国玉米大斑病菌生理小种种群动态分析 被引量:23
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作者 赵辉 高增贵 +2 位作者 张小飞 庄敬华 隋鹤 《沈阳农业大学学报》 CAS CSCD 北大核心 2008年第5期551-555,共5页
为明确近年来中国玉米大斑病菌(Setosphaeria turcica)的生理小种变化和种群消长情况,分析玉米大斑病逐年加重的原因。采用常规Ht单基因(Ht1、Ht2、Ht3和HtN)鉴别寄主,对2005年和2006年采自黑龙江、吉林、辽宁、河北、山东、陕西、山西... 为明确近年来中国玉米大斑病菌(Setosphaeria turcica)的生理小种变化和种群消长情况,分析玉米大斑病逐年加重的原因。采用常规Ht单基因(Ht1、Ht2、Ht3和HtN)鉴别寄主,对2005年和2006年采自黑龙江、吉林、辽宁、河北、山东、陕西、山西、河南、四川和北京-天津-唐山地区共10个省区28个地区的204个玉米大斑病菌菌株进行了生理小种鉴定;利用毒力频率法分析玉米大斑病菌的生理分化及其在中国的分布频率。结合前人研究结果,分析了近30年中国玉米大斑病菌生理小种变化趋势。共鉴定出0,1,2,3,12,13,23,123,23N共9个生理小种,其中0号小种和1号小种为优势小种,分别占供试菌株的62.25%和19.12%,其他小种占供试菌株的18.63%;所鉴定的204个菌株,对Ht1抗性基因的毒性频率最高,为33.33%;对HtN抗性基因的毒性频率最低,为1.47%;对Ht2和Ht3抗性基因的毒性频率分别为16.18%和6.37%。玉米大斑病菌生理分化明显,呈现复杂化,不断有新小种出现。Ht1和Ht2抗性基因在中国大部分玉米产区均有不同程度的抗性"丧失"。玉米大斑病菌新小种出现、0号和1号小种以外其他生理小种出现频率的的升高和品种抗性的"丧失"是玉米大斑病发生的重要原因。 展开更多
关键词 玉米大斑病菌 生理小种 毒性频率
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玉米大斑病菌ATMT突变体库的构建及其分析 被引量:15
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作者 王梅娟 李坡 +3 位作者 吴敏 范永山 谷守芹 董金皋 《中国农业科学》 CAS CSCD 北大核心 2012年第12期2384-2392,共9页
【目的】利用农杆菌(Agrobacterium tumefaciens)介导的遗传转化技术,对玉米大斑病菌(Setosphaeria turcica)进行转化,构建ATMT突变体库,为从分子水平上揭示病菌的致病机理奠定基础。【方法】以带有重组双元载体的农杆菌对玉米大斑病菌... 【目的】利用农杆菌(Agrobacterium tumefaciens)介导的遗传转化技术,对玉米大斑病菌(Setosphaeria turcica)进行转化,构建ATMT突变体库,为从分子水平上揭示病菌的致病机理奠定基础。【方法】以带有重组双元载体的农杆菌对玉米大斑病菌进行转化,利用潮霉素B进行筛选,对抗性稳定的转化子进行PCR检测,构建玉米大斑病菌ATMT突变体库;从突变体库中随机选取一定数量的突变体,对其菌落形态、菌丝及分生孢子发育、致病性等进行分析。【结果】获得了1 265株玉米大斑病菌T-DNA插入突变体;从中随机选取36株突变菌株,对其进行抗性筛选和PCR检测,发现潮霉素磷酸转移酶基因已整合进入野生型菌株的基因组中,且能够稳定遗传;与野生型菌株相比,在供试的菌株中,大部分菌株菌落形态和生长速率没有发生明显改变。生长速率明显减慢的菌株占总数的13.8%,明显加快的菌株占16.7%;发现了2株分生孢子形态发生明显改变的菌株,占菌株总数的5.6%;产孢量明显增大的菌株占5.6%,产孢量减少的菌株占13.5%;分生孢子萌发率发生明显改变的菌株占16.6%;发现了1株致病性明显增强的菌株,占菌株总数的2.8%。【结论】构建了玉米大斑病菌ATMT突变体库,并对突变体库进行了初步分析,将为克隆玉米大斑病菌生长发育和致病性相关基因奠定基础。 展开更多
关键词 玉米大斑病菌 ATMT 突变体库 T-DNA插入突变体
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玉米大斑病菌聚酮体合成酶基因StPKS功能分析 被引量:15
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作者 张鑫 曹志艳 +2 位作者 刘士伟 郭丽媛 董金皋 《中国农业科学》 CAS CSCD 北大核心 2011年第8期1603-1609,共7页
【目的】利用RNA干扰技术探讨玉米大斑病菌聚酮体合成酶基因StPKS的功能。【方法】将玉米大斑病菌StPKS基因片段连接到pSilent-1载体中,构建StPKS的RNA干扰载体pSilent-StPKS1-2。利用聚乙二醇(PEG)介导的方法将之转入玉米大斑病菌野生... 【目的】利用RNA干扰技术探讨玉米大斑病菌聚酮体合成酶基因StPKS的功能。【方法】将玉米大斑病菌StPKS基因片段连接到pSilent-1载体中,构建StPKS的RNA干扰载体pSilent-StPKS1-2。利用聚乙二醇(PEG)介导的方法将之转入玉米大斑病菌野生型菌株01-23的原生质体中,通过潮霉素筛选得到转化子,采用半定量RT-PCR方法分析转化子中StPKS的表达情况,显微观察转化子与野生型菌丝形态的差异。【结果】本研究构建了StPKS RNA干扰载体并进行了成功转化,经潮霉素抗性筛选和PCR验证最终获得了9个阳性转化子,其中5个转化子菌落颜色变浅。对转化子进行半定量PCR分析发现,5株转化子的StPKS表达量均有所下降;黑色素产量显著降低;菌丝呈不规则状,发生了膨大、变形、分枝等现象。【结论】StPKS在DHN黑色素合成途径中起作用,其表达量下降会减少黑色素的产生。 展开更多
关键词 玉米大斑病菌 聚酮体合成酶基因 DHN黑色素 RNA干扰
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DHN黑色素与玉米大斑病菌附着胞膨压形成的关系 被引量:15
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作者 曹志艳 贾慧 +1 位作者 朱显明 董金皋 《中国农业科学》 CAS CSCD 北大核心 2011年第5期925-932,共8页
【目的】探讨玉米大斑病菌胞内DHN黑色素在附着胞膨压产生过程中的作用,明确玉米大斑病菌的侵入机理。【方法】通过诱导玉米大斑病菌附着胞产生,确定附着胞形成的最佳条件,利用溶质排斥技术及incipient-cytorrhysis技术对玉米大斑病菌... 【目的】探讨玉米大斑病菌胞内DHN黑色素在附着胞膨压产生过程中的作用,明确玉米大斑病菌的侵入机理。【方法】通过诱导玉米大斑病菌附着胞产生,确定附着胞形成的最佳条件,利用溶质排斥技术及incipient-cytorrhysis技术对玉米大斑病菌野生型菌株01-23和黑色素缺失突变体△St3hnr附着胞细胞壁孔径大小和膨压进行测定。【结果】野生型菌株01-23细胞壁孔径范围是2.1—2.7 nm,膨压在5.4 MPa左右;黑色素缺失突变体△St3hnr细胞壁孔径范围是2.7—3.3 nm,膨压在4.1 MPa左右;缺乏黑色素的附着胞不能形成高膨压,丧失了穿透能力。【结论】黑色素层对溶质分子外渗的阻挡作用导致了附着胞高膨压的产生,膨压产生的机械穿透力在玉米大斑病菌穿透基质平面的过程中发挥了重要作用。 展开更多
关键词 玉米大斑病菌 附着胞 溶质排斥技术 incipient-cytorrhysis技术 膨压
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