玉米大斑病菌MAPK基因StIME2的基因组定位蛋白质结构预测及表达分析

doi:10.3864/j.issn.0578-1752.2015.13.007

摘要/Abstract

摘要： 【目的】确定玉米大斑病菌（Setosphaeria turcica）MAPK基因StIME2在基因组中的位置；系统解析目的蛋白质StIme2的结构特征；分析玉米大斑病菌StIME2在不同发育时期及不同胁迫条件下（温度、氧胁迫、高渗胁迫）的表达，为深入研究该基因的功能奠定基础。【方法】通过本地Blast搜索玉米大斑病菌基因组数据库，确定StIME2在基因组的精确位置；利用ProtParam在线分析StIme2蛋白的理化性质，利用SOMPA在线软件预测StIme2蛋白的二级结构。通过PHYRE2在线服务器对StIme2蛋白的三维结构进行预测。利用半定量RT-PCR方法分析不同发育时期及不同胁迫条件下StIME2的表达。【结果】玉米大斑病菌StIME2为一类与酿酒酵母中ScIME2具有较高同源性的基因，为在植物病原真菌中鲜有报道的MAPK基因。该基因的ID为98 105，位于scaffold_7正链的1 560 184—1 562 574位置，StIme2蛋白具有MAPK类蛋白激酶的特征性保守结构域，其二级结构主要以α-螺旋和无规则卷曲为主，β-折叠较少且主要存在于N端，其三级结构具有1个较小的N端域和1个较大的C端域；该基因在病菌分生孢子时期表达量最高，附着胞发育时期表达量最低。将病菌置于不同温度条件下处理，发现培养温度为28℃时StIME2表达量达到最高。在高渗胁迫条件下，随NaCl浓度的增加，StIME2的表达量逐渐增加，但在较高胁迫条件下（0.8 mol·L^-1 NaCl），该基因表达几乎被完全抑制。经H₂O₂胁迫处理后，StIME2的表达量随H₂O₂的浓度增加而增强，在10 mmol·L^-1 H₂O₂的处理下表达量最高。【结论】玉米大斑病菌StIME2位于scaffold_7正链的1 560 184—1 562 574位置。StIme2蛋白具有MAPK激酶的所有特征性保守结构域，为一类功能鲜有报道的MAPK蛋白激酶。该基因在玉米大斑病菌分生孢子时期表达量最高，推测可能在调控病菌分生孢子发育过程中起重要作用。该基因的表达水平可随培养温度的变化而变化，28℃表达量最高。该基因可能参与病菌的高渗胁迫及氧胁迫反应。

关键词: 玉米大斑病菌, StIME2, 生物信息学分析, 基因表达

Abstract: 【Objective】The objectives of this study are to determine the location in genome, investigate the protein structure of StIME2 of Setosphaeria turcica, and to compare the expression level of StIME2 at different developmental stages and under different stress conditions, such as temperature, oxygen stress, hyperosmolar stress, hence laying a foundation for future studying functions of StIME2.【Method】 Blast search tool was used to determine the exact location of StIME2 in genome of S. turcica. Physical and chemical properties of StIme2 was analyzed by employing online software ProtParam, and secondary structure of StIme2 was predicted by applying Software SOMPA. Three-dimensional structure of StIme2 was doped out using PHYRE2 server and the structure model was evaluated using online software SAVES and the aim was to obtain Laplace image (Ramachandran Plot). StIME2 expression level was detected at different developmental stages and stress conditions based on semi-quantitative RT-PCR analysis. 【Result】The identity of StIME2 was 98 105, located between 1 560 184 and 1 562 574 in the positive-strand of scaffold_7 in the genome of S. turcica. StIme2 shared characteristic conserved domains of MAPK-like protein kinase, α-helices and random coils, which were its main elements of secondary structure of StIme2. β strands were less and mainly located in N-terminal in this structure. Three-dimensional structure of the protein was composed of a smaller N-terminal and a bigger C-terminal. The expression level of StIME2 was the highest in conidium developmental periods, and was the lowest during appressorium development stage. Under different stress conditions, the expression level of StIME2 appeared the highest when the incubation temperature was at 28℃. In hyperosmotic stress conditions, the expression level of StIME2 increased with the increase of NaCl concentration, but the expression level of the gene was almost completely inhibited at higher stress conditions (0.8 mol·L^-1 NaCl). Under oxygen stress conditions, in H₂O₂ stress treatment, the expression level of StIME2 was heightened with the increase of the concentration of H₂O₂. And the expression level of the gene was the highest under 10 mmol·L^-1 H₂O₂. 【Conclusion】 StIME2 was located between 1 560 184 and 1 562 574 in the positive-strand of scaffold_7 in the genome of S. turcica. The characteristics of StIme2 were analyzed, and the result showed that StIme2 shared all conserved domains of MAPK kinase of plant pathogenic fungi. However, unlike three types of MAPK genes, which were widely reported in plant pathogens, StIme2 was rarely reported. The expression level of StIME2 in conidium developmental period in S. turcica was the highest. Thus, the authors speculated that StIME2 could play an important role in regulating the developmental processes in spore of the pathogen. Moreover, the expression level of StIME2 changed with the temperature and the highest expression level was at 28℃. The gene may be also involved in osmotic stress and oxygen stress reaction of the pathogen.

Key words: Setosphaeria turcica, StIME2, bioinformatics analysis, gene expression

巩校东，王玥，张盼, 范永山, 谷守芹，韩建民，董金皋. 玉米大斑病菌MAPK基因StIME2的基因组定位蛋白质结构预测及表达分析[J]. 中国农业科学, 2015, 48(13): 2549-2558.

GONG Xiao-dong, WANG Yue, ZHANG Pan, FAN Yong-shan, GU Shou-qin, HAN Jian-min, DONG Jin-gao. Analysis of the Genomic Location, Protein Structure Prediction and Expression of MAPK Gene StIME2 in Setosphaeria turcica[J]. Scientia Agricultura Sinica, 2015, 48(13): 2549-2558.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2015.13.007

https://www.chinaagrisci.com/CN/Y2015/V48/I13/2549

参考文献

[1] 孙淑琴, 温雷蕾, 董金皋. 玉米大斑病菌的生理小种及交配型测定. 玉米科学, 2005, 13(4): 112-113, 123.

Sun S Q, Wen L L, Dong J G. Identification of physiological races and mating type of Exserohilum turcicum. Journal of Maize Sciences, 2005, 13(4): 112-113, 123. (in Chinese)

[2] Degefu Y, Lohtander K, Paulin L. Expression patterns and phylogenetic analysis of two xylanase genes (htxyl1 and htxyl2) from Helminthosporium turcicum, the cause of northern leaf blight of maize. Biochimie, 2004, 86(2): 83-90.

[3] Zhao X H, Mehrabi R, Xu J R. Mitogen-activated protein kinase pathways and fungal pathogenesis. Eukaryotic Cell, 2007, 6(10): 1701-1714.

[4] Brefort T, Müller P, Kahmann R. The high-mobility-group domain transcription factor Rop1 is a direct regulator of prf1 in Ustilago maydis. Eukaryotic Cell, 2005, 4(2): 379-391.

[5] Yang S H, Sharrocks A D, Whitmarsh A J. Transcriptional regulation by the MAP kinase signaling cascades. Gene, 2003, 320(3): 3-21.

[6] Romeis T. Protein kinases in the plant defence response. Current Opinion in Plant Biology, 2001, 4: 407-414.

[7] 范永山, 刘颖超, 谷守芹, 桂秀梅, 董金皋. 植物病原真菌的MAPK基因及其功能. 微生物学报, 2004, 44(4): 547-551.

Fan Y S, Liu Y C, Gu S Q, Gui X M, Dong J G. Mitogen activated protein kinase genes and its functions in phytopathogenic fungus. Acta Microbiologica Sinica, 2004, 44(4): 547-551. (in Chinese)

[8] Idnurm A, Howlett B J. Pathogenicity genes of phytopathogenic fungi. Molecular Plant Pathology, 2001, 2(4): 241-255.

[9] Krens S F, Spaink H P, Snaar-Jagalska B E. Functions of the MAPK family in vertebrate-development. FEBS Letters, 2006, 580(21): 4984-4990.

[10] Chen R E, Thorner J. Function and regulation in MAPK signaling pathways: Lessons learned from the yeast Saccharomyces cerevisiae. Biochimica et Biophysica Acta, 2007, 1773(8): 1311-1340.

[11] Harel A, Bercovich S, Yarden O. Calcineurin is required for sclerotial development and pathogenicity of Sclerotinia sclerotiorum in an oxalic acid-independent manner. Molecular Plant-Microbe Interactions, 2006, 19(6): 682-693.

[12] Hamel L P, Nicole M C, Duplessis S, Ellis B E. Mitogen-activated protein kinase signaling in plant-interacting fungi: Distinct messages from conserved messengers. The Plant Cell, 2012, 24(4): 1327-1351.

[13] Xu J R. MAP kinases in fungal pathogens. Fungal Genetics and Biology, 2000, 31(3): 137-152.

[14] Garrido E, Voss U, Müller P, Castillo-Lluva S, Kahmann R, Pérez M J. The induction of sexual development and virulence in the smut fungus Ustilago maydis depends on Crk1, a novel MAPK protein. Genes & Development, 2004, 18(24): 3117-3130.

[15] 巩校东, 张晓玉, 田兰, 王星懿, 李坡, 张盼, 王玥, 范永山, 韩建民, 谷守芹, 董金皋. 玉米大斑病菌MAPK超家族的全基因组鉴定及途径模型建立. 中国农业科学, 2014, 47(9): 1715-1724.

Gong X D, Zhang X Y, Tian L, Wang X Y, Li P, Zhang P, Wang Y, Fan Y S, Han J M, Gu S Q, Dong J G. Genome-wide identification MAPK superfamily and establishment of the model of MAPK cascade pathway in Setosphaeria turcica. Scientia Agricultura Sinica, 2014, 47(9): 1715-1724. (in Chinese)

[16] 杨鹏雅, 李开绵, 周建国, 王文泉. 蓖麻WRKY基因的电子克隆及其生物信息学分析. 现代农业科学, 2009, 16(5): 22-25.

Yang P Y, Li K M, Zhou J G, Wang W Q. Identification and bioinformatical analysis of novel WRKY gene from Ricinus Communis. Modern Agricultural Sciences, 2009, 16(5): 22-25. (in Chinese)

[17] 武雪, 黄晓丽, 王喆之. 葡萄乙醇脱氢酶基因Ⅲ的电子克隆及生物信息学分析. 生物技术通报, 2009(5): 71-75.

Wu X, Huang X L, Wang Z Z. Electronic cloning and characterizations of ADHⅢ gene from vitisvinifera using bioinformatics tools. Biotechnology Bulletin, 2009(5): 71-75. (in Chinese)

[18] 张鑫, 曹志艳, 刘士伟, 郭丽媛, 董金皋. 玉米大斑病菌聚酮体合成酶基因StPKS功能分析. 中国农业科学, 2011, 44(8): 1603-1609.

Zhang X, Cao Z Y, Liu S W, Guo L Y, Dong J G. Functional analysis of polyketide synthase gene StPKS in Setosphaeria turcica. Scientia Agricultura Sinica, 2011, 44(8): 1603-1609. (in Chinese)

[19] Krylov D M,Nasmyth K,Koonin E V.Evolution of eukaryotic cell cycle regulation: stepwise addition of regulatory kinases and late advent of the CDKs.Current Biology, 2003, 13(2):173-177.

[20] Schindler K,Benjamin K R,Martin A,Boglioli A,Herskowitz I, Winter E.The Cdk-activating kinase Cak1p promotes meiotic S phase through Ime2p.Molecular and Cellular Biology, 2003, 23(23):8718-8728.

[21] Schindler K, Winter E.Phosphorylation of Ime2 regulates meiotic progression inSaccharomyces cerevisiae. The Journal of Biological Chemistry, 2006, 281(27):18307-18316.

[22] McDonald C M, Wagner M, Dunham M J, Shin M E, Ahmed N T, Winter E. The Ras/cAMP pathway and the CDK-Like Kinase Ime2 regulate the MAPK Smk1 and spore morphogenesis in Saccharomyces cerevisiae. Genetics, 2009, 181(2): 511-523.

[23] Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 2000, 24(1): 45-66.

[24] Xia Z, Dickens M, Raingeaud J, Davis R J, Greenberg M E. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science, 1995, 270(5240): 1326-1331.

[25] Johnson G L, Lapadat R L. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 2002, 298(5600): 1911-1912.

[26] Cook J G, Bardwell L, Thorner J. Inhibitory and activating functions for MAPK Kss1 in the S. cerevisiae filamentous-growth signalling pathway. Nature, 1997, 390: 85-88.

[27] Brückner S, Köhler T, Braus G H, Heise B, Bolte M, Mösch H U. Differential regulation of Tec1 by Fus3 and Kss1 confers signaling specificity in yeast development. Current Genetics, 2004, 46(6): 331-342.

[28] Yang H Y, Tatebayashi K, Yamamoto K, Saito H. Glycosylation defects activate filamentous growth Kss1 MAPK and inhibit osmoregulatory Hog1 MAPK. The EMBO Journal, 2009, 28(10): 1380-1391.

[29] Strudwick N, Brown M, Parmar V M, Schroder M. Ime1 and Ime2 are required for pseudohyphal growth of Saccharomyces cerevisiae on nonfermentable carbon sources. Molecular and Cellular Biology, 2010, 30(23): 5514-5530.

[30] Hutchison E A, Bueche J A, Glass N L. Diversification of a protein kinase cascade: IME-2 is involved in nonself recognition and programmed cell death in Neurospora crassa. Genetics, 2012, 192(2): 467-482.

[31] Garrido E, Pérez-Martín J. The crk1 gene encodes an Ime2-related protein that is required for morphogenesis in the plant pathogen Ustilago maydis. Molecular Microbiology, 2003, 47(3): 729-743.