玉米大斑病菌亲环素基因的克隆及表达规律分析
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摘要
利用简并引物PCR结合RACE技术获得S. turcica中亲环素基因的全长,并通过Real-time PCR技术检测该基因在病菌侵染结构发育过程中的表达模式。结果表明,玉米大斑病菌亲环素基因开放阅读框全长1 125 bp,3′UTR 154 bp,5′UTR 93 bp,编码374个氨基酸,将此基因命名为CyPs1,并将其cDNA序列提交GenBank,获得登录号EU679371.1,Protein ID为ACD62431.1。系统发育树分析显示,CyPs1与玉米小斑病菌(Bipolaris maydis)、蓝莓枯枝病菌(Neofusicoccum parvum)等物种的亲环素同源性可达到90%以上。该基因在病菌分生孢子萌发、附着胞形成及侵染阶段均有表达,至附着胞形成和侵染菌丝形成阶段,转录水平分别升至分生孢子时期的2倍和3倍。
Cyclophilin is a highly conserved protein family and involved in the development and function of pathogenic fungi. This study aimed to obtain the cyclophilin gene and clarify cyclophilin regulating mechanism during the morphogenesis and pathogenicity of Setosphaeria turcica. The full length of the cyclophilin gene was obtained through degenerate polymerase chain reaction(PCR) combined with rapid-amplification of c DNA ends(RACE) technique and its expression patterns were detected at different developmental stages of S. turcica based on quantitative real-time PCR(q RT-PCR) analysis. The main results were as follows, the full-length open reading frame(ORF) of the cyclophilin gene was 1 125 bp[with a 3′-untranslated region(UTR) of 154 bp and a 5′-UTR of93 bp] and encoded a protein of 374 amino acids. The gene was named as Cy Ps1 and submitted to Gen Bank with the accession no. EU679371.1 and Protein ID ACD62431.1. phylogenetic tree analysis showed that it contained more than 90% of the homologues in Bipolaria maydis and Neofusicoccum parvum respectively; in addition. Cy Ps1 continuously expressed during the developmental process of S. turcica, with the transcription levels at appressorium formation and invasive mycelial morphogenesis stages were respectively increased to 2 and 3 times of that at conidia.
Cyclophilin is a highly conserved protein family and involved in the development and function of pathogenic fungi. This study aimed to obtain the cyclophilin gene and clarify cyclophilin regulating mechanism during the morphogenesis and pathogenicity of Setosphaeria turcica. The full length of the cyclophilin gene was obtained through degenerate polymerase chain reaction(PCR) combined with rapid-amplification of c DNA ends(RACE) technique and its expression patterns were detected at different developmental stages of S. turcica based on quantitative real-time PCR(q RT-PCR) analysis. The main results were as follows, the full-length open reading frame(ORF) of the cyclophilin gene was 1 125 bp[with a 3′-untranslated region(UTR) of 154 bp and a 5′-UTR of93 bp] and encoded a protein of 374 amino acids. The gene was named as Cy Ps1 and submitted to Gen Bank with the accession no. EU679371.1 and Protein ID ACD62431.1. phylogenetic tree analysis showed that it contained more than 90% of the homologues in Bipolaria maydis and Neofusicoccum parvum respectively; in addition. Cy Ps1 continuously expressed during the developmental process of S. turcica, with the transcription levels at appressorium formation and invasive mycelial morphogenesis stages were respectively increased to 2 and 3 times of that at conidia.
引文
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[12]Wang P,Cardenas M E,Cox G M,et al.Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans[J].EMBO Reports,2001,2(6):511-518.
[13]Viaud M C,Balhadère P V,Talbot N J.A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection[J].Plant Cell,2002,14(4):917-930.
[14]杜茜,徐文静,赵洪锟,等.白僵菌亲环素蛋白基因Bb CPLA的克隆与分析[J].玉米科学,2008,16(6):117-120.Du Q,Xu W J,Zhao H K,et al.Cloning and analysis of a cyclophilin gene Bb CPLA from beauveria bassiana[J].Journal of Maize Sciences,2008,16(6):117-120.(in Chinese)
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[16]李志勇,郝志敏,司贺龙,等.玉米大斑病菌Ca M基因的克隆及三氟拉嗪的抑菌作用[J].植物病理学报,2008,38(3):277-282.Li Z Y,Hao Z M,Si H L,et al.Cloning Ca M(Calmodulin)gene and inhibitory effect of trifluoperazine on Setosphaeria turcica[J].Acta Phytopathologica Sinica,2008,38(3):277-282.(in Chinese)
[17]郝志敏,郝杰,李志勇,等.磷脂酶C特异性抑制剂U-73122对玉米大斑病菌分生孢子萌发及毒素活性的影响[J].华北农学报,2010,25(6):118-121.Hao Z M,Hao J,Li Z Y,et al.Effects of phospholipase C inhibitor U-73122 on conidial germination and HT-toxin activity of Setosphaeria turcica[J].Acta Agriculturae Boreali-Sinica,2010,25(6):118-121.(in Chinese)
[18]Hao Z M,Tong Y M,Han Y,et al.Molecular characterization of Stpka C2 and expression patterns of both PKA-c isoforms during the invasive growth of Setosphaeria turcica[J].Tropical Plant Pathology,2015,40(4):244-250.
[19]Zhang X C,Wang W D,Wang J S,et al.PPIase independent chaperone-like function of recombinant human Cyclophilin A during arginine kinase refolding[J].Febs Letters,2013,587(6):666-672.
[20]Walsh C T,Zydowsky L D,Mckeon F D.Cyclosporin A,the cyclophilin class of peptidylprolyl isomerases,and blockade of T cell signal transduction[J].Journal of Biological Chemistry,1992,267(19):13115-13118.
[21]Liu J,Jr F J,Lane W S,et al.Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes[J].Cell,1991,66(4):807-815.
[22]Lee Y K,Hong C B,Suh Y,et al.A c DNA clone for cyclophilin from Griffithsia japonica and phylogenetic analysis of cyclophilins[J].Molecules&Cells,2002,13(1):12-20.
[2]Sakaguchi A,Miyaji T,Tsuji G,et al.Kelch repeat protein Clakel2p and calcium signaling control appressorium development in Colletotrichum lagenarium[J].Eukaryotic Cell,2008,7(1):102-111.
[3]Lee S C,Lee Y H.Calcium/calmodulin-dependent signaling for appressorium formation in the plant pathogenic fungus Magnaporthe grisea[J].Molecules&Cells,1998,8(6):698-704.
[4]李俊霞,陈颕,王立安.钙信号途径参与小斑病菌致病过程的调控[J].微生物通报,2005,32(4):1-4.Li J X,Chen Y,Wang L A.Ca2+Signaling pathway involved in bipolaris maydis conidium germination and appressorium formation[J].Microbiology China,2005,32(4):1-4.(in Chinese)
[5]王立安,王源超,李昌文,等.Ca2+信号途径参与稻瘟病菌分生孢子萌发及附着胞形成的调控[J].菌物系统,2003,22(3):457-465.Wang L A,Wang Y C,Li C W,et al.Ca2+signaling pathway involved in Magnaporthe grisea conidia and the regulation of the formation of adherent cells[J].Bacterial System,2003,22(3):457-465.(in Chinese)
[6]Lee M H,Bostock R M.Induction,regulation,and role in pathogenesis of appressoria in Monilinia fructicola[J].Phytopathology,2006,96(10):1072-1080.
[7]Ahn I P,Suh S C.Calcium restores prepenetration morphogenesis abolished by Methylglyoxal-Bis-Guanyl Hydrazone in Cochliobolus miyabeanus infecting rice[J].Phytopathology,2007,97(3):331-337.
[8]Fisher G,Whittman Liebold B,Lang K,et al.Cyclophilin and peptidy-lprolyl cis-trans isomerase are probably identical proteins[J].Nature,1989,337(6206):476-478.
[9]Takahashi N,Hayano T,Suzuki M.Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin[J].Nature,1989,337(6206):473-475.
[10]Andreeva L,Heads R,Green C J.Cyclophilins and their possible role in the stress response[J].International Journal of Experimental Pathology,1999,80(6):305-315.
[11]Lu K P,Hanes S D,Hunter T.A human peptidyl-prolyl isomerase essential for regulation of mitosis[J].Nature,1996,380(6574):544-547.
[12]Wang P,Cardenas M E,Cox G M,et al.Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans[J].EMBO Reports,2001,2(6):511-518.
[13]Viaud M C,Balhadère P V,Talbot N J.A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection[J].Plant Cell,2002,14(4):917-930.
[14]杜茜,徐文静,赵洪锟,等.白僵菌亲环素蛋白基因Bb CPLA的克隆与分析[J].玉米科学,2008,16(6):117-120.Du Q,Xu W J,Zhao H K,et al.Cloning and analysis of a cyclophilin gene Bb CPLA from beauveria bassiana[J].Journal of Maize Sciences,2008,16(6):117-120.(in Chinese)
[15]Chen M,Jiang M,Shang J,et al.CYP1,a hypovirus-regulated cyclophilin,is required for virulence in the chestnut blight fungus[J].Molecular Plant Pathology,2011,12(3):239-246.
[16]李志勇,郝志敏,司贺龙,等.玉米大斑病菌Ca M基因的克隆及三氟拉嗪的抑菌作用[J].植物病理学报,2008,38(3):277-282.Li Z Y,Hao Z M,Si H L,et al.Cloning Ca M(Calmodulin)gene and inhibitory effect of trifluoperazine on Setosphaeria turcica[J].Acta Phytopathologica Sinica,2008,38(3):277-282.(in Chinese)
[17]郝志敏,郝杰,李志勇,等.磷脂酶C特异性抑制剂U-73122对玉米大斑病菌分生孢子萌发及毒素活性的影响[J].华北农学报,2010,25(6):118-121.Hao Z M,Hao J,Li Z Y,et al.Effects of phospholipase C inhibitor U-73122 on conidial germination and HT-toxin activity of Setosphaeria turcica[J].Acta Agriculturae Boreali-Sinica,2010,25(6):118-121.(in Chinese)
[18]Hao Z M,Tong Y M,Han Y,et al.Molecular characterization of Stpka C2 and expression patterns of both PKA-c isoforms during the invasive growth of Setosphaeria turcica[J].Tropical Plant Pathology,2015,40(4):244-250.
[19]Zhang X C,Wang W D,Wang J S,et al.PPIase independent chaperone-like function of recombinant human Cyclophilin A during arginine kinase refolding[J].Febs Letters,2013,587(6):666-672.
[20]Walsh C T,Zydowsky L D,Mckeon F D.Cyclosporin A,the cyclophilin class of peptidylprolyl isomerases,and blockade of T cell signal transduction[J].Journal of Biological Chemistry,1992,267(19):13115-13118.
[21]Liu J,Jr F J,Lane W S,et al.Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes[J].Cell,1991,66(4):807-815.
[22]Lee Y K,Hong C B,Suh Y,et al.A c DNA clone for cyclophilin from Griffithsia japonica and phylogenetic analysis of cyclophilins[J].Molecules&Cells,2002,13(1):12-20.
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