田头菇属真菌3-磷酸甘油醛脱氢酶稀有内含子特征分析
|
摘要
前期研究从杨柳田头菇(Agrocybe salicacola)YAASM0711的单核菌株M20中获得了含有GC-AG内含子的3-磷酸甘油醛脱氢酶(Glycerol-3-phosphate dehydrogenase,GPD)gpd完整编码基因,本研究通过特异性引物PCR扩增探讨该基因的组成结构及内含子的拼接特征.在杨柳田头菇和茶树菇(A.aegerita)野生群体中均获得了此基因,序列差异性分析显示该基因可被分为两类,并且在两个物种中均有分布,表明这种序列组成差异可能在两个物种分化之前就已经存在.另外,在M20菌株的菌丝体及YAASM0711菌株的不同发育时期均没有发现可选择性剪切现象.基因表达分析表明,gpd在幼嫩的子实体中表达量最高,约为菌丝体中表达量的5.5倍,推测与子实体的快速生长有关.本研究结果有助于提高对gpd功能及其在物种分化研究中作用的认识.
The rare introns with GC-AG border are found in some organisms including human,Caenorhabditis elegans and filamentous fungi with low detection rate of 0.6%-1.2%.In general,this type of introns seem to be related to the alternative splice,and their retention or removal regulate the function of the translated protein.It is necessary to obtain the glycerol-3-phosphate dehydrogenase(GPD) encoding gene from Agrocybe salicacola strain M20 and to understand its splicing characterization.This study cloned the complete gpd gene containing GC-AG intron from single spore isolation M20 of Agrocybe salicacola YAASM0711 by using PCR with genomic sequencing data.The population investigation showed two types of GC-AG introns amplified by special primers,both found in each of the two species A.salicacola and A.aegerita,which indicated that they probably occurred before the species divergence.In addition,alternative splice was not detected in the mycelia of M20 or any development stage of YAASM0711.Gene expression showed the highest mRNA level of gpd existing in young fruiting body,and probably playing an important role during the rapid growth of fruiting body.To the best of our knowledge,this was the first report on the GC-AG introns in gpd gene.The splicing characterization of glycerol-3-phosphate dehydrogenase GC-AG introns in genus Agrocybe will provide help for further understanding of GC-AG intron in function and evolution.
The rare introns with GC-AG border are found in some organisms including human,Caenorhabditis elegans and filamentous fungi with low detection rate of 0.6%-1.2%.In general,this type of introns seem to be related to the alternative splice,and their retention or removal regulate the function of the translated protein.It is necessary to obtain the glycerol-3-phosphate dehydrogenase(GPD) encoding gene from Agrocybe salicacola strain M20 and to understand its splicing characterization.This study cloned the complete gpd gene containing GC-AG intron from single spore isolation M20 of Agrocybe salicacola YAASM0711 by using PCR with genomic sequencing data.The population investigation showed two types of GC-AG introns amplified by special primers,both found in each of the two species A.salicacola and A.aegerita,which indicated that they probably occurred before the species divergence.In addition,alternative splice was not detected in the mycelia of M20 or any development stage of YAASM0711.Gene expression showed the highest mRNA level of gpd existing in young fruiting body,and probably playing an important role during the rapid growth of fruiting body.To the best of our knowledge,this was the first report on the GC-AG introns in gpd gene.The splicing characterization of glycerol-3-phosphate dehydrogenase GC-AG introns in genus Agrocybe will provide help for further understanding of GC-AG intron in function and evolution.
引文
1 Collins L,Penny D.Complex spliceosomal organization ancestral to extant eukaryotes[J].Mol Biol Evol,2005,22(2):1053-1066
2 Will CL,Luhrmann R.Spliceosomal UsnRNP biogenesis,structure and function[J].Curr Opin Cell Biol,2001,13(3):290-301
3 Sheth N,Roca X,Hastings ML,Roeder T,Krainer AR,Sachidanandam R.Comprehensive splice-site analysis using comparative genomics[J].Nucleic Acids Res,2006,34(4):3955-3967
4 Wu HJ,Gaubier-Comella P,Delseny M,Grellet F,Van Montagu M,Rouze P.Non-canonical introns are at least 10~9 years old[J].Nature Genet,1996,14:383-384
5 Farrer T,Roller AB,Kent WJ,Zahler AM.Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing[J].Nucleic Acids Res,2002,30(15):3360-3367
6 Dietrich RC,Fuller JD,Padgett RA.A mutational analysis of U12-dependent splice site dinucleotides[J].RNA,2005,14(11):1430-1440
7 Churbanov A,Winters-Hilt S,Eugene V,Koonin EV,Rogozin IB.Accumulation of GC donor splice signals in mammals[J].Biol Direct,2008,3:30
8 Burset M,Seledtsov IA,Solovyev VV.Analysis of canonical and noncanonical splice sites in mammalian genomes[J].Nucleic Acids Res,2000,28(21):4364-4375
9 Burset M,Seledtsov IA,Solovyev VV.Splicedb:database of canonical and non-canonical mammalian splice sites[J].Nucleic Acids Res,2001,29(1):255-259
10 Rep M,Duyvesteijn RGE,Gale L,Usgaard T,Cornelissen BJ,Ma LJ,Ward TJ.The presence of GC-AG introns in Neurospora crassa and other euascomycetes determined from analyses of complete genomes:Implications for automated gene prediction[J].Genomics,2006,87(3):338-347
11 Zhao CZ,Waalwijk C,de Wit PJGM,Tang DZ,Lee TVD.RNA-Seq analysis reveals new gene models and alternative splicing in the fungal pathogen Fusarium graminearum[J].BMC Genomics,2013,14:21
12 Thanaraj TA,Clark F.Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions[J].Nucleic Acids Res,2001,29(12):2581-2593
13 Sibley MH,Johnson JJ,Mello CC,Kramer JM.Genetic identification,sequence and alternative splicing of the Caenorhabditis elegans alpha 2(Ⅳ)collagen gene[J].J Cell Biol,1993,123(1):255-264
14 Fillinger S,Ruijter G,Tamas MJ,Visser J,Thevelein JM,d'Enfert C.Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans[J].Mol Microbiol,2001,39(1):145-157
15何莹莹,陈卫民,赵永昌,周德群.田头菇属真菌信息素受体基因保守区克隆及多态性分析[J].应用与环境生物学报,2013,19(5):794-799[He YY,Chen WM,Zhao YC,Zhou DQ.Cloning and diversity analysis of the conserved fragments of pheromone receptor genes in genus Agrocybe[J].Chin J Appl Environ Biol,2013,19(5):794-799
16 Chen WM,Chai HM,Zhou HM,Tian GT,Li SH,Zhao YC.Phylogenetic analysis of the Agrocybe aegerita multispecies complex in Southwest China inferred from ITS and mtSSU rDNA sequences and mating tests[J].Ann Microbiol,2012,62(4):1791-1801
17 Noel T,Labarere J.Isolation of DNA from Agrocybe aegerita for the construction of a genomic library in Escherichia coli[J].Mushroom Sci,1987,12:187-201.
18 Martin F,Aerts A,Ahren D,Brun A,Danchin EGJ,Duchaussoy F,Gibon J,Kohler A,Lindquist E,Pereda V,Salamov A,Shapiro HJ,Wuyts J,Blaudez D,Buee M,Brokstein P,Canback B,Cohen D,Courty PE,Coutinho PM,Delaruelle C,Detter JC,Deveau A,Difazio S,Duplessis S,Fraissinet-Tachet L,Lucic E,Frey-Klett P,Fourrey C,Feussner I,Gay G,Grimwood J,Hoegger PJ,Jain P,Kilaru S,Labbe J,Lin YC,Legue V,Le Tacon F,Marmeisse R,Melayah D,Montanini B,Muratet M,Nehls U,Niculita-Hirzel H,Secq MPOudot-Le;Peter M,Quesneville H,Rajashekar B,Reich M,Rouhier N,Schmutz J,Yin T,Chalot M,Henrissat B,Kues U,Lucas S,van de Peer Y,Podila GK,Polle A,Pukkila PJ,Richardson PM,Rouze P,Sanders IR,Stajich JE,Tunlid A,Tuskan G,Grigoriev IV.The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis[J].Nature,2008,452:88-92
19 Stajich JE1,Wilke SK,Ahren D,Au CH,Birren BW,Borodovsky M,Burns C,Canback B,Casselton LA,Cheng CK,Deng J,Dietrich FS,Fargo DC,Farman ML,Gathman AC,Goldberg J,Guigo R,Hoegger PJ,Hooker JB,Huggins A,James TY,Kamada T,Kilaru S,Kodira C,Kues U,Kupfer D,Kwan HS,Lomsadze A,Li W,Lilly WW,Ma LJ,Mackey AJ,Manning G,Martin F,Muraguchi H,Natvig DO,Palmerini H,Ramesh MA,Rehmeyer CJ,Roe BA,Shenoy N,Stanke M,TerHovhannisyan V,Tunlid A,Velagapudi R,Vision TJ,Zeng Q,Zolan ME,Pukkila PJ 2010.Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea(Coprinus cinereus)[J].Proc Natl Acad Sci USA,107:11889-11894
20 Meinhardt LW,Costa GG,Thomazella DP,Teixeira PJ,Carazzolle MF,Schuster SC,Carlson JE,Guiltinan MJ,Mieczkowski P,Farmer A,Ramaraj T,Crozier J,Davis RE,Shao J,Melnick RL,Pereira GA,Bailey BA.Genome and secretome analysis of the hemibiotrophic fungal pathogen,Moniliophthora roreri,which causes frosty pod rot disease of cacao:mechanisms of the biotrophic and necrotrophic phases[J].BMC Genomics,2014,15:164
21 Donnell KO,Ward TJ,Geiser DM,Kistler HC,Aoki T.Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade[J].Fungal Genet Biol,2004,41(6):600-623
22 Masters JN,Finch CE,Nichols NR.Rapid increase in glycerol phosphate dehydrogenase mRNA in adult rat brain:a glycocorticoid dependent stress response[J].Neuroendocrinology,1994,60(1):23-35
23 Nicols NR,Dokas L,Ting SM,Kumar S,de Vellis J,Shors TJ,Uenishi N,Thompson RF,Finch CE.Hippocampal responses to corticosterone and stress,one of which is the 35,000 M(r)protein,glycerol phosphate dehydrogenase[J].J Neuroendocrinol,1996,8(11):867-876
24 Albertyn J,Hohmann S,Thevelein JM,Prior BA.GPD1,which encodes glycerol-3-phosphate dehydrogenase,is essential for growth under osmotic stress in Saccharomyces cerevisiae,and its expression is regulated by the high-osmolarity glycerol response pathway[J].Mol Cell Biol,1994,14(6):4135-4144
25 Hirano T,Sato T,Enei H.Isolation of genes specifically expressed in the fruit body of the edible basidiomycete Lentinula edodes[J].Biosci Biotechnol Biochem,2004,68(2):468-472
26 Krynetski EY,Krynetskaia NF,Gallo AE,Murti KG,Evans WE.A novel protein complex distinct from mismatch repair binds thioguanylated DNA[J].Mol Pharmacol,2001,59(2):367-374
27 Otto GP,Wu MY,Kazgan N,Anderson OR,Kessin RH.Dictyostelium macroautophagy mutants vary in the severity of their developmental defects[J].J Biol Chem,2004,279(15):15621-15629
2 Will CL,Luhrmann R.Spliceosomal UsnRNP biogenesis,structure and function[J].Curr Opin Cell Biol,2001,13(3):290-301
3 Sheth N,Roca X,Hastings ML,Roeder T,Krainer AR,Sachidanandam R.Comprehensive splice-site analysis using comparative genomics[J].Nucleic Acids Res,2006,34(4):3955-3967
4 Wu HJ,Gaubier-Comella P,Delseny M,Grellet F,Van Montagu M,Rouze P.Non-canonical introns are at least 10~9 years old[J].Nature Genet,1996,14:383-384
5 Farrer T,Roller AB,Kent WJ,Zahler AM.Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing[J].Nucleic Acids Res,2002,30(15):3360-3367
6 Dietrich RC,Fuller JD,Padgett RA.A mutational analysis of U12-dependent splice site dinucleotides[J].RNA,2005,14(11):1430-1440
7 Churbanov A,Winters-Hilt S,Eugene V,Koonin EV,Rogozin IB.Accumulation of GC donor splice signals in mammals[J].Biol Direct,2008,3:30
8 Burset M,Seledtsov IA,Solovyev VV.Analysis of canonical and noncanonical splice sites in mammalian genomes[J].Nucleic Acids Res,2000,28(21):4364-4375
9 Burset M,Seledtsov IA,Solovyev VV.Splicedb:database of canonical and non-canonical mammalian splice sites[J].Nucleic Acids Res,2001,29(1):255-259
10 Rep M,Duyvesteijn RGE,Gale L,Usgaard T,Cornelissen BJ,Ma LJ,Ward TJ.The presence of GC-AG introns in Neurospora crassa and other euascomycetes determined from analyses of complete genomes:Implications for automated gene prediction[J].Genomics,2006,87(3):338-347
11 Zhao CZ,Waalwijk C,de Wit PJGM,Tang DZ,Lee TVD.RNA-Seq analysis reveals new gene models and alternative splicing in the fungal pathogen Fusarium graminearum[J].BMC Genomics,2013,14:21
12 Thanaraj TA,Clark F.Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions[J].Nucleic Acids Res,2001,29(12):2581-2593
13 Sibley MH,Johnson JJ,Mello CC,Kramer JM.Genetic identification,sequence and alternative splicing of the Caenorhabditis elegans alpha 2(Ⅳ)collagen gene[J].J Cell Biol,1993,123(1):255-264
14 Fillinger S,Ruijter G,Tamas MJ,Visser J,Thevelein JM,d'Enfert C.Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans[J].Mol Microbiol,2001,39(1):145-157
15何莹莹,陈卫民,赵永昌,周德群.田头菇属真菌信息素受体基因保守区克隆及多态性分析[J].应用与环境生物学报,2013,19(5):794-799[He YY,Chen WM,Zhao YC,Zhou DQ.Cloning and diversity analysis of the conserved fragments of pheromone receptor genes in genus Agrocybe[J].Chin J Appl Environ Biol,2013,19(5):794-799
16 Chen WM,Chai HM,Zhou HM,Tian GT,Li SH,Zhao YC.Phylogenetic analysis of the Agrocybe aegerita multispecies complex in Southwest China inferred from ITS and mtSSU rDNA sequences and mating tests[J].Ann Microbiol,2012,62(4):1791-1801
17 Noel T,Labarere J.Isolation of DNA from Agrocybe aegerita for the construction of a genomic library in Escherichia coli[J].Mushroom Sci,1987,12:187-201.
18 Martin F,Aerts A,Ahren D,Brun A,Danchin EGJ,Duchaussoy F,Gibon J,Kohler A,Lindquist E,Pereda V,Salamov A,Shapiro HJ,Wuyts J,Blaudez D,Buee M,Brokstein P,Canback B,Cohen D,Courty PE,Coutinho PM,Delaruelle C,Detter JC,Deveau A,Difazio S,Duplessis S,Fraissinet-Tachet L,Lucic E,Frey-Klett P,Fourrey C,Feussner I,Gay G,Grimwood J,Hoegger PJ,Jain P,Kilaru S,Labbe J,Lin YC,Legue V,Le Tacon F,Marmeisse R,Melayah D,Montanini B,Muratet M,Nehls U,Niculita-Hirzel H,Secq MPOudot-Le;Peter M,Quesneville H,Rajashekar B,Reich M,Rouhier N,Schmutz J,Yin T,Chalot M,Henrissat B,Kues U,Lucas S,van de Peer Y,Podila GK,Polle A,Pukkila PJ,Richardson PM,Rouze P,Sanders IR,Stajich JE,Tunlid A,Tuskan G,Grigoriev IV.The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis[J].Nature,2008,452:88-92
19 Stajich JE1,Wilke SK,Ahren D,Au CH,Birren BW,Borodovsky M,Burns C,Canback B,Casselton LA,Cheng CK,Deng J,Dietrich FS,Fargo DC,Farman ML,Gathman AC,Goldberg J,Guigo R,Hoegger PJ,Hooker JB,Huggins A,James TY,Kamada T,Kilaru S,Kodira C,Kues U,Kupfer D,Kwan HS,Lomsadze A,Li W,Lilly WW,Ma LJ,Mackey AJ,Manning G,Martin F,Muraguchi H,Natvig DO,Palmerini H,Ramesh MA,Rehmeyer CJ,Roe BA,Shenoy N,Stanke M,TerHovhannisyan V,Tunlid A,Velagapudi R,Vision TJ,Zeng Q,Zolan ME,Pukkila PJ 2010.Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea(Coprinus cinereus)[J].Proc Natl Acad Sci USA,107:11889-11894
20 Meinhardt LW,Costa GG,Thomazella DP,Teixeira PJ,Carazzolle MF,Schuster SC,Carlson JE,Guiltinan MJ,Mieczkowski P,Farmer A,Ramaraj T,Crozier J,Davis RE,Shao J,Melnick RL,Pereira GA,Bailey BA.Genome and secretome analysis of the hemibiotrophic fungal pathogen,Moniliophthora roreri,which causes frosty pod rot disease of cacao:mechanisms of the biotrophic and necrotrophic phases[J].BMC Genomics,2014,15:164
21 Donnell KO,Ward TJ,Geiser DM,Kistler HC,Aoki T.Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade[J].Fungal Genet Biol,2004,41(6):600-623
22 Masters JN,Finch CE,Nichols NR.Rapid increase in glycerol phosphate dehydrogenase mRNA in adult rat brain:a glycocorticoid dependent stress response[J].Neuroendocrinology,1994,60(1):23-35
23 Nicols NR,Dokas L,Ting SM,Kumar S,de Vellis J,Shors TJ,Uenishi N,Thompson RF,Finch CE.Hippocampal responses to corticosterone and stress,one of which is the 35,000 M(r)protein,glycerol phosphate dehydrogenase[J].J Neuroendocrinol,1996,8(11):867-876
24 Albertyn J,Hohmann S,Thevelein JM,Prior BA.GPD1,which encodes glycerol-3-phosphate dehydrogenase,is essential for growth under osmotic stress in Saccharomyces cerevisiae,and its expression is regulated by the high-osmolarity glycerol response pathway[J].Mol Cell Biol,1994,14(6):4135-4144
25 Hirano T,Sato T,Enei H.Isolation of genes specifically expressed in the fruit body of the edible basidiomycete Lentinula edodes[J].Biosci Biotechnol Biochem,2004,68(2):468-472
26 Krynetski EY,Krynetskaia NF,Gallo AE,Murti KG,Evans WE.A novel protein complex distinct from mismatch repair binds thioguanylated DNA[J].Mol Pharmacol,2001,59(2):367-374
27 Otto GP,Wu MY,Kazgan N,Anderson OR,Kessin RH.Dictyostelium macroautophagy mutants vary in the severity of their developmental defects[J].J Biol Chem,2004,279(15):15621-15629