蛹虫草CmKexin基因克隆和菌丝体中表达检测
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摘要
为了探讨蛹虫草类枯草杆菌蛋白酶(Subtilisin-like protease)的表达特性,以真菌蛹虫草菌丝体为研究对象,通过RT-PCR获得蛹虫草CmKexin基因的ORF序列,并进行序列分析。应用Northern杂交和Real-time PCR方法,检测蓝光照射后蛹虫草菌丝体中CmKexin基因的转录情况。结果获得蛹虫草CmKexin基因的ORF序列。对翻译蛋白质产物CmKexin进行生物信息学分析,表明该蛋白质含1个属蛋白转化酶的肽酶S8家族功能域(143~429)和1个前体蛋白转化酶P功能域(514~600),符合真菌类枯草杆菌蛋白酶的特征。蛹虫草菌丝体在黑暗预培养4 d后再蓝光照射,Northern Blotting和Real-time PCR检测均可得到相同的结果,即在持续的蓝光照射48~50 h时,出现CmKexin基因的瞬时大量转录。而其他时间内均未检测到该基因的大量转录。试验结果将为蛹虫草类枯草杆菌蛋白酶的利用提供理论依据。
In order to study the characteristics of subtilisin-like protease expression in the mycelia of fungus Cordyceps militaris,CmKexin gene ORF sequences of C. militaris was got by RT-PCR,then CmKexin sequence was analysed. The Real-time PCR and Northern Blotting were used to the CmKexin gene expression analysis in the C. militaris mycelia in blue light irradiation of different time. Results showed the CmKexin gene ORF sequence was obtained from C. militaris mycelia. The bioinformatics analysis for the translation products of CmKexin gene showed that there were 1 peptidase S8 family domain of protein convertases( 143-429) and 1 proprotein convertase P-domain( 514-600) in the protein. The two domains existed in CmKexin demonstrate the characteristics of subtilisinlike protease. Under the dark pre-culture in 96 h,a large amounts of transcript of CmKexin gene in the mycelia could be detected by Northern Blotting and Real-time PCR in the continuous blue light irradiation to 48-50 hours.But the transcription of CmKexin gene wasn't detected in the other time of C. militaris mycelia culture. These results would provide the basis for using the subtilisin-like protease in Cordyceps militaris.
In order to study the characteristics of subtilisin-like protease expression in the mycelia of fungus Cordyceps militaris,CmKexin gene ORF sequences of C. militaris was got by RT-PCR,then CmKexin sequence was analysed. The Real-time PCR and Northern Blotting were used to the CmKexin gene expression analysis in the C. militaris mycelia in blue light irradiation of different time. Results showed the CmKexin gene ORF sequence was obtained from C. militaris mycelia. The bioinformatics analysis for the translation products of CmKexin gene showed that there were 1 peptidase S8 family domain of protein convertases( 143-429) and 1 proprotein convertase P-domain( 514-600) in the protein. The two domains existed in CmKexin demonstrate the characteristics of subtilisinlike protease. Under the dark pre-culture in 96 h,a large amounts of transcript of CmKexin gene in the mycelia could be detected by Northern Blotting and Real-time PCR in the continuous blue light irradiation to 48-50 hours.But the transcription of CmKexin gene wasn't detected in the other time of C. militaris mycelia culture. These results would provide the basis for using the subtilisin-like protease in Cordyceps militaris.
引文
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[7]Nakayama K.Furin:a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety ofprecursor proteins[J].The Biochemical Journal,1997,327(Pt 3):625-635.
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[9]Holyoak T,Wilson M A,Fenn T D,et al.2.4 angstrom resolution crystal structure of the prototypical hormoneprocessing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor[J].Biochemistry,2003,42(22):6709-6718.
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[11]Jacob-Wilk D,Turina M,Kazmierczak P,et al.Silencing of Kex2 significantly diminishes the virulence of Cryphonectria parasitica[J].Phytopathology,2007,97(7,S):S50.
[12]Shi L M,Li R,Liao S H,et al.Prb1,a subtilisin-like protease,is required for virulence and phenotypical traits in the chestnut blight fungus[J].FEMS Microbiology Letters,2014,359(1):26-33.
[13]Wang J Y,Zhou H,Lu H,et al.Kexin-like endoprotease Kex B is required for N-glycan processing,morphogenesis and virulence in Aspergillus fumigatus[J].Fungal Genetics and Biology,2015,76:57-69.
[14]Mizutani O,Shiina M,Yoshimi A,et al.Substantial decrease in cell wallα-1,3-glucan caused by disruption of the kex B gene encoding a subtilisin-like processing protease in Aspergillus oryzae[J].Biosci Biotechnol Biochem,2016,15:1-11.
[15]Bader O,Schaller M,Klein S,et al.The KEX2 gene of Candida glabrata is required for cell surface integrity[J].Mol Microbiol,2001,41:1431-1444.
[16]Juntunen K,Makinen S,Isoniemi S,et al.A new Subtilase-Like protease deriving from Fusarium equiseti with high potential for industrial applications[J].Applied Biochemistry and Biotechnology,2015,177(2):407-430.
[17]Kertesz-Chaloupkova K,Walser P J,Granado J D,et al.Blue light overrides repression of asexual sporulation by mating type genes in the basidiomycete Coprinus cinereus[J].Fungal Genetics and Biology,1998,23(1):109.
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[19]Idnurm A,Rodriguez-Romero J,Corrochano L M,et al.The phycomyces mad A gene encodes a blue-light photoreceptor for phototropism and other light responses[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(12):4551.
[20]Velayos A,Blasco J L,Alvarez M I,et al.Blue-light regulation of phytoene dehydrogenase(car B)gene expression in Mucor circinelloides[J].Planta,2000,210(6):946.
[21]Froehlich A C,Liu Y,Loros J J,et al.White collar-1,a circadian blue light photoreceptor,binding to the frequency promoter[J].Science,2002,297(5582):819.
[22]高雅,金华燕,付鸣佳,等.蛹虫草组氨酸激酶基因(Cm HK)的序列分析和受蓝光影响的表达[J].应用与环境生物学报,2014,20(1):50-55.
[23]Zheng P,Xia Y,Xiao G,et al.Genome sequence of the insect pathogenic fungus Cordyceps militaris,a valued traditional hinese medicine[J].Genome Biology,2011,12(11):R116.
[24]Gong Z H,Su Y,Huang L,et al.Cloning and analysis of glyceraldehyde-3-phosphate dehydrogenase gene from Cordyceps militaris[J].African Journal of Agricultural Research,2009,4(4):402-408.
[25]Hatsuzawa K,Murakami K,Nakayama K.Molecular and enzymatic properties of furin,a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites[J].Biochem,1992,111(3):301.
[26]Zhong M,Benjannet S,Lazure C,et al.Functional analysis of human PACE4-A and PACE4-C isoforms:identification of a new PACE4-CS isoform[J].FEBS Letters,1996,396(1):36.
[27]Li A N,Xie C,Zhang J,et al.Cloning,expression,and characterization of serine protease from thermophilic fungus thermoascus aurantiacus var.levisporus[J].Journal of Microbiology,2011,49(1):121-129.
[28]Yu G,Liu J L,Xie L Q,et al.Characterization,cloning,and heterologous expression of a subtilisin-like serine protease gene Vl Pr1 from Verticillium lecanii[J].Journal of Microbiology,2012,50(6):946.
[29]Fan H J,Liu Z H,Zhang R S,et al.Functional analysis of a subtilisin-like serine protease gene from biocontrol fungus Trichoderma harzianum[J].Journal of Microbiology,2014,52(2):129-138.
[30]Yan L,Qian Y.Cloning and heterologous expression of SS10.A subtilisin-like protease displaying antifungal activity from Trichoderma harzianum[J].FEMS Microbiology Letters,2009,290(1):61.
[2]Bryant M K,Schardl C L,Hesse U,et al.Evolution of a subtilisin-like protease gene family in the grass endophytic fungus Epichlo festucae[J].BMC Evolutionary Biology,2009,19(9):168.
[3]Spreer A,Ruechel R,Reichard U.Characterization of an extracellular subtilisin protease of Rhizopus microsporus and evidence for its expression during invasive rhinoorbital mycosis[J].Medical Mycology,2006,44(8):723-731.
[4]Rosas-Garcia N M,Avalos-De-Leon O,Villegas-Mendoza J M,et al.Correlation between pr1 and pr2 gene content and virulence in Metarhizium anisopliae Strains[J].Journal of Microbiology and Biotechnology,2014,24(11):1495-1502.
[5]Wanyiri J W,Techasintana P,O'connor R M,et al.Role of Cp SUB1,a Subtilisin-Like protease,in Cryptosporidium parvum infection in vitro[J].Eukaryotic Cell,2009,8(4):470-477.
[6]Roebroek A J,Schalken J A,Bussemakers M J.Characterization of human c-fes/-fps reveals a new transcription unit(fur)in the immediately upstream region of the protooncogene[J].Molecular Biology Reports,1986,11(2):125.
[7]Nakayama K.Furin:a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety ofprecursor proteins[J].The Biochemical Journal,1997,327(Pt 3):625-635.
[8]Julius D,Brake A,Blair L,et al.Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor[J].Cell,1984,37(3):1089.
[9]Holyoak T,Wilson M A,Fenn T D,et al.2.4 angstrom resolution crystal structure of the prototypical hormoneprocessing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor[J].Biochemistry,2003,42(22):6709-6718.
[10]Fuller R S,Sterne R E,Thorner J.Enzyme required for yeast prohormone processing[J].Annual Reviews Physiol,1988,50(1):362.
[11]Jacob-Wilk D,Turina M,Kazmierczak P,et al.Silencing of Kex2 significantly diminishes the virulence of Cryphonectria parasitica[J].Phytopathology,2007,97(7,S):S50.
[12]Shi L M,Li R,Liao S H,et al.Prb1,a subtilisin-like protease,is required for virulence and phenotypical traits in the chestnut blight fungus[J].FEMS Microbiology Letters,2014,359(1):26-33.
[13]Wang J Y,Zhou H,Lu H,et al.Kexin-like endoprotease Kex B is required for N-glycan processing,morphogenesis and virulence in Aspergillus fumigatus[J].Fungal Genetics and Biology,2015,76:57-69.
[14]Mizutani O,Shiina M,Yoshimi A,et al.Substantial decrease in cell wallα-1,3-glucan caused by disruption of the kex B gene encoding a subtilisin-like processing protease in Aspergillus oryzae[J].Biosci Biotechnol Biochem,2016,15:1-11.
[15]Bader O,Schaller M,Klein S,et al.The KEX2 gene of Candida glabrata is required for cell surface integrity[J].Mol Microbiol,2001,41:1431-1444.
[16]Juntunen K,Makinen S,Isoniemi S,et al.A new Subtilase-Like protease deriving from Fusarium equiseti with high potential for industrial applications[J].Applied Biochemistry and Biotechnology,2015,177(2):407-430.
[17]Kertesz-Chaloupkova K,Walser P J,Granado J D,et al.Blue light overrides repression of asexual sporulation by mating type genes in the basidiomycete Coprinus cinereus[J].Fungal Genetics and Biology,1998,23(1):109.
[18]Lauter F R,Marchfelder U,Russo V E,et al.Photoregulation of cot-1,a kinase-encoding gene involved in hyphal growth in Neurospora crassa[J].Fungal Genetics and Biology,1998,23(3):300-310.
[19]Idnurm A,Rodriguez-Romero J,Corrochano L M,et al.The phycomyces mad A gene encodes a blue-light photoreceptor for phototropism and other light responses[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(12):4551.
[20]Velayos A,Blasco J L,Alvarez M I,et al.Blue-light regulation of phytoene dehydrogenase(car B)gene expression in Mucor circinelloides[J].Planta,2000,210(6):946.
[21]Froehlich A C,Liu Y,Loros J J,et al.White collar-1,a circadian blue light photoreceptor,binding to the frequency promoter[J].Science,2002,297(5582):819.
[22]高雅,金华燕,付鸣佳,等.蛹虫草组氨酸激酶基因(Cm HK)的序列分析和受蓝光影响的表达[J].应用与环境生物学报,2014,20(1):50-55.
[23]Zheng P,Xia Y,Xiao G,et al.Genome sequence of the insect pathogenic fungus Cordyceps militaris,a valued traditional hinese medicine[J].Genome Biology,2011,12(11):R116.
[24]Gong Z H,Su Y,Huang L,et al.Cloning and analysis of glyceraldehyde-3-phosphate dehydrogenase gene from Cordyceps militaris[J].African Journal of Agricultural Research,2009,4(4):402-408.
[25]Hatsuzawa K,Murakami K,Nakayama K.Molecular and enzymatic properties of furin,a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites[J].Biochem,1992,111(3):301.
[26]Zhong M,Benjannet S,Lazure C,et al.Functional analysis of human PACE4-A and PACE4-C isoforms:identification of a new PACE4-CS isoform[J].FEBS Letters,1996,396(1):36.
[27]Li A N,Xie C,Zhang J,et al.Cloning,expression,and characterization of serine protease from thermophilic fungus thermoascus aurantiacus var.levisporus[J].Journal of Microbiology,2011,49(1):121-129.
[28]Yu G,Liu J L,Xie L Q,et al.Characterization,cloning,and heterologous expression of a subtilisin-like serine protease gene Vl Pr1 from Verticillium lecanii[J].Journal of Microbiology,2012,50(6):946.
[29]Fan H J,Liu Z H,Zhang R S,et al.Functional analysis of a subtilisin-like serine protease gene from biocontrol fungus Trichoderma harzianum[J].Journal of Microbiology,2014,52(2):129-138.
[30]Yan L,Qian Y.Cloning and heterologous expression of SS10.A subtilisin-like protease displaying antifungal activity from Trichoderma harzianum[J].FEMS Microbiology Letters,2009,290(1):61.