香菇UDP-葡萄糖焦磷酸化酶转录水平及酶活性对不同碳氮源的响应特征
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摘要
以香菇新808为材料,利用苯酚-硫酸法和qPCR技术分析不同碳氮源下菌丝体多糖含量及尿苷二磷酸葡萄糖焦磷酸化酶基因相对表达量,并测定其酶活性.结果表明,与对照组相比,除硝酸铵处理外,其余处理均能提高香菇菌丝体生物量,以麦麸处理的生物量最大(0.63g).以小分子糖(蔗糖、麦芽糖和甘露糖)为碳源、有机复合物(麦麸和黄豆芽)为氮源时,UGP基因表达量明显上调,酶活性更高,菌丝胞内多糖含量也明显提高.其中蔗糖和麦麸处理下的UGP基因表达量、酶活性和菌丝胞内多糖含量最高.香菇菌丝UGP基因表达量、酶活性以及胞内多糖间存在明显的正相关关系.显示小分子碳源(蔗糖、麦芽糖和甘露糖)、有机氮源(麦麸和黄豆芽)有助于香菇菌丝多糖的合成,其中蔗糖和麦麸可作为香菇液体发酵优势碳源和氮源.
Different carbon and nitrogen sources were used to culture the Lentinula edodes Xin808 in this study.The contents of intracellular polysaccharide were determined by phenol-sulfuric acid.The expression level of UDP-glucose pyrophosphorylase,UGP gene were analyzed by real-time quantitative PCR,and enzyme activity of UGP were also determined.The results showed that,comparing to the control,besides NH_4NO_3 treatment group,the rest of carbon and nitrogen sources could increase the biomass of Letinous edodes,among which wheat bran exhibited as the best(0.63 g).The small molecule(sucrose,maltose and mannose)as carbon sources and organic compounds(wheat bran and yellow bean sprouts)as nitrogen sources,UGP gene relative expression level obviously were up-regulated,increased UGP activity,promoted the biosynthesis of mycelium intracellular polysaccharide.With sucrose as carbon source and wheat bran as nitrogen source,the relative expression level of UGP gene,the activity of UGP and mycelium polysaccharide content were the highest.The expression level of UGP gene,the activity of UGPase and mycelium intracellular polysaccharide of Letinous edodes showed high positive correlationbetween the three.The small molecule carbon sources(sucrose,maltose and mannose)and organic nitrogen sources(wheat bran and yellow bean sprouts)were helpful to the biosynthesis of mycelium polysaccharide.Sugar and wheat bran could be used as advantage carbon source and nitrogen source under Letinous edodes liquid fermentation.
Different carbon and nitrogen sources were used to culture the Lentinula edodes Xin808 in this study.The contents of intracellular polysaccharide were determined by phenol-sulfuric acid.The expression level of UDP-glucose pyrophosphorylase,UGP gene were analyzed by real-time quantitative PCR,and enzyme activity of UGP were also determined.The results showed that,comparing to the control,besides NH_4NO_3 treatment group,the rest of carbon and nitrogen sources could increase the biomass of Letinous edodes,among which wheat bran exhibited as the best(0.63 g).The small molecule(sucrose,maltose and mannose)as carbon sources and organic compounds(wheat bran and yellow bean sprouts)as nitrogen sources,UGP gene relative expression level obviously were up-regulated,increased UGP activity,promoted the biosynthesis of mycelium intracellular polysaccharide.With sucrose as carbon source and wheat bran as nitrogen source,the relative expression level of UGP gene,the activity of UGP and mycelium polysaccharide content were the highest.The expression level of UGP gene,the activity of UGPase and mycelium intracellular polysaccharide of Letinous edodes showed high positive correlationbetween the three.The small molecule carbon sources(sucrose,maltose and mannose)and organic nitrogen sources(wheat bran and yellow bean sprouts)were helpful to the biosynthesis of mycelium polysaccharide.Sugar and wheat bran could be used as advantage carbon source and nitrogen source under Letinous edodes liquid fermentation.
引文
[1]Lamerz A C,Haselhorst T,Bergfeld A K,et al.Molecular cloning of the Leishmania major UDP-glucose pyrophosphorylase,functional characterization,and ligand binding analyses using NMR spectroscopy[J].J Biol Chem,2006,281:16314.
[2]Chen R,Zhao X,Shao Z,et al.Rice UDP-glucose pyrophosphorylase1is essential for pollen callose deposition and its cosuppression results in a new type of thermosensitive genic male sterility[J].Plant Cell,2007,19:847.
[3]Kleczkowski L A,Geisler M,Ciereszko I,et al.UDP-glucose pyrophosphorylase.An old protein with new tricks[J].Plant Physiol,2007,134:912.
[4]Park J I,Ishimizu T,Suwabe K,et al.UDP-glucose pyrophosphorylase is rate limiting in vegetative and reproductive phases in Arabidopsis thaliana[J].Plant Cell Physiol,2010,51:981.
[5]Jiang S S,Lin T Y,Wang W B,et al.Characterization of UDP-glucose dehydrogenase and UDP-glucose pyrophosphorylase mutants of Proteus mirabilis:Defectiveness in polymyxin B resistance,swarming,and virulence[J].Antimicrob Agents Chemother,2010,54:2000.
[6]Hertzberg M,Aspeborg H,Schrader J,et al.Atranscriptional roadmap to wood formation[J].Proc Nat Acad Sci USA,2001,98:14732.
[7]Coleman H D,Ellis D D,Gilbert M,et al.Up-regulation of sucrose synthase and UDP-glucose pyrophosphorylase impacts plant growth and metabolism[J].Plant Biotechnol J,2006,4:87.
[8]Duan X,Chi Z,Wang L,et al.Influence of different sugars on pullulan production and activities ofα-phosphoglucose mutase,UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68[J].Carbohydra Polym,2008,73:587.
[9]Szeto C Y,Wong Q W,Leung G S,et al.Isolation and transcript analysis of two-component histidine kinase gene Le.nik1in Shiitake mushroom,Lentinula edodes[J].Mycol Res,2008,112:108.
[10]Yu S,Weaver V,Martin K,et al.The effects of whole mushrooms during inflammation[J].BMCImmunol,2009,10:121.
[11]Chandra L C,Smith B J,Clarke S L,et al.Differential effects of shiitake-and white button mushroom-supplemented diets on hepatic steatosis in C57BL/6mice[J].Food Chem Toxicol,2011,49:3074.
[12]Chen N Y,Hsu T H,Lin F Y,et al.Effects on cytokine-stimulating activities of EPS fromTremella mesenterica with various carbon sources[J].Food Chem,2006,99:92.
[13]Smiderle F R,Olsen L M,Ruthes A C,et al.Exopolysaccharides,proteins and lipids in Pleurotus pulmonarius,submerged culture using different carbon sources[J].Carbohydra Polym,2012,87:368.
[14]徐思炜,张君胜,周雯.香菇液体菌种培养条件优化[J].江苏农业科学,2014:290.
[15]张帆,钟威,穆虹,等.过量表达OsUgp2基因提高紫芝多糖含量[J].菌物学报,2011:442.
[16]陈霞连,杨华侨,黎佳欣,等.美容杜鹃RcHsfB3基因的克隆及表达分析[J].四川大学学报:自然科学版,2017,54:405.
[17]Ren A,Li M J,Shi L,et al.Profiling and quantifying differential gene transcription provide insights into Ganoderic Acid biosynthesis in Ganoderma lucidum in response to methyl jasmonate[J].Plos One,2013,8:e65027.
[18]Chen L,Gong Y,Cai Y,et al.Genome sequence of the edible cultivated mushroom Lentinulaedodes(Shiitake)reveals insights into lignocellulose degradation[J].Plos One,2016,11:1.
[19]Elisashvili V I,Kachlishvili E T,Wasser S P.Carbon and nitrogen effects on basidiomycetes exopolysaccharide production[J].Appl Biochem Microb,2009,45:531.
[20]Wu Z,Yang Z,Dan G,et al.Influences of carbon sources on the biomass,production and compositions of exopolysaccharides fromPaecilomyces hepiali,HN1[J].Biomass Bioenergy,2014,67:260.
[21]金月波,任一英,矫天育.碳源和氮源对金针菇菌丝体生物量及多糖产量的影响[J].辽宁农业职业技术学院学报,2008(03):1.
[22]石红萍.三角褐指藻尿苷二磷酸葡萄糖焦磷酸化酶在碳流分配中的功能[D].青岛:中国海洋大学,2015.
[23]Wang Q,Zhang X,Li F,et al.Identification of a UDP-glucose pyrophosphorylase from cotton(Gossypium hirsutum L.)involved in cellulose biosynthesis in Arabidopsis thaliana[J].Plant Cell Rep,2011,30:1303.
[24]Li M,Chen T,Gao T,et al.UDP-glucose pyrophosphorylase influences polysaccharide synthesis,cell wall components,and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose[J].Fungal Genet Biol,2015,82:251.
[25]Guo Y,Sagaram U S,Kim J S,et al.Requirement of the galU gene for polysaccharide production by and pathogenicity and growth in planta of Xanthomonas citri subsp.citri[J].Appl Environ Microb,2010,76:2234.
[26]Marc D J,Walter B,Jean F.Physiological and morphological effects of genetic alterations leading to a reduced synthesis of UDP-glucose in Saccharomyces cerevisiae[J].FEMS Microbiol Lett,1997,153:89.
[27]Ciereszko I,Johansson H,Kleczkowski L A.Sucrose and light regulation of a cold-inducible UDP-glucose pyrophosphorylase gene via a hexokinase-independent and abscisic acid-insensitive pathway in Arabidopsis[J].Biochem J,2001,354:67.
[28]Johansson H.Gene regulation of UDP-glucose synthesis and metabolism in plants[D].Sweden:Umea University,2003.
[2]Chen R,Zhao X,Shao Z,et al.Rice UDP-glucose pyrophosphorylase1is essential for pollen callose deposition and its cosuppression results in a new type of thermosensitive genic male sterility[J].Plant Cell,2007,19:847.
[3]Kleczkowski L A,Geisler M,Ciereszko I,et al.UDP-glucose pyrophosphorylase.An old protein with new tricks[J].Plant Physiol,2007,134:912.
[4]Park J I,Ishimizu T,Suwabe K,et al.UDP-glucose pyrophosphorylase is rate limiting in vegetative and reproductive phases in Arabidopsis thaliana[J].Plant Cell Physiol,2010,51:981.
[5]Jiang S S,Lin T Y,Wang W B,et al.Characterization of UDP-glucose dehydrogenase and UDP-glucose pyrophosphorylase mutants of Proteus mirabilis:Defectiveness in polymyxin B resistance,swarming,and virulence[J].Antimicrob Agents Chemother,2010,54:2000.
[6]Hertzberg M,Aspeborg H,Schrader J,et al.Atranscriptional roadmap to wood formation[J].Proc Nat Acad Sci USA,2001,98:14732.
[7]Coleman H D,Ellis D D,Gilbert M,et al.Up-regulation of sucrose synthase and UDP-glucose pyrophosphorylase impacts plant growth and metabolism[J].Plant Biotechnol J,2006,4:87.
[8]Duan X,Chi Z,Wang L,et al.Influence of different sugars on pullulan production and activities ofα-phosphoglucose mutase,UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68[J].Carbohydra Polym,2008,73:587.
[9]Szeto C Y,Wong Q W,Leung G S,et al.Isolation and transcript analysis of two-component histidine kinase gene Le.nik1in Shiitake mushroom,Lentinula edodes[J].Mycol Res,2008,112:108.
[10]Yu S,Weaver V,Martin K,et al.The effects of whole mushrooms during inflammation[J].BMCImmunol,2009,10:121.
[11]Chandra L C,Smith B J,Clarke S L,et al.Differential effects of shiitake-and white button mushroom-supplemented diets on hepatic steatosis in C57BL/6mice[J].Food Chem Toxicol,2011,49:3074.
[12]Chen N Y,Hsu T H,Lin F Y,et al.Effects on cytokine-stimulating activities of EPS fromTremella mesenterica with various carbon sources[J].Food Chem,2006,99:92.
[13]Smiderle F R,Olsen L M,Ruthes A C,et al.Exopolysaccharides,proteins and lipids in Pleurotus pulmonarius,submerged culture using different carbon sources[J].Carbohydra Polym,2012,87:368.
[14]徐思炜,张君胜,周雯.香菇液体菌种培养条件优化[J].江苏农业科学,2014:290.
[15]张帆,钟威,穆虹,等.过量表达OsUgp2基因提高紫芝多糖含量[J].菌物学报,2011:442.
[16]陈霞连,杨华侨,黎佳欣,等.美容杜鹃RcHsfB3基因的克隆及表达分析[J].四川大学学报:自然科学版,2017,54:405.
[17]Ren A,Li M J,Shi L,et al.Profiling and quantifying differential gene transcription provide insights into Ganoderic Acid biosynthesis in Ganoderma lucidum in response to methyl jasmonate[J].Plos One,2013,8:e65027.
[18]Chen L,Gong Y,Cai Y,et al.Genome sequence of the edible cultivated mushroom Lentinulaedodes(Shiitake)reveals insights into lignocellulose degradation[J].Plos One,2016,11:1.
[19]Elisashvili V I,Kachlishvili E T,Wasser S P.Carbon and nitrogen effects on basidiomycetes exopolysaccharide production[J].Appl Biochem Microb,2009,45:531.
[20]Wu Z,Yang Z,Dan G,et al.Influences of carbon sources on the biomass,production and compositions of exopolysaccharides fromPaecilomyces hepiali,HN1[J].Biomass Bioenergy,2014,67:260.
[21]金月波,任一英,矫天育.碳源和氮源对金针菇菌丝体生物量及多糖产量的影响[J].辽宁农业职业技术学院学报,2008(03):1.
[22]石红萍.三角褐指藻尿苷二磷酸葡萄糖焦磷酸化酶在碳流分配中的功能[D].青岛:中国海洋大学,2015.
[23]Wang Q,Zhang X,Li F,et al.Identification of a UDP-glucose pyrophosphorylase from cotton(Gossypium hirsutum L.)involved in cellulose biosynthesis in Arabidopsis thaliana[J].Plant Cell Rep,2011,30:1303.
[24]Li M,Chen T,Gao T,et al.UDP-glucose pyrophosphorylase influences polysaccharide synthesis,cell wall components,and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose[J].Fungal Genet Biol,2015,82:251.
[25]Guo Y,Sagaram U S,Kim J S,et al.Requirement of the galU gene for polysaccharide production by and pathogenicity and growth in planta of Xanthomonas citri subsp.citri[J].Appl Environ Microb,2010,76:2234.
[26]Marc D J,Walter B,Jean F.Physiological and morphological effects of genetic alterations leading to a reduced synthesis of UDP-glucose in Saccharomyces cerevisiae[J].FEMS Microbiol Lett,1997,153:89.
[27]Ciereszko I,Johansson H,Kleczkowski L A.Sucrose and light regulation of a cold-inducible UDP-glucose pyrophosphorylase gene via a hexokinase-independent and abscisic acid-insensitive pathway in Arabidopsis[J].Biochem J,2001,354:67.
[28]Johansson H.Gene regulation of UDP-glucose synthesis and metabolism in plants[D].Sweden:Umea University,2003.