耐热毛栓孔菌S0301同核体菌株的快速鉴定及产漆酶能力比较
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摘要
栓孔菌属(Trametes)真菌菌株漆酶同工酶种类多,产漆酶优势独特,是重要的工业漆酶来源之一.为建立方便快捷的获得同核体菌株的方法,以高产漆酶耐热毛栓孔菌Trametes trogii S0301为研究对象,在利用原生质体再生技术得到再生单菌、通过PCR克隆获得该菌株交配型基因b1、b2位点序列的基础上,设计b1、b2交配型基因特异引物并对各再生菌落中不同交配型基因类型进行识别,结合经典的锁状联合显微镜观察,共从45个性状稳定的再生菌株中获得了19个b1b2型异核体菌株,26个b1型同核体菌株,同核体菌株检出率为57.8%,且PCR鉴定结果和锁状联合观察结果一致.此外,还发现b1型同核体菌株菌丝细、菌落稀疏,有更快的蔓延速度,在不添加漆酶诱导剂Cu2+的固体培养基中表现出更强的产漆酶能力.本研究表明PCR克隆交配型基因的方法适用于同核体菌株的快速鉴定,同时b1型同核体菌株具有产漆酶的优势.
This study used heat-resistant Trametes trogii S0301 with high yield of laccase as the research object. The protoplast regeneration technology was used to obtain single regenerated strains, and PCR cloning was used to obtain the mating-type genes(b1 and b2); next, the specific primers for b1 and b2 genes were designed to identify the mating genotype in different regenerated colonies. As a classic standard, the clamp connection in each regenerated strain was detected using microscopic observation. In all, 45 stable regeneration strains were selected for further study. Among them, 19 b1 b2 heterokaryotic strains and 26 b1 homokaryotic strains were obtained using PCR detecting method, and the detection rate of homokaryotic strains was 57.8%, which was consistent with the results of the clamp connection observation. In this study, we also found that b1 homokaryotic strains showed different properties from b1 b2 heterokaryotic strains, such as thinner hypha, more rapid spread,and sparser colonies on the solid medium. In particular, b1 homokaryotic strains showed higher laccase-producing ability on the solid medium without Cu~(2+) as the laccase inducer, compared with the b1 b2 heterokaryotic strains. This study showed that the way of PCR cloning mating-type genes was suitable for the rapid identification of homokaryotic strains. Moreover, b1 homokaryotic strains had the advantage of producing laccase.
This study used heat-resistant Trametes trogii S0301 with high yield of laccase as the research object. The protoplast regeneration technology was used to obtain single regenerated strains, and PCR cloning was used to obtain the mating-type genes(b1 and b2); next, the specific primers for b1 and b2 genes were designed to identify the mating genotype in different regenerated colonies. As a classic standard, the clamp connection in each regenerated strain was detected using microscopic observation. In all, 45 stable regeneration strains were selected for further study. Among them, 19 b1 b2 heterokaryotic strains and 26 b1 homokaryotic strains were obtained using PCR detecting method, and the detection rate of homokaryotic strains was 57.8%, which was consistent with the results of the clamp connection observation. In this study, we also found that b1 homokaryotic strains showed different properties from b1 b2 heterokaryotic strains, such as thinner hypha, more rapid spread,and sparser colonies on the solid medium. In particular, b1 homokaryotic strains showed higher laccase-producing ability on the solid medium without Cu~(2+) as the laccase inducer, compared with the b1 b2 heterokaryotic strains. This study showed that the way of PCR cloning mating-type genes was suitable for the rapid identification of homokaryotic strains. Moreover, b1 homokaryotic strains had the advantage of producing laccase.
引文
1葛春梅,徐娟娟,孙芹英,张洁,蔡敬民,潘仁瑞.里氏木霉和鸡腿菇利用秸秆共发酵产木质降解酶[J].生物工程学报,2009,25(12):2008-2013[Ge CM,Xu JJ,Sun QY,Zhang J,Cai JM,Pan RR.Production of ligninase by co-fermentation of Coprinus comatus and Trichoderma reesei[J].Chin J Biotechnol,2009,25(12):2008-2013]
2秦改娟,王晓,陈青君,张国庆.不同配方培养料生产双孢蘑菇过程中主要木质纤维素降解酶及物料组分的变化[J].应用与环境生物学报,2017,23(6):1035-1041[Qin GJ,Wang X,Chen QJ,Zhang GQ.Changes of lignocellulolytic enzymes and material components in different compost formulas during the production of Agaricus bisporus[J].Chin J Appl Environ Biol,2017,23(6):1035-1041]
3刘刚,王义刚,吴淑芳,丁少军.鸡腿菇漆酶同工酶降解马尾松硫酸盐浆中残余木质素的研究[J].纤维素科学与技术,2013,21(4):239-245[Liu G,Wang YG,Wu SF,Ding SJ.Study on enzymatic delignification of Pinus massoniana kraft pulp by laccase isozymes of Coprinus comatus[J].J Cell Sci Technol,2013,21(4):239-245]
4郑飞,孟歌,安琪,吴雪君,司静,刘红霞.白腐真菌东方栓孔菌在两种液体培养基中产漆酶过程的生理学研究[J].菌物学报,2017,36(5):582-597[Zheng F,Meng G,An Q,Wu XJ,Si J,Liu HX.Physiological studies on laccase-producing process of white rot fungus Trametes orientalis incubated with two kinds of liquid media[J].Mycosystema,2017,36(5):582-597]
5司静,崔宝凯.绒毛栓孔菌液体培养过程中胞外酶活性的研究[J].基因组学与应用生物学,2012,31(1):70-77[Si J,Cui BK.Studies on activities of extracellular enzymes in liquid culture of Trametes pubescens[J].Genomics Appl Biol,2012,31(1):70-77]
6司静,崔宝凯,戴玉成.栓孔菌属漆酶高产菌株的初步筛选及其产酶条件的优化[J].微生物学通报,2011,38(3):405-416[Si J,Cui BK,Dai YC.Primary screening of effective Trametes strains with high laccaseproductivity and optimization of conditions on laccase production[J].Microbiol Chin,2011,38(3):405-416]
7司静,崔宝凯,戴玉成.东方栓孔菌在染料脱色中的应用及其脱色条件的优化[J].基因组学与应用生物学,2011,30(3):364-371[Si J,Cui BK,Dai YC.Application in dye decolorization and optimization of conditions in discoloration by Trametes orientalis[J].Genomics Appl Biol,2011,30(3):364-371]
8司静,李伟,崔宝凯,戴玉成.真菌漆酶性质、分子生物学及其应用研究进展[J].生物技术通报,2011,2:48-55[Si J,Li W,Cui BK,Dai YC.Advances of research on characteristics,molecular biology and applications of laccase from fungi[J].Biotechnol Bull,2011,2:48-55]
9林俊芳,刘志明,陈晓阳,郭丽琼,王杰.真菌漆酶的酶活测定方法评价[J].生物加工过程,2009,7(4):1-8[Lin JF,Liu ZM,Chen XY,Guo LQ,Wang J.Evaluation of assay methods for determining fungal laccase activity[J].Chin J Biopros Eng,2009,7(4):1-8]
10李江,袁月祥,闫志英,刘晓风,廖银章,李娜.毛栓菌原生质体制备和再生及单核菌株产漆酶特性[J].菌物学报,2012,31(1):102-109[Li J,Yuan YX,Yan ZH,Liu XF,Liao YZ,Li Na.The protoplast preparation and regeneration of Trametes hirsuta and laccase production of monokaryons[J].Mycosystema,2012,31(1):102-109]
11张美景,杨硕,陈艳萍,袁建华,孟庆长.玉米黑粉菌单倍体菌株的分离及交配型a位点的鉴别[J].江苏农业科学,2013,41(4):122-124[Zhang MJ,Yang S,Chen YP,Yuan JH,Meng QC.Isolation of haploid strain of maize black powders and the identification of mating type a sites[J].Jiangsu Agric Sci,2013,41(4):122-124]
12王冰.球孢白僵菌菌株交配型分子鉴定[J].哈尔滨师范大学自然科学学报,2014,30(1):62-64[Wang B.Molecular identification of strain mating type of Beauveria bassiana[J].Nat Sci J Harbin Norm Univ,2014,30(1):62-64]
13 Yan JP,Niu JZ,Chen DD,Chen YH,Irbis C.Screening of Trametes st rai ns for ef f icient decolor i zat ion of malach ite g reen at h ig h temperatures and ionic concentrations[J].Int Biodeterior Biodegrad,2014,87:109-115
14 Yan JP,Chen DD,Yang E,Niu JZ,Chen YH,Irbis C.Purification and characterization of a thermotolerant laccase isoform in T.trogii strain and its potential in decolorization[J].Int Biodeterior Biodegrad,2014,93:186-194
15 Fan FF,Zhuo R,Sun S,Wan X,Jiang ML,Zhang XY,Yang Y.Cloning and functional analysis of a new laccase gene from Trametes sp.48424which had the high yield of laccase and strong ability for decolorizing different dyes[J].Bioresour Technol,2011,102:3126-3137
16钟耀华,王晓利,汪天虹.丝状真菌高效表达异源蛋白研究进展[J].生物工程学报,2008,24(4):531-540[Zhong YH,Wang XL,Wang TH.Recent advances in the production of heterologous proteins in filamentous fungi[J].Chin J Biotechnol,2008,24(4):531-540]
17 Maestre-Reyna M,Liu WC,Jeng WY,Lee CC,Hsu CA,Wen TN,Wang AHJ,Shyur LF.Structural and functional roles of glycosylation in fungal laccase from Lentinus sp.[J].PLoS ONE,2015,10:1932-6203
18林范学,李宏盛,冯磊,梁保东,鲍大鹏.交配型对香菇单、双核菌丝体菌丝生长速度的影响[J].上海农业学报,2013,29(5):15-22[Lin FX,Li HS,Feng L,Liang BD,Bao DP.The relationships between mating types and mycelial growth rates of monokaryons and dikaryons in Lentinula edodes[J].Acta Agric Shanghai,2013,29(5):15-22]
19杨阳,马双新,贾慧,刘微微,曹志艳,董金皋.玉米大斑病菌两性交配型菌株的出现频率及其育性分析[J].植物保护学报,2015,42(6):921-926[Yang Y,Ma SX,Jia H,Liu WW,Cao ZY,Dong JG.The occurrence and fertility analysis of bisexual strains of Setosphaeria turcica[J].Acta Plant Prot,2015,42(6):921-926]
20高扬,章西,黄勃.蝉棒束孢交配型基因的分析和检测[J].中国生物防治学报,2015,31(6):860-867[Gao Y,Zhang X,Huang B.Analysis and molecular detection of Isaria cicadae mating-type gene[J].Chin JBiol Contr,2015,31(6):860-867]
21 Baccelli I,Gonthier P,Bernardi R.Gene expression analyses reveal a relationship between conidiation and cerato-platanin in homokaryotic and heterokaryotic strains of the fungal plant pathogen Heterobasidion irregulare[J].Mycol Prog,2015,14:40
22 Qu P,Aratani A,Syoji T,Toda T,Kubota M,Hyakumachi M.Use of single-protoplast isolates in the study of the mating phenomena of Rhizoctonia solani(Thanatephorus cucumeris)AG-1 IC and IA[J].Mycoscience,2008,49(2):132-137
2秦改娟,王晓,陈青君,张国庆.不同配方培养料生产双孢蘑菇过程中主要木质纤维素降解酶及物料组分的变化[J].应用与环境生物学报,2017,23(6):1035-1041[Qin GJ,Wang X,Chen QJ,Zhang GQ.Changes of lignocellulolytic enzymes and material components in different compost formulas during the production of Agaricus bisporus[J].Chin J Appl Environ Biol,2017,23(6):1035-1041]
3刘刚,王义刚,吴淑芳,丁少军.鸡腿菇漆酶同工酶降解马尾松硫酸盐浆中残余木质素的研究[J].纤维素科学与技术,2013,21(4):239-245[Liu G,Wang YG,Wu SF,Ding SJ.Study on enzymatic delignification of Pinus massoniana kraft pulp by laccase isozymes of Coprinus comatus[J].J Cell Sci Technol,2013,21(4):239-245]
4郑飞,孟歌,安琪,吴雪君,司静,刘红霞.白腐真菌东方栓孔菌在两种液体培养基中产漆酶过程的生理学研究[J].菌物学报,2017,36(5):582-597[Zheng F,Meng G,An Q,Wu XJ,Si J,Liu HX.Physiological studies on laccase-producing process of white rot fungus Trametes orientalis incubated with two kinds of liquid media[J].Mycosystema,2017,36(5):582-597]
5司静,崔宝凯.绒毛栓孔菌液体培养过程中胞外酶活性的研究[J].基因组学与应用生物学,2012,31(1):70-77[Si J,Cui BK.Studies on activities of extracellular enzymes in liquid culture of Trametes pubescens[J].Genomics Appl Biol,2012,31(1):70-77]
6司静,崔宝凯,戴玉成.栓孔菌属漆酶高产菌株的初步筛选及其产酶条件的优化[J].微生物学通报,2011,38(3):405-416[Si J,Cui BK,Dai YC.Primary screening of effective Trametes strains with high laccaseproductivity and optimization of conditions on laccase production[J].Microbiol Chin,2011,38(3):405-416]
7司静,崔宝凯,戴玉成.东方栓孔菌在染料脱色中的应用及其脱色条件的优化[J].基因组学与应用生物学,2011,30(3):364-371[Si J,Cui BK,Dai YC.Application in dye decolorization and optimization of conditions in discoloration by Trametes orientalis[J].Genomics Appl Biol,2011,30(3):364-371]
8司静,李伟,崔宝凯,戴玉成.真菌漆酶性质、分子生物学及其应用研究进展[J].生物技术通报,2011,2:48-55[Si J,Li W,Cui BK,Dai YC.Advances of research on characteristics,molecular biology and applications of laccase from fungi[J].Biotechnol Bull,2011,2:48-55]
9林俊芳,刘志明,陈晓阳,郭丽琼,王杰.真菌漆酶的酶活测定方法评价[J].生物加工过程,2009,7(4):1-8[Lin JF,Liu ZM,Chen XY,Guo LQ,Wang J.Evaluation of assay methods for determining fungal laccase activity[J].Chin J Biopros Eng,2009,7(4):1-8]
10李江,袁月祥,闫志英,刘晓风,廖银章,李娜.毛栓菌原生质体制备和再生及单核菌株产漆酶特性[J].菌物学报,2012,31(1):102-109[Li J,Yuan YX,Yan ZH,Liu XF,Liao YZ,Li Na.The protoplast preparation and regeneration of Trametes hirsuta and laccase production of monokaryons[J].Mycosystema,2012,31(1):102-109]
11张美景,杨硕,陈艳萍,袁建华,孟庆长.玉米黑粉菌单倍体菌株的分离及交配型a位点的鉴别[J].江苏农业科学,2013,41(4):122-124[Zhang MJ,Yang S,Chen YP,Yuan JH,Meng QC.Isolation of haploid strain of maize black powders and the identification of mating type a sites[J].Jiangsu Agric Sci,2013,41(4):122-124]
12王冰.球孢白僵菌菌株交配型分子鉴定[J].哈尔滨师范大学自然科学学报,2014,30(1):62-64[Wang B.Molecular identification of strain mating type of Beauveria bassiana[J].Nat Sci J Harbin Norm Univ,2014,30(1):62-64]
13 Yan JP,Niu JZ,Chen DD,Chen YH,Irbis C.Screening of Trametes st rai ns for ef f icient decolor i zat ion of malach ite g reen at h ig h temperatures and ionic concentrations[J].Int Biodeterior Biodegrad,2014,87:109-115
14 Yan JP,Chen DD,Yang E,Niu JZ,Chen YH,Irbis C.Purification and characterization of a thermotolerant laccase isoform in T.trogii strain and its potential in decolorization[J].Int Biodeterior Biodegrad,2014,93:186-194
15 Fan FF,Zhuo R,Sun S,Wan X,Jiang ML,Zhang XY,Yang Y.Cloning and functional analysis of a new laccase gene from Trametes sp.48424which had the high yield of laccase and strong ability for decolorizing different dyes[J].Bioresour Technol,2011,102:3126-3137
16钟耀华,王晓利,汪天虹.丝状真菌高效表达异源蛋白研究进展[J].生物工程学报,2008,24(4):531-540[Zhong YH,Wang XL,Wang TH.Recent advances in the production of heterologous proteins in filamentous fungi[J].Chin J Biotechnol,2008,24(4):531-540]
17 Maestre-Reyna M,Liu WC,Jeng WY,Lee CC,Hsu CA,Wen TN,Wang AHJ,Shyur LF.Structural and functional roles of glycosylation in fungal laccase from Lentinus sp.[J].PLoS ONE,2015,10:1932-6203
18林范学,李宏盛,冯磊,梁保东,鲍大鹏.交配型对香菇单、双核菌丝体菌丝生长速度的影响[J].上海农业学报,2013,29(5):15-22[Lin FX,Li HS,Feng L,Liang BD,Bao DP.The relationships between mating types and mycelial growth rates of monokaryons and dikaryons in Lentinula edodes[J].Acta Agric Shanghai,2013,29(5):15-22]
19杨阳,马双新,贾慧,刘微微,曹志艳,董金皋.玉米大斑病菌两性交配型菌株的出现频率及其育性分析[J].植物保护学报,2015,42(6):921-926[Yang Y,Ma SX,Jia H,Liu WW,Cao ZY,Dong JG.The occurrence and fertility analysis of bisexual strains of Setosphaeria turcica[J].Acta Plant Prot,2015,42(6):921-926]
20高扬,章西,黄勃.蝉棒束孢交配型基因的分析和检测[J].中国生物防治学报,2015,31(6):860-867[Gao Y,Zhang X,Huang B.Analysis and molecular detection of Isaria cicadae mating-type gene[J].Chin JBiol Contr,2015,31(6):860-867]
21 Baccelli I,Gonthier P,Bernardi R.Gene expression analyses reveal a relationship between conidiation and cerato-platanin in homokaryotic and heterokaryotic strains of the fungal plant pathogen Heterobasidion irregulare[J].Mycol Prog,2015,14:40
22 Qu P,Aratani A,Syoji T,Toda T,Kubota M,Hyakumachi M.Use of single-protoplast isolates in the study of the mating phenomena of Rhizoctonia solani(Thanatephorus cucumeris)AG-1 IC and IA[J].Mycoscience,2008,49(2):132-137
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