过表达漆酶基因Lelcc1的香菇菌株构建及表型分析
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摘要
真菌漆酶(laccase)是一种多酚氧化酶,在真菌生长发育中具有重要作用。本研究采用根癌农杆菌介导转化的方法,以香菇Lentinulaedodes菌株W1为受体菌株,在Leactin基因启动子调控下过表达Lelcc1基因;对其中7个单拷贝插入的转化子进行qRT-PCR分析,7个Lelcc1基因表达量较出发菌株W1均上调了1.5–8倍。进一步分析了这7个转化子的遗传稳定性;挑取了3个稳定的转化子进行表型分析,主要包括不同培养基中的生长速度、代料栽培过程中菌棒转色程度、以及透射电镜观察菌丝细胞的超微结构。发现转化子和受体菌株W1的生长速度无显著差异,但代料栽培过程中转化子较W1转色更快,菌棒表面颜色更深;透射电镜观察菌丝细胞的超微结构发现在细胞壁厚度、细胞膜形态等方面,其中两个超量表达转化子与W1间存在显著差异。结果表明,香菇Lelcc1基因可能参与了转色过程中色素合成或积累,也可能与细胞壁形成有关。
Fungal laccase is an important polyphenol oxidase which plays important roles in fungal development.In the present study, the shiitake mushroom Lentinula edodes strain W1 was used as acceptor, and a laccase gene Lelcc1 was overexpressed under the control of actin promoter. The vector was transferred into L. edodes mycelia via Agrobacterium tumefaciens-mediated transformation method. Seven transformants were single-copy inserted,and qRT-PCR analysis showed that the expression of Lelcc1 in these seven transformants was 1.5–8 times higher than that in wild type strain W1. The genetic stability of these seven transformants were further analyzed,and three stable transformants were selected for phenotypic analysis including growth rate in different media,browning degree of substitution cultivation bags, and the ultrastructure of mycelial cells observed by transmission electron microscopy. The results indicated that the mycelium growth rate of three overexpression transformants growing in different medium was not significantly different from that of wild type strain. In sawdust-based cultivation,the transformants browned faster and the surface color was darker than W1. Significant differences were detected in the cell wall thickness and cell membrane morphology between the two overexpressed transformants and W1.These results indicated that Lelcc1 gene of L. edodes might be involved in the synthesis or accumulation of pigments during the process of brown film formation, and might also be related to the formation of cell wall.
Fungal laccase is an important polyphenol oxidase which plays important roles in fungal development.In the present study, the shiitake mushroom Lentinula edodes strain W1 was used as acceptor, and a laccase gene Lelcc1 was overexpressed under the control of actin promoter. The vector was transferred into L. edodes mycelia via Agrobacterium tumefaciens-mediated transformation method. Seven transformants were single-copy inserted,and qRT-PCR analysis showed that the expression of Lelcc1 in these seven transformants was 1.5–8 times higher than that in wild type strain W1. The genetic stability of these seven transformants were further analyzed,and three stable transformants were selected for phenotypic analysis including growth rate in different media,browning degree of substitution cultivation bags, and the ultrastructure of mycelial cells observed by transmission electron microscopy. The results indicated that the mycelium growth rate of three overexpression transformants growing in different medium was not significantly different from that of wild type strain. In sawdust-based cultivation,the transformants browned faster and the surface color was darker than W1. Significant differences were detected in the cell wall thickness and cell membrane morphology between the two overexpressed transformants and W1.These results indicated that Lelcc1 gene of L. edodes might be involved in the synthesis or accumulation of pigments during the process of brown film formation, and might also be related to the formation of cell wall.
引文
Cai Y,Gong Y,Liu W,Hu Y,Chen L,Yan L,Zhou Y,Bian Y,2017.Comparative secretomic analysis of lignocellulose degradation by Lentinula edodes grown on microcrystalline cellulose,lignosulfonate and glucose.Journal of Proteomics,163:92-101
Chen L,Gong Y,Cai Y,Liu W,Zhou Y,Xiao Y,Xu Z,Liu Y,Lei X,Wang G,Guo M,Ma X,Bian Y,2016.Genome sequence of the edible cultivated mushroom Lentinula edodes(shiitake)reveals insights into lignocellulose degradation.PLoS One,11(8):e0160336
Hu Y,Cai Y,Yu J,Zhou Y,Li J,Bian Y,Gong Y,2018.Function analysis of HMG-box transcripion factor lelcrp1 in Lentinula edodes.Acta Microbiologica Sinica,58(12):2100-2109(in Chinese)
Giardina P,Faraco V,Pezzella C,Piscitelli A,Vanhulle S,Sannia G,2010.Laccases:a never-ending story.Cellular and Molecular Life Sciences,67(3):369-385
Jeon JR,Baldrian P,Murugesan K,Chang YS,2012.Laccase-catalyzed oxidations of naturally occurring phenols:from in vivo biosynthetic ways to green synthetic applications.Microbial Biotechnology,5(3):318-332
Li C,Gong W,Zhang L,Yang Z,Nong W,Bian Y,Kwan HS,Cheung MK,Xiao Y,2017.Association mapping reveals genetic loci associated with important agronomic traits in Lentinula edodes,shiitake mushroom.Frontiers in Microbiology,8:237
Li Y,2005.Research status and prospect of Lentinula edodes.Microbiology China,32(4):149-152(in Chinese)
Ma CJ,Wang GZ,Zhou SS,Luo Y,Gong YH,Bian YB,2018.Functional analyses of anthranilate synthase gene LetrpEin Lentinula edodes by RNAi mediated gene knockdown.Mycosystema,37(5):576-583(in Chinese)
Nagai M,Sato T,Watanabe H,Saito K,Kawata M,Enei H,2002.Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes,and decolorization of chemically different dyes.Applied Microbiology and Biotechnology,60(3):327-335
Nakade K,Watanabe H,Sakamoto Y,Sato T,2011.Gene silencing of the Lentinula edodes lcc1 gene by expression of a homologous inverted repeat sequence.Microbiological Research,166(6):484-493
Pfaffl MW,2001.A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Research,29(9):e45
Qin P,Li J,Gu Y,Zeng X,Xiang Q,2018.Transcriptional expression profiles of the laccase gene family in different development stages of Lentinus edodes.Chinese Journal of Applied and Environmental Biology,24(2):379-383(in Chinese)
Sakamoto Y,Nakade K,Sato S,Yoshimi A,Sasaki K,Konno N,Abe K,2018.Cell wall structure of secreted laccase-silenced strain in Lentinula edodes.Fungal Biology,122(12):1192-1200
Sakamoto Y,Nakade K,Yoshida K,Natsume S,Miyazaki K,Sato S,van Peer AF,Konno N,2015.Grouping of multicopper oxidases in Lentinula edodes by sequence similarities and expression patterns.AMB Express,5(1):63
Shui PR,Zheng XB,Lin JF,Guo LQ,2008.A simple and high quality RNA extraction method for edible fungi.Acta Edulis Fungi,15(1):32-36(in Chinese)
Siletti CE,Zeiner CA,Bhatnagar JM,2017.Distributions of fungal melanin across species and soils.Soil Biology and Biochemistry,113:285-293
Singh S,Malhotra AG,Pandey A,Pandey KM,2013.Computational model for pathway reconstruction to unravel the evolutionary significance of melanin synthesis.Bioinformation,9(2):94-100
Sugareva V,H?rtl A,Brock M,Hübner K,Rohde M,Heinekamp T,Brakhage AA,2006.Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus.Archives of Microbiology,186(5):345-355
Tang L,Jian H,Song C,Bao D,Shang X,Wu D,Tan Q,Zhang X,2013.Transcriptome analysis of candidate genes and signaling pathways associated with light-induced brown film formation in Lentinula edodes.Applied Microbiology and Biotechnology,97(11):4977-4989
Tang LH,Tan Q,Bao DP,Zhang XH,Jian HH,Li Y,Wang Y,2016.Comparative proteomic analysis of light-induced mycelial brown film formation in Lentinula edodes.BioMed Research International,2016:5837293
Zhang J,Chen H,Chen M,Ren A,Huang J,Wang H,Zhao M,Feng Z,2015.Cloning and functional analysis of a laccase gene during fruiting body formation in Hypsizygus marmoreus.Microbiological Research,179:54-63
Zhang Q,2016.Study on the change regularity of extracellular enzyme activity and agronony characters of Lentinula edodes.Master Thesis,Henan Institute of Science and Technology,Xinxiang.1-77(in Chinese)
胡悦,蔡英丽,喻晶晶,周雁,李军平,边银丙,龚钰华,2018.香菇HMG-box转录因子lelcrp1基因的功能.微生物学报,58(12):2100-2109
李月梅,2005.香菇的研究现状及发展前景.微生物学通报,32(4):149-152
马超君,王刚正,周莎莎,罗义,龚钰华,边银丙,2018.RNAi法分析香菇邻氨基苯甲酸合酶基因功能.菌物学报,37(5):576-583
秦澎,李津,辜运富,曾先富,向泉桔,2018.香菇生长发育过程中漆酶基因家族的转录表达.应用与环境生物学报,24(2):379-383
税丕容,郑晓冰,林俊芳,郭丽琼,2008.简便高质量的食用菌总RNA提取方法.食用菌学报,15(1):32-36
张权,2016.香菇胞外酶活性变化规律和农艺性状研究.河南科技学院硕士论文,新乡.1-77
Chen L,Gong Y,Cai Y,Liu W,Zhou Y,Xiao Y,Xu Z,Liu Y,Lei X,Wang G,Guo M,Ma X,Bian Y,2016.Genome sequence of the edible cultivated mushroom Lentinula edodes(shiitake)reveals insights into lignocellulose degradation.PLoS One,11(8):e0160336
Hu Y,Cai Y,Yu J,Zhou Y,Li J,Bian Y,Gong Y,2018.Function analysis of HMG-box transcripion factor lelcrp1 in Lentinula edodes.Acta Microbiologica Sinica,58(12):2100-2109(in Chinese)
Giardina P,Faraco V,Pezzella C,Piscitelli A,Vanhulle S,Sannia G,2010.Laccases:a never-ending story.Cellular and Molecular Life Sciences,67(3):369-385
Jeon JR,Baldrian P,Murugesan K,Chang YS,2012.Laccase-catalyzed oxidations of naturally occurring phenols:from in vivo biosynthetic ways to green synthetic applications.Microbial Biotechnology,5(3):318-332
Li C,Gong W,Zhang L,Yang Z,Nong W,Bian Y,Kwan HS,Cheung MK,Xiao Y,2017.Association mapping reveals genetic loci associated with important agronomic traits in Lentinula edodes,shiitake mushroom.Frontiers in Microbiology,8:237
Li Y,2005.Research status and prospect of Lentinula edodes.Microbiology China,32(4):149-152(in Chinese)
Ma CJ,Wang GZ,Zhou SS,Luo Y,Gong YH,Bian YB,2018.Functional analyses of anthranilate synthase gene LetrpEin Lentinula edodes by RNAi mediated gene knockdown.Mycosystema,37(5):576-583(in Chinese)
Nagai M,Sato T,Watanabe H,Saito K,Kawata M,Enei H,2002.Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes,and decolorization of chemically different dyes.Applied Microbiology and Biotechnology,60(3):327-335
Nakade K,Watanabe H,Sakamoto Y,Sato T,2011.Gene silencing of the Lentinula edodes lcc1 gene by expression of a homologous inverted repeat sequence.Microbiological Research,166(6):484-493
Pfaffl MW,2001.A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Research,29(9):e45
Qin P,Li J,Gu Y,Zeng X,Xiang Q,2018.Transcriptional expression profiles of the laccase gene family in different development stages of Lentinus edodes.Chinese Journal of Applied and Environmental Biology,24(2):379-383(in Chinese)
Sakamoto Y,Nakade K,Sato S,Yoshimi A,Sasaki K,Konno N,Abe K,2018.Cell wall structure of secreted laccase-silenced strain in Lentinula edodes.Fungal Biology,122(12):1192-1200
Sakamoto Y,Nakade K,Yoshida K,Natsume S,Miyazaki K,Sato S,van Peer AF,Konno N,2015.Grouping of multicopper oxidases in Lentinula edodes by sequence similarities and expression patterns.AMB Express,5(1):63
Shui PR,Zheng XB,Lin JF,Guo LQ,2008.A simple and high quality RNA extraction method for edible fungi.Acta Edulis Fungi,15(1):32-36(in Chinese)
Siletti CE,Zeiner CA,Bhatnagar JM,2017.Distributions of fungal melanin across species and soils.Soil Biology and Biochemistry,113:285-293
Singh S,Malhotra AG,Pandey A,Pandey KM,2013.Computational model for pathway reconstruction to unravel the evolutionary significance of melanin synthesis.Bioinformation,9(2):94-100
Sugareva V,H?rtl A,Brock M,Hübner K,Rohde M,Heinekamp T,Brakhage AA,2006.Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus.Archives of Microbiology,186(5):345-355
Tang L,Jian H,Song C,Bao D,Shang X,Wu D,Tan Q,Zhang X,2013.Transcriptome analysis of candidate genes and signaling pathways associated with light-induced brown film formation in Lentinula edodes.Applied Microbiology and Biotechnology,97(11):4977-4989
Tang LH,Tan Q,Bao DP,Zhang XH,Jian HH,Li Y,Wang Y,2016.Comparative proteomic analysis of light-induced mycelial brown film formation in Lentinula edodes.BioMed Research International,2016:5837293
Zhang J,Chen H,Chen M,Ren A,Huang J,Wang H,Zhao M,Feng Z,2015.Cloning and functional analysis of a laccase gene during fruiting body formation in Hypsizygus marmoreus.Microbiological Research,179:54-63
Zhang Q,2016.Study on the change regularity of extracellular enzyme activity and agronony characters of Lentinula edodes.Master Thesis,Henan Institute of Science and Technology,Xinxiang.1-77(in Chinese)
胡悦,蔡英丽,喻晶晶,周雁,李军平,边银丙,龚钰华,2018.香菇HMG-box转录因子lelcrp1基因的功能.微生物学报,58(12):2100-2109
李月梅,2005.香菇的研究现状及发展前景.微生物学通报,32(4):149-152
马超君,王刚正,周莎莎,罗义,龚钰华,边银丙,2018.RNAi法分析香菇邻氨基苯甲酸合酶基因功能.菌物学报,37(5):576-583
秦澎,李津,辜运富,曾先富,向泉桔,2018.香菇生长发育过程中漆酶基因家族的转录表达.应用与环境生物学报,24(2):379-383
税丕容,郑晓冰,林俊芳,郭丽琼,2008.简便高质量的食用菌总RNA提取方法.食用菌学报,15(1):32-36
张权,2016.香菇胞外酶活性变化规律和农艺性状研究.河南科技学院硕士论文,新乡.1-77