偏肿革裥菌系统发育与Lg-mnp2和Lg-mnp3基因克隆及结构特征
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摘要
白腐菌偏肿革裥菌Lenzites gibbosa能分泌降解木质素的锰过氧化物酶(MnPs),为进一步鉴定试验菌株,对菌株L. gibbosa CB1进行了ITS序列(Gen Bank登录号为JF279440)扩增与测序,并进行了基于ITS序列的比对与系统发育分析,结果表明CB1与桦革裥菌L. betulina和栓菌属Trametes等相关白腐菌的亲缘关系较近而聚类在一起,并与24个同种其他菌株的ITS序列覆盖度在85%～97%期间内的相似性都为99%,说明该菌株为偏肿革裥菌。为了获得该菌株多个编码MnP家族的基因,从而为研究MnP基因的表达调控奠定序列结构的基础,根据已知白腐菌MnPs基因保守区和已经扩增出的基因片段设计引物,以L. gibbosa CB1的基因组DNA和总RNA为模板,采用PCR、RT-PCR、RACE及染色体步移等方法,克隆到该菌株编码MnP2和MnP3的全长c DNA和DNA基因(Gen Bank登录号分别为JQ388597、JN571114; JQ411249、JN571116),分别命名为Lg-mnp2和Lg-mnp3。2个基因DNA全长分别为2 869、3 992 bp,都含有6个外显子和5个内含子;其启动子区域都含有TATA-Box、CAAT-Box、AP2、MRE等顺式作用元件,Lg-mnp2还含有AP1,Lg-mnp3还含有HSE;其c DNA基因分别含有50、52 bp的5'UTR,150、249 bp的3'UTR和1098、1 101 bp的完整开放阅读框ORF,分别编码了365、366个氨基酸的蛋白质多肽前体(Gen Bank登录号分别为AFC37493、AFC37494),成熟的Lg-mnp2蛋白含有339个氨基酸,比Lg-MnP1蛋白(Gen Bank登录号为ACO92620)多1个氨基酸;成熟的Lg-mnp3蛋白含有340个氨基酸,比Lg-MnP2蛋白多1个氨基酸。
The ITS sequence of the tested strain Lenzites gibbosa CB1 was amplified and sequenced( GenB ank Accession No.:JF279440) to identify it,and a phylogenetic analysis was performed. The results showed that L. gibbosa CB1 and L. betulina,sevaral Trametes spp.,and other related white-rot fungi had the closest genetic relationship and clustered together,the ITS sequence of L. gibbosa CB1 was 99% homologous in coverage of 85%-97% with 24 different L. gibbosa strains,indicating that strain CB1 was L. gibbosa. In order to obtain more genes encoding MnP family of this strain,and to establish the basis of sequence structure for the further study of expression regulation of MnP genes,two full length cD NA and DNA genes named Lg-mnp2 and Lg-mnp3 which encode for two isoenzymes of manganese peroxidase 2 and 3( MnP2 and MnP3) from L. gibbosa CB1( GenB ank Accession No. JQ388597 and JN571114; and JQ411249 and JN571116 for cD NA and DNA gene,respectively) were cloned by PCR,RT-PCR,RACE,and Genome Walking PCR with the genome DNA and total RNA as templates. The series of primers for various PCRs were designed based on the conserved sequences of the known MnP genes from other white-rot fungi and amplified DNA fragments of L. gibbosa CB1. The full length DNA of the two genes is 2 869 and 3 992 bp,respectively,both containing 6 exons and 5 introns. Both promoter regions contain diverse cis-acting elements such as TATA-Box,CAAT-Box,AP2,and MRE,while Lg-mnp2 contains AP1 and Lg-mnp3 contains HSE. Their cD NA genes contain 1 098,and 1 101 bp coding regions encoding for two preproteins of 365 and 366 amino acids( GenB ank Accession No. AFC37493 and AFC37494) and with 50 and 52 bp 5' UTR,and 150 and 249 bp 3'UTR,respectively. The mature Lg-mnp2 protein contains 339 amino acids,one more amino acid than Lg-MnP1 protein( GenB ank Accession No.ACO92620),and the mature Lg-mnp3 protein contains 340 amino acids,one amino acid more than Lg-MnP2 protein.
The ITS sequence of the tested strain Lenzites gibbosa CB1 was amplified and sequenced( GenB ank Accession No.:JF279440) to identify it,and a phylogenetic analysis was performed. The results showed that L. gibbosa CB1 and L. betulina,sevaral Trametes spp.,and other related white-rot fungi had the closest genetic relationship and clustered together,the ITS sequence of L. gibbosa CB1 was 99% homologous in coverage of 85%-97% with 24 different L. gibbosa strains,indicating that strain CB1 was L. gibbosa. In order to obtain more genes encoding MnP family of this strain,and to establish the basis of sequence structure for the further study of expression regulation of MnP genes,two full length cD NA and DNA genes named Lg-mnp2 and Lg-mnp3 which encode for two isoenzymes of manganese peroxidase 2 and 3( MnP2 and MnP3) from L. gibbosa CB1( GenB ank Accession No. JQ388597 and JN571114; and JQ411249 and JN571116 for cD NA and DNA gene,respectively) were cloned by PCR,RT-PCR,RACE,and Genome Walking PCR with the genome DNA and total RNA as templates. The series of primers for various PCRs were designed based on the conserved sequences of the known MnP genes from other white-rot fungi and amplified DNA fragments of L. gibbosa CB1. The full length DNA of the two genes is 2 869 and 3 992 bp,respectively,both containing 6 exons and 5 introns. Both promoter regions contain diverse cis-acting elements such as TATA-Box,CAAT-Box,AP2,and MRE,while Lg-mnp2 contains AP1 and Lg-mnp3 contains HSE. Their cD NA genes contain 1 098,and 1 101 bp coding regions encoding for two preproteins of 365 and 366 amino acids( GenB ank Accession No. AFC37493 and AFC37494) and with 50 and 52 bp 5' UTR,and 150 and 249 bp 3'UTR,respectively. The mature Lg-mnp2 protein contains 339 amino acids,one more amino acid than Lg-MnP1 protein( GenB ank Accession No.ACO92620),and the mature Lg-mnp3 protein contains 340 amino acids,one amino acid more than Lg-MnP2 protein.
引文
[1] GODFREY B J,MAYFIELD M B,BROWN J A,et al. Characterization of a gene encoding a manganese peroxidase from Phanerochaete chrysosporium[J]. Gene,1990,93(1):119-124.
[2] MAYFIELD M B,GODFREY B J,GOLD M H. Characterization of the mnp2 gene encoding manganese peroxidase isoenzyme 2 from the basidiomycete Phanerochaete chrysosporium[J]. Gene,1994,142(2):231-235.
[3] ALIC M,AKILESWARAN L,GOLD M H. Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium[J]. Biochimica et Biophysica Acta,1997,1338(1):1-7.DOI:10.1016/s0167-4838(96)00235-x.
[4] MACARENA S,LARRONDO L F,VASQUEZ M,et al. Incomplete processing of peroxidase transcripts in the lignin degrading fungus Phanerochaete chrysosporium[J]. FEMS Microbiology Letters,2005,242(1):37-44.
[5] MARTINEZ D,LARRONDO L F,PUTNAM N,et al. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78[J]. Nature Biotechnology,2004,22(6):695-700.
[6] LOBOS S,LARRONDO L,SALAS L,et al. Cloning and molecular analysis of a c DNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora[J]. Gene,1998,206(2):185-93.
[7] TELLO M,CORSINI G,LARRONDO L F,et al. Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora[J]. Biochimica et Biophysica Acta,2000,1490(1/2):137-144.DOI:10.1016/s0167-4781(99)00227-4.
[8] YAVER D S,WEBER B,MURRELL J. Global expression profiling of the lignin degrading fungus Ceriporiopsis subvermispora for the discovery of novel enzymes[J]. Applied Mycology and Biotechnology,2003,3(3):261-269.
[9] SUNDARAMOORTHY M,KISHI K,GOLD M H,et al. The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-resolution[J]. The Journal of Biological Chemistry,1994,269(52):32759-32767.
[10] MORGENSTERN I,ROBERTSON D L,HIBBETT D S. Characterization of three mnp genes of Fomitiporia mediterranea and report of additional class II peroxidases in the order hymenochaetales[J]. Applied Environment Microbiology,2010,76(19):6431-6440.
[11]闫洪波,池玉杰,吴书景.偏肿革裥菌Lg-mnp1基因克隆及表达研究[J].安徽农业科学,2014,42(11):3186-3190.
[12]闫洪波.偏肿拟栓菌锰过氧化物酶c DNA基因克隆及在毕赤酵母中的表达[D].哈尔滨:东北林业大学,2009.
[13]尹立伟,池玉杰,王雪童.灰树花的系统发育分析和主要木质素降解酶的测定[J].林业科学研究,2010,23(4):574-580.
[14] HEMMI T,IKEYA J. Studies on Lenzites gibbosa(Pers.)Hemmi n. comb. causing wood-rot of deciduous trees[J]. Japanese Transaction of Phytopathology,1939,9(1):1-15.
[15] TOMSOVSK Y M,KOLARIK M,PAZOUTOVA S,et al. Molecular phylogeny of European Trametes(Basidiomycetes,Polyporales)species based on LSU and ITS(nrDNA)sequences[J]. Nova Hedwigia,2006,82(3/4):269-280.
[16] RYU S H,KIM B,KIM M,et al. Molecular characterization of manganese peroxidases from white-rot fungus Polyporus brumalis[J]. Bioprocess Biosyst Eng,2014,37(3):393-400.
[17] KAZUMICHI F,MARI U,CHIE H,et al. Environmental control of lignin peroxidase,manganese peroxidase,and laccase activities in forest floor layers in humid Asia[J]. Soil Biology and Biochemistry,2013,57:109-115.
[2] MAYFIELD M B,GODFREY B J,GOLD M H. Characterization of the mnp2 gene encoding manganese peroxidase isoenzyme 2 from the basidiomycete Phanerochaete chrysosporium[J]. Gene,1994,142(2):231-235.
[3] ALIC M,AKILESWARAN L,GOLD M H. Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium[J]. Biochimica et Biophysica Acta,1997,1338(1):1-7.DOI:10.1016/s0167-4838(96)00235-x.
[4] MACARENA S,LARRONDO L F,VASQUEZ M,et al. Incomplete processing of peroxidase transcripts in the lignin degrading fungus Phanerochaete chrysosporium[J]. FEMS Microbiology Letters,2005,242(1):37-44.
[5] MARTINEZ D,LARRONDO L F,PUTNAM N,et al. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78[J]. Nature Biotechnology,2004,22(6):695-700.
[6] LOBOS S,LARRONDO L,SALAS L,et al. Cloning and molecular analysis of a c DNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora[J]. Gene,1998,206(2):185-93.
[7] TELLO M,CORSINI G,LARRONDO L F,et al. Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora[J]. Biochimica et Biophysica Acta,2000,1490(1/2):137-144.DOI:10.1016/s0167-4781(99)00227-4.
[8] YAVER D S,WEBER B,MURRELL J. Global expression profiling of the lignin degrading fungus Ceriporiopsis subvermispora for the discovery of novel enzymes[J]. Applied Mycology and Biotechnology,2003,3(3):261-269.
[9] SUNDARAMOORTHY M,KISHI K,GOLD M H,et al. The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-resolution[J]. The Journal of Biological Chemistry,1994,269(52):32759-32767.
[10] MORGENSTERN I,ROBERTSON D L,HIBBETT D S. Characterization of three mnp genes of Fomitiporia mediterranea and report of additional class II peroxidases in the order hymenochaetales[J]. Applied Environment Microbiology,2010,76(19):6431-6440.
[11]闫洪波,池玉杰,吴书景.偏肿革裥菌Lg-mnp1基因克隆及表达研究[J].安徽农业科学,2014,42(11):3186-3190.
[12]闫洪波.偏肿拟栓菌锰过氧化物酶c DNA基因克隆及在毕赤酵母中的表达[D].哈尔滨:东北林业大学,2009.
[13]尹立伟,池玉杰,王雪童.灰树花的系统发育分析和主要木质素降解酶的测定[J].林业科学研究,2010,23(4):574-580.
[14] HEMMI T,IKEYA J. Studies on Lenzites gibbosa(Pers.)Hemmi n. comb. causing wood-rot of deciduous trees[J]. Japanese Transaction of Phytopathology,1939,9(1):1-15.
[15] TOMSOVSK Y M,KOLARIK M,PAZOUTOVA S,et al. Molecular phylogeny of European Trametes(Basidiomycetes,Polyporales)species based on LSU and ITS(nrDNA)sequences[J]. Nova Hedwigia,2006,82(3/4):269-280.
[16] RYU S H,KIM B,KIM M,et al. Molecular characterization of manganese peroxidases from white-rot fungus Polyporus brumalis[J]. Bioprocess Biosyst Eng,2014,37(3):393-400.
[17] KAZUMICHI F,MARI U,CHIE H,et al. Environmental control of lignin peroxidase,manganese peroxidase,and laccase activities in forest floor layers in humid Asia[J]. Soil Biology and Biochemistry,2013,57:109-115.