基于DNA条形码的桑黄真菌分子鉴定
|
摘要
DNA条形码(DNA barcoding)技术是一种利用生物基因组内短遗传标签进行分类鉴定的方法。桑黄自古以来是一种名贵中药材,而市售桑黄主要依托简单的形态鉴定造成混淆众多。近年来大量研究表明,桑黄类真菌种类至少有7个种,并且其药效也不尽相同。【目的】为更准确、高效对桑黄类真菌进行鉴定,本研究以采集自西藏、吉林和浙江的3种桑黄子实体为材料,筛选最佳DNA条形码与相应PCR条件。【方法】利用组织分离获得3种桑黄纯培养菌株,分别编号为1713、1714和HS菌株。利用ITS、NL、rpb1、rpb2和ef1-α等7组DNA条形码分别对3种桑黄进行PCR扩增、测序、比对和发育树分析,并结合形态学特征,确定其最适DNA条形码和分类学地位。【结果】结果表明,ITS和NL条形码对3种桑黄均能够获得单一目的条带;rpb1Y、rpb2B和rpb2Y条形码特异性低,扩增后获得多个条带;而rpb1B和ef1-α条形码均无法获得条带。通过多序列比对,结合形态分析,确定1713菌株为桑黄纤孔菌(Inonotus sanghuang),而1714和HS菌株均为鲍姆纤孔菌(Inonotus baumii)。【结论】本研究确定ITS和NL为桑黄分子鉴定的最佳DNA条形码,其PCR最适退火温度范围分别为58～59℃和49～50℃,为桑黄真菌快速鉴定提供参考依据。
DNA barcoding is a taxonomic method that uses short genetic markers in an organism's genomic DNA to identify particular species.‘Sang Huang'is a kind of precious traditional Chinese medicinal materials since ancient times.It is easily confused in markets only based on morphological identification.In recent years,a large number of studies have shown that there were at least 7 fungal species named‘Sang Huang'with different medicinal activities.【Objective】In the present study,we focused on accurate and efficient identification of‘Sang Huang'mushrooms.Fruiting bodies of three‘Sang Huang'collected from Tibet,Jilin,and Zhejiang were used for screening of the optimum DNA barcodings and their PCR protocols.【Methods】Pure culture 1713,1714,and HS were obtained using the tissue separation method..PCR amplification were performed using seven pairs DNA barcodings of ITS,NL,rpb1,rpb2 and ef1-α.The optimal DNA barcodings were determined based on sequence alignment,phylogenetic tree construction,and morphological features.【Results】The results showed that single band product can be amplified using ITS and NL as the primers in the three strains.Multiple nonspecific bands were amplified usingrpb1Y,rpb2B and rpb2 Yprimers,while no band was obtained using rpb1 Band ef1-αprimers.Based on multiple sequence alignment and morphological features,strain 1713 was identified as Inonotus sanghuang,strains 1714 and HS were both Inonotus baumii.【Conclusion】It suggests that ITS and NL can be used as the optimal DNA barcodings to identify‘Sang Huang'mushrooms,and the optimal range of annealing temperature was 58～59℃,and 49～50℃,respectively.
DNA barcoding is a taxonomic method that uses short genetic markers in an organism's genomic DNA to identify particular species.‘Sang Huang'is a kind of precious traditional Chinese medicinal materials since ancient times.It is easily confused in markets only based on morphological identification.In recent years,a large number of studies have shown that there were at least 7 fungal species named‘Sang Huang'with different medicinal activities.【Objective】In the present study,we focused on accurate and efficient identification of‘Sang Huang'mushrooms.Fruiting bodies of three‘Sang Huang'collected from Tibet,Jilin,and Zhejiang were used for screening of the optimum DNA barcodings and their PCR protocols.【Methods】Pure culture 1713,1714,and HS were obtained using the tissue separation method..PCR amplification were performed using seven pairs DNA barcodings of ITS,NL,rpb1,rpb2 and ef1-α.The optimal DNA barcodings were determined based on sequence alignment,phylogenetic tree construction,and morphological features.【Results】The results showed that single band product can be amplified using ITS and NL as the primers in the three strains.Multiple nonspecific bands were amplified usingrpb1Y,rpb2B and rpb2 Yprimers,while no band was obtained using rpb1 Band ef1-αprimers.Based on multiple sequence alignment and morphological features,strain 1713 was identified as Inonotus sanghuang,strains 1714 and HS were both Inonotus baumii.【Conclusion】It suggests that ITS and NL can be used as the optimal DNA barcodings to identify‘Sang Huang'mushrooms,and the optimal range of annealing temperature was 58～59℃,and 49～50℃,respectively.
引文
[1]包海鹰,杨烁,李庆杰,图力古尔,李玉.“桑黄”的本草补充考证[J].菌物研究,2017(4):264-270
[2]戴玉成.药用担子菌---鲍氏层孔菌(桑黄)的新认识[J].中草药,2003(1):97-98
[3]Dai YC,Xu MQ.Studies on the medicinal polypore,Phellinus baumii,and its kin,P.linteus[J].Mycotaxon,1998,67(1):191-200
[4]戴玉成,崔宝凯.药用真菌桑黄种类研究[J].北京林业大学学报,2014(5):1-7
[5]Wu SH,Dai YC,T Hattori.Species clarification for the medicinally valuable'sanghuang'mushroom[J].Botanical Studies,2012,53:35-149
[6]张宇,郭良栋.真菌DNA条形码研究进展[J].菌物学报,2012(6):809-820
[7]周均亮,赵瑞琳.真菌DNA条形码技术研究进展[J].微生物学通报,2013(8):1468-1477
[8]Xu J.Fungal DNA barcoding[J].Genome,2016,59(11):913-932
[9]A Justo,O Miettinen,D Floudas,B Ortizsantana,ESj9kvist.A revised family-level classification of the Polyporales(Basidiomycota)[J].Fungal Biology,2017,121(9):798-824
[10]Tian XM,Yu HY,Zhou LW,Decock C,Vlasák J&Dai YC.Phylogeny and taxonomy of the Inonotus linteus complex[J].Fungal Diversity,2013,58(1):159-169
[11]O’Donnell K,Rooney AP,Mills GL,Kuo M,Weber NS&Rehner SA.Phylogeny and historical biogeography of true morels(Morchella)reveals an early Cretaceous origin and high continental endemism and provincialism in the Holarctic[J].Fungal Genetics and Biology,2011,48(3):252-265
[12]Du XH,Zhao Q,Yang ZL,Hansen K,Taskin H,Büyükalaca S,Dewsbury D,Moncalvo JM,Douhan GW,Robert VA,Crous PW,Rehner SA,Rooney AP,Sink S,O'Donnell K.How well do ITS rDNA sequences differentiate species of true morels(Morchella)[J].Mycologia,2012,104(6):1351-1368
[13]Du XH,Zhao Q,O'Donnell K,Rooney AP,Yang ZL.Multigene molecular phylogenetics reveals true morels(Morchella)are especially species-rich in China[J].Fungal Genetics Biology,2012,49(6):455-469
[14]王寿南,陈青君,张国庆,李兵,李伟聪,赵天健.一种野生多孔菌的分离、鉴定、培养条件及抗氧化活性[J].应用与环境生物学报,2017(1):82-88
[15]刘悦萍,王子健,崔凯,张国庆.一种野生香蘑的分离、鉴定、培养条件与产酶特性[J].应用与环境生物学报,2012(5):804-809
[16]O'Donnell K,Cigelnik E,Weber NS e,James M.Trappe Phylogenetic relationships among Ascomycetous truffles and the true and false morels inferred from 18Sand 28Sribosomal DNA sequence analysis[J].Mycologia,1997,89(1):48-65
[17]熊熊川,李小林,李强,杨志荣,郑林用.四川秋季发生的两种羊肚菌生境调查与鉴定[J].菌物学报,2016(1):29-38
[18]谢丽源,张勇,彭金华,邓科君,彭卫红,郭勇,贾定洪,甘炳成.桑黄真菌分子鉴定及遗传多样性分析[J].菌物学报,2010(3):347-356
[19]Guglielmo F,Bergemann SE,Gonthier P,Nicolotti G,Garbelotto M.A multiplex PCR-based method for the detection and early identification of wood rotting fungi in standing trees[J].Journal of Applied Microbiology,2007,103(5):1490-1507
[20]吴声华,黄冠中,陈愉萍,戴玉成,周丽伟.桑黄的分类及开发前景[J].菌物研究,2016(4):187-200
[21]周丽伟,戴玉成.中国多孔菌多样性初探:物种、区系和生态功能[J].生物多样性,2013(4):499-506
[22]蔡为明,金群力,郑社会,王建功,宋婷婷,余建妹.浙江野生桑黄的生长特征与初步鉴定结果[J].食药用菌,2012(4):235-236
[23]Dai YC.Hymenochaetaceae(Basidiomycota)in China[J].Fungal Diversity,2010,45(1):131-343
[24]Cui BK,Decock C.Phellinus castanopsidis sp.nov.(Hymenochaetaceae)from southern China,with preliminary phylogeny based on rDNA sequences[J].Mycological Progress,2013,12(2):341-351
[25]Han JG,Hyun MW,Kim CS,Jo JW,Cho JH,Lee KH,Kong WS,Han SK,Oh J,Sung GH.Species identity of Phellinus linteus(Sanghuang)extensively used as a medicinal mushroom in Korea[J].Journal of Microbiology,2016,54(4):290-295
[26]李雪玲.贝盖侧耳的系统发育地位---基于nrDNA-LSU和ITS序列分析的研究[J].北京林业大学学报,2005(3):67-71
[2]戴玉成.药用担子菌---鲍氏层孔菌(桑黄)的新认识[J].中草药,2003(1):97-98
[3]Dai YC,Xu MQ.Studies on the medicinal polypore,Phellinus baumii,and its kin,P.linteus[J].Mycotaxon,1998,67(1):191-200
[4]戴玉成,崔宝凯.药用真菌桑黄种类研究[J].北京林业大学学报,2014(5):1-7
[5]Wu SH,Dai YC,T Hattori.Species clarification for the medicinally valuable'sanghuang'mushroom[J].Botanical Studies,2012,53:35-149
[6]张宇,郭良栋.真菌DNA条形码研究进展[J].菌物学报,2012(6):809-820
[7]周均亮,赵瑞琳.真菌DNA条形码技术研究进展[J].微生物学通报,2013(8):1468-1477
[8]Xu J.Fungal DNA barcoding[J].Genome,2016,59(11):913-932
[9]A Justo,O Miettinen,D Floudas,B Ortizsantana,ESj9kvist.A revised family-level classification of the Polyporales(Basidiomycota)[J].Fungal Biology,2017,121(9):798-824
[10]Tian XM,Yu HY,Zhou LW,Decock C,Vlasák J&Dai YC.Phylogeny and taxonomy of the Inonotus linteus complex[J].Fungal Diversity,2013,58(1):159-169
[11]O’Donnell K,Rooney AP,Mills GL,Kuo M,Weber NS&Rehner SA.Phylogeny and historical biogeography of true morels(Morchella)reveals an early Cretaceous origin and high continental endemism and provincialism in the Holarctic[J].Fungal Genetics and Biology,2011,48(3):252-265
[12]Du XH,Zhao Q,Yang ZL,Hansen K,Taskin H,Büyükalaca S,Dewsbury D,Moncalvo JM,Douhan GW,Robert VA,Crous PW,Rehner SA,Rooney AP,Sink S,O'Donnell K.How well do ITS rDNA sequences differentiate species of true morels(Morchella)[J].Mycologia,2012,104(6):1351-1368
[13]Du XH,Zhao Q,O'Donnell K,Rooney AP,Yang ZL.Multigene molecular phylogenetics reveals true morels(Morchella)are especially species-rich in China[J].Fungal Genetics Biology,2012,49(6):455-469
[14]王寿南,陈青君,张国庆,李兵,李伟聪,赵天健.一种野生多孔菌的分离、鉴定、培养条件及抗氧化活性[J].应用与环境生物学报,2017(1):82-88
[15]刘悦萍,王子健,崔凯,张国庆.一种野生香蘑的分离、鉴定、培养条件与产酶特性[J].应用与环境生物学报,2012(5):804-809
[16]O'Donnell K,Cigelnik E,Weber NS e,James M.Trappe Phylogenetic relationships among Ascomycetous truffles and the true and false morels inferred from 18Sand 28Sribosomal DNA sequence analysis[J].Mycologia,1997,89(1):48-65
[17]熊熊川,李小林,李强,杨志荣,郑林用.四川秋季发生的两种羊肚菌生境调查与鉴定[J].菌物学报,2016(1):29-38
[18]谢丽源,张勇,彭金华,邓科君,彭卫红,郭勇,贾定洪,甘炳成.桑黄真菌分子鉴定及遗传多样性分析[J].菌物学报,2010(3):347-356
[19]Guglielmo F,Bergemann SE,Gonthier P,Nicolotti G,Garbelotto M.A multiplex PCR-based method for the detection and early identification of wood rotting fungi in standing trees[J].Journal of Applied Microbiology,2007,103(5):1490-1507
[20]吴声华,黄冠中,陈愉萍,戴玉成,周丽伟.桑黄的分类及开发前景[J].菌物研究,2016(4):187-200
[21]周丽伟,戴玉成.中国多孔菌多样性初探:物种、区系和生态功能[J].生物多样性,2013(4):499-506
[22]蔡为明,金群力,郑社会,王建功,宋婷婷,余建妹.浙江野生桑黄的生长特征与初步鉴定结果[J].食药用菌,2012(4):235-236
[23]Dai YC.Hymenochaetaceae(Basidiomycota)in China[J].Fungal Diversity,2010,45(1):131-343
[24]Cui BK,Decock C.Phellinus castanopsidis sp.nov.(Hymenochaetaceae)from southern China,with preliminary phylogeny based on rDNA sequences[J].Mycological Progress,2013,12(2):341-351
[25]Han JG,Hyun MW,Kim CS,Jo JW,Cho JH,Lee KH,Kong WS,Han SK,Oh J,Sung GH.Species identity of Phellinus linteus(Sanghuang)extensively used as a medicinal mushroom in Korea[J].Journal of Microbiology,2016,54(4):290-295
[26]李雪玲.贝盖侧耳的系统发育地位---基于nrDNA-LSU和ITS序列分析的研究[J].北京林业大学学报,2005(3):67-71