药用真菌桑黄分子鉴定及遗传多样性分析
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摘要
通过对不同来源的桑黄菌株进行分子鉴定和遗传多样性分析,为后续桑黄种质资源的研究、开发及利用提供参考。采用r DNA ITS序列分析技术,对收集到的国内22个桑黄菌株进行分子鉴定与遗传多样性分析。依据r DNA ITS序列计算遗传距离并构建系统发育树,结果显示收集到的桑黄菌株明确聚为3个独立类群,且3个类群的桑黄真菌存在明显的遗传分化。通过BLAST分析,成功鉴定出3类桑黄种质分别为:桑树桑黄(Sanghuangporus sanghuang)、杨树桑黄(Fuscoporia gilva)及丁香桑黄(Inonotus baumii)。不同来源的桑黄菌株间具有一定的遗传多样性,种间遗传趋异度显著高于种内。本研究结果提供了一种准确可靠且简单易行的桑黄真菌分子鉴定技术,可明确各桑黄真菌的种属来源及遗传结构组成,为桑黄真菌的进一步研究及开发应用奠定了基础。
To study the genetic diversity and genetic relationship of Phellinus baumii,and their relative species by ITS molecular marker technique,and to provide the reference for Phellinus baumii identification and breeding,the ribosomal DNA internal transcribed spacer( rDNA ITS) region was amplified and sequenced to identify the 22 species of Sanghuang which were collected from domestic and detect its genetic diversity. Based on r DNA ITS sequences,the genetic distance and phylogenetic tree were obtained. Results showed that there were distinct genetic divergences among 22 species of Sanghuang,which formed three separate species cluster in the topology tree obviously with high bootstrap value. Meanwhile,two counterfeit Sanghuang strain,Fuscoporia gilva and Inonotus baumii,were detected successfully,and the certified Sanghuang germplasms were distinguished as Sanghuangporus sanghuang unambiguously by BLAST analysis. The results of ITS analysis revealed that Phellinus baumii had the plentiful genetic diversity and the genetic relationships were consistent with morphological characters of taxonomy. ITS method was efficient for identification of Phellinus baumii,which could provide a scientific basis for the resource collection and identification of the species in Phellinus baumii.
To study the genetic diversity and genetic relationship of Phellinus baumii,and their relative species by ITS molecular marker technique,and to provide the reference for Phellinus baumii identification and breeding,the ribosomal DNA internal transcribed spacer( rDNA ITS) region was amplified and sequenced to identify the 22 species of Sanghuang which were collected from domestic and detect its genetic diversity. Based on r DNA ITS sequences,the genetic distance and phylogenetic tree were obtained. Results showed that there were distinct genetic divergences among 22 species of Sanghuang,which formed three separate species cluster in the topology tree obviously with high bootstrap value. Meanwhile,two counterfeit Sanghuang strain,Fuscoporia gilva and Inonotus baumii,were detected successfully,and the certified Sanghuang germplasms were distinguished as Sanghuangporus sanghuang unambiguously by BLAST analysis. The results of ITS analysis revealed that Phellinus baumii had the plentiful genetic diversity and the genetic relationships were consistent with morphological characters of taxonomy. ITS method was efficient for identification of Phellinus baumii,which could provide a scientific basis for the resource collection and identification of the species in Phellinus baumii.
引文
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[9]陈体强,吴锦忠,纪建英,等.桑黄栽培子实体成分分析及其显微形态观察[J].菌物研究,2005,3(1):30-34.CHEN T Q,WU J Z,JI J Y,et al.Composition analysis and micro-morphological observation of log-cultivated fruitbodies of Phellinus igniarius[J].Journal of Fungal Research,2005,3(1):30-34.(in Chinese with English abstract)
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[15]谢丽源,张勇,彭金华,等.桑黄真菌分子鉴定及遗传多样性分析[J].菌物学报,2010,29(3):347-356.XIE L Y,ZHANG Y,PENG J H,et al.Molecular identification and genetic diversity of a traditional Chinese medicinal fungus“sanghuang”[J].Mycosystema,2010,29(3):347-356.(in Chinese with English abstract)
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[17]WHITE T J,BRUNS T,LEE S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[M]//INNIS M A,GELFAND D H,SNINSKY J J,et al.PCR protocols:a guide to methods and applications.New York:Academic Press,1989:315-322.
[18]邵晨霞,靳磊,唐少军,等.7株桑黄遗传亲缘关系和栽培特性研究[J].中国食用菌,2017,36(3):40-43.SHAO C X,JIN L,TANG S J,et al.Study on genetic relationships and cultivate characteristic of 7 Inonotus sanghuang strains[J].Edible Fungi of China,2017,36(3):40-43.(in Chinese with English abstract)
[2]张小青,戴玉成.中国真菌志[M].北京:科学出版社,2005.
[3]吕英华,王建芳,李玉平,等.药用真菌桑黄的研究进展[J].蚕业科学,2009,35(1):204-210.LYU Y H,WANG J F,LI Y P,et al.Progress of studies on the medicinal fungus Phellinus spp.[J].Science of Sericulture,2009,35(1):204-210.(in Chinese with English abstract)
[4]孙焙龙,徐双阳,杨开,等.珍稀药用真菌桑黄的国内外研究进展[J].微生物学通报,2006,33(2):119-123.SUN P L,XU S Y,YANG K,et al.Advance of researches on Phellinus spp.a rare and precious medicinal fungus[J].Microbiology China,2006,33(2):119-123.(in Chinese with English abstract)
[5]杜萍,张春凤,崔宝凯,等.药用真菌桑黄的人工栽培技术研究[J].中国食用菌,2009,28(3):35-37.DU P,ZHANG C F,CUI B K,et al.Cultivation technique of the medicinal fungus Phellinus buamii.[J].Edible Fungi of China,2009,28(3):35-37.(in Chinese with English abstract)
[6]丁云云,刘锋,施超,等.桑黄化学成分及体外抗肿瘤活性研究[J].中国中药杂志,2016,41(16):3042-3048.DING Y Y,LIU F,SHI C,et al.Chemical constituents from Phellinus igniarius and their anti-tumor activity in vitro[J].China Journal of Chinese Materia Medica,2016,41(16):3042-3048.(in Chinese with English abstract)
[7]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,2014,36(5):1-6.DAI Y C,CUI B K.Progress on the species of medicinal fungus Inonotus sanghuang[J].Journal of Beijing Forestry University,2014,36(5):1-6.(in Chinese with English abstract)
[8]黄丽洋,石卉,王晓婷,等.野生桑黄菌株的分离、鉴定和次生代谢物分析[J].中草药,2013,44(23):3394-3399.HUANG L Y,SHI H,WANG X T,et al.Isolation and identification for strains from wild Phellinus and analysis on its secondary metabolites[J].Chinese Traditional and Herbal Drugs,2013,44(23):3394-3399.(in Chinese with English abstract)
[9]陈体强,吴锦忠,纪建英,等.桑黄栽培子实体成分分析及其显微形态观察[J].菌物研究,2005,3(1):30-34.CHEN T Q,WU J Z,JI J Y,et al.Composition analysis and micro-morphological observation of log-cultivated fruitbodies of Phellinus igniarius[J].Journal of Fungal Research,2005,3(1):30-34.(in Chinese with English abstract)
[10]吴声华,黄冠中,陈愉萍,等.桑黄的分类及开发前景[J].菌物研究,2016,14(4):187-200.WU S H,HUANG G Z,CHEN Y P,et al.Taxonomy and development prospects of Sanghuang(Sanghuangporus sanghuang)[J].Journal of Fungal Research,2016,14(4):187-200.(in Chinese with English abstract)
[11]高凯,杜明,吕英华,等.10株桑黄菌基于r DNA ITS序列的分子鉴定[J].蚕业科学,2010,36(4):584-589.GAO K,DU M,LYU Y H,et al.Molecular identification of10 Phellinus strains based on r DNA ITS sequences[J].Science of Sericulture,2010,36(4):584-589.(in Chinese with English abstract)
[12]LANDEWEERT R,LEEFLANG P,KUYPER T W,et al.Molecular identification of ectomycorrhizal mycelium in soil horizons[J].Applied&Environmental Microbiology,2003,69(1):327-333.
[13]KERNAGHAN G,SIGLER L,KHASA D.Mycorrhizal and root endophytic fungi of containerized Picea glauca seedlings assessed by r DNA sequence analysis[J].Microbial Ecology,2003,45(2):128-136.
[14]LEBLOND-BOURGET N,HERVE P,IRENE M,et al.16Sr RNA and 16S to 23S internal transcribed spacer analyses reveal inter and intraspecific Bifidobacterium phylogeny[J].International Journal of Systematic Bacteriology,1996,46(1):102-111.
[15]谢丽源,张勇,彭金华,等.桑黄真菌分子鉴定及遗传多样性分析[J].菌物学报,2010,29(3):347-356.XIE L Y,ZHANG Y,PENG J H,et al.Molecular identification and genetic diversity of a traditional Chinese medicinal fungus“sanghuang”[J].Mycosystema,2010,29(3):347-356.(in Chinese with English abstract)
[16]GESNETO A,LOGUERCIOLEITE C,GUERRERO R T.DNA extraction from frozen field-collected and dehydrated herbarium fungal basidiomata:performance of SDS and CTAB-based methods[J].Biotemas,2005,18(2):19-32.
[17]WHITE T J,BRUNS T,LEE S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[M]//INNIS M A,GELFAND D H,SNINSKY J J,et al.PCR protocols:a guide to methods and applications.New York:Academic Press,1989:315-322.
[18]邵晨霞,靳磊,唐少军,等.7株桑黄遗传亲缘关系和栽培特性研究[J].中国食用菌,2017,36(3):40-43.SHAO C X,JIN L,TANG S J,et al.Study on genetic relationships and cultivate characteristic of 7 Inonotus sanghuang strains[J].Edible Fungi of China,2017,36(3):40-43.(in Chinese with English abstract)