两株丝状真菌N3、N9的分类鉴定
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摘要
本实验室从一酸奶样品中分离到两株丝状真菌N3、N9。通过形态学观察、RAPD分析及5.8SrDNA及ITS区序列测定并结合出菇试验,初步确定了N3、N9菌株的分类地位以及它们与其近缘种的生物系统学关系。
1 分别观察了N3、N9菌株在PDA培养基上的菌落特征和菌丝体形态特征。结果表明:N3、N9菌株在PDA培养基上25℃培养7d后菌落生长旺盛,呈丝状辐射生长,表面呈绒絮状,圆形,平坦。菌丝浓密,粗壮,多气生菌丝,菌丝体上具有明显的锁状联合结构,故初步判定N3、N9菌株为担子菌。
2 比较了CTAB、SDS-CTAB和氯化苄这3种DNA提取方法提取8株担子菌DNA的效果。结果表明:CTAB法和SDS-CTAB法只适合于部分菌株的DNA提取,对另外一些菌株则提取不出谱带清晰的DNA;而氯化苄法适合于所有供试菌株的DNA提取,所有菌株的DNA凝胶电泳图谱带清晰,效果很好。同时研究比较了液氮研磨和提取液的pH值对氯化苄法提取DNA效果的影响。
3 运用RAPD技术对N3、N9菌株和6株担子菌标准菌株进行了比较。首先从40个10bp引物中筛选出18个有效引物,用于所有供试材料基因组DNA样品的扩增。这18个引物对8株供试菌株均能扩增出清晰的谱带。对RAPD结果进行聚类分析,构建了供试菌株基因型的亲缘关系图。从聚类图上可以看出,菌株N3、N9与糙皮侧耳(Pleurotus ostreatus)白平菇、糙皮侧耳澳黑平菇、姬菇(P.limpidus)河北33、毛头鬼伞(Coprinus comatus)聚为一类,而香菇(Lentinus edodes)、金针菇(Flammulina velutipes)聚为另一类。其中菌株N3、N9与白平菇的亲缘关系最近,与澳黑平菇、河北33的亲缘关系稍近一些,故可初步确定N3、N9菌株应属于侧耳属。
4 再次运用RAPD技术对N3、N9菌株和侧耳属7株菌株进行了聚类分析。以扩增条带清晰、多态性丰富和重现性好为原则,从40个10bp引物中筛选出了21个有效引物,用于所有供试材料基因组DNA样品的扩增。聚类结果表明:菌株N3、N9与白平菇相似程度最高,三
Two strains of filamentous fungi N3 and N9 were isolated from one yoghurt sample. By morphological observation, RAPD typing, nucleotide sequence analysis of their internal transcribed spacer regions and 5.8SrDNA as well as cultivating experiment, the taxonomy position of strains N3, N9 and biosystematics of them and their close relative species were identified.The colony and mycelial microscopic characters of N3, N9 on potato dextrose agar (PDA) medium were observed respectively. Colonial growth for 7d at 25 °C was characterized by the radial extension of mycelia on the surface of medium, creating a circular and smooth fungal colony. Their hyphae were dense, thick and aerial, and had distinct clamp connections structure, which was unique to basidiomycetes. Based on the characters above, strains N3 and N9 were primarily identified as basidiomycetes.Three kinds of methods (i. e. benzyl choeide, CTAB and SDS-CTAB method) were used to extract DNA from eight strains of basidiomycetes. The results showed that the benzyl choeide method was able to be used to extract the DNA of these basidiomycetes, and bands resulting from gel electrophoretic separations were very clear. However, both the CTAB and SDS-CTAB methods were only adequate to be used to extract DNA from some strains of basidiomycetes and the DNA bands of some strains were clear, but others were not obvious. The effects of both the pH values of extracts and the grinding to mycelia in liquid nitrogen on the result of extracting DNA were also discussed.Eight strains of basidiomycetes, i.e. N3, N9 and 6 standard strains, had been analyzed for their RAPD genotype. Among 40 arbitrary primers, 18 primers could get enough amplified bands for all strains. The genetic relationship had been evaluated by similarity cofficient obtained from these profiles. The cluster showed that all strains could be divided into two parts. The first part consisted of strains N3 and N9, all strains of Pleurotus and Coprinus comatus. The second part was Lentinus edodes and Flammulina
velutipes. N3, N9 and P. ostreatus were closely related. According the result, strains N3 and N9 were identified to the genus Pleurotus.RAPD was again employed to study the genetic variation of strains N3, N9 and 7 standard strains of Pleurotus. Twenty-one random primers selected from 40 ones were used for DNA amplification. Specific characteristic bands were produced by most of the 21 primers. The dendrogram resulated from hierarchical cluster analysis based on all bands amplified by the 21 primers showed that the isolates were distinctly classified into two large categories. The first group was composed of the strains N3, N9 and 4 isolates of Pleurotus (i.e. bai, aohei, hebei 33, heipingl). Furthermore, the strains N3, N9 and P. ostreatus had a close relationship. The second group was consisted of P. abalones, P. citrinopileatus and P. ferulae.The internal transcribed spacer regions and 5.8S of the ribosomal RNA gene from strains N3, N9 were amplified using the polymerase chain reaction and sequenced. Sequences from the above isolates were compared with published sequences of P. ostreatus, P. columbinus, P. spodoleucus and so on in Genbank database and phylogenetic trees were produced based on ITS sequence data by "Neighbour-joining" methods of the software Bioedit. The sequence analysis showed that the strain N3 and P. spodoleucus had the most close relationship with a bootstrap value of 100%. This suggested that they were probably conspecific. The strain N9 and P. floridanus were closely related with 99% bootstrap value, it indicated that N9 might belong to P. floridanus.The strains N3, N9 were cultivated for the formation of fruitbodies. The result showed that strains N3, N9 had the capability forming fruitbody and they were identified to the genus Pleurotus by the morphological character observation to their fruitbodies.
1 分别观察了N3、N9菌株在PDA培养基上的菌落特征和菌丝体形态特征。结果表明:N3、N9菌株在PDA培养基上25℃培养7d后菌落生长旺盛,呈丝状辐射生长,表面呈绒絮状,圆形,平坦。菌丝浓密,粗壮,多气生菌丝,菌丝体上具有明显的锁状联合结构,故初步判定N3、N9菌株为担子菌。
2 比较了CTAB、SDS-CTAB和氯化苄这3种DNA提取方法提取8株担子菌DNA的效果。结果表明:CTAB法和SDS-CTAB法只适合于部分菌株的DNA提取,对另外一些菌株则提取不出谱带清晰的DNA;而氯化苄法适合于所有供试菌株的DNA提取,所有菌株的DNA凝胶电泳图谱带清晰,效果很好。同时研究比较了液氮研磨和提取液的pH值对氯化苄法提取DNA效果的影响。
3 运用RAPD技术对N3、N9菌株和6株担子菌标准菌株进行了比较。首先从40个10bp引物中筛选出18个有效引物,用于所有供试材料基因组DNA样品的扩增。这18个引物对8株供试菌株均能扩增出清晰的谱带。对RAPD结果进行聚类分析,构建了供试菌株基因型的亲缘关系图。从聚类图上可以看出,菌株N3、N9与糙皮侧耳(Pleurotus ostreatus)白平菇、糙皮侧耳澳黑平菇、姬菇(P.limpidus)河北33、毛头鬼伞(Coprinus comatus)聚为一类,而香菇(Lentinus edodes)、金针菇(Flammulina velutipes)聚为另一类。其中菌株N3、N9与白平菇的亲缘关系最近,与澳黑平菇、河北33的亲缘关系稍近一些,故可初步确定N3、N9菌株应属于侧耳属。
4 再次运用RAPD技术对N3、N9菌株和侧耳属7株菌株进行了聚类分析。以扩增条带清晰、多态性丰富和重现性好为原则,从40个10bp引物中筛选出了21个有效引物,用于所有供试材料基因组DNA样品的扩增。聚类结果表明:菌株N3、N9与白平菇相似程度最高,三
Two strains of filamentous fungi N3 and N9 were isolated from one yoghurt sample. By morphological observation, RAPD typing, nucleotide sequence analysis of their internal transcribed spacer regions and 5.8SrDNA as well as cultivating experiment, the taxonomy position of strains N3, N9 and biosystematics of them and their close relative species were identified.The colony and mycelial microscopic characters of N3, N9 on potato dextrose agar (PDA) medium were observed respectively. Colonial growth for 7d at 25 °C was characterized by the radial extension of mycelia on the surface of medium, creating a circular and smooth fungal colony. Their hyphae were dense, thick and aerial, and had distinct clamp connections structure, which was unique to basidiomycetes. Based on the characters above, strains N3 and N9 were primarily identified as basidiomycetes.Three kinds of methods (i. e. benzyl choeide, CTAB and SDS-CTAB method) were used to extract DNA from eight strains of basidiomycetes. The results showed that the benzyl choeide method was able to be used to extract the DNA of these basidiomycetes, and bands resulting from gel electrophoretic separations were very clear. However, both the CTAB and SDS-CTAB methods were only adequate to be used to extract DNA from some strains of basidiomycetes and the DNA bands of some strains were clear, but others were not obvious. The effects of both the pH values of extracts and the grinding to mycelia in liquid nitrogen on the result of extracting DNA were also discussed.Eight strains of basidiomycetes, i.e. N3, N9 and 6 standard strains, had been analyzed for their RAPD genotype. Among 40 arbitrary primers, 18 primers could get enough amplified bands for all strains. The genetic relationship had been evaluated by similarity cofficient obtained from these profiles. The cluster showed that all strains could be divided into two parts. The first part consisted of strains N3 and N9, all strains of Pleurotus and Coprinus comatus. The second part was Lentinus edodes and Flammulina
velutipes. N3, N9 and P. ostreatus were closely related. According the result, strains N3 and N9 were identified to the genus Pleurotus.RAPD was again employed to study the genetic variation of strains N3, N9 and 7 standard strains of Pleurotus. Twenty-one random primers selected from 40 ones were used for DNA amplification. Specific characteristic bands were produced by most of the 21 primers. The dendrogram resulated from hierarchical cluster analysis based on all bands amplified by the 21 primers showed that the isolates were distinctly classified into two large categories. The first group was composed of the strains N3, N9 and 4 isolates of Pleurotus (i.e. bai, aohei, hebei 33, heipingl). Furthermore, the strains N3, N9 and P. ostreatus had a close relationship. The second group was consisted of P. abalones, P. citrinopileatus and P. ferulae.The internal transcribed spacer regions and 5.8S of the ribosomal RNA gene from strains N3, N9 were amplified using the polymerase chain reaction and sequenced. Sequences from the above isolates were compared with published sequences of P. ostreatus, P. columbinus, P. spodoleucus and so on in Genbank database and phylogenetic trees were produced based on ITS sequence data by "Neighbour-joining" methods of the software Bioedit. The sequence analysis showed that the strain N3 and P. spodoleucus had the most close relationship with a bootstrap value of 100%. This suggested that they were probably conspecific. The strain N9 and P. floridanus were closely related with 99% bootstrap value, it indicated that N9 might belong to P. floridanus.The strains N3, N9 were cultivated for the formation of fruitbodies. The result showed that strains N3, N9 had the capability forming fruitbody and they were identified to the genus Pleurotus by the morphological character observation to their fruitbodies.
引文
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[3] 曾东方.真菌分子系统学的产生和发展.中国食用菌,1999,18(2):3～4.
[4] 周与良,刑来君.真菌学.北京:高等教育出版社.1986:151～155.
[5] Simmons EG. Altemaria themes and variations. Mycotaxon, 1986, 25(11): 287-308.
[6] 黄亦存,沈崇尧,裘维藩.外生菌根的形态学、解剖学及分类学研究进展.真菌学报,1992,11(3):169～181.
[7] 曾东方,罗信昌.外生菌根蘑菇分离菌株的DNA鉴定方法.微生物学杂志,2001,21(2):26～29.
[8] 马富英,罗信昌.分子标记在食用菌遗传育种中的应用.菌物系统,2002,21(1):147～151.
[9] 周与良,邢来君.真菌的系统发育和进化方面的研究进展.微生物学通报,1982,9(5):216～220.
[10] 熊庆,刘作易.几种分子生物学方法在真菌中的应用.贵州农业科学,2002,30(3):69～73.
[11] Kohn LM. Developing new characters for fungal systematics: An experimental approach for determining the rank of resolution. Mycologia, 1992, 84(2): 139～153.
[12] Forster H, Oudemans P, Coffy MD. Mitochondrial and nuclear DNA diversity 'within six species of Phytophthora. Experimental Mycology, 1990, 14:18～31.
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