青霉PT95菌株的鉴定和培养条件的研究
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摘要
从山西汾阳混交林土壤中分离到一株能在菌核内积累β-胡萝卜素的青霉PT95菌株。本研究通过形态观察和RAPD分析，初步确定了PT95菌株的分类地位以及该菌株和其近缘种的生物系统学关系。在此基础上进一步研究了无机盐和碳氮源对PT95菌株菌核生物量和类胡萝卜素产率的影响。
1．分别观察了PT95菌株在查氏酵母膏琼脂(CYA)、麦芽汁琼脂(MEA)、25％甘油硝酸盐琼脂(G25N)和查氏琼脂(CA)上的菌落特征和个体形态特征。结果表明：PT95菌株在CYA培养基上25℃培养7d后能形成由大量的菌核和少量分生孢子构成的菌落，菌落表面呈放射状的沟纹和皱缩；菌核区颜色为粉红色，后变为深红色，产孢区颜色为青灰色；菌核球形或不规则形，直径约300μm；帚状枝为严格单轮生，分生孢子梗生自基质或气生菌丝；菌丝分枝较少；瓶梗3～8个轮生，安瓿形，排列较紧密，8μm～10μm×1.5μm～2.5μm；分生孢子椭圆形，长轴2.5μm～3.0μm。在CYA培养基上37℃不生长，5℃偶尔生长。经检索，PT95菌株属于汤姆青霉系。
2．比较了CTAB、SDS-CTAB、和氯化苄这3种DNA提取方法提取青霉DNA的效果。实验结果表明：CTAB法和SDS-CTAB法只能适合于部分青霉菌株的DNA提取。另外一些菌株则提取不出谱带清晰的DNA。而氯化苄法适合于供试的所有青霉菌株的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PT95菌株、4株PT95的突变株和6株标准青霉菌株进行了比较。从70个10bp引物中筛选出13个有效引物，用于所有供试材料基因组DNA样品的扩增。这13个引物对11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，构建了各供试菌株基因型的亲缘关系图。从聚类图上可以看出，PT95菌株与汤姆青霉系的3种标准菌聚为一类，而产黄青霉、娄地青霉和嗜松青霉聚为另一类，这与形态分类的结果
相一致。但PT95菌株在电泳图谱上与汤姆青霉系的3种己知标准菌均有一定的差异，根
据聚类图也可以清楚地将PT95菌株与其他菌株分开，这说明PT95菌株不同于任一汤姆青
霉系的已知菌，很可能是一新种。
4.研究了无机盐和碳氮源对青霉PT95菌株菌核生物量和类胡萝卜素产率的影响作
用，结果表明:供试的4种无机盐中，KZHPO;的单因子效应最好;KZHPo4+KCI+MgS04
表现出最好的正协同效应。5种碳源都能被PT95菌株利用，麦芽糖和蔗糖是最适碳源。在
以酵母膏为氮源的培养基上，PT95菌株的菌核生物量最高;而在以蛋自膝为氮源的培养基
上，类胡萝卜素产率最高;钱盐和尿素对菌核形成不利;硝酸钠是最好的无机氮源。培养
基中的含氮量保持在0.249几一0.489几，含碳量保持在5.269/L一21.059/L，有利于PT95菌
株形成菌核和积累色素;培养基的最适C闪比为25
A strain of Penicillium sp. PT95 was isolated from soil sample collected close to Fenyang, Shanxi Province. The strain can form abundant sclerotia in which β -carotene is accumulated. By morphological observation and RAPD typing, the taxonomy position of PT95 strain and biosystematics of it and its close relative species was identified. The effects of both various inorganic salts, carbon and nitrogen sources on sclerotia biomass and carotenoid yield in surface cultures of PT95 strain were also studied.
1.The colony and microscopic characters of PT95 strain in 4 kinds of media, i.e. Czapek Yeast Extract Agar (CYA), Malt Extract Agar (MEA), 25%Glycerol Nitrate Agar (G25N) and Czapek Agar (CA), were observed respectively. Colonies on CYA for 7d at 25℃ typically 32mm-40 mm diam, radially sulcate or winkled, usually enveloping abundant sclerotia and overlaid by scattered penicillin. Sclerotia, pink, becoming scarlet in age, globose or irregular, about 300μm diam. Conidiogenesis sparse, caesious; pinicilli monoverticillati strictly, conidiophores borne from surface or subsurface hyphae; phialides ampulliform, 8μm-10μm×1.5μm-2.5μm; codidia ellipsoidal, 2. 5μm -3. 0μm long. Usually no germination at 5℃; occasionally germination observed. No growth at 37℃. Based the characters above, PT95 strain was identified to P. thomii series.
2.Three kinds of methods (i. e. benzyl choeide method, CTAB method and SDS-CTAB method) were used to extract DNA from eleven strains of Penicillium, The results showed that the benzyl choeide method was able to be used to extract the DNA of those Penicillium strains, 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 of some strains of Penicillium, 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 of mycelia in liquid nitrogen on the result of DNA extracting were also discussed.
3. Eleven strains of Penicillium, i.e. PT95 strain and its 4 mutants as well as 6 standard strains bought from Institute of Microbiology, Academia Sinica, have been analyzed for their RAPD genotype. Among 70 arbitrary primers, 13 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 PT95 and all strains of Pinicillium thomii series, and the second part was P. chrysogenum, P. roqueforti and P. pinophilum. The genotypic typing of species seems to be related to morphological classification. But PT95 strain differed from all standard strains of P. thomii series in gel electrophoretic separations. According to cluster, PT95 strain could be obviously distinguished from other strains. The result showed that PT95 strain was not any species known of P. thomii series and probably was a new species.
4. This study examined the respective effect of various inorganic salts, carbon and nitrogen sources on sclerotia biomass and carotenoid yield in surface cultures of PT95 strain. Among inorganic salts tested, K2HPO4was more essential to the sclerotia formation and carotenogenesis of strain PT9S than KCl, MgSO4 or FeSO4 It was also shown that the combination of K2HPO4, KCl and MgSO4 could produce the best positive cooperation and give the highest sclerotia biomass (782mg/plate) and pigment yield (328
μg/plate). All of five carbon sources, i..e. glucose, sucrose, lactose, maltose and soluble starch, could be utilized by the strain PT95, and maltose was the best. Among 8 nitrogen sources, yeast extract favored the sclerotia formation, and peptone favored the pigment accumulation; amine salts and urea were unfavorable to form sclerotia. The medium containing 0.24 g/L -0.48g/L sodium nitrate-nitrogen was effective to both the sclerotia formation and the carotenoid production of strain PT95 when available maltose-carbon c
1．分别观察了PT95菌株在查氏酵母膏琼脂(CYA)、麦芽汁琼脂(MEA)、25％甘油硝酸盐琼脂(G25N)和查氏琼脂(CA)上的菌落特征和个体形态特征。结果表明：PT95菌株在CYA培养基上25℃培养7d后能形成由大量的菌核和少量分生孢子构成的菌落，菌落表面呈放射状的沟纹和皱缩；菌核区颜色为粉红色，后变为深红色，产孢区颜色为青灰色；菌核球形或不规则形，直径约300μm；帚状枝为严格单轮生，分生孢子梗生自基质或气生菌丝；菌丝分枝较少；瓶梗3～8个轮生，安瓿形，排列较紧密，8μm～10μm×1.5μm～2.5μm；分生孢子椭圆形，长轴2.5μm～3.0μm。在CYA培养基上37℃不生长，5℃偶尔生长。经检索，PT95菌株属于汤姆青霉系。
2．比较了CTAB、SDS-CTAB、和氯化苄这3种DNA提取方法提取青霉DNA的效果。实验结果表明：CTAB法和SDS-CTAB法只能适合于部分青霉菌株的DNA提取。另外一些菌株则提取不出谱带清晰的DNA。而氯化苄法适合于供试的所有青霉菌株的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PT95菌株、4株PT95的突变株和6株标准青霉菌株进行了比较。从70个10bp引物中筛选出13个有效引物，用于所有供试材料基因组DNA样品的扩增。这13个引物对11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，构建了各供试菌株基因型的亲缘关系图。从聚类图上可以看出，PT95菌株与汤姆青霉系的3种标准菌聚为一类，而产黄青霉、娄地青霉和嗜松青霉聚为另一类，这与形态分类的结果
相一致。但PT95菌株在电泳图谱上与汤姆青霉系的3种己知标准菌均有一定的差异，根
据聚类图也可以清楚地将PT95菌株与其他菌株分开，这说明PT95菌株不同于任一汤姆青
霉系的已知菌，很可能是一新种。
4.研究了无机盐和碳氮源对青霉PT95菌株菌核生物量和类胡萝卜素产率的影响作
用，结果表明:供试的4种无机盐中，KZHPO;的单因子效应最好;KZHPo4+KCI+MgS04
表现出最好的正协同效应。5种碳源都能被PT95菌株利用，麦芽糖和蔗糖是最适碳源。在
以酵母膏为氮源的培养基上，PT95菌株的菌核生物量最高;而在以蛋自膝为氮源的培养基
上，类胡萝卜素产率最高;钱盐和尿素对菌核形成不利;硝酸钠是最好的无机氮源。培养
基中的含氮量保持在0.249几一0.489几，含碳量保持在5.269/L一21.059/L，有利于PT95菌
株形成菌核和积累色素;培养基的最适C闪比为25
A strain of Penicillium sp. PT95 was isolated from soil sample collected close to Fenyang, Shanxi Province. The strain can form abundant sclerotia in which β -carotene is accumulated. By morphological observation and RAPD typing, the taxonomy position of PT95 strain and biosystematics of it and its close relative species was identified. The effects of both various inorganic salts, carbon and nitrogen sources on sclerotia biomass and carotenoid yield in surface cultures of PT95 strain were also studied.
1.The colony and microscopic characters of PT95 strain in 4 kinds of media, i.e. Czapek Yeast Extract Agar (CYA), Malt Extract Agar (MEA), 25%Glycerol Nitrate Agar (G25N) and Czapek Agar (CA), were observed respectively. Colonies on CYA for 7d at 25℃ typically 32mm-40 mm diam, radially sulcate or winkled, usually enveloping abundant sclerotia and overlaid by scattered penicillin. Sclerotia, pink, becoming scarlet in age, globose or irregular, about 300μm diam. Conidiogenesis sparse, caesious; pinicilli monoverticillati strictly, conidiophores borne from surface or subsurface hyphae; phialides ampulliform, 8μm-10μm×1.5μm-2.5μm; codidia ellipsoidal, 2. 5μm -3. 0μm long. Usually no germination at 5℃; occasionally germination observed. No growth at 37℃. Based the characters above, PT95 strain was identified to P. thomii series.
2.Three kinds of methods (i. e. benzyl choeide method, CTAB method and SDS-CTAB method) were used to extract DNA from eleven strains of Penicillium, The results showed that the benzyl choeide method was able to be used to extract the DNA of those Penicillium strains, 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 of some strains of Penicillium, 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 of mycelia in liquid nitrogen on the result of DNA extracting were also discussed.
3. Eleven strains of Penicillium, i.e. PT95 strain and its 4 mutants as well as 6 standard strains bought from Institute of Microbiology, Academia Sinica, have been analyzed for their RAPD genotype. Among 70 arbitrary primers, 13 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 PT95 and all strains of Pinicillium thomii series, and the second part was P. chrysogenum, P. roqueforti and P. pinophilum. The genotypic typing of species seems to be related to morphological classification. But PT95 strain differed from all standard strains of P. thomii series in gel electrophoretic separations. According to cluster, PT95 strain could be obviously distinguished from other strains. The result showed that PT95 strain was not any species known of P. thomii series and probably was a new species.
4. This study examined the respective effect of various inorganic salts, carbon and nitrogen sources on sclerotia biomass and carotenoid yield in surface cultures of PT95 strain. Among inorganic salts tested, K2HPO4was more essential to the sclerotia formation and carotenogenesis of strain PT9S than KCl, MgSO4 or FeSO4 It was also shown that the combination of K2HPO4, KCl and MgSO4 could produce the best positive cooperation and give the highest sclerotia biomass (782mg/plate) and pigment yield (328
μg/plate). All of five carbon sources, i..e. glucose, sucrose, lactose, maltose and soluble starch, could be utilized by the strain PT95, and maltose was the best. Among 8 nitrogen sources, yeast extract favored the sclerotia formation, and peptone favored the pigment accumulation; amine salts and urea were unfavorable to form sclerotia. The medium containing 0.24 g/L -0.48g/L sodium nitrate-nitrogen was effective to both the sclerotia formation and the carotenoid production of strain PT95 when available maltose-carbon c
引文
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[6] Pitt J I. An appraisal of identification methods for Penicillium species: novel taxonomic criteria based on temperature and water relations. Mycologia, 1973, 65:1134～1157.
[7] Simmons E G. Altermaria themes and variations. Mycotaxon, 1993, 46: 171-199.
[8] 马富英，罗信吕．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．
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[2] 韩建荣，徐军．青霉PT95菌株固态发酵产生类胡萝卜素的研究．微生物学报，1999，39(2)：148～153．
[3] Raper K B, Thom C. Manual of the Penicillia. Williams &Wilkins Company, Baltimore, 1949.
[4] Pitt J I. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press, London, 1979. 1～634.
[5] Pitt J I. A laboratory guide connom Penicillium species. Division of food processing, North Ryde, 1988.
[6] Pitt J I. An appraisal of identification methods for Penicillium species: novel taxonomic criteria based on temperature and water relations. Mycologia, 1973, 65:1134～1157.
[7] Simmons E G. Altermaria themes and variations. Mycotaxon, 1993, 46: 171-199.
[8] 马富英，罗信吕．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] Williams J G K, Kubelik.A R, Livakk. J, et al. DNA Polymorphism by arbitrary Primers are useful as genetic markers. Nucleic Acid Res, 1990, 18(22): 6531～6535.
[10] Welsh J, Mcclelland M. Fingerprinting genomes using PCR with arbitray Primers. Nucleic AcidRes, 1990, 18(22): 7213～7218.
[11] Okoli C, Shilling DG, Smith RL, et al. Genetic diversity in purple mutsedge and yellow mutsedge. Biological control, 1997, 8: 111-118.
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