: CMCC(F) Ja nos Varga (Aspergillus brasiliensis sp. nov.), (Aspergillus niger), [2] 2, 2008, Jan Houseknecht Biolog 5.8S rdna ITS Rep-PCR,

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1 微生物学通报 Microbiology China 研究报告 NOV 20, 2010, 37(11): by Institute of Microbiology, CAS 标准菌株 CMCC(F)98003 的多相复核鉴定 * 姚粟李辉程池 ( ) 摘要 : 采用形态学观察 Biolog 全自动微生物鉴定系统 ITS rdna 序列分析 ITS 区特殊位点碱基分析 β- 微管蛋白基因序列分析方法, 对中国药典 (CHP) 中指定使用的培养基质控参考菌株 CMCC(F)98003 进行了多相复核鉴定结果表明 : CMCC(F)98003 为黑曲霉, 与 CHP 收载的科学名称是一致的鉴于黑曲霉与巴西曲霉虽然较为接近, 但已成为两个不同的种, 其形态学系统发育学和代谢产物等特性均存在一定差异, 中国药典是否需要更换新的丝状真菌代表菌株, 以保持与国际上其他药典的一致性值得研究关键词 : CMCC(F)98003, 黑曲霉, 巴西曲霉, 多相鉴定 Polyphasic Identification Approaches for Reference Strain CMCC(F)98003 YAO Su LI Hui CHENG Chi * (China National Research Institute of Food & Fermentation Industries, China Center of Industrial Culture Collection, Beijing , China) Abstract: To identify reference strain CMCC(F)98003, which cited in Chinese Pharmacopoeia (CHP), polyphasic approaches including morphological features, Biolog identification system, ITS rdna sequence, specific positions based on the ITS region and β-tubulin gene sequences analysis were used. Results indicated that CMCC(F)98003 was identified as Aspergillus niger and consistent with the scientific name in CHP. Although A. brasiliensis is close to A. niger, they have been classified as two different species and their morphology, phylogenesis and metabolic profilies are distinct. To maintain the consistency of international Pharmacopoeia, whether CHP replaces the new representative of filamentous fungi strain is worth studying. Keywords: CMCC(F)98003, Aspergillus niger, Aspergillus brasiliensis, Polyphasic identification CMCC(F)98003, 2005,, ( 8 ), [1],,, 2007, 基金项目 : (No. 2007BAK36B06) * 通讯作者 :Tel: ; : [email protected] 收稿日期 : ; 接受日期 :

2 : CMCC(F) Ja nos Varga (Aspergillus brasiliensis sp. nov.), (Aspergillus niger), [2] 2, 2008, Jan Houseknecht Biolog 5.8S rdna ITS Rep-PCR, ATCC16404,, ATCC16404 IMI T, ATCC16888, ATCC16404 [3], ATCC [4], 2010 (WDCM) [5], 2010, [6] CMCC(F)98003 [7], Biolog ITS rdna β-, CMCC(F)98003, 1 材料 1.1 菌株 A. niger CMCC(F)98003, 1.2 培养基及试剂 (Czapek yeast extract agar, CYA): K 2 HPO 4 1 g, 10 ml, 5 g, 30 g, 15 g, 1000 ml : NaNO g, KCl 5.0 g, MgSO 4 7H 2 O 5.0 g, FeSO 4 7H 2 O 0.1 g, ZnSO 4 7H 2 O 0.1 g, CuSO 4 5H 2 O 0.05 g, 100 ml 2% (2% ME): 20 g (#LP0039B), 18 g ; 1000 ml, ; ph 5.5 ± 0.2 (Potato dextrose agar, PDA): 200 g, ; 1000 ml, 30 min;, 1000 ml; 20 g, 15 g 75% T/FF.IF FF-IF FF (100 μl) Biolog ; Taq DNA dntps DL2000 Marker ; GoldView ; K Merck ; 2 方法 2.1 形态学鉴定 CYA, 25 C 7 d, ;,,, 2.2 Biolog 全自动微生物鉴定系统鉴定 Biolog [8] 2.3 基因组 DNA 提取 CTAB DNA [9] 2.4 ITS rdna 区序列扩增及检测 CMCC(F)98003 DNA, ITS4 ITS5, ITS rdna ITS4: 5 -TCCTCCGCTTATTGATATGC-3 ; ITS5: 5 -GGAAGTAAAAGTCGTAACAAGG-3 [10] PCR 100 μl, μl; DNA 1 μl; dntps ( 2.5 mmol/l) 8 μl; (10 μl/l) 2.5 μl; Taq DNA (2.5 U/μL) 1.3 μl; 100 μl ITS : 94 C 5 min; 94 C 1 min, 55 C 1 min, 72 C 1 min, 30 ; 72 C 10 min; 4 C PCR 1%, Goldview 2.5 β- 微管蛋白基因序列扩增及检测 CMCC(F)98003 DNA, Bt2a Bt2b, β- Bt2a: 5 -GGTAACCAAATCG

6 序列测定分析及系统发育树的构建 PCR ABI3700 Chromas GenBank ITS rdna β-, Clustal X, MEGA 3.1 Neighbor-joining, 1000 Bootstraps 3 结果与分析 3.

niger A B C 图 1 25 C 培养 7 d 菌种的宏观形态和显微形态 Fig. 1 Macromorphology and micromorphology on 25 C, 7 d 注 : A: 菌落形态 ; B: 分生孢子梗形态 ( 100); C: 分生孢子形态 ( 400).

3 1602 微生物学通报 2010, Vol.37, No.11 GTGCTGCTTTC-3 ; Bt2b: 5 -ACCCTCAGTGTAGT GACCCTTGGC-3 [11] PCR, ITS rdna PCR PCR : 94 C 5 min; 94 C 1 min, 58 C 1 min, 72 C 1 min, 32 ; 72 C 10 min; 4 C PCR 1%, Goldview 2.6 序列测定分析及系统发育树的构建 PCR ABI3700 Chromas GenBank ITS rdna β-, Clustal X, MEGA 3.1 Neighbor-joining, 1000 Bootstraps 3 结果与分析 3.1 形态学鉴定 CMCC(F)98003, 1, 60 mm 70 mm; 2 mm; ;, ; ; ; ;, 50 μm 80 μm; 10 μm 15 μm, ;, 30 μm 40 μm, ;, (10 12) μm (2 3) μm; (6 8) μm (2 3) μm;, 3 μm 4 μm, A. niger A B C 图 1 25 C 培养 7 d 菌种的宏观形态和显微形态 Fig. 1 Macromorphology and micromorphology on 25 C, 7 d 注 : A: 菌落形态 ; B: 分生孢子梗形态 ( 100); C: 分生孢子形态 ( 400). Note: A: Colony morphology; B: Conidiophore morphology ( 100); C: Conidial morphology ( 400). 3.2 Biolog 全自动微生物鉴定系统鉴定 ATCC16888 ATCC16404, CMCC(F)98003 Biolog, ATCC16888 ATCC16404 β- -D- D- α- D-, CMCC(F)98003 ATCC16888, β- -D- D- D-, L- α-, 1 CMCC(F)98003 ATCC16888, ATCC16404 Carbon source Maltose β- -D- β-methyl-d-glucoside D- D-Trehalose L- L-Malic Acid α- α-ketoglutaric acid Amygdalin D- D-Fructose 表 1 碳源利用情况 Table 1 Carbon Source Utilization ATCC16888 ATCC16404 CMCC(F)98003 v + w + v + : : ; +: ; w: ( ) ; v:. Note: : No growth; +: Strong growth; w: Weak growth; v: Varied growth.

: CMCC(F)98003 1603 3.3 基因组提取 PCR 扩增与序列测定分析 ITS rdna, 2 [12] CMCC(F)98003 DNA 23 kb, PCR ITS4 ITS5 ITS rdna, Bt2a Bt2b β-, 630 bp 550 bp, 3 图 2 真核核糖体 DNA 操纵子的 ITS 区结构图 Fig.

position in ITS sequence Base position Aspergillus brasiliensis IMI 381727 Aspergillus niger ATCC 16888 140 166 172 173 384 C C T C T A T A A CMCC(F)98003 A T A A : 18S ITS1 TTACCG C 1.

3 ITS rdna and β-tubulin gene PCR amplification results of CMCC(F)98003 : M: DL2000 marker; 1: ITS rdna PCR ; 2: β- PCR.

4 : CMCC(F) 基因组提取 PCR 扩增与序列测定分析 ITS rdna, 2 [12] CMCC(F)98003 DNA 23 kb, PCR ITS4 ITS5 ITS rdna, Bt2a Bt2b β-, 630 bp 550 bp, 3 图 2 真核核糖体 DNA 操纵子的 ITS 区结构图 Fig. 2 Diagram of the organization of ITS region of the eukaryotic ribosomal DNA operon C C T C T CMCC(F)98003 ITS rdna IMI %, CMCC(F)98003 ATCC16888 表 2 ITS 序列特殊位点碱基差异 Table 2 Difference of base position in ITS sequence Base position Aspergillus brasiliensis IMI Aspergillus niger ATCC C C T C T A T A A CMCC(F)98003 A T A A : 18S ITS1 TTACCG C 1. Note: The first C in the border sequence (TTACCG) of 18S and ITS1 is here defined as position 1. 图 3 CMCC(F)98003 的 ITS rdna 区序列和 β- 微管蛋白基因序列 PCR 扩增结果 Fig. 3 ITS rdna and β-tubulin gene PCR amplification results of CMCC(F)98003 : M: DL2000 marker; 1: ITS rdna PCR ; 2: β- PCR. Note: M: DL2000 marker; 1: ITS rdna PCR amplification result; 2: β-tubulin gene PCR amplification result. GenBank ATCC16888(FJ195350) IMI (AJ280010) ITS rdna, CMCC(F)98003 ITS rdna (ITS rdna 627 bp) ITS rdna, 18S ITS1 TTACCG C 1, , 2, CMCC(F)98003 A T A A, ATCC16888, IMI 菌株系统发育分析 GenBank ITS rdna, A. flavus CBS T (AY819992), CMCC(F)98003 ITS rdna, 4 CMCC(F)98003 A. niger ATCC 16888, 100%, (A. niger ATCC T AY A. brasiliensis IMI T AJ A. tubigensis CBS T AJ A. costaricaensis CBS T DQ A. vadensis CBS T AY A. piperis CBS DQ900603), 98.6% 100%, GenBank 19 β-, A. flavus CBS T (AY819992), CMCC(F)98003 β-, 5 : CMCC(F)98003 A. niger CBS T, 100%, 94.7% ITS rdna, CMCC(F)98003

5 1604 微生物学通报 2010, Vol.37, No.11 图 4 基于 ITS 区 rdna 序列的黑色组曲霉的 NJ 系统发育树 Fig. 4 A Neighbor-joining tree of Aspergillus section Nigri. based on their ITS DNA sequences : Bootstrap 50% ;. Note: Numbers above branches are bootstrap values. Only values above 50% are indicated. Bar, genetic distance. 图 5 基于 β- 微管蛋白基因序列的黑色组曲霉的 NJ 系统发育树 Fig. 5 Neighbour-joining tree based on β-tubulin sequence data of Aspergillus section Nigri : Bootstrap 50%,. Note: Numbers above branches are bootstrap values. Only values above 50% are indicated. Bar, genetic distance. 4 讨论, 19,,, [13] Ehrlich, RFLP DNA RAPD AFLP ITS rdna β-

6 : CMCC(F) ,,, (ws/t ) : (SN/T ) (2008) ( ) ATCC16404 [6], CMCC(F)98003,,, (GB/T ) [14] (GB ) [15] (GJB ) [16] (QB/T ) [17], ATCC 6275 AS , 参考文献 [1]. 8. :, 2005, 68. [2] Ja nos Varga, Sa ndor Kocsube, Bea ta To th, et al. Aspergillus brasiliensis sp. nov., a biseriate black Aspergillus species with world-wide distribution. International Journal of Systematic and Evolutionary Microbiology, 2007(57): [3] Jan Houseknecht, Elena Stamenova, Sung-Oui Suh, et al. Reclassification of ATCC 16404TM and ATCC 9642TM as Aspergillus brasiliensis. Pharmaceutical Microbiology Forum Newsletter, 2008, 14(10): [4] ctdetails/tabid/452/default.aspx. [5] [6],. ATCC , 2010, 36(9): [7]. :, 2010 : ⅩⅨQ [8],,,. Biolog., 2006, 32(8): [9],,. CTAB DNA., 2003, 23(6): [10] Bruns T, Lee S, Talor J, et al. Application and direct sequencing of fungal ribosomal RNA genes for phylogenetics. San Diego: Academic Press, 1990: [11] Samson RA, Houbraken JAMP, Kuijpers AFA, et al. New ochratoxin or sclerotium producing speciesin Aspergillus section Nigri. Studies in Mycology, 2004(50): [12],. CICC S rdna., 2005, 31(1): [13] Samson R A, Noonim P, Meijer M, et al. Diagnostic tools to identify black Aspergilla. Studies in Mycology, 2007(59): [14]. GB/T [15]. GB [16]. GJB [17]. QB/T

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