小麦矮化腥黑穗病菌（Tilletia controversa K(?)hn）的分子检测
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摘要
小麦矮化腥黑穗病菌(Tilletia controversa kühn,简称TCK)引起的小麦矮化腥黑穗病(Dwarf bunt of wheat)是麦类黑穗病中危害最大，防治最难的一种病害，常对小麦生产造成巨大危害，同时也是我国一类检疫性病害。但是在我国日常的口岸检疫工作中我们发现小麦网腥黑穗病菌T．caries(TCT)和小麦光腥黑穗病菌T．foetida(TFL)与小麦矮腥黑穗病菌T．controversa(TCK)的冬孢子形态非常相似，这给检疫鉴定工作带来了很大的麻烦。几十年来，国内外一些学者主要从腥黑穗病菌的形态学、血清学、生化等方面进行了大量的研究探索，并取得了巨大的成果，但从分子生物学水平上对他们进行区分国内还未见报道。
利用两种分子生物学方法结合实时荧光PCR技术手段对TCK和它的两种近似种TCT和TFL进行了鉴别研究。利用RAPD方法，在目前筛选的引物中能够找出引物OperonA01(CAGGCCCTTC)鉴别出TCK和TFL，但不能鉴别出TCK和TCT；利用核糖体基因分析法，在核糖体基因的外部转录间隔区(externally transcribed spacer，ETS)和非转录间隔区(non- transcribed spacer，NTS)分别找到了TCK不同于TCT和TFL的特异性序列。并根据TCK的ETS上的特异性序列设计了Taqman探针，利用实时荧光PCR技术通过优化实时荧光PCR反应条件实现了从TCK、TCT、TFL中检测出TCK。
利用基于Taqman探针的实时荧光PCR技术可以快速、准确的实现对TCK的检测，可避免常规检测方法造成的误检、漏检，且这种检测方法相对于常规检测方法更为灵敏，整个检测过程只要2个小时，由于采用全封闭反应管及光电传导系统，不用跑凝胶电泳，因而降低了实验室常见的污染问题。
Dwarf bunt of wheat is the most serious and difficult controlled disease which often make; great damages in the production of wheat, it is also a kind of quarantined diseasem in China. But the teliospore morphology of T. caries (TCT), T. foetida (TFL) and T. controversa(TCK.) is found very similar. So it bring us great difficult on the quarantined identification. Many research have been done on the morphology, serology and biochemistry of TCK, TCT and TFL for many years and acquired great progresses. But there are no reports that have been found on the molecular biology in China.T. caries(TCT) > T. foetida(TFL)and T. controversa(TCK) have been differentiated by using two methods of molecular biology which combine Real-time Fluorescent PCR in this paper. Primer OperonA01 have been found to differentiated TCK and TFL but can ' t to differentiated TCK and TCT by using RAPD. Special sequences have been found at the externally transcribed spacer(ETS) and at the non-transcribed spacer (NTS) of ribosome gene by analytical method of ribosome gene sequence . Taqman probe have been designed according to the special sequences of TCK ETS sequence. TCK has been detected by using Real-time Fluorescent PCR.TCK can be deteced rapidly, accurately, relatively sensitive and effectively by Real-time Fluorescent PCR which based on probe Taqman .It reduced pollution due to adopting full-closed reaction tube and photoelectricity conduct system during the experiment.
利用两种分子生物学方法结合实时荧光PCR技术手段对TCK和它的两种近似种TCT和TFL进行了鉴别研究。利用RAPD方法，在目前筛选的引物中能够找出引物OperonA01(CAGGCCCTTC)鉴别出TCK和TFL，但不能鉴别出TCK和TCT；利用核糖体基因分析法，在核糖体基因的外部转录间隔区(externally transcribed spacer，ETS)和非转录间隔区(non- transcribed spacer，NTS)分别找到了TCK不同于TCT和TFL的特异性序列。并根据TCK的ETS上的特异性序列设计了Taqman探针，利用实时荧光PCR技术通过优化实时荧光PCR反应条件实现了从TCK、TCT、TFL中检测出TCK。
利用基于Taqman探针的实时荧光PCR技术可以快速、准确的实现对TCK的检测，可避免常规检测方法造成的误检、漏检，且这种检测方法相对于常规检测方法更为灵敏，整个检测过程只要2个小时，由于采用全封闭反应管及光电传导系统，不用跑凝胶电泳，因而降低了实验室常见的污染问题。
Dwarf bunt of wheat is the most serious and difficult controlled disease which often make; great damages in the production of wheat, it is also a kind of quarantined diseasem in China. But the teliospore morphology of T. caries (TCT), T. foetida (TFL) and T. controversa(TCK.) is found very similar. So it bring us great difficult on the quarantined identification. Many research have been done on the morphology, serology and biochemistry of TCK, TCT and TFL for many years and acquired great progresses. But there are no reports that have been found on the molecular biology in China.T. caries(TCT) > T. foetida(TFL)and T. controversa(TCK) have been differentiated by using two methods of molecular biology which combine Real-time Fluorescent PCR in this paper. Primer OperonA01 have been found to differentiated TCK and TFL but can ' t to differentiated TCK and TCT by using RAPD. Special sequences have been found at the externally transcribed spacer(ETS) and at the non-transcribed spacer (NTS) of ribosome gene by analytical method of ribosome gene sequence . Taqman probe have been designed according to the special sequences of TCK ETS sequence. TCK has been detected by using Real-time Fluorescent PCR.TCK can be deteced rapidly, accurately, relatively sensitive and effectively by Real-time Fluorescent PCR which based on probe Taqman .It reduced pollution due to adopting full-closed reaction tube and photoelectricity conduct system during the experiment.
引文
1 Duran R. and G.W. Fisher. The genus Tilletia. Wash. State Univ. Press. 1961, 34:138-142.
2 Goates B. J. and J.A. Hoffman. Nuclear behavior during teliospore germination and sporidial development in Tilletia caries, T. foetfda, and T. controversa. CJB 1987, 65:513-517.
3 章正．中美小麦矮腥黑穗病菌鉴定合作研究Ⅰ．自发荧光显微学特性研究．植物病理学报，1995，25：199—206．
4 章正．关于小麦矮腥黑穗病菌某些近似种的探讨．植物病理学报，1980，10：89—94．
5 Russell B W, Mills D. Electrophoretic karyotypes of Tilletia caries, T. controversa, and their F1 progeny:further evidence for conspecific statrus. Mol. Plant-Microbe Interact, 1993,61:66-74.
6 Russell B W, Mills D. Morphologieal physiological, and genetic evidence in support of a conspecife status for Tilletia caries, T. controversa, and T. foetida. Phytopathology, 1994,84:576-82.
7 Holton C. S. 1994. Ihheritance of chlamydospore and sorus characters in species and race hybrids of Tilletia caries and T. foetida. Phytopathology, 34:586-592.
8 刘作易．DNA指纹技术的发展极其在真菌分类上的应用．山地农业生物学报，2000，19(5)：460-469．
9 苏艳纯．18S rDNA和ITS rDNA的RFLP在疫霉菌分子系统学研究中的应用．[博士论文]．北京：北京农业大学，1994．
10 Mullis KB, Faloona F, Scharf SF et al.Specific enzymatic amplification of DNA in vitro:the ploymerase chain reaction. Cold Spring Harbor Symposium on Quantitative Biolo, 1986, 51: 263-267.
11 沛轩，杨朝国。PCR检验技术[M]．成都：四川大学出版社，1995．340．
12 Laroche A, Gaudet D A, Schaalje G B, et al. Grouping and identification of low temperature basidiomycetes using mating, RAPD and RFLP analysis[J].Mycilogical Research, 1995, 99 (3):297-310. 13 Pimentel G, et al. Genetic variability among isolates of Tilletia barclayana. T. indica and allied species. Mycologia,
1998,90:1017-1027.
14 Forster H, Oudemans P and Coffy M D. Mitochondrial and nuclear DNA diversity within six species of Phytophthora[J].Experimental Mycology, 1990(14):18-31.
15 Croft J H, Kevei F et al. Molecular and phenotypic characterization of mitochondrial DNA polymerphisms[J].Antomie Van Leeuwenhoek, 1997,72(4):337-347.
16 McDonald J G. Differentiation of tilletia species by rep-PCR genomic finger printing[J]. Plant-Disease. 2000,84:10, 1121-1125.
17 Zabeau M, Vos P. Selective restriction fragment amplification:A general method for DNA fingerprinting[P].European Patent Application:924026297,1992.
18 Vos P. AFLP:A new technique for DNA fingerprinting[J].Nucl Acid Res, 1996, (23): 4407-4414.
19 Majer D, Mithen R, Lewis B G, et al. The use of AFLP fingerprinting for detection of genetic variation in Fungi[J].Mycological Research, 1996, (100):1107-1111.
20 Rosendahl S, Taylor J W. Development of multiple genetic markers for studies of genetic variation in arbuscular mycorrhizal fungi using AFLP[J].Molecular Ecology, 1997, (6):821-829.
21 Patchara P P, Osborn T C, Williams P H et al. Genetic analysis and identification of amplified fragment length polymorphism market linded to the alml a virulence gene of Lepeosphaeria mucnlans[J]. Phytopatho, 1998(88):1068-1072.
22 Leissner C E W, Niessen M L and Vogel R. Use of the technique for the identification and discrimination of Fusarium graminerarum[J].Cereal Research Communications, 1997(25):555-556.
23 黄原．分子系统学原理、方法及应用．北京：中国农业出版社，1998．
24 Cassidy, Pukkila Inversion of riboso et al RNA genes within the genus Coprinus Curr, Genel, 1987,12:33-36.
25 Pieter Vos, Rene Hogers, M arjo Bleeker etal. AFLP:a new technique For DNA Fingerprinting Nucleic Acids Research, 1995,23. (21):4407-4414.
26 Russell P J, Wagner K D, et al,Mol. Genetics, 1984,196:275-282.
27 张星耀、赵仕光，扑春根．树木溃疡病原真菌类群分子遗传多样性研究之一—小穴壳菌属、疡壳孢属、壳囊孢属、盾壳霉属分类地位的分子证明．林业科学，1999，35(3)：34-40．
28 Lkuko Okabe, Chiharu Morikawa, Naoyuki Mastsumeto et al. Variation in Sclerotium rolfsiiisolates in Japan. Mycoscience, 1998,39:399-407.
29 SuSumu Takamatsu Tesuya Hiata, Yukio Sato:Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the poudery mildew fungi (Erysiphaceae). Myco, 1998,39:441-453.
30 Hausner G, Reid J, Klassen G R, On the subvision of Ophiostoma ceratocystis, s. based on partial ribosomal DNA sequendes[J].Bot, 1992,71:52-63.
31 郭成亮．腥黑穗菌属(Tilletia)部分种ITS rDNA限制性片段多态性(RFLP)的研究．[博士论文]．哈尔滨：东北林业大学，1997．
32 阎培生，罗信昌，周启．小耳属真菌rDNA特异性扩增片段RFLP研究．菌物系统，1999，18(1)：36-40．
33 黄河，毛伟敏，郑儒永．小克银汉rDNA PCR扩增产物的限制性片段K度多态性．菌物系统，1999，18(3)：259-269．
34 李英波。香菇均株的限制性片段长度多态性．真菌学报．1995，14(3)：209-217．
35 Anderson J B, Bailey S S, Variation in ribosomal and among biologyical specioes of Armillaria:a genus of root infecting fungi[J].Evolution, 1989,43(8):1652-1662.
36 Blanz P A, Gottschalk M. Systematic position of Sep to 7basidium, Gtaphiola and other basidiom ycetes as deduced on the basis of their 5S ribosomal RNA nucleotide sequences[J]. Dystem Microbiol, 1986,8:121-127.
37 Smith O P, et al. Development of a PCR-based method for identification of Tilletia indica, causal agent of Karnal bunt of wheat. Phytopathology, 1996,86:115-122.
38 Ferreira-MASV;Tooley-PW;Hatziloukas-E, et al Isolation of a species-specific mitochondrial DNA sequence for identification of tilletia indica, the Karnal bunt of wheat fungus[J]. Applied-and-Environmental-Microbiology, 1996,62:1,87-93.
39 Hausner G, Reid J.G.R, Klassen on the polygeny of Ophiostoma ceratocystiss, s. and Microasus and relationshios with Ophiostom a based in partial ribosom al DNA sequences[J]. Can. J. Bot, 1993.71:1249-1256.
40 程颖慧、章桂明、王颖等．小麦印度腥黑穗病菌PCR检测．植物检疫，2001，6(15)：321-325．
41 易建平、沈禹飞、陶庭典等．PCR技术在印度腥黑穗病菌检测和鉴定中的应用．上海农业学报，2002，18(2)：57-61．
42 Frederick R D, et al. An improved PCR method utilizing TaqMan for the detection and differentiation of Tilletia indica, the causal organism of Karnal bunt of Wheat, and a related ryegrass smut[J].phytopathology, 1998,88:S29.
43 Ursulla E.M. Giboson, Christial A. et al.A Novel Method for Real Time Quantitative RT-PCR. GENOME METHODS, 1996 5,6:995-1001.
44 Christian A. Heid, Junko Stevens. et al. Real Time Quantitative PCR.GENOME METHODS, 1996,6:986-994.
45 Nazarenlo I A, BHATNAGAR S.K, et al.A closed tube format for amplification and detection of DNA based on energy transfer[J].Nucleic Acids Research, 1997, 25(2): 2516-2521.
46 王小红，王升启．荧光定量PCR技术研究进展．国外医学分子生物分册，2001，23(1)：42—45．
47 陈忠斌，王升启，孙志贤．分子信标核酸检测技术研究进展．生物化学与生物物理进展，1998，25(6)：488—492．
2 Goates B. J. and J.A. Hoffman. Nuclear behavior during teliospore germination and sporidial development in Tilletia caries, T. foetfda, and T. controversa. CJB 1987, 65:513-517.
3 章正．中美小麦矮腥黑穗病菌鉴定合作研究Ⅰ．自发荧光显微学特性研究．植物病理学报，1995，25：199—206．
4 章正．关于小麦矮腥黑穗病菌某些近似种的探讨．植物病理学报，1980，10：89—94．
5 Russell B W, Mills D. Electrophoretic karyotypes of Tilletia caries, T. controversa, and their F1 progeny:further evidence for conspecific statrus. Mol. Plant-Microbe Interact, 1993,61:66-74.
6 Russell B W, Mills D. Morphologieal physiological, and genetic evidence in support of a conspecife status for Tilletia caries, T. controversa, and T. foetida. Phytopathology, 1994,84:576-82.
7 Holton C. S. 1994. Ihheritance of chlamydospore and sorus characters in species and race hybrids of Tilletia caries and T. foetida. Phytopathology, 34:586-592.
8 刘作易．DNA指纹技术的发展极其在真菌分类上的应用．山地农业生物学报，2000，19(5)：460-469．
9 苏艳纯．18S rDNA和ITS rDNA的RFLP在疫霉菌分子系统学研究中的应用．[博士论文]．北京：北京农业大学，1994．
10 Mullis KB, Faloona F, Scharf SF et al.Specific enzymatic amplification of DNA in vitro:the ploymerase chain reaction. Cold Spring Harbor Symposium on Quantitative Biolo, 1986, 51: 263-267.
11 沛轩，杨朝国。PCR检验技术[M]．成都：四川大学出版社，1995．340．
12 Laroche A, Gaudet D A, Schaalje G B, et al. Grouping and identification of low temperature basidiomycetes using mating, RAPD and RFLP analysis[J].Mycilogical Research, 1995, 99 (3):297-310. 13 Pimentel G, et al. Genetic variability among isolates of Tilletia barclayana. T. indica and allied species. Mycologia,
1998,90:1017-1027.
14 Forster H, Oudemans P and Coffy M D. Mitochondrial and nuclear DNA diversity within six species of Phytophthora[J].Experimental Mycology, 1990(14):18-31.
15 Croft J H, Kevei F et al. Molecular and phenotypic characterization of mitochondrial DNA polymerphisms[J].Antomie Van Leeuwenhoek, 1997,72(4):337-347.
16 McDonald J G. Differentiation of tilletia species by rep-PCR genomic finger printing[J]. Plant-Disease. 2000,84:10, 1121-1125.
17 Zabeau M, Vos P. Selective restriction fragment amplification:A general method for DNA fingerprinting[P].European Patent Application:924026297,1992.
18 Vos P. AFLP:A new technique for DNA fingerprinting[J].Nucl Acid Res, 1996, (23): 4407-4414.
19 Majer D, Mithen R, Lewis B G, et al. The use of AFLP fingerprinting for detection of genetic variation in Fungi[J].Mycological Research, 1996, (100):1107-1111.
20 Rosendahl S, Taylor J W. Development of multiple genetic markers for studies of genetic variation in arbuscular mycorrhizal fungi using AFLP[J].Molecular Ecology, 1997, (6):821-829.
21 Patchara P P, Osborn T C, Williams P H et al. Genetic analysis and identification of amplified fragment length polymorphism market linded to the alml a virulence gene of Lepeosphaeria mucnlans[J]. Phytopatho, 1998(88):1068-1072.
22 Leissner C E W, Niessen M L and Vogel R. Use of the technique for the identification and discrimination of Fusarium graminerarum[J].Cereal Research Communications, 1997(25):555-556.
23 黄原．分子系统学原理、方法及应用．北京：中国农业出版社，1998．
24 Cassidy, Pukkila Inversion of riboso et al RNA genes within the genus Coprinus Curr, Genel, 1987,12:33-36.
25 Pieter Vos, Rene Hogers, M arjo Bleeker etal. AFLP:a new technique For DNA Fingerprinting Nucleic Acids Research, 1995,23. (21):4407-4414.
26 Russell P J, Wagner K D, et al,Mol. Genetics, 1984,196:275-282.
27 张星耀、赵仕光，扑春根．树木溃疡病原真菌类群分子遗传多样性研究之一—小穴壳菌属、疡壳孢属、壳囊孢属、盾壳霉属分类地位的分子证明．林业科学，1999，35(3)：34-40．
28 Lkuko Okabe, Chiharu Morikawa, Naoyuki Mastsumeto et al. Variation in Sclerotium rolfsiiisolates in Japan. Mycoscience, 1998,39:399-407.
29 SuSumu Takamatsu Tesuya Hiata, Yukio Sato:Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the poudery mildew fungi (Erysiphaceae). Myco, 1998,39:441-453.
30 Hausner G, Reid J, Klassen G R, On the subvision of Ophiostoma ceratocystis, s. based on partial ribosomal DNA sequendes[J].Bot, 1992,71:52-63.
31 郭成亮．腥黑穗菌属(Tilletia)部分种ITS rDNA限制性片段多态性(RFLP)的研究．[博士论文]．哈尔滨：东北林业大学，1997．
32 阎培生，罗信昌，周启．小耳属真菌rDNA特异性扩增片段RFLP研究．菌物系统，1999，18(1)：36-40．
33 黄河，毛伟敏，郑儒永．小克银汉rDNA PCR扩增产物的限制性片段K度多态性．菌物系统，1999，18(3)：259-269．
34 李英波。香菇均株的限制性片段长度多态性．真菌学报．1995，14(3)：209-217．
35 Anderson J B, Bailey S S, Variation in ribosomal and among biologyical specioes of Armillaria:a genus of root infecting fungi[J].Evolution, 1989,43(8):1652-1662.
36 Blanz P A, Gottschalk M. Systematic position of Sep to 7basidium, Gtaphiola and other basidiom ycetes as deduced on the basis of their 5S ribosomal RNA nucleotide sequences[J]. Dystem Microbiol, 1986,8:121-127.
37 Smith O P, et al. Development of a PCR-based method for identification of Tilletia indica, causal agent of Karnal bunt of wheat. Phytopathology, 1996,86:115-122.
38 Ferreira-MASV;Tooley-PW;Hatziloukas-E, et al Isolation of a species-specific mitochondrial DNA sequence for identification of tilletia indica, the Karnal bunt of wheat fungus[J]. Applied-and-Environmental-Microbiology, 1996,62:1,87-93.
39 Hausner G, Reid J.G.R, Klassen on the polygeny of Ophiostoma ceratocystiss, s. and Microasus and relationshios with Ophiostom a based in partial ribosom al DNA sequences[J]. Can. J. Bot, 1993.71:1249-1256.
40 程颖慧、章桂明、王颖等．小麦印度腥黑穗病菌PCR检测．植物检疫，2001，6(15)：321-325．
41 易建平、沈禹飞、陶庭典等．PCR技术在印度腥黑穗病菌检测和鉴定中的应用．上海农业学报，2002，18(2)：57-61．
42 Frederick R D, et al. An improved PCR method utilizing TaqMan for the detection and differentiation of Tilletia indica, the causal organism of Karnal bunt of Wheat, and a related ryegrass smut[J].phytopathology, 1998,88:S29.
43 Ursulla E.M. Giboson, Christial A. et al.A Novel Method for Real Time Quantitative RT-PCR. GENOME METHODS, 1996 5,6:995-1001.
44 Christian A. Heid, Junko Stevens. et al. Real Time Quantitative PCR.GENOME METHODS, 1996,6:986-994.
45 Nazarenlo I A, BHATNAGAR S.K, et al.A closed tube format for amplification and detection of DNA based on energy transfer[J].Nucleic Acids Research, 1997, 25(2): 2516-2521.
46 王小红，王升启．荧光定量PCR技术研究进展．国外医学分子生物分册，2001，23(1)：42—45．
47 陈忠斌，王升启，孙志贤．分子信标核酸检测技术研究进展．生物化学与生物物理进展，1998，25(6)：488—492．
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