根癌农杆菌介导泡盛曲霉转化的研究
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摘要
丝状真菌具有良好的蛋白分泌能力,而且能对合成的真核蛋白正确地进行各种翻译后加工包括肽链的剪切和糖基化等,其糖基化修饰模式较酵母更类似于高等真核生物。由于在食品及食品加工中长期应用,许多菌种已被认定是安全的。与哺乳动物和昆虫等高等生物表达系统相比,丝状真菌培养方便、生长迅速、成本较低,表达的外源蛋白可以分泌到培养基中,下游的分离纯化简单。加之丝状真菌遗传转化系统研究的迅速发展,为在丝状真菌中表达外源蛋白奠定了基础。
本文以泡盛曲霉为研究对象,拟通过根癌农杆菌介导建立其遗传转化体系。通过分泌蛋白图谱分析和药物敏感性分析,确定了以潮霉素B抗性基因和乙酰胺酶基因amdS作为筛选标记的宿主菌;基于Gateway克隆技术,构建了由高效启动子、终止子和目的基因表达框组成的根癌农杆菌双元载体,筛选标记为潮霉素B抗性基因和乙酰胺酶基因amdS;通过根癌农杆菌介导,将构建的双元载体转化泡盛曲霉,获得了含有目的基因的转化子,经过PCR验证、SDS-PAGE蛋白电泳和Western蛋白杂交证明目的基因已转化进入泡盛曲霉,表明根癌农杆菌介导泡盛曲霉转化具有可行性,所得转化子遗传稳定性高,适合作为异源蛋白的表达体系。
Filamentous fungi could secrete a large amount of proteins, and they own many protein post-translational modification mechanisms such as peptide chain splicing, glycosylation and so on. Moreover, their glycosylation mode is much more similar with higher eukaryotes than yeast. Due to the long-term application in food industry, many species in filamentous fungi have been generally recognized as safe (GRAS). Compared to higher organisms such as mammalian and insect expression system, filamentous fungi are characteristic of simple nutrition, rapid growth, low costs and fast downstream separation and purification due to secreted expression. The great advances in fungal gene expression systems lay the foundation for genetic research in filamentous fungi.
In this paper, transformation system for Aspergillus awamori was established, which was mediated by Agrobacterium tumefaciens. Based on the analysis of secreted protein expression profile and drug sensitivity, the resistant genes to hygromycin B and acetamidase (amdS) was determined as selection markers. Then, using the Gateway cloning technique, Agrobacterium tumefaciens binary vectors were constructed containing effective promoter, terminator and target gene ORF, with hygromycin B resistance gene and acetamidase gene amdS as selection markers. After transformation the binary vectors with Aspergillus awamori mediated by Agrobacterium tumefaciens, several transformants were obtained. And these transformants were verified by PCR amplication, SDS-PAGE and Western blotting, suggesting that target gene had been transformated into Aspergillus awamori. The above data proved that the Agrobacterium-mediated transformation of Aspergillus awamori was feasible, with high genetic stability of transformants. Therefore, Aspergillus awamori was suitable as heterologous protein expression system.
本文以泡盛曲霉为研究对象,拟通过根癌农杆菌介导建立其遗传转化体系。通过分泌蛋白图谱分析和药物敏感性分析,确定了以潮霉素B抗性基因和乙酰胺酶基因amdS作为筛选标记的宿主菌;基于Gateway克隆技术,构建了由高效启动子、终止子和目的基因表达框组成的根癌农杆菌双元载体,筛选标记为潮霉素B抗性基因和乙酰胺酶基因amdS;通过根癌农杆菌介导,将构建的双元载体转化泡盛曲霉,获得了含有目的基因的转化子,经过PCR验证、SDS-PAGE蛋白电泳和Western蛋白杂交证明目的基因已转化进入泡盛曲霉,表明根癌农杆菌介导泡盛曲霉转化具有可行性,所得转化子遗传稳定性高,适合作为异源蛋白的表达体系。
Filamentous fungi could secrete a large amount of proteins, and they own many protein post-translational modification mechanisms such as peptide chain splicing, glycosylation and so on. Moreover, their glycosylation mode is much more similar with higher eukaryotes than yeast. Due to the long-term application in food industry, many species in filamentous fungi have been generally recognized as safe (GRAS). Compared to higher organisms such as mammalian and insect expression system, filamentous fungi are characteristic of simple nutrition, rapid growth, low costs and fast downstream separation and purification due to secreted expression. The great advances in fungal gene expression systems lay the foundation for genetic research in filamentous fungi.
In this paper, transformation system for Aspergillus awamori was established, which was mediated by Agrobacterium tumefaciens. Based on the analysis of secreted protein expression profile and drug sensitivity, the resistant genes to hygromycin B and acetamidase (amdS) was determined as selection markers. Then, using the Gateway cloning technique, Agrobacterium tumefaciens binary vectors were constructed containing effective promoter, terminator and target gene ORF, with hygromycin B resistance gene and acetamidase gene amdS as selection markers. After transformation the binary vectors with Aspergillus awamori mediated by Agrobacterium tumefaciens, several transformants were obtained. And these transformants were verified by PCR amplication, SDS-PAGE and Western blotting, suggesting that target gene had been transformated into Aspergillus awamori. The above data proved that the Agrobacterium-mediated transformation of Aspergillus awamori was feasible, with high genetic stability of transformants. Therefore, Aspergillus awamori was suitable as heterologous protein expression system.
引文
[1] Mishra, N.C.DNA-mediated genetic changes in Neurospora crassa[J]. Journal of general microbiology, 1979. 113(2): 255-9.
[2] Mishra, N.C.Characterization of the new osmotic mutants (os) which originated during genetic transformation in Neurospora crassa[J].Genetical Research (1977), 29: 9-19
[3] K. Kitamoto.Recent progress in research on koji mold [J]. Brew. Soc. Jpn. 95 (2000):404–406
[4] Archer, D.B. and J.F. Peberdy.The molecular biology of secreted enzyme production by fungi [J]. Critical Reviews in Biotechnology, 1997,17(4): 273-306.
[5] Punt PJ, van Biezen N, Conesa A, et al. Filamentous fungi as cell factories for heterologous protein production[J]. Trends in Biotechnology, 2002, 20: 200?206.
[6] Nevalainena H, Suominena P, Tamistob K. On the safety of Trichoderma reesei [J]. Journal of Biotechnology, 1994,37(3): 193-200.
[7] KMH Nevalainen,VSJ Teo,PL Bergquist. Heterologous protein expression in filamentous fungi[J] .Trends Biotechnol, 2005, 23 :468-474 .
[8] Meyer, V., M. Wedde, and U. Stahl.Transcriptional regulation of the Antifungal Protein in Aspergillus giganteus [J]. Molecular genetics and genomics , 2002. 266(5): 747-57.
[9] T. M. Klein, E. D. Wolf, R. Wu et al.High-velocity microprojectiles for delivering nucleic acids into living cells [J], Nature 1987,327, 70– 73
[10]陈赟娟,韦建福.丝状真菌遗传转化的研究进展[J].云南农业大学学报,2009,24(3): 448-454.
[11] Hinen, A., J. Hicks, and G. Fink. Transformation of yeast [J]. Proc. Natl. Acad. Sci. , 1978. 80: 3035-3039.
[12] Schiestl,R.H.,Petes,T.D. Integration of DNA Fragments by Illegitimate Recombination in Saccharomyces cerevisiae[J] .Proc. Natl. Acad. Sci. USA, 1991, 88 :7585-7589
[13] Bundock P, A den Dulk-Ras, A Beijersbergen et al.Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae[J].EMBO,1995,14(13): 3206–3214.
[14] de Groot, M.J., Bundock P., P. J. Hooykaas et al. Agrobacterium tumefaciens-mediated transformation of filamentous fungi[J]. Nature Biotechnology, 1998. 16(9): 839-42.
[15] Michielse,C.B.,P.J.J.Hooykaas,C.A.M.J.J.vanden Hondel, et al.Agrobacterium-mediated transformation as a tool for functional genomics in fungi[J]. Current Genetics, 2005. 48(1): 1-17.
[16]傅荣昭,孙勇如,贾士荣.植物遗传转化技术手册[M].北京,中国科学技术出版社,1994
[17] GW Bolton, EW Nester and MP Gordon.Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence [J], Science 23 May 1986,Vol. 232 no. 4753:983-985
[18] Stachel, S.E. and P.C. Zambryski. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens [J]. Cell,1986. 46(3):325-333.
[19] Liu, C.N., X.Q. Li, and S.B. Gelvin.Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens [J]. Plant Molecular Biology, 1992. 20(6):1071-87.
[20] Hooykaas, P. and A.G.M. Beijersbergen.The virulence system of Agrobacterium tumefaciens [J]. Annual Review of Phytopathology, 1994. 32(1):157-181.
[21] Fullner, K.J., J.C. Lara, and E.W. Nester.Pilus assembly by Agrobacterium T-DNA transfer genes [J]. Science,1996.273(5278): p. 1107.
[22] Lee, L.Y., S.B. Gelvin, and C.I. Kado. pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export[J]. Journal of Bacteriology,1999,181(1): 186.
[23] Piers, K.L., J.D. Heath, X. Liang, et al. Agrobacterium tumefaciens-mediated transformation of yeast [J]. Proceedings of the National Academy of Sciences of the United States of America, 1996. 93(4): 1613.
[24] Bevan, M. Binary Agrobacterium vectors for plant transformation[J]. Nucleic Acids Research,1984. 12(22): 8711.
[25]李娟,杨金奎,梁连铭等.丝状真菌遗传转化系统研究进展[J].江西农业大学学报.2006, 21(4): 517-520.
[26]黄卫,汪天虹.丝状真菌遗传转化系统研究进展[J].微生物学杂志,2000, 20(3): 40-44.
[27] Harkki,A., A. M?ntyl?, M. Penttil?, et al.Genetic engineering of Trichoderma to produce strains with novel cellulase profiles[J]. Enzyme and Microbial Technology,1991. 13(3): 227-233.
[28] Flipphi, M., J. Kocialkowska, and B. Felenbok. Characteristics of physiological inducers of the ethanol utilization (alc) pathway in Aspergillus nidulans[J]. Biochemical Journal, 2002. 364(Pt 1): 25.
[29] Punt, P.J., N. van Biezen, A. Conesa, et al. Filamentous fungi as cell factories for heterologous protein production [J]. Trends in Biotechnology, 2002. 20(5): 200-206.
[30] Verdoes, J.C., P.J. Punt, A.H. Stouthamer, et al. The effect of multiple copies of theupstream region on expression of the Aspergillus niger glucoamylase-encoding gene[J]. Gene, 1994. 145(2): 179-187.
[31] Fowler, T. and R.M. Berka.Gene expression systems for filamentous fungi[J]. Current Opinion in Biotechnology.1991. 2(5): 691-697.
[32] Gouka, R.J., P.J. Punt, and C.A. van den Hondel. Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels[J]. Applied and Environmental Microbiology, 1997. 63(2): 488.
[33] Berka,R.M.,W.Yoder,S.Takagi,et al. Aspergillus expression system [P].PCT. WO9515391A2. 1997:24-26
[34] Michielse, C.B., P.J.J. Hooykaas, C.A. Van Den Hondel, et al. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori[J]. Nature Protocols, 2008. 3(10): 1671-1678.
[35] Hamilton, C.M., A. Frary, C. Lewis, et al. Stable transfer of intact high molecular weight DNA into plant chromosomes[J]. Proceedings of the National Academy of Sciences, 1996. 93(18): 9975.
[36] Sambrook J , Fritsch EF , Maniatis T.分子克隆实验指南[M].第2版.金冬雁,黎孟枫,候云德.北京:科学出版社.1992:25-26
[37] Sambrook J , Fritsch EF , Maniatis T.分子克隆实验指南[M].第2版.金冬雁,黎孟枫,候云德.北京:科学出版社.1992:908
[38] Sambrook J , Fritsch EF , Maniatis T.分子克隆实验指南[M].第2版.金冬雁,黎孟枫,候云德.北京:科学出版社.1992:55-56
[39] Sambrook J , Fritsch EF , Maniatis T.分子克隆实验指南[M].第2版.金冬雁,黎孟枫,候云德.北京:科学出版社.1992:655
[40] Mulder, H.J., M. Saloheimo, M. Penttil?, et al. The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and up-regulates its own transcription [J]. Molecular Genetics and Genomics.2004,271(2): 130-140.
[41] Ye, L. and L. Pan. A Comparison of the Unfolded Protein Response in Solid-State with Submerged Cultures of Aspergillus oryzae[J]. Bioscience Biotechnology and Biochemistry, 2008. 72(11): p. 2998-3001.
[42] Galagan, J.E., S.E. Calvo, C. Cuomo, et al. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae[J]. Nature, 2005.438(7071): 1105-1115.
[43] Punt, P.J., R.P. Oliver, M.A. Dingemanse, et al. Transformation of Aspergillus based onthe hygromycin B resistance marker from Escherichia coli [J]. Gene, 1987. 56(1): 117-124.
[44] Mulder, H.J. and I. Nikolaev. HacA-Dependent Transcriptional Switch Releases hacA mRNA from a Translational Block upon Endoplasmic Reticulum Stress[J]. Eukaryotic Cell, 2009. 8(4): 665-675.
[45]朱锦辉,权军利,何玉科等.根癌农杆菌感受态细胞的制备以及质粒ProkⅡ对其转化的研究[J].西北农林科技大学学报(自然科学版). 2006年07期
[46]罗雯,刘阳.根癌农杆菌转化条件优化的研究[J].生物技术.2006年01期
[47]单世华,李春娟,张君诚等.农杆菌质粒DNA提取方法的改良与鉴定[J].生物技术,2003,13(4): 19-20.
[48]周小玲,沈微,饶志明.一种快速提取真菌染色体DNA的方法[J].微生物学通报,2004 31(4): 89-92
[2] Mishra, N.C.Characterization of the new osmotic mutants (os) which originated during genetic transformation in Neurospora crassa[J].Genetical Research (1977), 29: 9-19
[3] K. Kitamoto.Recent progress in research on koji mold [J]. Brew. Soc. Jpn. 95 (2000):404–406
[4] Archer, D.B. and J.F. Peberdy.The molecular biology of secreted enzyme production by fungi [J]. Critical Reviews in Biotechnology, 1997,17(4): 273-306.
[5] Punt PJ, van Biezen N, Conesa A, et al. Filamentous fungi as cell factories for heterologous protein production[J]. Trends in Biotechnology, 2002, 20: 200?206.
[6] Nevalainena H, Suominena P, Tamistob K. On the safety of Trichoderma reesei [J]. Journal of Biotechnology, 1994,37(3): 193-200.
[7] KMH Nevalainen,VSJ Teo,PL Bergquist. Heterologous protein expression in filamentous fungi[J] .Trends Biotechnol, 2005, 23 :468-474 .
[8] Meyer, V., M. Wedde, and U. Stahl.Transcriptional regulation of the Antifungal Protein in Aspergillus giganteus [J]. Molecular genetics and genomics , 2002. 266(5): 747-57.
[9] T. M. Klein, E. D. Wolf, R. Wu et al.High-velocity microprojectiles for delivering nucleic acids into living cells [J], Nature 1987,327, 70– 73
[10]陈赟娟,韦建福.丝状真菌遗传转化的研究进展[J].云南农业大学学报,2009,24(3): 448-454.
[11] Hinen, A., J. Hicks, and G. Fink. Transformation of yeast [J]. Proc. Natl. Acad. Sci. , 1978. 80: 3035-3039.
[12] Schiestl,R.H.,Petes,T.D. Integration of DNA Fragments by Illegitimate Recombination in Saccharomyces cerevisiae[J] .Proc. Natl. Acad. Sci. USA, 1991, 88 :7585-7589
[13] Bundock P, A den Dulk-Ras, A Beijersbergen et al.Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae[J].EMBO,1995,14(13): 3206–3214.
[14] de Groot, M.J., Bundock P., P. J. Hooykaas et al. Agrobacterium tumefaciens-mediated transformation of filamentous fungi[J]. Nature Biotechnology, 1998. 16(9): 839-42.
[15] Michielse,C.B.,P.J.J.Hooykaas,C.A.M.J.J.vanden Hondel, et al.Agrobacterium-mediated transformation as a tool for functional genomics in fungi[J]. Current Genetics, 2005. 48(1): 1-17.
[16]傅荣昭,孙勇如,贾士荣.植物遗传转化技术手册[M].北京,中国科学技术出版社,1994
[17] GW Bolton, EW Nester and MP Gordon.Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence [J], Science 23 May 1986,Vol. 232 no. 4753:983-985
[18] Stachel, S.E. and P.C. Zambryski. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens [J]. Cell,1986. 46(3):325-333.
[19] Liu, C.N., X.Q. Li, and S.B. Gelvin.Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens [J]. Plant Molecular Biology, 1992. 20(6):1071-87.
[20] Hooykaas, P. and A.G.M. Beijersbergen.The virulence system of Agrobacterium tumefaciens [J]. Annual Review of Phytopathology, 1994. 32(1):157-181.
[21] Fullner, K.J., J.C. Lara, and E.W. Nester.Pilus assembly by Agrobacterium T-DNA transfer genes [J]. Science,1996.273(5278): p. 1107.
[22] Lee, L.Y., S.B. Gelvin, and C.I. Kado. pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export[J]. Journal of Bacteriology,1999,181(1): 186.
[23] Piers, K.L., J.D. Heath, X. Liang, et al. Agrobacterium tumefaciens-mediated transformation of yeast [J]. Proceedings of the National Academy of Sciences of the United States of America, 1996. 93(4): 1613.
[24] Bevan, M. Binary Agrobacterium vectors for plant transformation[J]. Nucleic Acids Research,1984. 12(22): 8711.
[25]李娟,杨金奎,梁连铭等.丝状真菌遗传转化系统研究进展[J].江西农业大学学报.2006, 21(4): 517-520.
[26]黄卫,汪天虹.丝状真菌遗传转化系统研究进展[J].微生物学杂志,2000, 20(3): 40-44.
[27] Harkki,A., A. M?ntyl?, M. Penttil?, et al.Genetic engineering of Trichoderma to produce strains with novel cellulase profiles[J]. Enzyme and Microbial Technology,1991. 13(3): 227-233.
[28] Flipphi, M., J. Kocialkowska, and B. Felenbok. Characteristics of physiological inducers of the ethanol utilization (alc) pathway in Aspergillus nidulans[J]. Biochemical Journal, 2002. 364(Pt 1): 25.
[29] Punt, P.J., N. van Biezen, A. Conesa, et al. Filamentous fungi as cell factories for heterologous protein production [J]. Trends in Biotechnology, 2002. 20(5): 200-206.
[30] Verdoes, J.C., P.J. Punt, A.H. Stouthamer, et al. The effect of multiple copies of theupstream region on expression of the Aspergillus niger glucoamylase-encoding gene[J]. Gene, 1994. 145(2): 179-187.
[31] Fowler, T. and R.M. Berka.Gene expression systems for filamentous fungi[J]. Current Opinion in Biotechnology.1991. 2(5): 691-697.
[32] Gouka, R.J., P.J. Punt, and C.A. van den Hondel. Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels[J]. Applied and Environmental Microbiology, 1997. 63(2): 488.
[33] Berka,R.M.,W.Yoder,S.Takagi,et al. Aspergillus expression system [P].PCT. WO9515391A2. 1997:24-26
[34] Michielse, C.B., P.J.J. Hooykaas, C.A. Van Den Hondel, et al. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori[J]. Nature Protocols, 2008. 3(10): 1671-1678.
[35] Hamilton, C.M., A. Frary, C. Lewis, et al. Stable transfer of intact high molecular weight DNA into plant chromosomes[J]. Proceedings of the National Academy of Sciences, 1996. 93(18): 9975.
[36] Sambrook J , Fritsch EF , Maniatis T.分子克隆实验指南[M].第2版.金冬雁,黎孟枫,候云德.北京:科学出版社.1992:25-26
[37] Sambrook J , Fritsch EF , Maniatis T.分子克隆实验指南[M].第2版.金冬雁,黎孟枫,候云德.北京:科学出版社.1992:908
[38] Sambrook J , Fritsch EF , Maniatis T.分子克隆实验指南[M].第2版.金冬雁,黎孟枫,候云德.北京:科学出版社.1992:55-56
[39] Sambrook J , Fritsch EF , Maniatis T.分子克隆实验指南[M].第2版.金冬雁,黎孟枫,候云德.北京:科学出版社.1992:655
[40] Mulder, H.J., M. Saloheimo, M. Penttil?, et al. The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and up-regulates its own transcription [J]. Molecular Genetics and Genomics.2004,271(2): 130-140.
[41] Ye, L. and L. Pan. A Comparison of the Unfolded Protein Response in Solid-State with Submerged Cultures of Aspergillus oryzae[J]. Bioscience Biotechnology and Biochemistry, 2008. 72(11): p. 2998-3001.
[42] Galagan, J.E., S.E. Calvo, C. Cuomo, et al. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae[J]. Nature, 2005.438(7071): 1105-1115.
[43] Punt, P.J., R.P. Oliver, M.A. Dingemanse, et al. Transformation of Aspergillus based onthe hygromycin B resistance marker from Escherichia coli [J]. Gene, 1987. 56(1): 117-124.
[44] Mulder, H.J. and I. Nikolaev. HacA-Dependent Transcriptional Switch Releases hacA mRNA from a Translational Block upon Endoplasmic Reticulum Stress[J]. Eukaryotic Cell, 2009. 8(4): 665-675.
[45]朱锦辉,权军利,何玉科等.根癌农杆菌感受态细胞的制备以及质粒ProkⅡ对其转化的研究[J].西北农林科技大学学报(自然科学版). 2006年07期
[46]罗雯,刘阳.根癌农杆菌转化条件优化的研究[J].生物技术.2006年01期
[47]单世华,李春娟,张君诚等.农杆菌质粒DNA提取方法的改良与鉴定[J].生物技术,2003,13(4): 19-20.
[48]周小玲,沈微,饶志明.一种快速提取真菌染色体DNA的方法[J].微生物学通报,2004 31(4): 89-92