绿色木霉葡聚糖内切酶基因的克隆和表达研究
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摘要
纤维素酶主要分为三类:(1).葡聚糖外切酶(exo-1,4-β-D-glucana-ses)又称纤维二糖酶(cellobiohydrolases,简称CBH);(2).葡聚糖内切酶(endo-1,4-,β-D-glucanases,简称EG);(3).β—葡萄糖苷酶(1,4-β-D-glucosidases简称BG),三种组分协同作用,最终将纤维素水解生成葡萄糖;产纤维素酶的菌主要时有丝真菌,尤其是木霉、曲霉等,木霉产的纤维素酶主要是葡聚糖内切酶和葡聚糖外切酶,而曲霉主要是产β—葡萄糖苷酶能力较强。
纤维素酶的合成成受纤维素、乳糖(lactose)、纤维二糖(cellobiose)、槐二糖(sophorose)和L-山梨糖(L-Sorbose)等的诱导。本文用L-山梨糖诱导绿色木霉AS3.3711纤维素酶基因的转录,在诱导9-12h时,葡聚糖内切酶的mRNA转录量达到峰值,收集菌丝体提取总RNA,反转录获得cDNA。以单链cDNA为模板,PCR扩增得到了编码葡聚糖内切酶Ⅰ的cDNA基因(egl1)和编码葡聚糖内切酶Ⅲ的cDNA基因(egl3),去除egl1和egl3的信号肽序列后分别构建了重组质粒pET-egl1和pSE-egl3,并实现了其在大肠杆菌中的表达。SDS-PAGE分析大肠杆菌的表达产物,结果表明egl1基因在大肠杆菌中表达的蛋白分子量约46.2kDa;egl3基因在大肠杆菌中表达的蛋白质分子量约42kDa。CMC酶活检测发现大肠杆菌表达的EGI没有酶活,大肠杆菌表达的EGⅢ检测到胞内酶活,每毫升菌液所含葡聚糖内切酶的活力为0.04U/ml。
将含信号肽的egl1基因,克隆到酿酒酵母表达载体pYES2中,构建了重组质粒pYES2-egl1,转化酿酒酵母菌INVscl,β-D-半乳糖诱导EGI的表达,用刚果红染色法筛选重组转化子。酿酒酵母能够识别EGI自身携带的信号肽,而将表达产物分泌到胞外,故在CMC筛选平板上用刚果红染色法检测,有清晰的水解圈。用80%饱和度的硫酸铵从2ml菌液上清中沉淀酶蛋白,重悬于1ml的0.05M柠檬酸钠缓冲液(pH4.8),CMCase测定结果显示,酿酒酵母INVsvl表达的EGI最适反应温度为47℃,最适反应pH为5.2,在诱导70h时酶活达到最高值,约为0.08U/ml。
Cellulase system consists essentially of three types of enzymes:(1) exo-1,4-β-D-glucanases,also called cellobiohydrolases(CBH),(2) endo-1,4-,β-D-glucanases(EG),and(3)1,4-β-D-glucosidases (BG).Different cellulolytic enzymes cooperate in decomposing cellulose.Most of filamentous fungi produces cellulase,especially the Trichoderma spp which produces endoglucanases and cellobiohydrolases in high level,and Aspergillus which produces 1,4-β-D-glucosidases in high level.
The cellulase formation can be induced by cellulose,lactose, cellobiose,sophorose,L-Sorbose et al.L-Sorbose has previously been assumed to stimulate cellulase formation in an indirect manner.In this work,we induced the cellulase formation with L-Sorobse in Trichoderma viride AS3.3711.after induced for 9-12 huors,when the mRNA of egl1 and egl3 transcription culminates, we collected the mycelia through filtration and washed them twice with 0.9%NaCl,then the mycelia were used to extract total RNA,and the cDNA was obtained by reverse transcription.The cDNA clones encoding endoglucanaseⅠand endoglucanaseⅢwhoes signal peptide were deleted were isolated by PCR protocol. then constructed recombined plasmids pET-egl1 and pSE-egl3 to be expressed in E.coli.the results of analysing with SDS-PAGE were that Protein bands of 46kDa and 42kDa were present as expressed products of pET-egl1 and pSE-egl3 transformations,the enzyme activity of EGⅢculminates 0.04U/mL.
The egl1 with signal peptide was isolated from cDNA by PCR protocol,and then it was cloned into a yeast vector to construct a cellulases-producing Saccharomyces cerevisiae engineering strains with stable heredity.Recombinant yeast strains can secrete EGI by the guide of the signal peptide itself.So the transcription and expression of EGI can be screened by activity plate assays with Congo Red method respectively.The endoglucanase activity was assayed as CMCase activity with CMC-Na as a substrate.The results showed that The optimal reaction conditions of the enzyme were 47℃and pH5.2,the enzyme activity of EGI culminates 0.08U/mL when induced for 70h.
纤维素酶的合成成受纤维素、乳糖(lactose)、纤维二糖(cellobiose)、槐二糖(sophorose)和L-山梨糖(L-Sorbose)等的诱导。本文用L-山梨糖诱导绿色木霉AS3.3711纤维素酶基因的转录,在诱导9-12h时,葡聚糖内切酶的mRNA转录量达到峰值,收集菌丝体提取总RNA,反转录获得cDNA。以单链cDNA为模板,PCR扩增得到了编码葡聚糖内切酶Ⅰ的cDNA基因(egl1)和编码葡聚糖内切酶Ⅲ的cDNA基因(egl3),去除egl1和egl3的信号肽序列后分别构建了重组质粒pET-egl1和pSE-egl3,并实现了其在大肠杆菌中的表达。SDS-PAGE分析大肠杆菌的表达产物,结果表明egl1基因在大肠杆菌中表达的蛋白分子量约46.2kDa;egl3基因在大肠杆菌中表达的蛋白质分子量约42kDa。CMC酶活检测发现大肠杆菌表达的EGI没有酶活,大肠杆菌表达的EGⅢ检测到胞内酶活,每毫升菌液所含葡聚糖内切酶的活力为0.04U/ml。
将含信号肽的egl1基因,克隆到酿酒酵母表达载体pYES2中,构建了重组质粒pYES2-egl1,转化酿酒酵母菌INVscl,β-D-半乳糖诱导EGI的表达,用刚果红染色法筛选重组转化子。酿酒酵母能够识别EGI自身携带的信号肽,而将表达产物分泌到胞外,故在CMC筛选平板上用刚果红染色法检测,有清晰的水解圈。用80%饱和度的硫酸铵从2ml菌液上清中沉淀酶蛋白,重悬于1ml的0.05M柠檬酸钠缓冲液(pH4.8),CMCase测定结果显示,酿酒酵母INVsvl表达的EGI最适反应温度为47℃,最适反应pH为5.2,在诱导70h时酶活达到最高值,约为0.08U/ml。
Cellulase system consists essentially of three types of enzymes:(1) exo-1,4-β-D-glucanases,also called cellobiohydrolases(CBH),(2) endo-1,4-,β-D-glucanases(EG),and(3)1,4-β-D-glucosidases (BG).Different cellulolytic enzymes cooperate in decomposing cellulose.Most of filamentous fungi produces cellulase,especially the Trichoderma spp which produces endoglucanases and cellobiohydrolases in high level,and Aspergillus which produces 1,4-β-D-glucosidases in high level.
The cellulase formation can be induced by cellulose,lactose, cellobiose,sophorose,L-Sorbose et al.L-Sorbose has previously been assumed to stimulate cellulase formation in an indirect manner.In this work,we induced the cellulase formation with L-Sorobse in Trichoderma viride AS3.3711.after induced for 9-12 huors,when the mRNA of egl1 and egl3 transcription culminates, we collected the mycelia through filtration and washed them twice with 0.9%NaCl,then the mycelia were used to extract total RNA,and the cDNA was obtained by reverse transcription.The cDNA clones encoding endoglucanaseⅠand endoglucanaseⅢwhoes signal peptide were deleted were isolated by PCR protocol. then constructed recombined plasmids pET-egl1 and pSE-egl3 to be expressed in E.coli.the results of analysing with SDS-PAGE were that Protein bands of 46kDa and 42kDa were present as expressed products of pET-egl1 and pSE-egl3 transformations,the enzyme activity of EGⅢculminates 0.04U/mL.
The egl1 with signal peptide was isolated from cDNA by PCR protocol,and then it was cloned into a yeast vector to construct a cellulases-producing Saccharomyces cerevisiae engineering strains with stable heredity.Recombinant yeast strains can secrete EGI by the guide of the signal peptide itself.So the transcription and expression of EGI can be screened by activity plate assays with Congo Red method respectively.The endoglucanase activity was assayed as CMCase activity with CMC-Na as a substrate.The results showed that The optimal reaction conditions of the enzyme were 47℃and pH5.2,the enzyme activity of EGI culminates 0.08U/mL when induced for 70h.
引文
1.穆小民,吴显荣.纤维素酶分子生物学研究进展及趋向[J]。生物工程进展.1994,4.
2.Teeri T.Pentila M Keranen S.et al.1992.Structure.function and genetics of cellulases.P417-455.In Biotechnology of filamentous fungi.Butterworth-Heinemann,stoneham,Mass.
3.Lee R L.Paul J.2002.Microbial celulase utilization:fundamentals and biotechnonlgy.Microbial Mel Bioi Rev.Sept 506-577
4.Salohelmo,M.,et al.1988,Gene 63,11-21.
5.Chung Mong Chen.et al.1987.Bio/Technology 5:274-278.
6.Knowles,J.,et al.1987.TIBTECH 5:255-261.
7.Nisizawa T,Suzuki H,Nakayama M,Nisizawa K(1971)Inductive formation of cellulase by sophorose in Triehoderma viride.J Biochem 70:375-385
8.Sternberg D,Mandels GR(1979)Induction of cellulolytic enzymes in Triehoderma reesei by sophorose.J Bacteriol.139:761-769
9.Loewenberg JR(1984)Sophorose induction of an intracellular β-glucosidase in Trichoderma.Arch Microbiol 137:53-57
10.Hohn H-P,Sahm H(1983)Induction of cellulases in Trichoderma reesei.Enzyme Technology 55-68
11.Mikio Kawamori,Yasushi Morikawa,Seigo Takasawa.Induction and production of cellulases by L-sorbose in Trichoderma reesei.Appl Microbiol Biotcchnol(1986)24:449-453
12.Kawamori M,Morikawa Y,Takasawa S(1985b)Inductive formation of cellulases by L-sorbose in Trichoderma reesei.Appl Microbiol Biotechnol 22:235-236 crobiol 78:179-192
13.Bisaria VS,Mishra S.Regulatory aspects of cellulase biosynthesis and secretion.Crit Rev Biotechnol,1989,9:61-103
14.Masahiro Nogawa,Masahiro Goto,Hirofumi Okada,et al.L-Sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei.Curr Genet,2001,38:329-334
15.Beguin,P.et al.:CRCOrlt.Rev.Biotechnol.1987,6:1-162.
16.Llu,C.Q.Ph.D.thesis,University of New South Wales,1988.
17.HIROFUMI OKADA,KOHJI TADA,TADASHI SEKIYA,et al,.Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414.Applied and Environmental Microbilogy.1998,p.555-563
18.Teeri,T.T.(1987).The cellulolytic enzyme system of Trichoderma reesei.Molecular cloning,characterization and expression of the cellobiohydrolase genes.Espoo,Finland.VTT Publications 38.52 p.+app.41 p.
19.M iller,R.C.et al,.Biochemistry and Genetics of cellulose Degradation,eds.1988,p.235-248
20.Penttila,M.,Nevalainen,H.,Ratto,M.,Salminen,E.& Knowles,J.(1987a)A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei,Gene (Amst.)61,155-164.
21.Penttila,M.,AndrC,L.,Saloheimo,M.,Lehtovaara,P.& Knowles,J.(1987b)Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae,Yeast 3.175-185.
22.Penttila,M.E.,AndrC,L.,Lehtovaara,P.,Bailey,M.,Teeri,T.& Knowles,J.K.C.(1988)Efficient secretion of two fungal cellobiohydrolases in Saccharomyces cerevisiae.Gene(Amst.)63,103-112.
23.Stjlbrand,H.,Saloheimo,A,,Vehmaanpera,J.,Henrissat,B.& Penttila,M.(1995)Cloning and expression in Saccharornyces cerevisiae of a Trichoderma reesei β-mannanase gene containing a cellulose-binding domain,Appl.Environ.Microbiol.61.1090-1097.
24.Teeri,T.,Salovuori,I.& Knowles,J.(1983)The molecular cloning of the major cellulase gene from Trichoderma reesei,Biotechnology Ⅰ,696-699.
25.Teeri,T.T.,Lehtovaara,P.,Kauppinen,S.,Salovuori,I.& Knowles,J.(1987)Homologous domains in Trichoderma reesei cellulolytic enzymes:gene sequence and expression of cellobiohydrolase 11,Gene(Amst.)51,43-52.
26.van Arsdell,J.N.,Kwok,S.,Schweickart,V.L.,Ladner,M.B.,Gelfand,D.H.& Innis,M.A.(1987)Cloning,characterisation and expression in Saccharomyces cerevisiae of endoglucanase Ⅰ from Trichoderma reesei,Biotechnology 5,60-64.
27.SALOHEIMO,Tuna NAKARI-SETALA,et al.,cDNA cloning of a Trichoderma reesei cellulase and demonstration of endoglucanase activity by expression in yeast.Eur.J.Biochem.1997,249,584-591.
28.Shoemaker,S.,Schweickart,V.,Ladner,M.,Gelfand,D.,Kwok.S.,Myambo,K.and Innis,M.(1983).Molecular cloning of exo-cellobiohydrolase Ⅰ derived from Trichoderma reesei strain L27.Bio/Technol.1,691.695.
29.Teeri,T.,Salovuori,I.and Knowles,J.(1983).The molecular cloning of the major cellulase gene from Trichoderma reesei.Bio/Technol.1,696.699.
30.Teeri,T.T.,Lehtovaara,P.,Kauppinen,S.,Salovuori,I.,and Knowles,J.(1987).Homologous domains in Trichoderma reesei cellulolytic enzymes:gene scquence and expression of cellobiohydrolase Ⅱ.Gene 51,43.52.
31.Penttila,M.E.,Andre,L.,Saloheimo,M.,Lehtovaara,P.and Knowles,J.K.C.Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae.Yeast.1987,3,175-185.
32.Penttila,M.E.,Andre,L.,Lehtovaara,P.,Bailey,M.,Teeri,T.T.and Knowles,J.K.C.Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae.Gene.1988,63,103-112.
33.Ward,M.,Wu,S.,Dauberman,J.,Weiss,G.,Larenas,E.,Bower,B.,Rey,M.,Clarkson,K.and Bott,R.(1993).Cloning,sequence and preliminary structural analysis of a small,high pI endoglucanase EGⅢ from Trichoderma reesei.In:Suomincn,P.and Reinikainen,T.(Eds.).Trichoderma reesei cellulases and other hydrolases.Foundation for biotechnical and industrial fermentation research,Helsinki.Vol 8.pp.153-158.
34.Sternberg,D.Appl.Environ.Microbiol.1976,31,648-654.
35.Kri-shna-Murti,C.R.and Stone,B.A.Blochem.J.1961,78,715-723.
36.刘纯强,王祖农.纤维素酶基因克隆及应用前景.生物工程进展[J].1991,3
37.李艳,王正祥,诸葛健.酵母作为外源基因表达系统的研究进展,生物工程进展,2001,vol.21,No.2
38.ALBERT Hinnent JAMES B Hicks,GERALD R Fink Transformation of yeast[J]Pro Natl Acad Sci USA,1978,75(4):1 929-1 933
39.JEAN D BeRgs.Transformation of yeast by a replicating hybrid plamid[J].Nature 1 978、275(5676):104-1O9.
40.PETER M J Burgers,KIMBERLY J Perecival.Transformation of Yeast Spheroplasts without Cell Fusion[J].Aanallytical Biochemistry,1978,163:391 397.
41.HISUO Ito,YASUKI Fukuda,KOUSAKU Murata、et al.Transformation of Intact Yea～st Cells Treated with Alkali Cations[J].Journal of Bacteriology,1983.153(1):163-168
42.DANIEL Gietz ANDREW ST Jeant ROBIN A Woods,el-al.Improved method forhi efficiency transformation of intact yeast cells[J].Nucleic Acid Research,1992,20(6):1425.
43.Hitzeman R A,HzgieF E,LevineH L et at.Expresaion of a human gene for interferon in yeast.Nature.1981.293:717-722
44.Gekkssen G,Melber K,Janowicz Z A et al.Homologous gene expression in yeast J Gen Mole Microbiol.1991,62:79-93
45.Hirmen A,Buxton F,Chandhrui B et al.Gene expression in recombinant yeast.In Smith A(Ed)Gene expreseion in recombinant organisms.New York:Mal-cel Dekker,1994,121-193
46.E.V.Winzeler,R.W.Davis.Functional Analysis of the Yeast Genome.Current Opinion in Genetics&Development.1997,7(6):771-776
47.S.G.Oliver,M.K.Winson,D.B.Kell andF Baganz.Systematic Functional Analysis of the Yeast Genome.Trends in Biotechnology,1998,16(9):373-378
48.J.DeRisi,B.Hazel,P.Marc,et al.,Genome Microarray Analysis of Transcriptional Activation in Multidrug Resistance Yeast Mutants.FEBS Letters.2000,470(2):156-160
49.D.E.Bassett,M.A.Basrai,C.Connelly,et al.,Exploiting the Complete Yeast Genome Sequence.Current Opinionin Genetics&Development.1996,6(6):763-766
50.周璐,李越中,李健.酿酒酵母基因组学研究进展.生命科学[J].1999,11(2):87-91
51.C.Seoighe,K.H.Wolfe.Updated Map of Duplicated Regions in the Yeast Genome.Gene.1999,238(1):253-261
52.PiAkur.Origin of the Duplicated Regions in the Yeast Genomes.Trends in Genetics.2001,17(6):302-303
53.刘文,胡巍,王洪海.酵母表达基因工程产物特性分析[J].生物工程进展.2001,vol.21,No.2
54.宏新,李敏康.酵母分子生物学理论及应用研究进展.西北轻工业学院学报[J].2001,19(4):78-83
55.毛小洪.幕盎科.酵母完骼细胞快速高教转化法[J].生物工程学报,1990,6(2):905-910.
56.A.A.Voronovsky,C.A.Abbas,L.R.Fayura,B.V Kshanovska,K.V.Dmytruk,K.A.Sybirna and A.A.Sibirny.Development of a Transformation System for the Flavinogenic Yeast Candida famata.FEMS yeast Research.2002,2(3):381-388
57.R.D.Gietz,R.H.Schiesti,A.R.Willems and R.A.Woods.Studies on the Transformation of Intact Yeast Cells by the LiAc/ss-DNA/PEG Procedure.Yeast.1995,11(4):355-360
58.Hill,K.A.Donald and D.E.Griffiths.DMSO-Enhanced Whole Cell Yeast Transformation.Nuc.Acids Res.1991,19(20):5791-5796
59.秦玉静,刘世利,鲍晓明等.高效的酵母完整细胞转化法.生物技术[J].1997,7(3):37-38
60.R.H.Schiestl,R.D.Gietz.High Efficiency Transformation of Intact Cells Using Single Stranded Nucleic Acids as a Carrier.Curr.Genet.1989,16(5-6):339-346
61.胡又佳,高枫,朱春宝等.[J]PEG/LiAc转化酵母细胞方法的改进.生物技术.1998,8(5):22-26
62.Hinmen A,Hiclks JB,Fink G & Proe Natl Acad Sci,USA,1978,75:19-29
63.Steams T,Me E,et al.,Methods in Enzymology,1990,185-280
64.Gellissen G,Melber K,Janowicz Z A.et at.Herrologous gene expression in yeast.J Gen Mole Microbinl.1991a,62:79-93
65.Hirmen A,Buxton F,Chandhrui B.et al.Gene expression in recombinant yeast.In Smith A(Ed)Gene expreseion in recombinant organisms.New York:Marcel Dekker,1994,121-193
66.Kojo H,Greenberg B D,Sngino A.Yeast 2 μm plasmid DNA replication in vitro:Origin and direction.Proc Nad Acnd Scj USA,1981,78:7261-7265
67.Phaff H J.Biology of yeasts other than Saccharomyces In:Demain AL,Salomon N A.(Eds)Biology of industrial microorgnism Benjamin Cummings:Menlo Park(CA),1985, 537-562
68.肖志壮,王婷,汪天虹,等.瑞氏木霉内切葡聚糖酶Ⅲ基因的克隆及在酿酒酵母中的表达[J].微生物学报,2001,41(4):391-396.
69.张磊 吴兴泉 木霉纤维素酶基因的克隆与表达研究进展[J].纤维素科学与技术,2004(12)4
70.刘北东,杨谦,周麒,等.绿色木霉AS3.3711的葡聚糖内切酶Ⅲ基因的克隆与表达[J],环境科学,2004(25)5
71.刘北东.杨谦.周麒,等.,绿色木霉AS3.3711的葡聚糖内切酶V基因的克隆与表达[J]高技术通讯,2004(18)
72.刘北东.杨谦.周麒,等.绿色木霉AS3.3711的葡聚糖内切酶Ⅰ基因的克隆与表达[J].北京林业大学学报2004:(26)6
73.RONALD M.TEATHER,PETER J.WOOD Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rument.APPLIED AND ENVIRONMENTAL MICROBIOLOGY Apr.1982,p.777-780
74.T.K.CHOSE,Measurement of Cellulase activities.Pure & Appl.Chem,Vol.59,No.2,pp.257-268.1987.
75.汪家政,范明.蛋白质技术手册M].北京,中国科学出版社,2001
76.J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.分子科隆实验指南(第二版)M].北京,中国科学出版社,2002
77.pYES2 Catalog no.V825—20,Version J 8 April 2004 28—0053.
78.Kwon.I,Keisuke Ekion,Masatoshi Goto,et al.,Heterologous Expression and Characterization of Endoglucanase I(EGI)from Trichoderma viride HK-75.Biosci.Biotechnol.Biochem.,63(10),1714-1720,1999
79.Gietz.RD,Woods RA.Transformation of yeast by lithium acetate/ single-stranded carrier DNA/polyethylene glycol method.Methods Enzymol.2002;350:87-96.
80.K.A.lan G.Donald and David E.Griffiths.DMSO-enhanced whole cell yeast transformation James Hill.Nucleic Acids Research,Vol.19,No.20,5791
81.Gietz RD,Schiestl RH,Willems AR,Woods RA.Studies on the transformation of intact yeast cells by the LiAc/S S-DNA/PEG procedure.Yeast.1995 Apr 15;11(4):355-60.
2.Teeri T.Pentila M Keranen S.et al.1992.Structure.function and genetics of cellulases.P417-455.In Biotechnology of filamentous fungi.Butterworth-Heinemann,stoneham,Mass.
3.Lee R L.Paul J.2002.Microbial celulase utilization:fundamentals and biotechnonlgy.Microbial Mel Bioi Rev.Sept 506-577
4.Salohelmo,M.,et al.1988,Gene 63,11-21.
5.Chung Mong Chen.et al.1987.Bio/Technology 5:274-278.
6.Knowles,J.,et al.1987.TIBTECH 5:255-261.
7.Nisizawa T,Suzuki H,Nakayama M,Nisizawa K(1971)Inductive formation of cellulase by sophorose in Triehoderma viride.J Biochem 70:375-385
8.Sternberg D,Mandels GR(1979)Induction of cellulolytic enzymes in Triehoderma reesei by sophorose.J Bacteriol.139:761-769
9.Loewenberg JR(1984)Sophorose induction of an intracellular β-glucosidase in Trichoderma.Arch Microbiol 137:53-57
10.Hohn H-P,Sahm H(1983)Induction of cellulases in Trichoderma reesei.Enzyme Technology 55-68
11.Mikio Kawamori,Yasushi Morikawa,Seigo Takasawa.Induction and production of cellulases by L-sorbose in Trichoderma reesei.Appl Microbiol Biotcchnol(1986)24:449-453
12.Kawamori M,Morikawa Y,Takasawa S(1985b)Inductive formation of cellulases by L-sorbose in Trichoderma reesei.Appl Microbiol Biotechnol 22:235-236 crobiol 78:179-192
13.Bisaria VS,Mishra S.Regulatory aspects of cellulase biosynthesis and secretion.Crit Rev Biotechnol,1989,9:61-103
14.Masahiro Nogawa,Masahiro Goto,Hirofumi Okada,et al.L-Sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei.Curr Genet,2001,38:329-334
15.Beguin,P.et al.:CRCOrlt.Rev.Biotechnol.1987,6:1-162.
16.Llu,C.Q.Ph.D.thesis,University of New South Wales,1988.
17.HIROFUMI OKADA,KOHJI TADA,TADASHI SEKIYA,et al,.Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414.Applied and Environmental Microbilogy.1998,p.555-563
18.Teeri,T.T.(1987).The cellulolytic enzyme system of Trichoderma reesei.Molecular cloning,characterization and expression of the cellobiohydrolase genes.Espoo,Finland.VTT Publications 38.52 p.+app.41 p.
19.M iller,R.C.et al,.Biochemistry and Genetics of cellulose Degradation,eds.1988,p.235-248
20.Penttila,M.,Nevalainen,H.,Ratto,M.,Salminen,E.& Knowles,J.(1987a)A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei,Gene (Amst.)61,155-164.
21.Penttila,M.,AndrC,L.,Saloheimo,M.,Lehtovaara,P.& Knowles,J.(1987b)Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae,Yeast 3.175-185.
22.Penttila,M.E.,AndrC,L.,Lehtovaara,P.,Bailey,M.,Teeri,T.& Knowles,J.K.C.(1988)Efficient secretion of two fungal cellobiohydrolases in Saccharomyces cerevisiae.Gene(Amst.)63,103-112.
23.Stjlbrand,H.,Saloheimo,A,,Vehmaanpera,J.,Henrissat,B.& Penttila,M.(1995)Cloning and expression in Saccharornyces cerevisiae of a Trichoderma reesei β-mannanase gene containing a cellulose-binding domain,Appl.Environ.Microbiol.61.1090-1097.
24.Teeri,T.,Salovuori,I.& Knowles,J.(1983)The molecular cloning of the major cellulase gene from Trichoderma reesei,Biotechnology Ⅰ,696-699.
25.Teeri,T.T.,Lehtovaara,P.,Kauppinen,S.,Salovuori,I.& Knowles,J.(1987)Homologous domains in Trichoderma reesei cellulolytic enzymes:gene sequence and expression of cellobiohydrolase 11,Gene(Amst.)51,43-52.
26.van Arsdell,J.N.,Kwok,S.,Schweickart,V.L.,Ladner,M.B.,Gelfand,D.H.& Innis,M.A.(1987)Cloning,characterisation and expression in Saccharomyces cerevisiae of endoglucanase Ⅰ from Trichoderma reesei,Biotechnology 5,60-64.
27.SALOHEIMO,Tuna NAKARI-SETALA,et al.,cDNA cloning of a Trichoderma reesei cellulase and demonstration of endoglucanase activity by expression in yeast.Eur.J.Biochem.1997,249,584-591.
28.Shoemaker,S.,Schweickart,V.,Ladner,M.,Gelfand,D.,Kwok.S.,Myambo,K.and Innis,M.(1983).Molecular cloning of exo-cellobiohydrolase Ⅰ derived from Trichoderma reesei strain L27.Bio/Technol.1,691.695.
29.Teeri,T.,Salovuori,I.and Knowles,J.(1983).The molecular cloning of the major cellulase gene from Trichoderma reesei.Bio/Technol.1,696.699.
30.Teeri,T.T.,Lehtovaara,P.,Kauppinen,S.,Salovuori,I.,and Knowles,J.(1987).Homologous domains in Trichoderma reesei cellulolytic enzymes:gene scquence and expression of cellobiohydrolase Ⅱ.Gene 51,43.52.
31.Penttila,M.E.,Andre,L.,Saloheimo,M.,Lehtovaara,P.and Knowles,J.K.C.Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae.Yeast.1987,3,175-185.
32.Penttila,M.E.,Andre,L.,Lehtovaara,P.,Bailey,M.,Teeri,T.T.and Knowles,J.K.C.Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae.Gene.1988,63,103-112.
33.Ward,M.,Wu,S.,Dauberman,J.,Weiss,G.,Larenas,E.,Bower,B.,Rey,M.,Clarkson,K.and Bott,R.(1993).Cloning,sequence and preliminary structural analysis of a small,high pI endoglucanase EGⅢ from Trichoderma reesei.In:Suomincn,P.and Reinikainen,T.(Eds.).Trichoderma reesei cellulases and other hydrolases.Foundation for biotechnical and industrial fermentation research,Helsinki.Vol 8.pp.153-158.
34.Sternberg,D.Appl.Environ.Microbiol.1976,31,648-654.
35.Kri-shna-Murti,C.R.and Stone,B.A.Blochem.J.1961,78,715-723.
36.刘纯强,王祖农.纤维素酶基因克隆及应用前景.生物工程进展[J].1991,3
37.李艳,王正祥,诸葛健.酵母作为外源基因表达系统的研究进展,生物工程进展,2001,vol.21,No.2
38.ALBERT Hinnent JAMES B Hicks,GERALD R Fink Transformation of yeast[J]Pro Natl Acad Sci USA,1978,75(4):1 929-1 933
39.JEAN D BeRgs.Transformation of yeast by a replicating hybrid plamid[J].Nature 1 978、275(5676):104-1O9.
40.PETER M J Burgers,KIMBERLY J Perecival.Transformation of Yeast Spheroplasts without Cell Fusion[J].Aanallytical Biochemistry,1978,163:391 397.
41.HISUO Ito,YASUKI Fukuda,KOUSAKU Murata、et al.Transformation of Intact Yea～st Cells Treated with Alkali Cations[J].Journal of Bacteriology,1983.153(1):163-168
42.DANIEL Gietz ANDREW ST Jeant ROBIN A Woods,el-al.Improved method forhi efficiency transformation of intact yeast cells[J].Nucleic Acid Research,1992,20(6):1425.
43.Hitzeman R A,HzgieF E,LevineH L et at.Expresaion of a human gene for interferon in yeast.Nature.1981.293:717-722
44.Gekkssen G,Melber K,Janowicz Z A et al.Homologous gene expression in yeast J Gen Mole Microbiol.1991,62:79-93
45.Hirmen A,Buxton F,Chandhrui B et al.Gene expression in recombinant yeast.In Smith A(Ed)Gene expreseion in recombinant organisms.New York:Mal-cel Dekker,1994,121-193
46.E.V.Winzeler,R.W.Davis.Functional Analysis of the Yeast Genome.Current Opinion in Genetics&Development.1997,7(6):771-776
47.S.G.Oliver,M.K.Winson,D.B.Kell andF Baganz.Systematic Functional Analysis of the Yeast Genome.Trends in Biotechnology,1998,16(9):373-378
48.J.DeRisi,B.Hazel,P.Marc,et al.,Genome Microarray Analysis of Transcriptional Activation in Multidrug Resistance Yeast Mutants.FEBS Letters.2000,470(2):156-160
49.D.E.Bassett,M.A.Basrai,C.Connelly,et al.,Exploiting the Complete Yeast Genome Sequence.Current Opinionin Genetics&Development.1996,6(6):763-766
50.周璐,李越中,李健.酿酒酵母基因组学研究进展.生命科学[J].1999,11(2):87-91
51.C.Seoighe,K.H.Wolfe.Updated Map of Duplicated Regions in the Yeast Genome.Gene.1999,238(1):253-261
52.PiAkur.Origin of the Duplicated Regions in the Yeast Genomes.Trends in Genetics.2001,17(6):302-303
53.刘文,胡巍,王洪海.酵母表达基因工程产物特性分析[J].生物工程进展.2001,vol.21,No.2
54.宏新,李敏康.酵母分子生物学理论及应用研究进展.西北轻工业学院学报[J].2001,19(4):78-83
55.毛小洪.幕盎科.酵母完骼细胞快速高教转化法[J].生物工程学报,1990,6(2):905-910.
56.A.A.Voronovsky,C.A.Abbas,L.R.Fayura,B.V Kshanovska,K.V.Dmytruk,K.A.Sybirna and A.A.Sibirny.Development of a Transformation System for the Flavinogenic Yeast Candida famata.FEMS yeast Research.2002,2(3):381-388
57.R.D.Gietz,R.H.Schiesti,A.R.Willems and R.A.Woods.Studies on the Transformation of Intact Yeast Cells by the LiAc/ss-DNA/PEG Procedure.Yeast.1995,11(4):355-360
58.Hill,K.A.Donald and D.E.Griffiths.DMSO-Enhanced Whole Cell Yeast Transformation.Nuc.Acids Res.1991,19(20):5791-5796
59.秦玉静,刘世利,鲍晓明等.高效的酵母完整细胞转化法.生物技术[J].1997,7(3):37-38
60.R.H.Schiestl,R.D.Gietz.High Efficiency Transformation of Intact Cells Using Single Stranded Nucleic Acids as a Carrier.Curr.Genet.1989,16(5-6):339-346
61.胡又佳,高枫,朱春宝等.[J]PEG/LiAc转化酵母细胞方法的改进.生物技术.1998,8(5):22-26
62.Hinmen A,Hiclks JB,Fink G & Proe Natl Acad Sci,USA,1978,75:19-29
63.Steams T,Me E,et al.,Methods in Enzymology,1990,185-280
64.Gellissen G,Melber K,Janowicz Z A.et at.Herrologous gene expression in yeast.J Gen Mole Microbinl.1991a,62:79-93
65.Hirmen A,Buxton F,Chandhrui B.et al.Gene expression in recombinant yeast.In Smith A(Ed)Gene expreseion in recombinant organisms.New York:Marcel Dekker,1994,121-193
66.Kojo H,Greenberg B D,Sngino A.Yeast 2 μm plasmid DNA replication in vitro:Origin and direction.Proc Nad Acnd Scj USA,1981,78:7261-7265
67.Phaff H J.Biology of yeasts other than Saccharomyces In:Demain AL,Salomon N A.(Eds)Biology of industrial microorgnism Benjamin Cummings:Menlo Park(CA),1985, 537-562
68.肖志壮,王婷,汪天虹,等.瑞氏木霉内切葡聚糖酶Ⅲ基因的克隆及在酿酒酵母中的表达[J].微生物学报,2001,41(4):391-396.
69.张磊 吴兴泉 木霉纤维素酶基因的克隆与表达研究进展[J].纤维素科学与技术,2004(12)4
70.刘北东,杨谦,周麒,等.绿色木霉AS3.3711的葡聚糖内切酶Ⅲ基因的克隆与表达[J],环境科学,2004(25)5
71.刘北东.杨谦.周麒,等.,绿色木霉AS3.3711的葡聚糖内切酶V基因的克隆与表达[J]高技术通讯,2004(18)
72.刘北东.杨谦.周麒,等.绿色木霉AS3.3711的葡聚糖内切酶Ⅰ基因的克隆与表达[J].北京林业大学学报2004:(26)6
73.RONALD M.TEATHER,PETER J.WOOD Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rument.APPLIED AND ENVIRONMENTAL MICROBIOLOGY Apr.1982,p.777-780
74.T.K.CHOSE,Measurement of Cellulase activities.Pure & Appl.Chem,Vol.59,No.2,pp.257-268.1987.
75.汪家政,范明.蛋白质技术手册M].北京,中国科学出版社,2001
76.J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.分子科隆实验指南(第二版)M].北京,中国科学出版社,2002
77.pYES2 Catalog no.V825—20,Version J 8 April 2004 28—0053.
78.Kwon.I,Keisuke Ekion,Masatoshi Goto,et al.,Heterologous Expression and Characterization of Endoglucanase I(EGI)from Trichoderma viride HK-75.Biosci.Biotechnol.Biochem.,63(10),1714-1720,1999
79.Gietz.RD,Woods RA.Transformation of yeast by lithium acetate/ single-stranded carrier DNA/polyethylene glycol method.Methods Enzymol.2002;350:87-96.
80.K.A.lan G.Donald and David E.Griffiths.DMSO-enhanced whole cell yeast transformation James Hill.Nucleic Acids Research,Vol.19,No.20,5791
81.Gietz RD,Schiestl RH,Willems AR,Woods RA.Studies on the transformation of intact yeast cells by the LiAc/S S-DNA/PEG procedure.Yeast.1995 Apr 15;11(4):355-60.