作者声明 我郑重声明：本人恪守学术道德，崇尚严谨学风。所呈交的学位论文，是本人在导 师的指导下，独立进行研究工作所取得的结果。除文中明确注明和引用的内容外，本论文 不包含任何他人已发表过或者描写过的内容。论文为本人亲自描写，并对所写内容负责。 论文作者签名：陈武海 2013 年 月 日 分类号： 密级： UDC： _ 华东理工大学 学位论文 -TMT 和 FAD2 基因在大肠杆菌 与里氏木霉中的表达及鉴定 陈武海 指导教师姓名： 魏东芝 教授 王 玮博士 华东理工大学上海市梅陇路 130 号 申请学位级别： 博士 专 业 名 称： 论文定稿日期： 论文答辩日期： 学位授予单位： 华东理工大学 生物化工 学位授予日期： 答辩委员会主席 评阅人 ： _ ： _ _ _ _ EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY THESIS OF PHILOSOPHY DOCTOR EXPRESSIONAL ANALYSIS OF -TMT AND FAD2 GENES IN Escherichia coli AND Trichoderma reesei Specialty : Biochemistry Engineering Research field : Microbiology & Gene Technology PhD student : Tran Vu Hai Student ID : 010090147 Advisors : Prof PhD Wei Dong Zhi PhD Wang Wei Shanghai-China, May 4, 2013 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR I ABSTRACT Tocopherols, with antioxidant properties, are synthesized by photosynthetic organisms and play important roles in human and animal nutrition In the major oilseed crops, -tocopherol, the biosynthetic precursor to α-tocopherol, is the predominant form found in the leaves This suggests that the final step of the α-tocopherol biosynthetic pathway was catalyzed by γtocopherol methyltransferase The full-length -PfTMT was obtained from the total RNA of Perilla frutescens leaves by RT-PCR Sequence analysis indicates that -PfTMT consisted the open reading frame of 894 nucleotides encoding the protein of 34 kD polypeptide Our results demonstrated that the E Coli BL21(DE3) expression of the -PfTMT resulted in the α-tocopherol contents (and -tocopherol conversion yield) from 18% in the reaction products Transgenic Trichoderma reesei Rut-C30 strains, over-expressing the γ-PfTMT was also generated by Agrobacterium tumefaciensmediated transformation The presence of hph and γ-PfTMT gene in the transformants were confirmed by PCR analysis The expression of the γ-PfTMT gene of the transgenes was demonstrated by SDS-PAGE Furthermore, we demonstrated that the Trichoderma reesei RutC30 expression of the γ-PfTMT gene resulted in the tocopherol composition 5.9-fold increase in α-tocopherol content by using high-performance liquid chromatographic (HPLC) method The increase in the α-tocopherol content indicates that a regulatory function of the γ-PfTMT protein converts -tocopherol to α-tocopherol The full-length -BoTMT was obtained from the total RNA of Brassica oleracea leaves by RT-PCR Sequence analysis indicates that -BoTMT consisted the open reading frame of 1041 nucleotides encoding the protein of 39 kD polypeptide Our results demonstrated that the E Coli BL21(DE3) expression of the -BoTMT resulted in the α-tocopherol contents (and -tocopherol conversion yield) from 23% of the reaction products by using HPLC method Transgenic Trichoderma reesei Rut-C30 strains, over-expressing the γ-BoTMT gene was also generated by Agrobacterium tumefaciens-mediated transformation The presence of hph and γ-BoTMT gene in the transformants were confirmed by PCR analysis The expression of the γ-BoTMT gene of the transgenes was demonstrated by SDS-PAGE Fatty acids are the main groups of components of plant membrane lipid and seed storage lipid, and the major source of energy in plant According to bioinformation analysis of the cDNA II EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR sequence, the specific fragment of FAD2 from immature maize embryos was isolated by RTPCR Results of sequence analysis indicate that FAD2 fragment contains the open reading frame of 1,236 bp long coding for the 46 kD polypeptide Transgenic Trichoderma reesei Rut-C30 strains, over-expressing the FAD2 gene from maize were generated by Agrobacterium tumefaciens-mediated transformation The presence of hph and FAD2 gene in the transformants were confirmed by polymerase chain reaction (PCR) analysis The expression of the FAD2 gene of the transgenes from Trichoderma reesei and E coli BL21 were demonstrated by SDS-PAGE In this study, we developed novel plasmids containing three plasmids designated pBI121TMT, pCAMBIA1301S-FAD2 and pCAMBIA1301S-FAD2-TMT that incorporate modified and improved expression omega-3 and vitamin E content in seeds of the plant transformation The FAD2 and -PfTMT genes of each plasmid were driven by the constitutive CaMV 35S promoter which is mostly used for driving trangene expressions in both monocot and dicot plant transformation The binary vector pCAMBIA1301S-FAD2 and vector pBI121-TMT contains FAD2, -PfTMT genes respectively, whereby the binary vector pCAMBIA1301S-FAD2-TMT contains both FAD2 and -PfTMT gene All three plasmid vectors were introduced into A tumefaciens EHA105 by electroporation Keywords FAD2 gene; -TMT; Perilla frustescens; Brassica oleracea; tocopherol; HPLC; Trichoderma reesei; Agrobacterium tumefaciens EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR III CONTENTS ABSTRACT ……………………………………………………………………………… I CONTENTS……………………………………………………………………………… III LIST OF FIGURES……………………………………………………………………… VII LIST OF TABLE ……………………………………………………………………… IX NOMENCLATURE ……………………………………………………………………… X Chapter Biodiversity and phylogeny of Trichoderma ……………………………… 1.1 Characteristics of Trichoderma spp ………………………………………… 1.2 Tools for genetic manipulation of Trichoderma ……………………………… 1.3 Defense mechanisms and their exploitation …………………………………… 1.4 Trichoderma’s strategies for combat ………………………………………… 1.5 Regulatory mechanisms triggering the defense of Trichoderma …………… 1.6 Trichoderma as a protector of plant health …………………………………… 1.7 Secondary metabolites ………………………………………………………… 1.8 Trichoderma spp as industrial workhorses …………………………………… 1.9 Cellulases and plant cell wall-degrading enzymes …………………………… 1.10 Heterologous protein production ……………………………………………… 1.11 Food industry …………………………………………………………………… 1.12 Human pathogenic species …………………………………………………… 10 Chapter Agrobacterium-mediated transformation of Trichoderma reesei overexpressing the Perilla frutescens -tocopherol methyltransferase gene …………… 11 2.1 Introduction ……………………………………………………………………… 11 2.2 Materials and methods ………………………………………………………… 11 2.2.1 Strains, plasmid, media and major reagent …………………………………… 14 2.2.2 Primer design …………………………………………………………………… 14 2.2.3 RNA Isolation …………………………………………………………………… 14 2.2.4 Reverse Transcriptase Reactions PCR ………………………………………… 15 2.2.5 Transformation plasmid into DH5a strain of Escherichia coli ………………… 16 2.2.6 Vector construction ……………………………………………………………… 17 2.2 Transformation of A tumefaciens with plasmid DNA (binary vector system) … 17 2.2 7.1 Preparation of competent cells ………………………………………………… 19 IV EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR 2.2 7.2 Electroporation ………………………………………………………………… 19 2.2.8 Agrobacterium tumefaciens-mediated fungal transformation …………………… 19 2.2 Molecular analysis of transformants …………………………………………… 20 2.2.10 Expression -PfTMT gene in Trichoderma viride ……………………………… 21 2.2.11 Expression -PfTMT gene in E coli BL21 ……………………………………… 21 2.2.12 The enzyme activity assay of the recombinant -PfTMT ……………………… 21 2.2.13 Chemical analysis ……………………………………………………………… 22 2.3 Results ……………………………………………………………………………… 22 2.3.1 Characterization of -PfTMT gene ……………………………………………… 23 2.3.2 Agrobacterium-mediated fungal transformation ………………………………… 24 2.3.3 Molecular analysis of transformants …………………………………………… 27 2.3.4 Expression of -PfTMT in Trichoderma reesei ………………………………… 28 2.3.5 Expression of -PfTMT in E coli ……………………………………………… 29 2.3.6 The enzyme activity assay of the recombinant -PfTMT protein ………………… 30 2.4 Discussion ………………………………………………………………………… 33 2.5 Conclusion ………………………………………………………………………… 35 Chapter Agrobacterium-mediated transformation of Trichoderma reesei overexpressing the Brassica oleracea γ -tocopherol methyltransferase gene …………… 36 3.1 Introduction ……………………………………………………………………… 36 3.2 Materials and methods ………………………………………………………… 37 3.2.1 Strains, plasmid, media and major reagent …………………………………… 37 3.2.2 Primer design …………………………………………………………………… 38 3.2.3 Transformation plasmid into DH5a strain of Escherichia coli ………………… 38 3.2.4 Vector construction …………………………………………………………… 38 3.2.5 Agrobacterium tumefaciens-mediated fungal transformation ………………… 40 3.2.6 Molecular analysis of transformants …………………………………………… 41 3.2 Expression -BoTMT gene in Trichoderma reesei …………………………… 41 3.2.8 Expression -BoTMT gene in E coli BL21 ……………………………………… 42 3.2.9 The enzyme activity assay of the recombinant -BoTMT ……………………… 42 3.2.10 Chemical analysis …………………………………………………………… 42 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR V 3.3 Results ………………………………………………………………………… 43 3.3.1 Characterization of -BoTMT ………………………………………………… 43 3.3.2 Agrobacterium-mediated fungal transformation ……………………………… 44 3.3.3 Molecular analysis of transformants …………………………………………… 46 3.3.4 Expression of -BoTMT in Trichoderma reesei ……………………………… 47 3.3.5 Expression of -BoTMT in E coli ……………………………………………… 48 3.3.6 The enzyme activity assay of the recombinant -BoTMT protein ……………… 49 3.4 Discussion ………………………………………………………………………… 49 3.5 Conclusion ……………………………………………………………………… 51 Chapter Agrobacterium-mediated transformation of Trichoderma reesei overexpressing the FAD2 gene …………………………………………………………… 52 4.1 Introduction ……………………………………………………………………… 52 4.2 Materials and methods …………………………………………………………… 55 4.2.1 Strains, plasmid, media and major reagent ……………………………………… 55 4.2.2 Primer design …………………………………………………………………… 56 4.2.3 RNA Isolation, Reverse Transcriptase Reactions PCR ………………………… 56 4.2.4 Transformation plasmid into DH5a strain of Escherichia coli ………………… 56 4.2.5 Vector construction ……………………………………………………………… 57 4.2.6 Agrobacterium tumefaciens-mediated fungal transformation …………………… 58 4.2.7 Molecular analysis of transformants …………………………………………… 58 4.2.8 Expression FAD2 gene in E coli and Trichoderma reesei ……………………… 59 4.3 Results ……………………………………………………………………………… 60 4.3.1 Characterization of maize FAD2 gene …………………………………………… 60 4.3.2 Agrobacterium-mediated fungal transformation ………………………………… 62 4.3.3 Molecular analysis of transformants …………………………………………… 64 4.3.4 Expression of FAD2 in Trichoderma reesei ……………………………………… 65 4.4 Discussion ………………………………………………………………………… 66 4.5 Conclusion ………………………………………………………………………… 67 Chapter Novel plant transformation vectors containing γ-PfTMT and FAD2 genes 68 5.1 Introduction ……………………………………………………………………… 5.2 Materials and methods …………………………………………………………… 68 VI EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR 5.2.1 PCR Overlapping Extension ……………………………………………………… 70 5.2.2 Vector construction ……………………………………………………………… 70 5.2.3 Transformation of A.tumefacient with plasmid DNA …………………………… 74 5.2.3.1 Preparation of competent cell ………………………………………………… 74 5.2.3.2 Electroporation ……………………………………………………………… 75 5.3 Results and discussion …………………………………………………………… 75 5.3.1 PCR Overlapping Extension ……………………………………………………… 75 5.3.2 Construction of plasmid vector pCAMBIA1301S-FAD2, pCAMBIA1301S-TMT and pCAMBIA1301S-FAD2-TMT ……………………………………………………… 77 5.3.3 Transformation of A.tumefacient with plasmid DNA …………………………… 80 5.4 Conclusion ………………………………………………………………………… 81 Chapter Conclusion and Future direction …………………………………………… 82 6.1 Conclusion ………………………………………………………………………… 82 6.2 Future direction …………………………………………………………………… 83 REFERENCE ……………………………………………………………………………… 84 ACKNOWLEDGEMENT ………………………………………………………………… 112 AUTHOR’S INTRODUTION ……………………………………………………………… 113 APPENDIX ………………………………………………………………………………… 116 106 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR 189 Reese ET History of the cellulase program at the U S Army Natick Development Center Biotechnol Bioeng Symp 1976, 6: 9–20 190 Reino JL, Guerrero RF, Hernandez-Galan R, Collado IG Secondary metabolites from species of the biocontrol agent Trichoderma Phytochemistry 2008, 7: 89–123 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a new Δ-12 fatty acid desaturase (FAD2-4) gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana Plant Physiology and Biochemistry 2009, 47: 462-471 273 Zhong, YH, Wang XL, Wang TH, Jiang Q Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis Appl Microbiol Biotechnol 2007, 73: 1348–1354 274 http://genome.jgi-psf.org/Triat1/Triat1.home.html 275 http://genome.jgi-psf.org/Trire2/Trire2.home.html 276 http://nt.ars-grin.gov/ taxadescriptions/keys/TrichodermaIndex.cfm 277 http://www.indexfungo- rum.org/Names/Names.asp 278 http://www.isth.info 279 http://www.isth.info/biodiversity/index.php 115 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY-THESIS OF PHILOSOPHY DOCTOR ACKNOWLEDGEMENTS In January 2009, when I came to Shanghai to start my PhD research on Biochemistry Engineering I didn’t imagine that I would work in a wide range of new techniques that I never knew before With the completion of this booklet, which is the major outcome of my work, I would like to thank all of my colleague for their special contributions First of all, I would like to express my sincerest appreciation to my Professor Dr Wei Dong Zhi and his associate Dr Wang Wei in New World Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology for their excellent advice, great support, and encouragement throughout my doctorate studies I would like also to express my thanks to Lu Dan Dan, Zhao Xi Hua, Zhu Yao, Malisa B.e for his encouragements and advices during my study My thanks also go to the students in New World Institute of Biotechnology, East China University of Science and Technology Their friendship support and encourage during this research work I wish to express my special thanks to the Priority Program of Development and Application of Biotechnology in Agriculture and Rural Development Towards 2020-Vietnam for financial support and an opportunity to study abroad I am especially thankful to all of my current and past associates in the Cuu Long Rice Research Institute-Vietnam who have helped me in many ways, including Ass Dr Bui Ba Bong, Dr Le Van Banh, Ass Dr Tran Thi Cuc Hoa, Dr Nguyen Xuan Lai, Dr Luu Hong Man, Dr Pham Trung Nghia My thanks also go to the staff members in the Department of Biotechnology I especially appreciate my Mom and Dad for their continuing encouragement, support, and assistance for my family Without their encouragement, I would have given up My wife Le Khanh Linh and son Tran Minh Huy and daughter Tran Minh Thu are always encouraging me and supporting me when there is any problem occurred Shanghai-China, May 4, 2013 Author Tran Vu Hai 116 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR AUTHOR’S INTRODUCTION Personal information Name: Tran Vu Hai Date of birth: November 02nd, 1975 Place of birth: Tamky, Quangnam, Vietnam Office address: Biotechnology Department, Cuu Long Delta Rice Research Institute (CLRRI), Codo, Cantho, Vietnam Email: tranvuhai2005@yahoo.com Home address: Cuu Long Delta Rice Research Institute (CLRRI), ThoiLai, Cantho, Vietnam Nationality: Vietnamese, Sex: Male Marital status: Married, Religious: No Educational background Year Name of the School/University Degree 1986-1990 Tam ky Elementary School Elementary 1990-1993 An Nhon High School High School 1993-1998 University Forestry, University of Agriculture Vietnam of and Field of study B.Sc Agronomy National Hochiminh City, Hochiminh City, Vietnam 2000 - 2004 Cantho University, Cantho, Vietnam M.Sc Biotechnology 2008-up to East China University Science and Ph.D Biotechnology now Technology, Shanghai, China EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY-THESIS OF PHILOSOPHY DOCTOR 117 Duty 1998: Joining the Cuu Long Delta Rice Research Institute as a research assistant Training 2002: Attending a training course on genotype and environment (G x E) at the Cuu Long Delta Rice Research Institute (CLRRI), Vietnam 2004: Attending a training course: Assessment of Vitamin A status and analysis of vitamin A in Foods and Biological sample, Bangkok, Thailand Work experience 1998-2000: I actively involved in a project to develop rice varieties for high yield and pest resistance To pursue this aim, I have employed both traditional breeding methods (pedigree, backcross, tri- and tetra- crosses) and tissue culture like somaclonal variation and anther culture Within two years, I have selected 20 elite lines for yield testing trial and finally OM3925, the line that I have developed, have been released in 2004 and grown in large scale in most of provinces in Cuu Long delta 2000-2003: Besides my duty as plant breeder, I participated in the international project of “high iron densed-rice” that was sponsored by the International Food Policy Research Institute I carried out experiments to select rice cultivars, which performed well in multi-location trial and identified rice variety with high iron-content in rice grains 2003-2004: In my M.Sc thesis, I investigated the ability of transformation of two indica rice varieties KhangDan and IR68144 which possess high iron content with the genes for vitamin A (-carotene) by using Agrobacterium tumefaciens and mannose selection system 2004-2008: I researched crossing of rice by anther culture method and gene transformation method in Biotechnology Department of Cuu Long Delta Rice Research Institute I participated in the Vietnam project “Breeding new soybean varieties by genetic engineering” 2008-up to now: I am studying Ph.D training program in East China University Science and Technology, Shanghai, China 118 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR Published paper Pham Trung Nghia, Tran Vu Hai, Dong Thanh Liem, Tran Thi Cuc Hoa (2006) Effect of store of storage condition on total carotenoid content in golden rice grains Omonrice 14: 1-6 Tran Thi Cuc Hoa, Pham Ngoc Tu, Tran Vu Hai, Bui Ba Bong (2005) Breeding iron-dense rice in Vietnam 5th International Rice Genetics Symposium and 3rd International Rice Functional Genomics Symposium Tran Thi Cuc Hoa, Tran Vu Hai, La Cao Thang (2008) Transformation efficiencies of the soybean variety PC 19 [glycine max (L.) merrill] using Agrobacterium tumefaciens and the cotyledonary node method Omonrice 16:1-8 Tran Vu Hai et al (2012) Brassica rapa subsp pekinensis gamma-tocopherol methyltransferase (TMT) mRNA, complete cds NCBI, Accession number JQ031515.1 Tran Vu Hai et al (2011) Perilla frutescens gamma-tocopherol methyltransferase mRNA, complete cds NCBI, Accession number JN381069.1 Tran Vu Hai, Wei Wang, Dong Zhi Wei (2012) Cloning and characterization of a γtocopherol methyltransferase from Chinese cabbage African Journal of Biotechnology 11: 13907-13913 Tran Vu Hai, Wei Wang, Dong Zhi Wei (2012) Agrobacterium-mediated transformation of Trichoderma reesei overexpressing the γ-BoTMT gene Science and Technology Journal of Agriculture and Rural Development Ministry of Agriculture and Rural Development, Vietnam 24: 26-29 Tran Vu Hai, Wei Wang, Dong Zhi Wei (2013) Agrobacterium-mediated transformation of Trichoderma reesei overexpressing the FAD2 gene Omonrice 19:40-53 Tran Vu Hai, Wei Wang, Dong Zhi Wei (2013) Cloning and characterization of a tocopherol methyltransferase gene from Brassica oleracea and Perila frustescens National Biotechnololgy Conference, Vietnam Book No.1: 786-791 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY-THESIS OF PHILOSOPHY DOCTOR APPENDIX Potato Dextrose Agar (PDA) Peeled potato 200.0 g Glucose 20.0 g Yeast Extract 0.1 g Agar (for solid medium) 20.0 g Total volume 1.0 L Note: adjust pH to 7.0 and autoclave to sterilize LB medium Bacto-Tryptone 10 g Bacto-yeast extract NaCl 10 g Total volume g L Note: adjust pH to 7.0 and autoclave to sterilize Induction medium (IM) K2HPO4 10.0 mM KH2PO4 10.0 mM NaCl 2.5 mM MgSO4.7H2O 2.0 mM CaCl2 0.7 mM FeSO4.7H2O 9.0 µM (NH4) 2SO4 4.0 mM Glucose 10.0 mM Glycerol 0.5 % acetosyringone 200.0 M 2-(N-morpholino) ethanesulfonic acid (MES), pH 5.3 40.0 M Total volume 1.0 L 119 120 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR Note: adjust pH to 7.0 and autoclave to sterilize Glucose yeast medium (GYM) NH4H 2PO4 1.0 g KCl 0.2 g MgSO4.7H2O 0.2 g CuSO4.5H2O 1.0 ml (0.5%) ZnSO4.7H2O 1.0 ml (1%) Glucose 10.0 g Total volume 1.0 L Note: adjust pH to 7.0 and autoclave to sterilize Liquid minimal medium (LMM) Tryptone 5.00 g Yeast Extract 0.30 g Lactose 20.00 g (NH4) 2SO4 1.40 g Urea 0.30 g KH2PO4 2.00 g CaCl2 0.34 g MgSO4.7H2O 0.30 g mL 1.0 L Mandels trace solution FeSO4.7H2O 5.0 MnSO4.H2O 16.0 g ZnSO4.7H2O 1.4 g CoCl2.6H2O 2.0 g Total volume 1.0 L Total volume Note: adjust pH to 7.0 and autoclave to sterilize g [...]... 2-methyl-6-phytylquinol, the beginning ring methylation at position 3 to yield 2,3- EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY-THESIS OF PHILOSOPHY DOCTOR 13 dimethyl-5-phytylquinol, cyclization to yield -tocopherol, and finally a second ring methylation at position 5 to yield -tocopherol (Fig 2.2) Fig 2.2  -TMT enzymatic reaction  -TMT adds a methyl group to ring carbon 5 of tocopherol These investigations... pPK5-BoTMT was tested by electrophoresis ……………………… 44 Fig 3.6 PCR analysis of the hph and -BoTMT gene inserted in genomic DNA of T VIII EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR reesei Rut-C30 transformation …………………………………………………………… 45 Fig 3.7 Expression of the recombinant -BoTMT in T reesei Rut-C30 ………………… 47 Fig 3.8 Expression of the recombinant -BoTMT in E coli. .. enzyme mediated integration RNA Ribonucleic acid EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR RT-PCR Reverse transcription PCR SAM S-Adenosylmethionine SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis T reesei Trichoderma reesei XI 1 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY-THESIS OF PHILOSOPHY DOCTOR Chapter I BIODIVERSITY AND PHYLOGENY OF TRICHODERMA. .. industry [69] After the early mutation programs [61] and strain development, the protein emission capacity of industrial strains now reaches very high High 8 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR levels of cellulase and hemicellulase by using in the synthesis of a functional gene product can be achieved upon cultivation on cellulose, xylan, or a mixture of plant polymers... especially if they have generally regarded as safe status [169], as has T reesei [168] The manufactured in the production of calf chymosin use of T reesei as a producer of heterologous proteins begins more than 20 years ago [80, 249] At that time, the expression of immunologically active antibody fragments [172] in T reesei was achieved, and numerous enzymes and expression proteins followed Currently, T reesei. .. pPK5-PfTMT was tested by electrophoresis ……………………… 26 Fig 2.9 A PCR analysis of the hph gene inserted in genomic DNA of T reesei Rut-C30 transformation; B PCR analysis of the -PfTMT gene inserted in genomic DNA of T .reesei Rut-C30 transformation ………………………………………………………… 27 Fig 2.10 Expression of the recombinant -PfTMT in T reesei Rut-C30 ……………… 27 Fig 2.11 Purification of His-tagged -PfTMT fusion protein... tube in 4 ml of ice cold 10% glycerol Combine and aliquot into 2 tubes Repeat spin 6 Resuspend cells in each of the two tubes in 2ml of ice cold 10% glycerol Combine into one tube Repeat spin (use a balance tube with water or 10% glycerol) 7 Resuspend final pellet in 1.5 ml ice cold 10% glycerol EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY-THESIS OF PHILOSOPHY DOCTOR 19 8 Dispense 100ul aliquots into... signaling pathways of light response, heterotrimeric G-proteins, the cAMP pathway, sulfur metabolism, and oxidative stress are operative in Trichoderma [207, 241] In recently years, studying with Trichoderma has been promoted importantly by genomes sequencing of three strains representing the most crucially applications of this genus: The genome sequence of T reesei [147, 274], besides that, in spite of. .. the activity of the pathway of heterotrimeric G-protein signaling, two genes 6 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR have been found out with a show to biocontrol mechanisms relation in Trichoderma spp.: the class I (adenylate cyclase inhibiting) G-alpha subunits TGA1 of T atroviride and TgaA of T virens both the class III (adenylate cyclase activating) G-alpha... tested by restriction enzyme ………………………………… 78 Fig 5.6 The A tumefaciens EHA105 transformed with the recombinant vector ………… 80 Fig 5.7 PCR of A tumefaciens containing every plasmids pCAMBIA1301 -FAD2, pCAMBIA1301 -TMT, and pCAMBIA1301 -FAD2 -TMT transformants showing presence of FAD2, -BoTMT and -PfTMT gene …………………………………………………… 81 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR