Tài liệu Expression analysis of y tmt and fad2 gene in escherichia coli and transgenic trichoderma reesei

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 5 月 4 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 1. Biodiversity and phylogeny of Trichoderma ……………………………… 1 1.1. Characteristics of Trichoderma spp. ………………………………………….. 2 1.2. Tools for genetic manipulation of Trichoderma ……………………………… 3 1.3. Defense mechanisms and their exploitation …………………………………… 4 1.4. Trichoderma’s strategies for combat ………………………………………….. 4 1.5. Regulatory mechanisms triggering the defense of Trichoderma …………….. 5 1.6. Trichoderma as a protector of plant health …………………………………… 6 1.7. Secondary metabolites ………………………………………………………….. 6 1.8. Trichoderma spp. as industrial workhorses …………………………………… 7 1.9. Cellulases and plant cell wall-degrading enzymes …………………………….. 7 1.10. Heterologous protein production ……………………………………………… 8 1.11. Food industry …………………………………………………………………… 9 1.12. Human pathogenic species …………………………………………………….. 10 Chapter 2. 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. 7. 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. 9. 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 3. 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. 7. 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 4. 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 5. 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 6. Conclusion and Future direction …………………………………………….. 82 6.1. Conclusion …………………………………………………………………………. 82 6.2. Future direction …………………………………………………………………… 83 REFERENCE ……………………………………………………………………………….. 84 ACKNOWLEDGEMENT ………………………………………………………………….. 112 AUTHOR’S INTRODUTION ……………………………………………………………… 113 APPENDIX …………………………………………………………………………………. 116 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR VII LIST OF FIGURES Fig. 1.1. Expression of DsRed2 in transformed fungi ……………………………………. 5 Fig. 2.1. Chemical structures of α-, β-, γ-, and δ-tocopherols …………………………… 12 Fig. 2.2. -TMT enzymatic reaction. -TMT adds a methyl group to ring carbon 5 of tocopherol ………………………………………………………………………………….. 13 Fig. 2.3. Schematic diagram of the binary vectors pPK5-PfTMT ………………………… 18 Fig. 2.4. Schematic diagram of the expression vectors pET28a-TMT-Pf ………………… 18 Fig. 2.5. PCR amplification of -PfTMT gene …………………………………………….. 24 Fig. 2.6. Alignment of γ-TMT protein sequences from Perilla frutescence and three other organisms using ClustalW2 software ……………………………………………………… 24 Fig. 2.7. Colony morphology of T. reesei Rut-C30 transformants on solid media ……….. 25 Fig. 2.8. The plasmid 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 …………………………… 29 Fig. 2.12. Expression of the recombina -PfTMT in E. coli ……………………………… 30 Fig. 2.13. A. Separation of - and -tocopherol product standards; B. HPLC analysis of tocopherol production in T. reesei Rut-C30 untransformed control; C. HPLC analysis of -tocopherol production for T. reesei Rut-C30 transformation …………………………… 31 Fig. 2.14. HPLC analysis of -tocopherol production in E. coli. Cells; A. Separation of and -tocopherol product standards; B. E. coli BL21(DE3)/pET30a controls; C. E. coli BL21(DE3)/pET-PfTMT transformation ………………………………………………….. 32 Fig. 3.1. Schematic diagram of the binary vectors pPK5-BoTMT ………………………… 39 Fig. 3.2. Schematic diagram of the expression vectors pET28a-TMT-Bo ………………… 40 Fig. 3.3. PCR amplification of -BoTMT gene …………………………………………….. 46 Fig. 3.4. Colony morphology of T. reesei Rut-C30 transformants on solid media ………... 43 Fig. 3.5. The plasmid 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 ……………………………… 48 Fig. 3.9. HPLC analysis of -tocopherol production in E. coli. Cells …………………….. 49 Fig. 4.1. Schematic diagram of the binary vectors pPK5-FAD2 …………………………... 57 Fig. 4.2. Schematic diagram of the binary vectors pET-FAD2 ……………………………. 58 Fig. 4.3. PCR amplification of FAD2 gene from maize genomic DNA …………………… 60 Fig. 4.4. Alignment of FAD2 protein sequences from Zea mays and four other organisms using ClustalW2 software using ClustalW2 software ……………………………………… 61 Fig. 4.5. Colony morphology of T. reesei Rut-C30 transformants on solid media ……….. 62 Fig. 4.6. The plasmid pPK5-FAD2 was tested by electrophoresis ………………………… 63 Fig. 4.7. A. PCR analysis of the hph gene inserted in genomic DNA of T. reesei Rut-C30 transformation; B. PCR analysis of the FAD2 gene inserted in genomic DNA of T. reesei Rut-C30 transformation ……………………………………………………………………. 64 Fig. 4.8. Expression of the recombinant FAD2 ……………………………………………. 65 Fig. 5.1. Site directed mutagenesis using double stranded megaprimers ………………….. 71 Fig. 5.2. A. The binary vectors pCAMBIA1301-FAD2; B. The binary vectors pBI121TMT; C. The binary vectors pCAMBIA1301-FAD2-TMT ……………………………….. 73 Fig. 5.3. PCR amplification of -PfTMT gene from Perilla frutescens genomic DNA ….. 76 Fig. 5.4. The plasmids were tested by electrophoresis …………………………………….. 78 Fig. 5.5. The plasmids were 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 IX LIST OF TABLES Table 2. Primers used in -PfTMT gene …………………………………………………. 14 Table 3. Primers used in -BoTMT gene ………………………………………………… 38 Table 4. Primers used in FAD2 gene ……………………………………………………. 56 Table 5. Primers used in γ-PfTMT and FAD2 gene ……………………………………… 70 X EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR NOMENCLATURE -BoTMT Brassica oleracea γ -tocopherol methyltransferase -PfTMT Perilla frutescens -tocopherol methyltransferase -TMT -tocopherol methyltransferase A. tumefaciens Agrobacterium tumefaciens AMT Agrobacterium-mediated transformation AS Acetosyringone ATMT Agrobacterium tumefaciens-mediated transformation bp Base pair CaMV 35S promoter Cauliflower mosaic virus 35S promoter DEPC Diethylpyrocarbonate DNA Deoxyribo Nucleic acid E. coli Escheria coli EDTA Ethylenediaminetetraacetic acid FAD Fatty acids desaturases GUS -glucuronidase hph Hygromycin phosphotransferase HPLC High-performance liquid chromatography IM Induction medium IPTG Isopropyl -D-1-Thiogalactopyranoside kDa Kilodalton LB Luria Bertani MCS Multiple cloning site Nos-ter Nopaline synthase terminator OCS Octopine synthase terminator OD Optical density PCR Polymerase chain reaction PDA Potato dextrose agar REMI Restriction 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 Trichoderma was first proposed as a genus by Persoon in 1794 [181], and several species of Hypocrea have been linked in 1865 [248]. Nevertheless, the different species allocate to the genus Trichoderma/Hypocrea were not easy to distinguish morphologically. It was intended to reduce taxonomy to Trichoderma viride species. Therefore, the development of a conception for classification was introduced until 1969 [194, 204]. After that, multiple new species of Trichoderma/Hypocrea were observed, and the genus already consisted of more than 100 phylogenetically determined species in 2006 [54]. In some condition, misidentification of certain species has appeared in recently reports, for Trichoderma harzianum which has been applied to many different species [126]. Nevertheless, it is difficult to certainly correct these misunderstanding without studying the strains originally used. Therefore, we summarized the data using the names as originally published. In recently years, by development of an oligonucleotide barcode (TrichOKEY) and a customized similarity search tool (TrichoBLAST) safe classification of new species was significantly make easy [278, 53, 113]. An additional useful tool for the new characteristic isolated Trichoderma species are phenotype microarrays, which permit for analysis of carbon utilization patterns for 96 carbon sources [17, 119, 55]. The prolongation application to elucidate diversity and geographical occurrence of Trichoderma/ Hypocrea follow on detailed demonstration of the genus in the world [203, 30, 95, 276]. The Index Fungorum database [277] at present lists 471 different names for Hypocrea species and 165 records for Trichoderma. Even so, many of these names have been innovated long before molecular methods for species classification were obtainable. They are likely to have get obsolete in the meanwhile. Nowadays, the International Subcommission on Trichoderma/Hypocrea lists 104 species [279], which have been identified at the molecular level. 72 species of Hypocrea have been characterized in temperate Europe [95]. However, a appreciable number of putative Hypocrea strains and Trichoderma strains, for which sequences have been saved in GenBank, are still without safe characterization [54]. Species of the genus get a broad array of pigments from brightly greenish yellow to reddish, even though some of them are also colorless. Likewise, conidial pigmentation varies from colorless to severally green shades and even also gray or brown. Other species of pigments classification within the genus is 2 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR not easy because of the narrow class of modification of the uncomplicated morphology in Trichoderma [71]. 1.1. Characteristics of Trichoderma spp. Trichoderma spp. are universal colonizers of cellulosic materials and can thus frequently be create wherever decaying plant material is availability [121, 95] besides in the rhizosphere of plants, where they can induce systemic resistance opposed pathogens [82]. The searching for potent biomass degrading enzymes and organisms also run to insulation of these fungi from unexpected material, example of cockroaches [267], marine mussels and shellfish [200, 199], or termite guts [230]. Trichoderma spp. are identified by quick growth, mainly bright green conidia [71]. In spite of the early indicated connection between Trichoderma and Hypocrea [248], this anamorph teleomorph correlation of Trichoderma reesei and Hypocrea jecorina was only demonstrated more than 100 years later [125]. However, because all attempts to cross the attainable strains of this species had unsuccessful, T. reesei was then named a clonally, asexual derivative of H. jecorina. Trichoderma species brought more than ten up to a sexual cycle was published [217]. The further studies on molecular evolution of this species go to the finding of a detail sympatric agamospecies Trichoderma parareesei [51]. First of all, the industrial consideration of T. reesei, the usefulness of a sexual cycle was a groundbreaking finding and paves the way for elucidation of sexual growing in other members of the genus now. Trichoderma spp. are greatly successful colonizers of their habitats. It is presented both by their efficient utilization of the substrate at hand and their emission ability for enzymes and antibiotic metabolites. They are know-how deal with such other environments as the dark and the fermentation bioprocess fermentor or shake flask besides the rich and diversified of tropical rainforest ecology. Therefore, they react to their environment by regulation of growth, conidiation, enzyme production, and so adapt their lifestyle to present conditions such as light. Trichoderma has for a long duration of studying about the light effection on its physiology and evolution from 1957 and much paralleled which of Phycomyces blakesleeanus [207]. In addition, growth conidiation, enzyme production, and secondary metabolite biosynthesis, the cellulase gene expression has affected in fungi [207]. By a study on carbon source utilization using phenotype microarrays in different condition of light, the direct connection between light responsive and metabolic processes was advanced supported [67]. The research about the light EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY-THESIS OF PHILOSOPHY DOCTOR 3 affection for the molecular basis discovered interconnections between the 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 its significance in industrial cellulase production, its genome contain the fewest many genes encoding cellulolytic and hemicellulolytic enzymes. Analysis and explanation for genomes of two important biocontrol species as Trichoderma atroviride and Trichoderma virens [275] is still in research now. As the result, the genomes of two important species are significantly better than that of T. reesei, they comprise roughly 2000 genes. This substantial difference in genome sizes in the physiology of these fungi will be interesting on research. Further studies are milestones for Trichoderma, which provided intriguing insights into their lifestyle, physiology, and adaptive evolution at the molecular level [25, 147, 212, 133, 218]. 1.2. Tools for genetic manipulation of Trichoderma Because of the industrial application of T. reesei, the developmental genetic toolkit for this fungus is the great expansion of the genus, although also studying with other species is unlimited by technical obstacles and most tools can also be used for all species with slight transformation. Modification of many species is acceptable, and different advance such as protoplasting [75], transformation of Agrobacterium-mediated [271], or transformation of biolistic [139] were applied. The range of selectable marker cassettes such as hygromycin [142] and benomyl resistance [182, 213], the Aspergillus nidulans amdS gene, which possible growth on acetamide as sole nitrogen matterial [178] besides the auxotrophic markers, pyr4 [75], arg2 [10], and hxk1 [79] accept of multiple modification construction, which is now facilitated by the available of a T. reesei strain with perturbed nonhomologous endjoining pathway [78]. Sequential gene deletions despite a restriction number of selectable markers became able to by the use of a blaster cassette comprising direct repeats for homologous recombination and excision of the marker gene selection [84]. Besides knockout strategies for functional analysis of transgenic, also through over-expression or antisense knockdown [195, 156, 211] was studied for Trichoderma, and RNAi has been expressed to function in T. reesei [18]. The results indicated that, a sexual cycle in T. reesei [217] more boosts the adaptability of T. reesei was applied both industrial and 4 EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY- THESIS OF PHILOSOPHY DOCTOR basic research. 1.3. Defense mechanisms and their exploitation Fortunate colonization of a given habitat by any organism is crucially dependent on its capability to defend its ecological niche and to thrive and prosper in spite of contention for light, space, and nutrients in many fungi. In particular, Trichoderma is masters of this competition [89, 83, 255]. Their defense reaction comprised enzymatic, as well as chemical weapons, which make Trichoderma spp. efficient mycoparasites, antagonists, and biocontrol agent specific that can be taken advantage by using Trichoderma spp. or the metabolites secreted by these fungi as biological control of plant disease caused by pathogenic fungi [229, 254, 253, 166]. By that, Trichoderma spp. plays a very important factor role in the three way fundamental interaction both the plant and the pathogen [140, 262]. 1.4. Trichoderma’s strategies for combat After discovering of Trichoderma lignorum (later found to be T. atroviride) in 1932 [259], studying on antagonistic properties of Trichoderma spp. progressed quickly. At present, the most essential species in this field are T. atroviride, T. harzianum, T. virens, and Trichoderma asperellum [13], hence T. reesei can a little seen as a model organism used because of the determined molecular biological methods and available recombinant strains [216]. Trichoderma spp. are possible control ascomycetes, basidiomycetes, and oomycetes [155, 13], and have recently been reported [42, 129, 72]. In my laboratory, our team used the DsRed2 gene as a reporter to test the applicability of the newly constructed vectors in T. reesei. We constructed expression vectors pWEF31-red by insertion of the DsRed2 gene. The vectors were introduced into T. reesei by Agrobacterium-mediated transformation. Positive transformants, F1 (pWEF31red transformation) was selected and then screened for further observation of DsRed2 expression in both the conidia and mycelia under a fluorescence microscope (Fig. 1.1). The results indicated that both vectors were capable of expressing an exogenous gene in fungi. In their defensive actions, Trichoderma spp. produce a range of hydrolytic enzymes [123, 257], proteolytic enzymes [116, 235, 31], ABC transporter membrane pumps in the interaction with different plant-pathogenic fungi [198], diffusible or volatile metabolites [29, 62], and other secondary metabolites [190] as active measures against their hosts or they succeed by their impairing growth conditions of pathogens [13]. Interestingly, the accomplishment of these actions is not only independent of the surrounding temperature [162], but also crucial for the use