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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 葉開溫 | |
| dc.contributor.author | Rajendran Senthil kumar | en |
| dc.contributor.author | 庫瑪 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:21:43Z | - |
| dc.date.available | 2009-10-28 | |
| dc.date.copyright | 2009-10-28 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-10-19 | |
| dc.identifier.citation | Beuning (1991) Annual Queenstown Molecular Biology Meeting,.
Broadway, R. M. (1996) Dietary proteinase inhibitors alter complement of midgut proteinases. Arch. Insec Biochem. Physiol. 32, 39-53. Bolter, A. J. & Jongsma, M. A. (1995) Colorado potato beetles (Leptinotarsa decemlineata) adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. Journal of Insect Physiology 41, 1071-1078. Bown, D. P., Wilkinson, H. S. & Gatehouse, J. A. (1997) Differentially regulated inhibitor-sensitive and insensitive protease gene from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene family. Insecbiochemistry and Molecular Biology, 27, 625-638 Chen, M. S., Feng, G., Zen, K. C., Richardson, M., Valdes-Rodriguez, S., Reeck, G. R. and Kramer, K . J (1992) α-Amylases from three species of stored grain Coleoptera and their inhibition by wheat and corn proteinaceous inhibitors. Insect Biochem Mol Biol.; 22:261–268. Ding, L. C., Hu, C. Y., Yeh, K. W. and Wang, P. J. (1998) Development of insect-resistant transgenic cauliflower plants expressing the trypsin inhibitor gene isolated from local sweet potato. Plant Cell Reports 17: 854-860 Duan, X., Li, X., Xue, Q., Abo-El-Saad, M., Xu, D. and Wu, R. (1996) Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotechnology 14: 494-498. Gatehouse, A. M. R. (1999) Biotechnological applications of plant genes in the production of insect resistant crops. In S.L. Clement and S.S. Quisenberry (eds.), Global Plant Genetic Resources for Insect Resistant Crops, London, CRC Press, 263-280. Hilder, V. A., Gatehouse, A. M. R., Sheerman, S. E., Barker, R. F. and Boulder, D. (1987) A novel mechanism of insect resistance engineered into tobacco. Nature. 330:160–163. Hoffmann, M. P. , F. G. Zalom , L. T. Wilson , J. M. Smilanick , L. D. Malyj , J. Kiser , V. A. Hilder , and W. M. Barnes . (1992) Field evaluation of transgenic tobacco containing genes encoding Bacillus thuringiensis δ-endotoxin or cowpea trypsin inhibitor:efficacy against Helicoverpa zea (Lepidoptera:Noctuidae). J. Econ. Entomol. 85: 2516–2522. Irie, K., Hosoyama, H., Takeuchi, T., Iwabuchi, H., Watanabe, H., Abe, M. and Arai, S. (1989) Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases. Plant Mol. Biol., 30: 149–157. Jongma, M. A., Bakker, P. L., Peters, J., Bosch, D. & Stiekem, W.J. (1995) Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc. Nac.Aca. Scie USA 92, 8041-8045. Jongsma, M. A. & Bolter, C. (1997). The adaptation of insects to protease inhibitors. J. Inse.Phys 43, 885-895. Johnson, R. and Ryan, C. A. (1990) Wound-inducible potato inhibitor II genes: enhancement of expression by sucrose. Plant Mol. Biol., 14: 527–536. Lee, J. S., Brown, W. E., Graham, J. S., Pearce, G., Fox, E. A., Dreher, T. W., Ahern, K. G., Pearson, G. D. and Ryan, C. A. (1986) Molecular characterization and phylogenetic studies of a wound-inducible proteinase inhibitor I gene in Lycopersicon species. Proc. Natl. Acad. Sci. USA, 83: 7277–7281. Leple, J.C., Bonade-Bottino, M. Augustin, S., Pilate, G., Le Tan, V. D., Deleplanque, A., Cornu, D. and Jouanin, L. (1995) Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol. Breed., 1: 319–328. Maeshima, M., Sasaki, T. and Asahi, T. (1985) Characterization of major proteins in sweet potato tuberous roots. Phytochemistry 24: 1899–1902. McManus, M. T., White, D. W. R. and McGregor, P. G. (1994) Accumulation of chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgen. Res., 3: 50–58. Sane VA, Nath P, Aminuddin and Sane, P. V. (1997) Development of insect resistant.transgenic plants using plant genes expression of cowpea trypsin inhibitor in transgenic tobacco plants. Curr. Sci. 72: 741-747. Wu, Y., Llewellyn, D., Mathews, A., Dennis, E.S., (1997) Adaptation of Helicoverpa armigera (Lepidoptera:Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Mol Breeding.;3:371–380 Xu, R., Goldman, S., Coupe, S., and Deikman, J. (1996). Ethylene control of E4 transcription during tomato fruit ripening involves two cooperative cis elements .Plant Mol Biol 31, 1117-1127. Xu, Y., Chang, P., Liu, D., Narasimhan, M.L., Raghothama, K.G., Hasegawa, P.M., and Bressan, R.A. (1994) Plant defense genes are syngergistically induced by Ethylene and Methyl jasmonate. Plant Cell 6, 1077-1085. Yeh, K. W., Juan, R. H. and Su, J. C. (1991) A rapid and efficient method for RNA isolation from plants with high carbohydrate content. Focus 13: 102–103. Yeh, K.W., Chen, J.C., Lin, M.I., Chen, Y. M., and Lin C.Y. (1997). Functional activit of sporamin from sweet potato (Ipomoea batatas Lam): a tuber storage protein with trypsin inhibitory activity. Plant Mol. Biol 33, 565-570. . Yeh, K.W., Lin, M.I., Tuan, Y.M., Chen, C.Y., Kao, S.S. (1997) Sweet potato (ipomoea batatas) –trypsin inhibitor expressed in transgenic tobacco plants confers resistance against Spodoptera litura , Plant Cell Rep 16 (1997), pp 696-699. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45466 | - |
| dc.description.abstract | 甘薯的sporamin（胰蛋白酶抑制劑）基因已證實具有抗蟲效果，而芋頭中的cystatin（胱胺酸蛋白酶抑制劑）基因亦已證實具有抗線蟲以及抗黴菌等功效，本論文之主要目的是結合兩個基因轉殖於作物中，以創造一個具有抗蟲、抗黴菌及抗細菌之多重抗性 (mutiple resistance）的生技作物 (biotechnological crop）。
利用本實驗室所研發的合成啟動子（synthetic promoter), pMSPOA 驅動上述兩個蛋白酶抑制劑基因，然後轉殖於菸草中，經過hygromycin抗生素抗性篩選得到轉殖株後，利用南方轉印、北方轉印以及蛋白質活性分析確認兩個轉基因在菸草中之有效表達後，進行抗玉米穗蟲（Helicoverpa armigera Hubner）、軟腐病菌（Erwinia carotovora）及青枯病菌（Ralstonia solanacearum）之抗性試驗，實驗結果證實此轉基因對玉米穗蟲具有致死與延滯生長發育之效果，LT50（lethal 致死時間）分別在3.7天至5.8天之間，明顯較對照組減少。對於抗細菌及抗黴菌亦有明顯效果，細菌感染後之繁殖量，僅有對照組之25%，因此，此研究證實兩種蛋白脢抑制劑的共同效應確實可賦予作物具有抗蟲、抗細菌及抗黴菌的多重抗性功效。 | zh_TW |
| dc.description.abstract | Protease inhibitors provide a promising means of engineering plant resistance against attack by insects and pathogens. The major aim of this thesis was to develop a trait which confers dual broad spectrum resistance against insects and phytopathogens. On the research front, a variety of conventional and more novel methods have been employed to introduce multiple genes into plants, but all techniques suffer from certain drawbacks. This study was undertaken to determine the effect of two protease inhibitor genes under the control of two wound inducible synthetic promoter, pMPSOA, in tobacco W38. To achieve this purpose, Sporamin (trypsin inhibitor) from sweet potato and CeCPI (phytocystatin) from taro were stacked in a binary vector, using pMSPOA (a modified sporamin promoter) to drive both genes. Transgenic tobacco plants containing both the genes were selected for resistance to hygromycin, and confirmed by PCR. Furthermore, Northern blot analysis revealed that stable expression of transgenes. Progeny analysis of T0 plants confirmed the inheritance of transgene to the next generation. The polyphagous moth Helicoverpa armigera (Hubner) is one of the world’s most important agricultural pests. Larval of Helicoverpa armigera that ingested tobacco leaves either died or showed delayed growth and development relative to control larvae. Furthermore, second instar H. armigera larvae reared on transgenic lines showed reduced LT50 values of respectively, as compared to larvae collected from the wild type. Transgenic tobacco overexpressing the stacked genes exhibited also strong resistance against phytopathogens such as Erwinia carotovora, Ralstonia solanacearum, Pythium aphanidermatum, Alternaria alternata. Maceration of leaf tissue was analyzed from transgenic and wild type tissue revealed that bacterial populations were significantly reduced compared to wild type. Microscopic observation in the wild type and transgenic line showed that transgenic line exhibited limited the growth of hyphae and leaf remains green. Thus, stacking protease-inhibitor genes, driven by the wound and pathogen responsive pMSPOA promoter, is an effective strategy for engineering crops to obtain broad-spectrum resistance against insects and phytopathogens. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:21:43Z (GMT). No. of bitstreams: 1 ntu-98-D94b42006-1.pdf: 16283714 bytes, checksum: 9e680a2ae53d149c8d6682b523ec7899 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | Abstract 16
Chapter-1 20 General introduction 21 1.1Natural occurring protease inhibitors 21 1.2 Protease inhibitors as plant protective molecule 22 1.3 Plant proteinases 26 1.4 Plant proteinase inhibitors (PI’s) 27 1.5 Physiological role of proteinase inhibitors in plants 28 1.6 Role of protease inhibitors in defense mechanism 29 1.7 Effect of proteinase inhibitors on insect feeding 30 1.8 Crop protection against pests 35 1.9 Scope of the thesis 36 1.10 References 37 Chapter- 2 45 General methodology 46 2.1 General methods in nucleic acid amplification, cloning and sequencing 46 2.1.1 PCR amplification 46 2.1.2 Restriction enzyme digestion DNA 46 2.1.3 Alkaline dephosphorlation of 5’end 46 2.1.4 DNA Ligation 47 2.1.5 Preparation of heat-shock competent E. coli 47 2.1.6 Preparation of electro-competent Agrobacterium 48 2.1.7 Transformation of bacteria with recombinant plasmids 2.1.7.1 Escherichia coli 49 49 2.1.8 Isolation and purification of plasmid DNA 49 2.1.9 Preparation of bacterial glycerol’s 50 2.2 General methods for nuclei acid purification of plant cells 50 2.2.1 Purification of total DNA from tobacco cells 50 2.2.2 Purification of total RNA from tobacco leaf tissue 52 2.3 Gel electrophoresis 52 2.3.1 RNA gel electrophoresis and hybridization protocol 53 2.3.2 Extraction and purification of DNA from agarose gels 53 2.3.3 Northern transfer of RNA from agarose to gel nitrocellulose membrane 53 2.3.3.1 Radioactive DNA probes 54 2.4 Protein procedures 55 2.4.1 Extraction of proteins from plant material 55 2.4.2 Estimation of protein concentration 56 2.4.3 Polyacrylamide gel electrophoresis 56 2.4. 4 Trypsin inhibitory in-gel activity assay 57 2.4.5 Quantitative analysis of trypsin inhibitory activity 57 2.4.6 In-gel papain activity assay 59 2.4.7 Quantitative analysis of cysteine protease inhibitory activity 60 2.5 Generation of transgenic tobacco 62 2.5.1 Progeny analysis of transgenic tobacco plants 62 2.6.1 Leaf infiltration and CFU count 63 2.6.1.1 Ralstonia solnacearum 64 2.6.2 Fungal spore extraction 64 2.6.3 Vector construction 64 Chapter-3 Generation of transgenic tobacco harboring stacking gene construct 65 66 Abstract 66 3.1 Introduction 67 3.2 Sporamin 68 3.3 Taro cystatin 71 3.4 Synthetic promoter 72 3.5 Gene stacking 78 3.6 Materials and methods 80 3.6.1 Construction of binary vector 80 3.7 Generation of transgenic tobacco 80 3.8 Progeny analysis of transgenic tobacco plants 81 3.9 Genomic PCR and hygromycin resistance 82 3.10 Northern blot analysis 83 3.11 Results and discussion 85 3.12 References 87 Chapter -4 99 Overexpression of stacking gene confers enhance resistance against insect Helicoverpa armigera 100 Abstract 100 4.1 Introduction 100 4.2 Significance and objective of the research 106 4.3 Materials and methods 107 4.3.1 Plant material 107 4.3.2 Insect strain 107 4.3.3 Insect bioassay 107 4.3.4 Trypsin inhibitor assay 108 4.3.5 Percentage inhibition 108 4.3.6 Insect feeding assay on field trials 109 4.3.7 Statistical analysis 109 4.4 Results and discussion 110 4.5 References 113 Chapter -5 121 Pyramiding protease inhibitor genes in transgenic tobacco exhibit enhance resistance to phytopathogens 122 Abstract 122 5.1 Introduction 123 5.2 Phytocystatin 124 5.3 Biology of damping- off caused by Pythium aphanidermatum 128 5.4 Biology of soft rot disease caused by Erwinia carotovora 131 5.5 Significance and objective of research 133 5.6 Materials and methods 135 5.6.1 Plant material 135 5.6.2 Bacterial and fungal strains 135 5.6.2.1 Bacterial strain 135 5.6.2.2 Fungal strain 135 5.6.3 Antimicrobial assay 135 5.6.3.1 Bacterial inoculations 135 5.6.3.2 Fungal inoculation 136 5.6.4 Protease inhibitor assay 136 5.6.5 Percentage of inhibition 137 5.6.6 Histochemical staining and microscopy 137 5.6.7 Statistical analysis 138 5.7 Results and Discussion 139 5.8 References 146 Chapter -6 General discussion and future perspectives List of Tables 171 172 Table 3.1 Germination test and χ2 analysis for progeny seedlings obtained from Hygromycin resistance T0 plants……………………………….. 92 Table 4.1 Mortality effects (LT10, LT50 and LT90), percent survival rate and weight gains of 2nd instar larvae Helicoverpa armigera fed on T0 progeny from transgenic tobacco lines……………………………………………… 116 Table 4.2 Mortality effects (LT10, LT50 and LT90), percent survival rate and weight gains of 2nd instar larvae Helicoverpa armigera fed on T1 progeny from transgenic tobacco lines……………………………………………….. 117 Table 5. 1 Lesion size in wild type and T0 transgenic tobacco leaves from 1-3 days post inoculation. Averages of four repetitions are given in mm ……………………………………………………………………………………………….. 151 Table 5.2 Scale for assessment of resistance in tobacco plants to damping-off caused by Pythium aphanidermatum (modified from Liu et al., 1996)…………………………………………………………………………………………… 152 Table 5.3 Comparison of disease severity and disease incidence between wild type and T0 transgenic tobacco lines inoculated with Pythium aphanidermatum…………………………………………………………………… 153 Table 5.4 Comparison of disease severity and disease incidence between wild type and T0 transgenic tobacco lines inoculated with Pythium aphanidermatum……………………………………………………..…... 154 | |
| dc.language.iso | en | |
| dc.title | 利用轉殖菸草探討Pyramiding蛋白質酶抑制劑對廣泛昆蟲及
病源之抗性 | zh_TW |
| dc.title | Genetically pyramiding protease inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco (Nicotiana tabaccum) W38 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 楊文彬,陳志成,林秋榮,曾顯雄,黃鵬林,鄭秋萍 | |
| dc.subject.keyword | 蛋白脢抑制劑,甘薯胰蛋白酶,抑制蛋白,芋頭胱胺酸蛋白酶,抑制蛋白,廣泛昆蟲及病源之抗性,轉殖菸草, | zh_TW |
| dc.subject.keyword | Protease inhibitor,sporamin,tarocystatin,broad-spectrum resistance,transgenic tobacoo, | en |
| dc.relation.page | 180 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-10-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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