Poplar as the Woody Plant Model Species for Science and Industrial Applications: Overviews, Problems and Perspectives 著者 西口 満 year 2016 その他のタイトル 科学と産業利用に貢献する木本モデル植物としての ポプラ：概要、問題、展望 学位授与大学 筑波大学 (University of Tsukuba) 学位授与年度 2015 報告番号 12102甲第7781号 URL http://hdl.handle.net/2241/00143935 Poplar as the Woody Plant Model Species for Science and Industrial Applications: Overviews, Problems and Perspectives January 2016 Mitsuru NISHIGUCHI Poplar as the Woody Plant Model Species for Science and Industrial Applications: Overviews, Problems and Perspectives A Dissertation Submitted to the Graduate School of Life and Environmental Sciences, the University of Tsukuba in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biotechnology (Doctoral Program in Bioindustrial Sciences) Mitsuru NISHIGUCHI Table of Contents Table of Contents ............................................................................................................... i Abbreviations .................................................................................................................. iii Abstract ............................................................................................................................. 1 Chapter 1 Introduction .................................................................................................. 6 1.1 Taxonomy and distribution ............................................................................... 6 1.2 Industrial applications ...................................................................................... 7 1.3 New industrial applications .............................................................................. 9 1.4 Environmental applications ............................................................................ 10 1.5 Biology of poplars .......................................................................................... 12 1.6 Biodiversity and conservation ........................................................................ 14 1.7 Stress physiology ............................................................................................ 15 1.8 Genetics and genomics ................................................................................... 19 1.9 Genetic engineering ........................................................................................ 22 1.10 Populus nigra ................................................................................................. 28 1.11 Objectives ....................................................................................................... 31 Chapter 2 Elucidation of biological response to ionizing radiation in Populus nigra 34 2.1 Introduction .................................................................................................... 34 2.2 Materials and methods .................................................................................... 36 i 2.3 Results ............................................................................................................ 41 2.4 Discussion ....................................................................................................... 49 2.5 Conclusion ...................................................................................................... 54 Chapter 3 An improved transformation system for Populus nigra ............................. 56 3.1 Introduction .................................................................................................... 56 3.2 Materials and methods .................................................................................... 57 3.3 Results and discussion .................................................................................... 62 3.4 Conclusion ...................................................................................................... 67 Chapter 4 Conclusion .................................................................................................. 68 4.1 Conclusions and perspectives ......................................................................... 68 4.2 New plant breeding techniques (NPBT) ......................................................... 75 4.3 Risk assessment for transgenic poplars as living modified organisms ........... 76 4.4 The role of the poplar in science and its applications..................................... 80 Acknowledgements ........................................................................................................ 83 References ...................................................................................................................... 84 Publication lists ............................................................................................................ 107 Tables ............................................................................................................................ 108 Figures .......................................................................................................................... 120 ii Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid 8-oxoG 7,8-dihydro-8-oxoguanine BAP 6-benzylaminopurine bp base pairs Cas9 CRISPR-associated protein 9 CRISPR clustered regularly interspaced short palindromic repeats DDBJ DNA Data Bank of Japan DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EGFP enhanced green fluorescent protein EST expressed sequence tag FRET fluorescent resonance energy transfer GFP green fluorescent protein GUS β-glucuronidase Gy gray (the SI unit of absorbed dose) ha hectare (although not an SI unit, the commonly used metric unit of an area of land) LEA late embryogenesis abundant LMO living modified organism MES 2-(N-morpholino)ethanesulfonic acid MSBS Murashige and Skoog basal salts iii MSV Murashige and Skoog vitamin NCBI National Center for Biotechnology Information NPBT new plant breeding techniques NPTII neomycin phosphotransferase II PAR photosynthetically active radiation PCR polymerase chain reaction PHB poly-3-hydroxybutyrate RIM root-induction medium ROS reactive oxygen species RT-PCR reverse transcription PCR RT-qPCR reverse transcription quantitative real-time PCR SAM shoot apical meristem SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SIM shoot-induction medium TALEN transcription activator-like effector nuclease ZFN zinc finger nuclease iv Abstract Poplar trees (from the genus Populus) have been utilized for industrial and environmental applications such as wood production, biomass feedstock, and environmental conservation and restoration throughout the ages. The practical importance and the biological properties of poplars have enhanced many fields of study such as physiology, ecology, molecular biology, genetic engineering, forestry, chemistry, wood science and environmental science. Thereby, poplars have been recognized as a woody plant model species. For the advancement of science and applications of poplars as the woody plant model species, it was thought to be important to pioneer new fields of study on poplars and develop new technologies for the study. Thus, the objectives of this study are to elucidate the biological response to ionizing radiation as an abiotic stress in Populus nigra and improve the transformation system for P. nigra. Ionizing radiation is an environmental stress; however, little is known about its effects on poplars or the tolerance mechanism of woody plants to ionizing radiation. Therefore, in this study, we investigated the biological effects of γ-rays and the tolerance mechanism of P. nigra as a model species. Poplar plantlets irradiated with 50– 100 gray (Gy) of γ-rays were able to survive, although growth inhibition or morphological abnormalities were observed. A total dose of 200–300 Gy of γ-rays killed almost all poplar samples. A high dose of γ-rays also inhibited the regeneration of the shoots and roots. Comet assays demonstrated that the γ-rays damaged the nuclear DNA of the irradiated cells. To characterize the tolerance mechanism to ionizing radiation stress, six DNA repair-related cDNAs, PnLIG4, PnKU70, PnXRCC4, 1 PnRAD51, PnPCNA, and PnOGG1, were isolated and structurally analyzed. The expression of PnLIG4, PnKU70, PnXRCC4, PnRAD51, and PnPCNA was upregulated by γ-irradiation in a dose-dependent manner, while PnOGG1 was downregulated. The expression of PnLIG4, PnKU70, and PnRAD51 was also upregulated by the treatment of a DNA cleavage agent. Accordingly, it is concluded that the gene expression of PnLIG4, PnKU70, and PnRAD51 was directly induced by DNA strand breaks. A highly efficient transformation system for P. nigra was required to further study the DNA repair-related genes that were regulated in response to ionizing radiation stress. To improve the transformation system for P. nigra, it was aimed to construct a new binary vector, to shorten the time required and increase the efficiency of transformation. The newly designed binary vector has 11 restriction enzyme sites for DNA cloning and demonstrated higher resistance to selective antibiotics. P. nigra was transformed by Agrobacterium harboring the new vector fused to the enhanced green fluorescent protein gene. Successful transformation was confirmed by polymerase chain reaction (PCR), fluorescence microscopy, immunoblotting, Southern blotting, and resistance to kanamycin and G418. The period of transformation was shortened to a minimum of approximately 4 months by the direct regeneration of transgenic shoots from the Agrobacterium-infected stems. In the co-cultivation