WO2006109424A1 - Adn codant pour une proteine ayant pour fonction de former et de reguler la paroi cellulaire de fibre cellulosique dans un tronc d'arbre et leurs and promoteurs - Google Patents

Adn codant pour une proteine ayant pour fonction de former et de reguler la paroi cellulaire de fibre cellulosique dans un tronc d'arbre et leurs and promoteurs Download PDF

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WO2006109424A1
WO2006109424A1 PCT/JP2006/305402 JP2006305402W WO2006109424A1 WO 2006109424 A1 WO2006109424 A1 WO 2006109424A1 JP 2006305402 W JP2006305402 W JP 2006305402W WO 2006109424 A1 WO2006109424 A1 WO 2006109424A1
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dna
seq
trunk
protein
amino acid
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PCT/JP2006/305402
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English (en)
Japanese (ja)
Inventor
Shigeru Sato
Nanae Yamada
Shiho Nakamoto
Takashi Hibino
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Oji Paper Co., Ltd.
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Priority to JP2007512437A priority Critical patent/JPWO2006109424A1/ja
Publication of WO2006109424A1 publication Critical patent/WO2006109424A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates to DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk, a promoter DNA thereof, and use thereof.
  • the present invention also relates to DNA encoding plant transcription factors, promoter DNA fragments, and uses thereof.
  • wood fibers Most of the woody biomass in the trunk is composed of tissue cells called wood fibers.
  • the specific gravity of the wood fiber structure, cell wall structure and components (cellulose, hemicellulose, lignin), etc. are the main factors that determine the properties of wood fibers.
  • Non-Patent Document 1 Non-Patent Document 2
  • the fluctuation phenomenon due to the ground height is the vertical fluctuation of the material in the trunk
  • the fluctuation phenomenon by the species Is known as inter-species variation.
  • cellulose increases in cell wall components, while lignin and hemicellulose decrease.
  • Non-Patent Documents 1 to 3 Although details are unknown, it is thought that the difference in the properties of the wood fibers formed is caused by the difference in cell wall synthesis in each wood fiber-forming tissue. The gene groups responsible for cell wall synthesis with such different properties have not been identified. [0007] In recent years, gene analysis studies at the genome level using model plants such as herbs have been actively conducted. In herbs, only a few wood fibers are formed in specific organs at specific times.
  • Non-patent Document 4 Fibrous fiber cells differentiate from the formation layer tissue and are called wood fibers, whereas herbaceous fiber cells mainly divide the parenchyma force between the vascular bundles, and the inter-bundle fibers ( It is called interfascicular fiber).
  • wood fibers Fibrous fiber cells differentiate from the formation layer tissue and are called wood fibers
  • herbaceous fiber cells mainly divide the parenchyma force between the vascular bundles, and the inter-bundle fibers ( It is called interfascicular fiber).
  • interfascicular fiber the interfascicular fiber
  • Plant fiber formation-related genes and their regulatory factors cannot be distinguished and identified simply by homology search or motif search with known regulatory factors.
  • many enzymes are involved in the synthesis pathway of cellulose and lignin, which are the main components of wood fiber cell walls, and these genes form a large family in the plant genome (Non-patent Document 5).
  • Non-patent Document 5 Furthermore, within the family 1, there are those related to the synthesis of the primary cell wall and those related to the synthesis of the secondary cell wall, and the functioning places and roles are divided in various ways. Sequence motifs and homology search capabilities simply indicate high homology and cannot identify roles. For example, about 40 cellulose synthases (CesA) involved in cellulose synthesis are expected in Arabidopsis thaliana!
  • RSW1 Non-patent document 6
  • Ces A essential for cellulose synthesis of the primary wall
  • IRX3 Non-patent document 7
  • Ce sA essential for the synthesis of secondary wall cellulose
  • a homologous search factor NtLIMl identified in tobacco as a regulator of the biopropanoid biosynthesis system represented by lignin contained in wood fiber is known (Searching at http://www.ncbi.nlm.nih.gov/blast/) shows high homology with many LIM proteins. Even rat LI M proteins that do not form wood fibers are hit with a high homology score. In Arabidopsis, at least 10 LIM proteins and a high score (56-87%) are hit, but it is unclear which LIM protein is involved in the control of the lignin synthesis system in Arabidopsis. Thus, since the role of a regulatory factor cannot be identified only by homology with known regulatory factors, detailed expression analysis and the like must be performed. However, there is no knowledge of comprehensive expression analysis of regulatory factors in Kashiwagi.
  • a variety of wood fiber cell wall synthesis genes are expected to exist in woody plants, which are based only on the difference in the genes that act during fiber formation between grass and wood. For example, because the morphology of leaves and trunks is different between young and middle age, it is expected that multiple tree fiber cell wall synthetic genes that function according to age will function. Also, as mentioned above, vertical fluctuations due to drought height and fluctuations among varieties due to varieties occur, so that a plurality of tree fiber cell wall synthetic genes that are different within the same individual or between species function uniquely. ! I think. These tree fiber cell wall synthetic genes only show high homology with each other in sequence, and it is not possible to distinguish which belongs to which level of wood fiber cell wall synthetic genes. In order to make this distinction, detailed expression analysis at the wood fiber formation site is necessary. However, there is no such knowledge.
  • Non-Patent Documents 7 and 9 Also in woody plants, research on the analysis of silkworms at the genome level using poplar and pine has been conducted (Non-Patent Documents 7 and 9). However, the materials used are separate organs and tissue units such as roots and leaves' formation layers, and only report gene expression information in them. There is no comprehensive knowledge at the genome level regarding gene groups involved in various levels of wood fiber cell wall synthesis, their promoter information, and expression information by conducting expression analysis in different types of tree fiber-forming tissues on the ground.
  • the growth state of the afforestation tree is judged by appearance, and in order to know the quality as a pulp material, a trunk that is a pulp material by a chemical method after cutting is used.
  • Wood fiber analysis material analysis
  • As for the growth state it relies on experience and intuition, so it cannot grasp problems such as lack of nutrients that cannot be distinguished by appearance. And since the material is only divided after logging, what is the power of tree fiber formation (material) in planted trees during growth, what kind of tree fiber will be formed in the future? There is no policy of working (fertilization, thinning, etc.) to produce good materials.
  • trunk fiber formation material during growth is very important in consideration of plantation operations, selective breeding, and securing high-quality papermaking raw materials.
  • Patent Document 1 Patent No. 3444191
  • Non-Patent Document 1 Ken Shimaji ⁇ Akuji Sudo ⁇ Hirada Hiroshi "Wood Organization", Morikita Publishing, 1976, 111-21 Five
  • Non-Patent Document 2 Satoshi Furuno 'Osamu Sawabe's "Wood Science Course 2 Organization and Material", Kaiseisha, 1994, 109 -137
  • Non-Patent Document 3 Wood Science and Technology. 2001; 35: 229-243.
  • Non-Patent Document 4 Plant Physiology, 2001, 126: 477-479.
  • Non-Patent Document 5 Plant Physiology, 2000, 124: 495-498.
  • Non-Patent Document 6 Science 1998, 279: 717-720.
  • Non-Patent Document 7 Plant Cell 1999, 11: 769-780.
  • Non-Patent Document 8 Masami Iwabuchi ⁇ Kazuo Shinozaki “Dynamics of Plant Genomic Function” Syupurin Gaichi 'Fairark Tokyo, 2001, 1-34
  • Non-Patent Document 9 GenomeBiol. 2002; 3 (12): REVIEWS1033.
  • Non-Patent Document 10 Proc Natl Acad Sci U S A 2001 Dec 4; 98 (25): 14732-7.
  • woody biomass varies depending on the fineness and shape of wood fibers. By artificially controlling the formation of fine fibers and shapes of the wood fibers, woody biomass can be produced as an industrial raw material effectively and efficiently. In addition, the world still depends on fossil resources such as large quantities of oil and natural gas. Now, in order to change this situation with new technology, efficient utilization of trees as woody biomass is the key to power. In overseas countries, the establishment of technology for effective utilization of Kashiwagi biomass is positioned as an important issue at the beginning of this century. In fact, coniferous pine, broad-leaved poplar in the United States, spruce and poplar in Canada, and poplar in Scandinavia are the target species. In order to contribute to more effective and efficient production of woody biomass on a global scale, it is highly necessary to identify genes that control the formation of wood fiber cells in plants.
  • An object of the present invention is to provide a DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk, a DNA encoding a plant transcription factor, a promoter DNA thereof, a plasmid containing these, and a plasmid thereof Plant cells transformed by To provide microorganisms or plants.
  • Another object of the present invention is to provide a protein having a function of forming a tree fiber cell wall of a plant tree trunk and a DNA encoding a plant transcription factor as a test marker for a plant trunk tree part.
  • the present inventors use the prepared eucalyptus EST database to extract a group of genes involved in cell wall synthesis in a eucalyptus tree fiber-forming tissue by microarray analysis, and to determine the height of the ground. Expression variation was examined. As a result, we identified a cell wall synthetic gene group of Eucalyptus tree fiber-forming tissue whose expression is preferentially varied depending on the difference between the above-ground species. Furthermore, their promoter DNA was obtained. It is considered that the above gene group and promoter DNA can be used to control cell wall synthesis and morphogenesis of wood fiber-forming tissues and to control gene expression specific to wood fiber.
  • the gene group of the present invention has an expression level on the xylem side compared to the phloem side in the trunk base and central trunk of Eucalyptus genus Camaldrensis.
  • the expression level is decreased on the xylem side compared to the phloem side, and in the middle trunk, the xylem is compared with the phloem side.
  • the expression level increased on the xylem side compared to the phloem side, and in the trunk of the Eucalyptus globules, the xylem compared to the phloem side.
  • DNA encoding a protein having a function of forming a eucalyptus trunk tree fiber cell wall obtained by the present invention as well as techniques for comprehensively controlling them and expression information.
  • various characteristics such as high cellulose, low lignin, thick cell walls, thin ones, long fiber lengths, short ones, etc. depending on the characteristics of the new transgenic eucalyptus varieties obtained using these genes.
  • Quantitative and qualitative changes are expected.
  • the present invention relates to DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk, a promoter DNA fragment thereof, and use thereof, which provide the following [1] to [18] It is.
  • DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk according to any one of the following (a) to (e).
  • the expression level of at least one DNA described in the following (a) to (e) is detected, and the phloem side and xylem side are detected.
  • a method for examining the growth state of a stem trunk of a plant comprising a step of comparing the expression levels of the DNA.
  • SEQ ID NO: DNA comprising the nucleotide sequence set forth in any of 1-30 or 32-34
  • a promoter DNA comprising the nucleotide sequence of any of the following (a) to (c).
  • SEQ ID NO: 93- The nucleotide sequence according to any one of LOO
  • a recombinant vector comprising the DNA according to any one of [1] to [3] or [10] to [12]
  • the present inventors examined the transcriptional factors involved in eucalyptus tree fiber cell wall formation by microarray analysis, and the expression variation due to differences in the height and species of the ground. It was. As a result, we identified a group of transcription factors that regulate the expression of Eucalyptus tree fiber cell wall-forming genes, whose expression changes predominantly depending on the height of the ground. Furthermore, their promoter DNA was obtained.
  • the gene group of the present invention has an expression level on the xylem side compared to the phloem side in the trunk base and central trunk of the Eucalyptus genus Camaldrensis.
  • the expression level rises on the xylem side compared to the phloem side, and the phloem side and xylem on the trunk base Genes whose expression level is the same on the side of the eucalyptus genus, and that the expression level on the xylem side is higher than that on the phloem side of the eucalyptus genus Camaldrensis and Eucalyptus globules.
  • the expression (level) information of this gene group can be used for quantitative and qualitative advance prediction of plant growth state, wood fiber formation state, and material.
  • appropriate operations weeding, thinning, etc.
  • DNA encoding eucalyptus transcription factors obtained by the present invention, as well as technology and expression information for overall control of them.
  • these genes are used as one result.
  • High cellulose, low lignin, thick cell walls, thin cells, long fiber lengths, short ones Various quantitative and qualitative changes are expected.
  • it is expected to be used to distinguish the ground height, species, and other materials and growth status based on the expression information.
  • the present invention provides the following [1] to [17] regarding DNA encoding plant transcription factors, promoter DNA fragments thereof, and use thereof.
  • the expression level of at least one DNA described in the following (a) to (e) is detected, and the phloem side and xylem side are detected.
  • a method for examining the growth state of a stem trunk of a plant comprising a step of comparing the expression levels of the DNA.
  • SEQ ID NO: consisting of the base sequence described in any one of 101 to 107 or 109 to 111 DNA that hybridizes with DNA under stringent conditions
  • the expression level of at least one DNA described in the following (a) to (e) is detected, and the phloem side and xylem side are detected.
  • a method for examining the growth state of a stem trunk of a plant comprising a step of comparing the expression levels of the DNA.
  • a promoter DNA comprising the nucleotide sequence of any of the following (a) to (c).
  • a recombinant vector comprising the DNA according to any one of [1] to [3], [10] or [11]
  • the DNA of the present invention can be used as a test marker for trunk stem parts of plants.
  • the DNA of the present invention it is possible to examine the growth state of trunk trunks of trees, the formation state of wood fibers, the quality of norp quality, and the selection of plants having useful traits.
  • the present invention provides a DNA encoding a protein having a function of forming a tree fiber cell wall of a plant trunk (a protein involved in cell mouth synthesis and a protein involved in lignin synthesis) and a promoter DNA.
  • DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk in the present invention can be used for control of plant fiber fiber cell formation.
  • the promoter DNA of the DNA can be used to control expression specific to wood fiber tissue or wood fiber formation time.
  • the expression (level) information of DNA encoding a protein that functions to form the tree fiber cell wall of plant trunks is used for quantitative and qualitative prediction of plant growth, fiber formation, and materials. It is considered possible. Therefore, by using these, artificial modification of tree fiber cell morphogenesis, specific expression of protein's tree fiber cells, expression of plant growth state, state of fiber formation, amount of material • It can be used for qualitative advance prediction. As a result, the effectiveness of pulp production It will be possible to increase efficiency, reduce costs, and produce paper with new characteristics.
  • Controlling wood fiber formation in plants has various important implications in the fields of industry and agriculture. For example, modification of the wood fiber properties of Eucalyptus genus Camaldrensis is significant in terms of improving the fiber properties of fiber raw materials such as pulp by increasing the fiber length. In addition, increasing the cellulose and hemicellulose content of Eucalyptus globulae species increases the yield of pulp and the like, and improves the cooking efficiency, which is significant in terms of economy and profitability.
  • the plant from which the DNA of the present invention is derived is not particularly limited, for example, useful crops (including forage crops) such as cereals, vegetables, fruit berries, fiber raw material plants such as pulp, ornamental plants, etc.
  • useful crops including forage crops
  • examples include plants.
  • the plant such as eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower,
  • Examples include ivy, orange, grape, peach, pear, apple, tomato, Chinese cabbage, cabbage, Japanese radish, carrot, cabbage, cucumber, melon, parsley, orchid, chrysanthemum, lily and saffron.
  • examples of the DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree include the DNAs described in any of (a) to (e) below.
  • stringent hybridization conditions include conditions of 60 ° C, standing at room temperature in an O.lxSSC solution, or an equivalent stringency of no ibrida.
  • An example is the condition. Under such conditions, DNA that hybridizes with DNA having the nucleotide sequence ability described in any one of SEQ ID NOs: 1 to 34 can be isolated.
  • DNA is extracted from a plant, a gene library is constructed, and screening is performed under the same conditions, or the extracted DNA is represented by SEQ ID NOs: 1-34. From the base sequence described in any of the sequences, a contiguous neighboring sequence can be easily obtained by the TAIL-PCR method established by Liu et al. Using an arbitrary 20-mer sequence as a primer.
  • the present invention also provides a DNA encoding a protein having 50% or more homology with a protein having the amino acid sequence ability described in any of SEQ ID NOs: 35 to 68.
  • DNA can be isolated by those skilled in the art by generally known methods. For example, hybridization technology (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) technology (Saiki, RK. Et al., Science, 1985, 230, 1350. Sa iki, RK. Et al., Science 1988, 239, 487.).
  • the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1-34 or a part thereof as a probe, and the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1-34 It is a common practice for those skilled in the art to isolate a DNA having a high homology with a DNA comprising the base sequence described in any one of SEQ ID NOS: 1 to 34 using an oligonucleotide that specifically hybridizes as a primer. Is to get.
  • a hybridization reaction is preferably performed under stringent conditions.
  • stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5 X SSC or equivalent stringency hybridization conditions. It is expected that DNA with higher homology can be isolated under conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1X SSC.
  • the DNA thus isolated is considered to have high homology with the amino acid sequence encoded by the DNA having the nucleotide sequence described in any one of SEQ ID NOs: 1 to 34 at the amino acid level.
  • High homology means at least 50% or more of the entire amino acid sequence, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more. Refers to the identity of the sequence.
  • score 50
  • wo rdlength 3.
  • the DNA of the present invention includes an amino acid in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 35 to 68.
  • DNA encoding a protein consisting of a sequence is included.
  • Modification of amino acids in a protein is usually within 50 amino acids of all amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and even more preferably within 3 amino acids.
  • the amino acid modification can be performed using, for example, “Transformer Site—directed Mutagenesis Kit” or “ExSite PCR—Based 3 ⁇ 4ite—directed Mutagenesis KitJ (Clontech), for mutation or substitution, Deletion can be performed using “Quantum le ap Nested Deletion Kit J (Clontech)”.
  • the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, cDNA, chemically synthesized DNA, and the like.
  • Genomic DNA is, for example, a genomic DNA prepared according to the method described in the literature (Rogers and Bendich, Plant Mol. Biol, 1985, 5, 69.) as a saddle type, and the base sequence of the DNA of the present invention (for example, sequence No .: PCR (Saiki et al. Scienc) using a primer prepared on the basis of the nucleotide sequence described in any of 1 to 34) e, 1988, 239, 487.).
  • cDNA it is prepared by preparing mRNA from a plant by a conventional method (Maniatis et al. Molecular Cloning Cold Spring Harbor Laboratry Press), performing a reverse transcription reaction, and performing PCR using the same primers as described above. Is possible.
  • genomic DNA or cDNA a genomic DNA library or cDNA library is prepared by a conventional method.
  • the base sequence of the DNA of the present invention (for example, any one of SEQ ID NOs: 1-34) is prepared against this library. It can also be prepared by screening using a probe synthesized based on the base sequence described in (1).
  • the base sequence of the obtained DNA can be easily determined by using, for example, “Sequencer Model 373” (manufactured by ABI).
  • DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk is described below in the "root base of Eucalyptus genus Camaldrensis" and the center of the trunk.
  • DNA whose expression level is increased on the xylem side compared to the phloem side “ The expression level on the xylem side compared to the phloem side, ”
  • the expression level is higher on the xylem side compared to the phloem side, "in the central trunk of Eucalyptus genus Camaldrensis compared to the phloem side.
  • the expression level rises on the xylem side, and in the trunk trunk base, the DNA expression level is the same on the phloem side and the xylem side '', ⁇ the phloem in the stem of Eucalyptus genus Camaldrensis and Eucalyptus globules DNA whose expression level is higher on the xylem side than on the other side ”,“ The expression level rises on the xylem side compared to the phloem side, and the tree level on the eucalyptus globulus trunk, compared to the phloem side.
  • the timber of the trunk trunk is more of the fiber length (characteristic) than the timber of the trunk trunk. Is long It is known that thicker cell walls are thicker and fibril inclination is looser, and the wood fiber ratio and specific gravity are higher (mature material).
  • the xylem of the trunk of the trunk base Among the wood fiber formation, the wood fiber length is shorter, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, and the wood fiber rate It is known that the specific gravity is low (it is an immature material). Therefore, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side on the trunk base of a single pot” is expressed or increased in the immature wood formation site. It can also be expressed as “decreasing DNA”. In addition, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side in the central part of the eucalyptus trunk” is expressed in “ It can also be expressed as “DNA”.
  • the direction of the xylem of the Grobles trunk from the xylem of the Camaldrensis trunk is the fiber of the wood fiber formation (pulp quality) It is known that the length and diameter are thick and the cell wall is thick and the fibril inclination is loose and the wood fiber ratio and specific gravity are high. Also, the xylem of the Camaldrensis trunk is shorter than the globules trunk, but the diameter of the fiber is shorter, the cell wall is thinner, the fibril inclination is steeper, and the wood fiber ratio and specific gravity are lower. It has been known.
  • DNA that has an increased or decreased expression level on the xylem side compared to the phloem side, compared to the trunk of the Eucalyptus genus Camaldrensis ⁇
  • the thin cell walls are thin, the fibril inclination is steep, the wood fiber rate and specific gravity are low, and the expression level is increased or decreased at the wood fiber formation site with low quality pulp characteristics. '' It can also be expressed.
  • DNA with an increased or decreased expression level on the xylem side compared to the phloem side on the trunk of Eucalyptus globulus means that “wood fiber length is longer and diameter is thicker than the cell wall. It can be expressed as ⁇ DNA whose expression level is increased or decreased in the part that forms wood fiber with high quality pulp characteristics that thick fibril inclination is gentle, wood fiber rate and specific gravity are high '' .
  • the phloem side means the outside of the vascularization layer, which is a meristem, and preferably means the scab formation site on the phloem side.
  • the scab formation site is a site that is in the middle of or is capable of splitting into phloem cells, phloem tubules, parenchyma cells, and pelvic cells.
  • the xylem side means the inner side of the vascular bundle forming layer, preferably the xylem side wood fiber forming site.
  • the wood fiber formation site is a wood fiber cell, temporary conduit, conduit In other words, it is a part that is in the middle of separating into parenchymal cells or the like or has the ability to separate the cells.
  • the trunk base means a generally called breast height, for example, in the case of a 5- year-old plant of Eucalyptus, it indicates a ground height of less than about lm.
  • the central part of the trunk generally refers to the middle part of the culm height, for example, in the case of a eucalyptus fifth-year plant that has a culm height of 10 m or more, it refers to a height of about 5 m above the ground.
  • the DNA of the present invention or a partial DNA thereof can be used as a marker for examining the growth state of a trunk trunk (preferably a trunk of a trunk trunk or a middle section of a trunk) of a plant. That is, by using at least one of the above-mentioned DNAs or partial DNAs thereof, the growth state of the trunk trunk of the plant (preferably the trunk of the trunk trunk or the central part of the trunk) can be examined.
  • the trunk trunk portion of the plant is a xylem portion in which wood fiber formation is active. Furthermore, in the case of (2), it is determined that the trunk stem portion of the plant is a xylem portion in which immature wood is formed or a xylem in which immature material is formed in the future.
  • the immature wood means a wood fiber that is differentiated and formed from an immature formed layer structure, and the length of the wood fiber is shorter than that of the xylem of the center of the trunk collected under the same conditions.
  • the diameter is thin.
  • the cell wall is thin. The fibril inclination is steep, and the wood fiber ratio and specific gravity are low.
  • the trunk trunk of the plant is a xylem where mature wood is formed, or a xylem where mature wood is formed in the future.
  • matured wood means wood fibers that are formed by differentiation from a mature formation layer structure, and the length of the wood fibers is longer than that of the trunk of the trunk base collected under the same conditions.
  • the cell diameter is thick, the cell wall is thick, the fiber inclination is loose, and the wood fiber ratio and specific gravity are high and low.
  • the plant used in the test of the present invention is preferably Eucalyptus, more preferably Eucalyptus Camaldlensis or Eucalyptus Globulus.
  • the maturity qualitatively in the subject trunk Fibers are formed, or are formed in the future, and the amount of wood fibers ( It can be predicted that there will be a large (increase in specific gravity) or increase in the future (an average specific gravity of 450 to 500 kg / m 3 or more).
  • the material quality prediction as described above will clearly reveal the quality of the plant's growth, so if it is judged to be bad, fertilizer is applied in an oligotrophic area or weeding work is carried out if weeds are thick. It is possible to conduct operations quickly and appropriately, such as early implementation or thinning if the planting density is high.
  • material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance as shown in this application. .
  • the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis On the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), “the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis”
  • the expression level of at least one (preferably a plurality, more preferably all, the same below) of DNA whose expression level is increased on the xylem side relative to the xylem side is detected.
  • the expression level of the DNA on the trunk of the plant is higher on the xylem side than on the phloem side
  • DNA having an increased expression level on the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis Of (a) to (e), or any of the DNAs described in any of them.
  • SEQ ID NO: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66 Is a DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of any one of 67
  • the expression level is reduced on the xylem side in the trunk base of Eucalyptus genus Camaldrensis, compared to the phloem side, and in the middle part of the trunk, the xylem side is compared with the phloem side.
  • Examples of the DNA whose expression level is increased in (1) include the DNAs described in any one of the following (a) to (e).
  • the expression level is increased on the xylem side compared to the phloem side in the central trunk of Eucalyptus genus Camaldrensis, and the expression level is increased on the phloem side and xylem side in the trunk base.
  • “equivalent DNA” include the DNAs described in the following (a) to (e)!
  • the base sequence ability D ⁇ described in any of SEQ ID NOs: 7, 10, 18, 26, 29, or 34 includes the bases described in any of SEQ ID NOs: 7, 10, 18, 26, or 29.
  • DNA comprising the sequence is preferred DNA comprising the base sequence described in SEQ ID NO: 18, 26 or 29 is more preferred! /, But is not limited to these! /.
  • the present invention provides a method for examining the growth state (maturity) of two different trunks. In other words, it provides a method for examining which of two different trunks are mature or immature.
  • “two different trunk trunks” include two trunk trunks in two different plants and two different trunk trunks in one plant. The above method can also be used to select mature or immature trunk trunks from a plurality of trunk trunks.
  • trunk trunk trunk trunk The other trunk tree section (hereinafter referred to as trunk trunk section B) is a mature section. Compared with trunk trunk section B, trunk trunk section A is an immature section. It is determined that there is. Therefore, the following (5) or (6) is an index for selecting an excellent line called an elite tree or a plus tree, for example, at a breeding site. At present, selection of elite trees, etc. is performed using the numerical values such as the diameter of the spiders and diameters and the material analysis values after harvesting as indicators, and there is no technique (concept) to select with the above indicators.
  • the “DNA having the nucleotide sequence described in SEQ ID NO: 33 or 34” is preferably a DNA consisting of the nucleotide sequence described in SEQ ID NO: 33, but is not limited to these! /.
  • (6) At least one DNA in two different trunk trunks, "DNA whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the eucalyptus trunk center" As a result of comparing the expression level of the DNA in two different trunk trunks, the expression level of the DNA decreased in the trunk trunk A compared to the trunk trunk B.
  • the DNA whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the eucalyptus trunk center is as follows: (a) to (e )! Or any of the DNAs listed elsewhere.
  • the trunk trunk portion of the plant is a xylem portion in which wood fiber formation is active. Furthermore, in the case of (8) or (9), the length of the fiber of the trunk of the plant is shorter and the diameter of the cell wall is narrower than that of the grops of the trunk of the groprus collected under the same conditions. The fibril inclination angle is steep, the wood fiber ratio and specific gravity are low, and V, a wood fiber having a pulp characteristic is formed! It is determined that the xylem is formed.
  • the length of the tree fiber is longer and the cell wall is thicker than the length of the stem of the stem of the plant, compared to the xylem of the Camaldrensis trunk collected under the same conditions. It is a xylem where wood fibers with excellent pulp properties are formed, such as the ratio of loose fibrils is high and the specific gravity is high, or the xylem where wood fibers with the pulp properties are formed in the future It is determined.
  • the material quality prediction as described above makes it clear that the state of plant growth is good or bad, so if it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, early weeding work is performed. If the planting density is high, thinning can be performed quickly and appropriately.
  • material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance.
  • DNA having an increased expression level on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules includes the following (a ) To (e)!
  • DNA having the nucleotide sequence described in any of SEQ ID NO: 11, 12, 17, 21, 22 or 30 includes the nucleotide sequence described in any of SEQ ID NO: 11, 12, 17, 22 or 30.
  • DNA consisting of the base sequence described in any one of SEQ ID NOs: 11, 12, 17 or 22 is more preferred, but is not limited thereto.
  • the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side.
  • DNA consisting of the base sequence described in any of SEQ ID NO: 8, 13, 19, 23-25 or 33 is the sequence described in any of SEQ ID NO: 8, 13, 19, 24, 25 or 33 DNA consisting of a base sequence is preferred, but DNA consisting of the base sequence described in any one of SEQ ID NOS: 13, 19, or 25 is more preferred, but is not limited thereto.
  • the expression level is increased on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis, and the expression level on the phloem side and xylem side in the trunk of the Eucalyptus globules
  • Examples of the DNA having the same DNA include DNAs described in any of the following (a) to (e).
  • DNA consisting of the base sequence described in the IJ number: 1-6, 9, 14, 16, 20, 27, 28 or 32! DNA sequence ability described in any of 6, 9, 14, 16, 20, or 27 is also preferred by DNA. SEQ ID NO: 1, 3, 6, 14, or 16 Although DNA is more preferable, it is not limited to these.
  • the present invention provides a method for inspecting the growth state (pulp quality) of the stem portion of two different plants (preferably eucalyptus). In other words, it provides a method for inspecting which of two different plant trunks is superior or inferior in pulp quality. This method can also be used to select good or inferior trunk trunks from multiple plant trunk trunks.
  • (11) or (12) it is compared with the trunk trunk of one of the two different plants (hereinafter referred to as plant A).
  • the stem of the other plant (hereinafter referred to as plant B) is a xylem with excellent pulp quality.
  • the xylem has Therefore, the following (11) or (12) is an index for selecting an excellent line called an elite tree or a plus tree at a breeding site, for example. At present, selection of elite trees, etc., is based on apparent values such as height and diameter, and material analysis values after cutting. There is no technology (concept) to select with the above indicators.
  • the eucalyptus is compared with the xylem side of the trunk of the Eucalyptus globulus.
  • the expression level of at least one of the DNAs whose expression level is increased on the xylem side of the trunk of the genus Camaldrensis is detected, and the expression level of the DNA in the trunk xylem of two different plants.
  • the expression level of the DNA is higher in the plant A than in the plant B.
  • the Eucalyptus genus Camaldrensis is compared with the xylem side of the trunk of the Eucalyptus globules.
  • Examples of “DNA having an increased expression level on the xylem side of the trunk” include the following DNAs (a) to (e)!
  • DNA consisting of the nucleotide sequence set forth in any of SEQ ID NO: 11, 12, 17 or 22 is preferably the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 12 or 17 Any of the DNAs having base sequence strengths described in any one of these is more preferable, but not limited thereto.
  • the eucalyptus genus Camaldrensis is compared with the xylem side of the trunk of the Eucalyptus globulus.
  • the expression level of at least one DNA of ⁇ DNA with decreased expression level at the xylem of the trunk of the tree '' was detected, and the expression level of the DNA in the trunk of the two different plants was compared.
  • the expression level of the DNA is reduced in the plant A compared with the plant B.
  • the tree of the Eucalyptus genus Camaldlensis is compared with the tree side of the Eucalyptus globulus. Examples of the “DNA whose expression level is reduced on the part side” include the DNAs described in (a) to (e) below, or any of them.
  • a xylem corresponding to any of the above (1) to (12) or a plant having the xylem is selected, and the selected xylem or the xylem It is possible to produce a uniform pulp from plants that have a large amount of pulp, making it possible to increase the efficiency of pulp production, reduce costs, and produce paper with new characteristics.
  • a xylem corresponding to any of the following (1) to (12) or a plant having the xylem according to a method well known to those skilled in the art, between the selected xylems or having the xylem Pulp can also be produced after confirming whether the wood fiber state is uniform among plants.
  • the present invention also provides a DNA encoding a plant transcription factor and a promoter DNA.
  • DNA encoding a plant transcription factor in the present invention can be used to control the formation of plant fiber fibers.
  • the promoter DNA of the DNA can be used to control expression specific to wood fiber tissue or wood fiber formation time.
  • the expression (level) information of DNA encoding a protein having a function to control the formation of plant fiber in the tree trunk is based on the quantitative state of the plant growth state, tree fiber formation state, and material quality. It can be used for measurement. Therefore, by using these, artificial modification of tree fiber cell shape formation, tree fiber cell-specific expression of any gene / protein, plant growth state, tree fiber formation state, quantitative quantity of materials It can be used for qualitative advance prediction. As a result, more efficient pulp production, reduced costs, and paper with new characteristics Can be manufactured.
  • Controlling wood fiber formation in plants has various important implications in the fields of industry and agriculture. For example, modification of the wood fiber properties of Eucalyptus genus Camaldrensis is significant in terms of improving the fiber properties of fiber raw materials such as pulp by increasing the fiber length. In addition, increasing the cellulose and hemicellulose content of Eucalyptus globulae species increases the yield of pulp and the like, and improves the cooking efficiency, which is significant in terms of economy and profitability.
  • the plant from which the DNA of the present invention is derived is not particularly limited, for example, useful crops (including forage crops) such as cereals, vegetables and fruit straw, fiber raw material plants such as pulp, ornamental plants, etc.
  • useful crops including forage crops
  • examples include plants.
  • the plant such as eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower,
  • Examples include ivy, orange, grape, peach, pear, apple, tomato, Chinese cabbage, cabbage, Japanese radish, carrot, cabbage, cucumber, melon, parsley, orchid, chrysanthemum, lily and saffron.
  • examples of the DNA encoding a plant transcription factor include the DNAs described in any of the following (a) to (e).
  • stringent hybridization conditions include conditions of standing at 60 ° C in an O.lxSSC solution, or a stringency equivalent to this, An example is the condition. Under such conditions, DNA that hybridizes with DNA having the nucleotide sequence described in any one of SEQ ID NOs: 101 to 111 can be isolated.
  • DNA is extracted from a plant, a gene library is constructed, and screening is performed under the same conditions, or the extracted DNA is subjected to SEQ ID NO: 101-: L 11 From the base sequence described in any of the above, a contiguous neighboring sequence can be easily obtained by the TAIL-PCR method established by Liu et al. Using an arbitrary 20-mer sequence as a primer.
  • the present invention also provides a DNA encoding a protein having 50% or more homology with the protein having the amino acid sequence ability described in any of SEQ ID NOs: 112 to 122.
  • Such DNA is preferably DNA encoding a protein having the structural characteristics described in the Examples.
  • a DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 112 to 122 is generally isolated by a person skilled in the art by a known method. Is possible. For example, hybridization technology (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) technology (Sai ki, RK. Et al, Science, 1985, 230, 1350. Saiki, RK. Et al, Science 1988, 239, 487.).
  • DNA having a base sequence ability described in any of SEQ ID NOs: 101 to L11 or a part thereof as a probe, or a DNA having a base sequence ability described in any of SEQ ID NOs: 101 to 111 It is known to those skilled in the art to isolate a DNA having high homology with a DNA having a nucleotide sequence described in any one of SEQ ID NOS: 101 to 111 from a plant using an oligonucleotide that specifically hybridizes as a primer. It is about normal deeds.
  • a hybridization reaction is preferably performed under stringent conditions.
  • stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5 X SSC or equivalent stringency hybridization conditions. It is expected that DNA with higher homology can be isolated under conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1X SSC.
  • the DNA thus isolated has the SEQ ID NO: 101 at the amino acid level. It is considered to have a high homology with an amino acid sequence encoded by DNA having the nucleotide sequence ability described in any of ⁇ 111. High homology means at least 50% or more of the entire amino acid sequence, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, more preferably 95% or more, and most preferably 98% or more. Refers to sequence identity.
  • BLAST and Gapped BLAST programs the default parameters of each program are used. The technique is known (http://www.ncbi.nlm.nih.gov/).
  • the DNA of the present invention includes an amino acid sequence in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence of any one of SEQ ID NOs: 112 to 122.
  • Modification of amino acids in a protein is usually within 50 amino acids of all amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and even more preferably within 3 amino acids.
  • the amino acid modification can be performed using, for example, “Transformer Site—directed Mutagenesis Kit” or “ExSite PCR—Based 3 ⁇ 4ite—directed Mutagenesis KitJ (Clontech), for mutation or substitution, Deletion can be performed using “Quantum le ap Nested Deletion Kit J (Clontech)”.
  • the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, cDNA, chemically synthesized DNA, and the like.
  • Genomic DNA is, for example, a genomic DNA prepared according to the method described in the literature (Rogers and Bendich, Plant Mol. Biol, 1985, 5, 69.) as a saddle type, and the base sequence of the DNA of the present invention (for example, sequence It can be prepared by performing PCR (Saiki et al.
  • a genomic DNA library or cDNA library is prepared by a conventional method.
  • the nucleotide sequence of the DNA of the present invention (for example, any one of SEQ ID NOs: 101 to 111) is prepared. It is also possible to prepare by screening using a probe synthesized based on the base sequence described above. The base sequence of the obtained DNA can be easily determined by using, for example, “Sequencer Model 373j (manufactured by ABI)”.
  • the DNA encoding the plant transcription factor is described below in the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis.
  • the expression level rises in DNA ”,“ In the central trunk of Eucalyptus genus Camaldrensis, the expression level increases on the xylem side compared to the phloem side, and in the trunk base, The expression level on the xylem side is higher than that on the phloem side in the trunks of eucalyptus chamaldrensis and eucalyptus globulus.
  • DN A “ In the trunk of Eucalyptus genus Camaldrensis, the expression level increased on the xylem side compared to the phloem side, and in the trunk of the Eucalyptus globulus, the xylem side compared to the phloem side.
  • the xylem of the trunk of the trunk base Among the wood fiber formation, the wood fiber length is shorter, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, and the wood fiber rate It is known that the specific gravity is low (it is an immature material). Therefore, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side on the trunk base of a single pot” is expressed or increased in the immature wood formation site. It can also be expressed as “decreasing DNA”. In addition, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side in the central part of the eucalyptus trunk” is expressed in “ It can also be expressed as “DNA”.
  • the direction of the xylem of the Grobles trunk from the xylem of the Camaldrensis trunk is the fiber of the wood fiber formation (pulp quality) It is known that the length and diameter are thick and the cell wall is thick and the fibril inclination is loose and the wood fiber ratio and specific gravity are high. Also, the xylem of the Camaldrensis trunk is shorter than the globules trunk, but the diameter of the fiber is shorter, the cell wall is thinner, the fibril inclination is steeper, and the wood fiber ratio and specific gravity are lower. It has been known.
  • DNA that has an increased or decreased expression level on the xylem side compared to the phloem side, compared to the trunk of the Eucalyptus genus Camaldrensis ⁇
  • the thin cell walls are thin, the fibril inclination is steep, the wood fiber rate and specific gravity are low, and the expression level is increased or decreased at the wood fiber formation site with low quality pulp characteristics. '' It can also be expressed.
  • DNA with an increased or decreased expression level on the xylem side compared to the phloem side on the trunk of Eucalyptus globulus means that “wood fiber length is longer and diameter is thicker than the cell wall. It can be expressed as ⁇ DNA whose expression level is increased or decreased in the part that forms wood fiber with high quality pulp characteristics that thick fibril inclination is gentle, wood fiber rate and specific gravity are high '' .
  • the phloem side means the outside of the vascularization layer that is a meristem, and preferably the scab formation site on the phloem side.
  • the scab formation site is a site that is in the middle of or is capable of splitting into phloem cells, phloem tubules, parenchyma cells, and pelvic cells.
  • the xylem side means the inner side of the vascular bundle forming layer, preferably the xylem side wood fiber forming site.
  • the term “wood fiber forming site” refers to a site that is being divided into wood fiber cells, temporary conduits, conduits, parenchymal cells, etc., or that has the ability to separate them.
  • the trunk base means a generally called breast height, for example, in the case of a 5- year-old plant of Eucalyptus, it indicates a height of less than about lm above the ground.
  • the central part of the trunk generally refers to the middle part of the culm height, for example, in the case of a eucalyptus fifth-year plant that has a culm height of 10 m or more, it refers to a height of about 5 m above the ground.
  • the DNA of the present invention or a partial DNA thereof can be used as a marker for examining the growth state of a trunk of a plant (preferably, a xylem of a trunk trunk or a middle part of a trunk). That is, by using at least one of the above-mentioned DNAs or partial DNAs thereof, the growth state of the trunk trunk of the plant (preferably the trunk of the trunk trunk or the central part of the trunk) can be examined.
  • the trunk trunk portion of the plant is a xylem portion in which wood fiber formation is active. Further, in the case of (2), it is determined that the trunk trunk portion of the plant is a xylem portion where mature wood is formed, or a xylem where mature wood is formed in the future.
  • mature wood means wood fibers that are formed by differentiation from a mature formation layer structure, and the wood fiber length is longer than that of the trunk of the trunk base collected under the same conditions.
  • the cell wall is thick, the cell wall is thick, the fibril inclination is loose, and the wood fiber ratio and specific gravity are high.
  • immature wood means wood fibers that are differentiated and formed from an immature formed layer structure, and the length of the wood fiber is shorter than that of the wood part of the central trunk collected under the same conditions.
  • the cell wall is thin and the fibril inclination is steep, and the wood fiber rate and specific gravity are low.
  • the plant used in the test of the present invention is preferably Eucalyptus, more preferably Eucalyptus Camaldlensis or Eucalyptus Globulus.
  • the maturity qualitatively in the subject trunk Fibers are formed, or are formed in the future, and the amount of wood fibers ( It can be predicted that there will be a large (increase in specific gravity) or increase in the future (an average specific gravity of 450 to 500 kg / m 3 or more).
  • the material quality prediction as described above will clearly reveal the quality of the plant's growth, so if it is judged to be bad, fertilizer is applied in an oligotrophic area or weeding work is carried out if weeds are thick. It is possible to conduct operations quickly and appropriately, such as early implementation or thinning if the planting density is high.
  • material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance as shown in this application. .
  • the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis On the phloem side and the xylem side of the trunk of the plant (preferably the trunk trunk or the middle of the trunk), “the phloem in the trunk trunk and the middle trunk of the Eucalyptus genus Camaldrensis”
  • the expression level of at least one (preferably a plurality, more preferably all, the same below) of DNA whose expression level is increased on the xylem side relative to the xylem side is detected.
  • the expression level of the DNA on the trunk of the plant is higher on the xylem side than on the phloem side
  • DNA having an increased expression level on the xylem side compared to the phloem side in the trunk base and middle trunk of Eucalyptus genus Camaldrensis Of (a) to (e), or any of the DNAs described in any of them.
  • SEQ ID NO: 113-117 which encodes a protein having an amino acid sequence ability in which one or more amino acids are substituted, deleted, added, and Z or inserted in any one of the amino acid sequences described in any one of DNA
  • DNA having the base sequence ability described in any one of SEQ ID NOs: 102, 105 or 106 is preferable, and SEQ ID NO: 105 or DNA having a nucleotide sequence ability described in 106 is more preferable, but is not limited thereto.
  • the expression level is increased on the xylem side compared to the phloem side in the central trunk of Eucalyptus genus Camaldrensis, and the expression level is increased on the phloem side and xylem side in the trunk base.
  • “equivalent DNA” include the DNAs described in the following (a) to (e)!
  • DNA consisting of the nucleotide sequence of SEQ ID NO: 101, 107 or 109 to 111 DNA consisting of the nucleotide sequence of SEQ ID NO: 107 or 109 to 111 is preferred.
  • DNA having the nucleotide sequence set forth in SEQ ID NO: 107 or 109 is more preferred, but is not limited to these! /.
  • the present invention provides a method for examining the growth state (maturity) of two different trunks. In other words, it provides a method for examining which of two different trunks are mature or immature.
  • “two different trunk trunks” include two trunk trunks in two different plants and two different trunk trunks in one plant. The above method can also be used to select mature or immature trunk trunks from a plurality of trunk trunks.
  • trunk trunk A The xylem (hereinafter referred to as “trunk trunk B”) is a mature xylem, and compared to “trunk trunk timber B”, the trunk xylem A is determined to be an immature xylem.
  • Trunk trunk B is a mature xylem, and compared to “trunk trunk timber B”, the trunk xylem A is determined to be an immature xylem.
  • the following (3) is an index for selecting excellent lines called elite trees and brass trees, for example, in breeding sites. At present, selection of elite trees and the like is performed using an apparent numerical value such as the diameter of a spider and a material analysis value after cutting, and there is no technique (concept) for selecting with the above index.
  • DNA whose expression level is reduced on the xylem side of the trunk base as compared with the xylem side of the eucalyptus trunk center is the following (a) to (e): !, The DNA described in somewhere. (a) DNA encoding a protein having the amino acid sequence ability described in SEQ ID NO: 113 or 114
  • the “DNA having the nucleotide sequence ability described in SEQ ID NO: 102 or 103” is preferably a DNA having the nucleotide sequence ability described in SEQ ID NO: 102, but is not limited thereto.
  • the trunk stem portion of the plant is a xylem portion in which wood fiber formation is fostering.
  • the length of the fiber of the trunk of the plant is shorter and the diameter of the cell wall is narrower than that of the grops trunk of the groprus collected under the same conditions.
  • the fibril inclination angle is steep, the wood fiber ratio and specific gravity are low, and V, a wood fiber having a pulp characteristic is formed!
  • the length of the tree fiber is longer and the cell wall is thicker than the length of the stem of the trunk of the plant compared to the xylem of the Camaldrensis trunk collected under the same conditions. It is a xylem where wood fibers with excellent pulp properties are formed, such as the ratio of loose fibrils is high and the specific gravity is high, or the xylem where wood fibers with the pulp properties are formed in the future It is determined.
  • the material quality prediction as described above makes it clear that the state of plant growth is good or bad, so if it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, early weeding work is performed. If the planting density is high, thinning can be performed quickly and appropriately.
  • material prediction and operational policy decisions such as those described above rely heavily on on-site experience and intuition, or subsequent chemical analysis, and there are no scientific indicators that can be identified in advance.
  • DNA having an increased expression level on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules includes the following (a ) To (e)!
  • the “DNA having a nucleotide sequence ability described in SEQ ID NO: 103 or 105” is preferably a DNA having a nucleotide sequence ability described in SEQ ID NO: 105, but is not limited thereto.
  • the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side.
  • the expression level rises on the part side, and the expression level decreases on the xylem side compared to the phloem side in the trunk of Eucalyptus globules!
  • the DNA having the nucleotide sequence ability described in SEQ ID NO: 104 or 106 is preferably the DNA having the nucleotide sequence ability described in SEQ ID NO: 106, but is not limited thereto.
  • the expression level is increased on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis, and the expression level on the phloem side and xylem side in the trunk of the Eucalyptus globules
  • Examples of the DNA having the same DNA include DNAs described in any of the following (a) to (e).
  • the tree of the Eucalyptus genus Camaldrensis is compared to the phloem side.
  • the expression level of at least one DNA is detected in the eucalyptus globulus trunk, the expression level of which is reduced on the xylem side compared to the phloem side.
  • the growth state of the trunk stem part of two different plants is compared with the phloem side.
  • a method for inspecting pulp quality is provided. In other words, it provides a method for inspecting which of two different plant trunks is superior or inferior in pulp quality. This method can also be used to select good or inferior trunk trunks from multiple plant trunk trunks.
  • (11) it is compared with the trunk trunk of one of the two different plants (hereinafter referred to as plant A).
  • the trunk of the other plant (hereinafter referred to as plant B) is a xylem having an excellent pulp quality.
  • the trunk xylem A has an inferior pulp quality. It is determined that the xylem has. Therefore, the following (11) is an index when selecting excellent lines called elite trees and plus trees, for example, at breeding sites. At present, selection of elite trees, etc. is performed using the numerical values such as the diameter and the material analysis values after cutting, and there is no technology (concept) to select using the above indicators.
  • the eucalyptus is compared with the xylem side of the trunk of the Eucalyptus globulus.
  • the expression level of genus Camaldrensis is reduced at the xylem side of the trunk, and the expression level of at least one of the DNA is detected, and the expression level of the DNA in the trunk xylem of two different plants is detected.
  • the expression level of the DNA is reduced in plant A compared to plant B.
  • the Eucalyptus genus Camaldrensis is compared with the xylem side of the trunk of Eucalyptus globules.
  • Examples of the “DNA whose expression level is reduced on the xylem side of the trunk” include the following (a) to (e)!
  • a xylem corresponding to any of the above (1) to (8) or a plant having the xylem is selected, and the selected xylem or the xylem is selected. It is possible to produce a uniform norp from plants with xylem, making it possible to increase the efficiency of pulp production, reduce costs, and manufacture paper with new characteristics.
  • Pulp can also be produced after confirming whether the wood fiber state is uniform among plants having the. Further, if it does not fall under any of the following (1) to (8), it is obtained by collecting a xylem or a plant having the xylem to produce a pulp having an arbitrary composition.
  • the test of the present invention can be carried out by various known methods. For example, first, an RNA sample is prepared from a desired tissue of a test plant. Next, the amount of target RNA contained in the RNA sample is measured. The amount of RNA measured is then compared to a control. Examples of such methods include Northern blotting and RT-PCR.
  • the test of the present invention can also be performed by a DNA array method.
  • examples of the nucleotide immobilization (array) method include an oligonucleotide-based array developed by Aflfymetrix.
  • oligonucleotide array oligonucleotides are usually synthesized in situ.
  • photolithographic technology Alfymetrix
  • inkjet Rosetta Inpharmatics
  • the “substrate” means a plate-like material on which a nucleotide probe can be fixed.
  • the substrate of the present invention is not particularly limited as long as the nucleotide probe can be immobilized, but a substrate generally used in DNA array technology is preferably used. it can.
  • a DNA array consists of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate.
  • a force permeable membrane which is generally glass, can be used, for example, a trocellulose membrane.
  • the nucleotide probe immobilized on the substrate is not particularly limited as long as it can detect the expression of the DNA of the present invention. That is, the probe is a probe that hybridizes to the DNA of the present invention. If specific hybridization is possible, the nucleotide probe need not be perfectly complementary to the DNA of the present invention.
  • the length of the nucleotide probe to be bound to the substrate is not particularly limited, but is usually 10 to 100 bp, preferably 10 to 50 bp, more preferably 15 to 25 bp.
  • the DNA of the present invention is brought into contact with the substrate. Through this process, the DNA of the present invention is hybridized to the nucleotide probe.
  • the hybridization reaction solution and reaction conditions may vary depending on various factors such as the length of the nucleotide probe immobilized on the substrate, it can be generally performed by methods well known to those skilled in the art.
  • the intensity of hybridization between the DNA of the present invention and the nucleotide probe immobilized on the substrate is then detected.
  • This detection can be performed, for example, by reading the fluorescence signal of the fluorescent dye labeled on the DNA of the present invention with a scanner or the like.
  • the present invention also provides a promoter DNA of the DNA of the present invention.
  • the promoter DNA of the present invention includes a promoter DNA linked to a gene obtained by the present invention, in particular, a gene having a differential expression in the spatial position of an oak.
  • promoter-one DNA means DNA containing a specific base sequence necessary for the initiation of synthesis (transcription) of mRNA using DNA as a saddle type. It includes DNA created by artificial alterations such as replacement.
  • the promoter DNA of the present invention includes a protein having a function of forming a tree fiber cell wall of a plant trunk (a protein involved in cellulose synthesis and a protein involved in ligne synthesis).
  • Examples include promoter DNA of encoding DNA and promoter DNA of DNA encoding plant transcription factor.
  • Plant trunk tree fiber cells The promoter DNA of DNA encoding a protein having a function of forming a wall (a protein involved in cellulose synthesis and a protein involved in lignin synthesis) is shown in SEQ ID NO: 93-: LOO. Proteins that have the function of forming tree fiber cell walls of plant trunks (proteins involved in cellulose synthesis and proteins involved in lignin synthesis)
  • DNA encoding and its promoter DNA is as described in the Examples. Further, promoter DNAs of DNAs encoding plant transcription factors are shown in SEQ ID NOs: 156 to 166. The relationship between DNA encoding a plant transcription factor and its promoter DNA is also as described in the Examples.
  • the promoter of the present invention can be produced and used as follows, for example. Extract and purify DNA from the tissues of the desired eucalyptus plant. In preparing DNA, various methods can be used, and commercially available kits such as ISOPLANT kit (manufactured by Futtsubon Gene Co., Ltd.) can be used.
  • oligonucleotides were prepared from any two locations based on the base sequence of eucalyptus cDNA that had been successfully isolated by the present inventors and used as primers. By PCR, genomic DNA corresponding to the selected eucalyptus cDNA can be easily produced.
  • Upstream DNA of the gene is obtained by PCR using oligonucleotide primers created based on the base sequence of the gene (Inverse-PCR method or anchor PCR method ⁇ AIL-PCR method (supervised by Isao Shimamoto, PCR Experiment Protocol ”(plant cell engineering separate volume, plant cell engineering series 7) published by Shujunsha, July 1997), or by the hybridization method using the DNA sequence of the gene as a probe. Is possible.
  • a genomic DNA library can also be used for eucalyptus DNA.
  • various genome vectors TAC vectors (Liu et al. (1999), Proc. Natl. Acad. Sci. USA, vol96: p653 5) It is obtained by transforming E. coli.
  • Hybridization technology can be used for screening genomic DNA libraries.
  • the eucalyptus cDNA sequence that has been successfully isolated by the present inventors can be used.
  • the above genomic DNA library is screened to isolate a clone containing a DNA sequence homologous to the gene. Restriction Enzyme cleavage maps are created and nucleotide sequences are determined, the structure of the cloned DNA is clarified, and the sequence existing upstream of the gene is identified.
  • This upstream sequence preferably includes a TATA box sequence and is at least several hundred bp force of several kbp. This sequence is excised with an appropriate restriction enzyme and subcloned into another plasmid vector or the like as necessary.
  • the promoter activity of the above sequence can be analyzed as follows. For example, using a vector containing a reporter gene such as ⁇ , subcloning is performed so that the above sequence is linked upstream of the reporter gene.
  • the ⁇ vector uses E. coli / 3 dalc mouth-dase (GUS) as a reporter gene.
  • GUS E. coli / 3 dalc mouth-dase
  • this gene product uses 5-bromo-4-chloro-3-j8-D-glucronic acid (X-glucuric acid) as a substrate, it decomposes to produce indigotin, a blue precipitate. It is possible to monitor the current situation at the organizational level.
  • 4-methyHimbellif eryj8-D-glucronide When 4-methyHimbellif eryj8-D-glucronide (4MUG) is used as a substrate, gene expression can be quantified by fluorescence generated by the action of the gene product.
  • a chloramphee-cholacetyl transferase gene, a luciferase gene, a green fluorescein protein gene, and the like can be used as a reporter gene.
  • the chimeric gene construct prepared as described above can be introduced into plants such as Arabidopsis thaliana via agrobacterium and analyzed for its function.
  • pBI101 is used as a vector
  • a recombinant plasmid containing the chimeric gene is introduced into the MP90 strain of Agrobacterium tumefaciens, for example, using the electoporation method, and the resulting transformant
  • Arabidopsis thaliana plants are infected by the floral dip method (supervised by Isao Shimamoto et al., "Experimental protocol for model plants" (plant cell engineering separate volume, plant cell engineering series 4) Shujunsha published in April 1996).
  • Seeds obtained from the infected plant are sown in a medium containing a drug such as kanamycin based on the vector used to obtain a transformant that has become drug-resistant by gene transfer. Using this transformed substance, the expression of the reporter GUS gene is analyzed.
  • the promoter of the present invention or an expression vector containing the promoter can be used as follows.
  • An expression vector is constructed by inserting a chimeric gene in which genes involved in physical biosynthesis are linked, for example, into a ⁇ vector. This vector is introduced into, for example, Tabacco plants via agrobacterium.
  • the gene of the present invention is expected to be expressed at the site of tree fiber formation and to produce any secondary metabolite by the action of the promoter of the present invention. In this case, since there is no phenomenon that is expressed even in an unnecessary tissue like the 35S promoter, it is expected that other preferable traits do not appear.
  • the gene that can be controlled by the promoter of the present invention is not limited to the specific gene described above. It is also possible to modify the function of the promoter of the present invention by linking other expression control sequences to the promoter of the present invention. Examples of such expression control sequences include enhancer sequences, repressor sequences, and insulator sequences.
  • the promoter of the present invention includes several cis-element sequences that control the expression of genes involved in the above-ground specific location of trunk and cell wall biosynthesis as functional characteristics. For the purpose of using the cis element sequence contained in the promoter of the present invention, it is possible to insert a part of the promoter of the present invention into another promoter and modify the function of the promoter.
  • the present invention also provides DNA for suppressing the expression of a DNA encoding a protein involved in plant fiber fiber wall formation.
  • DNA for suppressing the expression of an endogenous gene is preferred in that the DNA encoding an antisense RNA complementary to the transcription product of the DNA of the present invention and the transcription product of the DNA of the present invention are specifically used.
  • RNA encoding RNA with ribozyme activity to cleave DNA encoding RNA that suppresses expression of DNA of the present invention by RNAi effect or co-suppression effect, dominant negative for transcript of DNA of the present invention
  • Examples thereof include DNA encoding a protein having various traits.
  • the above “suppression of endogenous gene expression” includes suppression of gene transcription and suppression of translation into Z or a protein that also encodes the gene force. Also included is a decrease in expression as well as complete cessation of expression of the gene.
  • antisense nucleic acids suppress the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kogaku 2 Nucleic acid IV gene replication and expression, Japan biochemicalization) The Society, Tokyo Chemistry, 1993, 319-347.)
  • the antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above.
  • an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of a gene is designed, it is considered effective for inhibiting the translation of the gene.
  • a sequence complementary to the coding region or the 3 ′ untranslated region can also be used.
  • DNA containing an antisense sequence of not only a translation region of a gene but also an untranslated region is also included in the antisense DNA used in the present invention.
  • the antisense DNA to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.
  • the DNA prepared in this way can be By using the method, it can be transformed into a desired plant.
  • the sequence of the antisense DNA is preferably a sequence complementary to an endogenous gene or a part of the plant to be transformed, but is completely complementary as long as the gene expression can be effectively suppressed. It doesn't have to be.
  • the transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene.
  • the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. .
  • the length of the antisense DNA usually used is shorter than 5 kb, preferably shorter than 2.5 kb.
  • Ribozyme refers to an RNA molecule that has catalytic activity.
  • research focused on ribozymes as enzymes that cleave RNA has enabled the design of ribozymes that cleave RNA in a site-specific manner.
  • Some ribozymes have a size of 400 nucleotides or more, such as the group I intron type and Ml RNA contained in RNase P, but the hammerhead type has an active domain of about 40 nucleotides called the hairpin type.
  • the self-cleaving domain of the hammerhead ribozyme has the ability to cleave the 3 'side of C15 in the sequence G13U14C15.
  • base pairing between U14 and A9 is important. It has been shown that A15 or U15 can also be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.) 0 Design a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site
  • a restriction enzyme-like RNA cleavage ribozyme that recognizes the sequence UC, UU, or UA in the target RNA can be generated (Koizumi, M.
  • Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that target-specific RNA cleavage ribozymes can also be generated from hairpin ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.).
  • the ribozyme designed to cleave the target is linked to a promoter and transcription termination sequence, such as the cauliflower mosaic virus 35S promoter, so that it is transcribed in plant cells. At this time, if an extra sequence is added to the 5 'or 3' end of the transcribed RNA, the activity of the ribozyme may be lost. In this case, the RNA containing the transcribed ribozyme It is possible to place another trimming ribozyme that acts on cis on the 5 'side or 3' side of the ribozyme part (T aira, K. et al, Protein Eng, 1990, 3, 733., Dzianott, AM.
  • RNAi RNA interference
  • RNAi refers to a phenomenon in which the expression of the introduced foreign gene and target endogenous gene and the deviation are suppressed when a double-stranded RNA having the same or similar sequence as the target gene sequence is introduced into the cell. .
  • the details of the RNAi mechanism are not clear, but it is thought that the target gene is degraded from the fact that the double-stranded RNA introduced first is broken down into small pieces and somehow serves as an indicator of the target gene. .
  • RNAi is known to be effective in plants (Chuang, CF.
  • RNAi RNA having a nucleotide sequence set forth in any of SEQ ID NOs: 1-34 or a sequence similar thereto. What is necessary is just to introduce
  • the gene used for RNAi is completely the same as the target gene.
  • the sequence identity is at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more. Further, the identity of the sequence can be determined by the method described above.
  • Suppression of endogenous gene expression can also be achieved by co-suppression caused by transformation of DNA having the same or similar sequence as the target gene sequence.
  • “Co-suppression” is a phenomenon in which when a gene having the same or similar sequence as a target endogenous gene is introduced into a plant by transformation, the expression of the introduced foreign gene and the target endogenous gene are both suppressed. Refers to that. The details of the mechanism of co-suppression are not clear, but at least part of the mechanism is thought to overlap with the RNAi mechanism. Co-suppression is also observed in plants! (Smyth, DR., Curr Biol, 1997, 7, R793., Martienssen, R., Curr Biol, 199 6, 6, 810.).
  • a vector prepared so that the DNA or a DNA having a similar sequence can be expressed.
  • the gene used for co-suppression need not be completely identical to the target gene, but at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of sequence identity. Have.
  • the sequence identity can be determined by the method described above.
  • the suppression of the expression of the endogenous gene in the present invention can also be achieved by transforming a gene encoding a protein having a dominant negative trait to the protein encoded by the target gene into a plant.
  • a gene encoding a protein having a dominant negative trait refers to a gene having a function of eliminating or reducing the activity of an endogenous wild-type protein inherent in a plant by expressing the gene. Refers to that.
  • the present invention also provides a recombinant vector containing the DNA.
  • the vector of the present invention is not particularly limited as long as it contains a promoter sequence that can be transcribed in plant cells and a terminator sequence that includes a polyadenylation site necessary for the stability of the transcript.
  • pUC Vectors that can be amplified in E. coli, such as derivatives, and shuttle vectors that can be amplified in both E. coli and agrobacterium, such as ⁇ (Clontech). I can get lost.
  • plant viruses such as cauliflower mosaic virus can be used as vectors for ⁇ IJ.
  • the vector of the present invention can be obtained, for example, by binding or inserting the promoter DNA of the present invention or a promoter DNA for constitutively or inducibly expressing a desired gene into a predetermined part of the vector. it can.
  • the method for inserting the promoter into the vector follows the method for inserting a normal gene into the vector.
  • An expression vector for gene expression can be obtained by functionally connecting a desired gene to the promoter of this recombinant vector.
  • promoters for constitutive expression include the cauliflower mosaic virus 35S promoter (Odell et al., Nature, 1985, 313, 810.) and the rice actin promoter (Zhang et al., Plant). Cell, 1991, 3, 1155.) and corn ubiquitin promoter (Cornejo et al., Plant Mol. Biol, 1993, 23, 567.).
  • promoters for inducible expression are expressed by external factors such as invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, UV irradiation, and spraying of specific compounds. There are known promoters.
  • Such promoters include, for example, the rice chitinase gene promoter (Xu et al., Plant Mol. Biol., 1996, 30, 387.) and tapa PR protein gene promoters (Ohshima et al., Plant Cell, 1990, 2, 95.), rice-derived “lipl9” gene promoter (Aguan et al., Mol.
  • Corn alcohol dehydrogenase promoter (Walker et al "Proc. Natl. Acad. Sci. USA, 1987, 84, 6624.)
  • rice chitinase gene promoter and tobacco PR protein gene The promoter of certain compounds such as salicylic acid, and “ ra bl6” is a plant hormone It is also induced by application of abscisic acid.
  • the above recombinant vector contains an appropriate selection marker gene, or a plasmid vector containing a selection marker gene and the cell. It is preferable to introduce.
  • Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene that is resistant to the antibiotic hygromycin, the neomycin phosphotransferase that is resistant to kanamycin or gentamicin, and the gene that is resistant to the herbicide phosphinothricin. Examples include cetyltransferase gene.
  • the present invention also provides a transformed plant cell into which the vector of the present invention has been introduced.
  • the cells into which the vector of the present invention is introduced are not particularly limited, for example, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower, ivy, orange, grape , Peach, pear, apple, tomato, cabbage, cabbage, radish, carrot, cabotya, cucumber, melon, parsley, orchid, chrysanthemum, lily, safran, etc. Trees such as poplar, cedar, cypress, bamboo, and yew are desirable.
  • the plant cells of the present invention include cells in plant bodies. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots.
  • a target gene can be introduced into a plant by using a plant virus as a vector.
  • plant viruses that can be used include cauliflower mosaic virus. That is, first, after preparing the recombinant by inserting the viral genome into a vector derived from E. coli, these target genes are inserted into the viral genome. insert. It is possible to introduce these target genes into a plant by excising the viral genome thus modified from the recombinant with a restriction enzyme and inoculating the plant (Hohn et al. (1982) Molecular Biology of Plant Tumors (Academic Press ⁇ New York) pp549, US Pat. No. 4,407,956). Vectors introduced into plant cells and plants are not limited to these, and include other possibilities.
  • plasmids for direct introduction into protoplasts, there are no restrictions on the required vectors.
  • a simple plasmid such as a pUC derivative can be used.
  • other DNA sequences may be required.
  • Ri plasmids when ⁇ or Ri plasmids are used for transformation of plant cells, the sequence of at least the right end of the T-DNA region of Ti and Ri plasmids, usually the sequences at both ends, must be inserted into the gene to be introduced. Must be connected to be adjacent to the.
  • Agrobacterium When Agrobacterium is used for transformation, the gene to be introduced needs to be cloned into a special plasmid, that is, an intermediate vector or a binary vector. Intermediate vectors are not replicated in Agrobacterium.
  • the intermediate vector is transferred into the genus Agrobataterium by a helper plasmid or electoral position.
  • the intermediate vector has a region that is homologous to the T-DNA sequence, and is incorporated into the Agrobacterium sputum or Ri plasmid by homologous recombination.
  • Agrobacterium used as a host must contain the vir region. Usually, vir or Ri plasmid contains vir region, and T-DNA can be transferred to plant cells by its function.
  • a binary vector can be replicated and maintained in an Agrobacterium, so when incorporated into an Agrobacterium by the helper plasmid or the electroporation method, the vir region of the host By function, T-DN A on the binary vector can be transferred to plant cells.
  • the present invention also provides a transformed plant regenerated from the transformed plant cell, a transformed plant that is a descendant or clone of the transformed plant, and the transformed plant.
  • Such a transformed plant is a useful transformed plant in which cell wall components and cell morphogenesis are modified by plant species.
  • the modification of the cell wall component in the present invention is not particularly limited, for example, high cellulose, low lignin, a thick cell wall, a thin cell, a long fiber length, a short one, various quantitative and qualitative. Change. Examples of the modification of the cell morphology include, but are not limited to, changes in cell elongation and changes in cell size (quantitative change in volume).
  • the transformed plant according to the present invention has, for example, a plant having a new value such as an increase in the amount of plant growth due to an increase in the amount of cell wall synthesis, a change in fiber cell morphology, an increase in useful components of crops depending on the plant species.
  • a plant having a new value such as an increase in the amount of plant growth due to an increase in the amount of cell wall synthesis, a change in fiber cell morphology, an increase in useful components of crops depending on the plant species.
  • Useful as It is also useful as a plant with new value, such as the development of new materials by controlling cell wall synthesis, increased digestion and absorption efficiency of forage crops, and changes in fiber cell morphology.
  • the "transformed sickle object” is a plant having the above-described transformed sickle cell, and includes, for example, a transformed plant regenerated from the transformed cell. Be turned.
  • the method of regenerating an individual from transformed plant cells varies depending on the type of plant cell. For example, the method of Fujimura et al. (Fujimura et al, Plant Tissue Culture Lett., 2, 74, 1995) is used for rice, and Shillito is used for maize. (Shillito et al., Bio / Technology, 7, 581, 1989), in potato, in Visser et al., Theor. Appl.
  • the present invention introduces into a host cell an expression vector having a gene group involved in plant fiber cell wall formation in plants, particularly trees, or homologues thereof, or a promoter region linked to these genes.
  • the method includes the steps of obtaining a transformed cell, regenerating a transformed plant from the transformed cell, obtaining a plant seed from the obtained transformed plant, and producing the plant from the plant seed.
  • the process of obtaining plant seeds from the transformation and the object is, for example, the cultivation of the transformation and the object. It refers to the process of collecting from the ground, transplanting it to a pot containing water-containing soil, growing it at a constant temperature, forming flowers, and finally forming seeds.
  • the process of producing a plant from a seed is, for example, when the seed formed on a transformed plant matures, is isolated and sown in soil containing water, and grows under a constant temperature and illuminance. This refers to the process of producing a plant body.
  • the presence of the introduced foreign DNA or nucleic acid in the transformed plant body is determined by a known PCR method or Southern hybridization method, or by analyzing the nucleotide sequence of the nucleic acid in the plant body. Can be confirmed.
  • transformation and extraction of DNA or nucleic acid from the object can be performed according to the known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor laboratory Press, 1989).
  • an amplification reaction is carried out using the nucleic acid extracted from the regenerated plant body as described above in a cage shape.
  • an amplification reaction can also be carried out in a reaction mixture in which a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of DNA of the present invention is used as a primer and these are mixed.
  • amplification reaction when DNA denaturation, annealing, and extension reactions are repeated several tens of times, an amplification product of a DNA fragment containing the DNA sequence of the present invention can be obtained.
  • the reaction solution containing the amplified product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and that the DNA fragments correspond to the DNA of the present invention. .
  • the present invention also provides a primer that amplifies all or a part of the base sequences set forth in SEQ ID NOs: 1-34 and 101-111.
  • the primer of the present invention can be used in the inspection method of the present invention.
  • the primer of the present invention is not particularly limited as long as it can amplify at least a part of the DNA of the present invention or its complementary strand. Is usually 15 bp to 100 bp, preferably 16 bp to 31 bp, and more preferably, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 31 nucleotides.
  • the 3 ′ region may be complementary, and a restriction enzyme recognition sequence or a tag may be added to the 5 ′ side.
  • the present invention provides a primer set for amplifying all or part of the base sequence described in SEQ ID NOs: 1-34, 101-: L11.
  • the primer set of the present invention includes:
  • PCR primer of the present invention can be prepared by those skilled in the art using, for example, an automatic oligonucleotide synthesizer.
  • the present invention also provides a reagent comprising the above primer set.
  • the reagent of the present invention can be used in the inspection method of the present invention.
  • Such reagents may include those used in the above-described detection methods.
  • the gene, primer, staining solution and the like of the present invention can be mentioned.
  • distilled water, salt, buffer solution, protein stabilizer, preservative and the like may be contained.
  • glucose is obtained from the lignocellulose obtained therefrom by an acid hydrolysis or enzymatic decomposition (cellulase) process, and then ethanol is obtained by alcohol fermentation.
  • acid hydrolysis or enzymatic decomposition cellulase
  • ethanol is obtained by alcohol fermentation.
  • biodegradable plastics polylactic acid
  • biomass derived from wood is expected to become the mainstream instead of cereal as food.
  • Lignin is also expected to be used for plastics and adhesives, although it will be necessary to overcome technical challenges in the future.
  • lignin is decomposed ichiologically in the pulp manufacturing process of the paper industry and is contained in the waste liquid (called black liquor). After extracting the necessary chemicals from the waste liquid, It is used as fuel in the factory. In other words, it is nothing else but to rely on woody biomass for a part of the fuel.
  • woody biomass is cultivated stably and on a large scale and circulated by afforestation, so that it can be used as a conventional raw material, as well as petroleum alternative energy by biomass conversion, and also cellulose or hemicellulose It is quite possible to create a new plastic from this (which is also technically possible). Furthermore, the spread of woody biomass will become a solution to energy security and environmental problems, and at the same time will lead to the development of new industries such as agriculture and forestry and the creation of employment opportunities.
  • eucalyptus tissue to be extracted For example, stem growth (secondary wall thickening zone; tissue rich in formation layer), leaves, roots, slant stimulation, stress application by salt solution exposure, etc. The gene expression from the situation is assumed.
  • the basic method of extraction is the method described by Hino et al. (Japanese Patent Application No. 6-219187).
  • Hino et al. Japanese Patent Application No. 6-219187.
  • an RNA extraction method using eucalyptus roots by hydroponics as a material will be described in detail.
  • 6Mo 2 O prepared with demineralized water, pH is 0.1M NaOH or KOH daily 6. Adjusted to 0. Furthermore, all the culture medium was changed every week. When stress treatment is performed, the culture solution supplemented with NaCl so that the final concentration is 50,100,200,300 mM in order from the first day to the fourth day of cultivation is controlled in the stress treatment area, and the culture solution without addition of NaCl is controlled. And On the fourth day, 10 g of the roots were cut into small pieces and ground in liquid nitrogen. This was transferred to a 50 ml centrifuge tube (manufactured by NUNC), 10 g of glass beads were added, and then ground for 5 minutes with a homogenizer.
  • NUNC 50 ml centrifuge tube
  • a methanol solution containing dithiothreitol (lmg / ml) was used for this, and solvent extraction of the ground sample was repeated until no coloration was observed in the supernatant (about 3 times). After extraction, the sample was lyophilized. This lyophilized sample was mixed with 25 ml of pH 9 lOOmM CHES buffer (20 milligrams of dithiothreitol and 10 mM vanadyl ribonucleoside compound solution added immediately before use) and incubated at 65 ° C for 30 minutes. A 5M sodium chloride solution and a 10% CTAB solution were added to the sample solution after the incubation.
  • the sodium chloride concentration in the sample solution after addition was 1.4 M, and the CTAB concentration was 1% (w / v).
  • RNA precipitate was collected by centrifugation.
  • 12M lithium chloride solution was added to a final concentration of 3M, mixed well, and then ice-cooled for 1 hour.
  • the RNA precipitate was collected by centrifugation, washed and dried, and finally dissolved in 100 L of water to obtain a total RNA fraction.
  • 610 g of total RNA was obtained from each of the roots of the stress-treated group and the control root.
  • PolyATtract mRNA Isolation Systemlll & IV kit Cat. # Z5300 & Z5310 Promega Corp. USA
  • 1.3 ⁇ g of the mRNA was obtained from 610 g of total RNA and 1.8 ⁇ g of mRNA was obtained from the control sample.
  • the eucalyptus mRNA derived from various tissues and situations obtained by the method shown in (1) was synthesized using the Clontech Smart cDNA library construction kit, and finally the eucalyptus mRNA was synthesized. -Dimid library.
  • Each cDNA library created in this way also had independent clone power of more than 10 X 10 6 pfo.
  • Library amplification was performed on a part of the library prepared, and the clone was analyzed without amplification.
  • genes related to cellulose and lignin synthesis (Proc Natl Acad Sci USA 2001 Dec 4; 98 (25): 14732-7., JP 2000-41685, JP 2002-95482, JP We searched for clones with high homology. As a result, 59 clones were identified. However, some of these clones include those involved in the formation of scab tissue and the like that are formed only by wood fiber formation and those that are not related to cell wall synthesis.
  • a eucalyptus oligo microarray was prepared from the eucalyptus EST database. The actual production was outsourced to Agilent Technologies, Inc. (Yokogawa Analytical Systems is the Japanese distributor). Details are described on the following website: o http: // www. Chem. Agilent ⁇ com / cag / country / JP / products / PL, ol494.htm
  • the eucalyptus oligo microarray produced in this way contains 8400 oligo DNAs, and can cover most of the genes that are expressed in eucalyptus trunks.
  • wood fibers Most of the woody biomass in the trunk is composed of tissue cells called wood fibers.
  • the main factors that determine the properties of wood fibers include the specific gravity of the wood fiber structure, the cell wall structure and components (cellulose, hemicellulose, lignin). These are formed by working together in a group of many genes that are not defined by a single gene or protein.
  • the properties of wood fibers are known to vary depending on the species of hardwood 'coniferous trees, high above ground'.
  • RNA extraction method shown in Example 1 the present inventors extracted total RNA from the trunk base and middle part of a 5-year-old Camaldrensis species, and Groplus species. At this time, the crust side (phloem) and the wood fiber-forming tissue side (xylem) of each plant were used. The total RNA power obtained from each individual was purified by the PolyATract mRNA Isolation System manufactured by Promega. Each cRNA obtained by synthesizing a total of 4 types of mRNA obtained in this way was labeled with 2 types of fluorescent dyes (cy3.cy5), and used as probes in oligo microarray analysis. An analysis was performed ( Figures 1-3).
  • the Sequence ID corresponds to SEQ ID NOs: 1-34 in order from the top.
  • CesA Cellulose synthase
  • SuSy Sucrose Synthase
  • EGase endo— 1,4— beta— glucanase
  • DL Dynamin-like protein
  • CAD cinnamyl alcohol dehydrogenase
  • OMT ⁇ — methyl transferase
  • 4CL 4— coumarate Co
  • CCR Cinnamoyl Co
  • reductase C4H
  • the microarray experiment 5y-cam5 X vs P is an experiment comparing the xylem and phloem of the middle part of the fifth-year eucalyptus / camaldolensis trunk, and 5y-caml X vs P
  • Experiment comparing phloem, 5y-glo X vs P, 5th grade Eucalyptus' Grobulus An experiment comparing the xylem and phloem of the base.
  • the experimental results are expressed as a positive multiple when the former is strong, and as a negative multiple when strong on the husk side, comparing the wood fiber-forming tissue side and the husk side.
  • the 34 gene groups were broadly classified into the following (1) to (3) based on the detailed expression patterns in the trunk base and the central part of the fifth-grade Camaldrensis species (Table 2, Four).
  • the 26 gene groups include gene groups (SEQ ID NOs: 1 to 6, 9) that have an expression differential power of 3 ⁇ 4 times or more on the xylem side compared to the phloem side at either the trunk base or the trunk trunk center. , 11-14, 1 6, 17, 19, 20, 22, 25, 27, 30), the trunk of the trunk or the center of the trunk of the tree! Gene group (SEQ ID NOs: 1, 3 to 6, 9, 11 to 14, 16, 17, 19, 22, 25, 30), stem base or trunk A group of genes (SEQ ID NOs: 1, 3-6, 11, 12, 14, 16, 17, 22, which are more than double the differential force on the xylem side compared to the phloem side in the! 25), a group of genes (SEQ ID NO: 1 ⁇ 3, 5, 12, 25) Power included.
  • the numerical value of the average fluorescence intensity obtained by the experiment was calculated by the formula of the trunk base or the central part or the central part of the trunk base. Based on the expression information of each gene that is significant, the value obtained by the formula of the trunk base and the central part is 1.1 or higher and the value of 0.001 is compared with the xylem side of the central trunk part.
  • the difference in expression between the xylem of the trunk trunk and the xylem of the trunk trunk is 1.5 times or more.
  • the above gene group (SEQ ID NO: 33) is included.
  • the 18 gene groups include gene groups (SEQ ID NOs: 1 to 6, 14, 16, 17, 19) that have a difference in expression between the xylem of the trunk trunk and the xylem of the trunk trunk of 1.5 times or more. -22, 25, 28, 30), gene group (SEQ ID NO: 1 to 6, 14, 16, 17, 22, 25, 30), and a group of genes (SEQ ID NOs: 3, 5, 25, 30) that have an expression differential power of at least twice that between the xylem of the trunk base and the xylem of the central trunk.
  • the six gene groups have gene groups (SEQ ID NOs: 7, 10, 18, 26, 29) whose expression difference on the xylem side is more than double in the middle of the trunk, compared to the phloem side, In the center of the trunk, the gene group (SEQ ID NO: 18, 26, 29) whose expression difference on the xylem side is 2.5 times or more compared to the phloem side is included.
  • the 34 gene groups are expressed in the following (1) to (3) from the detailed expression pattern in the trunk base of the fifth-grade Camaldorensis species or the middle of the trunk and the fifth-grade Groplus species. Broadly classified (Tables 3 and 4).
  • the six gene groups include genes that have a difference in expression of eucalyptus chamaldrensis and eucalyptus globulus stems that are more than 4 times different in the xylem compared to the phloem.
  • the numerical value of the average fluorescence intensity obtained by the experiment is calculated based on the xylem formula of the trunk (base or central part) of the Eucalyptus genus Camaldrensis Z Eucalyptus globulus trunk (base) xylem Z Eucalyptus chamaldorensis trunk (base or middle) xylem calculation.
  • the number of genes obtained by the expression of the xylem of the eucalyptus chamaldrensis tree and the eucalyptus globulus trunk Z As described above, P ⁇ 0.001 is significantly different from ⁇ the gene group in which the expression is increased on the xylem side of the Eucalyptus globulis tree compared to the xylem side of the Eucalyptus camaldrensis ''.
  • the average intensity is almost the same (the fluorescence intensity of the tree part of Eucalyptus genus Camaldrensis relative to the tree part of Eucalyptus globulus trunk is less than l.0 to ll times or P> 0.001)
  • ⁇ Yu -It was classified as a gene group with the same expression on the xylem side of the trunk of the genus Cali genus Camaldrensis and on the xylem side of the stem of the Eucalyptus globules.
  • the seven gene groups show that the difference in the expression on the xylem side is 2.5 times or more in the trunk of Eucalyptus genus Camaldrensis, or in the trunk of Eucalyptus globulus compared with the xylem side.
  • Gene groups (SEQ ID NOs: 8, 13, 19, 24, 25, 33) that have an expression difference of 2.5 times or more on the cervical side, in the trunk of the Eucalyptus genus Camaldrensis, on the xylem side compared to the phloem side Gene group (SEQ ID NOs: 13, 19, 25) Eucalyptus Kamal whose expression difference is 4 times or more, or in Eucalyptus globulus trunks, the expression difference on the phloem side is 4 times or more compared to the xylem side In the trunk of Drensis, the expression difference on the xylem side is more than 10 times compared to the phloem side, or the expression difference on the phloem side compared to the xylem side, A gene group (SEQ ID NO: 25) that is 10 times or more is included.
  • the 13 gene groups include gene groups (SEQ ID NOs: 1-6, 9, 14) that have a three-fold or more differential expression on the xylem side compared to the phloem side in the trunk of the Eucalyptus genus Camaldrensis.
  • Eucalyptus chamaldrensis trunks the gene group is more than double the expression difference on the xylem side compared to the phloem side (SEQ ID NO: 1, 3-6, 14) 16),
  • Eucalyptus genus Camaldrensis trunks include a group of genes (SEQ ID NOs: 1, 3, 5) whose expression difference on the xylem side is more than 10 times that of the phloem side It is.
  • Eucalyptus BAC genomic library was prepared according to the method of J. Agricultural Genomics 5 (http: ⁇ www.ncgr.orc / research / jag) by D. Peterson et al.
  • the prepared library was sorted into 120 384 well plates for each individual clone. From these plates, DNA was prepared for each plate, each column and each row of all plates, and used as a DNA pool for 3D screening by PCR.
  • the genomic clones of each gene were screened by the method.
  • the insert sequence of the BAC clone was determined by the method shown in Example 1. Using the obtained base sequence, the genomic region of each gene was determined based on the cDNA sequence (the cDNA self-sequences described in SEQ ID NOs: 3, 6, 11, 14, 19, 23, 24, 25 were also used). And the determined genome sequences are shown in SEQ ID NOs: 85 to 92, respectively). For clones that were full-length cDNA sequences including the translation initiation codon ATG (which encodes methionine in amino acids), the promoter region was determined based on that sequence. The determined promoter sequence is shown in SEQ ID NOs: 93-100.
  • the TATA-box sequence was determined by analyzing the sequence upstream from the 5 ′ end base of the full-length cDNA using the genetic information processing software “GENETYX” (Software Development Co., Ltd.). It is well known that the promoter region of higher eukaryotes has a TATA-box sequence usually about 10 to 25 bases upstream from the transcription start site! If the position of the determined TATA-bo X is close to the full-length cDNA 5 'terminal base (approx. LOObp), it is judged that cDNA is almost full length and that the upstream side of the terminal base is the promoter region. did. The length of the promoter was set to a maximum of 3 kb upstream from the 5′-terminal base of cDNA with reference to the maximum length (about 3 kb) known in model plants.
  • positions 1 to 660 are the amino acid coding region (SEQ ID NO: 35).
  • the 1st to 602nd positions are the amino acid coding region (SEQ ID NO: 36).
  • the 337th to 3273th positions are the amino acid code region (SEQ ID NO: 37).
  • positions 1 to 1407 are the amino acid coding region (SEQ ID NO: 38).
  • the 1st to 465th positions are the amino acid coding region (SEQ ID NO: 39).
  • the 87th to 3221th positions are the amino acid coding region (SEQ ID NO: 40).
  • the 1st to 699th positions are the amino acid coding region (SEQ ID NO: 41).
  • positions 1 to 1407 are the amino acid coding region (SEQ ID NO: 42).
  • positions 1 to 1122 are the amino acid coding region (SEQ ID NO: 43).
  • the 1st to 1005th positions are the amino acid coding region (SEQ ID NO: 44).
  • positions 313 to 1305 are the amino acid code region (SEQ ID NO: 45).
  • positions 1 to 465 are the amino acid coding region (SEQ ID NO: 46).
  • positions 1 to 762 are the amino acid coding region (SEQ ID NO: 47).
  • positions 375 to 1442 are the amino acid code region (SEQ ID NO: 48).
  • positions 1 to 774 are the amino acid coding region (SEQ ID NO: 49).
  • positions 1 to 498 are the amino acid coding region (SEQ ID NO: 50).
  • positions 68 to 1165 are the amino acid code region (SEQ ID NO: 51).
  • the 117th to 857th positions are the amino acid code region (SEQ ID NO: 52).
  • positions 84 to 968 are the amino acid coding region (SEQ ID NO: 53).
  • positions 1 to 207 are the amino acid coding region (SEQ ID NO: 54).
  • positions 56 to 223 are the amino acid coding region (SEQ ID NO: 55).
  • positions 1 to 621 are the amino acid coding region (SEQ ID NO: 56).
  • the 323rd to 1330th positions are the amino acid code region (SEQ ID NO: 57).
  • positions 240 to 1847 are the amino acid code region (SEQ ID NO: 58).
  • the 273rd to 1928th positions are the amino acid code region (SEQ ID NO: 59).
  • the 1st to 1134th positions are the amino acid coding region (SEQ ID NO: 60).
  • the 1st to 525th positions are the amino acid coding region (SEQ ID NO: 61).
  • the 1st to 1116th positions are the amino acid coding region (SEQ ID NO: 62).
  • the 1st to 195th positions are the amino acid coding region (SEQ ID NO: 63).
  • the first to 138th positions are the amino acid coding region (SEQ ID NO: 64).
  • positions 1 to 564 are the amino acid coding region (SEQ ID NO: 65).
  • the 1st to 711st positions are the amino acid coding region (SEQ ID NO: 66).
  • the 84th to 569th positions are the amino acid coding region (SEQ ID NO: 67).
  • the 1st to 249th positions are the amino acid coding region (SEQ ID NO: 68).
  • the 1st to 2013th positions of the nucleotide sequence represented by SEQ ID NO: 85 are the promoter region (SEQ ID NO: 93), 2350th to 2527th, 3105th to 3181th, 3371th to 3rd. 3422, 3539-3680, 4295-4561, 4924-5269, 5412-5549, 5793-5918, 6001-6213 6649th to 6907th, 6993th to 7192th, 7431th to 7784th, 8069th to 8650th are Etason in the code region, 2528th to 3104th, 3182-3370, 3423-3538, 3681-4294, 4562-4923, 5270-5411, 5550-5792, 5919 -6th, 6214th to 6648th, 6908th to 6992th, 7193th to 7430th, 7785th to 8068th are intron regions.
  • the 1st to 2920th positions of the nucleotide sequence represented by SEQ ID NO: 86 are the promoter region (SEQ ID NO: 94), 3068th to 3139th positions, 3941th to 4141th positions, 4275th to 4275th positions. 4396, 4488-4554, 4670-4808, 5291-5903, 6000-6137, 6426-6551, 6792-7004 Nos. 7091 to 7606, 7998 to 8348, 8695 to 9276 are Etasons in the code area, 3140 to 3940, 4142 to 4274.
  • the 1st to 2700th positions of the nucleotide sequence represented by SEQ ID NO: 87 are the promoter region (SEQ ID NO: 95). Nos. 3013 to 3415, 3508 to 3667, 4610 to 5039 are etasons in the coding region, 3416 to 3507, The 3668th to 4609th positions are intron regions.
  • the 1st to 2707th positions of the base sequence represented by SEQ ID NO: 88 are the promoter region (SEQ ID NO: 96), 3082th to 3170th position, 3365th to 3478th position, 3565th to 3rd position No. 3792, No. 4389 to No. 4828, No. 5124 to No. 5320 are Exons in the code area, No. 3171 to No. 3364, No. 3479 to No. 3564, No. 3793 to No. 4388 4829th to 5123rd position S intron region.
  • the 1st to 2835th positions of the nucleotide sequence represented by SEQ ID NO: 89 are the promoter region (SEQ ID NO: 97), the 2920th to 3011th positions, the 3142th to 3221th positions, the 3345th to 3rd positions.
  • 3489, 3643-3774, 4333-4623 are Exons in the code area, 3012-3141, 3222-3344, 3490-3642 , 3775th position-4332rd position force S intron region.
  • the 1st to 2469th positions of the nucleotide sequence represented by SEQ ID NO: 90 are the promoter region (SEQ ID NO: 98), 2792th to 2924th position, 3027th to 3181th position, 3850th to 3rd position.
  • No. 4035, No. 4202 to No. 4554, No. 5504 to No. 5684 are Exons in the code area, No. 2925 to No. 3026, No. 3182 to No. 3849, No. 4036 to No. 4201 , Positions 4555 to 5503 are S intron regions.
  • positions 1 to 1242 are the promoter region (SEQ ID NO: 99), positions 1482 to 2347, and positions 2452 to 3193 are exons in the coding region. 2nd to 2451th positions are intron regions.
  • the 1st to 2997th positions of the base sequence represented by SEQ ID NO: 92 are the promoter region (SEQ ID NO: 100), the 3270th to 3335th positions, the 3606th position to the 3757th position, the 403th position to the 403th position.
  • No. 4278, No. 4562 to No. 4690, No. 4797 to No. 5732, No. 5844 to No. 5971 are Sae Kwong in the code area, No. 3336 to No. 3605, No. 3758 to No. 4033 , 4279th to 4561th, 4691th to 4796th, 5733th to 584th are the intron regions.
  • Example 5 Selection of transcription factors Transcription factor genes involved in gene expression control (JL Riechmann et al., SCIENCE VOL 290 15 DECEM BER 2000, Kazuo Iwasaki, Kazuo Shinozaki, dynamism of plant genome function, using the annotation information that also obtained the database power shown in Example 1 ) And clones with high homology were searched. As a result, 71 clones were identified. However, these clones also contain transcription factors that are involved in the formation of scab tissue, etc., which is not limited to the formation of wood fibers.
  • RNA extraction method described in Example 1 the present inventors extracted total RNA from the stem base and trunk center of a 5-year-old Camaldrensis species and the trunk base of a Groplus species. At this time, the husk side (the phloem) and the wood fiber forming tissue side (the xylem) of each plant were used. From the total RNA obtained from each individual, mRNA was purified by PolyATract mRNA Isolation System manufactured by Promega. A total of 4 types of mRNA thus obtained were labeled with 2 types of fluorescent dyes (cy3.cy5), respectively, and used as probes in the oligo microarray analysis shown in Example 1 for hybridization. ( Figures 1-3).
  • the hybridization method including the label followed the analysis protocol presented by Agilent Technologies. Fluorescence intensity is calculated from the scanned image, analyzed with Rosetta analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%. Information on the expression of 71 clones selected in the trunk base of the drenches species and the center of the trunk, and in the trunk base of the groprus species was obtained.
  • the transcription factor group involved in eucalyptus tree fiber formation is expected to be significantly more strongly expressed on the wood fiber forming tissue side (xylem) than on the crust skin side (phloem).
  • xylem wood fiber forming tissue side
  • phloem crust skin side
  • 11 clones identified a group of genes involved in eucalyptus tree fiber cell wall formation (Table 5- 8).
  • a DNA encoding a protein consisting of the amino acid sequence described in any one of 112 to 122 has the following structural features (domains).
  • amino acid sequence set forth in SEQ ID NO: 112 is a CBF domain, a HAP2 domain,
  • amino acid sequence set forth in SEQ ID NO: 113 is a homeodomain
  • amino acid sequence set forth in SEQ ID NO: 114 is a Coprinusjnating domain
  • amino acid sequence set forth in SEQ ID NO: 115 is a LIM domain
  • amino acid sequence set forth in SEQ ID NO: 116 is a bZIP domain
  • amino acid sequence set forth in SEQ ID NO: 117 is SANT domain, Myb DNA binding domain, RE B1 domain, main,
  • amino acid sequence set forth in SEQ ID NO: 118 is the WRKY domain
  • amino acid sequence described in SEQ ID NO: 119 is homeodomain, leucine zipper domain, the amino acid sequence described in SEQ ID NO: 120 is UPF0023 domain, leucine zipper domain, the amino acid sequence described in SEQ ID NO: 121 is SANT domain, Myb DNA binding domain , RE B1 de, main
  • amino acid sequence set forth in SEQ ID NO: 122 is the Sina domain.
  • the Sequence ID corresponds to SEQ ID NO: 101 to L I 1 in order from the top.
  • the microarray experiment 5y-cam5 X vs P is an experiment comparing the xylem and phloem of the fifth grade eucalyptus 'Camardrensis trunk
  • 5y-caml X vs P is the fifth grade eucalyptus' camaldrenci
  • 5y-glo X vs P represents an experiment comparing the xylem and phloem of the 5th grade Eucalyptus base.
  • the experimental results are expressed as a positive multiple when the former is strong, and as a negative multiple when strong on the husk side, comparing the wood fiber-forming tissue side and the husk side.
  • the five gene groups include a gene group (SEQ ID NO: 102, which has an expression differential power of 3 ⁇ 4 times or more on the xylem side compared to the phloem side in either the trunk base or the trunk central part. 105, 106), a group of genes (SEQ ID NO: 105, 106) whose expression difference on the xylem side is 4 times or more compared to the phloem side in either the trunk base or the trunk central part.
  • Gene group whose expression is increased on the xylem side of the trunk trunk ” is significantly or the same as the average intensity (the fluorescence intensity ratio of the xylem at the center and the xylem at the base is 1.0). Less than -1.1 times or P> 0.001) was classified as a “gene group with similar expression on the xylem side of the trunk base and the xylem side of the trunk trunk”.
  • the two gene groups include a gene group (SEQ ID NOs: 102 and 103) having an expression differential power of ⁇ 3 times in the xylem of the trunk base and the xylem of the trunk central part.
  • the five gene groups have a gene group (SEQ ID NOs: 107, 109 to 111) in which the expression difference on the xylem side is 1.5 times or more in the central trunk portion compared to the phloem side, ⁇ In the center of the trunk, teacher A gene group (SEQ ID NOs: 107 and 109) having an expression differential power of ⁇ times or more on the xylem side compared to the xylem side is included.
  • the value of the average fluorescence intensity obtained from the experiment was calculated based on the trunk of the Eucalyptus chamaldrensis trunk (base or center) Z the trunk of the Eucalyptus globulus (base). It was calculated by the formula of the part or the xylem of the Eucalyptus globulus trunk (base) Z of the trunk of the eucalyptus chamaldrensis (base or middle part).
  • Some genes are not significant or have almost the same average intensity as "a group of genes whose expression increases on the xylem side of the Eucalyptus globulus tree compared to the xylem side of the Eucalyptus genus Camaldrensis" (The fluorescence intensity ratio of the tree part of Eucalyptus genus Camaldrensis compared to the tree part of Eucalyptus globulis
  • the expression difference on the xylem side was more than 4-fold in the trunk of Eucalyptus genus Camaldrensis, or the xylem side in the trunk of Eucalyptus globules
  • the gene (SEQ ID NO: 106) whose expression difference on the phloem side is 4 times or more is included.
  • the Eucalyptus BAC genomic library was screened by the method shown in Example 4. Oligonucleotide primer of SEQ ID NO: 123-144 synthesized based on cDNA sequence information in a DNA pool for 3D screening by PCR, based on cDNA sequence information (odd number of SEQ ID NO: 123-144 is forward primer, even number is reverse) Using primers, the genomic clones of each gene were screened by PCR. [0208] The insert sequence of the BAC clone was determined by the method shown in Example 1.
  • the genomic region and the promoter region of each gene were determined based on the cDNA sequence (SEQ ID NO: 101 to:
  • the genomic sequence determined based on the cDNA sequence described in L11 was determined. , Respectively, as shown in SEQ ID NOs: 145-155).
  • the promoter region was determined based on the full-length cDNA sequence including the translation initiation codon ATG (which encodes methionine in amino acids).
  • the determined promoter sequence is shown in SEQ ID NOs: 156-166.
  • the sequence upstream from the 5 'terminal base of the full-length cDNA was analyzed by genetic information processing software "GENETYX" (Software Development Co., Ltd.), and the TATA-box sequence was determined.
  • the promoter region of higher eukaryotes has a TATA-box sequence usually about 10 to 25 bases upstream from the transcription start site.
  • TATA-box sequence usually about 10 to 25 bases upstream from the transcription start site.
  • nucleotide sequence represented by SEQ ID NO: 101 the 87th to 662nd positions are the amino acid code region (SEQ ID NO: 112).
  • positions 262 to 1188 are the amino acid code region (SEQ ID NO: 113).
  • nucleotide sequence represented by SEQ ID NO: 103 the 257th to 1549th positions are the amino acid code region (SEQ ID NO: 114).
  • positions 159 to 722 are the amino acid code region (SEQ ID NO: 115).
  • positions 101 to 1444 are the amino acid code region (SEQ ID NO: 116).
  • the 163rd to 927th positions are the amino acid code region (SEQ ID NO: 117).
  • the 48th to 2300th positions are the amino acid code region (SEQ ID NO: 118).
  • the 23rd to 778th positions are the amino acid code region (SEQ ID NO: 119).
  • positions 40 to 1101 are the amino acid code region (SEQ ID NO: 120).
  • the 128th to 790th positions are the amino acid code region (SEQ ID NO: 121).
  • positions 269 to 1234 are the amino acid code region (SEQ ID NO: 122).
  • the 1st to 2680th positions of the nucleotide sequence represented by SEQ ID NO: 145 are the promoter region (SEQ ID NO: 156), the 2767th to 2796th positions, the 2884th to 2949th positions, and the 2nd 984th position.
  • -3142, 3265-3585 are etasons in the code area, 29797-2883, 2950-2983, 3143-3264, intron It is.
  • the 1st to 3000th positions of the nucleotide sequence represented by SEQ ID NO: 146 are the promoter region (SEQ ID NO: 157), positions 3262 to 3606, positions 3920 to 4040, and positions 5436. -5626th, 5829th-5960th, 6246th-6383th, Ekson in the force S code area, 3607th-3919th, 4041st-5435th, 5627th ⁇ No. 5828, No. 5961 ⁇ No. 6245 position S intron region.
  • the 1st to 1569th positions of the nucleotide sequence represented by SEQ ID NO: 147 are the promoter region (SEQ ID NO: 158), the 1826th to 2533th positions, the 4847th to 4967th positions, and the 5439th position.
  • -5632, 5709-5840, 5936-6072, 6158 are Etason in the code region, 2534-4846, 4968-5438, Intron regions are 5633 to 5708, 5841 to 5935, and 6073 to 6157.
  • the first to 2979th position of the nucleotide sequence represented by SEQ ID NO: 148 is the promoter region (SEQ ID NO: 159), 3241th to 3375th position, 3967th to 4063th position, 4159th position.
  • -No. 4202, No. 4293-No. 4282, No. 4539-No. 4736 are Eksons in the code area, No. 3376-No. 3966, No. 4064-No. 4158, No. 4203 Positions-4292, 4283-4538 positions S intron region.
  • the 1st to 1288th positions of the nucleotide sequence represented by SEQ ID NO: 149 are the promoter region (SEQ ID NO: 160), 1409th to 1964th position, 3755th to 3959th position, 4th 510th position -4585th, 4564th-4809th, 5066th-5446th Exon in the power code area, 1965-3754, 3960-4509, 4586-4586
  • the 4563rd and 4810th to 5065th positions are intron regions.
  • positions 1 to 1536 are the promoter region (SEQ ID NO: 161), positions 1723 to 1985, and positions 2217 to 2718 are in the code region. No. 1986 to No. 2216 are intron regions.
  • the 1st to 1658th positions of the nucleotide sequence represented by SEQ ID NO: 151 are the promoter region (SEQ ID NO: 162), the 1706th to 2167th positions, the 2407th to 2589th positions, the 2767th position.
  • -No. 3647, No. 3747-No. 3905, No. 4108-No. 4675 are Eksons in the code area, No. 2168-No. 2406, No. 2590-No. 2766, No. 3648-No.
  • the intron region is at positions 3746 and 3906-4107.
  • the 1st to 1979th positions of the nucleotide sequence represented by SEQ ID NO: 152 are the promoter region (SEQ ID NO: 163), positions 2002 to 2427, positions 2714 to 2793, positions 2984.
  • ⁇ Position 3233 is Xun Xun in the coding region
  • Positions 2428 to 2713 and Positions 2794 to 2983 are intron regions.
  • the 1st to 596th positions of the nucleotide sequence represented by SEQ ID NO: 153 are the promoter region (SEQ ID NO: 164), positions 637 to 770, positions 1143 to 1272, positions 1393 to 1 No. 1593, No. 2042 to No. 2209, No. 2939 to No. 3367 are exons in the code region, No. 771 to No. 1142, No. 1273 to No. 1392, No. 1594 to No. 2041 , Positions 2210 to 2938 are intron regions.
  • positions 1 to 2973 are the promoter region (SEQ ID NO: 165), positions 3101 to 3221, positions 3339 to 3468, and positions 4324.
  • ⁇ 4735 is the etason in the coding region, 3222 to 3338, and 3469 to 4323 is the intron region.
  • Sequence number 1 to position 1783 of the nucleotide sequence represented by SEQ ID NO: 155 is the promoter region Region (SEQ ID NO: 166), positions 2052 to 2270, positions 3339 to 3725, positions 4266 to 4625 are Xeon in the coding region, positions 2271 to 3338 , Positions 3726 to 4265 are intron regions.
  • the genes (SEQ ID NOs: 1-34, 101-: L11) expressed in the xylem forming tissues shown in Examples 3 and 6 are regulated in the xylem by their respective promoters. . Therefore, each promoter region can naturally be used for expression control in the xylem of an arbitrary gene. Therefore, reporter genes were connected downstream of these promoters and introduced into plants to confirm expression activity in xylem. In this example, the activity of the promoter region (SEQ ID NO: 93) of the cellulose synthase gene (SEQ ID NO: 3) was analyzed by the particle gun method.
  • the GUS gene was connected as a reporter downstream of the promoter region of the cellulose synthase gene (SEQ ID NO: 3) and introduced into Eucalyptus. Specifically, using the oligonucleotide primer of SEQ ID NO: 167, 168 synthesized based on the genome sequence information described in SEQ ID NO: 85, the BAC clone DNA is in a saddle shape and 5 ′ untranslated region by PCR. The promoter region containing was amplified, and after blunting, it was subcloned upstream of the GUS gene of the binary vector pBI121 vector (Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984) excluding the 35S promoter.
  • the binary vector pBI121 vector Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984
  • the above-described plasmid for introduction was introduced into a xylem-forming tissue with a particle gun, and the activity of the GUS gene was analyzed. 5th year eucalyptus growing vigorously Peeling the bark of the trunk and exposing it to the other side, the xylem formation tissue (Fig. 4A) force also cuts the xylem block (Fig. 4B), and the target for particle gun introduction did.
  • the introduction plasmid was coated on gold particles as described below. Immediately mix 50 ⁇ l of plasmid DNA with 51 (0.5 g / ⁇ 1) and 50 ⁇ l of 2.5 2 CaCa2, and add 20 ⁇ l of 0.1M spermidine. To precipitate gold particles.
  • the promoter sequence of the gene described in SEQ ID NOs: 1-34, 101-: L11, in which expression was observed in the xylem by the microarray experiment is the xylem in any gene. It can be used for expression control.
  • Transcription factor is controlled on / off ⁇ by binding to the promoter region of the gene under its control (JL Riechmann et al., SCIENCE VOL 290 15 DECEMBER 2000, Masaaki Iwabuchi, Kazuo Shinozaki, Plant genome function dynamism). Therefore, by analyzing the presence or absence of interaction (binding) with the promoter region of a gene, the relationship between control and control can be understood.
  • the transcription factor described in SEQ ID NO: 101-: L 11 (SEQ ID NO: 112-122) that controls the formation of tree cell walls and the tree described in SEQ ID NO: 1-34 (SEQ ID NO: 35-68) was analyzed the interaction of genes involved in cell wall formation with promoters.
  • the MYB transcription factor protein of SEQ ID NO: 117 and the homeodomain (HD-ZIP) transcription factor protein of SEQ ID NO: 119 are used as promoter regions (sequences of the gene involved in cellulose synthesis (CesA).
  • No. 93) shows the result of interaction between the lignin synthesis gene (CAD, OMT, C4H) promoter (SEQ ID NO: 96, 97, 99, respectively).
  • the MYB cDNA (SEQ ID NO: 106) was PCR-enhanced in a saddle shape using the primers shown in SEQ ID NOs: 169 and 170. Treat the amplified fragment with restriction enzymes Ndel and smal and insert it into pIVEX 1.3 vector (Roche) The reaction was performed at 24 ° C. for 24 hours using a cell-free translation system (wheat germ protein synthesis system kit RTS100; Roche). The reaction mixture (lul) was electrophoresed on SDS-PAGE, and expression of the target protein was confirmed by Western blotting using an ant His antibody.
  • HD-ZIP cDNA (SEQ ID NO: 108) was subjected to PCR using the primers described in SEQ ID NOs: 171 and 172.
  • the amplified DNA fragment was treated with restriction enzymes EcoRV and Sail, inserted into pET32a vector (Novagen), and transformed into E. coli BL21 strain.
  • pET32a vector Novagen
  • each probe was designed with both adjacent probe sequences and a 5 bp overlap, so a total of 40 probe pairs (sense / antisense) were artificially synthesized per promoter. At this time, the sense side DNA is
  • a (fluorescent dye) labeled For the analysis of the interaction, double-stranded DNA in which sense'antisense strands were used was used. For annealing, TAMRA-labeled sense oligo DNA (SEQ ID NO: 173 to 332) and unlabeled antisense oligo DNA (SEQ ID NO: 333 to 492) were adjusted to luM with 0.1M NaCl / TE (pH 8.0). Then, after denaturation treatment at 95 ° C for 5 minutes, it was performed while gradually returning to room temperature over 1 hour. Next, to remove unreacted single-stranded oligo DNA, add 10 mM MgCl and Exonuclease I, react at 37 ° C for 1 hour, and install the MERmaid SPIN Kit (Qbiogene).
  • the sense'antisense strand corresponds to the complementary strand of SEQ ID NO: 173 with SEQ ID NO: 333 (Probe No. 1), and thereafter SEQ ID NO: 174 and 334 (Probe No. 2) to SEQ ID NO: 332 Corresponding in order like 492 (Probe No.160).
  • the DNA-protein binding reaction was performed in 30 ul of reaction solution (25 mM Hepes / KOH (pH 7.9), 50 The reaction was performed with mM KC1, 0.5 mM DTT 5% glycerol, 2 mg / ml BSA 0.05 mg / ml poly (d to dC) '(d to dC), protein lul, 3 nM probe) at room temperature for 20 minutes.
  • FCS Fluorescence Correlation Spectroscopy
  • FCS Fluorescence Correlation Spectroscopy
  • standard Dye was measured 10 times X 5 times and reaction solution was measured 15 times X 5 times.
  • the translation time was calculated from this measured value, and the presence or absence of binding was verified. If binding is observed, the molecular weight of the labeled probe will increase, resulting in a longer translation time than the control.
  • the measured value of the empty vector expressed and each probe added to the reaction solution was used.
  • Fig. 5 shows the results of interaction analysis between the two transcription factors and the four promoters. From the results of the control experiment, it can be concluded that there is an interaction (bonding) when there is a translation time extension of at least 20%. From the results of analysis, in MYB, two sites of CesA promoter (SEQ ID NO: 181 and 341 double-stranded DNA, 199 and 359 double-stranded DNA), one CAD promoter (SEQ ID NO: 246 and 406), OMT promoter 1 (SEQ ID NOs: 274 and 434) and 2 sites of the C4H promoter (SEQ ID NOs: 296 and 456, 326 and 486).
  • CesA promoter SEQ ID NO: 181 and 341 double-stranded DNA, 199 and 359 double-stranded DNA
  • one CAD promoter SEQ ID NO: 246 and 406
  • OMT promoter 1 SEQ ID NOs: 274 and 434
  • 296 and 456, 326 and 486 296 and 456, 326
  • each of the cDNAs (SEQ ID NOs: 106 and 108) was subjected to PCR using the primers described in SEQ ID NOs: 169 to 172.
  • PBI121 vector (Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984) where the end of the amplified fragment was phosphorylated and blunt ended, and the GUS gene portion was removed beforehand using restriction enzymes. This was ligated downstream of the 35S plug motor and used as an introduction construct.
  • Agrobataterum LBA4404 strain
  • transformants were selected using LB agar medium (25 g / ml each of kanamycin, no, and idaromomycin, 1.0% Agar).
  • LB agar medium 25 g / ml each of kanamycin, no, and idaromomycin, 1.0% Agar.
  • Single colonies were picked from the transformed agrobataterum plates and cultured in LB liquid medium containing antibiotics (kanamycin, no, idaromomycin each g / m) (28.5 ° C, 240 rpm) o culture Thereafter, the agrobacterium was collected by centrifugation (5 ° C., 8000 rpm 5 min), the supernatant was removed, and MS liquid medium was added to gently suspend the precipitate. The suspension was collected in a 50 ml tube and diluted with MS liquid medium so that the OD550 of the suspension was 0.5. This suspension was used for the infection procedure.
  • FIG. 6 shows a transformed tobacco plant overexpressing the MYB transcription factor gene of SEQ ID NO: 106 and the homeodomain (HD-ZIP) transcription factor gene of SEQ ID NO: 108.
  • lignin content and fiber length were measured.
  • Lignin quantification total amount of acid-soluble 'insoluble lignin was determined by the Klason method (Wood Chemistry Experiments II. Chemistry P150-155, Patzlaff et al., 2003, Plant Journal). al., 1999, Kenaf Pro operties, Processing and Products. P149-167).
  • the ratio of lignin per bone dry weight averaged 14.7 ⁇ 1.8% (average of 5 individuals) compared to 19.3 ⁇ 1.1% of the control (wild strain).
  • the fiber length was 560.6 ⁇ 50.9um for the control, whereas it was 620.3 ⁇ 42.lum for the MYB-introduced individuals.
  • a decrease in the lignin ratio relative to the absolute dry weight indicates a relative increase in the cellulose ratio. Therefore, by overexpressing the MYB transcription factor, it was possible to modify the material with a long fiber length with a large amount of cellulose. This indicates that if the expression of this transcription factor is suppressed using a technique such as RNAi, the material can be altered to the opposite material (short fiber length with less cellulose).
  • Cell wall components, conduit distribution density, fiber length, etc. which are the main elements of wood fiber properties, are known as broad-leaved trees. .
  • This fluctuation phenomenon of wood fiber properties due to ground clearance has long been known as vertical fluctuation of the material in the trunk. Comparing each ground height (base ⁇ middle, crown), cellulose increases in cell wall components, and lignin and hemicellulose decrease in the center (above the chest and less than crown). .
  • morphological observation shows that the fiber length with a small conduit distribution density increases by about 20%.
  • the cell wall thickness increases from the crown to the base.
  • the bulk weight of the 5th-year eucalyptus plant was 432 kg / m 2 , the pulp yield was 43.8%, and the fiber length was 0.66 mm.
  • the central part the bulk weight was 525 kg / m 2 , the pulp yield was 53.4%, and the fiber length was 0.86 mm. From these results, it is confirmed in the sample of this example that the vertical fluctuation force is generally called. It was. In addition, it was confirmed that the center part having high fiber weight and high pulp yield and long fibers was superior as the material.
  • the materials of the test samples were examined.
  • the center of trunk of 2 individuals (Sl, 2) of 7th grade Eucalyptus maldorensis was used.
  • RNA extraction and microarray experiments were performed in Examples 1 to 3, and material analysis was performed according to the method described above.
  • the sample site to be examined can be at the trunk base, at the center, or anywhere else.
  • trunk center it is generally expected that the part will have the best material in the individual, so it can be said that the identified material is the best material in the individual.
  • trunk bases The opposite is true for trunk bases.
  • the expression was strong in caml (bad material, one), the gene group was S1, the expression was strong in cam5 (good material, V), and the gene group was highly expressed in S2. From this, it is determined that S2 has a better material than S1. (3) In the inspection of (4), S2 was judged to be closer to the center than S1, and the result was that S2 was superior in material quality. The result of direct comparison between the xylem was also the result that supported it.
  • S1 and S2 were analyzed by the method described in Example 11 (1).
  • S2 in test weight is S 1, 45.7% pulp yield in SI, 50.1% in S2, 0.69 mm fiber length by S1, was 0.75mm in S2 . Therefore, as expected based on the above-mentioned inspection results, S1 was a material close to the base, and S2 was a basic but somewhat central material. Also, comparing both, S2 was relatively better as expected. This As described above, the material of the sample to be tested can be discriminated alive by comparison based on the expression profile shown in the present application.
  • the cellulose of the Globules species generally increases cellulose, while lignin and hemicellulose decrease.
  • morphological observation shows an increase in fiber length when the conduit density is low.
  • the cell wall is thick. The details are unknown, but the nature of the wood fiber-forming tissue differs depending on the species, and it is thought that the difference in the nature of the wood fiber formed is caused.
  • the above-mentioned conventional method for refining kraft pulp using the trunk base of Eucalyptus genus Camaldrensis and the trunk base of Eucalyptus globulus are used.
  • the bulk weight was 432 kg / m 2
  • the pulp yield was 43.8%
  • the fiber length was 0.66 mm.
  • the gross base of Groplus had a bulk weight of 574 kg / m 2 , a pulp yield of 57.1%, and a fiber length of 0.95 mm. From these results, it is generally said that the variability between species varies. It was also confirmed in the sample. In addition, it was also confirmed that the GLOPLUS trunk base with high bulk weight and long fibers was superior as the material.
  • the material By comparing the expression profile of each sample obtained with the taxa in Tables 3 and 7, and examining whether it is similar to the profile of chamaldrensis or that of globus, The material (camaldrensis or glopuls) can be distinguished.
  • the sample site to be examined may be the trunk base, the center, or any other location.
  • Profile of 9 genes showing inverse correlation: 8 genes with camaldrense-like profile (X> P) and 1 gene with globulous-like profile (X P P) Camaldrensis-specific There are 14 genes that show X> P: 12 genes that are chamaldrensic (X> P) and 2 genes that are Globnores (X P).
  • C1 and G1 were analyzed by the method described in Example 11 (1). As a result, 534kg / m 2 at 449kg / m 2, G1 in test weight is C 1, 45.7% for C1 pulp yield 53.6% for G1, fiber length was 0.86mm in C1 0.69 mm, in G1 . Therefore, the material is based on the above inspection results As expected, CI was a camaldrensis-like material and G1 was a globus-like material. Also, comparing both, G1 was relatively better as expected. In this way, the species and material of the test sample can be discriminated alive by comparison based on the expression profile shown in the present application.
  • the present inventors Using the prepared eucalyptus EST database, the present inventors extracted a group of genes involved in cell wall synthesis in a eucalyptus tree fiber-forming tissue by microarray analysis, and the difference in the height and species of the ground. Expression variation was examined. As a result, we identified a cell wall synthetic gene group that is expressed / excited in eucalyptus tree fiber-forming tissues, and a gene group whose expression fluctuates predominantly due to differences in the height and species of the ground. Furthermore, these promoter DNAs were obtained and confirmed to function as promoters for xylem expression. The above gene group and expression information 'promoter DNA can be used to control cell wall synthesis and morphogenesis in wood fiber-forming tissues.
  • the present inventors examined the expression variation due to the difference in the height and species of the ground using the prepared eucalyptus EST database to extract a group of transcription factors involved in tree fiber formation by microarray analysis. .
  • a group of transcription factors involved in eucalyptus tree fiber formation identified a group of genes whose expression fluctuated predominantly depending on the height of the ground, and obtained their promoter DNA.
  • plant materials can be altered by introducing these transcription factors.
  • the above gene set and expression information 'promoter DNA can be used to control wood fiber formation (material).
  • FIG. 1 A graph showing the gene expression intensity of the 5-year-old ginger stem base of Eucalyptus camaldorensis. Probes for oligo microarray analysis by labeling each cRNA, which is synthesized from two types of mRNA extracted from the phloem side of the trunk base and the xylem side, in a cage shape with two fluorescent dyes (cy3.cy5) each And used for hybridization. The fluorescence intensity is calculated from the image obtained by scanning and analyzed with Rosetta's analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%. The relative intensity of expression of major gene groups at the base is shown.
  • + (black) in the vicinity of or above the upper part of the two calibration curves shows a significant increase in expression at the wood fiber formation site.
  • + (Light gray) in the middle of the two calibration curves shows a significant decrease in expression, and the two calibration curves are near or below the lower line + (dark gray) Indicates that there is no change in expression.
  • + (black) in the vicinity of or above the upper two of the two calibration curves shows a significant increase in expression at the wood fiber formation site, and is almost in the middle of the two calibration curves
  • + (dark gray) in the vicinity of or below the lower line of the two calibration curves indicates no change in expression.
  • ⁇ 3 It is a figure showing the gene expression intensity of Eucalyptus globulis spp.
  • Each cRNA synthesized from the phloem side and xylem side of the trunk is synthesized in a cage shape and labeled with two fluorescent dyes (cy3.cy5) for use as probes in oligo microarray analysis. I did a hybridization. Image power obtained by scanning Fluorescence intensity is calculated and analyzed with Rosetta analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%.
  • FIG. 4 is a graph showing the promoter activity of genes involved in wood fiber formation.
  • A shows the trunk from which the xylem-forming tissue block that is the target for particle gun introduction was excised.
  • B shows the formation of xylem by histological GUS assembly after gene transfer by particle gun. Indicates an organization block.
  • C and D are enlarged photographs of the blue spot where the GUS gene was expressed (the central part surrounded by a circle).
  • FIG. 5-a is a diagram showing the interaction (binding) between the promoter of the cell wall-forming gene group involved in tree fiber formation and the transcription factor group.
  • Each graph shows the analysis results of the MYB transcription factor and the four promoters.
  • the horizontal axis represents the number of the double-stranded DNA probe
  • the vertical axis represents the percentage increase in translation time relative to the control (probe and empty vector reaction solution).
  • the smaller probe number on the horizontal axis of each graph is the upstream region on the 5 ′ side of the promoter, and the larger one is closer to the transcription start point and the downstream region on the 3 ′ side of the promoter.
  • * Indicates a probe that has been recognized to interact.
  • FIG. 5-b is a diagram showing the interaction (binding) between the promoter of the cell wall-forming gene group involved in tree fiber formation and the transcription factor group.
  • Each graph shows the results of analysis of HD-ZIP transcription factors and four promoters.
  • the horizontal axis represents the number of the double-stranded DNA probe
  • the vertical axis represents the rate of increase in translation time relative to the control (probe and empty vector reaction solution) in%.
  • the smaller probe number on the horizontal axis of each graph is the upstream region on the 5 ′ side of the promoter, and the larger probe number is closer to the transcription start point and the downstream region on the 3 ′ side of the promoter.
  • the asterisk (*) indicates a probe that has been recognized to interact!
  • [6] It is a diagram showing a transformed tobacco plant into which a transcription factor has been introduced.
  • A is a transformant introduced with a MYB transcription factor
  • B is a transformant introduced with a homeodomain transcription factor.

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Abstract

En construisant et en utilisant une banque de données EST d'eucalyptus, des gènes participant à la synthèse de la paroi cellulaire dans un tissu formant les fibres cellulosiques d'eucalyptus et des facteurs transcriptionnels régulant l'expression de ces gènes sont extraits par analyse micro-réseau et leurs différences dans l'expression sont analysées en fonction de la hauteur à ciel ouvert et des essences. En conséquence, des gènes de synthèse de la paroi cellulaire exprimés dans le tissu formant les fibres cellulosiques d'eucalyptus et des facteurs transcriptionnels sont spécifiés et l'on a découvert qu'ils présentaient une variation coordonnée dominante de l'expression en fonction de la hauteur à ciel ouvert et des essences. En outre, leurs ADN promoteurs sont obtenus. On estime que les gènes et les AND promoteurs/données d'expression décrits ci-dessus peuvent être utilisés dans le contrôle de la synthèse de la paroi cellulaire par le tissu formant les fibres cellulosiques et la formation morphologique.
PCT/JP2006/305402 2005-03-31 2006-03-17 Adn codant pour une proteine ayant pour fonction de former et de reguler la paroi cellulaire de fibre cellulosique dans un tronc d'arbre et leurs and promoteurs WO2006109424A1 (fr)

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WO2007141705A2 (fr) * 2006-06-02 2007-12-13 Mondi Business Paper: South Africa Promoteurs végétaux
JP2009005684A (ja) * 2007-05-30 2009-01-15 Oji Paper Co Ltd 植物の形態改変方法
JP2009142219A (ja) * 2007-12-14 2009-07-02 National Agriculture & Food Research Organization アルコール発酵に適した新規酵母及びそれを用いたアルコール製造方法
WO2011010639A1 (fr) * 2009-07-22 2011-01-27 株式会社ブリヂストン Gène marqueur pour la formation de tube de latex, procédé pour cribler un adjuvant de formation de tube de latex, et procédé pour améliorer la formation de tubes de latex dans une plante
CN102090303A (zh) * 2010-11-10 2011-06-15 天津滨海国际花卉科技园区股份有限公司 好望角毛膏菜水培方法
WO2021193111A1 (fr) * 2020-03-27 2021-09-30 日本製紙株式会社 Copeaux de bois et leur utilisation

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MXPA05013190A (es) * 2003-06-06 2007-05-23 Arborgen Llc Factores de transcripcion.

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007141705A2 (fr) * 2006-06-02 2007-12-13 Mondi Business Paper: South Africa Promoteurs végétaux
WO2007141705A3 (fr) * 2006-06-02 2008-06-19 Mondi Business Paper South Afr Promoteurs végétaux
JP2009005684A (ja) * 2007-05-30 2009-01-15 Oji Paper Co Ltd 植物の形態改変方法
JP2009142219A (ja) * 2007-12-14 2009-07-02 National Agriculture & Food Research Organization アルコール発酵に適した新規酵母及びそれを用いたアルコール製造方法
WO2011010639A1 (fr) * 2009-07-22 2011-01-27 株式会社ブリヂストン Gène marqueur pour la formation de tube de latex, procédé pour cribler un adjuvant de formation de tube de latex, et procédé pour améliorer la formation de tubes de latex dans une plante
CN102090303A (zh) * 2010-11-10 2011-06-15 天津滨海国际花卉科技园区股份有限公司 好望角毛膏菜水培方法
CN102090303B (zh) * 2010-11-10 2013-02-20 天津滨海国际花卉科技园区股份有限公司 好望角毛膏菜水培方法
WO2021193111A1 (fr) * 2020-03-27 2021-09-30 日本製紙株式会社 Copeaux de bois et leur utilisation
JPWO2021193111A1 (fr) * 2020-03-27 2021-09-30
JP7053962B2 (ja) 2020-03-27 2022-04-12 日本製紙株式会社 木材チップ及びその用途

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