JPWO2006109424A1 - DNA encoding a protein having a function of forming and controlling a tree fiber cell wall of a plant trunk and its promoter DNA - Google Patents

Abstract

The present inventors use the prepared eucalyptus EST database to extract genes involved in cell wall synthesis and transcription factors controlling their expression in eucalyptus tree fiber-forming tissues by microarray analysis, and Expression analysis by species difference was performed. As a result, we identified cell wall synthesis genes and transcription factors that are expressed in eucalyptus tree fiber-forming tissues, and identified that they expressed and fluctuated predominantly depending on the ground height and species. Furthermore, promoter DNA of those groups was obtained. It is considered that the above gene group and expression information / promoter DNA can be used to control cell wall synthesis and morphogenesis of wood fiber-forming tissues.

Description

The present invention relates to a DNA encoding a protein having a function of forming a tree fiber cell wall of a plant 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.

  Mankind has long used woody biomass such as trunks, roots, leaves, and branches in a wide variety of industrial fields such as papermaking, construction, feed, and fuel. The world's woody biomass consumption is increasing every year, and by use, the consumption of wood-fired wood accounts for a majority, and it is still increasing in developing regions. Consumption of industrial materials such as lumber and wood chips for papermaking has also been increasing in the developing countries, although the consumption in developed regions has slightly decreased. In advanced areas, with the development of civilization, forests have been greatly reduced by diverting forests to farmland and using them as materials, but recently they have increased slightly due to tree planting activities. However, in the developing regions, once the population has grown due to commercial logging in developed countries, the demand for fuel and farmland has increased in recent years, and the forest area throughout the world has been rapidly decreasing. For these reasons, problems such as global warming due to a decrease in carbon dioxide fixation ability have been raised. In recent years, afforestation projects aiming at a stable supply of lumber and wood chips for papermaking have been actively conducted around the world while solving these problems.

  Industries that use woody biomass are re-recognized from the perspective of improving global environmental issues as they can continue to use sustainable resources in the future, and recycling industries that use carbon sources to replace current fossil resources As expected. As a concrete example, Japanese paper companies are actively promoting tree planting projects centering on early tropical trees such as Eucalyptus and Acacia in order to achieve a sustainable and stable supply of woody biomass. ing. As an example of the scale of the afforestation project, Oji Paper is aiming for an afforestation area of 300,000 ha by fiscal 2010, and is conducted in a wide area centering on the Pacific Rim such as Oceania and Southeast Asia. This is none other than the advancement of other industrial fields in the recycling-type material production (biomass recycling) of industrial use and regeneration of biomass through large-scale plantation at the business level.

  Furthermore, along with the progress of such afforestation projects, artificially controlling the formation of tree fibers in the trunk, which is the main woody biomass, the amount of cell wall components (cellulose, hemicellulose, lignin) as well as the quality Can be changed freely, and the fiber morphology (wood fiber cell elongation) can be freely changed. As a result, the quantity and quality of woody biomass can be increased, and future energy use and Application expansion as an industrial raw material is also expected.

  Most of the woody biomass in the trunk is composed of tissue cells called wood fibers. The specific gravity, cell wall structure and components (cellulose, hemicellulose, lignin), etc. of wood fiber tissue are the main factors that determine the properties of wood fibers. When used as industrial raw materials, various properties that meet the specifications of each product are required. . For example, paper made from wood fiber with high specific gravity has higher resistance to beating and tear strength compared to that with low specific gravity, but on the other hand, the density, tensile, rupture and folding strength of pulp sheet are not. Low.

  It is known that the nature of this wood fiber cell varies depending on the height of the ground, age, and species regardless of whether it is a hardwood or a conifer (Non-Patent Document 1, Non-Patent Document 2). The variation of wood fiber properties due to age is known as mature and immature wood for a long time, the variation due to ground height is known as vertical variation in the trunk of the material, and the variation due to species is known as variation between species. For example, when a matured material and an immature material are compared with each other, in the matured material, cellulose increases in cell wall components, and lignin and hemicellulose decrease. In addition, morphological observation shows that the conduit distribution density is small and the fiber length is increased. The same difference also occurs in the ground height and species difference (Non-Patent Documents 1 to 3). Although details are unknown, it is considered that a difference in the properties of the formed wood fibers occurs because the cell wall synthesis in each wood fiber forming tissue is different. The gene group responsible for cell wall synthesis with such different properties has not been identified.

  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. For example, in the case of Arabidopsis thaliana, which is the main model, almost no wood fibers are formed during the vegetative growth period, but are formed in a very small part within the flower stem of the reproductive growth stage (Non-patent Document 4). Tree fiber cells differentiate from the formation layer tissue and are called wood fibers, whereas herbaceous fiber cells differentiate mainly from parenchyma cells between vascular bundles, interfascicular fibers. be called. Thus, herb fiber cells are not the same as wood fiber cells that grow over many years. Therefore, in order to identify a gene group that is truly necessary for tree fiber formation, it must be analyzed using trees.

  Plant tree fiber formation-related genes and their regulatory factors cannot be distinguished / identified simply by homology search or motif search with known regulatory factors. For example, 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). Furthermore, in the family, 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 place and role are divided in various ways. From sequence motifs and homology searches, only high homology is shown, and the role and the like cannot be specified. For example, about 40 cellulose synthases (CesA) involved in cellulose synthesis are expected in the Arabidopsis genome. Among them, RSW1 (Non-Patent Document 6) has been identified as CesA essential for cellulose synthesis of the primary wall, and IRX3 (Non-patent Document 7) has been identified as CesA essential for the synthesis of secondary wall cellulose. The homology between the two amino acid sequences is 65% as a whole, partially exceeding 90% and very high. Thus, since the role cannot be identified simply by the sequence homology score, in order to specify the wood fiber cell wall synthesis gene group, a detailed expression analysis or the like is required.

Just as the genes of the cellulose / lignin synthesis system form a large number of families in the genome and are expressed in groups of various levels, their regulators also form a large number of families and diverse The expression is divided into groups of levels, and roles cannot be distinguished only by sequence motifs or homology searches. For example, 50 or more families and 1500 or more genes have been identified as regulators in the Arabidopsis genome (Non-patent Document 8). Among them, for example, there are about 190 regulatory factors belonging to the MYB family, and it is gradually being clarified that each is involved in a specific life phenomenon by detailed expression analysis and the like. Moreover, for example, a homologous search factor NtLIM1 (Patent Document 1) identified in tobacco as a regulator of a phenylpropanoid biosynthesis system represented by lignin contained in wood fiber is known search site ( http: //www.ncbi.nlm.nih.gov/blast/ ) shows high homology with many LIM proteins. Even rat LIM proteins that do not form wood fibers are hit with a high homology score. In Arabidopsis thaliana, hit with a high score (56-87%) with at least 10 LIM proteins, but it is unclear which LIM protein is involved in the regulation of the lignin synthesis system in Arabidopsis. Thus, since the role of a regulatory factor cannot be identified only by homology with a known regulatory factor, detailed expression analysis or the like must be performed. However, there is no knowledge of comprehensive expression analysis of regulatory factors in trees.

  As described above, the same wood fiber as the wood is not formed in the herb. In addition, a group of genes related to cellulose / lignin synthesis in the wood fiber cell wall cannot be identified merely by homology with known genes. That is, information relating to herbaceous fiber formation cannot be used as it is for tree fiber formation of trees. It is difficult for herbs to identify the genes of the cellulose and lignin synthesis systems that are truly involved in the synthesis of wood fiber cell walls. By analyzing the expression of wood fiber formation sites using trees, it is possible to identify genes required for cellulose / lignin synthesis in the wood fiber cell wall. However, there are few examples of using a tree to identify a cellulose / lignin synthesis system gene involved in wood fiber cell wall synthesis (Non-patent Documents 9 and 10).

  In addition to the difference in the gene group that works during fiber formation between herbaceous and woody, the existence of various wood fiber cell wall synthetic genes is also expected in woody plants. For example, since the form of leaves and trunks are different between young and middle-aged, it is expected that a plurality of tree fiber cell wall synthetic genes that function according to age function. In addition, as mentioned above, vertical fluctuations due to tree height and fluctuations between tree species due to tree species occur, so that a plurality of tree fiber cell wall synthetic genes that are different within the same individual or between species may function uniquely. is expected. Each of these wood fiber cell wall synthetic genes only shows high homology with each other in sequence, and it cannot be distinguished 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.

  Also in woody plants, tree analysis studies at the genome level using poplar and pine have been conducted (Non-patent Documents 7 and 9). However, the materials used are separate organs and tissue units such as roots, leaves and 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 and their promoter information and expression information by analyzing expression in different tree heights and species of wood fiber forming tissues.

  By the way, at the site of the afforestation project, the growth status of the afforestation trees is judged by appearance, and in order to know the quality of the pulp material, an analysis of the tree fiber, which is the pulp material, is carried out by a chemical method after logging. (Material analysis). As for the growth state, because it depends on experience and intuition, it is impossible to grasp problems such as lack of nutrients that cannot be distinguished by appearance. And since the material is only known after logging, we do not know what the state of the tree fiber formation (material) in the growing plantation tree is, what kind of tree fiber will be formed in the future, etc. There is no policy for the production of materials (fertilization, thinning, etc.). In addition, when selecting a good afforestation tree, it is only possible to use a growth amount (tree height / diameter) that can be judged on the appearance as long as it is growing. Thus, it is very important to predict tree fiber formation (materials) during growth in consideration of operations in plantations, selection / breeding, and securing high-quality papermaking raw materials.

  As described above, even in the same species / individual plant, there is a difference in the growth state such as the state of wood fiber formation, which cannot be discriminated in appearance. Therefore, an inspection marker for predicting wood fiber formation (material) during growth has been required. However, until now, no test marker has been developed for growth or wood fiber formation.

Prior art document information related to the invention of the present application will be described below.
Japanese Patent No. 3444191 Shimaji Ken, Sudo Shoji and Harada Hiroshi "Organization of Wood", Morikita Publishing, 1976, 111-215 Satoshi Furuno and Osamu Sawabe, "Wood Science Course 2, Organization and Materials", Kaiseisha, 1994, 109-137 Wood Science and Technology. 2001; 35: 229-243. Plant Physiology, 2001, 126: 477-479. Plant Physiology, 2000, 124: 495-498. Science 1998, 279: 717-720. Plant Cell 1999, 11: 769-780. Edited by Masaki Iwabuchi and Kazuo Shinozaki “Dynamics of Plant Genomic Function” Schupinger Fairlark Tokyo, 2001, 1-34 GenomeBiol. 2002; 3 (12): REVIEWS1033. Proc Natl Acad Sci USA 2001 Dec 4; 98 (25): 14732-7.

The nature of woody biomass varies depending on the cell walls and morphology of wood fibers. By artificially controlling the formation of the cell walls and morphology of the wood fibers, woody biomass can be produced as an industrial raw material effectively and efficiently. The world is still dependent on large quantities of fossil resources such as oil and natural gas. In order to change this situation with new technology, efficient utilization of trees as woody biomass is the key to this. In overseas countries, the establishment of technology for effective utilization of tree biomass is positioned as an important issue at the beginning of this century. In fact, coniferous pine, broadleaf poplars in the US, spruce and poplar in Canada, and poplars in Scandinavia are the target species. In order to contribute to more effective and efficient production of woody biomass on a global scale, there is a great need to identify genes that control tree fiber cell formation 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 them, and a transformation using the plasmid. The object is to provide a plant cell, microorganism or plant body. Another object of the present invention is to provide a use of 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 tree part.

  As a result of extensive research, the present inventors have obtained a gene group that forms the eucalyptus tree fiber cell wall and comprehensive analysis of expression and function using a genomic science approach to solve this problem. I came to the conclusion that I should do. In other words, by creating an expression gene database (EST database) that includes wood fiber-forming tissues and analyzing the expression at different ground high and species tree fiber formation sites, the target gene group that cannot be distinguished by sequence homology search The authors concluded that it would be desirable to use a method that comprehensively identifies and obtains and obtains and analyzes promoter DNA fragments of these genes. Furthermore, in order to give plants useful characteristics for human use by controlling artificial expression of improved genes using genetic engineering techniques, various new characteristics can be selectively expressed in appropriate plant tissues. It was concluded that the identification of tissue specific genes was necessary.

  In carrying out this research, analysis resources in Eucalyptus were developed. Specifically, EST databases were created for various gene libraries, trunks, leaves, and roots. In addition, Arabidopsis, which is widely used as a model plant all over the world, possesses gene libraries and mutants involved in cell wall biosynthesis, and these causative genes have already been analyzed.

  Using the eucalyptus EST database thus prepared, the present inventors examined extraction of genes involved in cell wall synthesis in eucalyptus tree fiber-forming tissues by microarray analysis, and expression variations due to differences in ground height and species. As a result, we identified a cell wall synthesis gene group of eucalyptus tree fiber forming tissue whose expression changes preferentially depending on the ground height and species. Furthermore, their promoter DNA was obtained. It is considered that the above gene group and promoter DNA can be used for control of cell wall synthesis and morphogenesis of wood fiber-forming tissue and wood fiber-specific gene expression.

  The gene group of the present invention, from a detailed expression pattern, "the gene group whose expression level is increased on the xylem side compared to the phloem side in the stem base and the center of the stem of Eucalyptus genus Camaldrensis", “In the stem base of Eucalyptus genus Camaldrensis, the expression level decreased on the xylem side compared to the phloem side, and the expression level increased on the xylem side in the middle of the stem compared to the phloem side. In the central stem of Eucalyptus genus Camaldrensis, the expression level increases on the xylem side compared to the phloem side, and the expression level is the same on the phloem side and xylem side in the trunk base In the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules, the gene group whose expression level is increased on the xylem side compared to the phloem side, in the trunk of Eucalyptus genus Camaldrensis Gene groups with increased expression levels on the xylem side compared to the phloem side, and decreased expression levels on the xylem side compared to the phloem side in the Eucalyptus globulus trunk, "Eucalyptus camal In the trunk of Drensis, the expression level increased on the xylem side compared to the phloem side, and in the trunk of the Eucalyptus globulus, the gene group with the same expression level on the phloem side and the xylem side ” It was divided roughly. It is considered that the expression (level) information of this gene group can be used for the quantitative and qualitative advance prediction of the plant growth state, the wood fiber formation state, and the material. As a result, in the afforestation business, appropriate operations (weeding, thinning, etc.) can be performed at the appropriate time based on the growth state, and as a result, the efficiency of pulp production, cost reduction, and new characteristics have been achieved. Paper can be manufactured.

  As a result of the DNA encoding the protein having the function of forming the tree fiber cell wall of the eucalyptus tree trunk obtained by the present invention, as well as the technology for comprehensively controlling them and the expression information / promoter DNA, Various quantitative and qualitative changes, such as high cellulose, low lignin, thick cell walls, thin cells, long fiber lengths, short ones, etc., depending on the characteristics of the new transgenic eucalyptus varieties obtained using the gene group There is expected. In addition, it is expected to be used for distinguishing materials such as ground height, seeds, and growth state based on expression information.

That is, the present invention provides the following [1] to [18] regarding 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.
[1] 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).
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 68
(B) DNA containing the base sequence described in any one of SEQ ID NOs: 1 to 34
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 35-68
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 34
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 35-68
[2] The DNA according to [1], wherein the plant is eucalyptus.
[3] A partial DNA of the DNA according to [1] or [2].
[4] A substrate on which the DNA according to any one of [1] to [3] is immobilized.
[5] On the phloem side and xylem side of the trunk of the plant, the expression level of at least one DNA described in the following (a) to (e) is detected, and the DNA on the phloem side and xylem side is detected. A method for examining a growth state of a stem part of a plant, comprising a step of comparing expression levels.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 64 or 66 to 68
(B) DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 30 or 32 to
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 35-64 or 66-68
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1-30 or 32-34
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 35-64 or 66-68
[6] A process of detecting the expression level of at least one DNA described in the following (a) to (e) in two different tree parts, and comparing the expression level of the DNA in two different tree parts A method for inspecting the growth state of two different stem parts including
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67
(B) DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, 8, 14, 16, 17, 19 to 22, 24, 25, 28, 30, 32, or 33
(C) SEQ ID NO: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67% of the protein consisting of the amino acid sequence and 50% or more DNA encoding a protein with homology
(D) DNA having the nucleotide sequence set forth in any one of SEQ ID NOs: 1-6, 8, 14, 16, 17, 19-22, 24, 25, 28, 30, 32, or 33, and stringent conditions DNA that hybridizes under
(E) SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67, one or more amino acids are substituted. DNA encoding proteins consisting of amino acid sequences deleted, added, and / or inserted
[7] The expression level of at least one DNA described in the following (a) to (e) is detected on the phloem side and the xylem side of the trunk of the plant, and the DNA on the phloem side and the xylem side is detected. A method for examining a growth state of a stem part of a plant, comprising a step of comparing expression levels.
(A) SEQ ID NO: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66, or 67. DNA
(B) DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, 8, 9, 11 to 14, 16, 17, 19 to 25, 27, 28, 30, 32, or 33
(C) SEQ ID NO: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66, or 67% of the protein consisting of the amino acid sequence described in 67 DNA encoding a protein having the above homology
(D) DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, 8, 9, 11 to 14, 16, 17, 19 to 25, 27, 28, 30, 32, or 33 and stringent DNA that hybridizes under mild conditions
(E) SEQ ID NOs: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66, or one or more amino acids in the amino acid sequence according to 67 DNA encoding a protein consisting of an amino acid sequence in which is substituted, deleted, added, and / or inserted
[8] A step of detecting the expression level of at least one DNA described in the following (a) to (c) in two different tree parts, and comparing the expression level of the DNA in two different tree parts A method for inspecting the growth state of two different stem parts including
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56
(B) DNA comprising the base sequence set forth in any one of SEQ ID NOs: 11, 12, 17, 21, or 22
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 11, 12, 17, 21, or 22
(E) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56 DNA to encode
[9] A protein encoded by the DNA of [1] or [2].
[10] A promoter DNA comprising the nucleotide sequence of any of the following (a) to (c).
(A) the base sequence according to any of SEQ ID NOs: 93 to 100 (b) consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a), and (A) a base sequence having a function as a promoter substantially the same as the promoter comprising the DNA of (a) (c) comprising a part of the base sequence of (a), and substantially the same as the promoter comprising the DNA of (a) A base sequence having the same function as a promoter [11], further comprising a base sequence that recognizes a transcription factor involved in specific expression in a plant fiber-forming tissue; Promoter DNA.
[12] The DNA according to any one of the following (a) to (e).
(A) DNA encoding an antisense RNA complementary to the transcription product of the DNA according to [1]
(B) DNA encoding an RNA having ribozyme activity that specifically cleaves the transcript of the DNA of [1]
(C) DNA encoding an RNA that suppresses the expression of the DNA according to [1] by an RNAi effect
(D) DNA encoding an RNA that suppresses the expression of the DNA according to [1] due to a co-suppression effect
(E) DNA encoding a protein having a dominant negative trait for the transcription product of the DNA according to [1]
[13] A recombinant vector having the DNA according to any one of [1] to [3] or [10] to [12].
[14] A microorganism carrying a plasmid comprising the vector according to [13].
[15] A transformed plant cell into which the vector according to [14] has been introduced.
[16] A transformed plant regenerated from the transformed plant cell according to [15].
[17] A transformed plant which is a descendant or clone of the transformed plant according to [16].
[18] A propagation material for the transformed plant according to [16] or [17].

  As a result of intensive studies, the present inventors have solved the problem by obtaining a transcription factor group that controls the expression of a gene group forming a eucalyptus tree fiber cell wall by a genomic scientific approach, It was concluded that a comprehensive analysis on expression and function should be performed. In other words, by creating an expression gene database (EST database) that includes wood fiber-forming tissues and analyzing the expression at different ground high and species tree fiber formation sites, the target gene group that cannot be distinguished by sequence homology search The authors concluded that it would be desirable to use a method that comprehensively identifies and obtains and obtains and analyzes promoter DNA fragments of these genes. Furthermore, in order to give plants useful characteristics for human use by controlling artificial expression of improved genes using genetic engineering techniques, various new characteristics can be selectively expressed in appropriate plant tissues. It was concluded that the identification of tissue specific genes was necessary.

  In carrying out this research, analysis resources in Eucalyptus were developed. Specifically, EST databases were created for various gene libraries, trunks, leaves, and roots. In addition, Arabidopsis, which is widely used as a model plant all over the world, possesses gene libraries and mutants involved in cell wall biosynthesis, and these causative genes have already been analyzed.

  The present inventors examined the expression variation by the difference in the height of the ground and the kind using the produced eucalyptus EST database, extraction of transcription factor groups involved in eucalyptus tree fiber cell wall formation by microarray analysis. As a result, we identified a group of transcription factors that regulate the expression of Eucalyptus tree fiber cell wall-forming genes, whose expression varies predominantly depending on the ground height and species. Furthermore, their promoter DNA was obtained.

  The gene group of the present invention, from a detailed expression pattern, "the gene group whose expression level is increased on the xylem side compared to the phloem side in the stem base and the center of the stem of Eucalyptus genus Camaldrensis", "In the central part of the stem of Eucalyptus genus Camaldrensis, the expression level is increased on the xylem side compared to the phloem side, and in the trunk base, the gene group with the same expression level on the phloem side and the xylem side", "In the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules, the gene group whose expression level is increased on the xylem side compared to the phloem side", " Compared with the gene group whose expression level is increased on the xylem side and the expression level is decreased on the xylem side compared to the phloem side in the trunk of the Eucalyptus globules, "Eucalyptus genus Camaldrensis In the trunk, the expression level increases on the xylem side compared to the phloem side, and in the trunk of the Eucalyptus globules, the gene group is roughly divided into 5 groups of “expression groups with the same expression level on the phloem side and the xylem side” It was. It is considered that the expression (level) information of this gene group can be used for the quantitative and qualitative advance prediction of the plant growth state, the wood fiber formation state, and the material. As a result, in the afforestation business, appropriate operations (weeding, thinning, etc.) can be performed at the appropriate time based on the growth state, and as a result, the efficiency of pulp production, cost reduction, and new characteristics have been achieved. Paper can be manufactured.

  Regarding the DNA encoding the eucalyptus transcription factor obtained by the present invention, as well as the technology for comprehensively controlling them and the expression information / promoter DNA, as one result, the genes obtained using these gene groups Various quantitative and qualitative changes such as high cellulose, low lignin, thick cell walls, thin cells, long fiber lengths, short ones, etc. are expected due to the characteristics of new varieties of eucalyptus. In addition, it is expected to be used for distinguishing materials such as ground height, seeds, and growth state based on expression information.

That is, the present invention provides the following [1] to [17] with respect to a DNA encoding a plant transcription factor, a promoter DNA fragment thereof, and use thereof.
[1] DNA encoding a plant transcription factor according to any one of the following (a) to (e):
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 112 to 122
(B) DNA comprising the base sequence described in any one of SEQ ID NOs: 101 to 111
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 112-122
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 101 to 111
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NOS: 112 to 122
[2] The DNA according to [1], wherein the plant is eucalyptus.
[3] A partial DNA of the DNA according to [1] or [2].
[4] A substrate on which the DNA according to any one of [1] to [3] is immobilized.
[5] On the phloem side and xylem side of the trunk of the plant, the expression level of at least one DNA described in the following (a) to (e) is detected, and the DNA on the phloem side and xylem side is detected. A method for examining a growth state of a stem part of a plant, comprising a step of comparing expression levels.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 112 to 118 or 120 to 122
(B) DNA comprising the base sequence set forth in any of SEQ ID NOs: 101 to 107 or 109 to 111
(C) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 112-118 or 120-122
(D) DNA that hybridizes under stringent conditions with the DNA consisting of the nucleotide sequence of SEQ ID NO: 101 to 107 or 109 to 111
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 112-118 or 120-122
[6] A process of detecting the expression level of at least one DNA described in the following (a) to (e) in two different tree parts, and comparing the expression level of the DNA in two different tree parts A method for inspecting the growth state of two different stem parts including
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 113 or 114
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(C) DNA encoding a protein having 50% or more homology with the protein comprising the amino acid sequence set forth in SEQ ID NO: 113 or 114
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 113 or 114
[7] The expression level of at least one DNA described in the following (a) to (e) is detected on the phloem side and the xylem side of the trunk of the plant, and the DNA on the phloem side and the xylem side is detected. A method for examining a growth state of a stem part of a plant, comprising a step of comparing expression levels.
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 113 to 117
(B) DNA comprising the base sequence according to any of SEQ ID NOs: 102 to 106
(C) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 113 to 117
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOS: 102 to 106
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 113 to 117
[8] A step of detecting the expression level of at least one DNA described in the following (a) to (c) in two different tree parts, and comparing the expression level of the DNA in two different tree parts A method for inspecting the growth state of two different stem parts including
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 116
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 105
(C) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence set forth in SEQ ID NO: 116
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 105
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 116
[9] A protein encoded by the DNA of [1] or [2].
[10] A promoter DNA comprising the nucleotide sequence of any of the following (a) to (c).
(A) the base sequence according to any one of SEQ ID NOs: 156 to 166 (b) consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a), and (A) a base sequence having a function as a promoter substantially the same as the promoter comprising the DNA of (a) (c) comprising a part of the base sequence of (a), and substantially the same as the promoter comprising the DNA of (a) A base sequence having the same function as a promoter [11], further comprising a base sequence that recognizes a transcription factor involved in specific expression in a plant fiber-forming tissue; Promoter DNA.
[12] A recombinant vector having the DNA of any one of [1] to [3], [10] or [11].
[13] A microorganism carrying a plasmid comprising the vector according to [12].
[14] A transformed plant cell into which the vector according to [12] has been introduced.
[15] A transformed plant regenerated from the transformed plant cell according to [14].
[16] A transformed plant that is a descendant or clone of the transformed plant according to [15].
[17] A propagation material for the transformed plant according to [15] or [16].

  By using the DNA of the present invention, artificially controlling plant fiber formation, in particular, changing the quantity and quality of cell wall components (cellulose, hemicellulose, lignin), which are the essence, and fiber morphology ( It has become possible to freely change the elongation of the wood fiber cells and the thickness of the cell wall, and to control the gene expression specific to the wood fiber. In other words, an increase in quantity and quality of woody biomass are expected, which can be expected to increase the efficiency of pulp production and papermaking and to produce paper with new characteristics. In addition, it can be expected to expand applications for future energy use and utilization as industrial raw materials.

  Moreover, the DNA of the present invention can be used as a test marker for a trunk part of a plant. By using the DNA of the present invention, it is possible to examine the growth state of the trunk part of the plant, the formation state of the wood fiber, the pulp quality, and the selection of the plant having a useful character.

  The present invention provides a DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk (a protein involved in cellulose synthesis and a protein involved in lignin synthesis) and a promoter DNA.

  It is considered that 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 the control of plant tree fiber cell formation. On the other hand, it is considered that the promoter DNA of the DNA can be used to control the expression specific to the wood fiber tissue or the wood fiber formation time. In addition, the expression (level) information of DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk is used for the quantitative and qualitative advance prediction of the plant growth state, tree fiber formation state, and material. It is considered possible. Therefore, by using these, artificial modification of tree fiber cell morphogenesis, tree fiber cell-specific expression of any gene / protein, plant growth state, tree fiber formation state, material quantitative / qualitative It is thought that it can be used for simple prior prediction. As a result, it is possible to increase the efficiency of pulp manufacture, reduce costs, and manufacture paper with new characteristics.

  Controlling wood fiber formation in plants has various important implications in the fields of industry and agriculture. For example, the modification of the wood fiber properties of the Eucalyptus genus Camaldrensis species 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, and examples thereof include useful agricultural crops (including forage crops) such as cereals, vegetables and fruit trees, fiber raw material plants such as pulp, and ornamental plants such as foliage plants. Can be mentioned. The plant is not particularly limited, eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugarcane, rapeseed, soybean, sunflower, Examples include cotton, orange, grape, peach, pear, apple, tomato, cabbage, cabbage, radish, carrot, pumpkin, cucumber, melon, parsley, orchid, chrysanthemum, lily, saffron and the like.

In the present invention, 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.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 68
(B) DNA containing the base sequence described in any one of SEQ ID NOs: 1 to 34
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 35-68
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 34
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 35-68

  In the present invention, stringent hybridization conditions include, for example, conditions of standing overnight at 60 ° C. in a 0.1 × SSC solution, or hybridization conditions of stringency equivalent thereto. Under such conditions, DNA that hybridizes with DNA consisting of the base sequence described in any one of SEQ ID NOs: 1 to 34 can be isolated.

  Specifically, DNA is extracted from a plant, a gene library is constructed, and screening is performed under the same conditions, or the extracted DNA is described in any one of SEQ ID NOs: 1 to 34 From the base sequence, an arbitrary 20-mer sequence can be used as a primer, and a contiguous neighboring sequence can be easily obtained by the TAIL-PCR method established by Liu et al.

  The present invention also provides a DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence set forth in any of SEQ ID NOs: 35-68. Such DNA can be isolated by those skilled in the art by generally known methods. For example, hybridization techniques (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) techniques (Saiki, RK. Et al., Science, 1985, 230, 1350., Saiki, RK. Et al., Science 1988, 239, 487.). That is, the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1 to 34 or a part thereof as a probe, and specifically to the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1 to 34 Isolation of a DNA having a high homology with a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 34 from a plant using a hybridizing oligonucleotide as a primer is usually possible for those skilled in the art. is there.

  In order to isolate such DNA, the hybridization reaction is preferably carried out under stringent conditions. In the present invention, stringent hybridization conditions refer to hybridization conditions of 6M urea, 0.4% SDS, 0.5 × SSC or equivalent stringency. It is expected that DNA with higher homology can be isolated under conditions of higher stringency, for example, 6M urea, 0.4% SDS, 0.1 × SSC. The DNA thus isolated is considered to have high homology with the amino acid sequence encoded by the DNA comprising the base 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 sequence identity.

  The identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 1990, 87, 2264-2268., Karlin, S. & Altschul, SF., Proc. Natl by Carlin and Arthur. Acad. Sci. USA, 1993, 90, 5873.). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul, SF. Et al., J Mol Biol, 1990, 215, 403.). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov/).

  In addition, the DNA of the present invention encodes a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 35-68. DNA to be included.

  In order to prepare the above DNA, methods well known to those skilled in the art include, for example, site-directed mutagenesis method (Kramer, W. & Fritz, HJ., Methods Enzymol, 1987, 154, 350 .)) To introduce a mutation.

  The 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 Site-directed Mutagenesis Kit" (manufactured by Clontech) for mutation or substitution, Deletion can be performed using “Quantum leap Nested Deletion Kit” (Clontech).

  Further, even if the base sequence is mutated, there are cases where the mutation does not accompany amino acid mutation (degenerate mutation), and such degenerate mutated DNA is also included in the present invention.

  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. For example, the genomic DNA prepared according to the method described in the literature (Rogers and Bendich, Plant Mol. Biol., 1985, 5, 69.) is used as a template for the nucleotide sequence of the DNA of the present invention (for example, SEQ ID NO: It can be prepared by performing PCR (Saiki et al. Science, 1988, 239, 487.) using a primer prepared based on the base sequence described in any one of 1 to 34). In the case of cDNA, it is prepared by preparing mRNA from a plant by a conventional method (Maniatis et al. Molecular Cloning Cold Spring Harbor Labortry Press), performing a reverse transcription reaction, and performing PCR using the same primers as described above. Is possible. For genomic DNA and cDNA, a genomic DNA library or cDNA library is prepared by a conventional method. For example, the base sequence of the DNA of the present invention (for example, any one of SEQ ID NOs: 1 to 34) is prepared against this library. It can also be prepared by screening using a probe synthesized based on the described base sequence). The base sequence of the obtained DNA can be easily determined by using, for example, “Sequencer Model 373” (manufactured by ABI).

  In the present invention, DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree trunk includes a xylem compared to the phloem side in the stem base and the center of the stem of Eucalyptus genus Camaldrensis described later. The expression level is lower on the xylem side compared to the phloem side and compared to the phloem side in the center of the trunk. In the central part of the stem of Eucalyptus genus Camaldrensis, the expression level is increased on the xylem side compared to the phloem side, and the DNA whose expression level is equivalent on the xylem side and xylem side "," DNA whose expression level is increased on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globulus ", "eucalyptus In the stem of Camaldorensis, the expression level is increased on the xylem side compared to the phloem side, and in the stem of the Eucalyptus globules, the expression level is decreased on the xylem side compared to the phloem side ”,“ In the stem of Eucalyptus genus Camaldrensis, the expression level is increased on the xylem side compared to the phloem side, and in the stem of Eucalyptus globules, the expression level is the same on the phloem side and xylem side. Is included.

  In the trunk, it is known that in the phloem and the xylem, wood fiber formation is performed only on the xylem side. Therefore, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side” can also be expressed as “DNA whose expression level is increased or decreased at the wood fiber formation site”. . In addition, in the xylem of the trunk base and the xylem in the middle of the trunk, the xylem in the middle of the trunk is longer than the xylem in the middle of the trunk. Is thick, cell wall is thick, fibril inclination is slow, wood fiber rate and specific gravity are high (mature material). Compared with the xylem of the center of the trunk, the xylem at the base of the trunk is shorter in the length of the fiber, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, 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 of the stem base of eucalyptus compared to the phloem side” is “DNA whose expression level is increased or decreased on the immature wood formation site” It can also be expressed as 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 tree trunk” is “DNA whose expression level is increased or decreased on the matured material formation site” Can also be expressed.

  Furthermore, in the xylem of the Camaldrensis trunk and the globules of the trunk, the globules trunk xylem is longer in the fiber length (pulp quality) than the chamaldrensis trunk xylem. Is thicker, the cell wall is thicker, the fibril tilt angle is lower, and the wood fiber rate and specific gravity are known to be higher. It is also known that the xylem of the Camaldrensis trunk is shorter, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, and the wood fiber ratio and specific gravity are lower than the globules trunk. It has been. Therefore, “DNA with increased or decreased expression level on the xylem side compared with the phloem side in the stem of Eucalyptus genus Camaldrensis” is “wood fiber length is short, diameter is thin, cell wall is thin. It can also be expressed as “DNA whose expression level is increased or decreased at a wood fiber formation site having a low-quality pulp property that fibril inclination is steep and wood fiber ratio and specific gravity are low”. In addition, “in the stem of Eucalyptus globulus, the DNA whose expression level is increased or decreased on the xylem side compared to the phloem side” is “wood fiber length is long, diameter is thick, cell wall is thick, fibrils. It can also be expressed as “DNA whose expression level is increased or decreased at a site where a wood fiber having a high-quality pulp characteristic of a low inclination angle and a high wood fiber ratio and specific gravity” is formed.

  In the present invention, the phloem side means the outside of the vascularization layer, which is a meristem, and preferably the bark formation site on the phloem side. Here, the bark forming site is a site that is differentiating into or has the differentiation ability of a mentor cell, a phloem tube, parenchyma cell, a renal cell, or the like. Moreover, in this invention, a xylem side means an inner side from a vascular bundle formation layer, Preferably it means the wood fiber formation site | part of a xylem side. Here, the wood fiber forming site is a site during differentiation into a wood fiber cell, a temporary conduit, a conduit, a parenchymal cell or the like or having the differentiation ability.

  In the present invention, the trunk base refers to a generally lower breast height, for example, a eucalyptus fifth-year plant refers to a ground height of less than about 1 m. The central portion of the trunk generally refers to the vicinity of the middle portion of the tree height. For example, in the case of a eucalyptus plant of 5 years above a tree 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 tree trunk part of a plant (preferably a tree base part or a tree part of a tree trunk central part). That is, by using at least one of the above DNAs or a partial DNA thereof, it is possible to examine the growth state of the trunk part of the plant (preferably the base part of the trunk or the central part of the trunk).

  More specifically, in the following cases (1) to (4), it is determined that the trunk portion of the plant is a xylem that is actively formed with fiber. Furthermore, in the case of (2), it is determined that the trunk portion of the plant is a xylem in which immature wood is formed or a xylem in which immature wood is formed in the future. Here, the immature wood means a wood fiber differentiated and formed from an immature formed layer structure, and has a short fiber length and a diameter compared with a xylem of the center of the trunk collected under the same conditions. Is thin, cell wall is thin, fibril inclination is steep, and wood fiber rate and specific gravity are low. In the case of (3) or (4), it is determined that the trunk portion of the plant is a xylem in which a mature material is formed or a xylem in which a mature material is formed in the future. Here, the matured wood means a wood fiber formed by differentiation from a mature formation layer structure, and the wood fiber length is longer and the diameter is larger than the xylem of the trunk base collected under the same conditions. Is thick, cell wall is thick, fibril inclination is gentle, and wood fiber rate and specific gravity are high.

  The plant used in the test of the present invention is preferably Eucalyptus, more preferably Eucalyptus camaldrensis species or Eucalyptus Globulus species.

In addition, by using the inspection method of the present invention, quantitative and qualitative advance prediction of materials can be performed. In afforestation projects, appropriate operations (weeding, thinning, etc.) can be carried out at appropriate times based on the growth state. For example, if there is a correlation between the expression pattern in the phloem and xylem of the trunk base in the present invention and the expression pattern obtained by the test method of the present invention (preferably Pearson's correlation coefficient is 0.5 or more, more preferably 0.7 or more Qualitatively immature fibers (unripe wood: short fibers, thin diameter, thin cell walls, steep fibril inclination, shrub fiber ratio and low specific gravity) are formed in the trunk portion of the test tree. It can be predicted that the amount of wood fibers (volume weight) will be small or will be small in the future (average volume weight 450 to 500 kg / m ³ or less). On the other hand, if there is a correlation between the expression pattern in the phloem and xylem of the central part of the trunk in the present invention and the expression pattern obtained by the test method of the present invention, the qualitatively mature fiber (Mature material: long fiber, thick diameter, thick cell wall, loose fibril inclination, high wood fiber rate and high specific gravity) are formed or are in the future formed state, the amount of wood fiber (volume weight ) Or in the future (average bulk weight of 450 to 500 kg / m ³ or more). Furthermore, the material prediction as described above reveals whether the plant growth state is good or bad. If it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, weeding work is performed early. Or, if the planting density is high, thinning can be performed quickly and appropriately. At present, the material prediction and the determination of the operation policy as described above depend largely on the experience and intuition of the field or the subsequent chemical analysis, and there is no scientific index that can be discriminated in advance as shown in the present application.

(1) At the phloem side and the xylem side of the trunk of the plant (preferably the trunk base or the center of the trunk) The expression level of at least one DNA (preferably a plurality, more preferably all, the same applies hereinafter) is detected, and expression of the DNA on the phloem side and xylem side is detected. As a result of comparing the levels, the expression level of the DNA in the trunk of the plant is increased on the xylem side compared with the phloem side. The DNA described in any of the following (a) to (e) is mentioned as “DNA whose expression level is increased on the xylem side compared with the phloem side”.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66 or 67
(B) DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, 8, 9, 11 to 14, 16, 17, 19 to 25, 27, 28, 30, 32, or 33
(C) SEQ ID NOs: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66 or 67 and a protein comprising 50% or more of the amino acid sequence DNA encoding a protein with homology of
(D) SEQ ID NOs: 1 to 6, 8, 9, 11, 14, 16, 17, 19 to 25, 27, 28, 30, 32, or 33. DNA that hybridizes under conditions
(E) SEQ ID NO: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66 or 67, wherein one or more amino acids are DNA encoding a protein consisting of a substituted, deleted, added, and / or inserted amino acid sequence
“SEQ ID NO: 1-6, 8, 9, 11-14, 16, 17, 19, 25-25, 27, 28, 30, 32, or 33” as the DNA : DNA consisting of the base sequence described in any one of 1 to 6, 9, 11 to 14, 16, 17, 19, 20, 22, 25, 27 or 30 is preferable. SEQ ID NOs: 1, 3 to 6, 9 Although DNA which consists of a base sequence in any one of 11-14, 16, 17, 19, 22, 25 or 30 is more preferable, it is not limited to these.

(2) At the phloem side and the xylem side of the plant trunk (preferably the trunk base), the expression level on the xylem side of the stem base of Eucalyptus genus Camaldrensis is reduced compared to the phloem side, At the center of the trunk, the expression level of at least one of the DNAs whose expression level is increased on the xylem side compared to the phloem side is detected, and the expression of the DNA on the phloem side and the xylem side is detected. As a result of comparing the levels, when the expression level of the DNA in the trunk of the plant is reduced on the xylem side compared to the phloem side, in the present invention, "in the stem base of Eucalyptus genus Camaldrensis, The expression level is decreased on the xylem side compared to the side, and the expression level is increased on the xylem side compared to the phloem side in the center of the trunk ”as the following (a) to ( e) DNAs described in any of the above.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 49
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 15
(C) DNA encoding a protein having 50% or more homology with the protein comprising the amino acid sequence set forth in SEQ ID NO: 49
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 15
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 49

(3) In the phloem side and the xylem side of the trunk of the plant (preferably the middle part of the trunk), the expression level is decreased on the xylem side compared to the phloem side in the above-mentioned “base of Eucalyptus camaldrensis” In the central part of the trunk, the expression level of at least one of the DNAs whose expression level is increased on the xylem side compared to the phloem side is detected, and the DNA on the phloem side and the xylem side is detected. As a result of comparing the expression level, the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side

(4) At the phloem side and the xylem side of the trunk of the plant (preferably the central part of the trunk), the expression level is increased on the xylem side compared to the phloem side in the central part of the stem of Eucalyptus genus Camaldrensis In the trunk base, the expression level of at least one of the DNAs having the same expression level on the phloem side and the xylem side was detected, and the expression levels of the DNA on the phloem side and the xylem side were compared. As a result, in the case where the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side, in the present invention, "in the central part of the stem of Eucalyptus genus Camaldrensis, compared with the phloem side Then, the expression level is increased on the xylem side, and in the trunk base, the DNA having the same expression level on the phloem side and the xylem side ”is the DNA according to any of the following (a) to (e): Is mentioned.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 41, 44, 52, 60, 63 or 68
(B) DNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 7, 10, 18, 26, 29 or 34
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of any one of SEQ ID NOs: 41, 44, 52, 60, 63 or 68
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 7, 10, 18, 26, 29, or 34
(E) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 41, 44, 52, 60, 63 or 68 DNA encoding
"DNA consisting of the base sequence described in any of SEQ ID NO: 7, 10, 18, 26, 29 or 34" is the base sequence described in any of SEQ ID NO: 7, 10, 18, 26 or 29 DNA comprising the nucleotide sequence of SEQ ID NO: 18, 26 or 29 is more preferred, but not limited thereto.
Furthermore, the present invention provides a method for examining the growth state (maturity) of two different trunk parts. That is, the present invention provides a method for inspecting which of two different trunk parts is mature or immature. In the present invention, “different two tree parts” includes a tree part in two different plants and two different tree parts in one plant. The above method can also be used to select a mature or immature tree part from a plurality of tree parts.
More specifically, in the case of (5) or (6) below, of the two different trunk parts, the other trunk part is compared with one of the trunk parts (hereinafter referred to as trunk part A). The xylem (hereinafter referred to as the trunk tree part B) is a mature xylem, and the trunk tree part A is determined to be an immature xylem compared to the trunk tree part B. Therefore, the following (5) or (6) 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 and the like is performed by using numerical values of appearance such as tree height and diameter and material analysis values after felling as an index, and there is no technique (concept) for selecting with the above index.

(5) The expression level of at least one of “different DNAs whose expression level is increased on the xylem side of the trunk base as compared with the xylem side of the central part of the eucalyptus tree trunk” in two different stem parts. In the present invention, as a result of comparing the expression level of the DNA in two different stem parts, the expression level of the DNA is increased in the stem part A compared to the stem part B. As the “DNA whose expression level is increased on the xylem side of the trunk base compared to the xylem side of the central part of the eucalyptus tree”, the DNA described in any of the following (a) to (e) is Can be mentioned.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 67 or 68
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 33 or 34
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence set forth in SEQ ID NO: 67 or 68
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 33 or 34
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 67 or 68
The “DNA consisting of the base sequence described in SEQ ID NO: 33 or 34” is preferably DNA consisting of the base sequence described in SEQ ID NO: 33, but is not limited thereto.

(6) The expression level of at least one of “different DNAs whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the central part of the eucalyptus tree trunk” in two different stem parts. In the present invention, as a result of comparing the expression level of the DNA in two different stem parts, the expression level of the DNA is reduced in the stem part A compared to the stem part B. As the “DNA whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the central part of the eucalyptus trunk”, the DNA described in any of the following (a) to (e) is Can be mentioned.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62 or 64
(B) DNA comprising the base sequence described in any one of SEQ ID NOs: 1-6, 8, 14, 16, 17, 19-22, 24, 25, 28, or 30
(C) SEQ ID NO: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62 or a protein having 50% or more homology with the protein comprising the amino acid sequence of any one of DNA encoding
(D) It hybridizes under stringent conditions with DNA consisting of the base sequence described in any one of SEQ ID NOs: 1 to 6, 8, 14, 16, 17, 19 to 22, 24, 25, 28, or 30. DNA
(E) SEQ ID NO: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62 or 64, one or more amino acids are substituted, deleted or added And / or DNA encoding a protein consisting of an inserted amino acid sequence
“SEQ ID NOS: 1-6, 8, 14, 16, 17, 19-22, 24, 25, 28 or 30” includes DNAs comprising the nucleotide sequences described in SEQ ID NOs: 1-6, 14 , 16, 17, 19, 20, 22, 25, or 30 is preferable, and any one of SEQ ID NOs: 1, 3 to 6, 14, 16, 17, 22, or 25 is preferable. Although DNA consisting of the described base sequences is more preferred, it is not limited thereto.
Further, in the present invention, in the following cases (7) to (10), it is determined that the trunk part of the plant is a xylem with vigorous tree fiber formation. Further, in the case of (8) or (9), the length of the tree fiber is shorter, the diameter is thinner, and the cell wall of the stem part of the plant is shorter than that of the globulus trunk collected under the same conditions. It is a xylem in which wood fibers having a pulp characteristic of being thin and having a steep fibril inclination and a low wood fiber ratio and specific gravity are formed, or a xylem in which wood fibers having the pulp characteristics are formed in the future. It is determined. Further, in the case of (10), the length of the tree fiber is longer, the diameter is thicker, the cell wall is thicker, and the fibril is larger than the xylem of the Camaldrensis trunk collected under the same conditions. Decided to be a xylem in which wood fibers having excellent pulp characteristics such as a gentle inclination and a high wood fiber ratio and specific gravity are formed, or a xylem in which wood fibers having the pulp characteristics will be formed in the future Is done.
In addition, by using the inspection method of the present invention, quantitative and qualitative advance prediction of materials can be performed. In afforestation projects, appropriate operations (weeding, thinning, etc.) can be carried out at appropriate times based on the growth state. For example, if there is a correlation between the expression pattern in the phloem and xylem of the Eucalyptus genus Camaldorensis trunk in the present invention and the expression pattern obtained by the test method of the present invention (preferably Pearson's correlation coefficient of 0.5 or more, more Preferably 0.7 or higher, the same applies below), and the quality of the wood fiber (low quality pulp: short fiber, thin diameter, thin cell wall, steep fibril inclination, shrub fiber rate and low specific gravity) is formed, or a future state formed, the amount of wood fiber (volume weight) becomes small or in the future less (average volume weight 450~500kg / m ³ or less) Can be predicted. On the other hand, if there is a correlation between the expression pattern in the phloem and xylem of the Eucalyptus globulus trunk in the present invention and the expression pattern obtained by the test method of the present invention, the test tree trunk is qualitatively superior. Fiber (high quality pulp: long fiber, thick diameter, thick cell wall, loose fibril inclination, high wood fiber rate and high specific gravity), or in the state of being formed in the future, the amount of wood fiber ( It can be predicted that there will be a lot of (bulk weight) or increase in the future (average bulk weight of 450 to 500 kg / m ³ or more). Furthermore, the material prediction as described above reveals whether the plant growth state is good or bad. If it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, weeding work is performed early. Or, if the planting density is high, thinning can be performed quickly and appropriately. At present, the material prediction and the determination of the operation policy as described above depend largely on the experience and intuition of the field or the subsequent chemical analysis, and there is no scientific index that can be discriminated in advance as shown in the present application.

(7) At the phloem side and the xylem side of the trunk of the plant (preferably the trunk base or the middle part of the trunk) As a result of detecting the expression level of at least one DNA among the “DNA whose expression level is increased” and comparing the expression level of the DNA on the phloem side and the xylem side, the expression level of the DNA on the trunk of the plant However, in the present invention, in the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules, the expression level on the xylem side compared to the phloem side Examples of “rising DNA” include DNAs described in any of the following (a) to (e).
(A) DNA encoding a protein comprising the amino acid sequence set forth in any of SEQ ID NOs: 45, 46, 51, 55, 56 or 64
(B) DNA comprising the base sequence set forth in any of SEQ ID NOs: 11, 12, 17, 21, 22, or 30
(C) DNA encoding a protein having 50% or more homology with the protein comprising the amino acid sequence of any one of SEQ ID NOs: 45, 46, 51, 55, 56 or 64
(D) DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 11, 12, 17, 21, 22, or 30
(E) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, 56 or 64 DNA encoding
"DNA consisting of the base sequence described in any of SEQ ID NO: 11, 12, 17, 21, 22 or 30" refers to the base sequence described in any of SEQ ID NO: 11, 12, 17, 22 or 30 A DNA consisting of the nucleotide sequence described in any one of SEQ ID NOS: 11, 12, 17 or 22 is more preferable, but it is not limited thereto.

(8) In the phloem side and the xylem side of the trunk of the plant (preferably the trunk base or the middle of the trunk), in the trunk of the Eucalyptus genus Camaldrensis, the expression level on the xylem side compared to the phloem side In the stem of Eucalyptus globules, the expression level of at least one of the DNAs whose expression level is decreased on the xylem side compared to the phloem side is detected, and the phloem side and the xylem side are detected. As a result of comparison of the expression level of the DNA in the plant, the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side. In the present invention, “the stem of Eucalyptus genus Camaldrensis” In the DNA, the expression level is increased on the xylem side compared to the phloem side, and the expression level is reduced on the xylem side compared to the phloem side in the trunk of the Eucalyptus globules `` (A) to (e) Any of the DNAs described above can be mentioned.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 42, 47, 53, 57-59 or 67
(B) DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 8, 13, 19, 23-25 or 33
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 42, 47, 53, 57 to 59 or 67
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 8, 13, 19, 23-25 or 33
(E) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 42, 47, 53, 57-59 or 67 DNA encoding
“DNA consisting of the base sequence described in any of SEQ ID NO: 8, 13, 19, 23-25 or 33” is described in any of SEQ ID NO: 8, 13, 19, 24, 25 or 33. DNA consisting of the base sequence is preferable, and DNA consisting of the base sequence described in any of SEQ ID NOs: 13, 19, or 25 is more preferable, but is not limited thereto.

(9) On the phloem side and xylem side of the trunk of the plant (preferably the trunk base or the middle of the trunk), the expression level is higher on the xylem side than on the phloem side in the stem of Eucalyptus genus Camaldrensis. In the trunk of Eucalyptus globules, the expression level of at least one of the DNAs having the same expression level on the phloem side and the xylem side is detected, and the expression of the DNA on the phloem side and the xylem side is detected. As a result of comparing the levels, when the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side, in the present invention, in the eucalyptus genus Camaldrensis trunk, The expression level is increased on the xylem side compared to the DNA of the Eucalyptus globules, and the expression level is the same on the phloem side and the xylem side ”as any of the following (a) to (e) The DNA of crab The
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 40, 43, 48, 50, 54, 61, 62 or 66
(B) DNA containing the base sequence described in any one of SEQ ID NOs: 1 to 6, 9, 14, 16, 20, 27, 28, or 32
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 40, 43, 48, 50, 54, 61, 62 or 66
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1-6, 9, 14, 16, 20, 27, 28, or 32
(E) SEQ ID NO: 35 to 40, 43, 48, 50, 54, 61, 62 or 66, wherein one or more amino acids are substituted, deleted, added, and / or inserted. DNA encoding a protein consisting of a specific amino acid sequence
Examples of the “DNA comprising the base sequence described in any one of SEQ ID NOs: 1-6, 9, 14, 16, 20, 27, 28 or 32” include SEQ ID NOs: 1-6, 9, 14, 16, 20 Or a DNA consisting of the base sequence described in any one of 27 is preferable, and a DNA consisting of the base sequence described in any one of SEQ ID NOs: 1, 3-6, 14 or 16 is more preferable, but is not limited thereto. is not.

(10) In the phloem side and the xylem side of the trunk of the plant (preferably the trunk base), the expression level is increased on the xylem side compared with the phloem side in the above-mentioned "eucalyptus genus camaldrensis trunk, In the trunk of the Eucalyptus globules, the expression level of at least one of the DNAs whose expression level is reduced on the xylem side compared to the phloem side is detected, and the DNA on the phloem side and the xylem side is detected. As a result of the comparison of the expression level of the plant, the expression level of the DNA in the trunk of the plant is decreased on the xylem side compared with the phloem side. The method of inspecting the growth state (pulp quality) of the trunk part of). That is, the present invention provides a method for inspecting which of the different trunk parts of two plants is superior in pulp quality or inferior. This method can also be used to select excellent or inferior trunk parts from a plurality of tree parts.
More specifically, in the case of the following (11) or (12), among the different tree trunk parts of two plants, the other is compared with the tree part of one plant (hereinafter referred to as plant A). The stem part of the plant (hereinafter referred to as plant B) is a xylem having an excellent pulp quality. Compared with the stem xylem B, the stem xylem A is a xylem having an inferior pulp quality. It is determined that there is. 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 and the like is performed by using numerical values of appearance such as tree height and diameter and material analysis values after felling as an index, and there is no technique (concept) for selecting with the above index.

(11) In the trunk part of two different plants (preferably, the root part of the trunk or the central part of the trunk part), "the xylem part of the Eucalyptus genus Camaldrensis compared to the xylem side of the Eucalyptus globules" As a result of detecting the expression level of at least one of the DNAs whose expression level is increased on the side and comparing the expression level of the DNA in the trunk part of two different plants, the expression level of the DNA is In the present invention, the expression level is increased on the xylem side of the stem of Eucalyptus genus Camaldrensis as compared to the xylem side of the stem of Eucalyptus globulis. Examples of the “DNA” include DNAs described in any of the following (a) to (e).
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51 or 56
(B) DNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 11, 12, 17 or 22
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51 or 56
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 11, 12, 17 or 22
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 45, 46, 51 or 56
The “DNA consisting of the base sequence described in any of SEQ ID NO: 11, 12, 17 or 22” is preferably a DNA consisting of the base sequence described in SEQ ID NO: 12 or 17, and any one of SEQ ID NO: 12 Although DNA which consists of a base sequence as described in is more preferable, it is not limited to these.

(12) In the trunk part of the two different plants (preferably, the root part of the trunk or the central part of the trunk part), “the xylem part of the Eucalyptus genus Camaldrensis as compared to the xylem part of the Eucalyptus globulus trunk” As a result of detecting the expression level of at least one of the DNAs whose expression level is reduced on the side and comparing the expression levels of the DNA in the trunk parts of two different plants, the expression level of the DNA is When decreased in plant A compared to plant B In the present invention, the expression level is decreased on the xylem side of the stem of Eucalyptus genus Camaldrensis as compared to the xylem side of the stem of Eucalyptus globulus. Examples of the “DNA” include DNAs described in any of the following (a) to (e).
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 55
(B) DNA comprising the base sequence set forth in SEQ ID NO: 21
(C) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence set forth in SEQ ID NO: 55
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 21
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 55

  By using the inspection method of the present invention, a xylem corresponding to any of the above (1) to (12) or a plant having the xylem is selected, and the selected xylem or a plant having the xylem is selected. It is possible to produce uniform pulp from the above, and it is possible to increase the efficiency of pulp production, reduce costs, and produce paper with new characteristics. In addition, after selecting a xylem corresponding to any of the following (1) to (12) or a plant having the xylem, it has a selected xylem or the xylem according to a method well known to those skilled in the art. Pulp can also be produced after confirming whether the wood fiber state is uniform among plants. Moreover, the xylem which does not correspond to either of the following (1)-(12) or the plant which has this xylem can be collected, and the pulp of arbitrary mixing | blendings can also be produced.

The present invention also provides a DNA encoding a plant transcription factor and a promoter DNA.
It is considered that the DNA encoding a plant transcription factor in the present invention can be used to control plant fiber formation. On the other hand, it is considered that the promoter DNA of the DNA can be used to control the expression specific to the wood fiber tissue or the wood fiber formation time. In addition, the expression (level) information of DNA encoding a protein that has a function to control the formation of tree fiber cells in plant trunks can be used for quantitative and qualitative prediction of plant growth state, tree fiber formation state, and material quality. It is considered that it can be used. Therefore, by using these, artificial modification of tree fiber cell morphogenesis, tree fiber cell-specific expression of any gene / protein, plant growth state, tree fiber formation state, material quantitative / qualitative It is thought that it can be used for simple prior prediction. As a result, it is possible to increase the efficiency of pulp manufacture, reduce costs, and manufacture paper with new characteristics.

  Controlling wood fiber formation in plants has various important implications in the fields of industry and agriculture. For example, the modification of the wood fiber properties of the Eucalyptus genus Camaldrensis species 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, and examples thereof include useful agricultural crops (including forage crops) such as cereals, vegetables and fruit trees, fiber raw material plants such as pulp, and ornamental plants such as foliage plants. Can be mentioned. The plant is not particularly limited, eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugarcane, rapeseed, soybean, sunflower, Examples include cotton, orange, grape, peach, pear, apple, tomato, cabbage, cabbage, radish, carrot, pumpkin, cucumber, melon, parsley, orchid, chrysanthemum, lily, saffron and the like.

In the present invention, the DNA encoding a plant transcription factor includes the DNA described in any of (a) to (e) below.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 112 to 122
(B) DNA comprising the base sequence according to any one of SEQ ID NOs: 101 to 111
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 112-122
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 101 to 111
(E) DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NOs: 112 to 122

  In the present invention, stringent hybridization conditions include, for example, conditions of standing overnight at 60 ° C. in a 0.1 × SSC solution, or hybridization conditions of stringency equivalent thereto. Under such conditions, DNA that hybridizes with DNA consisting of the base sequence set forth in any of SEQ ID NOs: 101 to 111 can be isolated.

  Specifically, DNA is extracted from a plant, a gene library is constructed, and screening is performed under the same conditions, or the extracted DNA is described in any one of SEQ ID NOs: 101 to 111 From the base sequence, an arbitrary 20-mer sequence can be used as a primer, and a contiguous neighboring sequence can be easily obtained by the TAIL-PCR method established by Liu et al.

  The present invention also provides a DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence set forth 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 a protein consisting of the amino acid sequence of SEQ ID NOs: 112 to 122 can be generally isolated by a known method by those skilled in the art. Is possible. For example, hybridization techniques (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) techniques (Saiki, RK. Et al., Science, 1985, 230, 1350., Saiki, RK. Et al., Science 1988, 239, 487.). That is, the DNA comprising the base sequence described in any one of SEQ ID NOs: 101 to 111 or a part thereof as a probe, and specifically to the DNA comprising the base sequence described in any of SEQ ID NOs: 101 to 111 Isolation of a DNA having a high homology with a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 101 to 111 from a plant using a hybridizing oligonucleotide as a primer is usually possible for those skilled in the art. is there.

  In order to isolate such DNA, the hybridization reaction is preferably carried out under stringent conditions. In the present invention, stringent hybridization conditions refer to hybridization conditions of 6M urea, 0.4% SDS, 0.5 × SSC or equivalent stringency. It is expected that DNA with higher homology can be isolated under conditions of higher stringency, for example, 6M urea, 0.4% SDS, 0.1 × SSC. The DNA thus isolated is considered to have high homology with the amino acid sequence encoded by the DNA comprising the base sequence described in any of SEQ ID NOs: 101 to 111 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 sequence identity.

  The identity of the amino acid sequence and nucleotide sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sei. USA, 1990, 87, 2264-2268., Karlin, S. & Altschul, SF., Proc. Natl by Carlin and Arthur. Acad. Sei. USA, 1993, 90, 5873.). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul, SF. Et al., J Mol Biol, 1990, 215, 403.). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov/).

  Further, the DNA of the present invention encodes a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of any one of SEQ ID NOs: 112 to 122 DNA to be included.

  In order to prepare the above DNA, methods well known to those skilled in the art include, for example, site-directed mutagenesis method (Kramer, W. & Fritz, HJ., Methods Enzymol, 1987, 154, 350 .)) To introduce a mutation.

  The 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 Site-directed Mutagenesis Kit" (manufactured by Clontech) for mutation or substitution, Deletion can be performed using “Quantum leap Nested Deletion Kit” (Clontech).

  Further, even if the base sequence is mutated, there are cases where the mutation does not accompany amino acid mutation (degenerate mutation), and such degenerate mutated DNA is also included in the present invention.

  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. For example, the genomic DNA prepared according to the method described in the literature (Rogers and Bendich, Plant Mol. Biol., 1985, 5, 69.) is used as a template for the nucleotide sequence of the DNA of the present invention (for example, SEQ ID NO: It can be prepared by performing PCR (Saiki et al. Science, 1988, 239, 487.) using a primer prepared based on the base sequence described in any of 101 to 111). In the case of cDNA, it is prepared by preparing mRNA from a plant by a conventional method (Maniatis et al. Molecular Cloning Cold Spring Harbor Labortry Press), performing a reverse transcription reaction, and performing PCR using the same primers as described above. Is possible. For genomic DNA and cDNA, a genomic DNA library or cDNA library is prepared by a conventional method. For example, the base sequence of the DNA of the present invention (for example, any one of SEQ ID NOs: 101 to 111) is prepared against this library. It can also be prepared by screening using a probe synthesized based on the described base sequence). The base sequence of the obtained DNA can be easily determined by using, for example, “Sequencer Model 373” (manufactured by ABI).

  In the present invention, the DNA encoding a plant transcription factor has an increased expression level on the xylem side compared to the phloem side in the stem base and the center of the stem of Eucalyptus genus Camaldrensis described later. DNA ”,“ DNA in Eucalyptus chamaldrensis that has an increased expression level on the xylem side compared to the phloem side, and an equivalent expression level on the phloem side and xylem side in the trunk base ”,“ DNA whose expression level is increased on the xylem side compared to the phloem side in the stems of Eucalyptus genus Camaldrensis and Eucalyptus globules ”,“ The phloem side in the stem of Eucalyptus genus Camaldrensis ” The expression level increased on the xylem side compared to the eucalyptus globulus, and the expression level decreased on the xylem side compared to the phloem side. " In Te, as compared with the phloem side elevated expression level in the xylem side, in trunks of Eucalyptus globulus, expression levels include equivalent DNA "in phloem side and xylem side.

  In the trunk, it is known that in the phloem and the xylem, wood fiber formation is performed only on the xylem side. Therefore, “DNA whose expression level is increased or decreased on the xylem side compared to the phloem side” can also be expressed as “DNA whose expression level is increased or decreased at the wood fiber formation site”. . In addition, in the xylem of the trunk base and the xylem in the middle of the trunk, the xylem in the middle of the trunk is longer than the xylem in the middle of the trunk. Is thick, cell wall is thick, fibril inclination is slow, wood fiber rate and specific gravity are high (mature material). Compared with the xylem of the center of the trunk, the xylem at the base of the trunk is shorter in the length of the fiber, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, 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 of the stem base of eucalyptus compared to the phloem side” is “DNA whose expression level is increased or decreased on the immature wood formation site” It can also be expressed as 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 tree trunk” is “DNA whose expression level is increased or decreased on the matured material formation site” Can also be expressed.

  Furthermore, in the xylem of the Camaldrensis trunk and the globules of the trunk, the globules trunk xylem is longer in the fiber length (pulp quality) than the chamaldrensis trunk xylem. Is thicker, the cell wall is thicker, the fibril tilt angle is lower, and the wood fiber rate and specific gravity are known to be higher. It is also known that the xylem of the Camaldrensis trunk is shorter, the diameter is thinner, the cell wall is thinner, the fibril inclination is steep, and the wood fiber ratio and specific gravity are lower than the globules trunk. It has been. Therefore, “DNA with increased or decreased expression level on the xylem side compared with the phloem side in the stem of Eucalyptus genus Camaldrensis” is “wood fiber length is short, diameter is thin, cell wall is thin. It can also be expressed as “DNA whose expression level is increased or decreased at a wood fiber formation site having a low-quality pulp property that fibril inclination is steep and wood fiber ratio and specific gravity are low”. In addition, “in the stem of Eucalyptus globulus, the DNA whose expression level is increased or decreased on the xylem side compared to the phloem side” is “wood fiber length is long, diameter is thick, cell wall is thick, fibrils. It can also be expressed as “DNA whose expression level is increased or decreased at a site where a wood fiber having a high-quality pulp characteristic of a low inclination angle and a high wood fiber ratio and specific gravity” is formed.

  In the present invention, the phloem side means the outside of the vascularization layer, which is a meristem, and preferably the bark formation site on the phloem side. Here, the bark forming site is a site that is differentiating into or has the differentiation ability of a mentor cell, a phloem tube, parenchyma cell, a renal cell, or the like. Moreover, in this invention, a xylem side means an inner side from a vascular bundle formation layer, Preferably it means the wood fiber formation site | part of a xylem side. Here, the wood fiber forming site is a site during differentiation into a wood fiber cell, a temporary conduit, a conduit, a parenchymal cell or the like or having the differentiation ability.

  In the present invention, the trunk base refers to a generally lower breast height, for example, a eucalyptus fifth-year plant refers to a ground height of less than about 1 m. The central portion of the trunk generally refers to the vicinity of the middle portion of the tree height. For example, in the case of a eucalyptus plant of 5 years above a tree 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 tree trunk part of a plant (preferably a tree base part or a tree part of a tree trunk central part). That is, by using at least one of the above DNAs or a partial DNA thereof, it is possible to examine the growth state of the trunk part of the plant (preferably the base part of the trunk or the central part of the trunk).

  More specifically, in the case of the following (1) or (2), it is determined that the trunk portion of the plant is a xylem that is actively forming the fiber. Furthermore, in the case of (2), it is determined that the trunk portion of the plant is a xylem in which a mature material is formed or a xylem in which a mature material is formed in the future. Here, the matured wood means a wood fiber formed by differentiation from a mature formation layer structure, and the wood fiber length is longer and the diameter is larger than the xylem of the trunk base collected under the same conditions. Is thick, cell wall is thick, fibril inclination is gentle, and wood fiber rate and specific gravity are high. In addition, immature wood means tree fibers that are differentiated and formed from immature formation layer structure, and the tree fiber length is shorter and the diameter is smaller than the xylem of the center of the trunk collected under the same conditions. It is thin, has a thin cell wall, has a steep fibril inclination, and has a low wood fiber rate and specific gravity.

  The plant used in the test of the present invention is preferably Eucalyptus, more preferably Eucalyptus camaldrensis species or Eucalyptus Globulus species.

In addition, by using the inspection method of the present invention, quantitative and qualitative advance prediction of materials can be performed. In afforestation projects, appropriate operations (weeding, thinning, etc.) can be carried out at appropriate times based on the growth state. For example, if there is a correlation between the expression pattern in the phloem and xylem of the trunk base in the present invention and the expression pattern obtained by the test method of the present invention (preferably Pearson's correlation coefficient is 0.5 or more, more preferably 0.7 or more Qualitatively immature fibers (unripe wood: short fibers, thin diameter, thin cell walls, steep fibril inclination, shrub fiber ratio and low specific gravity) are formed in the trunk portion of the test tree. It can be predicted that the amount of wood fibers (volume weight) will be small or will be small in the future (average volume weight 450 to 500 kg / m ³ or less). On the other hand, if there is a correlation between the expression pattern in the phloem and xylem of the central part of the trunk in the present invention and the expression pattern obtained by the test method of the present invention, the qualitatively mature fiber (Mature material: long fiber, thick diameter, thick cell wall, loose fibril inclination, high wood fiber rate and high specific gravity) are formed or are in the future formed state, the amount of wood fiber (volume weight ) Or in the future (average bulk weight of 450 to 500 kg / m ³ or more). Furthermore, the material prediction as described above reveals whether the plant growth state is good or bad. If it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, weeding work is performed early. Or, if the planting density is high, thinning can be performed quickly and appropriately. At present, the material prediction and the determination of the operation policy as described above depend largely on the experience and intuition of the field or the subsequent chemical analysis, and there is no scientific index that can be discriminated in advance as shown in the present application.

(1) At the phloem side and the xylem side of the trunk of the plant (preferably the trunk base or the center of the trunk) The expression level of at least one DNA (preferably a plurality, more preferably all, the same applies hereinafter) is detected, and expression of the DNA on the phloem side and xylem side is detected. As a result of comparing the levels, the expression level of the DNA in the trunk of the plant is increased on the xylem side compared with the phloem side. The DNA described in any of the following (a) to (e) is mentioned as “DNA whose expression level is increased on the xylem side compared with the phloem side”.
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 113 to 117
(B) DNA comprising the base sequence according to any of SEQ ID NOs: 102 to 106
(C) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 113 to 117
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOS: 102 to 106
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 113 to 117
The “DNA consisting of the base sequence described in any one of SEQ ID NOs: 102 to 106” is preferably DNA consisting of the base sequence described in any one of SEQ ID NOs: 102, 105 or 106, and SEQ ID NO: 105 or 106 Although DNA which consists of a base sequence as described in is more preferable, it is not limited to these.

(2) At the phloem side and the xylem side of the trunk of the plant (preferably at the center of the trunk), the expression level is increased on the xylem side compared to the phloem side in the center of the stem of Eucalyptus genus Camaldrensis In the trunk base, the expression level of at least one of the DNAs having the same expression level on the phloem side and the xylem side was detected, and the expression levels of the DNA on the phloem side and the xylem side were compared. As a result, when the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side Then, the expression level is increased on the xylem side, and in the trunk base, the DNA having the same expression level on the phloem side and the xylem side ”is the DNA according to any of the following (a) to (e): Is mentioned.
(A) DNA encoding a protein comprising the amino acid sequence set forth in any of SEQ ID NOs: 112, 118 or 120-122
(B) DNA comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 101, 107 or 109 to 111
(C) DNA encoding a protein having 50% or more homology with the protein comprising the amino acid sequence of SEQ ID NO: 112, 118 or 120 to 122
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence of SEQ ID NO: 101, 107 or 109 to 111
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 112, 118 or 120 to 122
The “DNA consisting of the base sequence described in any of SEQ ID NO: 101, 107 or 109 to 111” is preferably DNA consisting of the base sequence described in SEQ ID NO: 107 or 109 to 111. : DNA having the nucleotide sequence described in 107 or 109 is more preferred, but not limited thereto.
Furthermore, the present invention provides a method for examining the growth state (maturity) of two different trunk parts. That is, the present invention provides a method for inspecting which of two different trunk parts is mature or immature. In the present invention, “different two tree parts” includes a tree part in two different plants and two different tree parts in one plant. The above method can also be used to select a mature or immature trunk part from a plurality of trunk parts.
More specifically, in the case of the following (3), among the two different trunk parts, the other trunk part (hereinafter referred to as the trunk part A) is compared with the other trunk part (hereinafter referred to as the trunk part A). The tree part A) is a mature xylem, and the tree part A is determined to be an immature xylem compared to the tree part B. Therefore, the following (3) 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 and the like is performed by using numerical values of appearance such as tree height and diameter and material analysis values after felling as an index, and there is no technique (concept) for selecting by the above index.

(3) The expression level of at least one of “different DNAs whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the central part of the eucalyptus tree trunk” in two different stem parts. In the present invention, as a result of comparing the expression level of the DNA in two different stem parts, the expression level of the DNA is reduced in the stem part A compared to the stem part B. As the “DNA whose expression level is reduced on the xylem side of the trunk base compared to the xylem side of the central part of the eucalyptus trunk”, the DNA described in any of the following (a) to (e) is Can be mentioned.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 113 or 114
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(C) DNA encoding a protein having 50% or more homology with the protein comprising the amino acid sequence of SEQ ID NO: 113 or 114
(D) DNA that hybridizes under stringent conditions with the DNA consisting of the nucleotide sequence of SEQ ID NO: 102 or 103
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 113 or 114
The “DNA consisting of the base sequence described in SEQ ID NO: 102 or 103” is preferably a DNA consisting of the base sequence described in SEQ ID NO: 102, but is not limited thereto.
Further, in the present invention, in the following cases (4) to (7), it is determined that the trunk part of the plant is a xylem with a large amount of wood fiber formation. Further, in the case of (5) or (6), the length of the tree fiber of the plant is shorter, the diameter of the tree is smaller, and the cell wall is smaller than that of the globulus trunk collected under the same conditions. It is a xylem in which wood fibers having a pulp characteristic of being thin and having a steep fibril inclination and a low wood fiber ratio and specific gravity are formed, or a xylem in which wood fibers having the pulp characteristics are formed in the future. It is determined. In the case of (7), the length of the tree fiber is longer, the diameter is thicker, the cell wall is thicker, and the fibril of the stem part of the plant is longer than the xylem part of the camaldrensis trunk collected under the same conditions. Decided to be a xylem in which wood fibers having excellent pulp characteristics such as a gentle inclination and a high wood fiber ratio and specific gravity are formed, or a xylem in which wood fibers having the pulp characteristics will be formed in the future Is done.
In addition, by using the inspection method of the present invention, quantitative and qualitative advance prediction of materials can be performed. In afforestation projects, appropriate operations (weeding, thinning, etc.) can be carried out at appropriate times based on the growth state. For example, if there is a correlation between the expression pattern in the phloem and xylem of the Eucalyptus genus Camaldorensis trunk in the present invention and the expression pattern obtained by the test method of the present invention (preferably Pearson's correlation coefficient of 0.5 or more, more Preferably 0.7 or higher, the same applies below), and the quality of the wood fiber (low quality pulp: short fiber, thin diameter, thin cell wall, steep fibril inclination, shrub fiber rate and low specific gravity) is formed, or a future state formed, the amount of wood fiber (volume weight) becomes small or in the future less (average volume weight 450~500kg / m ³ or less) Can be predicted. On the other hand, if there is a correlation between the expression pattern in the phloem and xylem of the Eucalyptus globulus trunk in the present invention and the expression pattern obtained by the test method of the present invention, the test tree trunk is qualitatively superior. Fiber (high quality pulp: long fiber, thick diameter, thick cell wall, loose fibril inclination, high wood fiber rate and high specific gravity), or in the state of being formed in the future, the amount of wood fiber ( It can be predicted that there will be a lot of (bulk weight) or increase in the future (average bulk weight of 450 to 500 kg / m ³ or more). Furthermore, the material prediction as described above reveals whether the plant growth state is good or bad. If it is judged to be bad, fertilization is performed in an oligotrophic area, or if weeds are overgrown, weeding work is performed early. Or, if the planting density is high, thinning can be performed quickly and appropriately. At present, the material prediction and the determination of the operation policy as described above depend largely on the experience and intuition of the field or the subsequent chemical analysis, and there is no scientific index that can be discriminated in advance as shown in the present application.

(4) At the phloem side and the xylem side of the trunk of the plant (preferably the trunk base or the middle of the trunk), in the eucalyptus camaldrensis and eucalyptus globulus trunk, the xylem side compared to the phloem side As a result of detecting the expression level of at least one DNA among the “DNA whose expression level is increased” and comparing the expression level of the DNA on the phloem side and the xylem side, the expression level of the DNA on the trunk of the plant However, in the present invention, in the trunk of Eucalyptus genus Camaldrensis and Eucalyptus globules, the expression level on the xylem side compared to the phloem side Examples of “rising DNA” include DNAs described in any of the following (a) to (e).
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 114 or 116
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 103 or 105
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence set forth in SEQ ID NO: 114 or 116
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 103 or 105
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 114 or 116
The “DNA consisting of the base sequence described in SEQ ID NO: 103 or 105” is preferably a DNA consisting of the base sequence described in SEQ ID NO: 105, but is not limited thereto.

(5) In the phloem side and the xylem side of the trunk of the plant (preferably the trunk base or the middle of the trunk), the expression level is higher on the xylem side than the phloem side in the stem of Eucalyptus genus Camaldrensis. In the trunk of Eucalyptus globules, the expression level of at least one of the DNAs whose expression level is reduced on the xylem side compared to the phloem side is detected, and the phloem side and the xylem side As a result of comparing the expression level of the DNA in the plant, when the expression level of the DNA in the trunk of the plant is increased on the xylem side compared with the phloem side, in the present invention, "the stem of Eucalyptus genus Camaldrensis" In the DNA, the expression level is increased on the xylem side compared to the phloem side, and the expression level is decreased on the xylem side compared to the phloem side in the trunk of the Eucalyptus globules `` (A) to (e) DNA described in any one of them can be mentioned.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 115 or 117
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 104 or 106
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence set forth in SEQ ID NO: 115 or 117
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 104 or 106
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 115 or 117

  The “DNA consisting of the base sequence described in SEQ ID NO: 104 or 106” is preferably DNA consisting of the base sequence described in SEQ ID NO: 106, but is not limited thereto.

(6) In the phloem side and the xylem side of the trunk of the plant (preferably the trunk base or the middle of the trunk), the expression level is higher on the xylem side than in the phloem side in the trunk of the Eucalyptus genus Camaldrensis. In the trunk of Eucalyptus globules, the expression level of at least one of the DNAs having the same expression level on the phloem side and the xylem side is detected, and the expression of the DNA on the phloem side and the xylem side is detected. As a result of comparing the levels, when the expression level of the DNA in the trunk of the plant is increased on the xylem side compared to the phloem side, in the present invention, in the eucalyptus genus Camaldrensis trunk, The expression level is increased on the xylem side compared to the DNA of the Eucalyptus globules, and the expression level is the same on the phloem side and the xylem side ”as any of the following (a) to (e) The DNA of crab The
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 113
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102
(C) DNA encoding a protein having 50% or more homology with the protein comprising the amino acid sequence set forth in SEQ ID NO: 113
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 113

(7) In the phloem side and xylem side of the trunk of the plant (preferably the central part of the trunk), the expression level is increased on the xylem side compared to the phloem side in the above-mentioned "eucalyptus chamaldrensis trunk". , In the trunk of Eucalyptus globulus, the expression level of at least one of the DNAs whose expression level is decreased on the xylem side compared to the phloem side is detected, and the expression level on the phloem side and the xylem side is detected. As a result of comparing the expression level of DNA, the expression level of the DNA in the trunk of the plant is decreased on the xylem side compared to the phloem side. Furthermore, in the present invention, two different plants (preferably A method for inspecting the growth state (pulp quality) of a stem part of Eucalyptus is provided. That is, the present invention provides a method for inspecting which of the two different plant trunk parts is superior in pulp quality or inferior. This method can also be used to select excellent or inferior trunk parts from a plurality of tree parts.

  More specifically, in the case of the following (11), the other plant (hereinafter referred to as the stem tree portion of one plant (hereinafter referred to as plant A)) among the different tree trunk portions of the two plants (hereinafter referred to as plant A). The stem part of the plant B) is a xylem having an excellent pulp quality, and the stem part A is determined to be a xylem having an inferior pulp quality compared to the stem part B. The Therefore, the following (11) is an index for selecting excellent lines called elite trees and plus trees, for example, in breeding sites. At present, selection of elite trees and the like is performed by using numerical values of appearance such as tree height and diameter and material analysis values after felling as an index, and there is no technique (concept) for selecting by the above index.

(8) In the trunk part of two different plants (preferably the root part of the trunk or the central part of the trunk part), “the xylem part of the Eucalyptus genus Camaldrensis as compared to the xylem side of the Eucalyptus globulus trunk” As a result of detecting the expression level of at least one of the DNAs whose expression level is reduced on the side and comparing the expression levels of the DNA in the trunk parts of two different plants, the expression level of the DNA is When decreased in plant A compared to plant B In the present invention, the expression level is decreased on the xylem side of the stem of Eucalyptus genus Camaldrensis as compared to the xylem side of the stem of Eucalyptus globulus. Examples of the “DNA” include DNAs described in any of the following (a) to (e).
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 116
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 105
(C) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence set forth in SEQ ID NO: 116
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 105
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 116

  By using the inspection method of the present invention, a xylem corresponding to any of the above (1) to (8) or a plant having the xylem is selected, and the selected xylem or a plant having the xylem is selected. It is possible to produce uniform pulp from the above, and it is possible to increase the efficiency of pulp production, reduce costs, and produce paper with new characteristics. In addition, after selecting a xylem corresponding to any of the following (1) to (8) or a plant having the xylem, according to a method well known to those skilled in the art, the selected xylem or the xylem Pulp can also be produced after confirming whether the wood fiber state is uniform among plants. Moreover, the xylem which does not correspond to either of the following (1)-(8) or the plant which has this xylem can be collected, and the pulp of arbitrary mixing | blending can also be produced.

  The inspection 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 a method include Northern blotting or RT-PCR.

  The test of the present invention can also be performed by a DNA array method. In the present invention, an oligonucleotide-based array developed by Affymetrix can be exemplified as a nucleotide immobilization (array) method. In an oligonucleotide array, oligonucleotides are usually synthesized in situ. For example, in-situ synthesis methods for oligonucleotides using photolithographic technology (Affymetrix) and inkjet (Rosetta Inpharmatics) technology for immobilizing chemical substances are already known. Can be used.

  In the present invention, 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 can be preferably used. In general, a DNA array is composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface layer of the substrate is generally glass, but a porous membrane such as a nitrocellulose membrane can be used.

  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. The nucleotide probe need not be completely complementary to the DNA of the present invention if specific hybridization is possible. In 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, and more preferably 15 to 25 bp.

  In this method, 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, but can generally be performed by methods well known to those skilled in the art.

  In this method, 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. Examples of the promoter DNA of the present invention include promoter DNA linked to genes obtained by the present invention, particularly genes having a differential expression in the spatial position of trees. Here, “promoter DNA” means DNA containing a specific base sequence necessary for initiation of mRNA synthesis (transcription) using DNA as a template. DNA prepared by the artificial modification operation is included.

  More specifically, the promoter DNA of the present invention is a promoter DNA of a DNA encoding 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 lignin synthesis). And a promoter DNA of a DNA encoding a plant transcription factor. SEQ ID NOs: 93 to 100 show promoter DNAs of DNAs encoding proteins having a function of forming a tree fiber cell wall of a plant tree trunk (proteins involved in cellulose synthesis and proteins involved in lignin synthesis). The relationship between a DNA encoding a protein having a function of forming a tree fiber cell wall of a plant tree (a protein involved in cellulose synthesis and a protein involved in lignin synthesis) and its promoter DNA is as described in the Examples. is there. Moreover, the promoter DNA of DNA which codes a plant transcription factor is shown to sequence number: 156-166. 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 Nippon Gene Co., Ltd.) can be used.

  Using the obtained DNA as a material, based on the base sequence of the eucalyptus cDNA that has been already isolated by the present inventors, oligonucleotides were prepared from any two locations, and PCR was performed using this as a primer. It is possible to easily produce genomic DNA corresponding to the selected eucalyptus cDNA. The upstream DNA of the gene is a PCR method (inverse-PCR method or anchor PCR method / TAIL-PCR method (supervised by Isao Shimamoto et al. It can be isolated by "PCR experiment protocol" (plant cell engineering separate volume, plant cell engineering series 7) published by Shujunsha in July 1997) or by a hybridization method using the DNA sequence of the gene as a probe. .

  It is also possible to use a genomic DNA library for eucalyptus DNA. The genomic DNA library is a cloning vector such as cosmid vector, TAC vector (Liu et al. (1999), Proc. Natl. Acad. Sci. USA, vol96: p6535) as well as various vectors derived from λDNA from DNA extracted from Eucalyptus. And transformed into E. coli.

  Hybridization techniques can be used for screening genomic DNA libraries. As the probe, a eucalyptus cDNA sequence that has been successfully isolated by the present inventors can be used. The above genomic DNA library is screened using this probe, and a clone containing a DNA sequence homologous to the gene is isolated. The restriction enzyme cleavage map is prepared, the base sequence is determined, etc., the structure of the cloned DNA is clarified, and the sequence existing upstream of the gene is specified. The upstream sequence preferably contains a TATA box sequence and has a size of at least several hundred bp to 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 pBI101, subcloning is performed so that the above sequence is linked upstream of the reporter gene. The pBI101 vector uses E. coli β-glucuronidase (GUS) as a reporter gene. When this gene product uses 5-bromo-4-chloro-3-β-D-glucronic acid (X-gluc) as a substrate, it breaks down to produce indigotin, a blue precipitate. It can be monitored at the organizational level. In addition, when 4-methyl-umbelliferyl-β-D-glucronide (4MUG) is used as a substrate, gene expression can be quantified by fluorescence generated by the action of the gene product. In addition to the GUS gene, a chloramphenicol acetyltransferase gene, a luciferase gene, a green fluorescein protein gene, and the like can be used as the reporter gene.

  The chimeric gene construct prepared as described above can be introduced into plants such as Arabidopsis thaliana via Agrobacterium and the function thereof can be analyzed. When pBI101 is used as a vector, a recombinant plasmid containing a chimeric gene is introduced into, for example, the Agrobacterium tumefaciens MP90 strain using electroporation, and the resulting transformant is treated with, for example, a floral. Infect Arabidopsis thaliana plants by the dip method (supervised by Isao Shimamoto et al. "Experimental protocol for model plants" (plant cell engineering separate volume, plant cell engineering series 4) published by Shujunsha 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 transformed plant that has become drug-resistant by gene transfer. Using this transformed plant body, 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, for example, a pBI101 vector into a chimeric gene in which a target gene, for example, a gene involved in biosynthesis of certain secondary metabolites is linked downstream of the promoter of the present invention. This vector is introduced into, for example, a tobacco plant body via Agrobacterium. In the resulting transformed plant, it is expected that the gene of the present invention is expressed at the site of wood fiber formation, and any secondary metabolite can be produced 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 undesirable traits will 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, insulator sequences, and the like. The promoter of the present invention includes several cis-element sequences that control the expression of genes related to the above-ground specific position of the trunk as well as 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 also 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 a DNA for suppressing the expression of a DNA encoding a protein involved in plant fiber fiber cell wall formation. Preferred embodiments of the DNA for suppressing the expression of the endogenous gene include a DNA encoding an antisense RNA complementary to the transcription product of the DNA of the present invention, and a ribozyme that specifically cleaves the transcription product of the DNA of the present invention. DNA encoding RNA having activity, DNA encoding RNA that suppresses expression of the DNA of the present invention due to RNAi effect or co-suppression effect, and protein having a dominant negative trait for the transcription product of DNA of the present invention Examples include DNA to be encoded. The above “suppression of endogenous gene expression” includes suppression of gene transcription and / or suppression of translation from the gene into a protein encoded by the gene. Also included is a decrease in expression as well as complete cessation of expression of the gene.

  As a method for suppressing the expression of a specific endogenous gene in a plant, a method using an antisense technique is most often used by those skilled in the art. The antisense effect in plant cells was demonstrated for the first time by Ecker et al. Showing that antisense RNA introduced by electroporation exhibits an antisense effect in plants (Ecker, JR. & Davis, RW., Proc Natl Acad Sci USA, 1986, 83, 5372.). Since then, tobacco and petunia have been reported to have decreased target gene expression due to antisense RNA expression (van der Krol AR. Et al., Nature, 1988, 333, 866.). Sense technology has been established as a means to suppress gene expression in plants.

  There are several factors as described below for the action of an antisense nucleic acid to suppress the expression of a target gene. Inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally created by RNA polymerase, inhibition of transcription by hybridization with RNA undergoing synthesis, intron and exon Splicing inhibition by hybridization at splice point, splicing inhibition by hybridization with spliceosome formation site, inhibition of translocation from nucleus to cytoplasm by hybridization with mRNA, hybridization with capping site and poly (A) addition site Inhibition of splicing, translation initiation inhibition by hybridization with the translation initiation factor binding site, translation inhibition by hybridization with the ribosome binding site in the vicinity of the initiation codon, peptation by hybridization with the mRNA translation region and polysome binding site Elongation inhibition of de chain, and gene expression inhibition by hybrid formation at sites of interaction between nucleic acids and proteins, and the like. In this way, antisense nucleic acids suppress the expression of target genes by inhibiting various processes such as transcription, splicing, and translation (Hirashima and Inoue, Shinsei Kagaku Kogaku 2 Nucleic acid IV gene replication and expression, Japan biochemicalization) (Academic Society, Tokyo Chemical Doujin, 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. As one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the gene is designed, it is considered effective for inhibiting the translation of the gene. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, a DNA containing an antisense sequence of a non-translated region as well as a translated region of a gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The DNA thus prepared can be transformed into a desired plant by using a known method. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene or a part thereof possessed by the plant to be transformed, but is not completely complementary as long as the gene expression can be effectively suppressed. May be. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene. In order to effectively suppress the expression of the target gene using the antisense sequence, the length of the antisense DNA is at least 15 bases, 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.

  In addition, suppression of endogenous gene expression can be performed using ribozymes or DNA encoding ribozymes. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities, but research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA in a site-specific manner. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNase P, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191.)

  For example, the self-cleaving domain of the hammerhead ribozyme cleaves 3 ′ of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is important for its activity, and instead of C15, A15 or U15 However, it has been shown that it can be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA (Koizumi, M. et al., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191., Koizumi, M. et al., Nucl Acids Res, 1989, 17, 7059 .). For example, there are a plurality of sites that can be targets in the coding region of DNA that encodes a protein involved in the formation of plant fiber fiber walls.

  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 hairpin ribozymes can also produce target-specific RNA-cleaving ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.).

  A ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence 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, ribozyme activity may be lost. In such cases, RNA containing the transcribed ribozyme may be lost. It is also possible to place another trimming ribozyme that acts in cis on the 5 ′ side or 3 ′ side of the ribozyme portion to accurately excise only the ribozyme portion from the protein (Taira, K. et al., Protein Eng, 1990). , 3, 733., Dzianott, AM. & Bujarski, JJ., Proc Natl Acad Sci USA, 1989, 86, 4823., Grosshans, CA. & Cech, TR., Nucl Acids Res, 1991, 19, 3875. Taira, K. et al., Nucl Acids Res, 1991, 19, 5125.). It is also possible to increase the effect by arranging such structural units in tandem so that multiple sites in the target gene can be cleaved (Yuyama, N. et al., Biochem Biophys Res Commun, 1992 , 186, 1271.). Thus, the expression of the gene can be suppressed by specifically cleaving the transcript of the target gene in the present invention using a ribozyme.

  Inhibition of endogenous gene expression can also be carried out by RNA interference (RNAi) using double-stranded RNA having the same or similar sequence as the target gene sequence. RNAi refers to a phenomenon in which, when a double-stranded RNA having the same or similar sequence as a target gene sequence is introduced into a cell, the expression of the introduced foreign gene and target endogenous gene are both suppressed. Although the details of the RNAi mechanism are not clear, it is thought that the first introduced double-stranded RNA is decomposed into small pieces, and becomes an indicator of the target gene in some form, so that the target gene is decomposed. RNAi is known to have an effect in plants (Chuang, CF. & Meyerowitz, EM., Proc Natl Acad Sci USA, 2000, 97, 4985.). For example, in order to suppress the expression of DNA encoding a protein that controls the difference in wood fiber properties between Eucalyptus species by RNAi, the base sequence described in any one of SEQ ID NOs: 1-34 or similar thereto What is necessary is just to introduce | transduce into a target plant double stranded RNA which has the sequence | arrangement. The gene used for RNAi need not be completely identical to the target gene, but has at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity. . The sequence identity 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 the expression of the introduced foreign gene and target endogenous gene is both suppressed when a gene having the same or similar sequence as the target endogenous gene is introduced into a plant by transformation. Point to. The details of the mechanism of co-suppression are not clear, but at least part of the mechanism is thought to overlap with that of RNAi. Co-suppression is also observed in plants (Smyth, DR., Curr Biol, 1997, 7, R793., Martienssen, R., Curr Biol, 1996, 6, 810.). For example, in order to obtain a plant body in which a DNA encoding a protein that controls the formation of wood fibers in the plant body is co-suppressed, a vector prepared so that the DNA or a DNA having a similar sequence can be expressed. What is necessary is just to transform DNA into the target plant. The gene used for co-suppression does not have to be completely identical to the target gene, but at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more. Have. The sequence identity can be determined by the method described above.

  Furthermore, 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 character with respect to the protein encoded by the target gene into a plant. “A gene encoding a protein having a dominant negative character” 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. .

  The present invention also provides a recombinant vector containing the above 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 stabilization of the transcription product. Examples of amplifiable vectors include shuttle vectors that can be amplified by both E. coli and Agrobacterium, such as pBI101 (Clontech). Plant viruses such as cauliflower mosaic virus can also be used as vectors.

  The vector of the present invention can be obtained, for example, by binding or inserting a promoter DNA of the present invention or a promoter DNA for constitutively or inductively expressing a desired gene into a predetermined part of the vector. In addition, the method of inserting a promoter into a vector follows the method of inserting a normal gene into a vector. An expression vector for gene expression can be obtained by functionally connecting a desired gene to the promoter of this recombinant vector.

  Examples of promoters for constitutive expression include cauliflower mosaic virus 35S promoter (Odell et al., Nature, 1985, 313, 810.), rice actin promoter (Zhang et al., Plant Cell, 1991, 3, 1155.), corn ubiquitin promoter (Cornejo et al., Plant Mol. Biol., 1993, 23, 567.). In addition, promoters for inducible expression are known to be expressed by external factors such as infection and invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, ultraviolet irradiation, and spraying of specific compounds. Promoters and the like. Examples of such promoters include the rice chitinase gene promoter (Xu et al., Plant Mol. Biol., 1996, 30, 387.) expressed by infection and invasion of filamentous fungi, bacteria, and viruses, and tobacco PR. Promoter of protein gene (Ohshima et al., Plant Cell, 1990, 2, 95.), promoter of rice “lip19” gene induced by low temperature (Aguan et al., Mol. Gen Genet., 1993, 240, 1.) The rice hsp80 and hsp72 gene promoters induced by high temperature (Van Breusegem et al., Planta, 1994, 193, 57.) and the Arabidopsis rab16 gene induced by drought Promoter (Nundy et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 1406.), the promoter of parsley chalcone synthase gene induced by ultraviolet irradiation (Schulze-Lefert et al., EMBO) J., 1989, 8, 651.), induced under anaerobic conditions Alcohol dehydrogenase gene promoter of corn that (Walker et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 6624.), and the like. The rice chitinase gene promoter and tobacco PR protein gene promoter are also induced by specific compounds such as salicylic acid, and “rab16” is also induced by spraying the plant hormone abscisic acid.

  Further, in order to efficiently select cells transformed by introduction of the DNA of the present invention, the above recombinant vector may be introduced into cells together with a suitable selection marker gene or a plasmid vector containing a selection marker gene. preferable. Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene resistant to the antibiotic hygromycin, the neomycin phosphotransferase resistant to kanamycin or gentamicin, and the acetyltransferase gene resistant to the herbicide phosphinothricin. Etc.

  The present invention also provides a transformed plant cell into which the vector of the present invention has been introduced. The cell into which the vector of the present invention is introduced is not particularly limited, and examples thereof include rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower, cotton, orange, grape, peach , Pear, apple, tomato, cabbage, cabbage, radish, carrot, pumpkin, cucumber, melon, parsley, orchid, chrysanthemum, lily, saffron, etc., but eucalyptus, pine, red pepper, poplar, cedar Trees such as cypress, bamboo, and yew are desirable. In addition to cultured cells, the plant cells of the present invention include cells in the plant body. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots.

  Various techniques can be used to introduce the expression vector into a host plant cell. These methods include transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transforming factor, as well as protoplast transformation. Examples include direct introduction (electroporation method in which DNA is introduced into plant cells by electric pulse treatment of protoplasts, fusion method of liposomes and protoplasts, microinjection method, polyethylene glycol method, etc.), particle gun method, and the like.

  In addition, a target gene can be introduced into a plant by using a plant virus as a vector. Examples of plant viruses that can be used include cauliflower mosaic virus. That is, first, a viral genome is inserted into a vector derived from E. coli to prepare a recombinant, and then these target genes are inserted into the viral genome. These target genes can be introduced 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). The method of introducing a vector into a plant cell or plant body is not limited to these, but includes other possibilities.

  For direct introduction into protoplasts, there is no restriction on the required vector. For example, a simple plasmid such as a pUC derivative can be used. Depending on the method of introducing the gene of interest into the plant cell, other DNA sequences may be required. For example, when a Ti or Ri plasmid is used for transformation of a plant cell, the sequence of at least the right end of the T-DNA region of the Ti and Ri plasmid, most often the sequence of both ends, is inserted into the gene to be introduced. It must be connected so that it is adjacent.

  When Agrobacterium is used for transformation, it is necessary to clone the gene to be introduced 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 Agrobacterium by helper plasmid or electroporation. Since the intermediate vector has a region homologous to the sequence of T-DNA, it is incorporated into an Agrobacterium Ti or Ri plasmid by homologous recombination. Agrobacterium used as a host must contain a vir region. The Ti or Ri plasmid usually contains the vir region, and T-DNA can be transferred to plant cells by its function.

  On the other hand, binary vectors can be replicated and maintained in Agrobacterium, so when incorporated into Agrobacterium by a helper plasmid or electroporation, the host vir region causes Of T-DNA can be transferred to plant cells.

  The intermediate vector or binary vector thus obtained and microorganisms such as Escherichia coli and Agrobacterium are also objects of the present invention.

  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 a propagation material for the transformed plant. Such a transformed plant is a useful transformed plant in which cell wall components and cell morphogenesis are modified depending on the plant species. The modification of the cell wall component in the present invention is not particularly limited, and various quantitative and qualitative properties such as high cellulose, low lignin, a thick cell wall, a thin cell, a long fiber, a short one, etc. Changes. 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 in the present invention is useful as a plant having new value such as an increase in 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, etc., depending on the plant species. . Moreover, it is useful as a plant having new value such as development of a new material by controlling cell wall synthesis, increase of digestion and absorption efficiency of forage crops, and change of fiber cell morphology.

  In the present invention, the “transformed plant” is a plant having the above-described transformed plant cell, and includes, for example, a transformed plant regenerated from the transformed cell. The method of regenerating an individual from transformed plant cells varies depending on the type of plant cell. For example, in rice, the method of Fujimura et al. (Fujimura et al., Plant Tissue Culture Lett., 2,74,1995), in maize, Shillito et al. (Shillito et al., Bio / Technology, 7, 581,1989), in potato, Visser et al., Theor. Appl. Genet., 78, 589, 1989, in Arabidopsis Akama et al. (Akama et al., Plant Cell Rep., 12, 7, 1992) Eucalyptus includes a method of Toi et al. (Japanese Patent Application No. 11-127025). A transformed plant produced by these methods or a transformed plant obtained from its propagation medium (for example, seeds, tubers, cut ears, etc.) is the subject of the present invention.

  The present invention provides a transformed cell by introducing into a host cell an expression vector having a gene group involved in plant fiber cell wall formation, particularly a tree, homologues thereof, or a promoter region linked to these genes. And a step of regenerating a transformed plant from the transformed cell, obtaining a plant seed from the resulting transformed plant, and producing the plant from the plant seed.

  The step of obtaining plant seeds from a transformed plant is, for example, collecting the transformed plant from a rooting medium, transplanting it into a pot containing water-containing soil, growing it at a constant temperature, and It refers to the process of forming and finally forming seeds. The process of producing a plant from seeds is, for example, when the seed formed on the transformed plant is matured, isolated, sown in soil containing water, and grown under constant temperature and illuminance. This is a process for producing a plant body.

  The presence of the introduced foreign DNA or nucleic acid in the transformed plant body can be confirmed by a known PCR method or Southern hybridization method, or by analyzing the base sequence of the nucleic acid in the plant body. In this case, DNA or nucleic acid can be extracted from the transformed plant according to a known method by J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor laboratory Press, 1989).

  When the DNA of the present invention present in the plant body is analyzed using the PCR method, the amplification reaction is performed using the nucleic acid extracted from the regenerated plant body as a template as described above. Alternatively, an amplification reaction can be carried out in a reaction solution in which these are mixed using a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of the DNA of the present invention as a primer. In the amplification reaction, when the 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. When the reaction solution containing the amplification product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and the DNA fragments correspond to the DNA of the present invention. .

  Once a transformed plant into which the DNA of the present invention has been introduced into the chromosome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain propagation materials (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant and its descendants or clones, and mass-produce the plant based on them. It is.

  The present invention also provides a primer for amplifying all or part of the base sequence described in SEQ ID NOs: 1-34, 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, but its length is usually 15 bp to 100 bp, preferably 16 bp. It is ˜31 bp, and more preferably, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 31 nucleotides. In the primer of the present invention, the 3 ′ region may be complementary, and a restriction enzyme recognition sequence or a tag may be added to the 5 ′ side.

Furthermore, this invention provides the primer set which amplifies all or one part of the base sequence of sequence number: 1-34, 101-111. Specifically, as the primer set of the present invention,
(A) DNA consisting of the base sequence set forth in SEQ ID NO: 69 and DNA consisting of the base sequence set forth in SEQ ID NO: 70 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 3),
(B) DNA consisting of the base sequence set forth in SEQ ID NO: 71 and DNA consisting of the base sequence set forth in SEQ ID NO: 72 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 6),
(C) DNA consisting of the base sequence set forth in SEQ ID NO: 73 and DNA consisting of the base sequence set forth in SEQ ID NO: 74 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 11),
(D) DNA consisting of the base sequence set forth in SEQ ID NO: 75 and DNA consisting of the base sequence set forth in SEQ ID NO: 76 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 14),
(E) DNA consisting of the base sequence set forth in SEQ ID NO: 77 and DNA consisting of the base sequence set forth in SEQ ID NO: 78 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 19),
(F) DNA consisting of the base sequence set forth in SEQ ID NO: 79 and DNA consisting of the base sequence set forth in SEQ ID NO: 80 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 23),
(G) DNA consisting of the base sequence set forth in SEQ ID NO: 81 and DNA consisting of the base sequence set forth in SEQ ID NO: 82 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 24),
(H) DNA consisting of the base sequence set forth in SEQ ID NO: 83 and DNA consisting of the base sequence set forth in SEQ ID NO: 84 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 25),
(I) DNA consisting of the base sequence set forth in SEQ ID NO: 123 and DNA consisting of the base sequence set forth in SEQ ID NO: 124 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 101),
(J) DNA consisting of the base sequence set forth in SEQ ID NO: 125 and DNA consisting of the base sequence set forth in SEQ ID NO: 126 (a primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 102),
(K) DNA consisting of the base sequence set forth in SEQ ID NO: 127 and DNA consisting of the base sequence set forth in SEQ ID NO: 128 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 103),
(L) DNA consisting of the base sequence set forth in SEQ ID NO: 129 and DNA consisting of the base sequence set forth in SEQ ID NO: 130 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 104),
(M) DNA consisting of the base sequence set forth in SEQ ID NO: 131 and DNA consisting of the base sequence set forth in SEQ ID NO: 132 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 105),
(N) DNA consisting of the base sequence set forth in SEQ ID NO: 133 and DNA consisting of the base sequence set forth in SEQ ID NO: 134 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 106),
(O) DNA consisting of the base sequence set forth in SEQ ID NO: 135 and DNA consisting of the base sequence set forth in SEQ ID NO: 136 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 107),
(P) DNA consisting of the base sequence set forth in SEQ ID NO: 137 and DNA consisting of the base sequence set forth in SEQ ID NO: 138 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 108),
(Q) DNA consisting of the base sequence set forth in SEQ ID NO: 139 and DNA consisting of the base sequence set forth in SEQ ID NO: 140 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 109),
(R) DNA consisting of the base sequence set forth in SEQ ID NO: 141 and DNA consisting of the base sequence set forth in SEQ ID NO: 142 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 110),
(S) DNA consisting of the base sequence set forth in SEQ ID NO: 143 and DNA consisting of the base sequence set forth in SEQ ID NO: 144 (primer set for detecting all or part of the base sequence set forth in SEQ ID NO: 111),
Can be illustrated.

  In addition to the above primers, those skilled in the art can create a primer set having the same function by using the base sequences described in SEQ ID NOs: 1-34, 101-111. Such primers are also included in the primer of the present invention. The 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 inspection methods. For example, the gene, primer, staining solution and the like of the present invention can be mentioned. In addition, distilled water, salt, buffer solution, protein stabilizer, preservative and the like may be contained.

  By growing the transformed plant body in the present invention on a large scale by clone plantation, it is possible to stably supply biomass with cellulose as a core. At present, fossil resources are used in large quantities as raw materials and fuel (energy) in industrial production. In particular, for energy alternatives, wood biomass is directly combusted (using soot) on a daily basis in developing countries, but it is effective by converting it into a form (alcohol: ethyl alcohol) that is easier to use. It is considered that this is possible. In fact, ethanol is produced by alcoholic fermentation from waste molasses derived from sugarcane juice in Brazil, etc., and is used as an automobile fuel. In the United States and the EU, sweet potato and corn starch are once hydrolyzed into glucose. Examples of alcohol fermentation are increasing. In the United States in August 1999, the company announced that it will increase the energy use of biomass to 10% of the total primary energy by 2010, and one of its purposes is to use ethanol refined from biomass mixed with gasoline. . A specific example is an ethanol fuel called corn-based gasohol (the proportion of ethanol blended with gasoline is 10%), which is used in 20 states, mainly in the corn belt, It accounts for 1%, and in certain states where sugar millet can be used, it has a 40% share of gasoline. All US car manufacturers guarantee gasohol as fuel. Specifically, General Motors and Chrysler This use is recommended. In the EU, a plan to reduce the share of renewable energy to 12% of total energy with the aim of reducing greenhouse gas emissions by 10% in 1990 level in 2010 is in progress. The goal is to replace 5% of the fuel with biofuel (fuel derived from biological resources). Looking at the EU energy use plan, solar cells (contribution 1%), wind power generation (19%), and biomass cogeneration (80%) have shown great expectations for biomass. In 2015, the cultivation of energy bio-crop is expected to occupy the largest land use area. However, on the other hand, from the viewpoint of increasing food production, there is a limit to consuming large quantities of cereals and potatoes for industrial raw materials in the future. Therefore, for example, if eucalyptus having a high cellulose content can be grown in large quantities according to the present invention, lignocellulose obtained therefrom is used as a raw material, glucose is obtained by an acid hydrolysis or enzymatic decomposition (cellulase) process, and then ethanol is produced in large quantities by alcohol fermentation. It is possible to produce, and the basic technology of such a process has already been established. Furthermore, a technology for producing a biodegradable plastic (polylactic acid) using glucose as a raw material has already been established, and its practical application on an industrial production scale is being developed using starch of potatoes. However, in the future, biomass derived from trees is expected to become the mainstream instead of cereals as food. In addition, lignin is expected to be used for plastics and adhesives, although it is necessary to overcome technical problems in the future. In terms of energy, lignin is chemically decomposed 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, In other words, it depends on woody biomass for a part of the fuel.

In the present invention, woody biomass is cultivated stably and on a large scale and circulated through afforestation, so that it can be used as a conventional raw material, as well as petroleum alternative energy by biomass conversion, and also new plastic from cellulose or hemicellulose. It is also possible to create (this is also technically possible). In addition, the spread of woody biomass is a solution to energy security and environmental problems, and at the same time leads to the development of new industries such as agriculture and forestry and the creation of employment opportunities.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

  The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the following examples. Regarding experimental methods, unless otherwise stated, “Cloning and Sequence” (supervised by Watanabe, edited by Masahiro Sugiura, Rural Bunkasha (1989)) and “Molecular Cloning (Sambrook et al. (1989), Cold Spring Harbor” Laboratory press).

(Example 1) Preparation of eucalyptus EST database (1) RNA extraction from eucalyptus As for the eucalyptus tissue to be extracted, the hypertrophic growth part of the stem (secondary wall thickening zone; tissue rich in formation layer), leaf, root It assumes gene expression from various situations such as slant stimulation and stress application by salt solution exposure. The basic method of extraction is the method described in the method of Hino et al. (Japanese Patent Application No. 6-219187). Hereinafter, as an example, an RNA extraction method using eucalyptus roots by hydroponics as a material will be described in detail.

Eucalyptus (Eucalyptus camaldulensis) seedlings grown for 2 months were transplanted to a hydroponic culture tank. Hydroponics was performed using the culture solution of Hoagland-Arnon et al. The composition of the hydroponic culture solution is 5.0 mM KNO ₃ , 3.0 mM Ca (NO ₃ ) ₂ , 2.0 mM NH ₄ H ₂ PO ₄ , 2.0 mM MgSO ₄ , 47 μM H ₃ BO ₃ , 9.0 μM MnCl ₂ , 36 μM FeSO ₄ 3.1 μM ZnSO ₄ , 0.16 μM CuSO ₄ , 75 nM (NH ₄ ) 6 Mo ₇ O ₂₄ , prepared using demineralized water, and the pH was adjusted to 6.0 daily with 0.1 M NaOH or KOH. Furthermore, all the culture solutions were changed every week. When performing stress treatment, the culture solution added with NaCl so that the final concentration would be 50,100,200,300 mM in order from the first day to the fourth day of cultivation was used as the stress treatment group, and the NaCl-free culture solution was used as the control. . 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. To this, a methanol solution containing dithiothreitol (1 mg / ml) was used, 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 9 100 mM CHES buffer (20 milligrams 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 completion of the incubation. At this time, the sodium chloride concentration in the sample solution after addition was 1.4 M, and the CTAB concentration was 1% (w / v). The sample solution was mixed well and kept at 65 ° C. for 10 minutes, and then an equal volume of chloroform: isoamyl alcohol (= 24: 1) solution was added and gently and thoroughly mixed. After mixing, the supernatant was collected by centrifugation. To the supernatant, 55% volume of isopropanol was added and cooled on ice for 1 hour. A precipitate was obtained by centrifugation, dissolved in water, and extracted with phenol. A 10% volume of 3M sodium acetate solution and 60% volume of isopropanol were added to the supernatant after phenol extraction, and after mixing well, the precipitate was recovered by centrifugation. After the precipitate was dissolved in sterilized water, a 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 μL of water to obtain a total RNA fraction. As a result, 610 μg of total RNA could be obtained from each of the roots of the stress treatment group and the control roots. For purification of mRNA from the total RNA fraction, PolyATtract mRNA Isolation System III & IV kit (Cat. # Z5300 & Z5310 Promega Corp. USA) was used. As a result, 1.3 μg mRNA was obtained from 610 μg total RNA from the stress-treated sample, and 1.8 μg mRNA from the control sample.

(2) Construction of cDNA library The eucalyptus mRNA derived from various tissues and situations obtained by the method described in (1) was synthesized using the Clontech Smart cDNA library construction kit, and finally phage midri It was rally. The cDNA library thus prepared consisted of independent clones each of 1 × 10 ⁶ pfu or more. Library amplification was performed on a part of the library prepared, and clone analysis was performed without amplification.

(3) Decoding cDNA clones and constructing a database Randomly selecting clones from each tissue-derived eucalyptus phagemid cDNA library, purifying the plasmid, and then conducting an enzyme reaction with Amersham's dye terminator sequence kit Base sequence data was obtained using a high-speed sequencer.
In data analysis, after deleting a known sequence derived from a plasmid, a homologous sequence was extracted by a clustering operation using analysis software. After that, an approximate function prediction (annotation) was performed by comparison search with all databases of GenBank, one of the gene information databases in the United States.

(Example 2) Selection of a gene group presumed to be involved in cell wall formation Using the obtained annotation information, a gene involved in cellulose and lignin synthesis (Proc Natl Acad Sci USA 2001 Dec 4; 98 (25): 14732 -7., JP-A 2000-41685, JP-A 2002-95482, JP-A 2003-180372), and clones having high homology were searched. As a result, 59 clones were identified. However, these clones include not only the formation of wood fibers but also those involved in the formation of bark tissue and the like and those not related to cell wall synthesis.

(Example 3) Extraction of gene group involved in tree fiber cell wall formation (1) Preparation of Eucalyptus trunk specific oligo microarray Eucalyptus oligo microarray was prepared from the above-mentioned Eucalyptus EST database. The actual production was commissioned to Agilent Technologies (Yokogawa Analytical Systems in Japan). Details are described on the following website. http://www.chem.agilent.com/cag/country/JP/products/PCol494.htm
The eucalyptus oligo microarray produced in this way contains 8400 oligo DNAs and can cover most of the genes that are recognized in the eucalyptus trunk.

(2) Extraction of genes related to formation of tree fiber cell wall of Eucalyptus tree trunk by microarray analysis Most of the woody biomass in the tree trunk is composed of tissue cells called tree fibers. The main factors that determine the properties of the wood fiber include the specific gravity, cell wall structure and components (cellulose, hemicellulose, lignin) of the wood fiber structure. These are not defined by a single gene or protein, but formed by a large number of genes working together. On the other hand, it is known that the nature of wood fibers varies depending on the ground height and species regardless of whether they are broad-leaved trees or conifers. By performing EST data analysis and microarray gene expression analysis on such materials, it becomes possible to comprehensively capture a series of genes that work in concert during wood fiber formation, and the expression state of each constituent gene can be determined. It is also possible to capture.

  The 59 clones selected based on the annotation information include not only tree fiber formation but also those involved in the formation of bark tissue and the like and those not related to cell wall synthesis. Therefore, genes related to the formation of wood fiber cell walls were extracted from these by eucalyptus trunks of different ground heights and species, by microarray expression analysis.

  In accordance with the RNA extraction method described in Example 1, the present inventors extracted total RNA from the trunk base and middle part of a 5-year-old Camaldrensis species, and Globulus species as materials. At this time, the bark side (phloem) and the wood fiber forming tissue side (xylem) of each plant body were used. From the total RNA obtained from each individual, mRNA was purified by PolyATract mRNA Isolation System manufactured by Promega. Each cRNA synthesized using a total of 4 types of mRNAs obtained as described above as a template was labeled with 2 types of fluorescent dyes (cy3.cy5), and used as a probe in oligo microarray analysis for hybridization ( 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%. Expression information of 59 clones selected from the trunk base of the Drensis species and the central portion of the trunk and the trunk base of the Globus species was obtained.

  The expression of genes involved in the formation of eucalyptus tree fiber cell walls is expected to be significantly stronger on the wood fiber-forming tissue side (xylem) than on the bark side (phloem). Among the 59 selected clones, clones showing significantly strong expression on the wood fiber-forming tissue side were selected. As a result, 34 clones were identified as genes involved in eucalyptus tree fiber cell wall formation (Tables 1 to 4). ).

The Sequence ID corresponds to SEQ ID NOs: 1 to 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: O-methyltransferase, 4CL: 4-coumarate CoA ligase, CCR : Cinnamoyl CoA reductase, C4H: cinnamate 4-hydroxylase

  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 chamaldrensis trunk, and 5y-cam1 X vs P is the xylem part of the fifth-year eucalyptus chamaldrensis base 5y-glo X vs P represents an experiment comparing the xylem and phloem of the 5th grade Eucalyptus globulus base. The experimental results are expressed as a positive multiple when the former is strong and a negative multiple when it is strong on the bark 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 central part of the fifth-grade Camaldorensis species (Tables 2 and 4).

(1) 26 “groups of genes whose expression level is increased on the xylem side compared to the phloem side in the stem base and the center of the Eucalyptus genus Camaldrensis” (SEQ ID NOs: 1 to 6, 8, 9, 11-11, 16, 17, 17, 19-25, 27, 28, 30, 32, 33)
The 26 gene groups include gene groups (SEQ ID NOs: 1 to 6, 9, 11) in which the difference in expression on the xylem side is 3 times or more compared to the phloem side at either the trunk base or the middle of the trunk. -14, 16, 17, 19, 20, 22, 25, 27, 30), a gene whose expression difference on the xylem side is 4 times or more compared to the phloem side in either the trunk base or the middle trunk In the group (SEQ ID NO: 1, 3-6, 9, 11-14, 16, 17, 19, 22, 25, 30), the trunk base or the trunk center, on the xylem side compared to the phloem side In the gene group (SEQ ID NOs: 1, 3-6, 11, 12, 14, 16, 17, 22, 25), the stem base or the center of the trunk, the phloem side Gene group whose expression difference on the xylem side is 10 times or more (SEQ ID NO: 1, 3, 5, 12, 25) Is included.

  As a result of direct comparison of the gene expression at the xylem side of the trunk base and the central trunk of the 26 genes, two (vertical fluctuation classification group A-1 in Table 4) “Group of genes whose expression is increased on the xylem side of the trunk base” (SEQ ID NOs: 33 and 34), 6 (vertical variation classification group A-2 in Table 4), “xylem side of the trunk base” And “groups of genes with equivalent expression on the xylem side of the center of the trunk” (SEQ ID NOs: 9, 11, 13, 23, 27), 18 (vertical variation classification group A-3 in Table 4) Gene group whose expression is increased on the xylem side of the central part of the trunk ”(SEQ ID NOs: 1-6, 8, 14, 16, 17, 19-22, 24, 25, 28 30). More specifically, the numerical value of the average fluorescence intensity obtained by the experiment was calculated by the formula of the trunk base / central part or the central part / trunk base. Based on the expression information of each gene that is significant, the numerical value obtained by the formula of the stem base / central part is 1.1 or more and P <= 0.001, compared with the xylem side of the central part of the trunk. "A group of genes whose expression is increased on the side" and those with a numerical value of 1.1 or more and P <= 0.001 obtained by the formula of the center / tree trunk base as compared to the tree side of the trunk base Gene group whose expression is increased on the xylem side of the tree "is not significant or has almost the same average intensity (the fluorescence intensity ratio of the xylem in the center and the xylem in the base is 1.0 to less than 1.1 times or P> 0.001 ) Were classified as “gene groups with the same expression on the xylem side of the trunk base and the xylem side of the center of the trunk”.

The two gene groups include a gene group (SEQ ID NO: 33) in which the difference in expression between the xylem at the base of the trunk and the xylem at the center of the trunk is 1.5 times or more.
The 18 gene groups include gene groups (SEQ ID NOs: 1 to 6, 14, 16, 17, 19 to 22, wherein the expression difference between the xylem of the trunk base and the xylem of the middle of the trunk is 1.5 times or more. 25, 28, 30), a gene group (SEQ ID NOs: 1 to 6, 14, 16, 17, 22, 25, 30) in which the difference in expression between the xylem at the base of the trunk and the xylem at the center of the trunk is twice or more ), A gene group (SEQ ID NOs: 3, 5, 25, 30) in which the difference in expression between the xylem of the trunk base and the xylem of the center of the trunk is 3 times or more is included.

(2) One (vertical variation classification group D in Table 4) “Eucalium genus Camaldrensis stem base has a decreased expression level on the xylem side compared to the phloem side, Gene group whose expression level is increased on the xylem side compared to the xylem side ”(SEQ ID NO: 15)

(3) 6 (vertical variation classification group G in Table 4) “Eucalium genus Camaldrensis, in the middle of the trunk, the expression level increased on the xylem side compared to the phloem side, and in the trunk base, Gene groups with equivalent expression levels on the part side and xylem side "(SEQ ID NOs: 7, 10, 18, 26, 29, 34)
The six gene groups include a gene group (SEQ ID NO: 7, 10, 18, 26, 29) in which the expression difference on the xylem side is twice or more in the central part of the trunk compared to the phloem side, the trunk In the central part, a gene group (SEQ ID NO: 18, 26, 29) in which the expression difference on the xylem side is 2.5 times or more compared to the phloem side is included.
The 34 gene groups are roughly classified into the following (1) to (3) based on the detailed expression pattern in the trunk base of the fifth-year Camaldrensis species or the middle of the trunk and the fifth-year Globulus species. (Tables 3 and 4).

(1) Six “groups of genes whose expression levels are increased on the xylem side compared to the phloem side in the stems of Eucalyptus genus Camaldrensis and Eucalyptus globules” (SEQ ID NOs: 11, 12, 17 , 21, 22, 30)
The six gene groups include a gene group (SEQ ID NO: 11, wherein the expression difference on the xylem side is 4 times or more compared to the phloem side in any of Eucalyptus camaldrensis and Eucalyptus globulus trunks. 12, 17, 22, 30), a gene group (SEQ ID NO: 11, 12, 17, 22) whose expression difference is 6 times or more in any of the stems of Eucalyptus genus Camaldrensis and Eucalyptus globules, Eucalyptus In any of the trunks of Camaldrensis and Eucalyptus globules, the gene group (SEQ ID NOs: 11, 12, 17) whose expression difference is 8 times or more, in any of the stems of eucalyptus Camaldrensis and Eucalyptus globules And a gene group (SEQ ID NO: 12) having an expression difference of 10 times or more.

  As a result of direct comparison of the gene expression on the xylem side of the stem base of the Eucalyptus genus Camaldrensis or the central part of the stem and the xylem side of the Eucalyptus globulus, the six genes were found to be four (see Table 4). Inter-variation classification group A-1) “a group of genes whose expression is increased on the xylem side of Eucalyptus genus Camaldrensis compared to the xylem side of Eucalyptus globulus” (SEQ ID NO: 11, 12, 17, 22), 1 (expressed on the xylem side of Eucalyptus genus Camaldrensis, compared to the xylem side of Eucalyptus globulus trunk, group A-3) Are roughly classified into “gene group with decreased” (SEQ ID NO: 21). More specifically, the numerical value of the average fluorescence intensity obtained by the experiment is calculated based on the xylem formula of the stem (base or central part) of the Eucalyptus genus Camaldrensis / the xylem of the stem (base) of the Eucalyptus globules or the eucalyptus. The calculation was performed according to the formula of the xylem of the genus Globulus trunk (base) / the xylem of the eucalyptus chamaldrensis trunk (base or center). From the expression information of each gene, the value obtained from the tree part formula of Eucalyptus genus Camaldrensis / Eucalyptus globules is 1.1 or more and P <= 0.001. Compared to the gene group whose expression is increased on the xylem side of the stem of Eucalyptus genus Camaldrensis ”, the numerical value obtained by the formula of the stem of Eucalyptus globules / xylem genus Camaldrensis is 1.1 or more and Those with P <= 0.001 are not significant or mean intensity, as `` gene groups whose expression is increased on the xylem side of the Eucalyptus globulis tree compared to the xylem side of the Eucalyptus genus Camaldrensis '' Is approximately the same (the fluorescence intensity ratio of the tree part of Eucalyptus genus Camaldrensis is less than 1.0 to 1.1 times or P> 0.001 compared to the tree part of Eucalyptus globulus tree). Expression in xylem side and xylem side trunk Eucalyptus globulus of trunks Rudorenshisu is determined that equivalent genes "were classified.

  The four gene groups include a gene group (SEQ ID NOs: 12 and 17) in which the difference in expression between the xylem part of Eucalyptus genus Camaldrensis and the xylem part of Eucalyptus globules is 5 times or more. A gene group (SEQ ID NO: 12) in which the difference in expression between xylem and Eucalyptus globulus is 9 times or more is included.

(2) The expression level increased on the xylem side compared to the phloem side in the trunk of Eucalyptus genus Camaldrensis in 7 (variable classification group C between tree species in Table 4). , Genes whose expression level is reduced on the xylem side compared to the phloem side ”(SEQ ID NOs: 8, 13, 19, 23-25, 33)
The seven gene groups show that the difference in expression on the xylem side is more than 2.5 times in the stem of Eucalyptus genus Camaldrensis, or the phloem side in the stem of Eucalyptus globulus compared to the xylem side In the gene group (SEQ ID NO: 8, 13, 19, 24, 25, 33), Eucalyptus genus Camaldrensis stem difference in xylem side compared to phloem side Is a gene group (SEQ ID NOs: 13, 19, 25) Eucalyptus camaldrensis stem that has an expression difference of 4 times or more in the stem of Eucalyptus globulus compared to the xylem side In the gene group (SEQ ID NO: SEQ ID NO: 10 or more times the expression difference on the xylem side compared to the phloem side, or on the eucalyptus globulus trunk, the expression difference on the phloem side compared to the xylem side : 25) is included.

(3) 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 Eucalyptus globulus , Gene groups with the same expression level on the phloem side and the xylem side ”(SEQ ID NOs: 1 to 6, 9, 14, 16, 20, 27, 28, 32)
The 13 gene groups include gene groups (SEQ ID NOs: 1 to 6, 9, 14, and 3) that have a three-fold or more difference in expression on the xylem side compared to the phloem side in the stem of Eucalyptus genus Camaldrensis. 16, 20, 27), a gene group (SEQ ID NO: 1, 3-6, 14, 16) in which the difference in expression on the xylem side is twice or more in the stem of Eucalyptus genus Camaldrensis ), The stem of Eucalyptus genus Camaldrensis includes a gene group (SEQ ID NO: 1, 3, 5) in which the expression difference on the xylem side is 10 times or more compared to the phloem side.

(Example 4) Obtaining genomic clones and promoter DNA of genes involved in the formation of wood fiber cell walls (1) Screening of Eucalyptus BAC genomic library The method of http: //www.ncgr.orc/research/jag) was followed. 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. Using oligonucleotide primers of SEQ ID NOs: 69 to 84 synthesized based on cDNA sequence information (odd numbers of SEQ ID NOs: 69 to 84 are forward primers, and even numbers are reverse primers), and each of the DNA pools as templates by PCR. Screening of genomic clones of genes was performed.

(2) Determination of genome sequence 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 (based on the cDNA sequence described in SEQ ID NOs: 3, 6, 11, 14, 19, 23, 24, 25). The determined genomic sequences are shown in SEQ ID NOs: 85-92, respectively). For clones that were full-length cDNA sequences including the translation initiation codon ATG (amino acid encoded methionine), the promoter region was determined based on that sequence. The determined promoter sequences are shown in SEQ ID NOs: 93-100. Specifically, 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. 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. When the position of the determined TATA-box was close to the full-length cDNA 5 ′ terminal base (about 100 bp), it was judged that the cDNA was almost full length, and the upstream side of the terminal base was the promoter region. The length of the promoter was set to a maximum of 3 kb upstream from the cDNA 5 ′ terminal base with reference to the maximum length (about 3 kb) known in the model plant.

In the base sequence represented by SEQ ID NO: 1, the first to 660th positions are the amino acid coding region (SEQ ID NO: 35).
In the base sequence represented by SEQ ID NO: 2, the 1st to 602nd positions are the amino acid coding region (SEQ ID NO: 36).
In the base sequence represented by SEQ ID NO: 3, positions 337 to 3273 are the amino acid coding region (SEQ ID NO: 37).
In the base sequence represented by SEQ ID NO: 4, the 1st to 1407th positions are amino acid coding regions (SEQ ID NO: 38).
In the base sequence represented by SEQ ID NO: 5, the 1st to 465th positions are the amino acid coding region (SEQ ID NO: 39).
In the base sequence represented by SEQ ID NO: 6, positions 87 to 3221 are the amino acid coding region (SEQ ID NO: 40).
In the base sequence represented by SEQ ID NO: 7, positions 1 to 699 are amino acid coding regions (SEQ ID NO: 41).
In the base sequence represented by SEQ ID NO: 8, the 1st to 1407th positions are the amino acid coding region (SEQ ID NO: 42).
In the base sequence represented by SEQ ID NO: 9, the 1st to 1122nd positions are the amino acid coding region (SEQ ID NO: 43).
In the base sequence shown by SEQ ID NO: 10, the 1st to 1005th positions are the amino acid coding region (SEQ ID NO: 44).
In the base sequence represented by SEQ ID NO: 11, the 313th to 1305th positions are the amino acid coding region (SEQ ID NO: 45).
In the base sequence represented by SEQ ID NO: 12, the 1st to 465th positions are the amino acid coding region (SEQ ID NO: 46).
In the base sequence represented by SEQ ID NO: 13, positions 1 to 762 are the amino acid coding region (SEQ ID NO: 47).
In the base sequence represented by SEQ ID NO: 14, positions 375 to 1442 are the amino acid coding region (SEQ ID NO: 48).
In the base sequence represented by SEQ ID NO: 15, positions 1 to 774 are the amino acid coding region (SEQ ID NO: 49).
In the base sequence represented by SEQ ID NO: 16, positions 1 to 498 are the amino acid coding region (SEQ ID NO: 50).
In the base sequence represented by SEQ ID NO: 17, positions 68 to 1165 are the amino acid coding region (SEQ ID NO: 51).
In the base sequence represented by SEQ ID NO: 18, positions 117 to 857 are the amino acid coding region (SEQ ID NO: 52).
In the base sequence represented by SEQ ID NO: 19, positions 84 to 968 are the amino acid coding region (SEQ ID NO: 53).
In the base sequence represented by SEQ ID NO: 20, positions 1 to 207 are amino acid coding regions (SEQ ID NO: 54).
In the base sequence represented by SEQ ID NO: 21, positions 56 to 223 are the amino acid coding region (SEQ ID NO: 55).
In the base sequence represented by SEQ ID NO: 22, positions 1 to 621 are the amino acid coding region (SEQ ID NO: 56).
In the base sequence represented by SEQ ID NO: 23, the 323rd to 1330th positions are the amino acid coding region (SEQ ID NO: 57).
In the base sequence represented by SEQ ID NO: 24, positions 240 to 1847 are the amino acid coding region (SEQ ID NO: 58).
In the base sequence represented by SEQ ID NO: 25, the 273rd to 1928th positions are the amino acid coding region (SEQ ID NO: 59).
In the base sequence represented by SEQ ID NO: 26, the 1st to 1134th positions are the amino acid coding region (SEQ ID NO: 60).
In the base sequence represented by SEQ ID NO: 27, the 1st to 525th positions are the amino acid coding region (SEQ ID NO: 61).
In the base sequence represented by SEQ ID NO: 28, the 1st to 1116th positions are the amino acid coding region (SEQ ID NO: 62).
In the base sequence represented by SEQ ID NO: 29, the 1st to 195th positions are the amino acid coding region (SEQ ID NO: 63).
In the base sequence represented by SEQ ID NO: 30, the first to 138th positions are the amino acid coding region (SEQ ID NO: 64).
In the base sequence represented by SEQ ID NO: 31, positions 1 to 564 are the amino acid coding region (SEQ ID NO: 65).
In the base sequence represented by SEQ ID NO: 32, the first to 711th positions are the amino acid coding region (SEQ ID NO: 66).
In the base sequence represented by SEQ ID NO: 33, the 84th to 569th positions are the amino acid coding region (SEQ ID NO: 67).
In the base sequence represented by SEQ ID NO: 34, the 1st to 249th positions are the amino acid coding region (SEQ ID NO: 68).
The 1st to 2013th positions of the base sequence represented by SEQ ID NO: 85 are promoter regions (SEQ ID NO: 93), 2350th to 2527th, 3105th to 3181st, 3371th to 3rd 3422, 3539 to 3680, 4295 to 4561, 4924 to 5269, 5412 to 5549, 5793 to 5918, 6001 to 6213 6649th to 6907th, 6993th to 7192th, 7431th to 7784th, 8069th to 8650th are exons in the coding region, 2528th to 3104th, 3182-3370, 3423-3538, 3681-4294, 4562-4923, 5270-5411, 5550th to 5792th, 5919th to 6000th, 6214th to 6648th, 6908th to 6992th, 7193th to 7430th, 7785th to 8068th are intron regions It is.
The 1st to 2920th positions of the base sequence represented by SEQ ID NO: 86 are promoter regions (SEQ ID NO: 94), 3068th to 3139th positions, 394th 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 exons in the coding region, 3140 to 3940, 4142 to 4274, 4397th to 4487th, 4555th to 4669th, 4809th to 5290th, 5904th to 5999th, 6138th to 6425th, 6552 positions, second 6791 position, the 7005 position, second 7090 position, the 7607 position, second 7997 of, the 8349 positions - positions the 8694 is intron region.
The 1st to 2700th positions of the base sequence represented by SEQ ID NO: 87 are promoter regions (SEQ ID NO: 95), positions 3013 to 3415, positions 3508 to 3667, positions 4610 to 4610 The 5039th is an exon in the coding region, and the 3416th to 3507th positions and the 3668th to 4609th positions are intron regions.
The 1st to 2707th positions of the base sequence represented by SEQ ID NO: 88 are promoter regions (SEQ ID NO: 96), 3082th to 3170th positions, 3365th to 3478th positions, 3565th to 3565th positions. Exons in the coding region are 3792, 4389 to 4828, 5124 to 5320, 3171 to 3364, 3479 to 3564, 3793 to 4388 , Positions 4829 to 5123 are intron regions.
The 1st to 2835th positions of the base sequence represented by SEQ ID NO: 89 are promoter regions (SEQ ID NO: 97), 2920th to 3011th positions, 3142th to 3221st positions, 3345th to 3345th positions. Nos. 3489, 3463-377, 4333-4623 are exons in the coding region, 3012-3164, 3222-3344, 3490-3642 , Positions 3775 to 4332 are intron regions.
The 1st to 2469th positions of the base sequence represented by SEQ ID NO: 90 are promoter regions (SEQ ID NO: 98), 2792th to 2924th positions, 3027th to 3181st positions, 3850th position to 3850th positions. 4035, 4202 to 4554, 5504 to 5684 are exons in the coding region, 2925 to 3026, 3182 to 3849, 4036 to 4201 , Positions 4555 to 5503 are intron regions.
In the nucleotide sequence represented by SEQ ID NO: 91, 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. And positions 2348 to 2451 are intron regions.
The 1st to 2997th positions of the base sequence represented by SEQ ID NO: 92 are promoter regions (SEQ ID NO: 100), the 3270th to 3335th positions, the 3606th to 3757th positions, and the 4034th to 4th positions. Nos. 4278, 4562 to 4690, 4797 to 5732, 5844 to 5971 are exons in the coding region, 3336 to 3605, 3758 to 4033 , Positions 4279 to 4561, positions 4691 to 4796, positions 5733 to 5843 are intron regions.

(Example 5) Selection of transcription factor group Using annotation information obtained from the database shown in Example 1, transcription factor genes involved in gene expression control (JL Riechmann et al. SCIENCE VOL 290 15 DECEMBER 2000, Masaki Iwabuchi, Kazuo Shinozaki And clones with high homology with plant genome function). As a result, 71 clones were identified. However, these clones include not only wood fiber formation but also transcription factors involved in the formation of bark tissue and the like.

(Example 6) Extraction of transcription factor group involved in tree fiber formation Among 71 clones selected based on annotation information, not only tree fiber formation but also those involved in the formation of bark tissue etc. are included. Yes. Therefore, transcription factors related to tree fiber formation were extracted from these by eucalyptus trunks of different ground heights and species, by microarray expression analysis.

  In accordance with the RNA extraction method shown in Example 1, the present inventors extracted total RNA using the trunk base and the center of the trunk of the 5-year-old Camaldorensis species and the stem base of the Globulus species as materials. At this time, the bark side (phloem) and the wood fiber forming tissue side (xylem) of each plant body were used. From the total RNA obtained from each individual, mRNA was purified by PolyATract mRNA Isolation System manufactured by Promega. A total of four kinds of mRNA thus obtained were labeled with two kinds of fluorescent dyes (cy3.cy5), respectively, and used as probes in the oligo microarray analysis shown in Example 1 for hybridization (FIGS. 1 to 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's analysis software (Luminator Ver. 1.0), and all repeated experiments are integrated with a statistical reliability of 99.999%. Expression information of 71 clones selected at the trunk base of the drenches species and the center of the trunk, and at the trunk base of the globulus 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 bark side (phloem). Among the selected 71 clones, clones showing significantly strong expression on the side of the wood fiber forming tissue were selected. As a result, 11 clones were identified as a group of genes involved in eucalyptus tree fiber cell wall formation (Tables 5 to 8). ).

In the present specification, the identified cDNA sequences are shown in SEQ ID NOs: 101 to 111, and the amino acid sequences encoded from the respective cDNAs are shown in SEQ ID NOs: 112 to 122. In addition, DNA which codes the protein which consists of an amino acid sequence in any one of sequence number: 112-122 has the structural characteristics (domain) shown below.
The amino acid sequence set forth in SEQ ID NO: 112 is a CBF domain, a HAP2 domain,
The amino acid sequence set forth in SEQ ID NO: 113 is a homeodomain,
The amino acid sequence set forth in SEQ ID NO: 114 is the Coprinus_mating domain,
The amino acid sequence set forth in SEQ ID NO: 115 is a LIM domain,
The amino acid sequence set forth in SEQ ID NO: 116 is a bZIP domain,
The amino acid sequence set forth in SEQ ID NO: 117 is a SANT domain, Myb DNA binding domain, REB1 domain,
The amino acid sequence set forth in SEQ ID NO: 118 is the WRKY domain,
The amino acid sequence set forth in SEQ ID NO: 119 is homeodomain, leucine zipper domain,
The amino acid sequence set forth in SEQ ID NO: 120 is a UPF0023 domain, a leucine zipper domain,
The amino acid sequence described in SEQ ID NO: 121 is a SANT domain, Myb DNA binding domain, and REB1 domain. The amino acid sequence described in SEQ ID NO: 122 is a Sina domain.

The Sequence ID corresponds to SEQ ID NOs: 101 to 111 in order from the top.

  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 chamaldrensis trunk, and 5y-cam1 X vs P is the xylem part of the fifth-year eucalyptus chamaldrensis base 5y-glo X vs P represents an experiment comparing the xylem and phloem of the 5th grade Eucalyptus globulus base. The experimental results are expressed as a positive multiple when the former is strong and a negative multiple when it is strong on the bark side.

  The 11 gene groups were broadly classified into the following (1) and (2) based on the detailed expression patterns in the trunk base and central part of the fifth-grade Camaldrensis species (Tables 6 and 8).

  (1) Five “gene groups in which the expression level is increased on the xylem side compared to the phloem side in the stem base and the middle of the stem of Eucalyptus genus Camaldrensis” (SEQ ID NOs: 102 to 106)

  The five gene groups include a gene group (SEQ ID NO: 102, 105, 106) in which the difference in expression on the xylem side is twice or more compared to the phloem side at either the trunk base or the middle of the trunk, A gene group (SEQ ID NO: 105, 106) having an expression difference of 4 times or more on the xylem side compared to the phloem side is included in either the trunk base or the trunk trunk center.

  As a result of direct comparison of the gene expression of the 5 gene groups on the xylem side of the trunk base and the central part of the tree trunk, 3 (vertical fluctuation classification group A-2 in Table 8) Compared with the xylem side of the stem base of two genes (vertical variation classification group A-3 in Table 8), “gene groups with equivalent expression on the xylem side of the center of the trunk” (SEQ ID NOs: 104 to 106) Thus, it was roughly classified into “gene groups whose expression is increased on the xylem side of the center of the trunk” (SEQ ID NOs: 102 and 103). More specifically, the numerical value of the average fluorescence intensity obtained by the experiment was calculated by the formula of the trunk base / central part or the central part / trunk base. Based on the expression information of each gene that is significant, the numerical value obtained by the formula of the stem base / central part is 1.1 or more and P <= 0.001, compared with the xylem side of the central part of the trunk. "A group of genes whose expression is increased on the side" and those with a numerical value of 1.1 or more and P <= 0.001 obtained by the formula of the center / tree trunk base as compared to the tree side of the trunk base Gene group whose expression is increased on the xylem side of the tree "is not significant or has almost the same average intensity (the fluorescence intensity ratio of the xylem in the center and the xylem in the base is 1.0 to less than 1.1 times or P> 0.001 ) Were classified as “gene groups with the same expression on the xylem side of the trunk base and the xylem side of the center of the trunk”.

  The two gene groups include gene groups (SEQ ID NOs: 102 and 103) in which the difference in expression between the xylem at the base of the trunk and the xylem at the center of the trunk is twice or more.

  (2) The expression level increased in the xylem side compared to the phloem side in the middle part of the stem of Eucalyptus genus Camaldrensis in 5 (vertical variation classification group G in Table 8). Gene group with the same expression level on the part side and xylem side "(SEQ ID NOs: 101, 107, 109 to 111)

  In the gene group of 5 genes, the gene group (SEQ ID NO: 107, 109-111) in which the expression difference on the xylem side is 1.5 times or more compared to the phloem side in the center of the trunk, A gene group (SEQ ID NOs: 107 and 109) in which the expression difference on the xylem side is twice or more compared to the phloem side is included.

  The 11 gene groups are broadly divided into the following (1) to (3), based on the detailed expression patterns in the trunk base and middle part of the fifth-year Camaldorensis species, and the fifth-year Globulus species. (Tables 7 and 8).

  (1) Two “groups of genes whose expression levels are increased on the xylem side compared to the phloem side in the stems of Eucalyptus camaldrensis and Eucalyptus globulus” (SEQ ID NOs: 103 and 105)

  The two gene groups include a gene group (SEQ ID NO: 105) having an expression difference of 10 times or more on the xylem side compared to the phloem side in any of Eucalyptus camaldrensis and Eucalyptus globulus trunks. Is included.

  As a result of a direct comparison of the gene expression between the eucalyptus chamaldrensis tree base or central xylem side and the Eucalyptus globulus tree base side of two genes, one gene (between the tree species in Table 8) Variant classification group A-2) “genes whose expression on the xylem side of Eucalyptus genus Camaldrensis is equivalent to that on the xylem side of Eucalyptus globulus” (SEQ ID NO: 103), 1 (Table 8) "A gene whose expression level is reduced on the xylem side of the Eucalyptus genus Camaldrensis" compared to the xylem side of the Eucalyptus globulus tree (SEQ ID NO: 105). More specifically, the numerical value of the average fluorescence intensity obtained by the experiment is calculated based on the xylem formula of the stem (base or central part) of the Eucalyptus genus Camaldrensis / the xylem of the stem (base) of the Eucalyptus globules or the eucalyptus. The calculation was performed according to the formula of the xylem of the genus Globulus trunk (base) / the xylem of the eucalyptus chamaldrensis trunk (base or center). From the expression information of each gene, the value obtained from the tree part formula of Eucalyptus genus Camaldrensis / Eucalyptus globules is 1.1 or more and P <= 0.001. Compared to the gene group whose expression is increased on the xylem side of the stem of Eucalyptus genus Camaldrensis ”, the numerical value obtained by the formula of the stem of Eucalyptus globules / xylem genus Camaldrensis is 1.1 or more and Those with P <= 0.001 are not significant or mean intensity, as `` gene groups whose expression is increased on the xylem side of the Eucalyptus globulis tree compared to the xylem side of the Eucalyptus genus Camaldrensis '' Is approximately the same (the fluorescence intensity ratio of the tree part of Eucalyptus genus Camaldrensis is less than 1.0 to 1.1 times or P> 0.001 compared to the tree part of Eucalyptus globulus tree). Expression in xylem side and xylem side trunk Eucalyptus globulus of trunks Rudorenshisu is determined that equivalent genes "were classified.

  (2) 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 , Genes whose expression level is reduced on the xylem side compared to the phloem side ”(SEQ ID NOs: 104, 106)

  There are two gene groups, the difference in expression on the xylem side of the eucalyptus genus Camaldrensis is more than 4 times, or on the eucalyptus globulus tree, the phloem side compared to the xylem side. A gene (SEQ ID NO: 106) having an expression difference of 4 times or more in is included.

  (3) One (in the tree species variation classification group E in Table 8), the expression level increased on the xylem side compared to the phloem side in the stem of Eucalyptus genus Camaldrensis, , Gene groups with the same expression level on the phloem side and the xylem side ”(SEQ ID NO: 102)

(Example 7) Acquisition of genome clone and promoter DNA of transcription factor group involved in wood fiber formation (1) Screening of eucalyptus BAC genomic library Screening of the eucalyptus BAC genomic library was carried out by the method shown in Example 4. Oligonucleotide primer of SEQ ID NO: 123-144 synthesized based on cDNA sequence information using DNA pool for 3D screening by PCR as template (odd number of SEQ ID NO: 123-144 is forward primer, even number is reverse primer) The genomic clones of each gene were screened by the PCR 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 and the promoter region of each gene were determined based on the cDNA sequence (the genomic sequence determined based on the cDNA sequence described in SEQ ID NO: 101 to 111, SEQ ID NOs: 145 to 155). In addition, the promoter region was determined based on the full-length cDNA sequence including the translation initiation codon ATG (amino acid encoded methionine). The determined promoter sequences are shown in SEQ ID NOs: 156 to 166. Specifically, 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. 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. When the position of the determined TATA-box is close to the full-length cDNA 5 ′ terminal base (about 100 bp), it was judged that the cDNA was almost full length and that the upstream side of the terminal base was the promoter region. The length of the promoter was determined from the 5 ′ terminal base of the cDNA to the maximum of 3 kb upstream from the maximum length (about 3 kb) known in the model plant.

In the base sequence represented by SEQ ID NO: 101, positions 87 to 662 are the amino acid coding region (SEQ ID NO: 112).
In the base sequence represented by SEQ ID NO: 102, positions 262 to 1188 are the amino acid coding region (SEQ ID NO: 113).
In the base sequence represented by SEQ ID NO: 103, positions 257 to 1549 are the amino acid coding region (SEQ ID NO: 114).
In the base sequence represented by SEQ ID NO: 104, positions 159 to 722 are the amino acid coding region (SEQ ID NO: 115).
In the base sequence represented by SEQ ID NO: 105, positions 101 to 1444 are the amino acid coding region (SEQ ID NO: 116).
In the base sequence represented by SEQ ID NO: 106, the 163rd to 927th positions are the amino acid coding region (SEQ ID NO: 117).
In the base sequence represented by SEQ ID NO: 107, positions 48 to 2300 are the amino acid coding region (SEQ ID NO: 118).
In the base sequence represented by SEQ ID NO: 108, the 23rd to 778th positions are the amino acid coding region (SEQ ID NO: 119).
In the base sequence represented by SEQ ID NO: 109, positions 40 to 1101 are amino acid coding regions (SEQ ID NO: 120).
In the base sequence represented by SEQ ID NO: 110, positions 128 to 790 are the amino acid coding region (SEQ ID NO: 121).
In the base sequence represented by SEQ ID NO: 111, positions 269 to 1234 are the amino acid coding region (SEQ ID NO: 122).
The 1st to 2680th positions of the base sequence represented by SEQ ID NO: 145 are promoter regions (SEQ ID NO: 156), 2767th to 2796th positions, 2884th to 2949th positions, 2984th to 2894th positions. Positions 3142 and 3265 to 3585 are exons in the coding region, and positions 2797 to 2883, positions 2950 to 2983, and positions 3143 to 3264 are intron regions.
The 1st to 3000th positions of the nucleotide sequence represented by SEQ ID NO: 146 are promoter regions (SEQ ID NO: 157), the 3262th position to the 3606th position, the 3920th position to the 4040th position, the 5436th position to the 5th position. 5626, 5829 to 5960, 6246 to 6383 are exons in the coding region, 3607 to 3919, 4041 to 5435, 5627 to 5828. , Positions 5961 to 6245 are intron regions.
The 1st to 1569th positions of the nucleotide sequence represented by SEQ ID NO: 147 are the promoter regions (SEQ ID NO: 158), the 1826th position to the 2533th position, the 4847th position to the 4967th position, the 5439th position to the 5th position. 5632, 5709-5840, 5936-6072, 6158 are exons in the coding region, 2534-4846, 4968-5438, 5633 ~ 5708, 5841 ~ 5935, 6073 ~ 6157 are intron regions.
The 1st to 2979th positions of the base sequence represented by SEQ ID NO: 148 are promoter regions (SEQ ID NO: 159), the 3241th position to the 3375th position, the 3967th position to the 4063th position, the 4159th position to the 4159th position. Nos. 4202, 4293 to 4282, 4539 to 4736 are exons in the coding region, 3376 to 3966, 4064 to 4158, 4203 to 4292. , Positions 4283 to 4538 are intron regions.
The 1st to 1288th positions of the base sequence represented by SEQ ID NO: 149 are promoter regions (SEQ ID NO: 160), the 1409th to 1964th positions, the 3755th to 3959th positions, the 4510th to 45th positions. Nos. 4585, 4564 to 4809, 5066 to 5446 are exons in the coding region, 1965 to 3754, 3960 to 4509, 4586 to 4563 , Positions 4810 to 5065 are intron regions.
In the base sequence represented by SEQ ID NO: 150, positions 1 to 1536 are the promoter region (SEQ ID NO: 161), positions 1723 to 1985, and positions 2217 to 2718 are exons in the coding region. 1986 to 2216 are intron regions.
The 1st to 1658th positions of the base sequence represented by SEQ ID NO: 151 are promoter regions (SEQ ID NO: 162), the 1706th to 2167th positions, the 2407th position to the 2589th position, the 2767th position to the 2nd position. Expositions in the coding region are positions 3647, 3747-3905, 4108-4675, 2168-2406, 2590-2766, 3648-3746 , Positions 3906 to 4107 are intron regions.
The 1st to 1979th positions of the base sequence represented by SEQ ID NO: 152 are promoter regions (SEQ ID NO: 163), positions 2002 to 2427, positions 2714 to 2793, positions 2984 to 2984. Position 3233 is an exon in the coding region, and 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 promoter regions (SEQ ID NO: 164), the 637th position to the 770th position, the 1143th position to the 1272nd position, the 1393th position to the 1st position. 1593, 2042 to 2209, 2939 to 3367 are exons in the coding region, 771 to 1142, 1273 to 1392, 1594 to 2041 , Positions 2210 to 2938 are intron regions.
The 1st to 2937th positions of the base sequence represented by SEQ ID NO: 154 are the promoter region (SEQ ID NO: 165), the 3101st position to the 3221st position, the 3339th position to the 3468th position, the 4324th position to the 4th position. Position 4735 is an exon in the coding region, and positions 3222 to 3338 and positions 3469 to 4323 are intron regions.
In the nucleotide sequence represented by SEQ ID NO: 155, positions 1 to 1783 are promoter regions (SEQ ID NO: 166), positions 2052 to 2270, positions 3339 to 3725, positions 4266 to 4266. Position 4625 is an exon in the coding region, and positions 2271 to 3338 and positions 3726 to 4265 are intron regions.

(Example 8) Analysis of xylem specific promoter activity (1) Construction of construct for activity analysis Genes expressed in xylem forming tissues shown in Examples 3 and 6 (SEQ ID NOs: 1-34, 101- 111), the expression in the xylem is controlled 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. This example shows an example in which the activity of the promoter region (SEQ ID NO: 93) of the cellulose synthase gene (SEQ ID NO: 3) is analyzed by the particle gun method.
In order to confirm the activity as a xylem specific promoter, 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, a promoter containing a 5 ′ untranslated region by PCR using the oligonucleotide primer of SEQ ID NO: 167, 168 synthesized based on the genome sequence information set forth in SEQ ID NO: 85, using BAC clone DNA as a template The region was amplified, blunt-ended, and then subcloned upstream of the GUS gene of a binary vector pBI121 vector (Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984) excluding the 35S promoter.

(2) Analysis of xylem specific promoter activity As an analysis of promoter activity, the aforementioned plasmid for introduction was introduced into a xylem-forming tissue by a particle gun, and the activity of the GUS gene was analyzed. A xylem block (FIG. 4B) was excised from the xylem forming tissue (FIG. 4A) exposed on the surface where the bark of a vigorously growing 5-year-old eucalyptus tree was peeled, and used as a target for particle gun introduction. The introduction plasmid was coated on the gold particles as described below. To 50 μl of gold particles, 5 μl (0.5 μg / μl) of plasmid DNA and 50 μl of 2.5M CaCa2 were added and mixed immediately. Next, 20 μl of 0.1M spermidine was added and further mixed, and then gold particles were precipitated with a centrifuge. After washing with 100% ethanol, it was suspended in 60 μl of 100% ethanol, dispensed once (5-10 μl) onto a macrocarrier and dried. The introduction conditions were as follows: degree of vacuum: 27-28 inches Hg, rupture disk: 1800 psi, gold particle size: 1.6 μm, sample distance: 4-8 cm. After introduction treatment (bombardment) into the xylem-forming tissue block, the cells were cultured overnight in the dark at 25 ° C, and then the histochemical method (incubated with X-glu solution at 37 ° C for 24 hours, Kosugi et al., 1990, Plant Sci., 70: 133-140), GUS activity (blue color development) was detected. As a result, as shown in FIGS. 4B to 4D, blue spots indicating the expression of the GUS gene by the cellulose synthase gene promoter were observed. Therefore, this promoter was shown to have an activity expressed at the xylem formation site as a result of the microarray experiment.
As shown in this example, the promoter sequence of the genes described in SEQ ID NOs: 1-34, 101-111, whose expression was recognized in the xylem by the microarray experiment, is used for the expression control in the xylem of an arbitrary gene. Can be used.

(Example 9) Analysis of interaction between wood fiber-forming gene promoter and transcription factor Transcription factor regulates on / off of expression by binding to the promoter region of the gene under its control (JL Riechmann SCIENCE VOL 290 15 DECEMBER 2000, Masaki Iwabuchi, Kazuo Shinozaki, plant genome function dynamism). Therefore, by analyzing the presence or absence of interaction (binding) with the promoter region of the gene, the relationship between control and control can be understood.

  Therefore, transcription factors described in SEQ ID NOs: 101 to 111 (SEQ ID NOs: 112 to 122) that control the formation of tree cell walls and xylem cell wall formation described in SEQ ID NOs: 1 to 34 (SEQ ID NOs: 35 to 68) are involved. The interaction of the gene with the promoter was analyzed. In this example, as an example, for the MYB transcription factor protein of SEQ ID NO: 117 and the homeodomain (HD-ZIP) transcription factor protein of SEQ ID NO: 119, the promoter region of the gene (CesA) involved in cellulose synthesis (SEQ ID NO: 93) ) And the promoters (SEQ ID NOs: 96, 97, 99, respectively) of genes (CAD, OMT, C4H) involved in lignin synthesis are shown.

(1) Protein expression and purification by cell-free translation system In order to express MYB transcription factor protein by a cell-free translation system, using the primers described in SEQ ID NOs: 169 and 170, MYB cDNA (SEQ ID NO: 106) was obtained. PCR was performed on the template. The amplified fragment is treated with restriction enzymes NdeI and smaI, inserted into the pIVEX1.3 vector (Roche), and reacted at 24 ° C for 24 hours using a cell-free translation system (wheat germ protein synthesis kit RTS100; Roche). It was. 1 ul of this reaction solution was electrophoresed on SDS-PAGE, and expression of the target protein was confirmed by Western blotting using an anti-His antibody.

(2) Protein expression and purification by E. coli In order to express HD-ZIP transcription factor protein by E. coli, using primers of SEQ ID NOs: 171 and 172, and using HD-ZIP cDNA (SEQ ID NO: 108) as a template PCR was performed. The amplified DNA fragment was treated with restriction enzymes EcoRV and SalI, inserted into a pET32a vector (Novagen), and transformed into E. coli BL21 strain. When protein was expressed, it was inoculated into LB medium and cultured at 37 ° C. until OD ₆₀₀ = 0.8, and 1 mM IPTG was added and cultured at 25 ° C. for 4 hours. After collection, purification was performed using HisTrap HP (GE Healthcare), washed with 50 mM imidazole, and eluted with 400 mM imidazole. This purified protein was desalted using HiTrap Desalting (GE Healthcare).

(3) Probe preparation The 900 bp region from the side close to the transcription start point of each promoter was divided into 30 bp each to obtain an oligo DNA probe sequence. At that time, since each probe was designed to have a 5 bp overlap with the adjacent probe sequences, a total of 40 probe pairs (sense / antisense) were artificially synthesized per promoter. At this time, the DNA on the sense side was labeled with TAMRA (fluorescent dye). For the analysis of the interaction, double-stranded DNA in which the sense and antisense strands were annealed was used. For annealing, TAMRA-labeled sense oligo DNA (SEQ ID NO: 173-332) and unlabeled antisense oligo DNA (SEQ ID NO: 333-492) were adjusted to 1 uM with 0.1 M NaCl / TE (pH 8.0). Then, after denaturing treatment at 95 ° C. for 5 minutes, it was performed while gradually returning to room temperature over 1 hour. Next, in order to remove unreacted single-stranded oligo DNA, 10 mM MgCl ₂ and Exonuclease I were added and reacted at 37 ° C. for 1 hour, and the probe was purified using MERmaid SPIN Kit (Qbiogene). The correspondence between the sense and antisense strands is that the complementary strand of SEQ ID NO: 173 is SEQ ID NO: 333 (Probe No. 1), and thereafter SEQ ID NO: 174 and 334 (Probe No. 2) to SEQ ID NO: 332 and 492 Correspond in order as in (Probe No. 160).

(4) Measurement of DNA-protein binding reaction
DNA-protein binding reaction was carried out in 30 ul of reaction solution (25 mM Hepes / KOH (pH 7.9), 50 mM KCl, 0.5 mM DTT, 5% glycerol, 2 mg / ml BSA, 0.05 mg / ml poly (dI-dC) -(DI-dC), protein 1ul, 3nM probe) for 20 minutes at room temperature. The standard dye was measured for 10 seconds × 5 times and the reaction solution was measured for 15 seconds × 5 times by FCS (Fluorescence Correlation Spectroscopy) measurement using an intermolecular interaction analyzer “single molecule fluorescence analysis system MF20 (Olympus)”. The translation time was calculated from this measured value, and the presence or absence of binding was verified. When binding is observed, the molecular weight of the labeled probe is increased, resulting in a longer translation time than the control. For the translation time as a control, the measured value of the empty vector expressed and each probe added to the reaction solution was used.

(5) Interaction (binding) between wood fiber-forming gene promoter and transcription factor
FIG. 5 shows the result 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 (binding) when there is a translation time extension of at least 20% or more. From the analysis results, two sites of CesA promoter (double-stranded DNA of SEQ ID NO: 181 and 341, double-stranded DNA of 199 and 359), one site of CAD promoter (SEQ ID NO: 246 and 406), and OMT promoter in MYB 1 (SEQ ID NOs: 274 and 434) and 2 sites of the C4H promoter (SEQ ID NOs: 296 and 456, 326 and 486). In HD-ZIP, there are 8 CesA promoters (SEQ ID NOs: 176 and 336, 184 and 344, 187 and 347, 189 and 349, 191 and 351, 193 and 353, 194 and 354, 196 and 356), 4 locations (SEQ ID NOs: 216 and 376, 217 and 377, 220 and 380, 223 and 383), 5 locations of OMT promoters (SEQ ID NOs: 258 and 418, 264 and 424, 275 and 435, 276 and 436, 281 441) and 7 sites of the C4H promoter (SEQ ID NOs: 301 and 461, 307 and 467, 313 and 473, 315 and 475, 318 and 478, 319 and 479, 329 and 489).
From the above results, it was shown that the transcription factor group involved in the formation of the wood fiber of the present invention controls the expression of the gene group involved in the formation of the wood fiber cell wall.

(Example 10) Material modification using transcription factor The material modification effect by the transcription factor described in SEQ ID NOs: 101 to 111 (SEQ ID NOs: 112 to 122) that control the expression of wood fiber cell wall-forming genes is confirmed. Therefore, we created transformed tobacco. Tobacco is classified as a herb, but the basic tissue structure is very similar to that of trees, and is commonly used for genetic analysis of trees and verification of introduction effects (Gray-Mitsumune et al., Plant Mol. Biol. 1999, 39: 657-669, Patzlaff et al., 2003, Plant Journal 36: 743-754). In this example, the results of material modification of transformants are shown for the MYB transcription factor gene of SEQ ID NO: 106 and the homeodomain (HD-ZIP) transcription factor gene of SEQ ID NO: 108 as examples.

(1) Production of Material-modified Transformed Tobacco In order to construct an introduction construct, PCR was performed using the primers described in SEQ ID NOs: 169 to 172, using the cDNAs (SEQ ID NOs: 106 and 108) as templates. PBI121 vector (Bevan, M., Nucleic Acids Res. 12, 8711-8721, 1984) in which the end of the amplified fragment was phosphorylated and blunt-ended, and previously blunt-ended by removing the GUS gene part with a restriction enzyme, Ligation was performed downstream of the 35S promoter to obtain an introduction construct. These constructs were introduced into Agrobacterium (LBA4404 strain) by electroporation, and transformants were selected on LB agar medium (kanamycin and hygromycin 25 μg / ml, 1.0% Agar). Single colonies were taken from the transformed Agrobacterium plates and cultured overnight (28.5 ° C., 240 rpm) in LB liquid medium containing antibiotics (kanamycin and hygromycin 25 μg / m each). After culturing, the Agrobacterium broth was collected by centrifugation (5 ° C., 8000 rpm, 5 min), the supernatant was removed, MS liquid medium was added, and the precipitate was gently suspended. 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.

As a sample for infection, young leaves of tobacco grown aseptically in a growth medium (MS 3% Sucrose N7B5 * 1% Agar pH 5.6) were used. Leaves were placed in a petri dish containing sterilized water, the periphery of the leaves and the main vein were removed with a scalpel, and the remaining part was cut into approximately 7 mm squares. Lightly dehydrate the slices between sterile Kimtowels, pre-culture medium (MS, 3% Sucrose, NAA ^10-7 mM, BA = ^10-5 mM, 1% Agar, pH 5. 6) and pre-cultured overnight at 25 ° C.

  After immersing the pre-cultured leaf section in an agro-suspension suspension and gently shaking for 20 minutes, remove the moisture lightly with a sterile Kim towel and place the leaf back on the co-culture medium. Co-cultured at 25 ° C. for 2-3 days. The co-cultured sections were transferred to a selective medium with tweezers and cultured at 25 ° C. in the light. After the start of culture, transplant to a new selective medium every two weeks, and transplant to hormone free rooting medium (1 / 2MS 1.5% Sucrose 1% Agar pH5.6 Clf 200μg / ml Hyg30μg / ml) once the shoots have redifferentiated. The transformant was selected.

(2) Analysis of Material-modified Transformed Tobacco 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. As a material analysis of the transformant, lignin content and fiber length were measured. The amount of lignin (total amount of acid-soluble and insoluble lignin) was determined by the Klason method (Wood Chemistry Experiment II. Chemistry P150-155, Patzlaff et al., 2003, Plant Journal), and the fiber length was measured by the Jefrey method (Han et al. al., 1999, Kenaf Properties, Processing and Products. P149-167). As a result, in the MYB transcription factor-introduced individuals, the ratio of lignin per dry weight was 14.7 ± 1.8% (average of 5 individuals), which was lower than that of the control (wild strain) 19.3 ± 1.1%. In addition, the fiber length was 560.6 ± 50.9 um for the control, whereas it was as long as 620.3 ± 42.1 um 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 also indicates that if the expression of this transcription factor is suppressed using a technique such as RNAi, the material can be modified to the opposite material (short fiber length with less cellulose).

On the other hand, in the HD-ZIP transcription factor-introduced individuals, the ratio of lignin increased at an average of 23.1 ± 2.5% (average of 5 individuals), but the fiber length was shortened at 534.6 ± 34.3um. Thus, by overexpressing the HD-ZIP transcription factor, it was possible to modify the material with a short fiber length with less cellulose. At the same time, it was also shown that if the expression of this transcription factor is suppressed, it can be modified to the opposite material (long fiber length with much cellulose).
From the above results, the plant material alteration effect of the transcription factor shown in the present application was shown.

(Example 11) Growth and species inspection of plant trunks using tree fiber cell wall forming genes (1) Variation in material due to ground height of eucalyptus trunk (vertical variation)
Regarding cell wall components, conduit distribution density, fiber length, etc., which are the main elements of wood fiber properties, it is known to form tissues that vary depending on the ground height regardless of whether they are hardwoods or conifers. This fluctuation phenomenon of wood fiber properties due to the ground clearance has long been known as vertical fluctuation of the material in the trunk. Comparing each ground height (base, center, and crown), cellulose increases in cell wall components and lignin and hemicellulose decrease in the center (above the chest height and less than the crown). In addition, in the morphological observation, the conduit distribution density is low and the fiber length is increased by about 20%. Furthermore, the thickness of the cell wall increases from the crown to the base. Although the details are unknown, it is thought that the difference in the properties of the wood fiber formed depends on the maturity of the wood fiber forming organization of each ground (Ken Shimaji, Shoji Sudo, Hiroshi Harada) “Wood Organization”, Satoshi Furuno and Satoshi Sawabe “Wood Science Course 2, Organization and Materials”).

  In order to confirm the vertical fluctuation of the material generally referred to, the present inventors used a 5-year clone line (CPT1) of the Eucalyptus genus Camaldrensis as a material, and analyzed the material as in the following examples. Went. Generally, 5-year-old chamaldrensis is low-growth and immature wood fiber at the trunk base (immature wood: short fiber, thin diameter, thin cell wall, steep fibril inclination, shrub fiber rate and low specific gravity) It is known that in the central part, high growth and mature wood fibers (mature wood: long fibers, thick diameter, thick cell walls, loose fibril inclination, high wood fiber ratio and high specific gravity) are known to form Yes. This feature is common to 5-10 grade camaldrensis that is cut down.

The trunks of the base (less than 1 m above the ground) and the central part (about 5 m above the ground) of the fifth grade plant were used. The bark was removed from each tree trunk, cut into 25 mm-thick discs, dried thoroughly, crushed into chips, and mixed well with a chip mixer for 3 minutes for analysis. The pulp refining and material analysis methods are basically published by the Paper Pulp Technology Association [Paper Pulp Manufacturing Technology Series] Volume 1 "Craft Pulp", Volume 3 "Pulp Cleaning, Selection and Bleaching", Volume 9 "Paper The conventional method described in "Testing methods for pulp" was followed. The cooking was performed with 13.2% Na ₂ S at 170 ° C. for 90 minutes, and the pulp data was corrected based on the kappa number of 18.

As a result of the material analysis obtained by the above-described method, 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. On the other hand, in 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, the generally-known vertical fluctuation was confirmed in the sample of this example. In addition, it was confirmed that the center part having a long fiber with a high bulk weight and a high pulp yield was superior as the material.

(2) Inspection of materials based on difference in expression profile due to ground clearance (comparison of expression profiles)
According to the expression profile classification table according to the difference in ground height between the 34 genes shown in Example 3 and the 11 genes shown in Example 6, the materials of the test samples were examined. As a test sample, the center part of the trunk of 2 individuals (S1, 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. By comparing the expression profile of each sample obtained with the taxon of Table 2 and examining whether it is similar to the profile on the base side or the center side, the material of the sample to be tested is immature. It can be determined whether it is close (poor quality) or close to matured wood (excellent quality). The sample site to be examined may be at the trunk base, at the center, or anywhere else. When using the central part of the trunk, it is generally expected that the part will have the best material in the individual, so the identified material can be said to be the best material in the individual. The opposite is true for the trunk base.

(3) Examination of sample 1 (S1) (Kibu vs. phloem profile)
The expression profiles in S1 of 12 genes that were inversely correlated in Tables 2 and 6 and classified specifically for cam5 were as follows.
Profile of one gene showing inverse correlation: basic profile (X> P).
Eleven genes showing X> P specifically in cam5 (middle part): 9 genes with a basic profile (X = P) and 2 genes with a central profile (X> P).

  Overall, the total number of genes in the basic profile was 11 or 83.3%, and the total number of genes in the central profile was 2 or 16.7%. From the above results, it was found that the profile of S1 is very close to the base but somewhat central. Therefore, it is determined that the material of S1 is slightly better than cam1 (base).

(4) Examination of sample 2 (S2) (Kibu vs. phloem profile)
The expression profiles in S2 of 12 genes that were inversely correlated in Tables 2 and 6 and classified cam5 specific were as follows.
Profile of one gene showing inverse correlation: basic profile (X> P).
Eleven genes showing X> P specifically in cam5 (middle part): 6 genes with a basic profile (X = P) and 5 genes with a central profile (X> P).

  Taken together, the total number of genes in the basic profile was 7 or 58.3% of the total, and the total number of genes in the central profile was 5 or 41.7%. From this result, it was found that the profile of S2 includes a basic but fairly central element. Therefore, the material of S2 is determined to be approximately halfway between cam1 (base portion) and cam5 (center portion).

(5) Relative comparison of samples 1 and 2 (Kibu vs. Kibe profile)
In order to make a relative comparison between the materials of S1 and S2, a microarray experiment was performed between the xylem parts of S1 and S2. As a result, the expression profiles of 22 genes (2 + 18 + 2) that differed between xylem in Tables 2 and 6 were as follows.
Profiles of 2 genes with X> P and 1> 5: The genes of this group with strong expression in cam1 (the one with the poorer material) were S1> S2 and the expression of S1 was stronger in both genes.
20 genes with X> P and 1 <5: In this group with strong expression in cam5 (the material is better), there are 1 gene with S1> S2, 5 genes with S1 = S2, and S1 <S2. There were 14 genes, and many genes were expressed in S2.

  As a whole, the gene group with strong expression in cam1 (the one with poor material) was S1, and the gene group with strong expression in cam5 (the one with good material) was expressed more in S2. Therefore, S2 is better than S1. It is determined that it has an excellent material. (3) In the inspection of (4), it was a result that S2 judged to be closer to the central part than S1 was superior in material. The result of direct comparison between the xylem was also the result that supported it.

(6) Material analysis of test sample The materials of S1 and S2 were analyzed by the method described in Example 11 (1). As a result, 488kg / m ² at 449kg / m ^2, S2 in test weight is S1, 45.7% pulp yield S1, 50.1% in S2, 0.69 mm fiber length by S1, was 0.75mm in S2. Therefore, as expected based on the above-described inspection results, S1 is a material close to the base, and S2 is a material that is basic but somewhat central. Further, when both were compared, S2 was relatively superior as expected. In this way, the material based on the test sample can be discriminated alive by comparison based on the expression profile shown in the present application.

(7) Variation in material due to species of eucalyptus trunk (variation between tree species)
Cell wall components, conduit distribution density, fiber length, etc., which are the main elements of wood fiber properties, are known to form tissues that vary depending on the species, regardless of whether they are hardwoods or conifers (Ken Shimaji, Shoji Sudo, Hiroshi Harada “Wood Organization”, Satoshi Furuno and Osamu Sawabe “Wood Science Course 2, Organization and Materials”). This phenomenon of variation in wood fiber properties due to this species has long been known as variation in the material species.

  For example, comparing Eucalyptus / Camardolensis species and Globulus species, the Globulus species generally increase cellulose and decrease lignin and hemicellulose. In addition, morphological observation shows that the conduit distribution density is small and the fiber length is increased. Furthermore, the cell wall is thick. Although details are unknown, it is considered that the nature of the wood fiber-forming tissue differs depending on the species, and that the difference in the nature of the wood fiber formed is generated.

  In order to confirm the tree species variation of the generally-known material, the present inventors conducted a 5-year-old Eucalyptus genus Camaldrensis clone line (CPT1) and a Globus clone line (GAL1) as materials. Material analysis was performed by the method shown in Example 2. In general, in the trunk of 5th grade Camaldorensis, wood fiber is inferior to Globulus (low quality pulp: short fiber, thin diameter, thin cell wall, steep fibril inclination, shrub fiber rate and low specific gravity) It is known that the 5th grade Globulus trunk forms excellent pulp (long fibers, thick diameter, thick cell walls, loose fibril inclination, high wood fiber rate and high specific gravity). This feature is the same for 5-10 year-old camaldrensis and globules.

In the present invention, the above-mentioned standard method for kraft pulp refining was used, using the above-mentioned five-year-old Eucalyptus camaldrensis stem base and Eucalyptus globulus stem base (both below 1 m above ground). As a result of material analysis, at the base of the 5-year-old camaldrensis plant, the bulk weight was 432 kg / m ² , the pulp yield was 43.8%, and the fiber length was 0.66 mm. On the other hand, at the trunk of Globulus, the bulk weight was 574 kg / m ² , the pulp yield was 57.1%, and the fiber length was 0.95 mm. From these results, the generally-variable inter-species variation was also confirmed in the sample of this example. In addition, it was also confirmed that the Globulus trunk base with high bulk weight and long fibers was superior as the material.

(8) Inspection of materials based on expression profile differences between species (expression profile comparison)
According to the expression profile classification table according to the difference in species between the 34 genes shown in Example 3 and 11 genes shown in Example 6, the materials of the test samples were examined. As test samples, 7 individual eucalyptus maldolensis (C1: same as S1 in Example 11 (3)) and 7 individual eucalyptus globulus (G1) were used. RNA extraction and microarray experiments were carried out in accordance with the methods of Examples 1 to 3, and material analysis was carried out in accordance with the method of Example 11 (1). By comparing the expression profile of each sample obtained with the taxonomic groups in Tables 3 and 7 and examining whether it is similar to that of Camaldrensis or that of Globulus, the species and material of the test sample Can be discriminated. The sample site to be examined may be at the trunk base, at the center, or anywhere else.

(9) Examination of the Camaldrensis sample (C1) (Kibu vs. phloem profile)
The expression profiles in C1 of the 23 genes classified in the inverse correlation and cam-specific manner in Tables 3 and 7 were as follows.
Profiles of 9 genes showing inverse correlation: Eight genes showed a camaldrenetic profile (X> P) and 1 gene showed a Globulous profile (X <P).
14 genes showing X> P specifically for camaldrensis: 12 genes of camaldrensis (X> P) and 2 genes of Globulus (X = P).

  Taken together, the total number of genes with a camaldrenetic profile was 20 which was about 87.0% of the total, and the total number of genes with a Globulous profile was 3 which was 13.0%. From this result, it was found that the C1 profile is camaldrensis-like but contains a lot of globulish elements. Therefore, although the seed of C1 is certainly chamaldrensis, it is discriminated that the material is slightly more globules (good quality) than general chamaldrensis.

(10) Inspection of Globulus sample (G1) (Kibu vs. phloem profile)
The expression profiles in G1 of 23 genes that were inversely correlated in Tables 3 and 7 and were cam-specifically classified were as follows.
Profiles of 9 genes showing inverse correlation: 1 gene showed a camaldrenetic profile (X> P) and 8 genes showed a Globulous profile (X <P).
14 genes showing X> P specifically for camaldrensis: 4 genes were camaldrensis (X> P) and 10 genes were Globulous (X = P).

  Taken together, the total number of genes with the Camaldrensis profile was 5 (21.7%), and the total number of genes with the Globulus profile was 18 (78.3%). From this result, it was found that the profile of G1 is Globulous. Therefore, although the seed of G1 is certainly globules, it is determined that the material is camaldrenotic (lower quality) than general globules.

(11) Relative comparison between C1 and G1 (profile of xylem vs. xylem)
To directly compare the xylem profiles of C1 and G1, a microarray experiment was performed between the xylem of C1 and G1. As a result, the expression profiles of 6 genes (4 + 1 + 1) that differed between xylem in Tables 3 and 7 were as follows.
4 genes with X> P and cam> glo: 5 C1> G1 genes, 1 C1 = G1 gene, 2 C1 <G1 genes, and this group is highly expressed by camaldrensis This gene was highly expressed in C1.
Two genes with X> P and cam <glo: The genes of this group that are strongly expressed in Globulus are both C1 <G1 genes, and many genes were expressed in G1.

  As a whole, the gene group that is strongly expressed in camaldrensis was expressed in C1, and the gene group that was strongly expressed in Globulus was highly expressed in G1, so the results of direct comparison between the xylems were also shown in (9) and (10). Similar to the test, C1 was determined to be camaldrensis and G1 to be globules. Therefore, it can be estimated that G1 which is also a globulus material is excellent.

(12) Analysis of material The material of C1 and G1 was analyzed by the method as described in Example 11 (1). As a result, 534kg / m ² at 449kg / m ^2, G1 in test weight is C1, 45.7% for C1 pulp yield 53.6% for G1, fiber length was 0.86mm in C1 0.69 mm, in G1. Therefore, as expected based on the above-described inspection results, C1 is a camaldrensis-like material and G1 is a Globulous-like material as expected. Moreover, when both were compared, G1 was relatively superior as expected. In this manner, the species and material of the test sample can be discriminated alive by comparison based on the expression profile shown in the present application.

  Using the eucalyptus EST database thus prepared, the present inventors examined extraction of genes involved in cell wall synthesis in eucalyptus tree fiber-forming tissues by microarray analysis, and expression variations due to differences in ground height and species. As a result, we identified a cell wall synthetic gene group expressed in eucalyptus tree fiber-forming tissues, and identified a gene group whose expression fluctuates predominantly depending on the ground height and species. Furthermore, those promoter DNAs were obtained and confirmed to function as xylem expression promoters. The above gene group and expression information / promoter DNA can be used to control cell wall synthesis and morphogenesis of wood fiber-forming tissues.

  In addition, the present inventors examined the expression fluctuations due to the difference in the height of the ground and the species using the prepared eucalyptus EST database to extract transcription factor groups related to the formation of wood fibers by microarray analysis. As a result, we identified a group of transcription factors involved in eucalyptus tree fiber formation, identified a group of genes whose expression varies predominantly depending on the height of the ground and species, and obtained their promoter DNA. Furthermore, we showed that plant materials can be modified by introducing these transcription factors. The gene set and expression information / promoter DNA can be used to control wood fiber formation (material).

It is a figure which shows the gene expression intensity | strength of a 5-year-old tree trunk base of Eucalyptus genus Camaldrensis. Each cRNA synthesized using two kinds of mRNA extracted from the phloem side of the trunk base and the xylem side as a template is labeled with two fluorescent dyes (cy3.cy5), and used as a probe in oligo microarray analysis. Hybridization was performed. Fluorescence intensity is calculated from images obtained by scanning, 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 expression intensity of the major gene group was expressed. Among the two calibration curves in the figure, + (black) in the vicinity of the upper line or in the region above it is a significant increase in expression at the wood fiber formation site, which is almost in the middle of the two calibration curves + (Light gray) indicates that expression decrease is significantly observed, and + (dark gray) in the vicinity of the lower line or in the area below the two calibration curves indicates that expression is not changed. It is a figure which shows the gene expression intensity | strength of the 5-year-old tree trunk center part of Eucalyptus genus Camaldrensis. Each cRNA synthesized using two types of mRNA extracted from the phloem side and the xylem side of the center of the trunk as a template is labeled with two fluorescent dyes (cy3.cy5), and used as a probe in oligo microarray analysis. Hybridization was performed. 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%. The relative intensity of expression of major gene groups was expressed. Among the two calibration curves in the figure, + (black) in the vicinity of the upper line or in the region above it is a significant increase in expression at the wood fiber formation site, which is almost in the middle of the two calibration curves + (Light gray) indicates that expression decrease is significantly observed, and + (dark gray) in the vicinity of the lower line or in the area below the two calibration curves indicates that expression is not changed. It is a figure which shows the gene expression intensity | strength of a Eucalyptus globulus species tree trunk. Each cRNA synthesized using two kinds of mRNA extracted from the phloem side and xylem side of the trunk as a template is labeled with two fluorescent dyes (cy3.cy5), and used as a probe in oligo microarray analysis. Hybridization was performed. Fluorescence intensity is calculated from images obtained by scanning, 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 expression intensity of the major gene group was expressed. Among the two calibration curves in the figure, + (black) in the vicinity of the upper line or in the region above it is a significant increase in expression at the wood fiber formation site, which is almost in the middle of the two calibration curves + (Light gray) indicates that expression decrease is significantly observed, and + (dark gray) in the vicinity of the lower line or in the area below the two calibration curves indicates that expression is not changed. It is a figure which shows the promoter activity of the gene group involved in wood fiber formation. A shows a trunk obtained by excising a xylem-forming tissue block as a target for introducing a particle gun. B shows a xylem-forming tissue block subjected to a histochemical GUS assay after gene transfer using a particle gun. C and D are enlarged photographs of the blue spot where the GUS gene was expressed (center part circled). It is a figure which shows the interaction (binding | combination) of the promoter of a cell wall formation gene group involved in wood fiber formation, and a transcription factor group. Each graph shows the analysis results of the MYB transcription factor and the four promoters. In each graph, the horizontal axis represents the number of the double-stranded DNA probe, and the vertical axis represents the percent 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 promoter 5 'side, and the larger probe number is the downstream region on the promoter 3' side close to the transcription start point. * Indicates a probe that has been confirmed to interact. It is a figure which shows the interaction (binding | combination) of the promoter of a cell wall formation gene group involved in wood fiber formation, and a transcription factor group. Each graph shows the analysis results of the HD-ZIP transcription factor and four types of promoters. In each graph, the horizontal axis represents the number of the double-stranded DNA probe, and the vertical axis represents the percent 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 promoter 5 'side, and the larger probe number is the downstream region on the promoter 3' side close to the transcription start point. * Indicates a probe that has been confirmed to interact. It is a figure which shows the transformed tobacco plant body which introduce | transduced the transcription factor. A is a transformant introduced with a MYB transcription factor, and B is a transformant introduced with a homeodomain transcription factor.

Claims (29)

A 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).
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 68
(B) DNA containing the base sequence described in any one of SEQ ID NOs: 1 to 34
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 35-68
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 34
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 35-68 The DNA according to claim 1, wherein the plant is eucalyptus. A partial DNA of the DNA according to claim 1 or 2. A substrate on which the DNA according to claim 1 is immobilized. On the phloem side and xylem side of the trunk of the plant, the expression level of at least one DNA described in the following (a) to (e) is detected, and the expression level of the DNA on the phloem side and the xylem side is determined. A method for inspecting the growth state of a tree trunk part of a plant, comprising a step of comparing.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 64 or 66 to 68
(B) DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 30 or 32 to
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 35-64 or 66-68
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1-30 or 32-34
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 35-64 or 66-68 Detecting the expression level of at least one DNA described in the following (a) to (e) in two different tree parts, and comparing the expression level of the DNA in two different tree parts: A method for examining the growth status of two different stem parts.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67
(B) DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, 8, 14, 16, 17, 19 to 22, 24, 25, 28, 30, 32, or 33
(C) SEQ ID NO: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67% of the protein consisting of the amino acid sequence and 50% or more DNA encoding a protein with homology
(D) DNA having the nucleotide sequence set forth in any one of SEQ ID NOs: 1-6, 8, 14, 16, 17, 19-22, 24, 25, 28, 30, 32, or 33, and stringent conditions DNA that hybridizes under
(E) SEQ ID NOs: 35 to 40, 42, 48, 50, 51, 53 to 56, 58, 59, 62, 64, 66, or 67, one or more amino acids are substituted. DNA encoding proteins consisting of amino acid sequences deleted, added, and / or inserted On the phloem side and xylem side of the trunk of the plant, the expression level of at least one DNA described in the following (a) to (e) is detected, and the expression level of the DNA on the phloem side and the xylem side is determined. A method for inspecting the growth state of a tree trunk part of a plant, comprising a step of comparing.
(A) SEQ ID NO: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66, or 67. DNA
(B) DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, 8, 9, 11 to 14, 16, 17, 19 to 25, 27, 28, 30, 32, or 33
(C) SEQ ID NO: 35 to 40, 42, 43, 45 to 48, 50, 51, 53 to 59, 61, 62, 64, 66, or 67% of the protein consisting of the amino acid sequence described in 67 DNA encoding a protein having the above homology
(D) DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, 8, 9, 11 to 14, 16, 17, 19 to 25, 27, 28, 30, 32, or 33 and stringent DNA that hybridizes under mild conditions
(E) SEQ ID NOs: 35-40, 42, 43, 45-48, 50, 51, 53-59, 61, 62, 64, 66, or one or more amino acids in the amino acid sequence according to 67 DNA encoding a protein consisting of an amino acid sequence in which is substituted, deleted, added, and / or inserted Detecting the expression level of at least one DNA described in the following (a) to (c) in two different tree parts, and comparing the expression level of the DNA in two different tree parts: A method for examining the growth status of two different stem parts.
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56
(B) DNA comprising the base sequence set forth in any one of SEQ ID NOs: 11, 12, 17, 21, or 22
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 11, 12, 17, 21, or 22
(E) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 45, 46, 51, 55, or 56 DNA to encode A protein encoded by the DNA according to claim 1 or 2. Promoter DNA comprising any of the following base sequences (a) to (c):
(A) the base sequence according to any of SEQ ID NOS: 93 to 100 (b) consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a), and (A) a base sequence having a function as a promoter substantially the same as the promoter comprising the DNA of (a) (c) comprising a part of the base sequence of (a), and substantially the same as the promoter comprising the DNA of (a) Nucleotide sequence having the same promoter function as The promoter DNA according to claim 10, further comprising a base sequence that recognizes a transcription factor involved in specific expression in a plant fiber-forming tissue. The DNA according to any one of the following (a) to (e).
(A) DNA encoding an antisense RNA complementary to the transcription product of the DNA of claim 1
(B) DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcript of the DNA of claim 1
(C) DNA encoding an RNA that suppresses the expression of the DNA according to claim 1 by an RNAi effect
(D) a DNA encoding an RNA that suppresses the expression of the DNA according to claim 1 due to a co-suppression effect;
(E) a DNA encoding a protein having a dominant negative trait for the transcription product of the DNA of claim 1 A DNA encoding a plant transcription factor according to any one of the following (a) to (e):
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 112 to 122
(B) DNA comprising the base sequence described in any one of SEQ ID NOs: 101 to 111
(C) DNA encoding a protein having 50% or more homology with a protein comprising the amino acid sequence of SEQ ID NO: 112-122
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 101 to 111
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NOS: 112 to 122 The DNA according to claim 13, wherein the plant is eucalyptus. A partial DNA of the DNA according to claim 13 or 14. A substrate on which the DNA according to any one of claims 13 to 15 is immobilized. On the phloem side and xylem side of the trunk of the plant, the expression level of at least one DNA described in the following (a) to (e) is detected, and the expression level of the DNA on the phloem side and the xylem side is determined. A method for inspecting the growth state of a tree trunk part of a plant, comprising a step of comparing.
(A) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 112 to 118 or 120 to 122
(B) DNA comprising the base sequence set forth in any of SEQ ID NOs: 101 to 107 or 109 to 111
(C) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 112-118 or 120-122
(D) DNA that hybridizes under stringent conditions with the DNA consisting of the nucleotide sequence of SEQ ID NO: 101 to 107 or 109 to 111
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 112-118 or 120-122 Detecting the expression level of at least one DNA described in the following (a) to (e) in two different tree parts, and comparing the expression level of the DNA in two different tree parts: A method for examining the growth status of two different stem parts.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 113 or 114
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(C) DNA encoding a protein having 50% or more homology with the protein comprising the amino acid sequence set forth in SEQ ID NO: 113 or 114
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 102 or 103
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 113 or 114 On the phloem side and xylem side of the trunk of the plant, the expression level of at least one DNA described in the following (a) to (e) is detected, and the expression level of the DNA on the phloem side and the xylem side is determined. A method for inspecting the growth state of a tree trunk part of a plant, comprising a step of comparing.
(A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 113 to 117
(B) DNA comprising the base sequence according to any of SEQ ID NOs: 102 to 106
(C) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence of SEQ ID NO: 113 to 117
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of any one of SEQ ID NOS: 102 to 106
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 113 to 117 Detecting the expression level of at least one DNA described in the following (a) to (c) in two different tree parts, and comparing the expression level of the DNA in two different tree parts: A method for examining the growth status of two different stem parts.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 116
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 105
(C) DNA encoding a protein having 50% or more homology with the protein consisting of the amino acid sequence set forth in SEQ ID NO: 116
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 105
(E) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 116 A protein encoded by the DNA of claim 13 or 14. Promoter DNA comprising any of the following base sequences (a) to (c):
(A) the base sequence according to any one of SEQ ID NOS: 156 to 166 (b) consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a), and (A) a base sequence having a function as a promoter substantially identical to the promoter comprising the DNA of (a) (c) comprising a part of the base sequence of (a) and substantially comprising the promoter of the DNA of (a) Nucleotide sequence having the same promoter function as Furthermore, the promoter DNA according to claim 22, wherein a base sequence that recognizes a transcription factor involved in specific expression is functionally linked to a plant fiber-forming tissue. A recombinant vector having the DNA according to any one of claims 1 to 3, 10 to 15, 22 or 23. A microorganism carrying a plasmid comprising the vector according to claim 24. A transformed plant cell into which the vector according to claim 24 has been introduced. A transformed plant regenerated from the transformed plant cell according to claim 26. A transformed plant which is a descendant or clone of the transformed plant according to claim 27. The propagation material of the transformed plant body of Claim 27 or 28.