JP3914348B2 - Method for modifying cell wall components of plants - Google Patents

Description

【図面の簡単な説明】
【図１】 acw1変異体の根切片におけるウラニルアセテート・クエン酸鉛染色の結果を示す電子顕微鏡写真である。Aは野生型、Bは21度（許容温度）での変異体、Cは31度（非許容温度）での変異体における結果をそれぞれ示す。
【図２】 acw1変異体の下胚軸におけるウラニルアセテート・クエン酸鉛染色の結果を示す電子顕微鏡写真である。Aは野生型、Bは31度（非許容温度）での変異体における結果をそれぞれ示す。
【図３】 acw1変異体の根切片におけるPATAg染色の結果を示す電子顕微鏡写真である。Aは野生型、Bは31度（非許容温度）での変異体における結果をそれぞれ示す。
【図４】野生株とacw1変異体の細胞壁構成糖成分を分析した結果を示す。ＡはTFA可溶層の構成糖量を、ＢはTFA不溶画分（残さ；セルロース）の構成糖量を示す。 Rha；ラムノース、Fuc；フコース、Ara；アラビノース、Xyl；キシロース、Gal；ガラクトース、Glc；グルコース、Uronic acid；ウロン酸
【図５】野生株とacw1変異体のセルロース繊維を観察した結果を示す電子顕微鏡写真である。上は野生株のセルロース繊維、下はacw1変異体のセルロース繊維を示す。矢印は、強酸処理により太くなった繊維を示す。
【図６】野生型とacw1変異体のセルロースの重合度を解析するために行ったGPCのクロマトグラムの結果を示す。白丸は変異体、白三角は野生株の結果を示す。横軸は、カラムから溶出される時間を示し、縦軸は検出器で検出された値を示す。早い時間のピークほど分子量が大きい。
【図７】野生型とacw1変異体のクロロフォルム/メタノール層を、TFA加水分解し、薄層クロマトフラフィー（TLC）を行った結果を示す。レーン１はグルコースの対照、レーン２はガラクトースの対照、レーン３は野生株、レーン４は変異体、の結果を示す。明瞭なグルコースのスポットが観察される。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to modification of plant cell wall components using a gene encoding a cellulase-like protein involved in plant cell wall synthesis.
[0002]
[Prior art]
Modifying cell wall components in plants has various important implications in the fields of industry and agriculture. For example, the modification of plant cell wall components leads to an increase in the digestion and absorption efficiency of fiber raw material plants such as pulp by increasing the content of cellulose and hemicellulose, and useful agricultural and feed crops, in terms of economy and profitability. Meaningful. Moreover, it is also possible to produce raw material plants having new industrial value by changing the structure of the polysaccharide which is a cell wall component.
[0003]
Synthesis of cell walls and similar polysaccharide components is performed not only in bacteria, fungi, and plants, but also in animals. Despite its industrial importance, studies at the molecular level of cell wall synthesis have not been well analyzed. In recent years, analysis of plant cell wall synthesis at the molecular level has begun to be performed using molecular genetics techniques. As a gene involved in cell wall synthesis, for example, cellulose synthase has been reported (T. Arioli et al. Molecular analysis of cellulose biosynthesis in Arabidopsis. Science (1998) 279: 717-720). However, there are many genes that have not yet been isolated as genes involved in cell wall synthesis, and it is thought that there are unknown mechanisms for cell wall synthesis (reference: Y. Kawagoe and DP Delmer, Pathway and genes involved in cellulose biosynthesis; Genetic engineering 19 Plenum Press, New York, 1997; K. Nishitani, Construction and Restructuring of the cellulose-xyloglucan framework in the apoplast as mediated by the xyloglucan-related protein family-A hypotheticalscheme. Plant Res. 111: 159-166, 1998).
[0004]
On the other hand, cellulase is known as an enzyme that degrades the cell wall. Cellulase has been reported in bacteria, fungi, slime molds, plants, etc. as an enzyme that degrades β-1,4 linked polysaccharides (Endo β-1,4 glucanase) (Beguin, P. Annu. Rev. Microbiol. 44, 219-248. 1990). It has been shown that it can be divided into 6 or more classes from the active site determined from the hydrophobicity analysis of the amino acid sequence. Among them, plant cellulases belong to the E family (Beguin, P. Annu. Rev. Microbiol. 44, 219-248. 1990). E family cellulases such as bacteria retain a typical cellulose-binding domain and are secreted extracellularly and can degrade crystalline cellulose. Bacteria and the like use glucose, which is a degradation product, as a nutrient source.
[0005]
Cellulase in higher plants is said to be involved in the modification of the cell wall of the cell itself with growth, development, differentiation, etc. (DJ Cosgrove, How How plant cells extend? Plant Physiol. 102, 1-6, 1993). Therefore, cellulase needs to be secreted extracellularly, and all of the plant cellulases reported so far have signal peptides for secretion (Z. Shani, M. Dekel, G. Tsabary and O Shoseyov. Plant Mol. Biol. 34. 837-842, 1997. ML Tucker, R. Sexton, E. del Campillo and LN Lewis. Plant Physiol. 88: 1257-1262. 1988).
[0006]
Changes in the structure and properties of cell walls occur during plant growth and differentiation. For example, synthesis of a new cell wall or modification / reconstruction of an existing cell wall polysaccharide occurs, thereby changing the size, shape, function, etc. of the cell. So far, plant cellulases have been suggested to function on hemicellulose or cellulose with β-1,4 glucan chain as the backbone (Y.-S. Wong, GB Fincher and GA Maclachlan. J. Biol. Chem). 252, 1402-1407. 1977. T. Hayashi, Y.-S. Wong and GA Maclachlan. Plant Physiol. 75, 605-610, 1984). However, because there is a contradiction such as not retaining the cellulose binding domain necessary for degrading crystalline cellulose, it is not clear what cellulase actually functions in plants. Compared with cellulases such as bacteria, there is very little knowledge in higher plants.
[0007]
By the way, the present inventors have found a mutant having an abnormal cell or organ shape with a search number of # 66 in an Arabidopsis thaliana group treated with the chemical mutant EMS (1996). August 25, 7th Arabidopsis Workshop, March 27, 1997 Japanese Society of Plant Physiology, 1997 Annual Meeting, November 13, 1997 8th Arabidopsis Workshop, May 3, 1998 Japanese Plant Physiological Society Annual Meeting 1998). This mutation was named acw1 (altered cell wall 1), and the gene responsible for this mutation was named ACW1 gene. However, the ACW1 gene has not been isolated yet.
[0008]
[Problems to be solved by the invention]
It is an object of the present invention to modify plant cell wall components using a gene encoding an enzyme involved in plant cell wall synthesis.
[0009]
[Means for Solving the Problems]
The present inventors have succeeded in identifying and isolating a single gene responsible for the ACW1 mutation, which causes abnormal cell wall synthesis in plants, in a vast chromosomal region by using positional cloning techniques. The present inventors determined the base sequence of the isolated ACW1 gene, performed a database search for genes structurally related thereto, and found the same gene as the ACW1 gene on the database. This gene has been characterized as a homologue of a plant secretory cellulase known as a cell wall degrading enzyme.
[0010]
However, as a result of various analyzes conducted on the cell wall components of the acw1 mutant by the present inventors, surprisingly, the protein encoded by the ACW1 gene (hereinafter referred to as “ACW1 protein”) is not degraded by the cell wall. Rather, it has been found that it has a function related to synthesis. There have been no reports on cellulases involved in cell wall synthesis. The present inventors have found that the cell wall component of a plant can be modified by using the ACW1 gene from such properties of the ACW1 protein.
[0011]
The present invention relates to the modification of plant cell wall components using a gene encoding a cellulase-like protein involved in plant cell wall synthesis, more specifically,
(1) DNA selected from the following (a) to (d), which is used for modifying a cell wall component of a plant,
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1.
(B) having an amino acid sequence in which one or a plurality of amino acids are substituted, deleted and / or added in the amino acid sequence described in SEQ ID NO: 1, and functionally with the protein consisting of the amino acid sequence described in SEQ ID NO: 1 DNA encoding an equivalent protein.
(C) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 2.
(D) DNA that hybridizes with DNA consisting of the base sequence shown in SEQ ID NO: 2 and that encodes a protein functionally equivalent to the protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
(2) The DNA according to (1), wherein the cell wall component of the plant is a glucan chain,
(3) A method for modifying a cell wall component of a plant, which comprises using a DNA selected from (a) to (d) in (1),
(4) The method according to (3), wherein the cell wall component of the plant is a glucan chain,
(5) A transformed plant having a cell wall component modified by introduction and expression of a DNA selected from (a) to (d) in (1) compared to a wild-type plant,
(6) The plant body according to (5), wherein the cell wall component of the plant is a glucan chain,
About.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the use of DNA encoding cellulase-like proteins involved in plant cell wall synthesis to modify plant cell wall components. Specifically, the modification of plant cell wall components in the present invention is performed by producing a transformed plant body that retains a DNA encoding a cellulase-like protein involved in plant cell wall synthesis. The “ACW1” gene is preferred as the DNA used to modify cell wall components.
The nucleotide sequence of cDNA of the “ACW1” gene isolated by the present inventors is shown in SEQ ID NO: 2, and the nucleotide sequence of genomic DNA is shown in SEQ ID NO: 3.
[0013]
In the modification of the cell wall component of the plant of the present invention, DNA other than the “ACW1” gene can be used as long as it encodes a protein having a function equivalent to that of the “ACW1” protein. Examples of such DNA include an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence (SEQ ID NO: 1) of the “ACW1” protein, Examples include DNAs that encode functionally equivalent proteins. In the present invention, “functionally equivalent” means that a protein functions in plant cell wall synthesis, more specifically in glucan chain synthesis. Whether a protein functions in plant cell wall synthesis is determined, for example, by a functional complementation test by expressing the “ACW1” gene in a mutant strain, by controlling the expression of the “ACW1” gene, or by inhibiting the function of the “ACW1” protein. It can be detected as a change in plant cell wall synthesis, for example, a change in cell wall components (accumulation of amorphous glucan), a change in organ or cell morphology. Artificial modification of the amino acid sequence of the “ACW1” protein can be performed using, for example, “Transformer Site-directed Mutagenesis Kit” or “ExSite PCR-Based Site-directed Mutagenesis Kit” (Clontech) for mutation or substitution. In addition, if it is a deletion, it is possible to use “Quantum leap Nested Deletion Kit” (manufactured by Clontech). The number of amino acid modifications is usually within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and even more preferably within 3 amino acids. In addition, amino acid mutations may occur not only in the artificial world but also in the natural world.
[0014]
In the acw1 mutation, the present inventors found that the substitution of the base guanine at position 1355 of the ACW1 gene with adenine (which replaces the amino acid glycine at position 429 with serine) is a temperature-sensitive mutant protein (under low temperature conditions). It has been found that a wild-type phenotype is imparted to a plant but an abnormal phenotype is imparted under high temperature conditions (Example 1). In the present invention, it is also possible to use a protein that exhibits a function equivalent to that of “ACW1” at a specific temperature. Such a protein is useful in that its function can be controlled by adjusting the temperature.
[0015]
In the present invention, as long as it encodes a protein that is functionally equivalent to the “ACW1” protein, DNA derived from other plant species having a base sequence showing homology with the base sequence of the “ACW1” gene may be used. Is possible. Such DNA can be obtained, for example, by hybridization technology using all or a part of the base sequence of the “ACW1” gene as a probe (Southern 1975 J. Mol. Biol. 98: 503, Maniatis et al. Molecular Cloning Cold Spring harbor Laboratry Press) and PCR technology using oligonucleotides that specifically hybridize to the ACW1 gene as primers (supervised by Isao Shimamoto and Takuji Sasaki, “Plant PCR Experiment Protocol” (Cellular Engineering Supplement, Plant Cell Engineering Series 2) It can be isolated using Shujunsha (issued April 10, 1995). “Functionally equivalent” means that the protein functions in plant cell wall synthesis, more specifically in glucan chain synthesis, as described above. When DNA obtained by the hybridization technique or PCR technique encodes a protein having a function equivalent to that of the “ACW1” protein, it usually has high homology with the “ACW1” gene. High homology means 45% or more homology, preferably 60% or more homology, more preferably 75% or more homology, more preferably 90% or more homology at the level of amino acid encoded by each DNA. Sex, more preferably 95% homology or higher. Examples of plants other than Arabidopsis thaliana for isolating such genes include, but are not limited to, rice, corn, potato, tobacco, cotton, acacia, pine, cedar, eucalyptus, and poplar.
[0016]
In order to express these DNAs in plant cells, (i) a DNA molecule linked downstream of a promoter sequence that can be transcribed in plant cells, and (ii) transcription linked downstream of the gene, if necessary. An expression cassette containing a terminator sequence containing a polyadenylation site necessary for product stabilization is introduced into plant cells.
[0017]
The expression cassette may contain a promoter for constitutively or inducibly expressing the inserted DNA. Examples of promoters for constant expression include cauliflower mosaic virus 35S promoter (Odell et al. 1985 Nature 313: 810), rice actin promoter (Zhang et al. 1991 Plant Cell 3: 1155), maize And ubiquitin promoter (Cornejo et al. 1993 Plant Mol. Biol. 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, UV irradiation, and spraying of specific compounds. Promoters and the like. Examples of such promoters include the rice chitinase gene promoter (Xu et al. 1996 Plant Mol. Biol. 30: 387) expressed by infection and invasion of filamentous fungi, bacteria, and viruses, and the promoter of tobacco PR protein gene. (Ohshima et al. 1990 Plant Cell 2:95), low temperature induced rice “lip19” gene promoter (Aguan et al. 1993 Mol. Gen Genet. 240: 1), high temperature induced rice “ hsp80 ”and“ hsp72 ”gene promoters (Van Breusegem et al. 1994 Planta 193: 57), Arabidopsis thaliana“ rab16 ”gene promoter (Nundy et al. 1990 Proc. Natl. Acad. Sci. USA 87: 1406), Parsley Chalcone Synthase Gene Promoter Induced by UV Irradiation (Schulze-Lefert et al. 1989 EMBO J. 8: 651), Maize Induced under Anaerobic Conditions Le call dehydrogenase gene promoter (Walker et al 1987 Proc Natl Acad Sci USA 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.
[0018]
There is no particular limitation on the plant cell into which the expression cassette is introduced. Plant cells desiring to modify cell wall components can be used. Examples thereof include cells such as Arabidopsis, rice, corn, potato, tobacco, cotton, acacia, pine, cedar, eucalyptus, and poplar. Plant cells into which an expression cassette is introduced include cells in plants as well as cultured cells. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots. Introduction of a vector into a plant cell can be performed by, for example, an introduction method using Agrobacterium (Hood et al. 1993 Transgenic Res. 2: 218, Hiei et al. 1994 Plant J. 6: 271), electroporation method ( Tada et al. 1990 Theor. Appl. Genet 80: 475), polyethylene glycol method (Lazzeri et al. 1991 Theor. Appl. Genet 81: 437), particle gun method (Sanford et al. 1987 J. Part. Sci. Tech) 5:27) and various methods known to those skilled in the art can be used.
[0019]
The transformed plant cell can be regenerated to produce a plant body. The method of regeneration varies depending on the type of plant cell. For example, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) is mentioned for rice, and the method of Shillito et al. (Bio / Technology 7) for maize. : 581 (1989)) and Gorden-Kamm et al. (Plant Cell 2: 603 (1990)), and for potatoes, Visser et al. (Theor. Appl. Genet 78: 594 (1989)). In the case of tobacco, the method of Nagata and Takebe (Planta 99:12 (1971)) is mentioned, and in the case of Arabidopsis, the method of Akama et al. (Plant Cell Reports 12: 7-11 (1992)) is mentioned. As a result, the expression of the protein of the present invention is changed in the plant obtained from the plant or its propagation medium (for example, seeds, tubers, cuttings, etc.) compared to the wild-type plant. This modifies the cell wall components. “Modification of cell wall component” includes quantitative change, qualitative change and cell shape change of cell wall component. The cell wall component is preferably a glucan chain. Glucan chain modification is adjustment of glucan polymerization degree (number of polymerized sugars) (glucan polymerization degree includes, for example, conifer type, hardwood type, dicotyledon type and monocot type), crystallization Adjustment of the degree (the thickness of the fiber formed by bundling glucan, which is a determinant of fiber strength).
[0020]
Modification of cell wall components in plants has many advantages. An increase in cell wall components can lead to, for example, an increase in plant growth and an increase in fiber sources such as pulp, and qualitative changes in cell wall components can lead to the development of new materials and the increase in digestion and absorption efficiency of feed crops. be able to. The change in cell morphology can also be applied to the creation of ornamental plants with new aesthetic value.
[0021]
【Example】
EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples. Methods necessary for general gene recombination, such as DNA cleavage, ligation, E. coli transformation, gene sequencing, and hybridization, are attached to commercially available reagents and machinery used for each operation. Basic instructions were followed and experiments (eg “Molecular cloning (Maniatis T. et al. Cold Spring Harbor Laboratory Press)”). The growth, mating, and genomic DNA preparation of Arabidopsis thaliana using agar medium and soil were basically in accordance with an experimental document (for example, “Model Plant Experiment Protocol (Shyujunsha)”).
[0022]
[Example 1] Isolation of ACW1 gene
First, in order to determine the position of the ACW1 gene on the chromosome, mapping was performed using molecular markers. Since the acw1 (altered cell wall 1) mutant has been found in the Colombian ecotype of Arabidopsis thaliana, another ecotype, the Landsburg Electa species, was crossed to the next generation seeds. It was grown and selfed to obtain F2 generation seeds. Genomic DNA was extracted from plants (830 individuals) grown by germinating seeds of this F2 generation. Using these genomic DNAs as markers, “2I5,2.2” and “miP5-9,1.1” were used to calculate the recombination value of the genomic DNA by PCR. Specifically, two kinds of PCR primers “SEQ ID NO: 4 / 5-TCAAATAGATATATGTAATATGTTTCGC-3” and “SEQ ID NO: 5 / 5-CAATAAATAATTATACAAAACTTTCAAG-3” capable of amplifying “2I5,2.2” were synthesized, Using these, F2 generation genomic DNA was amplified using PCR and examined by agarose electrophoresis. This molecular marker shows a band at 112 bp for the Lansburg species and 110 bp for the Colombia species, and calculations based on this showed that recombination was observed on one chromosome between the ACW1 gene and the same marker. Next, two kinds of PCR primers “SEQ ID NO: 6 / 5-TACTTAATAAAGTAGGATAATGTCG-3” and “SEQ ID NO: 7 / 5-GAACATGAATTCATGTGTTACACTG-3” capable of amplifying “miP5-9,1.1” were synthesized. Was used to amplify F2 generation genomic DNA using PCR and examined by agarose electrophoresis. This molecular marker is known to show a band at 87 bp for the Landsburg species and 91 bp for the Colombia species, and as a result of calculation based on this, recombination was carried out on one chromosome between the ACW1 gene and the same marker. Admitted.
[0023]
As described above, it was confirmed that the region on the chromosome containing the ACW1 gene was sandwiched between two molecular markers “2I5, 2.2” and “miP5-9, 1.1”. Therefore, we decided to proceed with gene isolation by isolating genomic DNA fragments containing the molecular markers “2I5,2.2” and “miP5-9,1.1”, respectively. Specifically, a genomic DNA library (approximately 10000 clones) composed of 60-90kbp genomic DNA of Arabidopsis thaliana Colombia was screened using "2I5,2.2" and "miP5-9,1.1" as probes. , 2.2 "probe for 3 clones (named TAC6M17, 12J22, 13F4) and" miP5-9,1.1 "probe for 2 clones (named TAC15E4, 9P8). The DNA fragment (approximately 600b) on the “left” side (“2I5,2.2” side) of “TAC15E4” (this fragment is referred to as “15E4-L”) is subjected to the TAIL-PCR method (plant PCR experiment protocol (Syujun) ))). At this time, L1, L2, L3, and AD2 were used as primers in the TAIL-PCR method. Two types of PCR primers “SEQ ID NO: 8 / 5-GCTTATAAATGGGTATAAACCTATCAGC-3” and “SEQ ID NO: 9 / 5-TGCTTAATTCTCCGGTAACATTACCGGC” that can amplify “15E4-L” based on the base sequence of the isolated DNA fragment -3 "was synthesized, and the genomic DNAs of Landsberg and Colombia were amplified by PCR using the PCR method, and the nucleotide sequence was decoded. In Landsberg, 96 primers were used from the primer (SEQ ID NO: 8). It was found that the nucleotide sequence of the position was G, and that Colombia was T, and this DNA fragment could be used as a molecular marker. Therefore, when the distance between the ACW1 gene and this new marker was calculated in the aforementioned F2 plant, no recombination was found between chromosomes 1660.
[0024]
The TAC genomic DNA library is made with a vector (TAC vector) that can introduce the inserted DNA fragment directly into a plant via Agrobacterium. Therefore, TAC6M17, 12J22 was used using the hypocotyl callus transformation method. The gene was introduced into the acw1 mutant. The hypocotyl callus transformation is `` Kazuhito Akama, Hideaki Shiraishi, Shozo Ohta, Kenzo Nakamura, Kiyotaka Okada and Yoshiro Shimura; Efficient transformation of Arabidopsis thaliana: comparison of theefficiencies with various organs, plant ecotypes and Agrobacterium strains, '' Plant Cell Reports (1992) 12: 7-11 "and MP90 strain was used as an Agrobacterium. As a result, the phenotype recovered to the wild type in 2 individuals. That is, the ACW gene is contained in the overlapping region of the genomic DNA fragments contained in the “TAC6M17” and “12J22” clones.
[0025]
Therefore, the base sequence of the DNA fragment in this region was decoded. Four genes were expected to exist between the “2I5,1.1” marker and the above DNA region. Therefore, when the wild-type nucleotide sequences of these four hypothetical genes were compared with respect to the acw1 mutant, there was a mutation in the gene region corresponding to the cDNA (registration number U37702) registered as a cellulase homolog. There was found. Among these four genes, only the cellulase homologue was found to have a mutation in the base sequence in the acw1 mutant, indicating that the gene is the ACW1 gene.
[0026]
When full-length cDNA of ACW1 gene was isolated from λZAPII cDNA library (STRATAGENE), it contained 5 introns. Analysis of the amino acid sequence encoded by the gene revealed homology to cellulase. In general, plant cellulases do not have the cellulose-binding domain of fungal cellulases and have a signal sequence for secretion outside the membrane, but it is clear what they are targeting in vivo It has not been. The product of the ACW1 gene is also thought to work as a cellulase. However, (1) molecular weight is large (ACW1 is 69 kD, most others are about 54 kD), (2) no signal sequence, (3) a charged amino acid region on the N-terminal side, (4) membrane It is considered that it belongs to a new family different from other cellulases because of its characteristics such as having a transmembrane domain.
[0027]
[Example 2] Histochemical analysis of acw1 mutant
Aseptically inoculated in 1 / 2B5 medium, the roots and hypocotyls on day 7 were cut, and the samples were fixed at 4 ° C. in 3% glutaraldehyde solution. The sample was washed with phosphate buffer (pH 7.0) (2-4 ° C.). The buffer was changed 4 to 6 times at intervals of 10 to 20 minutes. Then 1% OsO _Four ・ 3% KMnO _Four The solution was fixed for 1 to 2 hours, and then washed with the phosphate buffer. The sample was immersed in an alcohol series (30, 50, 70, 90, 99.5, 99.5, 99.5% ethanol) for 10 to 20 minutes for dehydration. Samples were soaked in propylene oxide four times at 20 minute intervals and then soaked in epoxy resin (Quetol 812) for 3-6 hours. The sample was placed in an embedding mold, and an embedding resin containing an epoxy resin (Quetol 812), a curing agent (DDSA), and an accelerator (DMP-30) was poured. It was left in a 35 ° C incubator for 1 day, then in a 45 ° C incubator for 1 day, and then in a 60 ° C incubator for 1 day. After confirming sufficient hardening, it left still in a 45 degreeC thermostat for several days-one week. A sample was taken out from the mold, and a 0.1 μm ultrathin section sample was prepared with a microtome for electron microscope. This ultrathin slice sample was stained with uranyl acetate / lead citrate stain or PATAg, and then observed with a transmission electron microscope (the former was an amorphous polysaccharide such as hemicellulose or pectin, the latter was an amorphous sugar) Has the effect of staining).
[0028]
As a result of histochemical analysis by electron microscope, accumulation of amorphous glucan was observed in uranyl acetate / lead citrate staining in the root section of acw1 mutant (Fig. 1B, C), but not in the wild type. (FIG. 1A). Similar results were obtained for hypocotyl slices (FIG. 2). Similarly, accumulation of amorphous glucan was observed only in the acw1 mutant in the PATAg staining in the root section (FIG. 3). These amorphous glucans are transferred to longer glucan chains and crystallized. ACW1 cellulase gene was considered to be a novel cellulase having glucan transfer activity because of the accumulation of amorphous glucan. Although it is a cellulase of a higher plant that has been predicted to have only a function of degrading activity, it has been proved that the ACW1 gene product has a function related to synthesis rather than cell wall degradation. There is no other report of such cellulase in the ACW1 gene.
[0029]
[Example 3] Analysis of acw1 cell wall components
After growing at 21 ° C. for 2 weeks, the cell wall components of the above-ground part of the acw1 mutant grown at 31 ° C. for 2 weeks were examined. Preparation of the cell wall crude sample for cell wall component analysis was according to the method of Zablackis et al. (Zablackis et al, Plant Physiol, 107, 1129-1138 (1995)). First, a plant frozen in liquid nitrogen was ground in a mortar, phosphate buffer (pH 7.0) was added, and this was pipetted into a centrifuge tube. After crushing with polytron (130 rpm, 45 seconds, 3 times), it was centrifuged (9,500 rpm, 10 minutes, 4 ° C.). The supernatant was discarded, phosphate buffer was added, and the above disruption and centrifugation were performed again. This was repeated once more (total 3 times). In order to wash the precipitate, distilled water was added and centrifuged (9,500 rpm, 10 minutes, 4 ° C.). This was done a total of 4 times. 70% ethanol was added, well suspended and left overnight (4 ° C). After collection by centrifugation, chloroform / methanol (1: 1) solution was added and stirred with a stirrer (30 minutes). After centrifugation (9,500 rpm, 15 minutes, 4 ° C.), the supernatant was discarded, and extraction with chloroform / methanol (1: 1) was performed again. These chloroform / methanol (1: 1) extracts were concentrated to dryness using a concentrated evaporator (this was used for the thin layer chromatography of Example 5). Phenol / acetic acid / water (2: 1: 1) was added to the precipitate after centrifugation, and the mixture was stirred with a stirrer at room temperature for 1 hour and centrifuged (9,500 rpm, 15 min. 4 ° C.). This was repeated once more, then 100% ethanol was added, washed and centrifuged. After further washing twice with distilled water, amylase treatment (0.1 M phosphate buffer + α-amylase 2 mg / ml (Sigma) Aspergillus oryzae origin + 5 drops of toluene) was performed for 48 hours with gentle shaking. . After centrifugation, the supernatant was discarded and washed 5 times with distilled water. The finally collected precipitate was used as a cell wall crude purified sample. This was stored at −80 ° C. and the required amount was thawed and used for analysis.
[0030]
Sugar quantification was performed using this sample. First, an appropriate amount of the prepared cell wall sample was taken and lyophilized. 5 mg was weighed into a glass tube, 2.5 ml of 2 M trifluoroacetic acid (TFA) was added (0.5 ml TFA / 1 mg cell wall), and incubated in an oil bath at 121 ° C. for 1 hour to hydrolyze the amorphous sugar. After natural cooling to room temperature, the mixture was centrifuged (5,000 rpm, 10 minutes, tabletop centrifuge), and separated into supernatant and residue (precipitate). Distilled water was added to the residue, and the supernatant was collected by centrifugation and added to the previously collected supernatant. The supernatant was dried and dissolved in 1 ml of distilled water, and the amount of sugar was determined. 72% H in the residue ₂ SO _Four And left at room temperature for 2 hours. On the way, it was gently stirred to dissolve. Diluted 35 times with distilled water, boiled in boiling water for 2 hours, and completely hydrolyzed. After boiling, it was placed in ice and cooled, and the volume was measured with a graduated cylinder to determine the amount of sugar. The amount of sugar was determined by the phenol-sulfuric acid method for the total sugar amount and the m-hydroxybiphenyl method for the uronic acid amount.
[0031]
The result is shown in FIG. In the TFA soluble fraction, the amount of acw1 mutant glucose (1790.0 ± 86.4 nmol / mg cell wall) was almost the same as wt (wild type) (1907.6 ± 158.4 nmol / mg cell wall). However, in the TFA-insoluble fraction, the amount of acw1 mutant glucose (436.7 ± 20.0 nmol / mg cell wall) was less than wt (1342.5 ± 31.5 nmol / mg cell wall). What is insoluble in TFA is crystalline cellulose. Therefore, the glucose in the TFA insoluble fraction is derived from cellulose. This result showed that cellulose was decreased in the acw1 mutant. The amounts of acw1 mutant rhamnose and uronic acid (145.7 ± 7.0, 659.6 ± 31.7 nmol / mg cell wall, respectively) were higher than wt (90.3 ± 7.5, 441.6 ± 9.7 nmol / mg cell wall 1). A large amount of rhamnose and uronic acid suggests that pectin (Rhamnogalacturonan) is increasing. The change in stainability observed by electron microscopic observation seems to be due to an increase in pectin. The non-crystalline polysaccharide degraded by TFA was not changed except that an increase in pectin was suggested.
[0032]
[Example 4] Observation of cellulose fiber
The result of observation of cell walls of wt and acw1 with an electron microscope of the cellulose fibers is shown in FIG. The residue after the cell wall sample prepared in Example 3 was hydrolyzed with 2M TFA was observed with an electron microscope. Since amorphous polysaccharides such as pectin and hemicellulose are degraded by 2M TFA treatment, most of the residue is crystalline cellulose. The fiber width of wt was about 3.0 nm. The acw1 was about 2.4nm, thinner than wt. When the cellulose fiber is treated with a strong acid, the microfibril may be swollen or bundled to be observed to be very thick (FIG. 5, arrow). The fact that many bundles (17 nm) of wt were observed seems to be due to the strong acid treatment. However, in acw1 treated in the same way, few bundles were observed. The thickness was also about 12 nm. Although it cannot be said that the native state of microfibrils was observed due to the strong acid treatment, there was a difference in the state of microfibrils after the treatment. This seems to reflect the difference in the nature of the original microfibrils between wt and acw1.
[0033]
Cellulose is formed by bundling linear β-1,4 glucans and crystallizing them. Therefore, the degree of polymerization can be determined by examining the molecular weight. Then, next, in order to analyze the polymerization degree of cellulose, the molecular weight was measured by gel filtration analysis. The cellulose sample used for the measurement was prepared by the method of Edashige et al. (Edashige et al, Holzforshung, 49, 197-202 (1995)). Appropriate amount of cell wall crude purified sample, 0.05M CDTA, 0.05M Na ₂ CO _Three , 1N KOH, 4N KOH, 4M KOH + 3% Borate was extracted sequentially. The residue (crystalline cellulose) was nitrated with a fuming nitric acid + phosphorous oxide (V) solution prepared in advance. The reaction product (nitrocellulose) was collected by suction filtration and washed with ice water. The sample was collected by suction filtration again, boiled lightly with a little water. Furthermore, after suction filtration, it was dried at 60 ° C. For gel filtration analysis, the sample was dissolved in tetrahydrofuran and 50 μl was injected with a syringe. As the column, Shimadzu LC-6A was used.
[0034]
As a result, the degree of polymerization (DP) of microfibrils was distributed over a wide range of several hundred to 5,000 in wt (FIG. 6), and the average was about 1,000. However, the distribution of acw1 was very narrow, with almost one peak, and the average was about 5,000.
[0035]
From the above facts, it was shown that the ACW1 gene is an essential gene for cellulose synthesis, and that the amount and quality of cellulose (fiber width, degree of polymerization, etc.) are changed by mutation of this gene.
[0036]
[Example 5] Analysis of chloroform / methanol extraction layer
The chloroform / methanol fraction was treated with 2M TFA and then analyzed by thin layer chromatography (TLC) (FIG. 7). In thin-layer chromatography, first, an appropriate amount of 2M TFA was added to the dried chloroform / methanol extracted layer prepared in Example 3, and incubated at 121 ° C. for 1 hour to hydrolyze it. The treatment liquid was dried, distilled water and chloroform were added and stirred well, centrifuged and the supernatant (aqueous layer) was taken and dried. An appropriate amount of distilled water was added again, and a part was spread on a silica plate for thin layer chromatography with a syringe and developed with a solvent (n-butanol / pyridine / distilled water = 8: 5: 4). After development, the plate was dried, sprayed with sulfuric acid and incubated on a hot plate. When sugar spots appeared black (development), they were removed from the hot plate and allowed to cool naturally.
[0037]
As a result of this analysis, spots of galactose were observed for both wt and acw1 (FIG. 7, lanes 3 and 4). This is considered to be derived from galactose-lipid constituting the chloroplast membrane. In acw1, glucose spots were also clearly observed (lane 4 in FIG. 7). It was not observable with wt.
[0038]
Moreover, in order to analyze the component of an extract, the gas chromatography analysis by the alditol acetate method was performed. A portion of the chloroform / methanol extract of Example 3 was dried to dryness and sodium borohydride (10 mg / ml, NH _Four (In OH) was dissolved in 250 μl and incubated at room temperature for 1 hour. After adding a few drops of acetic acid, 250 μl of acetic acid / methanol (1: 9) was added. It was made to dry while warming in a water bath (40 degreeC). This was done a total of 4 times. Further, 250 μl of methanol was added and dried in the same manner. This was also done a total of 4 times. To the dried sample, 50 μl of acetic anhydride and 50 μl of pyridine were added and incubated in an oil bath at 121 ° C. for 20 minutes for acetylation. After cooling, 200 μl of toluene was added and dried twice. Then, 500 μl of dichloromethane and 500 μl of distilled water were added and stirred well, followed by centrifugation to remove the lower layer (dichloromethane layer) and dry it. It was dissolved in an appropriate amount of acetone and subjected to gas chromatography (Shimadzu GC14A SP233 column).
[0039]
The TFA soluble layer may be obtained by treating the dried sample with the above method. TFA residue (sulfuric acid hydrolysis) sample is Ba (OH) ₂ After neutralization with, the concentrated product was used for treatment.
[0040]
As a result of gas chromatography analysis, most of the sugars in the extract were galactose and glucose. As a result of examining the molar ratio of galactose to glucose, wt was galactose 88.9 mol (%), glucose 11.1 mol (%), and acw1 was galactose 70.0 mol (%), glucose 30.0 mol (%). The glucose amount of acw1 was about 3 times higher in molar ratio than wt.
[0041]
Glucan that is increasing in the chloroform / methanol fraction is expected to be glucan. Therefore, the molecular weight distribution was analyzed by gel permeation chromatography (GPC) analysis. The chloroform / methanol fractions of wt and acw1 were analyzed after saponification with NaOH.
[0042]
As a result, peaks corresponding to 1, 3, 4, 6 sugars were obtained in both cases. The area ratio of each peak of wt was 36.1%, 14.8%, 28.4%, 20.7%. The area ratio of each peak of acw1 was 26.9%, 15.2%, 30.0%, 27.9%. In acw1, the amount of 3, 4, 6 sugars increased compared to wt. The increase of 6 sugars was the highest with 7.2%. It was 1.6% with 4 sugars. Increased glucose was expected to exist as oligosaccharides of 4,6 sugars.
[0043]
Then, the coupling | bonding mode of the oligosaccharide of the glucose estimated next was investigated by the methylation analysis method. An appropriate amount of the cell wall crude purified sample was taken, and 0.5 ml of DMSO was added. The mixture was stirred at room temperature for 2 hours, 4M Na-DMSO was added, and the mixture was further stirred for 2 hours. The reaction solution was added to a small amount of triphenylmethane powder and confirmed to turn red. Then, the mixture was placed in ice and 50 μl of methyl iodide was added and stirred for 1 hour. Thereafter, 0.5 ml of distilled water was added and nitrogen gas was blown. The reaction solution was applied to a Sep · Pak C-18 column and eluted with 2 ml of 100% acetonitrile and 4 ml of 100% ethanol, and the purified solution was dried at room temperature. 250 μl of 2M TFA was added and hydrolyzed at 121 ° C. for 1 hour to dry the reaction solution, and 300 μl of isopropanol was added and dried again. Furthermore, 95% ethanol 220μl and NaBD _Four (NH _Four After adding 200 μl of OH) and reacting at room temperature for 1 hour, a few drops of acetic acid were dropped into the reaction solution, and 250 μl of acetic acid methanol was added to dryness. This process was repeated four more times. The process of adding 250 μl of methanol to dryness was repeated three times. Next, 50 μl of acetic anhydride is added and reacted (acetylated) at 121 ° C. for 3 hours. After cooling to room temperature, 500 μl of distilled water is added and Na ₂ CO _Three Was added until no more air bubbles were added, and 500 μl of dichloromethane was further added and mixed well. Centrifugation was performed at low speed, and the dichloromethane layer was removed to dryness. The product was dissolved in an appropriate amount of acetone and subjected to gas chromatography / mass spectrometry (GC-MS). Shimadzu QP-2000A and JEOL.JMS-DX303 were used for gas chromatography / mass spectrometry.
[0044]
As a result, terminal bond, 2-bond, 3-bond, 4-bond, and 6-bond were detected in galactose. On the other hand, only terminal binding and 4-bond were detected for glucose. When the alkali saponified extract is treated with β-1,4 glucanase, glucose monosaccharides are produced (data not shown). From the above results, it was clarified that in the acw1 mutant, the oligosaccharide having β-1,4-linked glucose was accumulated in the chloroform / methanol fraction more than wt (about 3 times).
[0045]
As a result, it was first clarified that an oligosaccharide having glucose β-1,4 bonded to a lipid or the like becomes a substrate for cellulose synthesis.
[0046]
【The invention's effect】
The present invention provides the use of DNA encoding a cellulase-like protein for modifying plant cell wall components. Modification of plant cell walls is due to, for example, increasing the supply efficiency of fiber raw material plants such as pulp, developing new materials by controlling cell wall synthesis, increasing useful components of crops, increasing digestive absorption efficiency of feed crops, and increasing the amount of cell wall synthesis This can be useful in the fields of agriculture, industry, and horticulture because it can bring about the creation of ornamental plants with new aesthetic value by increasing the amount of plant growth and changing the cell morphology accompanying modification of cell wall components.
[0047]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is an electron micrograph showing the result of uranyl acetate / lead citrate staining in a root section of acw1 mutant. A shows the results for the wild type, B for the mutant at 21 degrees (permissible temperature), and C for the mutant at 31 degrees (non-permissible temperature).
FIG. 2 is an electron micrograph showing the results of uranyl acetate / lead citrate staining on the hypocotyl of the acw1 mutant. A shows the results for the wild type and B for the mutants at 31 degrees (non-permissive temperature).
FIG. 3 is an electron micrograph showing the result of PATAg staining in a root section of acw1 mutant. A shows the results for the wild type and B for the mutants at 31 degrees (non-permissive temperature).
FIG. 4 shows the results of analyzing cell wall constituent sugar components of wild strains and acw1 mutants. A shows the constituent sugar amount of the TFA-soluble layer, and B shows the constituent sugar amount of the TFA-insoluble fraction (residue; cellulose). Rha; rhamnose, Fuc; fucose, Ara; arabinose, Xyl; xylose, Gal; galactose, Glc; glucose, Uronic acid; uronic acid
FIG. 5 is an electron micrograph showing the results of observation of cellulose fibers of a wild strain and an acw1 mutant. The upper shows the cellulose fiber of the wild strain, and the lower shows the cellulose fiber of the acw1 mutant. Arrows indicate fibers that are thickened by the strong acid treatment.
FIG. 6 shows the results of chromatograms of GPC performed for analyzing the degree of polymerization of cellulose of wild type and acw1 mutant. White circles show the results of mutants, and white triangles show the results of wild strains. The horizontal axis indicates the time for elution from the column, and the vertical axis indicates the value detected by the detector. The earlier the peak, the higher the molecular weight.
FIG. 7 shows the results of TFA hydrolysis and thin layer chromatography (TLC) of chloroform / methanol layers of wild-type and acw1 mutants. Lane 1 shows the results for the glucose control, Lane 2 for the galactose control, Lane 3 for the wild type, and Lane 4 for the mutant. A clear glucose spot is observed.

Claims (4)

A method for promoting glucan chain synthesis in a cell wall of a plant, which comprises expressing a DNA selected from the following (a) to (d):
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1.
(B) A DNA encoding a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1, and promoting the synthesis of the glucan chain.
(C) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 2.
(D) DNA encoding a protein having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 1 and promoting the synthesis of the cell wall.   The method according to claim 1, wherein the glucan chain is cellulose. A transformed plant in which glucan chain synthesis in the cell wall is promoted by introduction and expression of DNA selected from the following (a) to (d) as compared to a wild-type plant.
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1.
(B) A DNA encoding a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1, and promoting the synthesis of the glucan chain.
(C) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 2.
(D) DNA encoding a protein having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 1 and promoting the synthesis of the cell wall. The plant according to claim 3 , wherein the glucan chain is cellulose.

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