JP6103631B2 - High-efficiency production method for wood using transformed plants - Google Patents

Description

  The present invention relates to a high-efficiency production method of wood in a plant transformed with a transcriptional control factor involved in wood formation of rice or a related plant.

In recent years, the use of renewable energy has attracted attention due to factors such as the global warming due to excessive carbon dioxide emission and a sense of crisis over the depletion of fossil fuels, and the accompanying increase in the price of fossil fuels. In particular, plant-derived biofuels such as bioethanol and biodiesel are highly expected as fuels for internal combustion engines because they do not need to capture carbon dioxide generated by combustion as a new release (carbon neutral). However, conventional biofuels (first-generation biofuels) are produced using raw materials such as corn starch and rapeseed oil, and the supply and demand of food is becoming tight as the world population increases. It is hard to say that it is sustainable. Therefore, there is a demand for practical application of second generation bioethanol using non-edible cellulose as a raw material. Cellulose is one of the main components constituting wood (secondary cell wall, secondary wall) and is a polymer in which glucose is repeatedly β-1,4 bonded. Since glucose is obtained by treatment with cellulase derived from fungi or the like, ethanol can be obtained by fermenting it with yeast. Cellulose is contained in large quantities in wood, and strengthening the wood of plants can be an important development goal. If wood quality can be increased, the value of wood can be increased.
NST transcription factor genes discovered by the present inventors are well known as genes that control plant wood formation (Non-patent Document 1). NST transcription factor is a gene found in the model experimental plant Arabidopsis thaliana, and the nst1 nst3 double mutant does not form any wood (secondary cell wall) except for the canal. In such a plant, cellulose having low crystallinity is contained only in the primary cell wall and the enzymatic saccharification by cellulase is enhanced (Patent Document 1, Non-Patent Document 2), but since there is no secondary cell wall, the amount of cellulose per individual is It is assumed that the final sugar yield will be reduced. In addition, the strength of the stem is greatly reduced and it cannot stand upright. Conversely, if NST transcription factors are overexpressed throughout the plant, ectopic wood formation occurs in all cells, and plant growth is significantly inhibited.
Patent documents 2 and non-patent documents 3 are cited as prior arts for enhancing biomass for biofuels. In this document, the human sugar nucleotide (UDP-galactose) transporter is expressed in tobacco to achieve an overall increase in biomass, but it is not focused on the wood, but mainly the primary cell wall. Receive an increasing impression.
Patent Document 3 and Non-Patent Document 4 claim a technique for thickening the secondary cell wall, but what is actually demonstrated is a special condition in cultured cells, and has not been verified in a plant body.
Thus, it is not easy to selectively enhance wood quality, and a reliable method has not yet been established.

PCT / JP2007 / 053673 JP 2010-51252 A JP 2010-233536 A

The Plant Cell, 2007 19,270-280 Journal of Biotechnology, 2009 142,279-284 Journal of Bioscience and Bioengineering, 2010 109,159-169 The Plant Cell, 2007 21,1155-1165 Plant Biotechnology Journal, 2006 4,325-332 Tappi, 1961 44,793-798

  In the present invention, an object is to provide a technique for increasing the production capacity of a secondary cell wall (wood) inherent in a plant and increasing the amount of wood produced per cultivated individual. Since wood contains a large amount of cellulose, which is a raw material for second-generation bioethanol, if the wood can be strengthened, the amount of bioethanol produced can generally be increased, contributing to the realization of a low-carbon society.

In order to selectively enhance the wood composed of the secondary cell walls, it is considered necessary to selectively express only in the wood fiber cells, for example, by strengthening a gene that fundamentally controls the formation of the wood. In the process of comprehensively analyzing the NAC family transcription factor group, which is a plant-specific transcription factor, in Arabidopsis thaliana, the present inventors have used the NST transcription factor group (NST1, NST1, NST2 and NST3) were discovered (Non-patent Document 1).
Among these, the nst1 nst3 double mutant of Arabidopsis thaliana no longer formed wood in the flower stems and hypocotyl wood fiber cells, greatly reducing the strength of the flower stems and unable to stand upright. However, in the same way, wood loss does not occur in the canal where the wood is formed, and the survival of the plant was not affected (Non-patent Document 1). Conversely, when these genes are excessively expressed in the whole plant, ectopic wood formation occurs in all cells, and plant growth is significantly inhibited (Non-patent Document 1).
The present inventors paid attention to the woody forming ability of NST transcription factor, and extensively examined its orthologous gene, and when conducting functional analysis of OsNST1, OsNST2 gene identified as an orthologous gene of NST transcription factor in rice, Similar to the Arabidopsis NST transcription factor, when its function was suppressed in rice, the secondary cell wall decreased and showed drooping leaf traits. In addition, in Arabidopsis thaliana, which is a dicotyledonous plant, when ectopically expressed, ectopic wood formation occurred and it was confirmed that the growth of Arabidopsis thaliana was significantly inhibited. Moreover, the ectopic formation of the wood that occurred at this time was much more prominent than when the NST transcription factor of Arabidopsis thaliana was expressed ectopically (Fig. 1), and the transcription activation ability for NST binding sequences was compared. However, the OsNST2 transcription factor was significantly higher than the Arabidopsis NST3 transcription factor (FIG. 2).
Therefore, we thought that the secondary cell wall could be enhanced by replacing the Arabidopsis thaliana NST transcription factor with the rice OsNST transcription factor, and linked the OsNST2 transcription factor to the NST3 promoter that specifically induces gene expression in the wood, and nst1 The OsNST2 transcription factor was expressed by introducing it into the nst3 double mutant (hereinafter this plant is referred to as OsNST2 complementary line). As a result, not only did the double mutant phenotype (cannot stand upright) recovered, but when the lower 10 cm of the flower stem was sampled, the dry weight increased and the bending strength was stronger than the wild strain (FIG. 3).
Furthermore, it was found that the tree formation represented by the deposition of the lignin-like substance occurred up to the central part of the flower stalk which is not normally converted into a tree (FIG. 3). The remarkable ability to form wood or enhance the cell wall exhibited by OsNST transcription factor, which is a monocotyledonous NST transcription factor in dicotyledonous plants, is surprising to the present inventors. Represents speciality. Such characteristics observed in dicotyledonous plants containing tree resources that are overwhelmingly valuable as woody biomass can be expected to be extremely useful. For the obtained OsNST2 complementary line (ProNST3: OsNST2 nst1 nst3), a portion of 3 cm to 10 cm was obtained from the base of the flower stalk and subjected to saccharification, and the amounts of glucose and xylose obtained per fresh weight were compared. It was found that about 45% glucose per fresh weight and about 77% more monosaccharides were obtained with xylose than the wild type strain (FIG. 4). In addition, when the OsNST2 complementary line was subjected to the general “caustic soda pretreatment + cellulase saccharification” for bioethanol production, it was found that about 20% more glucose than the wild strain was obtained (FIG. 4). ).
Obtaining the above knowledge, the present invention was completed.

That is, the present invention is as follows.
[1] A nucleic acid encoding a transcriptional regulatory factor having the activity of controlling wood formation according to any one of the following (a) to (d) is arranged downstream of a promoter that specifically induces gene expression in woody cells. A method for enhancing the woody quality of a plant, comprising transforming the plant with the constructed construct;
(A) a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 1,
(B) a nucleic acid encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
(C) a nucleic acid comprising the base sequence represented by SEQ ID NO: 2,
(D) A nucleic acid comprising a base sequence that hybridizes with a complementary sequence of the base sequence shown in SEQ ID NO: 2 under stringent conditions.
[2] A nucleic acid encoding a transcriptional regulatory factor having the activity of controlling wood formation according to any one of the following (a) to (d) is disposed downstream of a promoter that specifically induces gene expression in wood forming cells. A transformed plant with enhanced wood, or a progeny thereof, characterized by being transformed with the constructed construct;
(A) a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 1,
(B) a nucleic acid encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
(C) a nucleic acid comprising the base sequence represented by SEQ ID NO: 2,
(D) A nucleic acid comprising a base sequence that hybridizes with a complementary sequence of the base sequence shown in SEQ ID NO: 2 under stringent conditions.
[3] The transformed plant or the progeny thereof according to [2] above, wherein the promoter that specifically induces gene expression in woody cells is the promoter of NST1 or NST3 gene.
[4] Using the transformed plant according to the above [2] or [3] or its progeny, a treatment including a step of obtaining a saccharification treatment by obtaining at least one part thereof is performed, Monosaccharide production method.
[5] Using the transformed plant or the progeny thereof according to [2] or [3], at least one part thereof is obtained and subjected to a treatment including a pretreatment step with caustic soda and a saccharification step with cellulase. A method for producing glucose, characterized in that

  The amount of wood that can be produced from one individual can be increased by the wood-enhanced plant produced using the “OsNST2 gene” of the present invention. If the wood quality can be increased, not only can bioethanol production be increased, but the value as wood can be increased.

An overexpression of Arabidopsis and rice NST transcription factors in Arabidopsis. OsNST2 gene overexpression (b) causes woody ectopic formation more significantly than Arabidopsis thaliana NST1 gene overexpression (a). Difference in transcriptional activation ability of NST transcription factor between Arabidopsis thaliana and rice to NST binding sequence (SNBE). OsNST2 has higher transcriptional activation ability than Arabidopsis NST3. OsNST2 complementary line dry weight, bending strength, fiber cell appearance, lignin amount Sulfate saccharification and enzymatic saccharification on the OsNST2 complementary line

Hereinafter, the present invention will be described in more detail.
1. About the plant which strengthened the wood in this invention (1) About the gene which strengthens the wood in this invention The gene which provides the high woody production ability with respect to a plant in this invention is typically represented by sequence number 2 derived from rice. The corresponding amino acid sequence is represented by SEQ ID NO: 1. This gene has been proved that its Arabidopsis ortholog gene is a master transcriptional regulator of wood formation.
In the present invention, when the `` OsNST2 gene '' derived from rice is overexpressed in a typical dicotyledon, Arabidopsis thaliana, the ability to cause ectopic wood formation is exceptionally high for the first time. I found it. The gene can be expressed by connecting it to a wood cell-specific promoter, particularly a dicotyledon-derived promoter that works specifically for wood. Based on finding out that it can be fully utilized. The “OsNST2 gene” has a low homology of about 70% with NST gene groups such as Arabidopsis thaliana, which are dicotyledonous plants. Therefore, mutations have been introduced into homologous genes in some other plant genomes, preferably grasses, or in part. It is apparent that similar genes having a homology of 80% or more, preferably 90% with the “OsNST2 gene” can be used in the same manner. Moreover, as long as the same function is hold | maintained, the fragment may be sufficient. Such a gene has a homology of 80% or more, preferably 90% or more, more preferably 95% or more with SEQ ID NO: 2 in the base sequence, and 80% or more with SEQ ID NO: 1 in the amino acid sequence encoded by it. Preferably 90% or more, more preferably 95% or more.
That is, a gene that expresses the woody enhancing trait of the present invention can be generally expressed as follows.
(A) a gene encoding the amino acid sequence represented by SEQ ID NO: 1,
(B) a gene encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
(C) a gene consisting of the base sequence shown in SEQ ID NO: 2,
(D) A gene comprising a base sequence that hybridizes with a complementary sequence of the base sequence shown in SEQ ID NO: 2 under stringent conditions.
Here, 1 or several refers to the presence of 1 to 20, more preferably 1 to 10 mutations. Stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a highly homologous nucleic acid, that is, a complementary strand of DNA consisting of a nucleotide sequence having a homology of 90% or more, preferably 95% or more, is hybridized with the nucleotide sequence shown in SEQ ID NO: 2 and has a lower homology. A condition in which the complementary strand of the nucleic acid does not hybridize is mentioned. Such stringent conditions are the conditions for increasing the Tm value in normal hybridization operations described in T. Maniatis et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring Harbor Laboratory, etc. It is obvious to the contractor. For example, 6 × SSC containing 0.5% SDS, 5 × Denhartz [Denhardt's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400] and 100 μg / ml salmon sperm DNA (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), and the like.
In the following description of the present invention, the name “OsNST2 gene” that is typical as a gene that imparts the “woody enhancing trait” of the present invention will be used.

(2) Method for producing plant having woody-enhancing trait in the present invention The promoter for expressing the OsNST2 gene, a transcriptional regulator used in the present invention, is a “promoter that specifically induces gene expression in woody cells” Specifically, the NST3 promoter or its ortholog gene promoter, an artificial promoter in which the NST binding sequence (SNBE) is repeated and placed upstream of the core promoter, a promoter of secondary cell wall cellulose synthase, and enzymes involved in lignin synthesis Promoter of enzymes involved in xylan synthesis. In particular, promoters of NST1 and NST3 genes derived from Arabidopsis thaliana are preferred.
A construct (recombinant vector) is constructed downstream of a promoter that specifically expresses the OsNST2 gene in wood-forming cells, and transformed into a wild strain or a plant in which the function of the NST transcription factor or its ortholog transcription factor gene is suppressed. To make.
A typical method for introducing a gene or a recombinant vector into a plant is the Agrobacterium method, but the PEG-calcium phosphate method, electroporation method, liposome method, particle gun method, microinjection method, etc. can be applied. .

(3) Types of target plants It was demonstrated in the present invention that the “OsNST2 gene” used in the present invention is a gene that can be expressed across species and can express a “woody enhancing trait”. The target plant can be generally applied to dicotyledonous plants such as potatoes, beans, and solanaceae, but dicotyledonous plants such as poplar, eucalyptus, and acacia are particularly preferable from the viewpoint of biomass resources. However, the present invention is not limited to these plants, and can also be applied to general breeding of monocotyledonous energy plants such as Miscanthus, Elianthus, Danchiku, Sorghum and Susuki.

(4) Trait characteristics of transformed plants by “OsNST2 gene” (4-1) Increase in dry weight The lower 10 cm of the flower stems of OsNST2 complementary line Arabidopsis plants transformed by using “OsNST2 gene” were sampled and analyzed. As a result, the dry weight increased by about 50% by weight compared to the wild strain, and the bending strength became stronger than that of the wild strain (FIG. 3).

(4-2) Increase in amount of lignin Furthermore, it can be seen from FIG. 3 that lignin-like substances are deposited up to the central part of the flower stalk, which does not normally become wood (FIG. 3).
The “quantification of lignin amount” was performed by a general “acetyl bromide method (Johnson et al., Non-patent Document 6)”. Specifically, lignin is acid-hydrolyzed after starch removal treatment with amylase or amyloglucosidase, and the amount of lignin is measured with a spectrophotometer. As a result, the OsNST2 complementary line was significantly higher than the wild type by about 40% (FIG. 3).

(4-3) Manufacture of monosaccharides by sulfate saccharification treatment "Sulphate saccharification treatment" is a well-known technique for extracting most neutral sugars from cell wall components, which is hydrolyzed after heating and carbonization in the presence of sulfuric acid. Monosaccharides are obtained as raw materials such as bioethanol.
After obtaining the bottom 10 cm of the flower stalk of the resulting transformed plant to obtain a purified cell wall powder, saccharification was performed, and the amounts of glucose and xylose obtained per fresh weight were compared. It was found that about 45% glucose and about 80% more xylose were obtained than the strain (FIG. 4).

(4-4) Production of glucose by "caustic soda pretreatment + cellulase saccharification" As a general method for bioethanol production, the "caustic soda pretreatment + cellulase saccharification" method is well known. Specifically, lignin is produced by caustic soda treatment. The residue from which the cell wall has been removed is subjected to an enzymatic saccharification reaction with cellulase.
The glucose-containing solution obtained by this enzymatic saccharification becomes a bioethanol raw material by adding yeast.
In the Example, after obtaining the cell wall purified powder of the lower 10 cm part of the flower stem, it was found that about 20% more glucose than the wild strain can be obtained as a result of the “caustic soda pretreatment + cellulase saccharification” treatment. (Figure 4).

2. Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention are those that specifically indicate the source. Except for, a person skilled in the art can easily and surely implement the method based on known documents and the like. For example, genetic engineering techniques are described in J. Sambrook, EFFritsch & T. Maniatis, Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); DMGlover et al. Ed. , DNA Cloning, 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995), etc., or substantially similar methods or modification methods It can be carried out. In addition, various proteins and peptides used in the present invention, or DNAs encoding them, can be obtained from existing databases (URL: http://www.arabidopsis.org/ or http: //www.ncbi.nlm.nih. gov / sites / entrez? db = pubmed etc.).
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

  Examples of the present invention are shown below, but the present invention is not limited to these examples.

(Example 1) Production of nst1 nst3 double mutant Arabidopsis thaliana plant transformed with ProNST3: OsNST2 (1-1) Construction of transformation vector ProNST3: OsNST2 A transformation vector was constructed as follows.
In the p35SSRDXG vector described in Non-Patent Document 5, SRDX was omitted, and a mutation was introduced into the HindIII recognition sequence and EcoRI recognition sequence outside the attL1 and attL2 sites was designated as p35SG v2. This vector is cut with HindIII and SalI, and the following oligonucleotides are annealed and inserted.
5'-AGCTTAATTAAGGCGCGCCGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGGGTACCG-3 '(SEQ ID NO: 3)
5'-TCGACGGTACCCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCGGCGCGCCTTAATTA-3 '(SEQ ID NO: 4)
The plasmid generated in this step was designated as pNOS_Entry. Next, the NST3 promoter was amplified by PCR using the following oligonucleotide pair as a primer, and the pNOS_Entry vector was cleaved with AscI and BamHI and inserted.
5'-TAACAAGGCGCGCCGATTCTACACATTCACAAAGTTTACTAC-3 '(SEQ ID NO: 5)
5'-ATTCCTGGATCCATTAACGAAGATAGCAATATATTTTTGGG-3 '(SEQ ID NO: 6)
The plasmid generated in this step was designated as pNST3_NOS_Entry. Next, this vector was cleaved with SmaI, and the OsNST2 gene was amplified and inserted by PCR using the following oligonucleotide pair as a primer.
5'-GATGAGCATATCGGTGAACGGGCAGTCGGT-3 '(SEQ ID NO: 7)
5'-TTATACGTTATTCATGGTCGTCAAGTCTGC-3 '(SEQ ID NO: 8)

(1-2) Transformation of Arabidopsis thaliana nst1 nst3 double mutant with ProNST3: OsNST2 using floral dip method The plasmid obtained in Example (1-1) above was transformed into soil bacteria (Agrobacterium tumefaciens strain GV3101 (C58C1Rifr)). It was introduced into the pMP90 (Gmr) (koncz and Schell 1986) strain by electroporation. The introduced bacteria were cultured in 200 ml of LB medium for 2 days.
Subsequently, the cells were collected from the culture solution, suspended in 500 ml of infiltration medium, and immersed in Arabidopsis thaliana nst1 nst3 double mutant for 2 minutes, and then grown as usual to harvest T1 seeds. After that, T1 seeds were sterilized with 50% bleach and 0.02% Triton X-100 solution for 7 minutes, rinsed three times with sterilized water, and sown in MS selective medium containing 30 mg / l hygromycin.
Transformed plants (ProNST3: OsNST2 nst1 nst3, T1) that grow on the hygromycin plate are selected, replanted in soil, grown, seeded and sown to obtain the next generation (T2) Arabidopsis plants It was.

(Example 2) ProNST3: OsNST2 nst1 nst3 Arabidopsis plant trait evaluation (2-1) ProNST3: OsNST2 nst1 nst3 Arabidopsis plant flower stem strength measurement ProNST3: OsNST2 nst1 nst3 nst3 Arabidopsis plant obtained in the above Example (1-2) About 70 days after sowing in T2 plants, the lower 3 cm of the flower stem was collected, immersed in a methanol solution, boiled at 80 ° C. for 10 minutes, and then rinsed 3 times with distilled water. Thereafter, the protein in the flower stem was decomposed for 18 hours at 37 ° C. in a 50 mM phosphate buffer (pH 7.5) and a 5% ethanol 0.02% pronase solution, and then the breaking load was measured with a bending tester. As a result, ProNST3: OsNST2 nst1 nst3 (sample number: 70) was found to have a rupture load approximately twice as high as that of the wild strain (sample number: 70) (FIG. 3).

(2-2) Section observation of flower stems of ProNST3: OsNST2 nst1 nst3 Arabidopsis plants Flower stems were collected about 40 days after sowing in the ProNST3: OsNST2 nst1 nst3 Arabidopsis T1 plants obtained in the above Example (1-2). At a height of about 5 cm, a 50 μm-thick cross section was prepared using a vibration microtome, and lignin was stained with 2% phloroglucinol hydrochloride. As a result, in the wild strain, staining was observed only in vascular fiber cells and vascular cells, whereas in ProNST3: OsNST2 nst1 nst3 Arabidopsis thaliana, clear staining was also observed in internal parenchyma, and lignin-like substances accumulated in different places. (Figure 3).

(Example 3) Cell wall component analysis of ProNST3: OsNST2 nst1 nst3 Arabidopsis plant (3-1) Comparison of dry weight of cell wall component Seed in ProNST3: OsNST2 nst1 nst3 Arabidopsis T2 plant obtained in Example (1-2) above About 80 days later, a 3 cm to 10 cm portion was sampled from the base and the fresh weight was measured. Next, the flower stalk was immediately cut into a length of 1 cm or less, soaked in methanol, boiled at 80 ° C. for 10 minutes, and then rinsed three times with distilled water. Next, the protein was degraded with 50 mM phosphate buffer (pH 7.5), 5% ethanol 0.02% pronase solution for 18 hours and at 37 ° C. for 18 hours. Next, it was immersed in a 50% chloroform / 50% methanol solution and boiled at 70 ° C., and then replaced with a new 50% chloroform / 50% methanol solution. This operation was repeated three times, then immersed in an acetone solution, boiled at 70 ° C., and then replaced with a new acetone solution. This operation was repeated 3 times, followed by rinsing 3 times with a 100% ethanol solution, followed by drying at 70 ° C. for 2 days, and the dry weight was measured. As a result, ProNST3: OsNST2 nst1 nst3 showed that the dry weight of the cell wall component increased by about 50% per fresh weight compared to the wild type strain (FIG. 3).

(3-2) Comparison of lignin content The dried cell wall residue was crushed at 2000 rpm for 10 minutes using Shake Master Neo to obtain cell wall powder. 100 mg of the resulting cell wall powder was suspended in 1 ml of 50 mM maleic acid, 2 mM calcium chloride, 0.02% sodium azide solution, and starch was gelatinized at 70 ° C. for 10 minutes, followed by 4 ml of 50 mM maleic acid, 2 mM chloride. Calcium, 0.02% sodium azide solution was added and allowed to stand at room temperature for 10 minutes. Next, 1 ml of 500 U α-amylase (E-PANAA), 0.33 U amyloglucosidase (E-AMGIF), 50 mM maleic acid, 2 mM calcium chloride, 0.02% sodium azide solution was added, and then at 37 ° C. for 18 hours. The starch was hydrolyzed and destarched. The cell wall powder suspension subjected to destarching was rinsed 3 times with 10 ml of distilled water and twice with 100% ethanol, and then dried at 70 ° C. for 2 days to obtain a purified cell wall powder. 2 mg of the obtained cell wall purified powder was suspended in 1 ml of 75% acetic acid and 25% acetyl bromide solution, and then lignin was hydrolyzed at 50 ° C. for 2 hours. Next, 3 ml of 100% acetic acid was added and stirred, and then centrifuged at 2000 × g for 15 minutes to separate the supernatant and the residue. To 0.4 ml of the obtained supernatant, 0.5 ml of 100% acetic acid and 0.3 ml of 0.3 M NaOH solution were added and stirred. Then, 0.1 ml of 0.5 M hydroxyammonium chloride solution was added and stirred, then the spectrophotometer Absorbance was measured at 280 nm and the lignin content was measured. As a result, in ProNST3: OsNST2 nst1 nst3, the amount of lignin increased by about 40% per dry weight compared to the wild type strain (FIG. 4).

(3-3) Production of monosaccharides by sulfate saccharification treatment 10 mg of cell wall purified powder obtained by the same treatment as in (3-2) above was suspended in 0.2 ml of 72% sulfuric acid solution and stirred every 15 minutes. The cell wall was carbonized at room temperature for 1 hour. Next, 5.6 milliliters of distilled water was added to dilute the sulfuric acid concentration to 4%, and the cell wall was hydrolyzed at 120 ° C. for 60 minutes. The suspension after hydrolysis is centrifuged at 2000 × g for 15 minutes to separate the supernatant and the residue, and the resulting supernatant is adjusted with calcium carbonate so that the pH is 5 to 7, and is then added to the solution. The amount of glucose and the amount of xylose contained were examined using a glucose test kit and a xylose test kit. As a result, it was found that ProNST3: OsNST2 nst1 nst3 can obtain as many monosaccharides as 45% for glucose and about 80% for xylose per fresh weight compared to the wild type strain (FIG. 4).

(3-4) Production of glucose by “caustic soda pretreatment + cellulase saccharification” 2 mg of cell wall purified powder obtained by the same treatment as in the above (3-2) was suspended in 0.5 ml of 2% NaOH solution, and 95 ° C. 10 Pre-treatment for minutes was performed. Next, rinsing was performed three times with 1 ml of distilled water and twice with 1 ml of 100% ethanol, and then dried at 70 ° C. for 24 hours to obtain a delignified cell wall residue. Add 1 ml of 5 mM citrate buffer (pH 4.8), 0.02% sodium azide, 10 μg Accellulase 1500 solution to the resulting delignified cell wall residue, and shake the enzyme saccharification reaction at 50 ° C. and 200 rpm for 24 hours. To determine the amount of glucose obtained. As a result, it was found that ProNST3: OsNST2 nst1 nst3 can obtain about 20% more glucose per fresh weight than the wild type strain (FIG. 4). From these facts, it was demonstrated that the plant expressed with OsNST2 developed in the present invention is an excellent plant as a biofuel raw material plant with an increased secondary cell wall amount and enhanced wood quality.

Claims (4)

A nucleic acid encoding a transcriptional regulatory factor having a woody-forming regulatory activity according to any of the following (a) to (d) downstream of a promoter of the NST1 or NST3 gene that induces gene expression specifically in woody cells A method for enhancing the wood quality of a plant, comprising transforming the plant with a construct in which
(A) a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 1,
(B) a nucleic acid encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
(C) a nucleic acid comprising the base sequence represented by SEQ ID NO: 2,
(D ) a nucleic acid having 90% or more identity with the gene consisting of the base sequence shown in SEQ ID NO: 2 . A nucleic acid encoding a transcriptional regulatory factor having a woody-forming regulatory activity according to any of the following (a) to (d) downstream of a promoter of the NST1 or NST3 gene that induces gene expression specifically in woody cells A transformed plant with enhanced wood, or a progeny thereof, characterized by being transformed with a construct in which
(A) a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 1,
(B) a nucleic acid encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
(C) a nucleic acid comprising the base sequence represented by SEQ ID NO: 2,
(D) a nucleic acid having 90% or more identity with the gene consisting of the base sequence shown in SEQ ID NO: 2 .   A method for producing a monosaccharide, wherein the transformed plant according to claim 1 or 2 or a progeny thereof is used, and a treatment including a step of obtaining at least a part thereof and subjecting to a sulfated saccharification treatment is performed.   A process for producing glucose, wherein the transformed plant according to claim 2 or its progeny is used, and at least a part thereof is obtained and subjected to a pretreatment step with caustic soda and a saccharification step with cellulase. Method.

Images