JP5764357B2 - Method for evaluating glycation of plants - Google Patents

Description

  The present invention relates to a method for improving the saccharification efficiency of a plant, a plant suitable as a raw material for biomass ethanol, a method for producing biomass ethanol, and a method for evaluating the saccharification property of a plant.

  Brown midrib (bm or bmr, hereinafter referred to as bmr) mutants whose stems and leaves are brown are long known for maize. To date, C4 such as pearl millet, sorghum, and Sudangrass are also used. It has also been found in grasses that perform type photosynthesis. The bmr phenotype has been shown to be associated with reduced expression and activity of cinnamyl alcohol dehydrogenase (CAD) and caffeic acid 3-O-methyltransferase (COMT) involved in lignin biosynthesis. In many bmr lines, the lignin content is reduced as compared to the wild type (see, for example, Non-Patent Document 1). In addition, it is known that the reduction in lignin content has various effects on plants such as a reduction in yield and stem strength (see, for example, Non-Patent Document 2).

CAD is an enzyme belonging to the Zn ²⁺ -dependent medium chain dehydrogenase / reductase (MDR) superfamily, and is an enzyme that catalyzes a reaction for synthesizing cinnamyl alcohol from cinnamyl aldehyde together with the coenzyme NADPH. Cinnamyl alcohols are precursors of lignin. CAD is a dimer and has two Zn ²⁺ ions per subunit. Of the two Zn ²⁺ ions, one is catalytic zinc and the other is structural zinc (see, for example, Non-Patent Documents 3 and 4). Although the four cysteine residues coordinated to structural zinc in CAD are highly conserved, their functional role has not yet been clarified.

  The bmr line has different characteristics such as lignin content, yield, and height depending on its genetic background. For example, there are several bmr lines in sorghum for feed, but in many bmr lines, the dry matter yield is lower than the wild type and the digestibility of NDF (neutral detergent fiber) is improved. The content of ADL (acid detergent insoluble lignin) is not different from the wild type in the bmr6 strain (cad2 mutation), but is lower than the wild type in the bmr12 strain (comt mutation) (for example, Non-Patent Document 5 and 6).

  The sorghum bmr6 line is a mutant having a mutation in SbCAD2 and, like the bmr2 line and bmr12 line, the clathon lignin content in the plant body is lower than the wild type, and the saccharification efficiency is increased. ing. In the bmr6 strain, a plurality of alleles having different mutations in SbCAD2 have been reported (for example, see Non-Patent Document 7). Specifically, in the bmr6-ref mutant, a stop codon is formed by replacing the 2800th base cytosine with thymine. Therefore, SbCAD2 in the bmr6-ref mutant is synthesized only up to the 131st amino acid, and is a mutant that lacks most of the nucleotide binding site, the factor binding site, and the substrate binding site. In the bmr6-3 mutant, the 3875th base guanine is replaced with adenine. Due to the mutation, the 191st glycine (G) forming the glycine-rich motif G (X) GGV (L) G present at the cofactor binding site and highly conserved in the MDR protein is replaced with serine (S). Has been. Furthermore, in the bmr6-27 mutant, guanine is inserted after the 4288th base, resulting in a frame shift. As a result, SbCAD2 in the bmr6-27 mutant is completely different from wild-type SbCAD2 in the 31 amino acid region on the C-terminal side, and is 4 amino acid residues shorter than the wild-type due to the formation of a stop codon and is located outside the active site. It is a mutant with a broken secondary structure. Further, when the composition ratio of lignin monomer was examined, the ratio of S-lignin content to G-lignin content was much smaller in the bmr6-ref mutant than in the wild-type sorghum, and due to the mutation of SbCAD2. It was confirmed that the composition ratio of the lignin monomer in the plant body was affected (see Non-Patent Document 8).

  In addition, data suggesting that the improvement in saccharification efficiency in the bmr line is not necessarily due to a decrease in the lignin content has been reported. For example, according to Non-Patent Document 9, among bmr6 strains of sorghum, bmr6-ref mutant and bmr6-3 mutant have reduced clathon lignin content compared to wild type, and are precursors of lignin monomers In contrast, the bmr6-ref mutant showed no significant change in the saccharification efficiency from the wild type, whereas the coniferaldehyde content was increased. The saccharification efficiency was significantly improved in the bmr6-3 mutant in which the degree of content reduction and precursor accumulation was smaller than that in the bmr6-ref mutant.

  On the other hand, in sorghum, the ethanol conversion efficiency when using wild type, bmr6 strain, bmr12 strain, and double mutants having both cad2 mutation and comt mutation as biomass resources is higher than that of bmr6 strain and bmr12 strain, respectively. In addition, it has been reported that the double mutant has higher ethanol conversion efficiency and a stronger negative correlation between the clathon lignin content and the ethanol conversion efficiency (see, for example, Non-Patent Document 10). .) According to the report, it is said that the clason lignin content greatly affects the enzymatic saccharification of lignocellulose.

  In addition, analysis of CAD function-deficient mutant lines has also been performed in plants other than sorghum. For example, Arabidopsis thaliana CAD has two types of paralogs (AtCAD4, AtCAD5), and all of them have been reported to be mutants in which mRNA is not normally synthesized by insertion of T-DNA. (For example, refer nonpatent literature 11.). In maize bm1 mutants, the genetic analysis revealed that the bmr phenotype was strongly linked to the region of ZmCAD2 on the chromosome, and the CAD enzyme activity was also reduced compared to the wild type. However, no mutation has been found in the mutant ZmCAD2 gene itself (see, for example, Non-Patent Document 12).

  For a long time, it was thought that mutants corresponding to bmr did not exist in rice (see, for example, Non-Patent Document 13), but recently the cause of rice gh2 (gold hull and internode 2) mutant phenotypes. Has been reported to be in the homologous gene (OsCAD2) of the CAD gene responsible for the bmr phenotype (see Non-Patent Document 14). However, in the rice gh2 line, unlike the bmr lines of other plants such as corn and sorghum, it has been reported that there is no brown coloration in the middle of the leaf. It is said that this is a mutant phenotype not found in grasses that perform C3-type photosynthesis.

Sattler SE, et al. Plant Science (2010) 178 (3): p229-238 Pedersen J, et al. Crop Science (2005) 45 (3): p812-819 Bomati E, et al. The Plant Cell (2005) 17 (5): p1598-1611 Youn B, et al. Organic and Biomolecular Chemistry (2006) 4 (9): p1687-1697 Oliver A, et al. Crop Science (2005) 45 (6): p2234-2239 Oliver A, et al. Crop Science (2005) 45 (6): p2240-2245 Saballos A, et al. Genetics (2009) 181 (2): p783-795 Sattler S, et al. Plant Physiology (2009) 150 (2): p584-595 Saballos A, et al. BioEnergy Research (2008) 1 (3): p193-204 Dien B, et al. BioEnergy Research (2009) 2 (3): p153-164 Sibout R, et al. Plant physiology (2003) 132 (2): p848-860 Halpin C, et al. The Plant Journal (1998) 14 (5): p545-553 Barriиre Y, et al. Comptes Rendus Biologies (2004) 327 (9-10): p847-860 Zhang K, et al. Plant physiology (2006) 140 (3): p972-983 NREL (NATIONAL RENEWABLE ENERGY LABOLATORY), “Determination of Structural Carbohydrates and Lignin in Biomass (Laboratory Analytical Procedure (LAP)”, 2008, NREL / TP-510-42618. Lin SY, et al. (Ed), “Method in lignin chemistry”, 1994, Springer-Verlag (Uni Publishing). Edited by Ryuichi Yamamoto and two others, “Rice Breeding Manual”, (1996) Yokendo, p180-184. Terada R., et al. Plant Physiol (2007) 144: p846-856. Kashiwagi, T., et al. Plant physiology (2004) 134 (2): p676-683. Sunohara, H ,. et al., Breeding Science (2006) 56 (3): p261-268. Ookawa, T. et al., Nature Communications (2010) 1 (8): p132-143.

When used for feed or as a biomass resource, the lower the lignin content in the plant body, the easier it is for cellulose-degrading enzymes to access the cellulose half-covered with lignin, and the cellulose degradation (saccharification) efficiency improves. It is considered.
However, as described in Non-Patent Document 2 and the like, when the lignin content in the plant body is reduced, the yield is reduced, and the rigidity (fall resistance) of the plant body is reduced. There is also a case.
In addition, although it is clear that plants with high saccharification efficiency are more preferable for feed and biomass resources, the relationship between the composition and properties of lignocellulose constituting the plant and saccharification efficiency is not clear. Whether a body or a part is excellent in saccharification efficiency can be determined only by actually performing saccharification treatment, and it is difficult to evaluate considering the existence of various pretreatment methods and saccharifying enzymes There is also the problem of being.

  The present invention provides a method for improving the saccharification efficiency of a plant, a plant used in the method, a method for producing biomass ethanol using the plant, and a method for evaluating the saccharification property of a plant. Objective.

  As a result of intensive studies to solve the above problems, the present inventors have found that the ratio of the acid-soluble lignin content to the clathon lignin content in the plant ([acid-soluble lignin content] / [classon lignin content] ) Has a high correlation with the saccharification efficiency of the plant body, and the present invention has been completed.

That is, the present invention
( 1 ) The ratio of the acid-soluble lignin content to the clathon lignin content in the plant body ([acid-soluble lignin content] / [classon lignin content]) is used as an index, and the ratio is lower than that of the plant body. A plant body having a higher ratio is evaluated as having a higher saccharification efficiency , a method for evaluating the saccharification property of a plant body,
Is provided.

In the method for improving saccharification efficiency of a plant of the present invention, saccharification efficiency can be improved by using a plant having a specific lignin composition as a raw material for saccharification treatment.
Moreover, the plant of this invention is excellent in saccharification efficiency rather than a wild-type plant. For this reason, the method for producing biomass ethanol of the present invention using the plant as a raw material can produce biomass ethanol at a higher efficiency and at a lower cost than when using a wild-type plant as a raw material.
Further, by using the method for evaluating the saccharification property of the plant body of the present invention, before performing the saccharification treatment, the raw material candidate plant body has high saccharification efficiency and is suitable as a feed plant or biomass resource. It can be evaluated whether or not.

FIG. 2 is a photograph of wild-type rice (left) and gh2-2 mutant (right) spikes (FIG. 1A), anthers (FIG. 1B), leaves (FIG. 1C), and internodes (FIG. 1D). In Example 1, it is the figure which showed the result of having measured the dry weight of each organ of a wild type rice and gh2-2 mutant. In Example 1, it is the figure which showed the result of having measured the total lignin content per dry weight of each organ of a wild type rice and gh2-2 mutant. In Example 1, it is the figure which showed the result of having measured the cellulose content per dry weight of each organ of a wild type rice and gh2-2 mutant. In Example 1, it is the figure which showed the result of having measured the xylan content per dry weight of each organ of a wild type rice and gh2-2 mutant. In Example 1, it is the figure which showed the result of having measured the ash content per dry weight of each organ of a wild type rice and gh2-2 variant. In Example 1, it is the figure which showed the relationship between the saccharification efficiency in the whole rice straw of wild-type rice and gh2-2 mutant, and the reaction time of a cellulose-degrading enzyme reaction. In Example 1, it is the figure which showed the result of having calculated the saccharification efficiency of each organ of wild-type rice and gh2-2 mutant 48 hours after the reaction start. In Example 1, it is the figure which showed the result of having calculated the glucose production efficiency per dry weight of each organ of wild-type rice and gh2-2 mutant 48 hours after the reaction start. In Example 1, it is the figure which showed the relationship between the acid-soluble lignin content and saccharification efficiency in each organ of wild type rice and gh2-2 mutant. In Example 1, it is the figure which showed the result of having carried out the path | pass analysis using the covariance structure analysis about the correlation with content of each component, and a saccharification rate. In Example 1, it is the figure which showed the result of the multiple regression analysis which made the ASL / KL ratio and cellulose content in each organ of a wild type rice and gh2-2 mutant an independent variable, and made saccharification efficiency a dependent variable. The vertical axis is an actual measurement value of saccharification efficiency, and the horizontal axis is a predicted value of saccharification efficiency calculated from a regression equation (the following equation (2)) obtained as a result of multiple regression analysis. In Example 1, it is the figure which showed the measurement result of the push-down resistance value (N / tilling) of a wild type rice and gh2-2 mutant. In Example 1, it is the figure which showed the measurement result of the section modulus (mm < ³ >) between the nodes of wild type rice and gh2-2 mutant. In Example 1, it is the figure which showed the measurement result of the bending stress (g / mm < ² >) between nodes of a wild-type rice and gh2-2 mutant. In Example 1, it is the figure which showed the calculation result of the moment (kg * cm) at the time of a fracture of wild type rice and gh2-2 mutant.

  In the present invention and the present specification, the saccharification efficiency of a plant means the efficiency with which cellulose in the plant is converted to glucose. Moreover, to evaluate the saccharification property of a plant body means to evaluate the level of saccharification efficiency of the plant body.

<Evaluation method for easy saccharification of plants>
The method for evaluating the saccharification property of the plant of the present invention is the ratio of the acid-soluble lignin content to the clathon lignin content in the plant ([acid-soluble lignin content] / [classon lignin content], hereinafter “ASL / KL ratio”) is used as an index. As shown in Example 1 to be described later, the saccharification efficiency of the plant body does not have a statistically significant correlation with the total lignin content or the clason lignin content in the plant body, but with the ASL / KL ratio. High correlation. For this reason, the saccharification efficiency at the time of carrying out the saccharification process of the said plant body can be evaluated by using ASL / KL ratio in a plant body as a parameter | index. In addition, it is the knowledge which the present inventors discovered for the first time that the saccharification efficiency of a plant body has a high correlation with ASL / KL ratio.

  Specifically, it can be evaluated that a plant having a high ASL / KL ratio has a higher saccharification efficiency than a plant having a low ASL / KL ratio. For example, when there are a plurality of plants to be evaluated, the ASL / KL ratio of each plant is measured and compared with each other to evaluate which plant has higher saccharification efficiency before actually performing the saccharification treatment. be able to.

  In the case of feed crops, the higher the saccharification efficiency, the better the digestion efficiency. Similarly, as a biomass resource used as a source of raw materials such as saccharides for synthesizing compounds such as ethanol, plant bodies that can efficiently obtain more saccharides by saccharification treatment can obtain a smaller amount of saccharides. Preferred over no plant. Therefore, the method for evaluating the saccharification property of the plant body of the present invention evaluates whether the candidate plant body is suitable as a feed crop or biomass resource, or as a feed crop or biomass resource from a plurality of types of candidate plants. It can be suitably used when selecting a more suitable plant.

  The plant body to be evaluated may be the whole plant individual, but can also be evaluated for each organ. For example, it is also possible to compare stems and leaves derived from the same type of plant body and evaluate which organ has high saccharification efficiency and is suitable as a biomass resource.

  The type of plant body to be evaluated is not particularly limited as long as it is a plant body containing clathon lignin and acid-soluble lignin, and may be a monocotyledonous plant or a dicotyledonous plant. Moreover, the plant (C3 plant) which performs C3 type | mold photosynthesis may be sufficient, and the plant (C4 plant) which performs C4 type | mold photosynthesis may be sufficient.

The ASL / KL ratio in the plant body can be calculated from the measurement results of the acid-soluble lignin content and the clason lignin content in the plant body. The acid-soluble lignin content and the clason lignin content in the plant can be measured by conventional methods (see, for example, Non-Patent Documents 15 and 16). More specifically, for example, it can be measured by the following method. First, starch is removed from the sample by using a plant or its organs that have been chopped or pulverized as a sample, and solubilizing the starch in the sample, followed by filtration. The washed and dried filtration residue is mixed with a mineral acid aqueous solution such as a sulfuric acid solution, followed by a hydrolysis reaction. Next, the sample after the hydrolysis reaction is subjected to filtration and the like, and a liquid component (filtrate) containing soluble components such as monosaccharides and acid-soluble lignin and a solid component containing insoluble components such as clathon lignin and ash ( Filter residue). The residue after the washed and dried filtration residue is baked at a high temperature of about 600 ° C. is ash. Therefore, the clathon lignin content contained in the filtration residue can be obtained by subtracting the ash content from the filtration residue amount before firing. On the other hand, the acid-soluble lignin content in the filtrate is calculated from the following formula (1) according to the Beer rule from the absorbance at 205 nm of the filtrate.
Acid-soluble lignin content (g · L ⁻¹ ) = absorbance / [light path (cm) × absorption count (L · g ⁻¹ · cm ⁻¹ )] (1)

<Method for improving saccharification efficiency of plant>
Acid-soluble lignin is more easily decomposed by pretreatment such as acid treatment than clason lignin, which is insoluble in many solvents including acid solvents. For this reason, the higher the content of acid-soluble lignin in lignocellulose, that is, the higher the ASL / KL ratio, the easier the cellulose-degrading enzyme can access the pretreated plant, and the cellulose degradation (saccharification) efficiency. Will improve. Therefore, the saccharification efficiency of the plant body can be improved by increasing the ASL / KL ratio in the plant body.

  That is, in the method for improving saccharification efficiency of a plant according to the present invention, the plant subjected to saccharification treatment has a function-deficient mutation in at least one lignin biosynthesis-related gene, and ASL in the plant A / KL ratio is higher than that of a plant body not having the function-deficient mutation. The “plant having no function-deficient mutation” means a plant having substantially the same genetic background except for the presence or absence of the function-deficient mutation. Specifically, for example, a plant body before the function-deficient mutation is introduced.

  In the present invention and the present specification, the lignin biosynthesis-related gene means a gene in which the protein encoded by the gene is related to the lignin biosynthesis metabolic pathway. Specific examples of lignin biosynthesis-related genes include CAD gene, COMT gene, 4-hydroxycinnamoyl-CoA ligase (4CL) gene, ferulic acid 5 hydroxylase (F5H) gene, and lignin monomer involved in lignin monomer biosynthesis. And a gene encoding peroxidase (PO) and laccase (LAC) involved in the polymerization of. In the present invention, the plant subjected to saccharification treatment may have a function-deficient mutation only in one kind of lignin biosynthesis-related gene, or may have a function-deficient mutation in two or more kinds of lignin biosynthesis-related genes. It may be. When the lignin biosynthesis-related gene is a gene encoding an enzyme, the function-deficient mutation means a gene mutation in which the enzyme activity is deleted or reduced.

  In the method for improving the saccharification efficiency of a plant of the present invention, it is preferable that a saccharification treatment is performed on a plant having at least a function-deficient mutation in the CAD gene. By reducing the enzyme activity of CAD (EC 1.1.1.195) in the plant body, the ASL / KL ratio can be increased more effectively. As a function-deficient mutation possessed by a CAD gene, if the enzyme activity of a mutant CAD encoded by a CAD gene into which the mutation is introduced is lower than that of a CAD gene encoded by a CAD gene into which the mutation has not been introduced, It is not particularly limited, and may be a missense mutation, a nonsense mutation, or a frameshift mutation.

  In the method for improving the saccharification efficiency of a plant of the present invention, the function-deficient mutation of the CAD gene is preferably a missense mutation, and at least one of four cysteine residues coordinated to structural zinc in CAD. Is more preferably a mutation in which the other cysteine residue is substituted with another amino acid residue (hereinafter sometimes referred to as “cysteine mutation”), and among the four cysteine residues coordinated to the structural zinc, More preferably, the mutation is such that the third cysteine residue from the N-terminal side is substituted with another amino acid residue, and 3 of the four cysteine residues coordinated to structural zinc in CAD are 3 from the N-terminal side. Particularly preferred is a mutation in which the second cysteine residue is substituted with a phenylalanine residue. The inventors found for the first time a mutation in which the third cysteine residue from the N-terminal side of the four cysteine residues coordinated to structural zinc in CAD was substituted with another amino acid residue. Mutation.

  The kind of plant body in which saccharification efficiency is improved is not particularly limited as long as it is a plant body containing clathon lignin and acid-soluble lignin, and may be a monocotyledonous plant or a dicotyledonous plant. Good. Moreover, the plant which performs C3 type | mold photosynthesis may be sufficient, and the plant which performs C4 type | mold photosynthesis may be sufficient. In the method for improving the saccharification efficiency of the plant of the present invention, the plant whose saccharification efficiency is improved is preferably a plant that performs C3 type photosynthesis, more preferably a monocotyledon that performs C3 type photosynthesis, Rice is particularly preferred.

  As shown in Example 1 described later, in rice having a cysteine mutation, a part or all of the middle wing of the leaf is colored reddish brown. Therefore, in the method for improving the saccharification efficiency of the plant body of the present invention, it is preferable to perform saccharification treatment on a plant that performs C3-type photosynthesis in which part or all of the middle part of the leaf is colored reddish brown, It is more preferable to perform saccharification treatment on rice in which part or all of the middle wings of the leaves are colored reddish brown. In addition, in plants which perform C3 type photosynthesis, such as rice, the lignin biosynthesis defect | mutation mutant which has the character by which one part or all part of the middle part of the leaf was colored reddish brown is not known until now.

  The method for saccharification treatment of the plant body is not particularly limited as long as it is a method capable of decomposing cellulose in the plant body, and is a method usually used for decomposing cellulose in plant bodies such as corn. It can be used by appropriately selecting from the above. For example, an acid saccharification method in which a plant body is immersed in an acidic solution such as a sulfuric acid solution may be used, or an enzyme saccharification method using a cellulolytic enzyme may be used. In the acid saccharification method, the enzymatic saccharification method is more preferable because the yield of monosaccharides decreases due to excessive decomposition and the ethanol fermentation inhibition by excessive decomposition products such as furfural occurs. In the case of acid saccharification, the reaction conditions such as the temperature and pH of the reaction solution in the saccharification treatment, the reaction time, etc. In the case of the enzymatic saccharification method, it can be appropriately adjusted in consideration of the characteristics of the cellulolytic enzyme used. In addition, it is preferable that the plant body used for a saccharification process is shredded or grind | pulverized previously.

  When using the enzymatic saccharification method, it is preferable to immerse the plant in an acidic solution (for example, a solution having a pH of 4 to 6) in advance before the saccharification treatment. By soaking in an acidic solution, the acid-soluble lignin in the plant body is eluted, so that the cellulose-degrading enzyme can easily attack the cellulose in the plant body. As a result, the saccharification efficiency can be further improved. Plants previously immersed in an acidic solution can be washed with an appropriate buffer to adjust the pH, and then an enzyme reaction can be performed by adding a cellulolytic enzyme. When a saccharification treatment of a plant body in an acidic solution is performed using a cellulose-degrading enzyme having enzyme activity under acidic conditions, the immersion treatment in an acidic solution before saccharification treatment can be omitted.

<Manufacturing method of biomass ethanol>
The method for producing biomass ethanol of the present invention has a function-deficient mutation in at least one lignin biosynthesis-related gene as a biomass resource, and the ASL / KL ratio in the plant body contains the function-deficient mutation. It is characterized by using a plant body higher than the plant body which does not have. As a result of introducing a function-deficient mutation into a lignin biosynthesis-related gene, a plant body having an increased ASL / KL ratio has good saccharification efficiency and is therefore suitable as a biomass resource for producing biomass ethanol.

  By introducing a function-deficient mutation into at least one lignin biosynthesis-related gene, the plant body in which the ASL / KL ratio in the plant body is higher than that of the plant body not having the function-deficient mutation, The plant mentioned in the method for improving the saccharification efficiency of the plant can be used. In the method for producing biomass ethanol of the present invention, as a biomass resource, a mutation in which at least one cysteine residue of four cysteine residues coordinated to structural zinc in CAD is substituted with another amino acid residue ( It is preferable to use a plant having a cysteine mutation, more preferably a plant performing C3 type photosynthesis having a cysteine mutation, and more preferably rice having a cysteine mutation. As a cysteine mutation provided in a plant used as a biomass resource, the third cysteine residue from the N-terminal side of the four cysteine residues coordinated to structural zinc in CAD is substituted with another amino acid residue. The mutation is preferably a mutation in which the third cysteine residue from the N-terminal side of the four cysteine residues coordinated to the structural zinc in CAD is substituted with a phenylalanine residue. .

  Bioethanol can be produced by saccharifying a plant used as a biomass resource and then synthesizing ethanol from the saccharide in the obtained saccharified solution. The saccharification treatment can be performed by the method mentioned in the method for improving the saccharification efficiency of the plant body. In addition, the ethanol synthesis reaction is not particularly limited, and can be appropriately selected from techniques usually used when producing bioethanol such as alcohol fermentation using yeast.

<Plants with increased ASL / KL ratio in plants>
In the plant of the present invention, in the genome of at least one or more cells constituting the plant body, at least one cysteine residue of the four cysteine residues coordinated to structural zinc in CAD is other than that of the CAD gene. It has a mutation (cysteine mutation) substituted with an amino acid. The cysteine mutation provided in the plant of the present invention is a mutation in which the third cysteine residue from the N-terminal side of the four cysteine residues coordinated to structural zinc in CAD is substituted with another amino acid residue. Preferably, the mutation is such that the third cysteine residue from the N-terminal side of the four cysteine residues coordinated to structural zinc in CAD is substituted with a phenylalanine residue.

  The kind of plant having a cysteine mutation is not particularly limited as long as it is a plant having a CAD gene, and may be a monocotyledonous plant or a dicotyledonous plant. Moreover, the plant which performs C3 type | mold photosynthesis may be sufficient, and the plant which performs C4 type | mold photosynthesis may be sufficient.

  The four cysteine residues that coordinate to structural zinc in CAD are highly conserved in various plant species. For example, rice CAD2 (OsCAD2), Arabidopsis CAD5 (AtCAD5), Aspen CAD (PotCAD), Teidamatsu CAD (PtCAD), Corn CAD (ZmCAD2), Sorghum Bmr6 (SbCAD2), Iwahiba CAD (Smah CAD) In the case of tobacco CAD (NtCAD19), sugarcane CAD (SoCAD), the cysteine residues at positions 100, 103, 106 and 114 from the N-terminal, and in Arabidopsis CAD4 (AtCAD4) from the N-terminal 101, 104, 107, And the cysteine residues at the 115th, and the CAD4 (SbCAD4) of Sorghum are the 98th, 101st, 104th and 112th cysteine residues from the N-terminal. Furthermore, other alcohol dehydrogenases other than CAD are highly conserved, suggesting that substitution of these cysteines is likely to cause changes in enzyme function. Examples of the plant of the present invention include a plant having a mutation in which at least one of these cysteine residues is substituted with another amino acid residue.

  The plant of the present invention is preferably a plant that performs C3-type photosynthesis having the mutation, more preferably a monocotyledon that performs C3-type photosynthesis having the mutation, and is a rice having a cysteine mutation. Is particularly preferred. Among them, rice having a mutation in which at least one cysteine residue coordinated to structural zinc in OsCAD2 is substituted with another amino acid residue is preferable, and structural zinc in OsCAD2 Rice having a mutation in which the third cysteine residue from the N-terminal side of the four cysteine residues coordinated to is substituted with another amino acid residue is preferred. Specifically, among the four cysteine residues coordinated to the structural zinc in OsCAD2 described in Example 1 below, the third cysteine residue from the N-terminal side (the 106th amino acid residue from the N-terminal side) ) Rice having a mutation substituted with a phenylalanine residue (gh2-2 mutant).

  In the plant of the present invention, the third cysteine residue from the N-terminal side of the four cysteine residues coordinated to structural zinc in CAD may be substituted with an amino acid residue other than a phenylalanine residue. Good. For example, in rice, a plant having a gene encoding a polypeptide consisting of an amino acid sequence in which the 106th phenylalanine in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than cysteine is also included in the present invention. Plant.

  In addition, as a plant of this invention, as long as the CAD gene does not impair the effect that the ASL / KL ratio in the plant body by a cysteine mutation is raised, it may have mutations other than a cysteine mutation.

  A plant having a cysteine mutation can be produced by a technique known in the art used for producing a mutant by causing a point mutation in a specific region in a genome in a plant. Specific examples include seed irradiation with gamma rays, seed treatment with the chemical mutation agent ethyl methyl sulfate or sodium azide, or fertilized egg treatment with MNU (N-methyl-N-nitrosorea) (for example, non-patent literature). 17).

When producing a plant having a cysteine mutation, for example, a vector incorporating a polynucleotide having the following base sequence can be used.
(A) A base sequence encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
(B) A base sequence encoding a polypeptide consisting of an amino acid sequence in which the 106th phenylalanine in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than cysteine.
(C) a polypeptide comprising an amino acid sequence in which one or several amino acids other than the 106th phenylalanine in the amino acid sequence represented by SEQ ID NO: 1 have been deleted, substituted or added, and a plant A base sequence encoding a polypeptide having a function of increasing the ratio of the acid-soluble lignin content to the clason lignin content in the body ([acid-soluble lignin content] / [classon lignin content]).
(D) A clason in a plant having a homology of 80% or more (preferably 85% or more, more preferably 90% or more) with any one of the base sequences (a) to (c) A base sequence encoding a polypeptide having a function of increasing the ratio of the acid-soluble lignin content to the lignin content ([acid-soluble lignin content] / [classon lignin content]).

  For example, by recombining the polynucleotide having the base sequence (a) with the OsCAD2 gene in the rice genome using gene targeting by homologous recombination (see, for example, Non-Patent Document 18), the structural zinc in OsCAD2 is converted into the structural zinc. Rice having a mutation in which the third cysteine residue from the N-terminal side of the four coordinated cysteine residues (the 106th amino acid residue from the N-terminal side) is substituted with a phenylalanine residue is By recombination with a polynucleotide having the base sequence of b), the third cysteine residue from the N-terminal side of the four cysteine residues coordinated to the structural zinc in OsCAD2 is other than phenylalanine residue. Rices having mutations substituted with amino acid residues can be produced respectively. Similarly, by substituting with the polynucleotide having the base sequence (c) or (d), for example, the third cysteine residue from the N-terminal side of the four cysteine residues coordinated to the structural zinc in OsCAD2 A cysteine residue (the 106th amino acid residue from the N-terminal side) is substituted with a phenylalanine residue, and rice having other mutations can be produced.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
<Production of rice having cysteine mutation in CAD gene>
In addition to a gh2 (gold hull and internode 2) -like phenotype that shows reddish brown color in the cocoons and cocoons, from the MNU-treated rice mutant lines that have been selected and fixed for mutation, red brown in the middle leaf of the upper leaves Mutant strains showing the coloration were selected. When the nucleotide sequence of the lignin biosynthesis-related gene of the selected mutant was examined, it was found that the OsCAD2 gene had a mutation. The amino acid sequence of the protein OsCAD2 encoded by the OsCAD2 gene is shown in SEQ ID NO: 1. In the mutant, the second base of the base sequence TGC encoding the 106th cysteine residue of SEQ ID NO: 1 is substituted from guanine to thymine, and the 106th cysteine residue of OsCAD2 is phenylalanine due to the mutation. It was replaced with a residue. Furthermore, in the transformant in which wild type OsCAD2 was incorporated into this mutant, the mutant phenotype was recovered, so that the mutant was confirmed to be a novel allele of the OsCAD2 function-deficient mutant (gh2). Hereinafter, the selected mutant is referred to as gh2-2 mutant.

<Cultivation and yield of rice>
Wild-type rice and gh2-2 mutant were cultivated in a paddy field and the above-ground part of the plant (rice) was harvested 50 days after heading. FIG. 1 shows photographs of wild-type rice (left) and gh2-2 mutant (right) spikes (FIG. 1A), cocoons (FIG. 1B), leaves (FIG. 1C), and internodes (FIG. 1D). The gh2-2 mutant had ears, pods, and internodes that were darker brown than the wild-type rice, and the leaves were colored reddish brown.
The harvested plant body was naturally dried indoors for about one week and then threshed. Thereafter, the straw and rice husk were dried in a dry oven at 50 ° C. for 2 weeks. A part of the plant body after threshing was collected separately into leaf blades, leaf sheaths, cocoons, ears (not including cocoons), and rice husks to prepare samples for organ-specific analysis, and then dried. It was dried in an oven at 50 ° C. for 2 weeks. The dried soot and the like were each chopped to about 1 cm with scissors and then pulverized using a cutter mill (product name: MKCM-5, equipped with 3 mm screen, manufactured by Masuko Sangyo Co., Ltd.).

  The result of measuring the dry weight of the obtained ground sample is shown in FIG. In FIG. 1, “WT” is the result of wild-type rice, and “gh” is the result of the gh2-2 mutant. In addition, “WS” is the result of the entire cocoon, “LB” is the result of the leaf blade, “LS” is the result of the leaf sheath, “C” is the result of the cocoon, “P” is the result of the ear, “H” indicates the result of rice husk, respectively. Furthermore, “*” indicates a statistical difference between wild-type rice and gh2-2 mutant at 5% level, “**” at 1% level, and “***” at 0.1% level. “NS” indicates that there was no significant difference between the two. As a result, the yield of wild-type rice and the gh2-2 mutant is not significantly different in the whole rice straw, and the introduction of a cysteine mutation in the CAD gene does not significantly change the whole rice straw. all right.

<Preparation of measurement sample>
Starch in the obtained ground sample was measured according to a standard total starch analysis method (AOAC method 996.11, AACC method 76.13) using Total Starch Kit (product name: K-TSTA, manufactured by Megazyme). It was removed by filtration after solubilization. The filtration residue was washed with sterilized water and then dried at 40 ° C., and used as samples for the subsequent cell wall component analysis and enzyme saccharification test.

<Cell wall component analysis>
Cellulose, xylan (hemyl cellulose), clathon lignin, acid-soluble lignin, and ash in the sample were quantified according to the methods described in Non-Patent Documents 15 and 16. In addition, the quantitative value of each component was finally calculated as weight% per sample dry matter amount. The total lignin content was calculated as the sum of the clason lignin content and the acid-soluble lignin content.

  First, 3 mL of 72% sulfuric acid was added to and mixed with 300 mg of the sample, and then warm bathed at 30 ° C. for 60 minutes. Further, 84 mL of water was added and mixed, and then subjected to an autoclave (121 ° C., 60 minutes) to decompose polysaccharides such as cellulose and xylan in the sample into monosaccharides by an acid hydrolysis reaction. The sample after the reaction was cooled to near room temperature and then left in ice. Then, the sample after acid hydrolysis reaction was isolate | separated into the filtrate and the filtration residue by performing suction filtration.

  From the obtained filtration residue, clathon lignin and ash were quantified. Specifically, the filtration residue was washed with sterilized water, collected in a crucible, dried at 105 ° C. overnight, and then weighed (weight A). Then, after further baking at 600 ° C. for 4 hours, the weight was measured again (weight B). The residue after firing is ash (weight B), and the fired residue is clathon lignin (weight A minus weight B).

The absorbance at 205 nm of the obtained filtrate was measured with a spectrophotometer. From the obtained absorbance, the acid-soluble lignin content in the filtrate was determined from the above formula (1) according to the Beer rule. Incidentally, extinction coefficient was used 110L ^· g ^{-1 · cm} -1.

  Cellulose and xylan were quantified from the obtained filtrate. Specifically, calcium carbonate was added to the filtrate to adjust the pH to 5.0 to 6.0, and then the supernatant was passed through a 0.2 μm filter as a sample for high performance liquid chromatography (HPLC). The prepared HPLC sample was injected into HPLC, HPLC was performed under the following conditions, and glucose and xylose were separated and detected. The detected glucose and xylose were quantified by a calibration curve method using a sample. The value obtained by multiplying the quantitative value of gulose by 0.9 was defined as the quantitative value of cellulose, and the value obtained by multiplying the quantitative value of xylose by 0.88 was defined as the quantitative value of xylan.

HPLC conditions;
Column: Aminex HPX-87P (300 mm × Φ7.8 mm, manufactured by Bio-Rad),
Column oven temperature: 85 ° C
Mobile phase: water,
Flow rate: 0.6 mL / min,
Detector: RI (differential refraction) detector,
Detector internal temperature: 40 ° C
Autosampler temperature: 4 ° C
Sampling amount: 50 μL,

  The measurement results are shown in FIGS. FIG. 3 shows the result of the total lignin content, FIG. 4 shows the result of the cellulose content, FIG. 5 shows the result of the xylan content, and FIG. 6 shows the result of the ash content. 3 to 6, “WT”, “gh”, “WS”, “LB”, “LS”, “C”, “P”, “H”, “*”, “**”, “**”. The meanings of “*” and “NS” are the same as those in FIG. As shown in FIG. 2, although there was no significant difference in the leaf sheath, the total lignin content of the gh2-2 mutant was about 10% in the leaf blade, about 20% in the cocoon, and in the ear and rice husk than the wild type rice. It has decreased by about 14%, and the entire cocoon has also decreased by about 14%. On the other hand, the content of cellulose, xylan, and ash, which are other main components of lignin, was not significantly different between wild-type rice and gh2-2 mutants when viewed as a whole.

<Enzyme saccharification test>
The reaction solution (1.9% Acremonium cellulase (manufactured by Meiji Seika Co., Ltd.), 50% acetate buffer (pH 4.8), 0.02% sodium azide) was added to 25 mg of the sample obtained by removing starch from the ground sample. 1 mL was added, and the enzyme reaction was performed with shaking at 50 ° C. and 180 rpm. In order to measure the glucose content in the reaction solution, a part of the reaction solution was fractionated after 0, 2, 6, 24, 30, and 48 hours after the start of the reaction. For the other samples, the reaction solution after 48 hours from the start of the reaction was used. In any sample, the collected reaction solution was measured after stopping the enzyme reaction by allowing the vessel to bathe in boiling water for 5 minutes.
Glucose in the reaction solution collected for measurement was quantified using Glucose CII Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The saccharification efficiency was obtained by dividing the obtained quantitative value by the glucose content contained in 25 mg of the sample calculated from the result of quantification by HPLC.

  FIG. 7 is a graph showing the relationship between the saccharification efficiency of wild-type rice and the gh2-2 mutant in the whole rice straw and the reaction time of the cellulose-degrading enzyme reaction, and FIG. 8 shows the relationship between the wild-type rice and gh2 after the completion of the reaction. FIG. 9 shows the results of calculating the saccharification efficiency of each organ of the -2 mutant, and FIG. 9 shows the glucose production efficiency per dry weight of each organ of the wild-type rice and the gh2-2 mutant after the completion of the reaction ( It is the figure which showed the result of having calculated the dry matter saccharification rate. As a result, in rice straw as a whole, gh2-2 mutant has higher saccharification efficiency and easier saccharification than wild-type rice, consistently from the early stage of cellulolytic enzyme reaction to 48 hours after the end of the reaction. I understood. More specifically, the glycation efficiency of the gh2-2 mutant was about 1.22 times that of wild-type rice, and the dry matter saccharification rate was about 1.23 times that of wild-type rice. Moreover, although there were some somewhat for each organ, it was found that the gh2-2 mutant had significantly higher saccharification efficiency than wild-type rice in any organ.

<Calculation of correlation coefficient between saccharification efficiency and content of each component>
When the correlation coefficient between the saccharification efficiency and the content of each component was calculated, as shown in Table 1, the saccharification efficiency was hardly correlated with the contents of cellulose, xylan and ash. The correlation coefficient with the total lignin content was slightly higher at -0.57, but not significant, and the correlation coefficient with the clason lignin content was -0.69, which was significant. However, a very high correlation was not observed. On the other hand, it was found that the correlation coefficient between the acid-soluble lignin content containing only about 3% of the whole and the saccharification efficiency was as high as 0.87. These results strongly suggested that the quality (composition) of lignin affects the saccharification efficiency more than the total lignin content in the plant body.

  Therefore, the relationship between the acid-soluble lignin content and saccharification efficiency was examined in more detail. FIG. 10 is a graph plotting the relationship between the content of acid-soluble lignin and the saccharification efficiency in each organ of wild-type rice and gh2-2 mutant. As a result, in rice husks (H), leaf sheaths (LS), and leaf blades (LB), the gh2-2 mutant having a higher content of acid-soluble lignin had improved saccharification efficiency over wild-type rice. However, in whole pods (WS), pods (C), and panicles (P), the gh2-2 mutant has higher saccharification efficiency than wild-type rice, although there is no obvious difference in the content of acid-soluble lignin. It was remarkably improved. From these results, it was not possible to explain that the glycation efficiency was improved in the gh2-2 mutant compared to the wild-type rice only from the acid-soluble lignin content, and there was a high correlation between the acid-soluble lignin content and the saccharification efficiency. This suggests that it may not reflect a direct causal relationship.

<Analysis of saccharification efficiency and content of each component by path analysis>
In order to investigate the correlation between the content of each component and saccharification efficiency in more detail, an attempt was made to estimate the influence of the content of each component on the saccharification efficiency. The analysis has an indirect effect on the saccharification efficiency, whether the clathon lignin content or acid-soluble lignin content is directly acting, or the qualitative factor of the lignin composition in the plant, the ratio of both. In order to clarify whether this is the case, we performed not only the direct effect from the explanatory variable to the dependent variable, but also the path analysis that can determine the indirect effect through another explanatory variable. In this example, the path analysis was performed using structural equation modeling (SEM), and the magnitude of each variable was quantitatively clarified. SEM is also called covariance structure analysis.

  First, assuming that the acid-soluble lignin content and the clason lignin content have both a direct action on saccharification efficiency and an indirect action through the ratio of both (ASL / KL ratio), A model (path diagram) of the causal relationship between the component content and the saccharification rate was created. Although this model showed an extremely high explanation rate that 93% of the variation in saccharification efficiency can be explained by the content of each cell wall component, instability such as large variation in the estimated value of the path coefficient indicating the magnitude of action. Was further rejected in the test of whether the entire model fits the structure of the actual data (showing that the model is statistically significantly different from the actual data structure).

  Therefore, after reviewing the relationship between the content of each component, the direct effect of acid-soluble lignin on saccharification efficiency in the previous model was almost 0 (standardized path coefficient = 0.03). Therefore, when the model was reconstructed on the assumption that acid-soluble lignin does not have a direct effect on saccharification efficiency, the accuracy of path coefficient estimation and the fitness of the model to actual data were greatly improved, and good results were obtained. Obtained. The created path diagram is shown in FIG. As a result of the analysis, it is the ASL / KL ratio (standardized path coefficient = 0.84) and the cellulose content (standardized path coefficient = −0.57) that have a significant direct effect on saccharification efficiency. It was shown that the amount and the content of acid-soluble lignin had no significant direct effect on saccharification efficiency, as did the xylan content and ash content. Especially, it became clear that the direct effect on the saccharification efficiency of ASL / KL ratio is large. From the above results, the saccharification efficiency is more greatly affected by the ASL / KL ratio than the total lignin content in the plant body. Therefore, even if the total lignin content in the plant body is not reduced, the ASL / KL ratio. It was shown that it is possible to change the saccharification property of lignocellulose while suppressing the disadvantage caused by the decrease in the total lignin content.

Based on the measurement data, as a result of multiple regression analysis with the ASL / KL ratio and cellulose content in each organ of wild type rice and gh2-2 mutant as independent variables and saccharification efficiency as a dependent variable, the coefficient of determination (R ² ) Was as high as 0.93 (FIG. 12). From this, it is possible to predict the saccharification efficiency of cellulose by measuring the ASL / KL ratio and the cellulose content in the biomass sample, that is, easy saccharification using the ASL / KL ratio and the cellulose content in the biomass sample as indices. It is clear that sex can be evaluated. In addition, when a saccharification process is performed on the same conditions as a present Example, it is thought that the predicted value of saccharification efficiency can be calculated by following formula (2). By making the amount of cellulose used in the enzymatic saccharification reaction constant, a predicted value of saccharification efficiency can be calculated from only the ASL / KL ratio.
Saccharification efficiency (predicted value [%]) = 227.9 × [ASL / KL ratio] −2.4 × [cellulose content (%)] + 87.0 (2)

<Lodging resistance test>
In order to compare the lodging resistance of wild-type rice and the gh2-2 mutant, the pushing resistance value and the moment at fracture were measured. The measurement of the push-down resistance value was performed on the plant body (rice) on the 30th day after heading, and the moment at break was performed on the plant body (rice) on the 40th day after heading.
The pushing resistance value is a force (N) required to push down the plant body by 45 °. The measurement was performed using a push-down resistance measuring instrument (manufactured by Daiki Rika Kogyo Co., Ltd.) according to the method of Kashiwagi and Ishimaru (Non-patent Document 19). Specifically, after the plant body is trimmed so that its height is 40 cm from the ground, it is necessary to apply a force perpendicular to the plant at a position 20 cm from the ground so that the plant tilts to 45 °. The force was pushed down to obtain the resistance value. The pushing resistance value was divided by the measured number of tillers to remove the influence of the thickness of the strain. In addition, the moment (kg · cm) at the time of fracture between the first to fifth nodes of each plant was measured using a device incorporating a digital force gauge (Non-patent Document 20), and the outer diameter between the nodes measured simultaneously. The section modulus (mm ³ ) and bending stress (g / mm ² ) were calculated according to the method of Ookawa et al.

FIG. 13 shows the measurement result of the pushing resistance value (N / parting), FIG. 14 shows the measurement result of the section modulus (mm ³ ) between the nodes, and FIG. 15 shows the bending stress (g / mm ² ) of the nodes. FIG. 16 shows the measurement results, and the calculation results of the moment (kg · cm) at the time of fracture are shown. As a result, there is no statistically significant difference between the gh2-2 mutant and the wild-type rice, and there is no significant change in the strength of the plant. all right. Although the gh2-2 mutant had a lower bending stress and a larger section modulus than the wild type rice, a statistically significant difference was observed between the gh2-2 mutant and the wild type rice. The bending stress was only between the third nodes, and the section modulus was only between the second and third nodes. From these results, when the total lignin content in the plant body is reduced, the strength of the plant body may be reduced, but by increasing the ASL / KL ratio in the plant body, It was suggested that saccharification efficiency could be increased.

  The method for improving the saccharification efficiency of the plant of the present invention can be used in the fields of plant variety improvement and biomass ethanol production.

Claims (1)

The ratio of the acid-soluble lignin content to the clathon lignin content in the plant body ([acid-soluble lignin content] / [classon lignin content]) is used as an index, and the ratio is lower than that of the plant body having a low ratio. A method for evaluating the glycation-enhancing property of a plant, characterized in that a plant having a higher saccharification efficiency is evaluated as having higher saccharification efficiency .