Method for regulating and controlling saccharification efficiency of rice by XAT4 gene

Abstract

The invention relates to the field of biomass energy, and discloses a method for regulating and controlling the saccharification efficiency of rice by using an XAT4 gene, which comprises the following steps: mutagenizing rice by using EMS to construct a mutant library, and screening mutants with abnormal saccharification efficiency; identifying a mutation site of the XAT4 gene in the mutant by combining Mutmap positioning with sequencing analysis, so as to obtain the rice mutant with XAT4 function deletion; analyzing the cell wall structure and components of the rice mutant, and obtaining an improved body when the xylan side chain modification degree and the lignin content of the rice mutant are reduced compared with those of wild rice; and determining the saccharification efficiency of the improved body to obtain the rice material with the saccharification efficiency regulated and controlled. The XAT4 function-deficient rice mutant is obtained through EMS mutagenesis, the xylan side chain modification degree and the lignin content are confirmed to be reduced through screening and cell wall component analysis, the saccharification efficiency is finally determined to be improved, and the purpose of regulating and controlling the structure and components of the rice cell wall from the gene level is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomass energy technology, specifically a method for regulating rice saccharification efficiency using the XAT4 gene. Background Technology

[0002] Against the backdrop of global energy structure transformation, biomass energy, as an important component of renewable energy, has become a key area for achieving green and circular development. Lignocellulose, as the main carrier of biomass energy, is widely found in crop straw and forestry waste. It can be enzymatically converted into monosaccharides such as glucose, and then used to produce clean energy such as bioethanol.

[0003] Lignocellulose possesses a highly crystalline structure, with its internal cellulose, hemicellulose, and lignin forming a complex cross-linked network, which has become a core bottleneck restricting the large-scale application of biomass energy. In the normal cell wall structure, cellulose chains are arranged in a stable crystalline pattern, and its crystallinity (CrI) is inversely correlated with enzymatic hydrolysis efficiency. The branching substitution of hemicellulose directly affects the formation of the interaction forces between cellulose and hemicellulose. The stable chemical bonds formed between lignin monomers and hemicellulose, as well as between cellulose, further enhance the degree of lignification of the cell wall. This results in low enzymatic hydrolysis efficiency of lignocellulose due to its strong structural stability. At the same time, pretreatment that destroys its structure requires a large amount of energy, forming a technical bottleneck restricting the utilization of biomass energy. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for regulating rice saccharification efficiency using the XAT4 gene, solving the problems of low enzymatic hydrolysis efficiency and high energy consumption caused by the stable structure of lignocellulose.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for regulating rice saccharification efficiency using the XAT4 gene, comprising the following steps:

[0006] A mutant library was constructed using EMS mutagenesis in rice, and mutants with abnormal saccharification efficiency were screened.

[0007] By combining Mutmap localization with sequencing analysis, the mutation site of the XAT4 gene in the mutant was identified, and rice mutants with loss of XAT4 function were obtained.

[0008] The cell wall structure and composition of rice mutants were analyzed. When the degree of xylan side chain modification and the lignin content of rice mutants were reduced compared with wild-type rice, improved mutants were obtained.

[0009] The saccharification efficiency of the improved rice was determined to obtain rice materials with saccharification efficiency regulated.

[0010] By adopting the above technical solution, rice mutants lacking XAT4 function were obtained through EMS mutagenesis. Screening and cell wall component analysis confirmed that the degree of xylan side chain modification and lignin content were reduced, and the saccharification efficiency was ultimately improved. This achieved the goal of regulating the structure and composition of rice cell walls at the gene level, thus eliminating the need for high-intensity physicochemical pretreatment. By directly weakening the cross-linking between hemicellulose and lignin and reducing the crystallinity of cellulose through gene mutation, the substrate accessibility during enzymatic hydrolysis was increased. This solved the problem of low enzymatic hydrolysis efficiency and high energy consumption caused by the stable structure of lignocellulose, providing an economical and environmentally friendly technical path for the efficient utilization of biomass energy.

[0011] Preferably, the EMS mutation involves soaking rice seeds in a 0.5-1.5% EMS solution, treating them at 25-30°C for 8-16 hours, rinsing them with water, germinating them to obtain M1 generation plants, and then harvesting M2 generation seeds through self-pollination to construct a mutant library.

[0012] Preferably, the screening of mutants with abnormal saccharification efficiency includes: taking straw from M2 generation plants, pretreating it with dilute acid or dilute alkali, adding cellulose complex enzyme solution for enzymatic hydrolysis; determining the monosaccharide yield by high performance liquid chromatography, and comparing it with wild-type rice to screen mutants with a higher monosaccharide yield than wild-type rice.

[0013] Preferably, the Mutmap localization combined with sequencing analysis involves extracting genomic DNA from mutant and wild-type rice, mixing them to construct a sequencing library; after whole-genome resequencing, locating candidate genomic regions using the Mutmap algorithm; and performing PCR amplification and Sanger sequencing on the XAT4 gene coding region to determine the mutation site of the XAT4 gene.

[0014] Preferably, the analysis of the cell wall structure and composition of the rice mutant involves a loss-of-function mutation in the XAT4 gene, which reduces the degree of xylan side chain modification in the rice mutant and weakens the cross-linking of hemicellulose and lignin.

[0015] Preferably, the determination of saccharification efficiency includes pretreating rice straw with acid and alkali, enzymatically hydrolyzing it with a cellulose complex enzyme solution, and determining the monosaccharide yield.

[0016] Preferably, the acid-base pretreatment is a treatment with 1% sulfuric acid solution at 121°C or a treatment with 1% sodium hydroxide solution at 50°C.

[0017] Preferably, the method also includes using gene editing technology to target and edit the coding region of the rice XAT4 gene, constructing a recombinant vector containing an sgRNA sequence and a Cas9 coding sequence, and transforming rice using Agrobacterium-mediated transformation or gene gun transformation to obtain rice mutants with downregulated or lost XAT4 gene expression.

[0018] Preferably, the rice material with saccharification efficiency regulation is used in biomass conversion, pulp and paper industries.

[0019] This invention provides a method for regulating rice saccharification efficiency using the XAT4 gene. It has the following beneficial effects:

[0020] 1. This invention obtains a rice mutant with XAT4 loss of function through EMS mutagenesis. Screening and cell wall component analysis confirmed that its xylan side chain modification degree and lignin content were reduced. Finally, the saccharification efficiency was improved. This invention achieves the goal of regulating the structure and composition of rice cell wall at the gene level, thus eliminating the need for high-intensity physicochemical pretreatment. By directly weakening the cross-linking of hemicellulose and lignin and reducing the crystallinity of cellulose through gene mutation, the substrate accessibility during enzymatic hydrolysis is increased. This solves the problem of low enzymatic hydrolysis efficiency and high energy consumption caused by the stable structure of lignocellulose, and provides an economical and environmentally friendly technical path for the efficient utilization of biomass energy.

[0021] 2. This invention utilizes chemical mutagenesis and molecular localization techniques to achieve targeted screening of XAT4 gene mutants, avoiding the vector construction and genetic transformation steps of traditional gene editing technologies, thus reducing the technical threshold and operating costs.

[0022] 3. This invention reduces xylan side chain modification and lignin content by regulating the function of the XAT4 gene, thereby weakening the cross-linking structure of lignocellulose, making rice straw exhibit higher substrate accessibility during enzymatic hydrolysis and improving saccharification efficiency.

[0023] 4. The rice material provided by this invention can reduce the amount of acid and alkali reagents and high-temperature energy consumption in biomass conversion, and can reduce the consumption of pulping chemical reagents in the pulp and paper industry, thus having both energy-saving and environmental protection benefits, and promoting the green development of related industries. Attached Figure Description

[0024] Figure 1 This is a predicted transmembrane region diagram of the rice XAT4 protein according to the present invention;

[0025] Figure 2 The phenotypic diagram of the rice mutant xat4 of this invention;

[0026] Figure 3 This is a cross-sectional scan of the stem and leaves of the rice mutant xat4 of this invention;

[0027] Figure 4 This is a diagram showing the results of immunofluorescence observation of a cross-section of the stem of the rice mutant xat4 of this invention;

[0028] Figure 5 This is a graph showing the cell wall composition determination results of the mutant xat4 of this invention;

[0029] Figure 6 The images show the crystallinity determination and cellulose microfibril scanning results of the mutant xat4 of this invention.

[0030] Figure 7 The graph shows the results of enzymatic digestion scanning and saccharification efficiency determination of the mutant xat4 of this invention;

[0031] Figure 8 This is a flowchart of the method for regulating rice saccharification efficiency using the XAT4 gene proposed in this invention. Detailed Implementation

[0032] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Please see the appendix Figure 1 - Appendix Figure 8 This invention provides a method for regulating rice saccharification efficiency using the XAT4 gene, comprising the following steps:

[0034] A mutant library was constructed using EMS mutagenesis in rice, and mutants with abnormal saccharification efficiency were screened.

[0035] Furthermore, the EMS mutation involves soaking rice seeds in a 0.5–1.5% EMS solution, treating them at 25–30°C for 8–16 hours, rinsing them with water, germinating them to obtain M1 generation plants, and then harvesting M2 generation seeds through self-pollination to construct a mutant library.

[0036] Furthermore, the screening of mutants with abnormal saccharification efficiency includes: taking straw from M2 generation plants, pretreating it with dilute acid or dilute alkali, adding cellulose complex enzyme solution for enzymatic hydrolysis; determining the monosaccharide yield by high performance liquid chromatography, and comparing it with wild-type rice to screen mutants with a higher monosaccharide yield than wild-type rice.

[0037] Specifically, in this embodiment, when constructing a rice mutant library through EMS mutagenesis and screening for mutants with abnormal saccharification efficiency, EMS mutagenesis is first performed. Generally, rice seeds are selected as the mutagenesis target and soaked in a 0.5–1.5% EMS solution at a controlled temperature of 25–30°C for 8–16 hours. This process utilizes ethyl methanesulfonate as a chemical mutagen, inducing base substitutions, frameshifts, and other mutations in the DNA sequence through a chemical reaction with the rice seed genomic DNA, thus laying the foundation for obtaining a rich variety of mutant types. After the above treatment, the rice seeds are rinsed with clean water to remove residual EMS solution, and then the seeds are germinated to obtain M1 generation plants. Since the mutations generated by the mutagenesis in the M1 generation plants are mostly heterozygous, the M1 generation plants need to be self-pollinated to harvest M2 generation seeds. The M2 generation seeds are then used to construct a mutant library. At this point, homozygous mutant lines can be isolated from the M2 generation seeds, facilitating subsequent screening for stable genetic phenotypes.

[0038] In the screening of mutants with abnormal saccharification efficiency, some embodiments used M2 generation plant straw as experimental material. Specifically, the straw was pretreated using either dilute acid or dilute alkali. For example, when using dilute acid pretreatment, an acidic solution of appropriate concentration, such as sulfuric acid solution, could be used to destroy some of the lignocellulose structure in the straw; when using dilute alkali pretreatment, the alkaline environment was used to weaken the cross-linking between cellulose, hemicellulose, and lignin, such as sodium hydroxide solution. After pretreatment, a cellulose complex enzyme solution was added to the treated straw for enzymatic hydrolysis. The cellulose complex enzyme solution contains multiple enzyme components, such as cellulase and hemicellulase, which can synergistically decompose the lignocellulose in the straw and convert it into monosaccharides.

[0039] High-performance liquid chromatography (HPLC) was used to determine monosaccharide yield, enabling the separation and detection of different monosaccharide components and their contents. The monosaccharide yield of M2 generation rice straw was compared with that of wild-type rice straw after the same treatment. In one possible approach, mutants with higher monosaccharide yields than wild-type rice were screened. These mutants, due to genomic mutations, exhibit altered straw lignocellulose structure or composition, making them more susceptible to enzymatic conversion and thus showing abnormal saccharification efficiency, such as higher than the wild type. This process identifies candidate mutants with abnormal saccharification efficiency, providing a material basis for further research on the regulation of rice saccharification efficiency by the XAT4 gene. This screening process effectively enriches rice mutants related to saccharification efficiency, facilitating the subsequent discovery of key regulatory genes using methods such as Mutmap mapping.

[0040] By constructing a mutant library through EMS mutagenesis and screening operations, rice materials exhibiting differences in saccharification efficiency due to random gene mutations can be obtained. This provides initial research material for further identification of XAT4 gene function and analysis of its molecular mechanism regulating rice saccharification efficiency, and allows for the screening of mutants with research value at the population level.

[0041] By combining Mutmap localization with sequencing analysis, the mutation site of the XAT4 gene in the mutant was identified, and rice mutants with loss of XAT4 function were obtained.

[0042] Furthermore, the Mutmap localization combined with sequencing analysis involves extracting genomic DNA from mutant and wild-type rice, mixing them to construct a sequencing library, resequencing the whole genome, locating candidate genomic regions using the Mutmap algorithm, and performing PCR amplification and Sanger sequencing on the XAT4 gene coding region to determine the mutation site of the XAT4 gene.

[0043] Specifically, when identifying the mutation site of the XAT4 gene in mutants using Mutmap localization combined with sequencing analysis, genomic DNA extraction is performed first. Generally, fresh leaves from both mutant and wild-type rice are selected as materials, and genomic DNA is extracted using the CTAB method to ensure the integrity and purity of the DNA meets the requirements for subsequent sequencing. Specifically, the leaves are ground and added to CTAB buffer, followed by water bath, chloroform-isoamyl alcohol extraction, and ethanol precipitation to obtain high-quality genomic DNA.

[0044] Next, sequencing libraries are constructed and whole-genome resequencing is performed. In some embodiments, mutant and wild-type genomic DNA are mixed in equimolar amounts to construct a hybrid sequencing library. In one possible implementation, DNA is fragmented into 300-500 bp fragments using ultrasonic disruption, followed by end repair, A-tailing, and adapter ligation, and then amplified by PCR to obtain the sequencing library. The constructed library is then placed on a high-throughput sequencing platform for whole-genome resequencing to obtain massive amounts of sequencing data.

[0045] The Mutmap algorithm is used to analyze sequencing data to locate candidate genomic regions. Specifically, bioinformatics software is used to align sequencing reads to a rice reference genome, and the single nucleotide polymorphism (SNP) frequencies of mutant and wild-type genomes are calculated. Since mutations induced by EMS exhibit specific segregation patterns in a population, the Mutmap algorithm can locate candidate genomic regions associated with the target trait by analyzing the distribution of SNPs across the genome. Generally, regions with SNP frequencies higher than 0.5 are selected as candidate regions, which may contain mutation sites in the XAT4 gene.

[0046] PCR amplification and Sanger sequencing were performed on the coding region of the XAT4 gene within the candidate genomic region. Alternatively, specific primers, such as primers XAT4-seq-F (SEQ ID NO:1) and XAT4-seq-R (SEQ ID NO:2), were designed flanking the coding region based on the XAT4 gene sequence information in the rice genome database. Using mixed DNA or mutant single-plant DNA as templates, PCR amplification was performed. The reaction system included DNA template, PCR buffer, dNTPs, primers, and Taq enzyme. The reaction conditions were 94℃ pre-denaturation, 94℃ denaturation, 55-60℃ annealing, and 72℃ extension for 35 cycles, with a final extension at 72℃ for 10 minutes. The amplified PCR products were purified and sequenced using Sanger sequencing. The sequences were compared with the wild-type XAT4 gene sequence to determine the specific mutation sites in the XAT4 gene, such as base deletions, substitutions, or insertions, ultimately obtaining rice mutants with XAT4 loss of function.

[0047] By combining Mutmap localization with sequencing analysis, the mutation sites of the XAT4 gene in mutants can be identified efficiently and accurately, providing an important foundation for subsequent research on the function of the XAT4 gene and the mechanism regulating rice saccharification efficiency. Combining whole-genome resequencing with bioinformatics analysis can rapidly narrow down the range of candidate genes and improve the efficiency and accuracy of mutation site identification.

[0048] The cell wall structure and composition of rice mutants were analyzed. When the degree of xylan side chain modification and the lignin content of rice mutants were reduced compared with wild-type rice, improved mutants were obtained.

[0049] Furthermore, the analysis of the cell wall structure and composition of the rice mutant revealed a loss-of-function mutation in the XAT4 gene, which reduced the degree of xylan side chain modification in the rice mutant and weakened the cross-linking of hemicellulose and lignin.

[0050] Specifically, in this embodiment, the analysis of the cell wall structure and composition of rice mutants is first based on the characteristics of the loss-of-function mutation in the XAT4 gene. Normally, the XAT4 gene encodes xylan-arabinosyltransferase, and its loss-of-function mutation directly affects the enzyme's catalytic activity, leading to the inhibition of xylan side chain modification. This mutation reduces the synthesis of xylan side chains in rice mutants, thereby weakening the cross-linking between hemicellulose and lignin. This is the core theoretical basis for analyzing cell wall structure and composition.

[0051] In practice, some embodiments use immunofluorescence to detect the degree of xylan side chain modification. For example, sections are prepared from the second internode tissue of rice stems, and LM10 and LM11 antibodies are used for staining to identify unsubstituted or poorly substituted xylan, respectively. When the XAT4 gene function is lost, the degree of xylan arabinose side chain modification in the mutant is reduced, resulting in a significant decrease in the fluorescence intensity of LM10 and LM11 antibodies compared to the wild type, thus visually reflecting the change in xylan structure.

[0052] For the analysis of lignin content, the acid hydrolysis-ultraviolet spectrophotometry method is usually used. The specific steps are as follows: rice mutant straw is crushed, then extracted sequentially with potassium phosphate buffer and chloroform-methanol mixture to remove soluble substances. The remaining residue is then acid-hydrolyzed, and the released lignin monomers are quantified by ultraviolet spectroscopy at 280 nm. Because the XAT4 mutation weakens the cross-linking between hemicellulose and lignin, the amount of lignin deposition in the mutant is reduced, and the measurement results show that its lignin content is about 10% to 15% lower than that of the wild type.

[0053] For cell wall structure observation, scanning electron microscopy (SEM) can be used to analyze stem cross-sections. When the XAT4 gene is lost, the xylem vessel structure of the mutant may collapse, and the mechanically supporting tissue cells may become loosely arranged. This is directly related to the changes in cell wall mechanical properties caused by weakened hemicellulose-lignin cross-linking. At the same time, the arrangement of cellulose microfibrils also becomes disordered, further confirming the improvement in cell wall structure.

[0054] Through the above multi-dimensional analysis, when it is confirmed that the xylan side chain modification degree and lignin content of the rice mutant are significantly reduced compared with the wild type, the mutant can be identified as a cell wall structure and composition improver. This process not only verifies the regulatory effect of XAT4 gene loss on cell wall from a biochemical perspective, but also provides a structural basis for improved saccharification efficiency—the reduced xylan side chain modification degree and decreased lignin content can increase the accessibility of cellulase, enabling the mutant to exhibit a higher monosaccharide yield in subsequent enzymatic hydrolysis.

[0055] This analytical process establishes a complete technical logic chain by linking molecular mechanisms with phenotypic characteristics, ensuring the scientific rigor and accuracy of mutant screening.

[0056] The saccharification efficiency of the improved rice varieties was determined to obtain rice materials with saccharification efficiency control. The determination of saccharification efficiency involved acid-base pretreatment of rice straw, followed by enzymatic hydrolysis using a cellulose complex enzyme solution, and the determination of monosaccharide yield. The acid-base pretreatment consisted of treatment with a 1% sulfuric acid solution at 121℃ or a 1% sodium hydroxide solution at 50℃.

[0057] Specifically, in this embodiment, when determining the saccharification efficiency of the modified rice straw, the straw was first pretreated to disrupt the lignocellulose structure. Generally, a 1% sulfuric acid solution was used for high-pressure treatment at 121°C for 20 minutes, or a 1% sodium hydroxide solution was used for shaking treatment at 50°C for 2 hours. Acid pretreatment hydrolyzes hemicellulose glycosidic bonds through hydrogen ion hydrolysis, while alkali pretreatment disrupts lignin-hemicellulose crosslinks through hydroxide ion destruction. Both methods increase the enzyme accessibility of the straw.

[0058] After pretreatment, the straw is centrifuged and washed, then enzymatically hydrolyzed with a cellulose complex enzyme solution. In one possible implementation, the enzyme solution contains β-glucanase, cellulase, and xylanase, and the reaction is carried out with shaking at 50°C and pH 4.8 for 48 hours. This synergistically decomposes cellulose and hemicellulose, converting them into monosaccharides such as glucose and xylose.

[0059] After enzymatic hydrolysis, the monosaccharide yield in the supernatant was determined by high-performance liquid chromatography (HPLC). The specific procedures included: centrifuging the hydrolysate and filtering it through a 0.22 μm filter membrane; separating the monosaccharides using an amino chromatography column; using acetonitrile-water as the mobile phase; and detecting with a differential refractive index detector. Due to the reduced xylan side chain modification and lignin content in the modified form, the release of monosaccharides during enzymatic hydrolysis was significantly increased. For example, with 1% sodium hydroxide pretreatment, the glucose yield was more than three times higher than that of the wild type.

[0060] By comparing the monosaccharide yields of the improved mutant with those of the wild type, the extent of the improvement in saccharification efficiency can be clearly identified, thereby obtaining rice materials with saccharification efficiency control. This allows for the simulation of an industrial saccharification process, with reproducible pretreatment conditions and enzymatic hydrolysis systems, providing data support for the practical application of the mutants.

[0061] It also includes using gene editing technology to target the coding region of the rice XAT4 gene, constructing a recombinant vector containing sgRNA and Cas9 coding sequences, and transforming rice using Agrobacterium-mediated transformation or gene gun transformation to obtain rice mutants with downregulated or lost XAT4 gene expression.

[0062] Specifically, in this embodiment, when using gene editing technology to target and edit the rice XAT4 gene, an sgRNA sequence is first designed for the coding region of the XAT4 gene. Generally, the sgRNA sequence targets the transmembrane region or catalytic domain of the XAT4 gene, for example, selecting the coding sequence corresponding to amino acids 150-200. Bioinformatics tools are used to analyze GC content and assess off-target risks to ensure the specificity of the sgRNA sequence.

[0063] When constructing the recombinant vector, the sgRNA expression cassette and the Cas9 coding sequence are integrated into the vector backbone. Alternatively, the pCAMBIA series plasmids are used as the vector backbone, and the sgRNA expression cassette is driven by the Osa-U6 promoter to achieve stable transcription of sgRNA in rice cells. Specifically, the vector is digested with restriction endonucleases, and the target fragment is ligated using DNA ligase. After sequencing verification, the recombinant vector is obtained.

[0064] In the genetic transformation process, recombinant vectors are introduced into rice cells using either Agrobacterium-mediated transformation or gene gun transformation. In some embodiments, mature embryos of the japonica rice variety Nipponbare are selected as recipient material, sterilized, and inoculated into an induction medium to obtain callus tissue. If Agrobacterium-mediated transformation is used, the recombinant vector is transformed into an Agrobacterium strain, a bacterial solution is prepared, and the callus tissue is infected. Co-culture allows the recombinant vector to enter the rice cells. If gene gun transformation is used, high-pressure gas is used to inject gold powder particles encapsulating the recombinant vector into the callus tissue cells.

[0065] Transgenic callus tissue was screened, differentiated, and rooted to obtain transgenic plants. Molecular identification of the transgenic plants was performed, and the mutation type of the XAT4 gene was analyzed by PCR amplification and sequencing to screen for rice mutants with downregulated or absent XAT4 gene expression. Because the XAT4 gene was edited, the modification degree of the xylan-arabinose side chain in the mutants was reduced, and the cross-linking between hemicellulose and lignin was weakened. After saccharification efficiency determination, rice materials with regulated saccharification efficiency could be obtained.

[0066] Gene editing technology, through targeted modification of the XAT4 gene, has achieved directional improvement of the structure and components of rice cell walls. This complements the EMS mutagenesis method and provides a variety of technical options for obtaining XAT4 loss-of-function mutants.

[0067] Through the coordinated steps, XAT4-deficient rice mutants were obtained via EMS mutagenesis. Screening and cell wall component analysis confirmed reduced xylan side chain modification and lignin content, ultimately leading to improved saccharification efficiency. This achieved the goal of regulating rice cell wall structure and composition at the gene level, eliminating the need for intensive physicochemical pretreatment. By directly weakening the cross-linking between hemicellulose and lignin and reducing cellulose crystallinity through gene mutation, substrate accessibility during enzymatic hydrolysis was increased. This solved the problem of low enzymatic hydrolysis efficiency and high energy consumption caused by the stable structure of lignocellulose, providing an economical and environmentally friendly technical path for the efficient utilization of biomass energy.

[0068] Rice materials with saccharification efficiency regulation are used in biomass conversion, pulp and paper industries.

[0069] Specifically, rice materials with saccharification efficiency regulation can be directly used in the enzymatic conversion of lignocellulose raw materials such as straw in the field of biomass conversion. Due to the reduced degree of cross-linking between hemicellulose and lignin in their cell wall structure and the decreased crystallinity of cellulose, efficient saccharification can be achieved during enzymatic hydrolysis without relying on high-intensity acid-base pretreatment, thereby significantly improving the production efficiency of bioethanol and other biomass fuels, while reducing energy consumption and chemical reagent costs in the pretreatment stage.

[0070] In the pulp and paper industry, this rice material, due to its reduced lignin content and improved hemicellulose structure, can reduce the amount of chemical reagents such as sulfides used in the pulping process, thereby reducing pulping energy consumption and environmental pollution. At the same time, the change in cell wall structure improves fiber separation efficiency, increases pulp yield, and optimizes paper fiber properties, making it suitable for high-quality pulp production and environmentally friendly papermaking processes.

[0071] The following is a further description with reference to the embodiments:

[0072] Example 1:

[0073] Bioinformatics analysis was performed on the XAT4 protein in rice. Transmembrane region prediction was performed on the XAT4 protein (TMHMMServer 2.0 (http: / / www.cbs.dtu)). The results showed that XAT4 has a transmembrane region. Figure 1 ).

[0074] Example 2:

[0075] Obtaining the xat4 mutant. The mutant was derived from the Nipponbare ethyl methanesulfonate EMS-induced mutant library. After five generations of continuous self-pollination, the mutant traits were stably inherited, exhibiting characteristics such as plant brittleness and dwarfing. Figure 2To identify the controlling genes causing plant fragility and height variation, the mutant was backcrossed with the wild type. Mutmap identification was performed on the F2 segregating population from the backcross using plants exhibiting fragility. Based on candidate genes, and referring to the LOC_Os06g49320 locus information in the rice genome database (http: / / rice.uga.edu / cgi-bin / sequence_display.cgi?orf=LOC_Os06g49320.1), a pair of amplification primers, XAT4-seq-F (SEQ ID NO:1) and XAT4-seq-R (SEQ ID NO:2), were designed flanking the XAT4 sequence for PCR amplification. The resulting sequence containing the complete XAT4 coding region was used for sequence alignment with the wild-type CDS sequence. The CDS sequence of the XAT4 gene in the wild type is shown in SEQ ID NO:3, the protein sequence in SEQ ID NO:4, and the CDS sequence of the xat4 mutant in SEQ ID NO:5. It is evident that a base substitution occurred in the XAT4 gene, leading to premature termination of transcription and translation.

[0076] Example 3:

[0077] Rice stem and leaf sections were prepared and observed using scanning electron microscopy. The stems and the last leaf from the second internode (from top to bottom) of the main tiller, two weeks after heading, were selected as materials and vacuum-preserved in 2.5% glutaraldehyde. Cross-sectional scanning electron microscopy was then performed to observe cell structure and morphology. The results showed that the xylem of the mutant xat4 underwent significant collapse, and the mechanically supporting tissue cells were loosely arranged. Figure 3 ).

[0078] Example 4:

[0079] Rice stem section preparation and chemiimmunoassay. LM10 (Anti-xylan, which can recognize unsubstituted and relatively low-substituted xylan in several species) and LM11 (Anti-xylan / arabinoxylan, which can recognize unsubstituted and relatively low-substituted xylan in several species) were used to determine structural differences in xylan. Sample sections were prepared and immersed in PBS (pH 7.4) solution containing monoclonal antibodies LM10 and LM11 diluted 50-fold for 1.5 h, followed by rinsing with PBS (pH 7.4) buffer at least three times. The rinsed sections were then incubated in rabbit secondary antibody diluted 300-fold for 1.5 h, rinsed with PBS buffer and deionized water, and the fluorescence of the sections was observed under a fluorescence microscope. All images were observed and captured under the same conditions. Results showed that the immunofluorescence intensity of LM10 and LM11 in mutant xat4 was significantly lower than that in WT ( Figure 4 ).

[0080] Example 5:

[0081] Cell wall composition determination. Two weeks after heading, the second internode of the main tiller (from top to bottom) was freeze-dried, and cell wall composition was determined. Soluble sugars, lipids, and starches were removed from the samples sequentially using potassium phosphate buffer, chloroform-methanol (1:1, v / v), and dimethoxy-sulfone-water (9:1, v / v). The remaining samples were suspended in 4M KOH containing 1.0 mg / mL sodium borohydride for 1 h at 25°C. The supernatant was hemicellulose. Total hemicellulose content was determined by the DNS method. The remaining air was considered as total cellulose, and the total cellulose content was determined by the anthrone method. Total lignin content was determined by acid hydrolysis. The results showed that the total hemicellulose and lignin content of the mutant xat4 were significantly decreased, while the cellulose content did not change significantly. Figure 5 ).

[0082] Example 6:

[0083] Cell wall monosaccharide content determination. Two weeks after heading, the second internode of the main tiller (from top to bottom) was freeze-dried, and the monosaccharide content was determined. Quantitative analysis was performed using a Thermo Field (1510-04201, USA) multifunctional enzyme labeler. All experiments were biologically replicated three times. Thermo ICS 5000+ ion chromatography system (ICS 5000+, ThermoFisher Scientific, USA) and electrochemical detector were used to detect the monosaccharide components Fuc (emphasis), Rha (rhamnose), Ara (arabinose), Gal (galactose), Glc (glucose), Xyl (xylose), Man (mannose), Fru (frucose), Rib (ribose), Gal-ua, Guel-UA, Glc-ua, and Man-ua. The results showed that the arabinose and xylose contents of the mutant xat4 were significantly decreased (Table 1).

[0084] Example 7:

[0085] Cellulose crystallinity determination. Following the cellulose extraction method described above, 0.2 g of dried cellulose was weighed and placed in a diffraction cell. Nickel-wave Cu target Kα rays (γ = 0.154056 nm) were generated under conditions of 40 kV and 18 mA. The scanning speed was 0.0197° / s, and the scanning range was 10-45°. 200 peaks (I...) were used to determine the crystallinity. 200 The intensity of (θ=22.5°) and 200–110 peaks (I am Minimum strength estimation crystallinity index (Crl) between θ = 18.5°: Crl = 100 × (I 200 -I am ) / I 200The samples were scanned according to the experimental conditions to obtain diffraction intensity curves, and the crystallinity was calculated based on the relevant area ratios in the X-ray diffraction intensity curves. The results showed that the crystallinity of cellulose in the mutant xat4 was significantly reduced. Figure 6 ).

[0086] Example 8:

[0087] Cellulose microfibril sectioning and scanning electron microscopy observation. Stems from the second internode (from top to bottom) of the main tiller, two weeks after heading, were selected as material and vacuum-preserved in 2.5% glutaraldehyde. Cell structure and morphology were observed using scanning electron microscopy. Lignin was removed by culturing in 8% acidic chlorite (1 mM HCl plus 1 g sodium chlorite) at 50℃ for 48 h, followed by treatment with 1 M KOH for 1 h to remove amorphous polysaccharides. Cellulose arrangement was observed after washing with distilled water and drying. Results showed that the cellulose microfibrils of the mutant xat4 were disordered. Figure 6 ).

[0088] Example 9:

[0089] Scanning electron microscopy observation of enzymatic hydrolysis treatment of rice stem biomass. Stems from the second internode (from top to bottom) of the main tiller, two weeks after heading, were selected as material, cut into uniformly sized segments, and subjected to acid-base pretreatment. Pretreatment: 10 mL of 1% H₂SO₄ was added to 0.5 g of sample, and the mixture was autoclaved at 121℃ for 20 min, then shaken at 150 rpm for 2 h at 50℃, centrifuged at 3000 g for 5 min, washed three times with 10 mL of distilled water, and the sample was collected for enzymatic hydrolysis. NaOH pretreatment: 10 mL of 1% NaOH was added to 0.5 g of sample, and the mixture was autoclaved at 150 rpm at 50℃. -1 Shake at a constant speed for 2 hours, centrifuge at 3000g for 5 minutes, wash three times with 10mL distilled water, collect the sample, and perform enzymatic digestion. Enzymatic digestion: 10mL mixed cellulase reaction buffer (containing 6×10⁻⁶ β-glucanase) 4 U, cellulase 600U, xylanase 10×10 4 U) was enzymatically hydrolyzed at 50℃ and 150 rpm. All experiments were performed in triplicate. Scanning electron microscopy showed that the xat4 mutant exhibited more pronounced cell fragmentation and greater cell loss after hydrolysis with cellulose complex enzyme. Figure 7 ).

[0090] Example 10:

[0091] Saccharification efficiency determination. The plant stems were completely dried, crushed, filtered through a 100-mesh sieve, and subjected to acid-base pretreatment. Pretreatment: 10 mL of 1% H₂SO₄ was added to 0.5 g of sample, and the mixture was steam-pressed at 121 °C for 20 min, then at 50 °C and 150 r·min. -1Shake for 2 hours, centrifuge at 3000g for 5 minutes, wash the remaining residue three times with 10mL distilled water, and collect the remaining residue for enzymatic hydrolysis. NaOH pretreatment: Add 10mL of 1% NaOH to 0.5g of sample, and incubate at 50℃ with a speed of 150r·min. -1 Shake at a constant speed for 2 hours, then centrifuge at 3000g for 5 minutes. Wash the remaining residue three times with 10mL of distilled water, and collect the residue for enzymatic hydrolysis. Enzymatic hydrolysis: 10mL of mixed cellulase reaction buffer (containing 6×10⁻⁶ β-glucanase) 4 U, cellulase 600U, xylanase 10×10 4 U) was shaken at 50℃ and 150 rpm for 48 h, centrifuged at 3000g for 10 min, and the supernatant was collected. The content of glucose monosaccharide in xylose monosaccharide was determined by high performance liquid chromatography-mass spectrometry. All experiments were performed in triplicate. The results showed that the mutant xat4 exhibited a more significant improvement in saccharification efficiency ( Figure 7 ).

[0092] Table 1: Determination of Monosaccharide Content

[0093]

[0094] Table 2: Primer sequences used in the experiment

[0095]

[0096] Example 11:

[0097] Improve plant saccharification efficiency by downregulating or eliminating the function of the plant XAT4 gene.

[0098] Downregulation or deletion of XAT4 gene function can be achieved by using RNA interference (RNAi) or gene editing technology (CRISPR-CAS9) to downregulate the activity of XAT4 in plants, thereby improving saccharification efficiency.

[0099] The selected plants are preferably Arabidopsis thaliana, poplar, eucalyptus, rice (Oryza sativa), wheat (Triticum aestivum), pine (Pinus), or Jatropha carcass L., with rice being more preferred. However, this can also be extended to other plants, using RNA interference (RNAi) or gene editing technology (CRISPR-CAS9) to downregulate XAT4 activity in the plant, thereby improving saccharification efficiency.

[0100] Transmembrane region analysis of the XAT4 protein was performed using TMHMM Server 2.0 (http: / / www.cbs.dtu). The XAT4 protein possesses a transmembrane domain.

[0101] Phenotypic analysis of the mutant xat4 revealed that its growth and development were affected, and the entire plant exhibited brittleness. Section analysis of the second internode and the last leaf of the mutant xat4 showed collapsed xylem vessels, and the mechanical supporting tissue of the mutant xat4 exhibited significant cellular loosening.

[0102] Chemoimmunoassay was performed on the xat4 mutant. Chemoimmunoassay was performed on the second internode of rice stems 14 days after heading (antibodies: LM10, LM11). The weak fluorescence of LM10 indicated a low xylan content in the xat4 mutant. The weak fluorescence of LM11 indicated impaired xylan biosynthesis and a low arabinoxylan content in the xat4 mutant.

[0103] The cell wall composition of the mutant xat4 was analyzed. Stem segments of the xat4 mutant were collected, and the main cell wall components and monosaccharide composition were analyzed. The results showed that the hemicellulose and lignin contents of xat4 decreased by 19% and 14%, respectively, but the cellulose content was unaffected. Monosaccharide analysis showed that the arabinose and xylose contents of xat4 decreased by 35% and 15%, respectively.

[0104] Cellulose crystallinity analysis was performed on the mutant xat4. X-ray diffraction was used to determine the cellulose crystallinity of xat4, and the results showed that the cellulose crystallinity of xat4 decreased by 5%.

[0105] The arrangement of cellulose microfibrils in the mutant xat4 was observed. Scanning electron microscopy revealed that the cellulose microfibrils of xat4 were arranged in a disordered manner.

[0106] The saccharification efficiency of the mutant xat4 was analyzed. Xylan cross-links with cellulose and lignin, hindering the entry of cellulases and affecting saccharification efficiency. The resulting mutant xylan structure underwent significant changes, thus leading to substantial variations in its saccharification efficiency. After enzymatic hydrolysis of the mutant with a mixed cellulose enzyme, the monosaccharide content was measured to calculate the saccharification efficiency. The mutant showed a significant increase in saccharification efficiency. Results indicated that under 1% H₂SO₄ pretreatment, the glucose and xylose yields of mutant xat4 increased by 1.03-fold and 1.15-fold, respectively; under 1% NaOH treatment, the glucose and xylose yields of mutant xat4 increased by 3.43-fold and 2.34-fold, respectively.

[0107] An application of XAT4-downregulated plants in biomass conversion, pulp and paper industries was obtained through the above preparation method.

[0108] The utilization efficiency of biomass energy largely depends on the properties of raw materials. Improving the availability of raw materials or reducing their processing costs can both improve the utilization efficiency of biomass energy. This invention focuses on these two aspects. The resulting mutant material has reduced hemicellulose and lignin content, as well as reduced arabinose and xylose content, thus reducing the cross-linking between xylan and lignin, and between xylan and cellulose. The crystallinity of cellulose is reduced, and the accessibility between cellulose complex enzymes and cellulose is increased. This improves saccharification efficiency while reducing material processing costs. Most existing methods focus on changing the pretreatment methods of materials, which have limited effect on improving saccharification efficiency. This invention fundamentally changes the biomass characteristics of the material, resulting in a fundamental improvement in saccharification efficiency, saving energy and reducing labor intensity.

[0109] SEQ ID NO: 1

[0110] CAACCTCTGGTTGCCATCGC

[0111] SEQ ID NO: 2

[0112] GGACACTTGTCTATGACACGATC

[0113] SEQ ID NO: 3

[0114] ATGAAGCACCATCATGTGGCGCGGGGGCGCGCCGAGCCGCGGCGGATGGGCAACGCCGCCATGGTCATCACCATGCTCCTCTCCCTCTGCGTCCTCACCTACATCAAGGCTCGCTACTGCTCCACCCCCTTCCCCAAGGCGGCGGAGGAGATGGAGGTGGTGGAGATCGACGAGGATTACGACAGCACGAGGTACAAGATGACGGGGCCGATAGGGGAGGAGGACTTCGACCCGACCAGGCCGACGTGCTACGTCACGAGCAAGCGGTCGGAGCGGTGCGCCGCCGTGGGCGACATCCGCGTCGACGGCAACCACTCCAAGATCTACATCAACCCGCTCGACAAGGAGTGGAGAACCAAGCCGTACGCGCGCCTCCACGACGCCGTCGCCATGGACGACGTGCGGGAGTTCACGCTCGTCCCCTTCGGCGGCGCCAACCACACCGCCGTGCCGCCGCTCTGCACGCGCAACCACTCCGTCCCGGCGTTCCTCTTCTCCAGCGGCGGCTTCGCCGGCAACCTGTACCATGACTACACCGACGTGCTCGTCCCGCTCTTCACCAGCACCAACCACTTCGGCGGCGAGGTGCAGTTCTTGCTCAGCGGGATCAAGGATTGGTGGCTCGACAAGTTCACGCCGCTGTTCCGGCAGCTCTCCCGGTACGACGTCATCGACGTCGACAACGACCAGGAGGTGCACTGCTTCCCGCGGATCTTCATCGGCGCCACGTTCCACCGCGCCATGGGCATCGACCCGGCGCGCTCCCCGGGCGGCGTCACCGTCGCCGACTTCAAGCGCCTCCTCCGCCGCACCTTCCGCCTGGAGCGCGCCGTCGCGTCGCGCACGGGCGCGCCGCGGCGCGACAAGCCGCGCCTCCTCATCATCTCCCGCAAGAGCTCGCGCCGCTTCCTCAACGAGCGCGCCATGGCGCACGCCGCCGCGCTCGCCAGGTTCGACGTGCGCATCGCCGAGCCGGACAACCACACCGACATGCCCAACTTCGCGCGCCTCGTCAACTCCGCCGACGTGATGATGGGCGTCCATGGCGCCGGCCTCACCAACATGGTGTTCCTCCCCAGCCGCGCCGTGCTCATCCAGGTGGTGCCATTCGGGGGGCTCGAATGGCTCACCCGTGTCACCTTCAAGGATCCAGCCAAGGACATGGATGTCAACTACATGGAGTACAATGTCTCCTTCGACGAGAGCTCGCTGAGAGAGCTCTACCCGAGAGACCATTTCTACATCCAGCATCCATACGATGTGCACAAGAAAGGCTGGGACGCCATCAAGACGGTGTACCTGGACAAGCAGAATGTGGAGCTCAACCTCACCAAGCTCACCAACACGCTGGAACGGGCGCGCGACTTCTTGCCAGAGCCCTGA

[0115] SEQ ID NO:4

[0116] MKHHHHVARGRAEPRRMGNAAMVITMLLSLCVLTYIKARYCSTPFPKAAEEMEVVEIDEDYDSTRYKMTGPIGEEDFDPTRPTCYVTSKRSERCAAVGDIRVDGNHSKIYINPLDKEWRTKPYARLHDAVAMDDVREFTLVPFGGANHTAVPP LCTRNHSVPAFLFSSGGFAGNLYHDYTDVLVPLFTSTNHFGGEVQFLLSGIKDWWLDKFTPLFRQLSRYDVIDVDNDQEVHCFPRIFIGATFHRAMGIDPARSPGGVTVADFKRLLRRTFRLERAVASRTGAPRRDKPRLLIISRKSSRRFLN ERAMAHAAALARFDVRIAEPDNHTDMPNFARLVNSADVMMGVHGAGLTNMVFLPSRAVLIQVVPFGGLEWLTRVTFKDPAKDMDVNYMEYNVSFDESSLRELYPRDHFYIQHPYDVHKKGWDAIKTVYLDKQNVELNLTKLTNTLERARDFLPEP*

[0117] SEQ ID NO: 5

[0118] ATGAAGCACCATCATGTGGCGCGGGGGCGCGCCGAGCCGCGGCGGATGGGCAACGCCGCCATGGTCATCACCATGCTCCTCTCCCTCTGCGTCCTCACCTACATCAAGGCTCGCTACTGCTCCACCCCCTTCCCCTAG

[0119] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for regulating rice saccharification efficiency using the XAT4 gene, characterized in that, Includes the following steps: A mutant library was constructed using EMS mutagenesis in rice, and mutants with abnormal saccharification efficiency were screened. By combining Mutmap localization with sequencing analysis, the mutation site of the XAT4 gene in the mutant was identified, and rice mutants with loss of XAT4 function were obtained. The cell wall structure and composition of rice mutants were analyzed. When the degree of xylan side chain modification and the lignin content of rice mutants were reduced compared with wild-type rice, improved mutants were obtained. The saccharification efficiency of the improved rice was determined to obtain rice materials with saccharification efficiency regulated.

2. The method for regulating rice saccharification efficiency using the XAT4 gene according to claim 1, characterized in that: The EMS mutation involves soaking rice seeds in a 0.5–1.5% EMS solution, treating them at 25–30°C for 8–16 hours, rinsing them with water, germinating them to obtain M1 generation plants, and then harvesting M2 generation seeds through self-pollination to construct a mutant library.

3. The method for regulating rice saccharification efficiency using the XAT4 gene according to claim 2, characterized in that: The screening of mutants with abnormal saccharification efficiency includes: taking straw from M2 generation plants, pretreating it with dilute acid or dilute alkali, adding cellulose complex enzyme solution for enzymatic hydrolysis; determining the monosaccharide yield by high performance liquid chromatography, and comparing it with wild-type rice to screen mutants with higher monosaccharide yield than wild-type rice.

4. The method for regulating rice saccharification efficiency using the XAT4 gene according to claim 1, characterized in that: The Mutmap localization combined with sequencing analysis involves extracting genomic DNA from mutant and wild-type rice, mixing them to construct a sequencing library, resequencing the whole genome, locating candidate genomic regions using the Mutmap algorithm, and performing PCR amplification and Sanger sequencing on the XAT4 gene coding region to determine the mutation site of the XAT4 gene.

5. The method for regulating rice saccharification efficiency using the XAT4 gene according to claim 1, characterized in that: The analysis of the cell wall structure and composition of the rice mutants revealed a loss-of-function mutation in the XAT4 gene, which reduced the degree of xylan side chain modification in the rice mutants and weakened the cross-linking of hemicellulose and lignin.

6. The method for regulating rice saccharification efficiency using the XAT4 gene according to claim 1, characterized in that: The determination of saccharification efficiency includes pretreating rice straw with acid and alkali, followed by enzymatic hydrolysis with a cellulose complex enzyme solution, and then determining the monosaccharide yield.

7. The method for regulating rice saccharification efficiency using the XAT4 gene according to claim 6, characterized in that: The acid-base pretreatment is either a 1% sulfuric acid solution at 121°C or a 1% sodium hydroxide solution at 50°C.

8. The method for regulating rice saccharification efficiency using the XAT4 gene according to claim 6, characterized in that: It also includes using gene editing technology to target the coding region of the rice XAT4 gene, constructing a recombinant vector containing sgRNA and Cas9 coding sequences, and transforming rice using Agrobacterium-mediated transformation or gene gun transformation to obtain rice mutants with downregulated or lost XAT4 gene expression.

9. The method for regulating rice saccharification efficiency using the XAT4 gene according to any one of claims 1-8, characterized in that: The rice materials with saccharification efficiency regulation are used in biomass conversion, pulp and paper industries.

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