Application of knocking out m6a demethylase pagalk bh10a / b gene in promoting poplar lignification

Abstract

The application discloses knockout m6A demethylase PagALKBH10A / B application. The application knocks out ALKBH10A and ALKBH10B genes in poplar through gene editing to obtain mutant AlkB, the width of xylem and vascular cambium of the stem of the mutant AlkB is higher than that of WT, the degree of xylem lignification of the mutant AlkB is also higher than that of WT, further transcriptome analysis shows that the expression level of genes related to lignin and hemicellulose synthesis of the mutant AlkB is improved, and component analysis also shows that the content of lignin and cellulose in the mutant AlkB is higher than that in WT. PagALKBH10A / B The gene is a target gene for regulating poplar lignin synthesis, and has good application value for improving the performance of poplar wood.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to the application of knocking out the m6A demethylase PagALKBH10A / B gene in promoting poplar woodification. Background Technology

[0002] Wood is an essential raw material for energy, papermaking, and construction, and is also an important renewable resource worldwide. The formation of wood is a highly ordered developmental process. Its structure is mainly composed of cellulose microfibrils as the framework, with hemicellulose and lignin as binding and filling components. It participates in the formation of plant secondary cell walls. The accumulation and ratio of lignin and hemicellulose affect the degree of lignification of wood, which in turn affects the mechanical properties of wood such as hardness, compressive strength, and bending strength.

[0003] Poplar (Populus spp.) is the most widely planted fast-growing tree species in the world due to its rapid growth, strong adaptability, wide distribution, high survival rate after afforestation, and short rotation cycle. However, in its development and utilization, although poplar grows rapidly and has a short maturity cycle, its generally low lignin and hemicellulose content results in a low degree of lignification in its stems, which limits the application scenarios of poplar timber and greatly restricts the economic value and industrial application scope of poplar plantations. Furthermore, my country currently faces significant deficiencies in the reserves and utilization of various timber types, with a high dependence on imported timber and a current situation of timber shortage.

[0004] In summary, discovering key genes that can regulate the synthesis of prostaglandins and hemicellulose in poplar trees and the lignification process, and using certain molecular techniques to directionally improve the wood composition and mechanical properties, and to cultivate high-quality new poplar varieties, is a research direction that meets practical application needs. It is of great significance and value for solving the current problem of wood shortage, developing diversified wood resources, expanding the application fields of poplar wood, and improving the economic benefits of artificial poplar forest industry. Summary of the Invention

[0005] To overcome the defects and shortcomings of the existing technology, this invention aims to provide the application of knocking out the m6A demethylase PagALKBH10A / B gene in promoting poplar woodification.

[0006] The first objective of this invention is to provide a method for knocking down poplar trees. ALKBH10A Genes and ALKBH10B The application of genes in promoting poplar lignification, as described ALKBH10A The gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO.2. ALKBH10B The gene is a gene that encodes a protein with an amino acid sequence as shown in SEQ ID NO.4.

[0007] Preferably, the aforementioned ALKBH10A The nucleotide sequence of the gene is shown in SEQ ID NO.1. ALKBH10B The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0008] Preferably, the promotion of poplar woodlization is to increase the synthesis of lignin and hemicellulose in the poplar stem.

[0009] Preferably, the poplar trees to be knocked out ALKBH10A Genes and ALKBH10B The gene was knocked out of poplar trees using CRISPR / Cas9 multi-gene editing technology. ALKBH10A Genes and ALKBH10B Gene.

[0010] Preferably, the knockout vector used in the CRISPR / Cas9 multi-gene editing technology targets... ALKBH10A The nucleotide sequence of the gene-specific target is shown in SEQ ID NO. 5 or SEQ ID NO. 6; the knockout vector used in the CRISPR / Cas9 multi-gene editing technology targets... ALKBH10B The nucleotide sequences of the gene-specific target sites are shown in SEQ ID NO.7 or SEQ ID NO.8.

[0011] A second objective of this invention is a method for obtaining poplar trees with a high degree of lignification, comprising knocking out [the fibrous tissue] from the poplar tree. ALKBH10A Genes and ALKBH10B The steps of gene generation, as described ALKBH10A The gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO. 2. ALKBH10B The gene is a gene that encodes a protein with an amino acid sequence as shown in SEQ ID NO.4.

[0012] Preferably, the aforementioned ALKBH10A The nucleotide sequence of the gene is shown in SEQ ID NO.1. ALKBH10B The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0013] Preferably, the poplar trees that are knocked out ALKBH10A Genes and ALKBH10B Genes were knocked out using CRISPR / Cas9 multi-gene editing technology. ALKBH10A Genes and ALKBH10B Gene.

[0014] Preferably, the knockout vector used in the CRISPR / Cas9 multi-gene editing technology targets... ALKBH10AThe nucleotide sequence of the gene-specific target is shown in SEQ ID NO. 5 or SEQ ID NO. 6; the knockout vector used in the CRISPR / Cas9 multi-gene editing technology targets... ALKBH10B The nucleotide sequences of the gene-specific target sites are shown in SEQ ID NO.7 or SEQ ID NO.8.

[0015] The beneficial effects of this invention are: This invention removes the poplar tree's... ALKBH10A Genes and ALKBH10B The resulting mutants of the gene exhibited increased diameter at root, elevated expression levels of genes involved in lignin and hemicellulose synthesis, promoting lignin and hemicellulose synthesis, increasing the lignin and hemicellulose content in the stem, and thus promoting the lignification of the poplar stem. This indicates that the gene in poplar... ALKBH10A Genes and ALKBH10B Genes can serve as molecular targets for the genetic improvement of poplar trees, by knocking out genes in poplar trees. ALKBH10A Genes and ALKBH10B Genetics can be used to cultivate poplar varieties with high lignification, which is of great significance and value for solving the current timber shortage problem, developing diversified timber resources, expanding the application fields of poplar timber, and improving the economic benefits of artificial poplar forest industry. Attached Figure Description

[0016] Figure 1 It is a poplar tree ALKBH10A and ALKBH10B A schematic diagram of the gene editing vector construction (A) and the mutation status of the mutant AlkB (B).

[0017] Figure 2 This study analyzed the phenotypic characteristics of poplar main stems after 4 months of soil culture. A represents the phenotypic observation of the poplar main stems (scale bar: 5 cm); B represents the statistical results of the fresh weight of the poplar main stems (scale bar: 5 cm); and C represents the statistical results of the poplar plant height and diameter at ground level. Data in B and C are the mean (±SD) of five biological replicates. ** indicates that the same index differed significantly between the two poplar lines (p<0.05).

[0018] Figure 3 This is a section analysis of poplar main stem sections after 4 months of soil culture; where A is a safranin-fast green staining image of poplar main stem sections, with scale bars of 200 μm and 100 μm for the upper and lower side views, respectively; B is the statistical data of poplar main stem sections, with data representing the average of five biological replicates (±SD). ** indicates that the same index differs significantly between the two poplar lines (p<0.05).

[0019] Figure 4This study is a phenotypic analysis of the main stems of poplar trees after 6 months of soil culture. Among them, A is the phenotypic observation of the main stems of poplar trees after 6 months of soil culture, with scale bars of 5 cm and 1 cm for the upper and lower side views, respectively. B is the statistical analysis of the diameter of different regions of the main stems of poplar trees after 6 months of soil culture. The data are the average (±SD) of three biological replicates.

[0020] Figure 5 The images show observations of xylem vessel sections after 3 months of soil culture. In the images, A is a scanning electron microscope image of xylem vessels of the WT and AlkB lines at different scales, with scale bars of 100 μm, 50 μm, and 20 μm on the left, middle, and right sides, respectively. B is an image of toluidine blue staining results of xylem vessels of the WT and AlkB lines, with a scale bar of 200 μm.

[0021] Figure 6 This section presents PCA analysis of the AlkB and WT transcriptome results and expression pattern analysis of differentially expressed genes; where A is the PCA analysis of the AlkB and WT transcriptome results, B is the heatmap analysis of differentially expressed genes, and C is a bar chart classifying differentially expressed genes in the AlkB and WT transcriptomes according to their expression status.

[0022] Figure 7 The figures show the results of GO enrichment analysis (A) and KEGG metabolic pathway enrichment analysis (B) of differentially expressed genes in the AlkB and WT transcriptomes.

[0023] Figure 8 This represents the change in the expression levels of lignin synthesis pathway-related genes in AlkB relative to WT. Among them, A is a schematic diagram of the lignin synthesis pathway drawn based on the results of transcriptomics and quantitative PCR; B is a heatmap made based on the expression level information of lignin synthesis pathway-related genes in the transcriptomics results; the numbers on the right are the gene numbers assigned by the reference transcriptomics; and C is the verification of the changes in the expression abundance of lignin synthesis-related genes with high expression levels by quantitative PCR.

[0024] Figure 9 This represents the changes in the expression levels of genes related to the hemicellulose synthesis pathway; where A is a simulation diagram of the hemicellulose chain, B is a heatmap created based on the expression level information of genes related to the hemicellulose synthesis pathway from the transcriptome results, the numbers on the right are the gene numbers assigned by the reference transcriptome, and C is the verification of the changes in the expression abundance of hemicellulose synthesis-related genes with high expression levels by real-time PCR.

[0025] Figure 10 This is a comparison of the acid-soluble lignin, acid-insoluble lignin, and total lignin content in AlkB and WT.

[0026] Figure 11This is an infrared spectral analysis result of lignin (A) and hemicellulose (B) in the stems of AlkB and WT plants.

[0027] Figure 12 It is the hemicellulose in the stems of AlkB and WT plants. 1 H NMR spectrum.

[0028] Figure 13 The results are the analysis results of AlkB and WT stem cell wall cellulose crystallinity (A) and saccharification efficiency (B). Detailed Implementation

[0029] The technical solution of the present invention will be clearly described below using 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.

[0030] The wild-type poplar used in the following examples is 84K poplar ( Populus alba x Populus glandulosa (hereinafter referred to as 84K, WT or wild type), which is propagated and preserved in the laboratory by tissue culture.

[0031] Example 1: Obtaining gene-edited plants Simultaneous knockout of poplar trees using CRISPR / Cas9 multi-gene editing vector ALKBH10A (The nucleotide sequence of this gene is shown in SEQ ID NO.1, encoding the protein shown in SEQ ID NO.2) and ALKBH10B (The nucleotide sequence of this gene is shown in SEQ ID NO.3, encoding the protein shown in SEQ ID NO.4.) To improve editing efficiency, targeting... ALKBH10A and ALKBH10B The CDS front end is designed with two target points, which are overlapped to target different promoters and target points (target 1 and target 2). ALKBH10A The target sites, with nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6 respectively; target 3 and target 4 are the target sites. ALKBH10B The target sites (nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively) and gRNA were linked to form expression cassettes. Then, using a golden gate ligation method, the four expression cassettes were assembled onto a binary vector to complete the construction of the multi-gene editing vector. Figure 1 (A in the middle).

[0032] The constructed gene-editing vector was transformed into Agrobacterium GV3101, and a poplar plant was obtained through Agrobacterium-mediated genetic transformation. ALKBH10A and ALKBH10BThe homozygous double-mutant poplar trees with both genes knocked out were edited and named AlkB. After comparison with the WT (84K) genome, the mutation status of the AlkB gene-edited plants is as follows: Figure 1 As shown in B, where for ALKBH10A The first target (target 1) designed did not work. ALKBH10A Insertion of A and T bases occurred on the two DNA strands at the second target site (target 2); ALKBH10B Both targets were effective. ALKBH10B On one DNA strand, an A base insertion (first target site) and a GG base deletion (second target site) occurred at two target sites, while on the other DNA strand, a 406 bp fragment was cleaved between the two target sites.

[0033] Example 2: Phenotypic analysis of WT and AlkB plants Poplar strains (WT and AlkB plants) were propagated through plant tissue culture. Poplar plants with complete root systems, uniform growth, and a height of about 8 cm were selected. After hardening off for 5 days, they were transplanted into the soil for cultivation. Physiological data of the poplar plants were collected at 4 months and 6 months after transplanting.

[0034] Plant height measurement: Measure the height of the plant from the soil surface to the terminal bud; Ground diameter measurement: Use vernier calipers to measure the diameter 5 cm above the soil surface.

[0035] (1) Phenotypic analysis of poplar trees after 4 months of soil culture Experimental results showed that after 4 months of cultivation in soil, the stems of AlkB plants were thicker than those of WT plants. Figure 2 (A) Statistical results of the fresh weight of poplar main stem showed that the dry weight and fresh weight of AlkB plant stems were significantly higher than those of WT, indicating a higher biomass ( Figure 2 (B in the text). Four months after planting in the soil, the statistical results of the plant height and diameter at ground level of the poplar seedlings showed that the plant height of AlkB was not significantly different from that of WT, but the diameter at ground level of AlkB was significantly higher than that of WT. Figure 2 (C in the text). Overall, the rate of radial stem development in AlkB was significantly better than that in WT.

[0036] To further analyze the differences in stem development between AlkB and WT, histological sections of AlkB and WT stems were prepared and stained with safranin and fast green. Safranin stained highly lignified plant cells red, while fast green stained fibrous cells containing sapstone green. Safranin-fast green stained sections ( Figure 3Observations in section A) show that AlkB has a wider xylem than WT, and a larger area of ​​the xylem is stained red with safranin, indicating a higher degree of lignification in its xylem. Observations of the vascular cambium between the phloem and xylem show (…). Figure 3 In the A section, AlkB cambium cells have more layers than WT. Statistical analysis of slice data using ImageJ showed that the width of the xylem and highly lignified xylem in AlkB plants was significantly greater than that in wild WT, and the width of its vascular cambium was also significantly greater than that in WT. Figure 3 (B in the text). The more active vascular cambium may be the reason why AlkB has a better radial growth capacity than WT. The higher degree of lignification in the xylem of AlkB indicates that there is more lignin synthesis and deposition in the plant.

[0037] (2) Phenotypic analysis of poplar trees after 6 months of soil culture After 6 months of cultivation in soil, both WT and AlkB plants reached approximately two meters in height, with no significant difference in plant height between the two. However, the stem diameter of AlkB was still wider than that of WT. Figure 4 (A) The main stem of the plant was divided into ten equal-length stem segments, each approximately 10 cm long, and the diameters of eleven nodes were compared from bottom to top. The results showed that the diameter of AlkB stem was generally higher than that of WT ( Figure 4 The B in the figure further confirms that AlkB has superior radial growth capability.

[0038] The above results indicate that, under the same culture conditions and for the same culture time, the AlkB line has a wider xylem than the WT line; and the AlkB plant has a higher biomass accumulation than the WT line.

[0039] (3) The xylem vessel density of AlkB is higher than that of the wild type. Furthermore, histological sections of the stems of AlkB and WT were prepared and stained with toluidine blue. The differences in xylem vessels between AlkB and WT were compared by scanning electron microscopy and optical microscopy after toluidine blue staining. Scanning electron microscopy showed that AlkB had more xylem vessels per unit area than WT at different scales. Figure 5 (A) After toluidine blue staining, the xylem was observed on a larger scale using an optical microscope, revealing that the xylem vessel density of AlkB was significantly higher than that of the wild type (A). Figure 5 (B in the middle).

[0040] Example 3: RNA-seq analysis of AlkB and WT To further investigate the reasons for the differences in lignification between AlkB and WT at the molecular level, WT and AlkB plants were propagated through plant tissue culture. Poplar plants with complete root systems, uniform growth, and a height of approximately 8 cm were selected. After 5 days of hardening off, they were transplanted into soil and cultured for 3 months. Stem samples were then taken for transcriptome sequencing (RNA-seq) to detect differences in gene expression. Three biological replicates were designed for both AlkB and WT samples: ALK_ND1-ALK_ND3 and WT_ND1-WT_ND3.

[0041] 1. Transcriptome sequencing showed that AlkB transcripts were generally upregulated compared to WT. Principal component analysis (PCA) cluster plots showed that the three replicates from the two groups were distributed in two distinct regions, indicating good reproducibility among biological replicates. Figure 6 (A in the original text). Using fold change (FC) ≥2 and p-value <0.05 as the threshold criteria for screening differentially expressed genes, the results showed that a total of 1025 differentially expressed genes existed. Heatmap analysis of differentially expressed genes showed that the expression of differentially expressed genes in AlkB generally showed an upregulated trend compared to WT. Figure 6 (B in the text). A bar chart showing differentially expressed genes categorized according to their expression status is provided. Figure 6 In the C group, AlkB showed upregulation in 705 genes and downregulation in 320 genes compared to WT. This indicates that the poplar RNA m6A demethylase gene... ALKBH10A and ALKBH10B The absence of this component generally led to an upregulation of transcriptional levels in gene-edited plants.

[0042] 2. Enrichment of differentially expressed genes in the KEGG and GO databases The GO database primarily displays the characteristics and tendencies of genes in molecular function, biological processes, and cellular composition, helping to understand the specific biological processes in which genes participate. The KEGG database focuses on the metabolic and signal transduction pathways in which genes participate, showing the synergistic effects and regulatory relationships of genes in overall cellular biochemical reactions and physiological processes.

[0043] Differentially expressed genes were enriched in the GO and KEGG databases to explore their main functions. GO enrichment analysis showed that differentially expressed genes were mainly enriched in the cell cycle, cell wall synthesis, and xylan synthesis modules. Figure 7The result (A) is consistent with the previous phenotypic analysis showing increased vascular cambium thickness and enhanced stem lignification. KEGG metabolic pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the phenylpropanone metabolic pathway (…). Figure 7 The results of the B), GO enrichment analysis and KEGG metabolic pathway enrichment analysis were consistent with the results of the previous phenotypic analysis showing increased vascular cambium thickness and enhanced stem lignification. It is speculated that the differentially expressed genes upregulated in AlkB are mainly involved in the synthesis of xylem cell walls, mainly involving the synthesis regulation of lignin and hemicellulose, which led to the phenotype of xylem widening and increased lignification observed in AlkB stem sections.

[0044] 3. Changes in the AlkB lignin synthesis pathway Vascular plants maintain the mechanical strength of their cell walls and impart hydrophobicity to their vascular systems for water conduction through lignin synthesis. Lignin is the main polymer of the cell wall, primarily formed by the oxidative polymerization of three lignin precursors: p-coumarol, coniferyl alcohol, and sinigrin. These lignin precursors cross-link in the cell wall to produce p-hydroxyphenyl (H), guaiacol (G), and eugenol (S) lignin monomers, respectively. In gymnosperms, lignin is mainly composed of G-type lignin, while in angiosperms, it is mainly composed of both G- and S-type lignin. Gymnosperms and dicotyledons contain only small amounts of H-type lignin, while monocotyledons have a higher H-type lignin content than the former two. Three P450 enzymes are involved in lignin monomer synthesis: C4H, C3H, and F5H. C4H catalyzes the 4-position hydroxylation of the benzene ring in cinnamic acid to produce p-coumaric acid. Cinnamic acid is the first aromatic compound in the phenylpropane pathway, while p-coumaric acid is a common precursor for all phenolic metabolites. C3H hydroxylates the benzene ring at the 3-position of the coumaroyl ester derivative, transferring the phenylpropane precursor to the synthetic pathways of G- and S-type lignin monomers. F5H, as a key branching enzyme, hydroxylates the G-type lignin precursor at the C-5 position, leading to the formation of S-type lignin monomers in angiosperms. The lignin synthetic pathway can be found in [reference needed]. Figure 8 A.

[0045] Based on the results of differentially expressed genes enriched in the phenylpropanone metabolic pathway through transcriptional sequencing, genes related to the lignin synthesis pathway (such as...) were extracted from the omics data. PLA , F5H , 4CL , HCT , C4H , CCoAOMT , COMT , CCR , CAD The expression levels of related genes were analyzed and a heatmap was created. The results showed that, compared to WT, AlkB gene-edited plants exhibited an overall upregulation trend in genes along the lignin synthesis pathway. Figure 8 (B in the original text). The expression of the most abundant lignin synthesis gene was verified using quantitative real-time PCR, and the results were compared with those obtained from transcriptome data. Figure 1 As a result, the expression levels of genes related to the AlkB lignin synthesis pathway were generally upregulated. Figure 8 (C in the original text). A schematic diagram of the lignin synthesis pathway was drawn based on the results of transcriptomics and quantitative PCR. Figure 8 The A in the figure clearly shows that AlkB is upregulated compared to WT for genes related to the lignin synthesis pathway.

[0046] 4. Changes in the AlkB hemicellulose synthesis pathway Hemicellulose is an important component of plant cell walls, maintaining their integrity and flexibility through interactions with other polymers within the cell wall. Its structure varies among plant species, including configurations such as xylan, xyloglucan, mannan, glucomannan, and β-1,3 / β-1,4 glucan. In angiosperms, the main type of hemicellulose in the secondary cell wall is xylan (e.g., ...). Figure 9 As shown in A), the study indicates that... IRX9 / IRX9L , IRX10 / IRX10L , IRX14 / IRX14L It participated in the synthesis of the xylan backbone. PARVUS , IRX8 , FRA8 and FRA9 It participates in the synthesis of the tetrasaccharide reduction terminal structure of the xylan backbone. Glucuronic acid substitution and methylation of glucuronic acid on the hemicellulose side chain are respectively influenced by… GUX and GXM Regulation, RWA and ESK1 It can affect the modification of acetyl groups on the xylan side chain.

[0047] Based on the results of differentially expressed genes enriched in xylan synthesis from transcriptome sequencing, xylan synthesis pathways related to main chain synthesis (such as those related to...) were extracted from the transcriptome data. IRX10 , IRX14 , IRX9 Gene-related), reduction end synthesis ( PARVUS , IRX8 , FRA8 and FRA9 ), glucuronic acid side chain modification ( GUX , GXM ) and acetyl side chain modification ( ESK1 , RWA The expression levels of genes related to xylan backbone synthesis, reduced end synthesis, glucuronic acid side chain modification, and acetyl side chain modification were analyzed and a heatmap was created. The results showed that the expression levels of genes related to xylan backbone synthesis, reduced end synthesis, glucuronic acid side chain modification, and acetyl side chain modification were all upregulated in AlkB gene-edited plants. Figure 9(B in the original text). One of the xylan synthesis genes with the highest expression abundance was selected for quantitative verification. The quantitative results (…). Figure 9 Consistent with the transcriptome heatmap, the expression levels of hemicellulose synthesis genes were generally upregulated in AlkB gene-edited plants compared to wild-type plants (C).

[0048] The synthesis pathways of hemicellulose and lignin in AlkB plants were significantly upregulated. Combined with physiological analysis showing widening of the xylem and cambium, and increased lignification of the xylem, it is speculated that the poplar RNA m6A demethylase gene... ALKBH10A and ALKBH10B The absence of this substance leads to changes in the m6A modification level of transcripts of cell wall synthesis-related genes in AlkB plants, which in turn enhances the expression of related genes by affecting transcription or transcript stability.

[0049] Example 4: Analysis of the differences in wood properties between AlkB and WT plants 1. Cell wall component analysis Plant physiology and transcriptomics analysis of AlkB and WT plants revealed significant changes in the stems of AlkB plants compared to WT plants, including a more active cambium, more lignified xylem, and denser xylem vessels. To determine the changes in stem properties between AlkB and WT plants, stem samples were taken and acid-hydrolyzed to analyze differences in cell wall composition. Monosaccharide analysis showed that the main sugars in the stems of both AlkB and WT plants were glucose, xylose, and galacturonic acid (Table 1), representing cellulose, hemicellulose, and pectin in the stem cell walls of poplar stem cells, respectively. Unlike the transcriptomics analysis, which showed an overall upregulation of xylan synthesis-related genes in AlkB plants, monosaccharide analysis revealed a lower relative xylose content in the cell walls of AlkB gene-edited plants compared to WT (Table 1). This may be due to the faster synthesis rate of other cell wall components, diluting the relative abundance of xylose. It is worth noting that the monosaccharide analysis shows the relative content of different monosaccharides in each component of the cell wall. However, due to the enhanced radial growth of the stem, the absolute synthesis of each component of the cell wall in AlkB plants is significantly increased compared with WT, so the absolute content of xylose is increased.

[0050] Table 1. Cell wall monosaccharide analysis fructose 0.22±0.04 0.21±0.06 Rhamnose 1.88±0.44 2.28±0.07 Arabic sugar 1.05±0.13 1.13±0.09 Galactose 2.20±0.11 2.26±0.06 glucose 64.64±0.71 64.68±0.57 Xylose 19.42±0.39 18.69±0.39 Galacturonic acid 10.44±0.49 10.55±0.18 Glucuronic acid 0.15±0.06 0.20±0.03 Monosaccharide analysis of hemicellulose extracted from the stems (Table 2) showed a significant decrease in galactose and glucose content in the hemicellulose of AlkB plants, indicating a reduction in the low-content heterosaccharides in the hemicellulose of gene-edited plants. The degree of lignification of hemicellulose in AlkB stems was also higher than that in WT, and the proportion of glucuronic acid xylan in hemicellulose gradually increased, which was further supported by the increased xylose and glucuronic acid content.

[0051] Table 2 Hemicellulose Monosaccharide Analysis Rhamnose 0.71±0.27 0.46±0.20 Arabic sugar 2.52±0.07 2.66±0.18 Galactose 2.70±0.10 2.36±0.12 glucose 1.97±0.17 1.53±0.03 glucose 88.81±0.52 89.35±0.40 Glucuronic acid 3.29±0.22 3.64±0.07 Results of lignin content determination ( Figure 10 The results showed that the acid-soluble lignin content in AlkB plants was significantly lower than that in WT plants, while the content of acid-insoluble lignin was significantly higher than that in WT plants. Since acid-insoluble lignin accounted for a relatively small proportion of the total lignin content, the total lignin content of AlkB stems was still significantly higher than that in WT plants, indicating that the lignification degree of AlkB plants was significantly higher than that in WT plants.

[0052] 2. Infrared spectroscopy analysis FTIR spectroscopy can detect chemical bonds in test samples, and infrared spectroscopy can be used to analyze the compositional differences of stem cell walls in AlkB and WT plants. The scanning results of coarse stem samples from AlkB and WT plants are shown below. Figure 11 A) shows that 1735 cm -1 The signal peak at 1633 cm⁻¹ represents acetyl substitution on poplar hemicellulose. -1 The absorption peak is for water. The C=C bond of lignin in the AlkB infrared spectrum is at 1510 cm⁻¹. -1 The peak produced at this location is higher than that at WT, indicating that AlkB plants have a higher lignin content than WT. The methyl and acetyl groups of hemicellulose are located at 1381 cm⁻¹. -1 and 1248 cm -1 An absorption peak was generated at 1160 cm⁻¹. -1 The peak at 1055 cm⁻¹ is characteristic of arabinose; the glycosidic bonds of xylan are at 1055 cm⁻¹. -1 The presence of a signal indicates that xylan is the main component of poplar hemicellulose. Infrared spectroscopy analysis was performed on the hemicellulose extracted from the stems of AlkB and WT plants, and the peak diagrams (...) Figure 11 The results shown in B) indicate that the main configuration of poplar hemicellulose is xylan with acetyl substitution.

[0053] 3. Analysis of the degree of acetyl substitution in hemicellulose Infrared spectroscopy and cell wall component analysis not only showed an increase in lignin content in the stems of AlkB plants, but also indicated acetyl modification on poplar hemicellulose. Combined with transcriptome analysis showing upregulation of genes related to hemicellulose acetyl modification, this further enhanced the degree of acetyl substitution (DS) in the hemicellulose of AlkB and WT plants. AC The determination was performed. Considering the damage of alkaline extraction to the acetyl groups of xylan side chains, a mild DMSO extraction method was used to better preserve the side chain modifications of hemicellulose. The hemicellulose extracted by DMSO was scanned. 1 ¹H NMR spectroscopy was used to analyze the degree of acetyl substitution in xylan. Xylan enzymatic hydrolysis products... 1H NMR spectrum ( Figure 12 The results showed that xylose residues and acetyl groups produced signal peaks at 3.0–5.5 ppm and 2.0–2.25 ppm, respectively, with the signal intensity of AlkB acetyl groups being higher than that of WT. Based on the integrated results of xylose residue and acetyl group signals, the DS values ​​of AlkB plants and WT were calculated. AC The percentages were 4.48% and 3.45%, respectively. The increased degree of acetyl substitution in the hemicellulose of AlkB plants may have altered the cell wall structure.

[0054] 4. Lignin monomer determination By cleaving the β-O-4 bonds in lignin polymers using thioglycolysis, the released H-, G-, and S-type lignin monomers can be quantified, verifying the changes in the lignin S / G ratio in AlkB plants. The results (Table 3) show that poplar pith is mainly composed of S- and G-type lignin, containing only a very small amount of H-type lignin. The increase in the total amount of the three lignin monomers in AlkB plants indicates a higher lignin content than WT. Studies have shown that vascular cells in plants have a higher content of G-type lignin than fiber cells. The decreased lignin S / G ratio in gene-edited plants compared to wild-type plants supports the finding of increased xylem vessel density in gene-edited plants compared to wild-type plants, as shown in the sections.

[0055] Table 3. Determination of lignin monomers H 1.29±0.18 1.96±0.12 G 73.20±15.10 89.55±10.31 S 124.02±26.38 140.80±17.54 Total H+G+S 198.50±41.67 232.31±27.86 S / G ratio 1.69±0.01 1.57±0.04* 2.7 Cellulose crystallinity and saccharification efficiency XRD analysis was performed on the stem powder to analyze the changes in the crystallinity of poplar cell wall cellulose. Characteristic peaks of the cellulose I crystalline region were observed at 2θ = 16.14°, 22.56°, and 34.80°, while a valley in the amorphous region appeared at 2θ = 18.64°. Figure 13 (A in the text). Calculations showed that the cellulose crystallinity indices of AlkB and WT were 45.26% and 49.11%, respectively. Lower crystallinity makes it easier for cellulase to act on cellulose, and the reduced cellulose crystallinity of AlkB compared to WT may improve its saccharification efficiency. Further analysis of the saccharification efficiency of the stem powders from the two lines, with glucose yield measurements at 24 h, 36 h, 48 h, 60 h, and 72 h, showed (…). Figure 13 In the AlkB strain (B), the saccharification efficiency of the gene-edited plants was significantly lower than that of the WT strain at all time points. The low crystallinity of cellulose in the AlkB strain did not effectively improve its saccharification efficiency, which is presumably due to the significantly increased lignin content in the xylem of AlkB.

Claims

1. Remove poplar trees ALKBH10A Genes and ALKBH10B The application of genes in promoting poplar lignification is characterized by, The aforementioned ALKBH10A The gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO.

2. ALKBH10B The gene is a gene that encodes a protein with an amino acid sequence as shown in SEQ ID NO.

4.

2. The application according to claim 1, characterized in that, The aforementioned ALKBH10A The nucleotide sequence of the gene is shown in SEQ ID NO.

1. ALKBH10B The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. The application according to claim 1, characterized in that, The aforementioned promotion of poplar woodlization refers to increasing the synthesis of lignin and hemicellulose in poplar stems.

4. The application according to claim 1, characterized in that, The aforementioned poplar trees to be knocked out ALKBH10A Genes and ALKBH10B The gene was knocked out of poplar trees using CRISPR / Cas9 multi-gene editing technology. ALKBH10A Genes and ALKBH10B Gene.

5. The application according to claim 4, characterized in that, The CRISPR / Cas9 multi-gene editing technology uses a knockout vector targeting... ALKBH10A The nucleotide sequence of the gene-specific target is shown in SEQ ID NO. 5 or SEQ ID NO. 6; the knockout vector used in the CRISPR / Cas9 multi-gene editing technology targets... ALKBH10B The nucleotide sequence of the gene-specific target is shown in SEQ ID NO.7 or SEQ ID NO.

8.

6. A method for obtaining poplar trees with a high degree of lignification, characterized in that, Including knocking down poplar trees ALKBH10A Genes and ALKBH10B The steps of gene generation, as described ALKBH10A The gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO.

2. ALKBH10B The gene is a gene that encodes a protein with an amino acid sequence as shown in SEQ ID NO.

4.

7. The method according to claim 6, characterized in that, The aforementioned ALKBH10A The nucleotide sequence of the gene is shown in SEQ ID NO.

1. ALKBH10B The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

8. The method according to claim 6, characterized in that, The aforementioned knockout of poplar trees ALKBH10A Genes and ALKBH10B Genes were knocked out using CRISPR / Cas9 multi-gene editing technology. ALKBH10A Genes and ALKBH10B Gene.

9. The method according to claim 8, characterized in that, The CRISPR / Cas9 multi-gene editing technology uses a knockout vector targeting... ALKBH10A The nucleotide sequence of the gene-specific target is shown in SEQ ID NO. 5 or SEQ ID NO. 6; the knockout vector used in the CRISPR / Cas9 multi-gene editing technology targets... ALKBH10B The nucleotide sequence of the gene-specific target is shown in SEQ ID NO.7 or SEQ ID NO.8.

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