Use of irx10-ct protein in de novo synthesis of xylan

Abstract

The application discloses application of IRX10-CT protein in xylan de novo synthesis reaction. The amino acid sequence of the IRX10-CT protein is shown in the 25th-417th amino acid sequence. Experiments prove that the IRX10-CT protein has the function of catalyzing xylan de novo synthesis of plant cell walls, can adjust the degradable degree of the plant cell walls by changing the xylan content, helps to reduce the production cost of bioenergy, and plays a significant role in the production process of energy. The IRX10-CT protein can synthesize xylan in vitro in plant cells, provides a new processing mode for industrial production of xylan, and plays a significant role in energy saving and sustainable development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to application of IRX10-CT protein in de novo synthesis of xylan in plants in vivo and / or in vitro. BACKGROUND

[0002] Plant cell wall is a unique structure of plant cells, and plays a very important role in the growth and development of plants, such as providing mechanical support, water transport, etc. Xylan is an important component of plant cell wall, which can cross-link with cellulose and lignin and other polymers to form a complex network structure, and ultimately determines the structure and function of cell wall. Deficiency in xylan synthesis will lead to decreased plant support, slow growth and development, and even death, etc.

[0003] The xylan backbone is connected by β-1, 4-glycosidic bond of xylose. Studies have shown that IRX10 (Irregular xylem), IRX9 and IRX14 in GT47 (Glycosyltransferase) and GT43 families in Arabidopsis thaliana are involved in the synthesis of the main chain, but the synthesis mechanism is not clear.

[0004] Plant cell wall is not only the material basis for plant growth and development, but also affects all aspects of our life as food, industrial raw materials, etc. Therefore, how plants synthesize cell wall is an important scientific problem, and analyzing its synthesis mechanism and biological function has important theoretical and application value. Establishing polysaccharide synthetic biology technology is one of the important topics of contemporary biotechnology. Polysaccharide molecular design requires thorough analysis of polysaccharide biosynthesis mechanism, but this aspect of research has not made a breakthrough, and the initiation mechanism of polysaccharide synthesis is even less known. Therefore, revealing the biosynthesis mechanism of plant cell wall polysaccharide can provide a theoretical basis for artificial synthesis using synthetic biology technology, and also provide targets for molecular design and improvement of important agronomic traits such as lodging resistance of crops.

[0005] As an important plant cross-linking polysaccharide, xylan has excellent properties of renewable and low cost, and its in vitro synthesis plays an important role in human production and life. In industry, xylan is converted into ethanol by fermentation in yeast in vivo, which can be used for the production of biofuels; xylan can form furfural after dehydration, which can be used as a processing raw material for resins, pesticides, etc.; xylan has good film-forming properties and degradability, and can be used for manufacturing food packaging; xylan is hydrolyzed into xylose in dilute acid, which can be used to prepare xylitol, sweeteners, etc. In the pharmaceutical industry, xylan has the effects of anti-inflammatory and tumor inhibition; can be used as an antibacterial agent to inhibit the proliferation of Escherichia coli and Staphylococcus aureus; can be used as a health food to specifically promote the proliferation of Bifidobacterium in the human intestinal tract, promote gastrointestinal function, and also can reduce serum cholesterol, lower blood pressure, and protect the liver. Therefore, how to quickly and effectively synthesize xylan in vitro has important significance and application value. SUMMARY

[0006] The object of the present application is to provide the use of IRX10 protein or its related biomaterials.

[0007] To achieve the above object, in a first aspect, the present application provides the use of a protein or its related biomaterial in the preparation of xylan,

[0008] The protein can be A1), A2) or A3) as follows:

[0009] A1) the protein with an amino acid sequence shown in positions 25-417 of SEQ ID NO: 1 in the sequence listing or the protein with an amino acid sequence of SEQ ID NO: 1 in the sequence listing;

[0010] A2) a protein having more than 80% identity with the protein of A1) and being related to xylan synthesis, which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues to the amino acid sequence of the protein of A1);

[0011] A3) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein of A1) or A2);

[0012] The biomaterial can be any one of B1) to B7) as follows:

[0013] B1) a nucleic acid molecule encoding the protein;

[0014] B2) an expression cassette containing the nucleic acid molecule of B1);

[0015] B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2);

[0016] B4) a recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

[0017] B5) a transgenic plant cell line comprising the nucleic acid molecule of B1), or a transgenic plant cell line comprising the expression cassette of B2);

[0018] B6) a transgenic plant tissue comprising the nucleic acid molecule of B1), or a transgenic plant tissue comprising the expression cassette of B2);

[0019] B7) a transgenic plant organ comprising the nucleic acid molecule of B1), or a transgenic plant organ comprising the expression cassette of B2).

[0020] The xylans can include xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose, xyloheptaose, xylooctaose, xylo nonaose, xylo dec aose, xylo undecaose and / or xylo dodecaose.

[0021] Further, in the above-mentioned use, the protein of A2) can be a protein with an amino acid sequence as set forth in SEQ ID NO: 4, 1-393.

[0022] Further, in the above-mentioned use, the protein of A2) or A3) can be a1) or a2)

[0023] a1) a protein with an amino acid sequence as set forth in SEQ ID NO: 3;

[0024] a2) a protein with an amino acid sequence as set forth in SEQ ID NO: 4.

[0025] In the present application, SEQ ID NO: 1 consists of 417 amino acid residues; SEQ ID NO: 3 consists of 416 amino acid residues; and SEQ ID NO: 4 consists of 416 amino acid residues.

[0026] The above-mentioned protein can be artificially synthesized, or can be obtained by first synthesizing a gene encoding the protein and then performing biological expression.

[0027] The protein tag refers to a polypeptide or protein fused and expressed with a target protein by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and / or a SUMO protein tag, etc.

[0028] Table 1: Sequences of tags

[0029] Tag Residue Sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0030] To achieve the above object, in a second aspect, the present application provides the use of the above-mentioned protein as a xylose glycosyltransferase or in the preparation of an enzyme preparation having xylose glycosyltransferase activity.

[0031] To achieve the above object, in a third aspect, the present application provides a method for preparing xylan, comprising a step of catalyzing the synthesis of xylan from xylose using the above-mentioned protein.

[0032] Further, the above-mentioned method can be a method for preparing xylan in vitro from a plant.

[0033] In the above-mentioned method, the catalysis of the synthesis of xylan can occur in a cell-free system or in a non-plant cell system.

[0034] The in vitro preparation of a plant can include a cell-free system or a non-plant cell system.

[0035] The non-plant cell system can specifically include a yeast cell, an Escherichia coli cell, or other non-plant cells.

[0036] To achieve the above object, in a fourth aspect, the present application provides the use of the above-mentioned protein or its related biological material, which can be any of the following:

[0037] D1) the use of the above-mentioned protein or its related biological material in the synthesis of xylan;

[0038] D2) the use of the above-mentioned protein or its related biological material in the preparation of a product for the synthesis of xylan;

[0039] D3) the use of the above-mentioned protein or its related biological material in the regulation of the content of xylan in the cell wall of a plant;

[0040] D4) the use of the above-mentioned protein or its related biological material in the preparation of a product for regulating the content of xylan in the cell wall of a plant;

[0041] D5) the use of the above-mentioned protein or its related biological material in the regulation of the synthesis of xylan in the cell wall of a plant;

[0042] D6) the use of the above-mentioned protein or its related biological material in the preparation of a product for regulating the synthesis of xylan in the cell wall of a plant.

[0043] Further, in the above-mentioned use, B1) the nucleic acid molecule can be a DNA molecule as shown in any one of b1) to b5) below:

[0044] b1) the coding sequence of the coding strand is a DNA molecule as shown in SEQ ID No. 2 from position 73 to position 1254;

[0045] b2) the coding sequence of the coding strand is a DNA molecule as depicted in SEQ ID No. 2;

[0046] b3) the coding sequence of the coding strand is a DNA molecule as depicted in positions 73-1254 of SEQ ID No. 5;

[0047] b4) the coding sequence of the coding strand is a DNA molecule as depicted in SEQ ID No. 5;

[0048] b5) a DNA molecule having 80% or more identity to the DNA molecule as defined in b1), b2), b3) or b4) and encoding a protein as defined in claim 1.

[0049] In the present application, the identity refers to the identity of the amino acid sequence or the nucleotide sequence. The identity of the amino acid sequence can be determined using the homology search site on the internet, such as the BLAST page of the NCBI homepage. For example, the identity (%) of one pair of amino acid sequences can be calculated by searching using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, in the advanced BLAST 2.1, and then the value of the identity (%) can be obtained.

[0050] In the present application, the identity of 80% or more can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0051] Further, in the above-mentioned use, the modulating the content of xylan in the plant cell wall can be increasing the content of xylan in the plant cell wall.

[0052] Further, in the above-mentioned use, the plant can be any one of the following:

[0053] E1) a monocotyledonous plant;

[0054] E2) a plant of the order Poales;

[0055] E3) a plant of the family Poaceae;

[0056] E4) a plant of the genus Oryza

[0057] E5) rice.

[0058] To achieve the above object, the fifth aspect of the present application provides the above-mentioned protein or its related biological material.

[0059] The present application has the following beneficial effects:

[0060] The protein IRX10-CT or protein 10D of the present application has the function of catalyzing the synthesis of plant cell wall xylan, and can be used to regulate the content of plant cell wall xylan, and can adjust the degradability of plant cell wall by changing the content of xylan, which helps to reduce the production cost of bioenergy and plays a significant role in the production process of energy.

[0061] The protein provided by the present application can synthesize xylan in vitro in plant cells, which provides a new processing method for industrial production of xylan and plays a significant role in energy saving and sustainable development. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 It is a phenotype graph of bc18 genetic material, and the scale is 12 cm.

[0063] Figure 2 It is a schematic diagram of the structure of IRX10 protein.

[0064] Figure 3 It is a graph for measuring the content of xylose in the stem cell wall of bc18 genetic material and wild type Nipponbare.

[0065] Figure 4 It is a graph for measuring the content of xylan in the stem cell wall of bc18 genetic material and wild type Nipponbare; wherein: A is a gel electrophoresis graph, and B is the quantitative result of the graph A.

[0066] Figure 5 It is a protein electrophoresis graph of the fusion protein IRX10-CT-myc-His.

[0067] Figure 6 It is the catalytic activity of the fusion protein IRX10-CT-myc-His on xylotriose detected by PACE method. 10D and 10R represent two mutant forms of IRX10-CT-myc-His protein.

[0068] Figure 7 It is the catalytic activity of the fusion protein IRX10-CT-myc-His on xylotriose detected by MALDI-TOF method.

[0069] Figure 8 It is the kinetic curve of the catalytic reaction of the fusion protein IRX10-CT-myc-His on xylotriose. The gradient concentration of xylotriose (mM) is taken as the abscissa, and the release rate of UDP (pmol min -1 ) is taken as the ordinate.

[0070] Figure 9 The catalytic activity of the fusion protein IRX10-CT-myc-His towards UDP-xylose was detected using the PACE method. 10D and 10R represent two mutant forms of the IRX10-CT-myc-His protein.

[0071] Figure 10 The catalytic activity of the fusion protein IRX10-CT-myc-His on UDP-xylose was detected by MALDI-TOF method.

[0072] Figure 11 The kinetic curve of the fusion protein IRX10-CT-myc-His catalytic reaction of UDP-xylose is shown in Figure 1. The horizontal axis is the gradient concentration of UDP-xylose (mM), and the vertical axis is the UDP release rate (fmol min -1 ) is the vertical axis. DETAILED DESCRIPTION

[0073] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0074] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0075] The japonica rice variety “Nipponbare” (WT, also known as wild-type plants) used in the following examples was purchased from the China National Rice Research Institute.

[0076] The pPICZαC vector in the following examples was purchased from Invitrogen. The vector has coding sequences of His and myc tags and expresses foreign proteins with His and myc tags.

[0077] The yeast strain X33 used in the following examples was purchased from Invitrogen.

[0078] Example 1. Obtaining a Rice Brittle Stalk Mutant and IRX10-CT Protein

[0079] 1.1. Identification of a brittle rice mutant and its IRX10 protein sequence analysis

[0080] 1.1.1 Phenotype of the rice brittle stalk mutant

[0081] Rice brittle culm 18 (abbreviated as bc18) is a spontaneous mutant of japonica rice variety "Nipponbare". Compared with the wild type rice variety "Nipponbare", the mutant bc18 has the following characteristics: (1) brittle culm (the mechanical strength of the stem is significantly reduced, and the structure of the xylem vessel is abnormal); (2) dwarf plant.

[0082] The phenotype of the above genetic material is shown in Figure 1 .

[0083] 1.1.2, IRX10 protein sequence analysis of the rice brittle culm mutant

[0084] Through map-based cloning, the target gene IRX10 and its point mutation position are determined. Sequencing shows that compared with the wild type rice plant (Nipponbare), the 2103th base in the IRX10 genomic sequence of the rice brittle culm mutant bc18 is mutated from G to A. This base mutation causes the 295th arginine in the IRX10 protein sequence to be mutated into lysine, and the rest of the amino acid sequence is the same as that of the wild type rice plant.

[0085] The protein sequence of IRX10 protein is shown in SEQ ID NO: 1 of the sequence listing, and the nucleotide sequence of IRX10 gene is shown in SEQ ID NO: 2. The structural diagram of IRX10 protein is shown in Figure 2 . SP represents the signal peptide domain, TM represents the transmembrane domain, and exostosin domain represents the main functional domain of IRX10 protein. Among them, the 1st-23rd of SEQ ID NO: 1 is SP, the 5th-24th is TM, and the 50th-345th is the main functional domain of IRX10 protein. Among them, the 1st-69th of SEQ ID NO: 2 is the coding gene of SP, the 13th-72th is the coding gene of TM, and the 148th-1035th is the coding gene of the main functional domain of IRX10 protein.

[0086] 1.2, detection of xylan content in the mutant bc18 and the wild type plant

[0087] Take the mutant bc18 and the wild type plant (japonica rice variety "Nipponbare") grown for 3 months respectively, and take at least 10 main tillers of each material, and detect the xylan content in the cell wall. The specific operation is as follows:

[0088] 1.2.1, preparation of ethanol insoluble matter

[0089] The second internode below the panicle of 3-month-old rice was selected, dried until the weight no longer changed, and crushed into powder with a tissue grinder. The uniform powder was obtained through a 200-mesh sieve. 100 mg of the powder was weighed into a 10-mL centrifuge tube, 6 mL of 70% ethanol solution was added, shaken and mixed, and stood for 1 h or more. Centrifugation was performed at 3,000 rpm for 10 min, the supernatant was discarded, and the above steps were repeated until the supernatant was colorless. 6 mL of chloroform:methanol (1:1, v / v) solution was added for washing, shaken and mixed, centrifuged at 3,000 rpm for 10 min, and the supernatant was discarded. The above steps were repeated until the supernatant was colorless. 1 mL of acetone was added and shaken uniformly, centrifuged at 3,000 rpm for 10 min, the supernatant was discarded, and repeated once and then dried in an oven to obtain the ethanol-insoluble material (AIR) of the plant cell wall. 6 mL of 0.1 M sodium acetate solution (pH 5.0) was added to the AIR powder, and placed in a 80°C water bath for heating for 20 min. After cooling, 100 μL of 50 μg / mL amylase and 5 μL of pullulanase were added, and shaken in a 37°C shaking bed for 24 h or more. The starch removal was detected by starch-iodine potassium reagent. The sample was boiled in a water bath for 10 min to inactivate the amylase, centrifuged at 3,000 rpm for 10 min, and the supernatant was discarded. 6 mL of distilled water was added for washing twice, 1 mL of acetone was added for resuspension, and dried in an oven. The starch-removed AIR powder was obtained.

[0090] 1.2.2, Monosaccharide content detection

[0091] About 2 mg of the starch-removed AIR powder was weighed, 5 replicates were taken for each sample, and 40 μg of inositol was added as an internal standard. 250 μL of 2 M trifluoroacetic acid was added, and heated at 121°C for 90 min. The sample was cooled in an ice bath, centrifuged at 12,000 rpm for 10 min, 200 μL of the supernatant was transferred to a glass tube, dried in a fume hood, washed with isopropyl alcohol, and dried. 200 μL of sodium borohydride solution (10 mg / mL, dissolved in 1 M ammonia water) was added, and reacted for 90 min. 150 μL of glacial acetic acid was added for neutralization, and then washed with acetic acid / methanol (1:9, v / v) and methanol solution and dried. 50 μL of acetic anhydride and pyridine was added, shaken and mixed, heated at 121°C for 20 min for acetylation reaction. After drying, 1 mL of ethyl acetate and 4 mL of distilled water were added for extraction, mixed, and centrifuged at 2,000 rpm for 10 min. 100 μL of the upper ethyl acetate phase was taken into a sample tube, and 200 μL of acetone was added. The cell wall monosaccharides were separated and the content was determined by gas chromatography-mass spectrometry.

[0092] The results are shown in Figure 3WT represents the wild-type plant, which contains 271.6 μg of xylose per mg of ethanol-insoluble cell wall; bc18 represents the mutant plant, which contains 190.5 μg of xylose per mg of ethanol-insoluble cell wall. The results showed that the xylose content in the cell wall of the bc18 mutant was significantly reduced.

[0093] 1.2.3. Xylan content detection

[0094] Weigh about 1 mg of AIR powder with starch removed, add 100 μL of 1M NaOH solution, and place it in a shaker at 37°C for more than 16 hours. Add 100 μL of 1M HCl solution to neutralize to pH 6.0. Centrifuge at 12,000 rpm for 10 minutes. Take 100 μL of supernatant to a 1.5 mL centrifuge tube, add 140 μL of 50 mM sodium acetate solution (pH 6.0) and 10 μL of xylanase M6 (purchased from Megazyme), and react at 37°C for 24 hours. Boil for 10 minutes to terminate the reaction. Centrifuge at 12,000 rpm for 10 minutes, take the supernatant, and obtain the oligoxylose sample.

[0095] The oligosaccharide structure was detected by polysaccharide analysis by carbohydrate gelelectrophoresis (PACE). To each 5 μL sample, add 5 μL of 8-aminonaphthalene-1,3,6-trisulfonic acid solution (0.2 M, dissolved in aqueous acetic acid (acetic acid:water = 3:17)) and 5 μL of sodium cyanoborohydride solution (1 M, dissolved in DMSO). Mix well and incubate at 37°C for 16 hours for fluorescent labeling. Freeze-dry in a vacuum freeze dryer for at least 3 hours. Add 10 μL of 6 M urea to dissolve the sample. The sample was separated by electrophoresis on a 20% polyacrylamide gel in 0.1 M Tris-boric acid (pH 8.2) buffer at 200 V for 20 minutes, followed by 1,000 V for 90 minutes. Standards were prepared as follows: 5 μL each of 0.1 M mono-, di-, tri-, tetra-, penta-, and hexasaccharides were added to 30 μL of 8-aminonaphthalene-1,3,6-trisulfonic acid and 30 μL of sodium cyanoborohydride. Labeling and electrophoretic detection were performed as described above.

[0096] See the results Figure 4 Lane Marker represents the standard xylose to xylopentaose; Lane WT represents the wild-type plant. Positional comparison with the xylan standard reveals the presence of xylose and xylobiose signals. Lane bc18 represents the mutant plant, where xylose and xylobiose signals are also detected. Quantitative comparison of the signals reveals that the xylan signal in the mutant is significantly lower than that in the wild-type.

[0097] 1.3. Acquisition of IRX10-CT protein

[0098] The DNA molecule encoding the nucleotide sequence of the coding strand shown in SEQ ID NO: 2 from 73 to 1251 is named as IRX10-CT gene, and the amino acid sequence of the protein encoded by the IRX10-CT gene is shown in SEQ ID NO: 1 from 25 to 417, and the protein shown in SEQ ID NO: 1 from 25 to 417 is named as IRX10-CT protein.

[0099] Example 2, Preparation of IRX10-CT protein

[0100] 2.1, Construction of recombinant plasmid

[0101] Step 1, Extraction of total RNA from japonica rice variety "Nipponbare" and reverse transcription into cDNA.

[0102] Step 2, PCR amplification using primer pair F1 and R1 with the cDNA extracted in step 1 as template, and gel recovery of the product.

[0103] F1: 5'-AGAGGCTGAAGCATCGAT GAATTC AGAGGTGCGGCAGG-3' (underlined is the recognition site of restriction enzyme EcoRI);

[0104] R1: 5'-TTCTGAGATGAGTTTTTGTTCTAGAAACCAAGGCTTCAGG-3' (underlined is the recognition site of restriction enzyme XbaI).

[0105] Step 3, Double digestion of pPICZαC vector with restriction enzymes EcoRI and XbaI, and recovery of the vector skeleton of about 3600 bp.

[0106] Step 4. The gel recovery product of Step 2 and the vector backbone of Step 3 were ligated using a one-step ligation system (NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid pPICZαC-IRX10-CT. According to the sequencing result, the structure of the recombinant plasmid pPICZαC-IRX10-CT was described as follows: the fragment between the EcoRI and XbaI recognition sites of the pPICZαC vector (the small fragment between the EcoRI and XbaI recognition sites of the pPICZαC vector) was replaced by a double-stranded DNA molecule with the nucleotide sequence of SEQ ID NO: 2 from 73 to 1251 (IRX10-CT gene). In the recombinant plasmid pPICZαC-IRX10-CT, the exogenous sequence IRX10-CT forms a coding gene of a fusion protein IRX10-CT-myc-His with part of the nucleotides on the vector backbone, and the recombinant plasmid pPICZαC-IRX10-CT expresses the fusion protein IRX10-CT-myc-His. The amino acid sequence of the fusion protein IRX10-CT-myc-His is shown in SEQ ID NO: 3. Among them, SEQ ID NO: 3 from 1 to 393 is IRX10-CT, from 396 to 405 is a myc tag, and from 411 to 416 is a His tag.

[0107] The aspartic acid at positions 287 and 288 of SEQ ID NO: 3 was mutated to asparagine, as a mutant protein of IRX10-CT-myc-His, named 10D; the nucleotide sequence of the coding gene of the mutant protein 10D consists of 1179 nucleotides, which is only different from SEQ ID NO: 2 from 73 to 1251 in the sequence table in that the base at position 931 of SEQ ID NO: 2 in the sequence table is mutated to A, and the base at position 934 is mutated to A. The coding gene of 10D is a DNA molecule with a nucleotide sequence shown in SEQ ID NO: 5 from 73 to 1254.

[0108] The arginine at position 271 of SEQ ID NO: 3 was mutated to lysine, as a mutant protein of IRX10-CT-myc-His, named 10R; the nucleotide sequence of the coding gene of the mutant protein 10R consists of 1179 nucleotides, which is only different from SEQ ID NO: 2 from 73 to 1251 in the sequence table in that the base at position 884 of SEQ ID NO: 2 in the sequence table is mutated to A.

[0109] The recombinant plasmid pPICZαC-IRX10-CT was constructed by replacing the IRX10-CT gene with the 10D or 10R gene, respectively, to generate the recombinant plasmids pPICZαC-10D and pPICZαC-10R. The recombinant plasmid pPICZαC-10D expresses the 10D-myc-His fusion protein, the amino acid sequence of which is shown in Sequence Number 4 in the sequence listing; the recombinant plasmid pPICZαC-10R expresses the 10R-myc-His fusion protein.

[0110] 2.2 Preparation of recombinant bacteria

[0111] The recombinant plasmid pPICZαC-IRX10-CT and the mutant forms pPICZαC-10D and pPICZαC-10R were electroporated into Pichia pastoris strain X33 (Invitrogen) to obtain recombinant strains X33 / pPICZαC-IRX10-CT, X33 / pPICZαC-10D, and X33 / pPICZαC-10R. The specific procedures are as follows:

[0112] 2.2.1 Preparation of competent yeast cells

[0113] Pichia pastoris strain X33 was streaked onto a YPDS plate and cultured at 30°C for two days. A single colony was picked and activated using YPD medium. The colony was transferred to 100 mL of YPD medium and cultured at 30°C overnight until the bacterial concentration reached OD 600nm The value is 1.3-1.5. Centrifuge at 1,500 rpm and 4°C for 5 minutes, discard the supernatant, wash twice with 100 mL of pre-cooled sterile distilled water, centrifuge and discard the supernatant, resuspend the bacteria with 4 mL of pre-cooled 1 M sorbitol, centrifuge and discard the supernatant, and finally resuspend with 200 μL of pre-cooled 1 M sorbitol.

[0114] 2.2.2 Electroporation of Competent Yeast Cells

[0115] 5 μg of pPICZαC-IRX10-CT and mutant forms pPICZαC-10D and pPICZαC-10R recombinant plasmids were added to the restriction endonuclease Pme I, incubated at 37°C for 8 hours, and the plasmids were recovered by ethanol precipitation and dissolved in 10 μL of ultrapure water. Then, 80 μL of the above-mentioned yeast competent cells were added, transformed by electroporation, and the cells were suspended in 1 M sorbitol and incubated at 30°C for 1 hour. Finally, the cells were plated on bleomycin-resistant YPD plates and cultured at 30°C for 3 days.

[0116] 2.3 Inducible Expression and Purification of Fusion Protein IRX10-CT-myc-His

[0117] 2.3.1. Inducible expression of the fusion protein IRX10-CT-myc-His

[0118] Select the single colonies on the YPD solid plate and culture them in 25 mL of BMGY medium. Shake the culture at 30°C overnight. 600nm When the OD value is 2-6, centrifuge at 2,000 rpm for 5 minutes, discard the supernatant, collect the cells, and resuspend the cells in 100-200 mL BMMY medium to adjust the OD value. 600nm The value is 1.0. Protein expression was induced, and methanol was added every 24 hours to a final concentration of 0.5%. 1 mL of the sample was taken for detection. The protein sample was precipitated with methanol / ammonium acetate overnight, and then separated by SDS-polyacrylamide gel electrophoresis and detected by Western blotting. The strain with the highest expression level and the optimal expression time were selected for large-scale induction. The culture medium used in the experiment and the detailed operation process can be found in EasySelect. TM PichiaExpression Kit (Invitrogen).

[0119] 2.3.2 Purification of the fusion protein IRX10-CT-myc-His

[0120] use The Pure system was used to purify the fusion protein IRX10-CT-myc-His. The specific steps are as follows: centrifuge at 4,000 rpm and 4°C for 20 minutes, take the supernatant, and filter through a 0.22 μm filter membrane. The protein sample was passed through a HisTrap crude FF column equilibrated with a buffer (50 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 20 mM imidazole, 10% glycerol), and then eluted with a (0-0.5) M gradient concentration of imidazole solution. Desalting was performed with a HiTrap desalting column, and ultrafiltration and concentration were performed to obtain the fusion protein IRX10-CT-myc-His. Mutant proteins 10D-myc-His and 10R-myc-His were expressed according to the above method. The fusion protein was detected by Western Blotting using an anti-His tag antibody as a specific antibody. The results are shown as follows. Figure 5 , the target protein can be detected. IRX10 means the sample loaded is IRX10-CT-myc-His; 10D means the sample loaded is 10D-myc-His; 10R means the sample loaded is 10R-myc-His

[0121] Example 3: Application of the fusion protein IRX10-CT-myc-His in in vitro synthesis of xylan backbone

[0122] 3.1. Use of fusion protein IRX10-CT-myc-His in extension of xylan backbone

[0123] 3.1.1. Assay of activity of fusion protein IRX10-CT-myc-His on oligomeric xylose

[0124] Take 0.1 mM xylotetraose (purchased from Megazyme) as the reaction substrate, 1 mM UDP-xylose as the reaction donor, 1 mM magnesium chloride as the metal cofactor, and sodium phosphate solution (50 mM, dissolved in water, pH 6.8) as the reaction buffer, and add 5 μg of the purified fusion protein IRX10-CT-myc-His in Example 2, and catalyze the reaction at 25°C for 16 hours. Take 5 μL of the product, and analyze the enzyme activity product according to the PACE method in 1.2.3 in Example 1.

[0125] The results are shown in Figure 6 IRX10-CT protein can react the substrate xylotetraose into multiple band signals, presumably xylotetrose to xylododecaose. The activity of mutant protein 10D does not change significantly, and 10R completely loses the reaction activity. After boiling the three proteins to inactivate, only the signal of xylotetraose can be seen in the product, proving that the reaction activity is completely lost.

[0126] The above enzyme activity product is analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). Take 1 μL CMBT on the sample well, and dry until it is a white solid. Mix 2 μL of the sample with an equal volume of matrix DHB, and take 1 μL to the sample well. Use ABI 4700 Proteomics Analyzer for detection, and the laser wavelength used is 355 nm, and the frequency is 200 Hz. The obtained data is analyzed by Data Explorer V4.5.

[0127] The results are shown in Figure 7 The ion signal peaks in the product spectrum are equally spaced by 132, proving that the enzyme activity product is xylotetrose to xylodecaose, which are five oligomeric xyloses, wherein the ion signal peak of xylotetrose is m / z 920.2, the ion signal peak of xylotetrose is m / z 1052.2, the ion signal peak of xylotetrose is m / z 1184.2, the ion signal peak of xylotetrose is m / z 1316.2, the ion signal peak of xylotetrose is m / z 1448.2, and the ion signal peak of xylotetrose is m / z 1580.2.

[0128] The above experiment proves that the fusion protein IRX10-CT-myc-His has the enzyme activity of xylose glycosyltransferase.

[0129] 3.1.2. Assay of enzyme activity kinetics curve of fusion protein IRX10-CT-myc-His on xylotetraose

[0130] The enzyme activity kinetics curve of the fusion protein IRX10-CT-myc-His on xylotriose was determined by using the Glo assay kit (product of Promega). The specific steps are as follows: xylotriose was used as the substrate, and a series of concentration gradients of the substrate were set: 0 mM, 0.125 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM, 2 mM, and the enzyme activity reaction system was the same as the above step 1. Then the UDP content released by the fusion protein IRX10-CT-myc-His catalyzing different concentrations of substrates was determined by using the Glo assay kit. Finally, the concentration of xylotriose was used as the horizontal coordinate, and the release rate of UDP was used as the vertical coordinate to obtain the enzyme activity kinetics curve of the fusion protein IRX10-CT-myc-His on xylotriose. The data were analyzed by using Origin v9.1 software, and the Km value of 0.10±0.02 mM was calculated. The enzyme activity kinetics curve is shown in Figure 8 .

[0131] 3.2, Application of the fusion protein IRX10-CT-myc-His in the initial xylan backbone reaction

[0132] 3.2.1, Determination of the activity of the fusion protein IRX10-CT-myc-His on UDP-xylose

[0133] 1 mM UDP-xylose was used as the substrate, 1 mM magnesium chloride was used as the metal cofactor, 10% glycerol was used as the additive, 50 mM sodium phosphate solution (dissolved in water, pH 6.8) was used as the reaction buffer, 5 μg of the purified fusion protein IRX10-CT-myc-His was added, and the catalytic reaction was carried out at 25°C for 36 hours. 5 μL of the product was taken, and the PACE method in Example 1 was used to analyze the enzyme activity product.

[0134] The results are shown in Figure 9 According to the positions of the standard products (xylotriose to xylotriose) in the gel, it can be preliminarily determined that there are xylotriose in the product, including xylotriose, xylotriose, xylotriose and xylotriose. The activity of the mutant protein 10D did not change significantly, and 10R completely lost the reaction activity. After boiling the three proteins to inactivate, no xylotriose signal was detected in the product, and only background signal was present, proving that the reaction activity was completely lost.

[0135] The above enzyme activity product was detected according to the MALDI-TOF method in 3.1.1. The results are shown in Figure 10As shown, the ion signal peaks in the product spectrum are equally spaced by 132, proving that the enzyme active product is the seven oligomeric xyloses from xylobiose to xyllooctaose. The ion signal peak of xylobiose is m / z 524.2, the ion signal peak of xylotriose is m / z 656.2, the ion signal peak of xylotetraose is m / z 788.2, the ion signal peak of xylopentaose is m / z 920.2, the ion signal peak of xylohexaose is m / z 1052.2, the ion signal peak of xyloheptaose is m / z 1184.2, and the ion signal peak of xyllooctaose is m / z 1316.2.

[0136] 3.2.2, Determination of the enzyme activity kinetics curve of the fusion protein IRX10-CT-myc-His on UDP-xylose

[0137] The Glo assay kit was used to determine the enzyme activity kinetics curve of the fusion protein IRX10-CT-myc-His on UDP-xylose. The specific steps are as follows: UDP-xylose was used as the substrate, and a series of concentration gradients were set for the substrate: 0 mM, 0.125 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM, 2 mM, and 4 mM. The enzyme activity reaction system was synchronized with Step 1, and then the Glo assay kit was used to determine the UDP content released by the fusion protein IRX10-CT-myc-His catalyzing different concentrations of substrates. Finally, the concentration of UDP-xylose was taken as the abscissa, and the release rate of UDP was taken as the ordinate, to obtain the enzyme activity kinetics curve of the fusion protein IRX10-CT-myc-His on UDP-xylose. The data were analyzed by Origin v9.1 software, and the Km value was calculated to be 1.75 ± 0.31 mM. The enzyme activity kinetics curve is shown in Figure 11 .

[0138] The results prove that the IRX10-CT protein is relatively stable for xylan elongation activity, and the experiment has good repeatability.

[0139] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although specific examples are given in the present application, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application. SEQUENCE LISTING <110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences <120> Application of IRX10-CT protein in de novo synthesis of xylan <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 417 <212> PRT <213> Oryza sativa L. <400> 1 Met Arg Arg Trp Val Leu Ala Ile Ala Ile Leu Ala Ala Ala Val Cys 1 5 10 15 Phe Phe Leu Gly Ala Gln Ala Gln Glu Val Arg Gln Gly His Gln Thr 20 25 30 Glu Arg Ile Ser Gly Ser Ala Gly Asp Val Leu Glu Asp Asp Pro Val 35 40 45 Gly Arg Leu Lys Val Tyr Val Tyr Asp Leu Pro Ser Lys Tyr Asn Lys 50 55 60 Lys Leu Leu Lys Lys Asp Pro Arg Cys Leu Asn His Met Phe Ala Ala 65 70 75 80 Glu Ile Phe Met His Arg Phe Leu Leu Ser Ser Ala Val Arg Thr Phe 85 90 95 Asn Pro Glu Glu Ala Asp Trp Phe Tyr Thr Pro Val Tyr Thr Thr Cys 100 105 110 Asp Leu Thr Pro Ser Gly Leu Pro Leu Pro Phe Lys Ser Pro Arg Met 115 120 125 Met Arg Ser Ala lie Glu Leu lie Ala Thr Asn Trp Pro Tyr Trp Asn 130 135 140 Arg Ser Glu Gly Ala Asp His Phe Phe Val Thr Pro His Asp Phe Gly 145 150 155 160 Ala Cys Phe His Tyr Gin Gin Gin Lys Ala lie Gly Arg Gly lie Leu 165 170 175 Pro Leu Leu Gin Arg Ala Thr Leu Val Gin Thr Phe Gly Gin Lys Asn 180 185 190 His Val Cys Leu Lys Asp Gly Ser lie Thr lie Pro Pro Tyr Ala Pro 195 200 205 Pro Gin Lys Met Gin Ala His Leu lie Pro Pro Asp Thr Pro Arg Ser 210 215 220 lie Phe Val Tyr Phe Arg Gly Leu Phe Tyr Asp Thr Ser Asn Asp Pro 225 230 235 240 Glu Gly Gly Tyr Tyr Ala Arg Gly Ala Arg Ala Ser Val Trp Glu Asn 245 250 255 Phe Lys Asn Asn Pro Leu Phe Asp lie Ser Thr Asp His Pro Pro Thr 260 265 270 Tyr Tyr Gin Asp Met Gin Arg Ser Val Phe Cys Leu Cys Pro Leu Gly 275 280 285 Trp Ala Pro Trp Ser Pro Arg Leu Val Glu Ala Val Val Phe Gly Cys 290 295 300 Ile Pro Val Ile Ile Ala Asp Asp Ile Val Leu Pro Phe Ala Asp Ala 305 310 315 320 Ile Pro Trp Glu Glu Ile Gly Val Phe Val Ala Glu Glu Asp Val Pro 325 330 335 Lys Leu Asp Ser Ile Leu Thr Ser Ile Pro Thr Asp Val Ile Leu Arg 340 345 350 Lys Gln Arg Leu Leu Ala Asn Pro Ser Met Lys Gln Ala Met Leu Phe 355 360 365 Pro Gln Pro Ala Gln Ala Gly Asp Ala Phe His Gln Ile Leu Asn Gly 370 375 380 Leu Ala Arg Lys Leu Pro His Gly Glu Asn Val Phe Leu Lys Pro Gly 385 390 395 400 Glu Arg Ala Leu Asn Trp Thr Ala Gly Pro Val Gly Asp Leu Lys Pro 405 410 415 Trp <210> 2 <211> 1254 <212> DNA <213> Rice (Oryza sativa L.) <400> 2 atgaggaggt gggtcttggc cattgccatt cttgctgctg ctgtatgctt cttccttgga 60 gctcaggccc aggaggtgcg gcagggccac cagacagaga ggatctcagg aagtgctggt 120 gatgtgttgg aagatgaccc tgttgggagg cttaaggtct atgtctatga tctcccaagc 180 aagtacaaca agaagctgct gaagaaggat cctaggtgcc tgaaccacat gtttgccgct 240 gagattttca tgcatcggtt cctgttgtca agcgctgtcc gaacttttaa tcccgaggaa 300 gctgattggt tctacacacc ggtgtacact acatgcgacc tgactccctc cggtcttccc 360 ttgcccttca aatccccaag aatgatgcgc agcgcaattg agctgattgc aacaaattgg 420 ccttactgga atagatcaga gggggctgat catttctttg ttacaccaca tgactttggc 480 gcttgcttcc actatcagga agaaaaggca attggacgtg gaatcctccc attgcttcag 540 cgtgccaccc tggttcagac ctttggacaa aagaaccatg tctgcttgaa ggacggctcg 600 atcaccattc cgccatatgc acccccacag aaaatgcagg ctcatcttat tcccccagac 660 acccctcggt ctatctttgt atatttccgt ggtctgttct acgataccag caatgatcct 720 gaggtggat actatgcaag aggtgcccgc gcgtcggttt gggagaatt caagaacaac ccgctgtttg acatctcaac cgatcaccca cccacgtact acgaagatat gcagagatct gtgttctgct tgtgcccatt gggctgggct ccatggagcc ccagactggt ggaagctgtg gttttcggtt gtattccggt gtcattgca gatgacattg tcctcccctt tgctgatgct 960 atccctggg aggagattgg cgtgtttgtc gccgaggagg atgttccgaa gctggacagt atcctgacat ccataccaac agatgttatc ctgaggaagc agaggcttct cgcgaacccg tcgatgaagc aggccatgct gttcccccag cctgctcagg caggagatgc attccatcag atactgaatg gtctcgctcg caagcttcca catggcgaaa acgtcttctt gaagcccggg gagagggccc tgaactggac tgctggaccg gtgggcgacc tgaagccttg gtag 1254 <210> 3 <211> 416 <212> PRT <213> Artificial Sequence <400> 3 Glu Val Arg Gln Gly His Gln Thr Glu Arg Ile Ser Gly Ser Ala Gly 1 5 10 15 Asp Val Leu Glu Asp Asp Pro Val Gly Arg Leu Lys Val Tyr Val Tyr 20 25 30 Asp Leu Pro Ser Lys Tyr Asn Lys Lys Leu Leu Lys Lys Asp Pro Arg 35 40 45 Cys Leu Asn His Met Phe Ala Ala Glu Ile Phe Met His Arg Phe Leu 50 55 60 Leu Ser Ser Ala Val Arg Thr Phe Asn Pro Glu Glu Ala Asp Trp Phe 65 70 75 80 Tyr Thr Pro Val Tyr Thr Thr Cys Asp Leu Thr Pro Ser Gly Leu Pro 85 90 95 Leu Pro Phe Lys Ser Pro Arg Met Met Arg Ser Ala Ile Glu Leu Ile 100 105 110 Ala Thr Asn Trp Pro Tyr Trp Asn Arg Ser Glu Gly Ala Asp His Phe 115 120 125 Phe Val Thr Pro His Asp Phe Gly Ala Cys Phe His Tyr Gln Glu Glu 130 135 140 Lys Ala Ile Gly Arg Gly Ile Leu Pro Leu Leu Gln Arg Ala Thr Leu 145 150 155 160 Val Gln Thr Phe Gly Gln Lys Asn His Val Cys Leu Lys Asp Gly Ser 165 170 175 Ile Thr lie Pro Pro Tyr Ala Pro Pro Gin Lys Met Gin Ala His Leu 180 185 190 Ile Pro Pro Asp Thr Pro Arg Ser lie Phe Val Tyr Phe Arg Gly Leu 195 200 205 Phe Tyr Asp Thr Ser Asn Asp Pro Glu Gly Gly Tyr Tyr Ala Arg Gly 210 215 220 Ala Arg Ala Ser Val Trp Glu Asn Phe Lys Asn Asn Pro Leu Phe Asp 225 230 235 240 Ile Ser Thr Asp His Pro Pro Thr Tyr Tyr Glu Asp Met Gin Arg Ser 245 250 255 Val Phe Cys Leu Cys Pro Leu Gly Trp Ala Pro Trp Ser Pro Arg Leu 260 265 270 Val Glu Ala Val Val Phe Gly Cys Ile Pro Val Ile lie Ala Asp Asp 275 280 285 Ile Val Leu Pro Phe Ala Asp Ala Ile Pro Trp Glu Glu Ile Gly Val 290 295 300 Phe Val Ala Glu Glu Asp Val Pro Lys Leu Asp Ser Ile Leu Thr Ser 305 310 315 320 Ile Pro Thr Asp Val Ile Leu Arg Lys Gin Arg Leu Leu Ala Asn Pro 325 330 335 Ser Met Lys Gin Ala Met Leu Phe Pro Gin Pro Ala Gin Ala Gly Asp 340 345 350 Ala Phe His Gin He Leu Asn Gly Leu Ala Arg Lys Leu Pro His Gly 355 360 365 Glu Asn Val Phe Leu Lys Pro Gly Glu Arg Ala Leu Asn Trp Thr Ala 370 375 380 Gly Pro Val Gly Asp Leu Lys Pro Trp Ser Lys Glu Gin Lys Leu He 385 390 395 400 Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His His 405 410 415 <210> 4 <211> 416 <212> PRT <213> Artificial Sequence <400> 4 Glu Val Arg Gin Gly His Gin Thr Glu Arg He Ser Gly Ser Ala Gly 1 5 10 15 Asp Val Leu Glu Asp Asp Pro Val Gly Arg Leu Lys Val Tyr Val Tyr 20 25 30 Asp Leu Pro Ser Lys Tyr Asn Lys Lys Leu Leu Lys Lys Asp Pro Arg 35 40 45 Cys Leu Asn His Met Phe Ala Ala Glu He Phe Met His Arg Phe Leu 50 55 60 Leu Ser Ser Ala Val Arg Thr Phe Asn Pro Glu Glu Ala Asp Trp Phe 65 70 75 80 Tyr Thr Pro Val Tyr Thr Thr Cys Asp Leu Thr Pro Ser Gly Leu Pro 85 90 95 Leu Pro Phe Lys Ser Pro Arg Met Met Arg Ser Ala Ile Glu Leu Ile 100 105 110 Ala Thr Asn Trp Pro Tyr Trp Asn Arg Ser Glu Gly Ala Asp His Phe 115 120 125 Phe Val Thr Pro His Asp Phe Gly Ala Cys Phe His Tyr Gln Glu Glu 130 135 140 Lys Ala Ile Gly Arg Gly Ile Leu Pro Leu Leu Gln Arg Ala Thr Leu 145 150 155 160 Val Gln Thr Phe Gly Gln Lys Asn His Val Cys Leu Lys Asp Gly Ser 165 170 175 Ile Thr Ile Pro Pro Tyr Ala Pro Pro Gln Lys Met Gln Ala His Leu 180 185 190 Ile Pro Pro Asp Thr Pro Arg Ser Ile Phe Val Tyr Phe Arg Gly Leu 195 200 205 Phe Tyr Asp Thr Ser Asn Asp Pro Glu Gly Gly Tyr Tyr Ala Arg Gly 210 215 220 Ala Arg Ala Ser Val Trp Glu Asn Phe Lys Asn Asn Pro Leu Phe Asp 225 230 235 240 Ile Ser Thr Asp His Pro Pro Thr Tyr Tyr Glu Asp Met Gln Arg Ser 245 250 255 Val Phe Cys Leu Cys Pro Leu Gly Trp Ala Pro Trp Ser Pro Arg Leu 260 265 270 Val Glu Ala Val Val Phe Gly Cys Ile Pro Val Ile Ile Ala Asn Asn 275 280 285 Ile Val Leu Pro Phe Ala Asp Ala Ile Pro Trp Glu Glu Ile Gly Val 290 295 300 Phe Val Ala Glu Glu Asp Val Pro Lys Leu Asp Ser Ile Leu Thr Ser 305 310 315 320 Ile Pro Thr Asp Val Ile Leu Arg Lys Gln Arg Leu Leu Ala Asn Pro 325 330 335 Ser Met Lys Gln Ala Met Leu Phe Pro Gln Pro Ala Gln Ala Gly Asp 340 345 350 Ala Phe His Gln Ile Leu Asn Gly Leu Ala Arg Lys Leu Pro His Gly 355 360 365 Glu Asn Val Phe Leu Lys Pro Gly Glu Arg Ala Leu Asn Trp Thr Ala 370 375 380 Gly Pro Val Gly Asp Leu Lys Pro Trp Ser Lys Glu Gln Lys Leu Ile 385 390 395 400 Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His His 405 410 415 <210> 5 <211> 1254 <212> DNA <213> Artificial Sequence <400> 5 atgaggaggt gggtcttggc cattgccatt cttgctgctg ctgtatgctt cttccttgga 60 gctcaggccc aggaggtgcg gcagggccac cagacagaga ggatctcagg aagtgctggt 120 gatgtgttgg aagatgaccc tgttgggagg cttaaggtct atgtctatga tctcccaagc 180 aagtacaaca agaagctgct gaagaaggat cctaggtgcc tgaaccacat gtttgccgct 240 gagattttca tgcatcggtt cctgttgtca agcgctgtcc gaacttttaa tcccgaggaa 300 gctgattggt tctacacacc ggtgtacact acatgcgacc tgactccctc cggtcttccc 360 ttgcccttca aatccccaag aatgatgcgc agcgcaattg agctgattgc aacaaattgg 420 ccttactgga atagatcaga gggggctgat catttctttg ttacaccaca tgactttggc 480 gcttgcttcc actatcagga agaaaaggca attggacgtg gaatcctccc attgcttcag 540 cgtgccaccc tggttcagac ctttggacaa aagaaccatg tctgcttgaa ggacggctcg 600 atcaccattc cgccatatgc acccccacag aaaatgcagg ctcatcttat tcccccagac 660 acccctcggt ctatctttgt atatttccgt ggtctgttct acgataccag caatgatcct 720 gagggtggat actatgcaag aggtgcccgc gcgtcggttt gggagaattt caagaacaac 780 ccgctgtttg acatctcaac cgatcaccca cccacgtact acgaagatat gcagagatct 840 gtgttctgct tgtgcccatt gggctgggct ccatggagcc ccagactggt ggaagctgtg 900 gttttcggtt gtattccggt gatcattgca aataacattg tcctcccctt tgctgatgct 960 atcccctggg aggagattgg cgtgtttgtc gccgaggagg atgttccgaa gctggacagt 1020 atcctgacat ccataccaac agatgttatc ctgaggaagc agaggcttct cgcgaacccg 1080 tcgatgaagc aggccatgct gttcccccag cctgctcagg caggagatgc attccatcag 1140 atactgaatg gtctcgctcg caagcttcca catggcgaaa acgtcttctt gaagcccggg 1200 gagagggccc tgaactggac tgctggaccg gtgggcgacc tgaagccttg gtag 1254

Claims

1. The use of protein in the preparation of xylan, characterized by: The protein is a1) or a2), a1) a protein whose amino acid sequence is shown in Sequence 3 in the sequence listing; a2) a protein whose amino acid sequence is shown in Sequence 4 in the sequence listing; The preparation of xylan is to prepare xylan in vitro using UDP-xylose as a substrate.

2. Use of a biomaterial related to the protein of claim 1 in the preparation of xylan, wherein the preparation of xylan is in vitro preparation of xylan using UDP-xylose as a substrate, wherein the biomaterial is any one of the following B1) to B7): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2); B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2).

3. Use of the protein according to claim 1 as a xylose glycosyltransferase or in the preparation of an enzyme preparation, wherein the xylose glycosyltransferase or enzyme preparation uses UDP-xylose as a substrate.

4. A method for preparing xylan, characterized in that: The method comprises the step of using the protein according to claim 1 to catalyze the synthesis of xylan from UDP-xylose.

Images