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WO2018130060A1 - Application de la protéine bs1-ct dans la régulation de la désacétylation du xylane dans une paroi cellulaire végétale - Google Patents

Application de la protéine bs1-ct dans la régulation de la désacétylation du xylane dans une paroi cellulaire végétale Download PDF

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WO2018130060A1
WO2018130060A1 PCT/CN2017/117896 CN2017117896W WO2018130060A1 WO 2018130060 A1 WO2018130060 A1 WO 2018130060A1 CN 2017117896 W CN2017117896 W CN 2017117896W WO 2018130060 A1 WO2018130060 A1 WO 2018130060A1
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protein
xylan
plant cell
cell wall
acetylation
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Chinese (zh)
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周奕华
张保才
张兰军
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中国科学院遗传与发育生物学研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the invention relates to the field of biotechnology, and particularly relates to the application of the BS1-CT protein in regulating the deacetylation reaction of plant cell wall xylan.
  • O-acetylation modification is a relatively common modification of plant cell wall polysaccharides. In addition to cellulose, beta-1,3-1,4-linked glucan and some glycoproteins, O-acetylation modifications are present on almost all other cell wall polysaccharides.
  • O-acetylation may affect the cell wall structure by affecting the physiological and biochemical properties of cell wall polysaccharides. Studies have found that acetylation affects the interaction of polysaccharides with polar molecules. The mode of acetylation modification on xylan affects the binding mode of xylan to cellulose, which in turn affects cell wall structure. The degree of O-acetylation of plant cell wall polysaccharides showed dynamic changes with plant tissues, organs and growth stages, indicating that O-acetylation modification is closely related to plant growth and development, and is strictly regulated in plants. Therefore, the regulation mechanism of plant cell wall polysaccharide O-acetylation modification is important for plants to maintain cell wall structure and normal growth.
  • a first object of the invention is to provide a protein.
  • the protein provided by the present invention is a protein of the following a) or b) or c), which is named as a BS1-CT protein:
  • amino acid sequence is the protein shown in positions 25-382 of SEQ ID NO:1;
  • the label shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in positions 25-382 of SEQ ID NO:1 in the Sequence Listing.
  • substitution and/or deletion and/or addition of the one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.
  • the protein in the above c) can be artificially synthesized, or the encoded gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding the protein in the above c) can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in positions 73-1149 of SEQ ID NO: 2, and/or performing one or several base pair errors.
  • amino acid sequence of the fusion protein in the above b) is shown in SEQ ID NO:3.
  • a second object of the invention is to provide a biological material associated with the BS1-CT protein.
  • the biomaterial related to the BS1-CT protein provided by the present invention is any one of the following A1) to A12):
  • A1 a nucleic acid molecule encoding a BS1-CT protein
  • A2) an expression cassette comprising the nucleic acid molecule of A1);
  • A3 a recombinant vector comprising the nucleic acid molecule of A1);
  • A4 a recombinant vector comprising the expression cassette of A2);
  • A5 a recombinant microorganism comprising the nucleic acid molecule of A1);
  • A6 a recombinant microorganism comprising the expression cassette of A2)
  • A7 a recombinant microorganism comprising the recombinant vector of A3);
  • A8 a recombinant microorganism comprising the recombinant vector of A4)
  • A9 a transgenic plant cell line comprising the nucleic acid molecule of A1);
  • A10 a transgenic plant cell line comprising the expression cassette of A2)
  • A11 a transgenic plant cell line comprising the recombinant vector of A3);
  • a transgenic plant cell line comprising the recombinant vector of A4).
  • nucleic acid molecule of A1) is a gene represented by 1) or 2) or 3) as follows:
  • the coding sequence thereof is a cDNA molecule or a DNA molecule as shown in SEQ ID NO: 73-1149;
  • a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by 1) or 2) and which encodes a BS1-CT protein.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • nucleotide sequence encoding BS1-CT of the present invention can readily mutate the nucleotide sequence encoding BS1-CT of the present invention using known methods, such as directed evolution and point mutation methods.
  • Those artificially modified nucleotides having a nucleotide sequence encoding 75% or more of BS1-CT, as long as they encode BS1-CT and have the same function, are derived from the nucleotide sequence of the present invention and Equivalent to the sequence of the invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, or 85% or more, or 90% or more of the nucleotide sequence of a protein consisting of the amino acid sequence shown in positions 25-382 of the coding sequence 1 of the present invention. , or a nucleotide sequence of 95% or greater identity. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • the above 75% or more of the identity may be 80%, 85%, 90% or 95% or more.
  • the stringent conditions are: hybridization in a solution of 2 ⁇ SSC, 0.1% SDS at 68 ° C and washing the membrane twice, 5 min each time, in a solution of 0.5 ⁇ SSC, 0.1% SDS, Hybridization and washing at 68 ° C for 2 times each time for 15 min; or, in a solution of 0.1 ⁇ SSPE (or 0.1 ⁇ SSC), 0.1% SDS, hybridization at 65 ° C and washing the membrane.
  • the vector may be a plasmid, a cosmid, a phage or a viral vector.
  • the microorganism may be yeast, bacteria, algae or fungi such as Agrobacterium.
  • the transgenic plant cell line, the transgenic plant tissue, and the transgenic plant organ do not include the propagation material.
  • a third object of the invention is to provide a novel use of the BS1-CT protein.
  • the present invention provides the use of a BS1-CT protein as an acetyl esterase.
  • a fourth object of the present invention is to provide a novel use of a BS1-CT protein or a biological material related to a BS1-CT protein.
  • the present invention provides the use of a BS1-CT protein or a biological material associated with a BS1-CT protein for regulating the level of acetylation of a plant cell wall xylan.
  • the invention also provides the use of a BS1-CT protein or a biological material associated with a BS1-CT protein for the preparation of a product that modulates the level of acetylation of a plant cell wall xylan.
  • the phytochemical modification level of the plant cell wall xylan is to catalyze the deacetylation reaction of the plant cell wall xylan.
  • the invention also provides the use of a BS1-CT protein or a biological material associated with a BS1-CT protein for regulating the level of acetylation of xylan.
  • the invention also provides the use of a BS1-CT protein or a biological material associated with a BS1-CT protein for the preparation of a product that modulates the level of acetylation of xylan.
  • the xylan acetylation modification level is a catalytic xylan deacetylation reaction.
  • the xylan is acetylated to the acetylation of the xylan O-2 position and/or the O-3 position.
  • a fifth object of the present invention is to provide a product for regulating the deacetylation of xylan.
  • the active ingredient of the product provided by the present invention is a fusion protein of BS1-CT protein or BS1-CT protein.
  • a final object of the invention is to provide a method of modulating xylan deacetylation.
  • the method for modulating xylan deacetylation comprises the step of treating xylan with a fusion protein of BS1-CT protein or BS1-CT protein.
  • the xylan is deacetylated to regulate the deacetylation of plant cell wall xylan
  • the xylan is acetyl xylan.
  • FIG. 1 is a schematic view showing the structure of the BS1 protein.
  • TM represents the transmembrane domain
  • GDSL represents the major domain of the BS1 protein
  • AG represents the antigenic fragment of the BS1 antiserum.
  • Figure 2 is an electropherogram of the fusion protein BS1-CT-His solution.
  • Figure 3 is a comparison of the activity of the fusion protein BS1-CT-His for different commercial acetylated monosaccharides using an acetic acid assay kit and a quadrupole rod-time-of-flight mass spectrometer (LC-QTOF-MS) method, respectively.
  • the left panel uses the five acetylated monosaccharides as the abscissa and the acetic acid release rate ( ⁇ mol min -1 mg -1 ) as the ordinate.
  • the right graph uses the mass-to-charge ratio as the abscissa and the relative abundance of acetylated xylose as the ordinate.
  • Tri-Ac-Mexyl represents triacetylmethylxylose
  • Di-Ac-Mexyl represents diacetylmethylxylose
  • Ac-Mexyl represents monoacetylmethylxylose.
  • BS1 is an experimental group to which the fusion protein BS1-CT-His is added
  • Mock is a control group to which no fusion protein BS1-CT-His is added.
  • Figure 4 is a kinetic curve of the reaction of the fusion protein BS1-CT-His to acetylated xylose as determined by an acetic acid assay kit.
  • the gradient concentration of triacetylmethylxylose (mM) was plotted on the abscissa and the acetic acid release rate ( ⁇ mol min -1 mg -1 ) was plotted on the ordinate.
  • Figure 5 is a comparison of the activity of the fusion protein BS1-CT-His on acetyl xylan extracted from rice and acetyl oligo-xylose produced by endoxylanase.
  • the left panel uses the acetyl xylan extracted from rice as the abscissa, and the amount of acetic acid released by the reaction ( ⁇ mol mg -1 acetyl xylan) as the ordinate.
  • the figure on the right shows the amount of acetic acid released by the reaction ( ⁇ mol mg -1 acetyl oligo-xylose) as the ordinate on the abscissa of acetyl oligo-xylose produced by xylan endonuclease.
  • BS1 is an experimental group to which the fusion protein BS1-CT-His is added
  • Mock is a control group to which no fusion protein BS1-CT-His is added.
  • Figure 6 shows the deacetylation activity of the fusion protein BS1-CT-His on rice acetyl oligo-xylose by LC-QTOF-MS method.
  • the relative abundance of acetyl oligo-xylose was taken as the abscissa with four acetyl oligo-xyloses (trimeric xylose DP3, tetradose xylose DP4, pentapoly xylose DP5 and hexameric xylose DP6).
  • Y-axis. - represents the control group
  • + represents the addition of the fusion protein BS1-CT-His experimental group.
  • Figure 7 is a kinetic curve of the deacetylation reaction of the fusion protein BS1-CT-His on rice oligo-xylose.
  • the gradient concentration of acetyl oligo-xylose (mg mL -1 ) was plotted on the abscissa and the acetic acid release rate ( ⁇ mol min -1 mg -1 ) was plotted on the ordinate.
  • Figure 8 is a NMR method for detecting the deacetylation activity of the fusion protein BS1-CT-His on the mutant bs1 acetyl oligosaccharide.
  • the indica variety "Golden Clear” (WT, also known as wild type plant) in the following examples was purchased from the China Rice Research Institute.
  • the vector pCAMBIA1300 in the following examples was purchased from CAMIA Corporation, Australia.
  • Agrobacterium EHA105 in the following examples was purchased from CAMIA Corporation, Australia.
  • the pDONR207 vector in the following examples was purchased from Life Technologies, Inc., Cat. No. 12213-013.
  • the pGEM-T-easy vector in the following examples was purchased from Invitrogen.
  • the pPICZ ⁇ C vector in the following examples was purchased from Invitrogen, which has a His-tagged coding sequence and expresses a His-tagged foreign protein.
  • the yeast strain X33 in the following examples was purchased from Invitrogen.
  • the deacetylation reaction in the following examples refers to a reaction in which a acetylation-modified cell wall polysaccharide is specifically deacetylated by an acetyl esterase.
  • Example 1 Obtainment of rice fragile sheath mutant and BS1-CT protein
  • the rice brittle sheath mutant brittle sheath1 (abbreviated as bs1 or mutant bs1) is a spontaneous mutant material of the indica variety "Golden Cherry". Compared with the indica variety "Golden Clear", the mutant bs1 is mainly characterized by: (1) the sheath becomes brittle (the mechanical strength of the sheath is significantly decreased, the xylem conduit structure is abnormal); and (2) the plant becomes shorter.
  • the rice fragile sheath mutant is only the second intron of the BS1 gene.
  • the first base from the 5' end is mutated from G to A, and the rest of the genome is compared with the wild type rice plant (Golden).
  • the sequence is identical to the wild type rice plants. This base mutation causes the second intron of the BS1 gene to be not normally cleaved, resulting in early termination of translation of the BS1 protein.
  • the amino acid sequence of the BS1 protein is shown in SEQ ID NO: 1 of the Sequence Listing, and the nucleotide sequence of the BS1 gene is shown in SEQ ID NO: 2 of the Sequence Listing.
  • a schematic diagram of the structure of the BS1 protein is shown in Figure 1.
  • TM represents the transmembrane domain
  • GDSL represents the major domain of the BS1 protein
  • AG represents the antigenic fragment of the BS1 antiserum.
  • Mutant bs1 and wild-type plants which were grown for 4 weeks, were randomly selected. Six plants were randomly selected from each line, mixed and powdered to detect the acetyl group on the acetyl xylan in the cell wall of the plant to be tested. content. The specific operations are as follows:
  • the plant seedlings grown for 4 weeks were lyophilized to no change in weight, ground to a powder, and sieved through a 200 mesh sieve to remove coarse particles.
  • the mixture was washed three times with a 70% aqueous solution of ethanol, and washed three times with an equal volume of a mixed chloroform-methanol mixture, and the precipitate was collected by centrifugation at 12,000 rpm for 10 minutes after each rinsing.
  • the obtained precipitate was washed with acetone and dried to obtain an alcohol-insoluble residue (AIR) which is mainly composed of a cell wall component.
  • AIR alcohol-insoluble residue
  • AIR plant stem alcohol-insoluble matter
  • the precipitate was rinsed with 5 mL of MES/Tris buffer (Tris, Sigma, 77-86-1; MES, Sigma, 1266615-59-1), and the supernatant was discarded. Then, 20 mL of MES/Tris buffer and 40 U of amylase (Megazyme, K-TDFR-100A) were added, and after reacting at 97 ° C for 35 min, the mixture was transferred to a 60 ° C water bath for 1 h to remove the starch. After centrifugation at 2500 rpm for 15 minutes, the supernatant was discarded, and the precipitate was rinsed three times with 5 mL of acetone and dried under vacuum.
  • MES/Tris buffer Tris, Sigma, 77-86-1; MES, Sigma, 1266615-59-1
  • the extraction was carried out overnight at 70 ° C with 20 mL of DMSO, and after repeating twice, the supernatant was transferred to a new tube.
  • Five volumes of ethanol were added: methanol: water (7:2:1, v/v, pH 2-3), and precipitated at 4 ° C for 3 days to give an acetyl xylan precipitate.
  • the precipitate collected by centrifugation at 2500 rpm for 15 minutes was rinsed three times with absolute ethanol and dried under vacuum to obtain an acetyl xylan component.
  • acetyl xylan 1 mg was dissolved in 100 ⁇ L of 1N NaOH, and reacted at 28 ° C, 200 rpm for 1 h. It was further neutralized by adding 100 ⁇ L of 1N HCl, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken for testing. The amount of acetic acid released by the reaction in the supernatant was determined using an acetic acid assay kit (Megazyme, K-ACET) (the acetic acid released from the reaction was derived from the acetyl group in the cell wall, representing a portion of the acetyl group involved in the reaction in the cell wall).
  • Blank control (A2-A0) - (A1-A0) (A1-A0) / (A2-A0).
  • the acetyl content (the amount of acetic acid released) on the acetyl xylan in the cell wall of the mutant bs1 was significantly increased compared to the wild type plant.
  • the gene shown in positions 73-1149 of SEQ ID NO: 2 is named as BS1-CT gene, and the amino acid sequence of the protein encoded by BS1-CT gene is shown in positions 25-382 of SEQ ID NO: 1, and positions 25-382 of SEQ ID NO: 1.
  • the protein was named BS1-CT protein.
  • the cDNA extracted by the step 1 is used as a template, and the primer pair consisting of F2 and R2 is subjected to PCR amplification, and the PCR amplification product is recovered and linked to the T vector.
  • F2 5'-TCTCGAGAAGAGAGAGGCTGAAGCAGAGGGGAAGGTGAACGGGA-3';
  • R2 5'-TTCTAGACCTGAAGATTGGAAGATCGGTTGG-3'.
  • the T vector of step 2 was digested with restriction endonucleases XhoI and XbaI, and the digested product was recovered.
  • the pPICZ ⁇ C vector was digested with restriction endonucleases XhoI and XbaI to recover a vector backbone of about 3600 bp.
  • the digested product of step 3 is ligated with the vector backbone of step 4 to obtain a recombinant plasmid pPICZ ⁇ C-BS1-CT.
  • the recombinant plasmid pPICZ ⁇ C-BS1-CT was structurally described as follows: Sequence 2 of the sequence listing was inserted between the XhoI and XbaI restriction sites of the pPICZ ⁇ C vector from the 5' end of the 5' end to the 7th to 1146th nucleotides. A double-stranded DNA molecule as shown.
  • the exogenous insert and the partial nucleotide on the vector backbone form the coding gene of the fusion protein BS1-CT-His
  • the recombinant plasmid pPICZ ⁇ C-BS1-CT expresses the fusion protein BS1-CT-His.
  • the amino acid sequence of the fusion protein BS1-CT-His is shown in SEQ ID NO: 3 of the Sequence Listing.
  • the recombinant plasmid pPICZ ⁇ C-BS1-CT was introduced into yeast strain X33 to obtain a recombinant strain.
  • the pPICZ ⁇ C vector was introduced into yeast strain X33 to obtain a control strain.
  • the strain with the highest expression rate and the optimal expression time were selected for large-scale induction.
  • the medium used in the experiment and the detailed operation flow See EasySelect TM Pichia Expression Kit (Invitrogen) .
  • the electrophoresis pattern of the fusion protein BS1-CT-His solution is shown in Fig. 2.
  • the size of the fusion protein BS1-CT-His is about 60 kDa. There was no target band in the control strain.
  • the fusion protein BS1-CT-His was purified by the system. The specific steps are as follows: adding ammonium sulfate to a large amount of induced protein solution, the final concentration of ammonium sulfate is 1M, centrifugation at 12000 rpm for 10 min, and the supernatant is taken to flow through the buffer (1 M ammonium sulfate, 50 mM Tris-HCl, pH 7.0) equilibrated HiTrap phenyl FF (HS) column, eluted with (1-0) M gradient ammonium sulfate solution, desalted by HiTrap desalting column to obtain purified fusion protein BS1-CT-His.
  • the acetylated monosaccharide sample is as follows: 1,2,3,4,6-5-O-acetyl- ⁇ -D-glucose (Glc) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 604-69-3); , 2,3,4,6-5-O-acetyl- ⁇ -D-galactopyranosyl (Gal) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 4163-60-4); 1,2,3 , 5-4-O-acetyl- ⁇ -L-arabinofuranose (Ara) (Tianjin Xiens Biochemical Technology Co., Ltd., 79120-81-3); 1,2,3,4,6-5-O -acetyl- ⁇ -D-mannopyranose (Man) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 4026-35-1); methyl 2,3,4-3-O-acetyl- ⁇ -D - Xylpyranose (Xy
  • the activity of the fusion protein BS1-CT-His against acetylated monosaccharide was determined by using the acetic acid assay kit (Megazyme, K-ACET), and the fusion protein BS1-CT-His was used as the control group (Mock).
  • the specific steps are as follows: 2 mM acetylated monosaccharide was used as the reaction substrate, 50 mM Tris-HCl was used as the reaction buffer, 2 ⁇ g of the purified fusion protein BS1-CT-His was added, and the reaction was catalyzed at 37 ° C for 2 h, and then the reagent was determined by acetic acid.
  • the cartridge detects the amount of acetic acid released by the reaction (the acetic acid released from the reaction comes from the acetyl group in the cell wall, which represents a part of the acetyl group involved in the reaction in the cell wall), and the acetic acid release rate ( ⁇ mol min -1 mg -1 ) is calculated based on the amount of acetic acid released. .
  • the specific procedure was as follows: 10 ⁇ L of the supernatant was taken in a UV capable 96-well flatness plate, and 94 ⁇ L of water was added. Subsequently, 42 ⁇ L (2.5:1) of the solution of Solution 1 and Solution 2, Solution 3 and Solution 4 were sequentially added. The respective absorbance values A0, A1 and A2 at 340 nm are read separately. A standard curve is drawn using solution 5, and the amount of acetic acid released in the sample is calculated using the following formula:
  • Blank control (A2-A0) - (A1-A0) (A1-A0) / (A2-A0).
  • the deacetylation activity of the fusion protein BS1-CT-His on acetylated xylose was determined by quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) method, and the fusion protein BS1-CT-His was used as the control group.
  • LC-QTOF-MS quadrupole-time-of-flight mass spectrometry
  • the enzyme activity curve of the fusion protein BS1-CT-His to triacetylmethylxylose (Xyl) was determined using an acetic acid assay kit. The specific steps are as follows: using triacetylmethylxylose (Xyl) as a substrate, the substrate is set to a series of concentration gradients: 0.5 mM, 1 mM, 1.5 mM, 2.0 mM, 4.0 mM, 6 mM, 8 mM, 10 mM, 14 mM, 20 mM, 25 mM, the enzyme activity reaction system is the same as step 1, and then the acetic acid assay kit is used to determine the amount of acetic acid released by the fusion protein BS1-CT-His catalyzing different concentrations of the substrate, and finally the triacetylmethyl xylose (Xyl)
  • the enzymatic activity curve of the fusion protein BS1-CT-His to triacetylmethylxylose (Xyl) was
  • Acetyl xylan was extracted from wild type rice plants (Golden Clear) and mutant bs1, respectively.
  • the specific steps are the same as those in the second step of the first embodiment.
  • the extracted acetyl xylan was prepared as acetyl oligosaccharide.
  • the specific steps are as follows: 1 mg of acetyl xylan extracted by the above method is weighed, and 200 ⁇ L of 50 mM NaAc is used as a buffer (pH 6.0), and 8 U ⁇ -Xylanase M6 endoxylanase (Megazyme, E-XYRU6) is added.
  • the acetyl oligo-xylose was treated, centrifuged at 12,000 rpm for 10 min, the supernatant was taken, and lyophilized to obtain acetyl oligo-xylose.
  • the deacetylation activity of the fusion protein BS1-CT-His against acetyl xylan and acetyl oligo-xylose was determined by the acetic acid assay kit, and the fusion protein BS1-CT-His was used as the control group (Mock).
  • the specific steps are as follows: 1 mg of wild type rice plants (Golden Clear) and 4 parts of mutant bs1 acetyl xylan and acetyl oligosaccharide, 50 mM Tris-HCl (pH 7.0) as reaction buffer, and 2 ⁇ g were added.
  • the purified fusion protein BS1-CT-His was catalyzed at 37 °C for 2 h, and the amount of acetic acid released by the reaction was determined by an acetic acid assay kit (the acetic acid released from the reaction was derived from the acetyl group in the cell wall, representing a part of the acetyl group involved in the reaction in the cell wall. base).
  • the specific steps are the same as in step 2.
  • the four-stage rod-time-of-flight mass spectrometer (LC-QTOF-MS) method was used to determine the fusion protein BS1-CT-His for different acetyl oligo xylose (trimeric xylose DP3, tetrameric xylose DP4, pentapoly xylose DP5). And the deacetylation activity of hexameric xylose DP6), while the fusion protein BS1-CT-His was not used as a control group (Mock).
  • the enzyme activity curve of the fusion protein BS1-CT-His against the mutant bs1 acetyl xylooligosaccharide was determined using an acetic acid assay kit.
  • the measurement method is the same as that in step two.
  • the acetyl oligo-xylose extracted from the mutant bs1 was used as a substrate, and the substrate was set to a series of concentration gradients: 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, 0.8 mg/mL, 1.0 mg/ mL, 1.5mg/mL, 2.0mg/mL, 3.6mg/mL, 7.2mg/mL, then use the acetic acid assay kit to determine the amount of acetic acid released by the fusion protein BS1-CT-His catalyzed by different concentrations of substrate, and finally acetyl The concentration of oligo-xylose was plotted on the abscissa and the release rate of acetic acid was plotted on the ordinate.
  • the enzymatic activity curve of the fusion protein BS1-CT-His to acetyl oligo-xylose was obtained. Data analysis was performed using Origin v8.0 software, and the Km value was calculated to be 1.58 ⁇ 0.52 mg / mL. The results of the enzyme activity mechanics curve are shown in Figure 7.
  • the acetyl xylan in the mutant bs1 was extracted, and acetyl oligo-xylose was prepared, and the preparation method was the same as that in the third step.
  • the Agilent standard pulse sequence gHSQCAD was used to detect 13C-1H related single bonds in the cell wall.
  • the acquisition range of all 1H-13C HSQC spectra is: F2 (1H) direction spectrum width 10ppm, F1 (13C) spectrum width 200ppm.
  • the data matrix of 2048 ⁇ 512 (F2 ⁇ F1) is obtained, and the sampling parameters are: the receiving gain is 30, the scanning frequency is 64 times/FID, and the Interscan delay (d1) is 1 s.
  • the DMSO solvent peaks (dC 39.5 ppm and dH 2.49 ppm) were used to calibrate the spectra. Processing and analysis of NMR data was performed using MestReNova 10.0.2 software.
  • the invention provides the application of the BS1-CT protein in regulating the deacetylation reaction of plant cell wall xylan. It has been proved by experiments that the BS1-CT protein of the invention has the function of catalyzing the deacetylation reaction of plant cell wall xylan, and can be used for regulating the acetylation modification level of plant cell wall xylan, and transforming biomass resources into energy. It plays a major role in helping to reduce the production cost of bioenergy and has important economic value.

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Abstract

La présente invention concerne une application de la protéine BS1-CT dans la régulation de la désacétylation du xylane dans une paroi cellulaire végétale. La protéine BS1-CT est la protéine a) ou b) ou c) suivante : a) une protéine ayant une séquence d'acides aminés telle que représentée aux positions 25 à 382 de la SEQ ID NO : 1 ; b) une protéine de fusion obtenue par marquage de l'extrémité N-terminale et/ou de l'extrémité C-terminale de la protéine représentée aux positions 25 à 382 de la SEQ ID NO : 1 ; et c) une protéine obtenue par la mise en oeuvre d'une substitution et/ou d'une délétion et/ou d'une addition d'un ou de plusieurs résidus d'acides aminés sur la séquence d'acides aminés représentée aux positions 25 à 382 de la SEQ ID NO : 1. Des expériences démontrent que : la protéine BS1-CT possède une fonction de catalyse de la désacétylation du xylane dans une paroi cellulaire végétale, peut être utilisée pour réguler le taux d'acétylation du xylane dans une paroi cellulaire végétale, jouera un rôle important dans la transformation des ressources de biomasse en énergie, est avantageuse pour réduire les coûts de production de bioénergie, et présente une valeur économique importante.
PCT/CN2017/117896 2017-01-12 2017-12-22 Application de la protéine bs1-ct dans la régulation de la désacétylation du xylane dans une paroi cellulaire végétale WO2018130060A1 (fr)

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US20040123343A1 (en) * 2000-04-19 2004-06-24 La Rosa Thomas J. Rice nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement
US20140130203A1 (en) * 2000-04-19 2014-05-08 Thomas J. La Rosa Rice nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement
WO2008034648A1 (fr) * 2006-04-05 2008-03-27 Metanomics Gmbh Procédé de production d'un produit chimique fin
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