WO2018130060A1

WO2018130060A1 - Application of bs1-ct protein in regulating deacetylation of xylan in plant cell wall

Info

Publication number: WO2018130060A1
Application number: PCT/CN2017/117896
Authority: WO
Inventors: 周奕华; 张保才; 张兰军
Original assignee: 中国科学院遗传与发育生物学研究所
Priority date: 2017-01-12
Filing date: 2017-12-22
Publication date: 2018-07-19
Also published as: CN108299548B; CN108299548A

Abstract

An application of BS1-CT protein in regulating deacetylation of xylan in a plant cell wall. The BS1-CT protein is the following protein a) or b) or c): a) a protein having an amino acid sequence as shown at positions 25-382 of SEQ ID NO: 1; b) a fusion protein obtained by tagging the N-terminus and/or C-terminus of the protein shown at positions 25-382 of SEQ ID NO: 1; and c) a protein obtained by carrying out substitution and/or deletion and/or addition of one or several amino acid residues on the amino acid sequence shown at positions 25-382 of SEQ ID NO: 1. Experiments demonstrate that: the BS1-CT protein has a function of catalyzing deacetylation of xylan in a plant cell wall, can be used for regulating the acetylation level of xylan in a plant cell wall, will play a significant role in transformation of biomass resources into energy, is beneficial to reduce production costs of bioenergy, and has an important economic value.

Description

Application of BS1-CT protein in the regulation of plant cell wall xylan deacetylation

Technical field

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.

Background technique

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.

Studies have found that acetyltransferase is present in plants and is involved in acetylation modification during plant cell wall polysaccharide synthesis, but the mechanism of plant deacetylation modification in this process has not been reported. The mechanism of deacetylation modification of cell wall polysaccharides is the basis for genetic improvement of plant cell wall structure and growth and development phenotype.

In the process of converting biomass resources into bioenergy ethanol, microbial fermentation is required, and the acetyl group released by plant cell wall polysaccharides causes environmental acidification, which affects the fermentation process, and the optimization conditions increase the cost of ethanol production. Therefore, the regulation of cell wall polysaccharide deacetylation helps to reduce the production cost of bioenergy and has important economic value.

Invention disclosure

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:

a) the amino acid sequence is the protein shown in positions 25-382 of SEQ ID NO:1;

b) a fusion protein obtained by ligating the N-terminus and/or C-terminus of the protein shown in positions 25-382 of SEQ ID NO: 1;

c) A protein having the same function obtained by subjecting the amino acid sequence shown at positions 25-382 of SEQ ID NO: 1 to one or several amino acid residues by substitution and/or deletion and/or addition.

In order to facilitate the purification of the protein in a), 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.

Table 1, the sequence of labels

标签label	残基Residues	序列sequence
Poly-ArgPoly-Arg	5-6(通常为5个)5-6 (usually 5)	RRRRRRRRRR
Poly-HisPoly-His	2-10(通常为6个)2-10 (usually 6)	HHHHHH HHHHHH

FLAGFLAG	88	DYKDDDDKDYKDDDDK
Strep-tag IIStrep-tag II	88	WSHPQFEKWSHPQFEK
c-mycC-myc	1010	EQKLISEEDLEQKLISEEDL

The protein in c) above, the 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. A sense mutation, and/or a coding sequence for the tag shown in Table 1 attached at its 5' end and/or 3' end.

The 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);

A12) A transgenic plant cell line comprising the recombinant vector of A4).

In the above biological material, the nucleic acid molecule of A1) is a gene represented by 1) or 2) or 3) as follows:

1) the coding sequence thereof is a cDNA molecule or a DNA molecule as shown in SEQ ID NO: 73-1149;

2) a cDNA molecule or a genomic DNA molecule having a 75% or more identity with a defined nucleotide sequence and encoding a BS1-CT protein;

3) 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.

Wherein, 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.

One of ordinary skill in the art 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.

The term "identity" as used herein 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.

In the above biological material, 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.

In the above biological material, the vector may be a plasmid, a cosmid, a phage or a viral vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi such as Agrobacterium.

In the above biological material, 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.

In the above application, 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.

In the above application, the xylan acetylation modification level is a catalytic xylan deacetylation reaction.

In the above application, 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 provided by the present invention comprises the step of treating xylan with a fusion protein of BS1-CT protein or BS1-CT protein.

In the above product or method, the xylan is deacetylated to regulate the deacetylation of plant cell wall xylan;

The deregification of the plant cell wall xylan to catalyze the deacetylation of plant cell wall xylan.

In the above application or product or method, the xylan is acetyl xylan.

DRAWINGS

Figure 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, and 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 (μmol 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, and Ac-Mexyl represents monoacetylmethylxylose. BS1 is an experimental group to which the fusion protein BS1-CT-His is added, and 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 (μmol 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 (μmol mg ^-1 acetyl xylan) as the ordinate. The figure on the right shows the amount of acetic acid released by the reaction (μmol 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, and 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 (μmol 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 best way to implement the invention

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

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 pPICZαC 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

I. Acquisition of rice fragile sheath mutant and analysis of its BS1 protein sequence

1. Phenotype of rice fragile sheath mutant

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.

2. Sequence analysis of BS1 protein in rice fragile sheath mutant

By sequencing, 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, and AG represents the antigenic fragment of the BS1 antiserum.

2. Detection of acetyl content in mutant bs1 and wild type plants

Mutant bs1 and wild-type plants (Jinqing), 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:

1. Extraction of acetyl xylan

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. 400 mg of plant stem alcohol-insoluble matter (AIR) was weighed, and 40 mL of a 1% (mass fraction) ammonium oxalate solution was added thereto, and reacted at 37 ° C overnight to remove the pectin component. The sample was centrifuged, and the precipitate was collected, rinsed with 5 mL of a 11% (volume fraction) peroxyacetic acid solution, and the supernatant was discarded. Then 20 mL of a 11% (volume fraction) peroxyacetic acid solution was added and reacted at 85 ° C for 30 minutes. Centrifuge at 2500 rpm for 15 minutes and discard the supernatant to obtain a delignified sample. 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. 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.

2, acetyl content detection

1 mg of acetyl xylan was dissolved in 100 μL of 1N NaOH, and reacted at 28 ° C, 200 rpm for 1 h. It was further neutralized by adding 100 μL 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). 10 μL of the supernatant was taken in a UV capable 96-well flatness plate and 94 μL of water was added. Subsequently, 42 μL (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:

Sample = (A2-A0)-(A1-A0)(A1-A0)/(A2-A0)-Blank;

Blank control = (A2-A0) - (A1-A0) (A1-A0) / (A2-A0).

the result shows. 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.

Third, the acquisition of BS1-CT protein

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.

Example 2 Preparation of BS1-CT protein

First, the construction of recombinant plasmid

1. Extract the total RNA of the indica variety "Golden Clear" and reverse-transcribe it into cDNA.

2. 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'.

3. The T vector of step 2 was digested with restriction endonucleases XhoI and XbaI, and the digested product was recovered.

4. The pPICZαC vector was digested with restriction endonucleases XhoI and XbaI to recover a vector backbone of about 3600 bp.

5. The digested product of step 3 is ligated with the vector backbone of step 4 to obtain a recombinant plasmid pPICZαC-BS1-CT. According to the sequencing results, the recombinant plasmid pPICZαC-BS1-CT was structurally described as follows: Sequence 2 of the sequence listing was inserted between the XhoI and XbaI restriction sites of the pPICZαC vector from the 5' end of the 5' end to the 7th to 1146th nucleotides. A double-stranded DNA molecule as shown. In the recombinant plasmid pPICZαC-BS1-CT, the exogenous insert and the partial nucleotide on the vector backbone form the coding gene of the fusion protein BS1-CT-His, and the recombinant plasmid pPICZαC-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.

Second, the preparation of recombinant bacteria

The recombinant plasmid pPICZαC-BS1-CT was introduced into yeast strain X33 to obtain a recombinant strain.

The pPICZαC vector was introduced into yeast strain X33 to obtain a control strain.

Induction expression and purification of fusion protein BS1-CT-His

1. Induced expression of fusion protein BS1-CT-His

Select the monoclonal in 25mL BMGY medium for culture. When the OD value of the bacterial solution reaches 2-6, centrifuge at 1500rpm for 5min, discard the supernatant, collect the bacteria, and resuspend the cells with 100-200mL BMMY medium, adjust the resuspension. The bacterial concentration is such that the OD600 value is about 1.0. Protein expression was initially induced and samples were taken at the following time points: 0h, 6h, 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h. Protein samples were treated with methanol/ammonium acetate precipitation, SDS-polyacrylamide gel electrophoresis and Western detection. Comparing the results of monoclonal expression, 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.

2. Purification of fusion protein BS1-CT-His

use

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.

Example 3 Application of Fusion Protein BS1-CT-His in Deacetylation of Xylan in Vitro

1. Substrate specificity of the fusion protein BS1-CT-His for acetylated monosaccharides

1, acetylated monosaccharide sample

The acetylated monosaccharide sample is as follows: 1,2,3,4,6-5-O-acetyl-β-D-glucose (Glc) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 604-69-3); , 2,3,4,6-5-O-acetyl-β-D-galactopyranosyl (Gal) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 4163-60-4); 1,2,3 , 5-4-O-acetyl-α-L-arabinofuranose (Ara) (Tianjin Xiens Biochemical Technology Co., Ltd., 79120-81-3); 1,2,3,4,6-5-O -acetyl-β-D-mannopyranose (Man) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 4026-35-1); methyl 2,3,4-3-O-acetyl-β-D - Xylpyranose (Xyl) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 13007-37-9) (referred to as triacetylmethyl xylose).

2. Determination of the activity of the fusion protein BS1-CT-His on acetylated monosaccharides

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 μg 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 (μmol min ^-1 mg ^-1 ) is calculated based on the amount of acetic acid released. . The specific procedure was as follows: 10 μL of the supernatant was taken in a UV capable 96-well flatness plate, and 94 μL of water was added. Subsequently, 42 μL (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:

Sample = (A2-A0)-(A1-A0)(A1-A0)/(A2-A0)-Blank;

Blank control = (A2-A0) - (A1-A0) (A1-A0) / (A2-A0).

The result is shown in Figure 3A. The results showed that the acetic acid release rate (μmol min ^-1 mg ^-1 ) of the experimental group added with the fusion protein BS1-CT-His was significantly higher than that of the control group, indicating that the fusion protein BS1-CT-His of the present invention is Glc, Gal, Ara, Man and Xyl all have higher deacetylation activity.

Deacetylation activity of acetylated xylose and determination of enzyme activity curve of fusion protein BS1-CT-His

1. Determination of deacetylation activity of acetylated xylose by fusion protein BS1-CT-His

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. (Mock). 2 mM triacetylmethylxylose (Xyl) was used as a reaction substrate, and 50 mM Tris-HCl was used as a reaction buffer, and 2 μg of the purified fusion protein BS1-CT-His was added for a reaction time of 16 hours. The protein in the reaction system was filtered using a 10 KDa concentrated centrifuge tube. The reaction solution was transferred to a liquid chromatography sample bottle and detected by a machine. The experimental data was analyzed using Agilent Mass Hunter Qualitative Analysis B.07.00.

The result is shown in Figure 3B. The results showed that the fusion protein BS1-CT-His of the present invention can specifically catalyze the removal of two acetyl groups from triacetylmethyl xylose to form diacetylmethylxylose and monoacetylmethyl, respectively, compared with the control group. Xylose.

2. Determination of the enzyme activity mechanical curve of the fusion protein BS1-CT-His to triacetylmethylxylose (Xyl)

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 obtained by taking the concentration as the abscissa and the release rate of acetic acid as the ordinate. Data analysis was performed using Origin v8.0 software, and the Km value was calculated to be 4.4 ± 0.78 mM. The enzyme activity mechanics curve is shown in Figure 4.

Determination of deacetylation activity of acetyl xylan and acetyl oligo-xylose extracted from rice by fusion protein BS1-CT-His

1. Preparation of acetyl xylan and acetyl oligo-xylose

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 μL of 50 mM NaAc is used as a buffer (pH 6.0), and 8 U β-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.

2. Determination of deacetylation activity of acetyl xylan and acetyl oligo-xylose by fusion protein BS1-CT-His

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 μg 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 results are shown in Figure 5. Since the acetyl xylan content in the cell wall of the mutant bs1 was higher than that in the wild type, the amount of acetic acid released from the acetyl xylan in the mutant bs1 was also higher than that in the wild type (Fig. 5 left panel). The acetyl oligo-xylose obtained by treating an equivalent amount of 1 mg of wild type and mutant bs1 acetyl xylan with an equivalent amount of β-Xylanase M6 endoxylanase was carried out under the same enzyme reaction system as above. The amount of acetic acid released after the reaction was detected was higher than that of acetyl xylan (Fig. 5 right panel). It is indicated that the fusion protein BS1-CT-His has higher activity with acetyl oligo-xylose as a reaction substrate.

3. Determination of deacetylation activity of different acetyl oligo-xylose by fusion protein BS1-CT-His

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). 2 mM acetyl oligo-xylose was used as a reaction substrate, and 50 mM Tris-HCl was used as a reaction buffer, and 2 μg of the purified fusion protein BS1-CT-His was added for a reaction time of 16 hours. The protein in the reaction system was filtered using a 10 KDa concentrated centrifuge tube. The reaction solution was transferred to a liquid chromatography sample bottle and detected by a machine. The experimental data was analyzed using Agilent Mass Hunter Qualitative Analysis B.07.00.

The results are shown in Figure 6. It can be seen from the figure that the fusion protein BS1-CT-His has significant activity against triacetyl xylan (trimeric xylose) and is more likely to act on short-chain acetyl oligo-xylose.

4. Determination of Enzymatic Activity Mechanics Curve of Mutant Bs1 Acetyloligo-Xylose by Fusion Protein BS1-CT-His

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.

4. NMR detection of the fusion protein BS1-CT-His on the acetylation site of plant xylan by nuclear magnetic resonance spectroscopy

1. Preparation of acetyl oligo-xylose in mutant bs1

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.

2. Determination of acetylation modification sites of acetyl oligo-xylose by NMR experiment

Take 1 mg of acetyl oligosaccharide in mutant bs1 as a substrate, add 50 μg of purified fusion protein BS1-CT-His in 50 mM Tris (pH 7.0) buffer, and react at 37 ° C for 16 h, while not The fusion protein was added as a control group, the inactivated protein was heated for 15 min, centrifuged at 12,000 rpm for 10 min, and the supernatant was transferred to a nuclear magnetic resonance (NMR) sample tube. In the NMR experiment, the proton resonance frequency was 599.90 MHz, the 1H-NMR and HSQC-NMR experimental temperatures were set to 298 K, and the probe used was a 5-mm HCN triple resonance cryoprobe. 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 result is shown in Figure 8. The results showed that the acetylation of O-2 and O-3 in acetyl oligo-xylose in mutant bs1 was significantly lower than that in the control group, indicating that the fusion protein BS1-CT-His specifically participates in xylan O-2 and Deacetylation modification of O-3.

Industrial application

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.

Claims

Protein, which is a protein of the following a) or b) or c):

a) the amino acid sequence is the protein shown in positions 25-382 of SEQ ID NO:1;

b) a fusion protein obtained by ligating the N-terminus and/or C-terminus of the protein shown in positions 25-382 of SEQ ID NO: 1;

c) A protein having the same function obtained by subjecting the amino acid sequence shown at positions 25-382 of SEQ ID NO: 1 to one or several amino acid residues by substitution and/or deletion and/or addition.
The biomaterial related to the protein of claim 1 is any one of the following A1) to A12):

A1) a nucleic acid molecule encoding the protein of claim 1;

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);

A12) A transgenic plant cell line comprising the recombinant vector of A4).
The related biological material according to claim 2, wherein: A1) the nucleic acid molecule is a gene represented by 1) or 2) or 3) as follows:

1) the coding sequence thereof is a cDNA molecule or a DNA molecule as shown in SEQ ID NO: 73-1149;

2) a cDNA molecule or genomic DNA molecule having 75% or more of the identity to the defined nucleotide sequence and encoding the protein of claim 1;

3) A cDNA molecule or genomic DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by 1) or 2) and which encodes the protein of claim 1.
Use of the protein of claim 1 as an acetyl esterase.
Use of the protein of claim 1 or the related biological material of claim 2 or 3 for regulating the level of acetylation of plant cell wall xylan.
Use of the protein of claim 1 or the related biomaterial of claim 2 or 3 in the manufacture of a product that modulates the level of acetylation of plant cell wall xylan.
The use according to claim 5 or 6, wherein the level of acetylation acetylation of the plant cell wall is catalyzed to catalyze the deacetylation of the plant cell wall xylan.
The use according to claim 7, characterized in that the xylan is acetylated to the acetylation of the xylan O-2 position and/or the O-3 position.
Use of the protein of claim 1 or the related biomaterial of claim 2 or 3 for regulating the level of acetylation of xylan.
Use of the protein of claim 1 or the related biomaterial of claim 2 or 3 in the manufacture of a product that modulates the level of acetylation of xylan.
The use according to claim 9 or 10, characterized in that the level of the xylan acetylation modification is a catalytic xylan deacetylation reaction.
The use according to claim 11, characterized in that the xylan is acetylated to the acetylation of the xylan O-2 position and/or the O-3 position.
A product for regulating acetylation of xylan, the active ingredient of which is the protein of claim 1 or a fusion protein thereof.
A method of modulating xylan deacetylation comprising the step of treating xylan with the protein of claim 1 or a fusion protein thereof.
The product of claim 13 or the method of claim 14, wherein the xylan is deacetylated to modulate plant cell wall xylan deacetylation.
A product or method according to claim 15 wherein said modulating plant cell wall xylan deacetylation is to catalyze deacetylation of plant cell wall xylan.