CN108299548B

CN108299548B - Application of BS1-CT protein in regulation and control of plant cell wall xylan deacetylation reaction

Info

Publication number: CN108299548B
Application number: CN201710020745.8A
Authority: CN
Inventors: 周奕华; 张保才; 张兰军
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2017-01-12
Filing date: 2017-01-12
Publication date: 2020-06-09
Anticipated expiration: 2037-01-12
Also published as: WO2018130060A1; CN108299548A

Abstract

The invention discloses application of BS1-CT protein in regulation and control of plant cell wall xylan deacetylation reaction. The BS1-CT protein is the protein of a) or b) or c) as follows: a) the amino acid sequence is a protein shown in 25 th to 382 th positions of the sequence 1; b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the 25 th to 382 th positions of the sequence 1; c) and (b) the protein obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the 25 th to 382 th positions of the sequence 1. Experiments prove that: the BS1-CT protein has the function of catalyzing deacetylation reaction of plant cell wall xylan, can be used for regulating and controlling acetylation modification level of plant cell wall xylan, plays an important role in the process of converting biomass resources into energy, is beneficial to reducing production cost of biological energy, and has important economic value.

Description

Application of BS1-CT protein in regulation and control of plant cell wall xylan deacetylation reaction

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of BS1-CT protein in regulation and control of plant cell wall xylan deacetylation reaction.

Background

O-acetylation is a more common form of modification in plant cell wall polysaccharides, with the exception of cellulose, β -1,3-1, 4-linked glucans and some glycoproteins, O-acetylation is present on almost all other cell wall polysaccharides.

The O-acetylation modification may affect the cell wall structure by affecting the physiological and biochemical properties of the cell wall polysaccharide. Acetylation has been found to affect the interaction of polysaccharides with polar molecules. The acetylation modification mode on xylan affects the binding mode of xylan and cellulose, and further affects the cell wall structure. The O-acetylation modification degree of the plant cell wall polysaccharide is dynamically changed along with plant tissues, organs and growth and development stages, which shows that the O-acetylation modification is closely related to the growth and development states of plants and is strictly regulated and controlled in plants. Therefore, the regulation mechanism of the plant cell wall polysaccharide O-acetylation modification has important significance for maintaining the cell wall structure and normal growth of plants.

Research has found that the presence of acetyltransferase in plants is involved in acetylation modification in the synthesis process of plant cell wall polysaccharide, but the mechanism of whether plant deacetylation modification exists in the process is not reported. The analysis of the deacetylation modification mechanism of cell wall polysaccharide is the basis for genetic improvement of plant cell wall structure and growth and development phenotype.

In the process of converting biomass resources into ethanol as a biological energy source, microbial fermentation is required, acetyl released by plant cell wall polysaccharide causes environmental acidification to influence the fermentation process, and the optimized conditions increase the cost of ethanol production. Therefore, the cell wall polysaccharide deacetylation regulation is beneficial to reducing the production cost of biological energy, and has important economic value.

Disclosure of Invention

It is a first object of the present invention to provide a protein.

The protein provided by the invention is the protein of a) or b) or c) as follows, and is named as BS1-CT protein:

a) the amino acid sequence is a protein shown in 25 th to 382 th positions of the sequence 1;

b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the 25 th to 382 th positions of the sequence 1;

c) and (b) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the 25 th to 382 th positions of the sequence 1.

In order to facilitate the purification of the protein in a), a tag as shown in Table 1 can be connected to the amino terminal or the carboxyl terminal of the protein shown in 25 th to 382 th positions of the sequence 1 in the sequence table.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of c) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in the c) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding the protein of c) above can be obtained by deleting one or more codons of amino acid residues from the DNA sequence shown in the 73 rd to 1149 th positions of the sequence No. 2, and/or performing missense mutation of one or more base pairs, and/or connecting the coding sequence of the tag shown in the above Table 1 to the 5 'end and/or the 3' end thereof.

The amino acid sequence of the fusion protein of b) is shown in SEQ ID No. 3.

It is a second object of the present invention to provide a biomaterial related to the BS1-CT protein.

The biological material related to the BS1-CT protein provided by the 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 a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

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

1) the coding sequence is cDNA molecule or DNA molecule shown in 73-1149 of sequence 2;

2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes BS1-CT protein;

3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes 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, etc.

The nucleotide sequence encoding BS1-CT of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence encoding BS1-CT are derived from and identical to the nucleotide sequence of the present invention as long as they encode BS1-CT and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown at positions 25-382 of the coding sequence 1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above biological material, the stringent conditions are hybridization and membrane washing at 68 ℃ for 2 times, 5min each, in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing at 68 ℃ for 2 times, 15min each, in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

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 comprise propagation material.

The third purpose of the invention is to provide a new application of the BS1-CT protein.

The invention provides an application of a BS1-CT protein as acetyl esterase.

The fourth purpose of the invention is to provide a new application of the BS1-CT protein or the biological material related to the BS1-CT protein.

The invention provides application of a BS1-CT protein or a biological material related to the BS1-CT protein in regulation and control of plant cell wall xylan acetylation modification level.

The invention also provides application of the BS1-CT protein or the biological material related to the BS1-CT protein in preparing products for regulating and controlling the acetylation modification level of xylan on plant cell walls.

In the application, the regulation and control of the plant cell wall xylan acetylation modification level is the catalysis of plant cell wall xylan deacetylation reaction.

The invention also provides application of the BS1-CT protein or the biological material related to the BS1-CT protein in regulating and controlling the acetylation modification level of xylan.

The invention also provides application of the BS1-CT protein or the biological material related to the BS1-CT protein in preparing a product for regulating and controlling the acetylation modification level of xylan.

In the application, the regulation and control of the xylan acetylation modification level is to catalyze xylan deacetylation reaction.

In the application, the acetylation of the xylan is acetylation of O-2 position and/or O-3 position of the xylan.

A fifth object of the invention is to provide a product for modulating the deacetylation of xylan.

The active component of the product provided by the invention is BS1-CT protein or BS1-CT protein fusion protein.

It is a final object of the invention to provide a method for modulating the deacetylation of xylans.

The method for regulating deacetylation of xylan provided by the 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 modulation of xylan deacetylation is modulation of plant cell wall xylan deacetylation; the regulation of plant cell wall xylan deacetylation is the catalysis of plant cell wall xylan deacetylation.

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

Experiments prove that: the BS1-CT protein has the function of catalyzing deacetylation reaction of plant cell wall xylan, can be used for regulating and controlling acetylation modification level of plant cell wall xylan, plays an important role in the process of converting biomass resources into energy, is beneficial to reducing production cost of biological energy, and has important economic value.

Drawings

FIG. 1 is a schematic structural diagram of the BS1 protein.

FIG. 2 is an electrophoretogram of a fusion protein BS1-CT-His solution.

FIG. 3 shows the comparison of the activity of the fusion protein BS1-CT-His on different commercial acetylated monosaccharides detected by the acetic acid determination kit and the quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) method respectively. The left panel uses five acetylated monosaccharides as abscissa and acetic acid release rate (μmol min)^-1mg^-1) Is the ordinate. The right graph has mass to charge ratio as abscissa and relative abundance of acetylated xylose as ordinate. Tri-Ac-Mexyl represents triacetyl methyl xylose, Di-Ac-Mexyl represents diacetyl methyl xylose, and Ac-Mexyl represents monoacetyl methyl xylose. BS1 is an experimental group added with the fusion protein BS1-CT-His, and Mock is a control group without the fusion protein BS 1-CT-His.

FIG. 4 is a kinetic curve of the response of the fusion protein BS1-CT-His to acetylated xylose determined using the acetic acid assay kit. Gradient concentrations of triacetyl methyl xylose (mM) as abscissa and acetic acid release rate (. mu. mol min)^-1mg^-1) Is the ordinate.

FIG. 5 shows the activity of the fusion protein BS1-CT-His on acetylxylan extracted from rice and acetylxylooligosaccharide produced by endoxylanase. Left panel with acetyl groups extracted from riceXylan is plotted as abscissa, in terms of amount of acetic acid released in the reaction (μmol mg)^-1Acetylxylan) as ordinate. The right panel shows the amount of acetic acid released by the reaction (μmol mg) using the acetyloligoxylose produced by endoxylanase as the abscissa^-1Oligo (acetyl) xylose) as ordinate. BS1 is an experimental group added with the fusion protein BS1-CT-His, and Mock is a control group without the fusion protein BS 1-CT-His.

FIG. 6 shows that LC-QTOF-MS is used to detect the deacetylation activity of the fusion protein BS1-CT-His on the rice acetyl xylooligosaccharide. Taking four kinds of acetyl xylooligosaccharide (trimeric xylose DP3, tetrameric xylose DP4, pentameric xylose DP5 and hexameric xylose DP6 respectively) as abscissa and taking the relative abundance of acetyl xylooligosaccharide as ordinate. -represents the control group and + represents the experimental group with the addition of the fusion protein BS 1-CT-His.

FIG. 7 is a kinetic curve of deacetylation reaction of rice xylooligosaccharide by fusion protein BS 1-CT-His. Acetyloligoxylose (mg mL) in gradient concentration^-1) In abscissa, the release rate of acetic acid (. mu.mol min)^-1mg^-1) Is the ordinate.

FIG. 8 shows that the fusion protein BS1-CT-His has deacetylation activity on the mutant BS1 acetylxylooligosaccharide by NMR method.

Detailed Description

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

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

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The japonica rice variety "jinqing" (WT, also known as wild type plant) in the following examples was purchased from rice institute in china.

The vector pCAMBIA1300 in the following examples was purchased from CAMIA, Australia.

Agrobacterium EHA105 in the examples described below was purchased from CAMIA, Australia.

The pDONR207 vector in the following examples was purchased from Life Technologies, Inc., cat # 12213-.

pGEM-T-easy vector in the following examples was purchased from Invitrogen.

The pPICZ α C vector in the following examples was purchased from Invitrogen, and has a His-tagged coding sequence to express a His-tagged foreign protein.

Yeast strain X33 in the following examples was purchased from Invitrogen.

The deacetylation reaction in the following examples is a reaction in which acetylation of cell wall polysaccharides modified by acetylation is specifically affected by acetyl esterase.

Example 1 preparation of rice crispy sphingoid variant and BS1-CT protein

First, the acquisition of rice crisp sphingoid variant and the analysis of its BS1 protein sequence

1. Phenotype of rice fragile sheath mutant

The rice crisp sheath mutant brittleth 1 (abbreviated as bs1 or mutant bs1) is a spontaneous mutation material of japonica rice variety "Jinjinqing". Compared with the japonica rice variety 'jinqing', the mutant bs1 mainly shows that: (1) the leaf sheath becomes brittle (the mechanical strength of the leaf sheath is remarkably reduced, and the xylem duct structure is abnormal); (2) the plant becomes short.

2. Sequence analysis of BS1 protein of rice crisp sphingoid variant

Sequencing showed that: compared with a wild rice plant (jinqing), the rice crispy sheath mutant is only that the first base of the second intron of the BS1 gene is mutated from G to A from the 5' end, and the rest sequences of the genome are all the same as the wild rice plant. This base mutation results in the second intron of the BS1 gene not being normally cleaved, thereby causing premature termination of translation of the BS1 protein.

The protein sequence of the BS1 protein is shown as the sequence 1 in the sequence table, and the nucleotide sequence of the BS1 gene is shown as the sequence 2 in the sequence table. The structural schematic diagram of the BS1 protein is shown in FIG. 1. TM represents the transmembrane domain, GDSL represents the major domain of the BS1 protein, and AG represents an antigenic fragment of the BS1 antiserum.

II, detection of acetyl content in mutant bs1 and wild plants

Respectively taking the mutant bs1 and wild type plants (japonica rice variety 'jinqing') which grow for 4 weeks, randomly selecting 6 plants from each plant line, mixing and powdering, and detecting the acetyl content on acetyl xylan in the cell walls of the plants to be detected. The specific operation is as follows:

1. extraction of acetylxylan

The young plants grown for 4 weeks were freeze-dried until the weight did not change, ground into powder, and sieved with a 200 mesh sieve to remove coarse particles. Washing with 70% ethanol water solution for three times, washing with equal volume of mixed chloroform-methanol solution for three times, centrifuging at 12000rpm for 10min after each rinsing, and collecting precipitate. The obtained precipitate was washed with acetone and dried to obtain an Alcohol-insoluble fraction (AIR) mainly containing cell wall components. 400mg of plant Stem alcohol insoluble substance (AIR) was weighed, 40mL of 1% (mass fraction) ammonium oxalate solution was added, and the mixture was reacted at 37 ℃ overnight to remove the pectin component. The sample was centrifuged and the precipitate was collected, rinsed with 5mL of 11% (volume fraction) peracetic acid solution, and the supernatant was discarded. Then 20mL of 11% (volume fraction) peracetic acid solution was added and reacted at 85 ℃ for 30 minutes. Centrifugation was carried out at 2500rpm for 15 minutes, and the supernatant was discarded to obtain a delignified sample. The pellet was rinsed with 5mL MES/Tris buffer (Tris, Sigma, 77-86-1; MES, Sigma, 1266615-59-1) and the supernatant discarded. Then 20mL of MES/Tris buffer and 40U of amylase (Megazyme, K-TDFR-100A) were added thereto, and after reacting at 97 ℃ for 35min, the mixture was transferred to a 60 ℃ water bath and reacted for 1h to remove starch. After centrifugation at 2500rpm for 15 minutes, the supernatant was discarded, and 5mL of acetone was added to rinse the precipitate three times and dried in vacuo. After two repetitions of extraction with 20mL of DMSO at 70 ℃ overnight, the supernatant was transferred to a new tube. 5 volumes of ethanol were added: methanol: precipitating with water (7:2:1, v/v, pH 2-3) at 4 deg.C for 3 days to obtain acetyl xylan precipitate. And centrifuging at 2500rpm for 15 minutes, rinsing the collected precipitate with absolute ethyl alcohol for three times, and drying in vacuum to obtain the acetyl xylan component.

2. Acetyl content detection

1mg of acetylxylan was dissolved in 100. mu.L of 1N NaOH and reacted at 28 ℃ for 1 hour at 200 rpm. Then 100. mu.L of 1N HCl was added for neutralization, and the mixture was centrifuged at 12,000rpm for 10 minutes to obtain a supernatant to be assayed. 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 by the reaction is derived from acetyl groups in the cell wall and represents a part of the acetyl groups involved in the reaction in the cell wall). Take 10. mu.L of supernatant in a UV capable 96 well plate and add 94. mu.L of water. Then 42. mu.L (2.5:1) of a mixture of solution 1 and solution 2, solution 3 and solution 4 were added in that order. The corresponding absorbance values A0, A1 and A2 at 340nm were read, respectively. A standard curve was plotted using solution 5 and the amount of acetic acid released in the sample was calculated using the following equation:

sample ═ (a2-a0) - (a1-a0) (a1-a0)/(a2-a0) -Blank;

blank control (a2-a0) - (a1-a0) (a1-a0)/(a2-a 0).

The results show that. The acetyl content (amount of acetic acid released) on acetylxylan in the cell wall of mutant bs1 was significantly increased compared to wild type plants.

III, obtaining BS1-CT protein

The gene shown in 73 th to 1149 th positions of the sequence 2 is named as BS1-CT gene, the amino acid sequence of the protein coded by the BS1-CT gene is shown as 25 th to 382 th positions of the sequence 1, and the protein shown in 25 th to 382 th positions of the sequence 1 is named as BS1-CT protein.

Example 2 preparation of BS1-CT protein

Construction of recombinant plasmid

1. Extracting the total RNA of japonica rice variety "jinqing" and making reverse transcription to obtain cDNA.

2. And (3) taking the cDNA extracted in the step 1 as a template, carrying out PCR amplification by using a primer pair consisting of F2 and R2, recovering a PCR amplification product, and connecting a T vector.

F2：5'-TCTCGAGAAGAGAGAGGCTGAAGCAGAGGGGAAGGTGAACGGGA-3'；

R2：5'-TTCTAGACCTGAAGATTGGAAGATCGGTTGG-3’。

3. The T vector of step 2 is double digested with restriction enzymes XhoI and XbaI, and the digested product is recovered.

4. The pPICZ α C vector was double digested with restriction enzymes XhoI and XbaI, and the vector backbone of about 3600bp was recovered.

5. And (2) connecting the enzyme digestion product in the step (3) with the vector skeleton in the step (4) to obtain a recombinant plasmid pPICZ α C-BS 1-CT., and structurally describing the recombinant plasmid pPICZ α C-BS1-CT according to a sequencing result, wherein a double-stranded DNA molecule shown by 73 th-1146 th nucleotides from the 5' end of the sequence 2 in the sequence table is inserted between XhoI and XbaI enzyme digestion sites of the pPICZ α C vector, in the recombinant plasmid pPICZ α C-BS1-CT, an encoding gene of the fusion protein BS1-CT-His is formed by the exogenous insertion sequence and part of the nucleotides on the vector skeleton, the recombinant plasmid pPICZ α C-BS1-CT expresses the fusion protein BS1-CT-His, and the amino acid sequence of the fusion protein BS1-CT-His is shown as the sequence 3 in the sequence table.

Second, preparation of recombinant bacterium

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

The control strain was obtained by introducing the pPICZ α C vector into yeast strain X33.

Thirdly, induced expression and purification of fusion protein BS1-CT-His

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

Selecting a single clone to culture in 25mL BMGY medium, when the OD value of the bacterial liquid reaches 2-6, centrifuging at 1500rpm for 5min, discarding the supernatant, collecting the thallus, suspending the thallus by using 100-200mL BMMY medium, and adjusting the concentration of the suspended thallus to make the OD600 value about 1.0. Induction of protein expression was initiated and samples were taken at the following time points: 0h, 6h, 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96 h. Protein samples were treated with methanol/ammonium acetate precipitation, SDS-polyacrylamide gel electrophoresis and Western detection. Comparing the expression results of the monoclonals, selecting the strain with the highest expression quantity and the optimal expression time to carry out mass induction. The media used in the experiments and the detailed procedure are described in easy select^TMPichia Expression Kit (Invitrogen). The electrophoretogram of the fusion protein BS1-CT-His solution is shown in FIG. 2, and the size of the fusion protein BS1-CT-His is about 60 kDa. Whereas the control strain had no band of interest.

2. Purification of the fusion protein BS1-CT-His

By using

Pure system for fusion protein BS1-And purifying by CT-His. The method comprises the following specific steps: ammonium sulfate was added to the mass-induced protein solution, the solution was centrifuged at 12000rpm for 10min at a final concentration of 1M ammonium sulfate, the supernatant was taken, passed through a HiTrap phenyl FF (HS) column equilibrated with a buffer (1M ammonium sulfate, 50mM Tris-HCl, pH7.0), eluted with a gradient concentration of (1-0) M ammonium sulfate solution, and desalted using a HiTrap desalting column to obtain the purified fusion protein BS 1-CT-His.

Example 3 application of fusion protein BS1-CT-His in vitro regulation and control of xylan deacetylation reaction

First, the substrate specificity of the fusion protein BS1-CT-His to acetylated monosaccharide

1. Acetylated monosaccharide samples

Acetylated monosaccharide samples were 1,2,3,4, 6-5-O-acetyl- β -D-glucose (Glc) (Beijing Kaisnlai medicine science and technology Co., Ltd., 604-69-3), 1,2,3,4, 6-5-O-acetyl- β -D-galactopyranose (Gal) (Beijing Kaisnlai medicine science and technology Co., Ltd., 4163-60-4), 1,2,3, 5-4-O-acetyl- α -L-arabinofuranose (Ara) (Tianjin Xienci biochemistry science and technology Co., Ltd., 79120-81-3), 1,2,3,4, 6-5-O-acetyl- β -D-mannopyranose (Man) (Beijing Kaisen medicine science and technology Co., Ltd., 4026-35-1), methyl 2,3, 4-3-O-acetyl- β -D-xylopyranose (Xsenyl) (Beijing Kaisei medicine science and technology Co., Ltd., 4029-13007).

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

The activity of the fusion protein BS1-CT-His on acetylated monosaccharides was determined using an acetic acid assay kit (Megazyme, K-ACET), while the control group (Mock) was prepared without the addition of the fusion protein BS 1-CT-His. The method comprises the following specific steps: respectively taking 2mM acetylated monosaccharide as reaction substrates, taking 50mM Tris-HCl as reaction buffer, adding 2 mu g of purified fusion protein BS1-CT-His, carrying out catalytic reaction at 37 ℃ for 2h, detecting the amount of acetic acid released by the reaction by using an acetic acid measuring kit (the acetic acid released by the reaction is from acetyl in a cell wall and represents a part of acetyl involved in the reaction in the cell wall), and calculating the release rate of the acetic acid (mu mol min) according to the amount of the released acetic acid^-1mg^-1). The method comprises the following specific steps: 10 μ L of the supernatant was taken in a UV capable 96 well plate and 94 μ L of water was added. Then 42. mu.L (2.5:1) of a mixture of solution 1 and solution 2, solution 3 and solution 4 were added in that order. The corresponding absorbance values A0, A1 and A2 at 340nm were read, respectively. A standard curve was plotted using solution 5 and the amount of acetic acid released in the sample was calculated using the following equation:

sample ═ (a2-a0) - (a1-a0) (a1-a0)/(a2-a0) -Blank;

blank control (a2-a0) - (a1-a0) (a1-a0)/(a2-a 0).

The results are shown in FIG. 3A. The results show that: the acetic acid release rate (. mu.mol min) of the experimental group to which the fusion protein BS1-CT-His was added was compared with that of the control group^-1mg^-1) The obvious improvement shows that the fusion protein BS1-CT-His has higher deacetylation activity on Glc, Gal, Ara, Man and Xyl.

Secondly, determining deacetylation activity of the fusion protein BS1-CT-His on acetylated xylose and enzyme activity mechanical curve

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

The deacetylation activity of the fusion protein BS1-CT-His on the acetylated xylose was determined by a quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) method, and the control group (Mock) was prepared without the addition of the fusion protein BS 1-CT-His. 2mM of triacetyl methyl xylose (Xyl) was used as a reaction substrate, 50mM of Tris-HCl was used as a reaction buffer, and 2. mu.g of purified fusion protein BS1-CT-His was added thereto for 16 hours. The protein in the reaction system was filtered using a10 kDa concentrating centrifuge tube. Transferring the reaction liquid to a liquid chromatogram sampling bottle, and detecting on a machine. The experimental data was analyzed using an active Mass HunterQualitative Analysis B.07.00.

The results are shown in FIG. 3B. The results show that: compared with a control group, the fusion protein BS1-CT-His can specifically catalyze triacetyl methyl xylose to remove two acetyl groups, and respectively generate diacetyl methyl xylose and monoacetyl methyl xylose.

2. Determination of enzyme activity mechanical curve of fusion protein BS1-CT-His on triacetyl methyl xylose (Xyl)

The enzyme activity mechanical curve of the fusion protein BS1-CT-His to the triacetyl methyl xylose (Xyl) is measured by adopting an acetic acid measuring kit. The method comprises the following specific steps: triacetyl methyl xylose (Xyl) was used as substrate, and a series of concentration gradients were set for the substrate: 0.5mM, 1mM, 1.5mM, 2.0mM, 4.0mM, 6mM, 8mM, 10mM, 14mM, 20mM and 25mM, and the enzyme activity reaction system is the same as the step 1, then the amount of acetic acid released by the fusion protein BS1-CT-His to catalyze substrates with different concentrations is measured by using an acetic acid measuring kit, and finally the concentration of the triacetyl methyl xylose (Xyl) is used as an abscissa, and the release rate of the acetic acid is used as an ordinate, so that an enzyme activity curve of the fusion protein BS1-CT-His to the triacetyl methyl xylose (Xyl) is obtained. Data analysis was performed using Origin v8.0 software and Km values were calculated to be 4.4. + -. 0.78 mM. The enzyme activity kinetics curve is shown in FIG. 4.

Thirdly, the fusion protein BS1-CT-His measures the deacetylation activity of acetyl xylan and acetyl xylooligosaccharide extracted from rice

1. Preparation of acetyl xylan and acetyl xylooligosaccharide

Acetyl xylan was extracted from wild type rice plants (jinqing) and mutant bs1, respectively. Same as 1 in step two of example 1.

Weighing 1mg of acetylxylan extracted by the method, taking 200 mu L of 50mM NaAc as a buffer solution (pH6.0), adding 8U β -Xylanase M6 endoxylanase (Megazyme, E-XYRU6) to process to obtain acetylxylooligosaccharide, centrifuging at 12000rpm for 10min, taking supernatant, and freeze-drying to obtain the acetylxylooligosaccharide.

2. Determination of deacetylation activity of fusion protein BS1-CT-His on acetyl xylan and acetyl xylooligosaccharide

The deacetylation activity of the fusion protein BS1-CT-His to acetyl xylan and acetyl xylooligosaccharide was determined by using an acetic acid assay kit, and the fusion protein BS1-CT-His was not added as a control group (Mock). The method comprises the following specific steps: respectively weighing 4 parts of wild rice plant (jinqing) and mutant BS1 acetylxylan and acetylxylooligosaccharide, 1mg of wild rice plant (jinqing), 4 parts of mutant BS1 acetylxylan and acetylxylooligosaccharide, 50mM Tris-HCl (pH7.0) as reaction buffer, adding 2 mu g of purified fusion protein BS1-CT-His, carrying out catalytic reaction at 37 ℃ for 2h, and measuring the amount of acetic acid released by the reaction by using an acetic acid measurement kit (the acetic acid released by the reaction is from acetyl in a cell wall and represents a part of acetyl participating in the reaction in the cell wall). The concrete steps are the same as step 2 in the first step.

The result is shown in figure 5. because the acetyl content of the acetylxylan in the cell wall of the mutant BS1 is higher than that of the wild type, after the reaction is finished, the acetic acid amount released by the acetylxylan in the mutant BS1 is also higher than that of the wild type (the left figure of figure 5). Alternatively treating 1mg of the acetylxylan in the wild type and the mutant BS1 with β -Xylanase M6 endoxylanase to obtain acetylxylooligosaccharide, then carrying out the reaction in the same enzyme activity reaction system, and detecting that the acetic acid amount released after the reaction is higher than that of the acetylxylan (the right figure of figure 5). The fusion protein BS1-CT-His has higher activity by using the acetylxylooligosaccharide as a reaction substrate.

3. Determination of deacetylation activity of fusion protein BS1-CT-His on different acetyl xylooligosaccharide

The deacetylation activity of the fusion protein BS1-CT-His on different acetyl xylooligosaccharide (xylotrimer DP3, xylotetramer DP4, xylopentamer DP5 and xylohexamer DP6) was determined by a quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) method, and the fusion protein BS1-CT-His was not added as a control group (Mock). 2mM acetyl xylooligosaccharide is taken as a reaction substrate, 50mM Tris-HCl is taken as a reaction buffer solution, 2 mu g of purified fusion protein BS1-CT-His is added, and the reaction time is 16 h. The protein in the reaction system was filtered using a10 kDa concentrating centrifuge tube. Transferring the reaction liquid to a liquid chromatogram sampling bottle, and detecting on a machine. The experimental data was analyzed using an active Mass Hunter Qualitative Analysis B.07.00.

The results are shown in FIG. 6. As can be seen from the figure: the fusion protein BS1-CT-His has obvious activity on the trimeric acetyl xylan (trimeric xylose), and is more prone to act on short-chain acetyl oligomeric xylose.

4. Determination of enzyme activity mechanical curve of fusion protein BS1-CT-His on mutant BS1 acetyl xylooligosaccharide

And (3) determining the enzyme activity mechanical curve of the fusion protein BS1-CT-His to the mutant BS1 acetyl xylooligosaccharide by using an acetic acid determination kit. The measuring method is the same as that of step 4 in the second step. Taking the acetyl xylooligosaccharide extracted from the mutant bs1 as a substrate, and setting a series of concentration gradients for the substrate: 0.1mg/mL, 0.2mg/mL, 0.5mg/mL, 0.8mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 3.6mg/mL, 7.2mg/mL, then using an acetic acid determination kit to determine the amount of acetic acid released by the fusion protein BS1-CT-His to catalyze substrates with different concentrations, and finally using the concentration of the acetyloligoxylose as a horizontal coordinate and the release rate of the acetic acid as a vertical coordinate to obtain an enzyme activity mechanical curve of the fusion protein BS1-CT-His to the acetyloligoxylose. Data analysis was performed using Origin v8.0 software and Km values were calculated to be 1.58. + -. 0.52 mg/mL. The results of the enzyme activity curves are shown in FIG. 7.

Fourthly, detecting the influence of the fusion protein BS1-CT-His on the plant xylan acetylation site by using nuclear magnetic resonance technology NMR

1. Preparation of acetyl xylooligosaccharide in mutant bs1

And (3) extracting acetyl xylan in the mutant bs1, preparing acetyl xylooligosaccharide, and extracting and preparing 1 in the third step.

2. Determination of acetylation modification sites of acetylxylooligosaccharide by NMR experiment

Taking 1mg of acetylxylooligosaccharide in the mutant BS1 as a substrate, adding 50 μ g of purified fusion protein BS1-CT-His into 50mM Tris (pH7.0) buffer, reacting at 37 ℃ for 16h, heating for 15min to inactivate protein by taking no fusion protein as a control group, centrifuging at 12000rpm for 10min, and transferring the supernatant to a Nuclear Magnetic Resonance (NMR) sample tube. In the NMR experiment, the proton resonance frequency was 599.90MHz, the experimental temperature of 1H-NMR and HSQC-NMR was set at 298K, and the probe used was a low-temperature probe of 5-mm HCN triple response. The Agilent standard pulse sequence gHSQCAD was used to detect 13C-1H associated single bonds in the cell wall. The acquisition range of all 1H-13C HSQC spectrograms is as follows: the spectral width of F2(1H) is 10ppm, and the spectral width of F1(13C) is 200 ppm. A 2048 × 512(F2 × F1) data matrix is acquired, and sampling parameters: the receive gain is 30, the number of scans is 64/FID, and the Interscan delay (d1) is 1 s. DMSO solvent peaks (dC 39.5ppm and dH 2.49ppm) were used to calibrate the spectra. NMR data was processed and analyzed using MestReNova 10.0.2 software.

The results are shown in FIG. 8. The results show that: the O-2 site and O-3 site acetylation modification of the acetyl xylooligosaccharide in the mutant BS1 are obviously lower than those of the control group, which indicates that the fusion protein BS1-CT-His is specifically involved in the deacetylation modification of the O-2 site and O-3 site of xylan.

Sequence listing

<110> institute of genetics and developmental biology of Chinese academy of sciences

Application of <120> BS1-CT protein in regulation and control of plant cell wall xylan deacetylation reaction

<160>3

<210>1

<211>382

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>1

Met Gly Ala Val Arg Gly Ile Leu Val Val Ala Val Val Leu Ala Val

1 5 10 15

Ala Ala Ile Leu Ala Gly Ala Ala Glu Gly Lys Val Asn Gly Lys Ala

20 25 30

Lys Gly Lys Tyr Arg Ala Leu Phe Asn Phe Gly Asp Ser Leu Ala Asp

35 40 45

Ala Gly Asn Leu Leu Ala Asn Gly Val Asp Phe Arg Leu Ala Thr Ala

50 55 60

Gln Leu Pro Tyr Gly Gln Thr Phe Pro Gly His Pro Thr Gly Arg Cys

65 70 75 80

Ser Asp Gly Arg Leu Val Val Asp His Leu Ala Asp Glu Phe Gly Leu

85 90 95

Pro Leu Leu Pro Pro Ser Lys Leu Lys Asn Ser Ser Phe Ala His Gly

100 105 110

Ala Asn Phe Ala Ile Thr Gly Ala Thr Ala Leu Asp Thr Pro Tyr Phe

115 120 125

Glu Ala Lys Gly Leu Gly Ala Val Val Trp Asn Ser Gly Ala Leu Leu

130 135 140

Thr Gln Ile Gln Trp Phe Arg Asp Leu Lys Pro Phe Phe Cys Asn Ser

145 150 155 160

Thr Lys Val Glu Cys Asp Glu Phe Tyr Ala Asn Ser Leu Phe Val Val

165 170 175

Gly Glu Phe Gly Gly Asn Asp Tyr Asn Ala Pro Leu Phe Ala Gly Lys

180 185 190

Gly Leu Glu Glu Ala Tyr Lys Phe Met Pro Asp Val Ile Gln Ala Ile

195 200 205

Ser Asp Gly Ile Glu Gln Leu Ile Ala Glu Gly Ala Arg Glu Leu Ile

210 215 220

Val Pro Gly Val Met Pro Thr Gly Cys Phe Pro Val Tyr Leu Asn Met

225 230 235 240

Leu Asp Glu Pro Ala Asp Gly Tyr Gly Pro Gln Ser Gly Cys Val Arg

245 250 255

Arg Tyr Asn Thr Phe Ser Trp Val His Asn Ala His Leu Lys Arg Met

260 265 270

Leu Glu Lys Leu Arg Pro Lys His Pro Asn Val Arg Ile Ile Tyr Gly

275 280 285

Asp Tyr Tyr Thr Pro Val Ile Gln Phe Met Leu Gln Pro Glu Lys Phe

290 295 300

Gly Phe Tyr Lys Gln Leu Pro Arg Ala Cys Cys Gly Ala Pro Gly Ser

305 310 315 320

Val Ala Lys Ala Ala Tyr Asn Phe Asn Val Thr Ala Lys Cys Gly Glu

325 330 335

Ala Gly Ala Thr Ala Cys Asp Asp Pro Ser Thr His Trp Ser Trp Asp

340 345 350

Gly Ile His Leu Thr Glu Ala Ala Tyr Gly His Ile Ala Arg Gly Trp

355 360 365

Val Tyr Gly Pro Phe Ala Asp Gln Pro Ile Phe Gln Ser Ser

370 375 380

<210>2

<211>1149bp

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>2

atgggggcag ttcgggggat tttggtcgtg gcggtggttc ttgcggtggc ggcgattctt 60

gctggggcgg cggaggggaa ggtgaacggg aaggcgaagg ggaagtacag ggcgctgttc 120

aacttcgggg actcgctggc cgacgccggc aacctcctcg ccaacggcgt cgacttccgc 180

ctcgctaccg cccagctccc ctacggccag accttccccg gccaccccac cggccgctgc 240

tccgacggcc gcctcgtcgt cgaccacctc gccgacgagt tcggcctgcc gctgctgccg 300

ccgtccaagc tcaagaactc cagcttcgct cacggcgcca acttcgccat caccggcgcc 360

accgcgctcg acacccccta cttcgaggcc aaggggctcg gcgccgtcgt ctggaactcc 420

ggcgccctcc tcacccaaat ccagtggttc cgcgatctca agcccttctt ctgcaactcc 480

accaaggtgg aatgcgatga attctatgcg aattcgctct tcgtcgtcgg cgagtttggt 540

ggcaacgact acaatgcgcc gctgtttgcg gggaagggcc ttgaggaggc ctacaagttc 600

atgccggatg tcatccaggc tatctccgat ggcatcgagc aattgattgc tgagggcgca 660

agggagctga ttgtacccgg tgtgatgccc actggatgct tccctgtcta cttgaacatg 720

ctcgatgagc cggccgatgg gtatggcccc cagagcggct gcgtccgtcg gtacaacaca 780

ttctcatggg tgcacaatgc acatctcaag cgcatgcttg agaagctccg gcccaagcac 840

cccaatgtga ggatcatata tggcgattac tacacgcctg ttatccagtt catgcttcag 900

cccgagaagt ttggatttta caagcagcta cctagggcat gctgcggggc tcctgggtcc 960

gttgcgaagg ccgcttacaa cttcaatgtc acagccaaat gtggtgaggc tggtgcaacc 1020

gcgtgtgatg atccatcaac ccattggagc tgggatggca ttcacctgac agaggcggct 1080

tacggtcaca ttgccagagg ttgggtatat ggtcctttcg ctgaccaacc gatcttccaa 1140

tcttcatga 1149

<210>3

<211>381

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>3

Glu Gly Lys Val Asn Gly Lys Ala Lys Gly Lys Tyr Arg Ala Leu Phe

5 10 15

Asn Phe Gly Asp Ser Leu Ala Asp Ala Gly Asn Leu Leu Ala Asn Gly

20 25 30

Val Asp Phe Arg Leu Ala Thr Ala Gln Leu Pro Tyr Gly Gln Thr Phe

35 40 45

Pro Gly His Pro Thr Gly Arg Cys Ser Asp Gly Arg Leu Val Val Asp

50 55 60

His Leu Ala Asp Glu Phe Gly Leu Pro Leu Leu Pro Pro Ser Lys Leu

65 70 75 80

Lys Asn Ser Ser Phe Ala His Gly Ala Asn Phe Ala Ile Thr Gly Ala

85 90 95

Thr Ala Leu Asp Thr Pro Tyr Phe Glu Ala Lys Gly Leu Gly Ala Val

100 105 110

Val Trp Asn Ser Gly Ala Leu Leu Thr Gln Ile Gln Trp Phe Arg Asp

115 120 125

Leu Lys Pro Phe Phe Cys Asn Ser Thr Lys Val Glu Cys Asp Glu Phe

130 135 140

Tyr Ala Asn Ser Leu Phe Val Val Gly Glu Phe Gly Gly Asn Asp Tyr

145 150 155 160

Asn Ala Pro Leu Phe Ala Gly Lys Gly Leu Glu Glu Ala Tyr Lys Phe

165 170 175

Met Pro Asp Val Ile Gln Ala Ile Ser Asp Gly Ile Glu Gln Leu Ile

180 185 190

Ala Glu Gly Ala Arg Glu Leu Ile Val Pro Gly Val Met Pro Thr Gly

195 200 205

Cys Phe Pro Val Tyr Leu Asn Met Leu Asp Glu Pro Ala Asp Gly Tyr

210215 220

Gly Pro Gln Ser Gly Cys Val Arg Arg Tyr Asn Thr Phe Ser Trp Val

225 230 235 240

His Asn Ala His Leu Lys Arg Met Leu Glu Lys Leu Arg Pro Lys His

245 250 255

Pro Asn Val Arg Ile Ile Tyr Gly Asp Tyr Tyr Thr Pro Val Ile Gln

260 265 270

Phe Met Leu Gln Pro Glu Lys Phe Gly Phe Tyr Lys Gln Leu Pro Arg

275 280 285

Ala Cys Cys Gly Ala Pro Gly Ser Val Ala Lys Ala Ala Tyr Asn Phe

290 295 300

Asn Val Thr Ala Lys Cys Gly Glu Ala Gly Ala Thr Ala Cys Asp Asp

305 310 315 320

Pro Ser Thr His Trp Ser Trp Asp Gly Ile His Leu Thr Glu Ala Ala

325 330 335

Tyr Gly His Ile Ala Arg Gly Trp Val Tyr Gly Pro Phe Ala Asp Gln

340 345 350

Pro Ile Phe Gln Ser Ser Gly Leu Glu Gln Lys Leu Ile Ser Glu Glu

355 360 365

Asp Leu Asn Ser Ala Val Asp His His His His His His

370 375380

Claims

1. The protein is the protein of the following a) or b):

b) and (b) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein represented by positions 25 to 382 of the sequence No. 1.

2. The protein-related biomaterial according to claim 1, which is any one of the following a1) to A8):

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

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising the recombinant vector of a 4).

3. The related biological material according to claim 2, wherein: A1) the coding sequence of the nucleic acid molecule is a DNA molecule shown in 73-1149 of the sequence 2.

4. Use of a protein according to claim 1 or a related biomaterial according to claim 2 or 3 to modulate the level of modification of xylan acetylation in plant cell walls;

or the use of a protein according to claim 1 or a related biomaterial according to claim 2 or 3 for the preparation of a product for modulating the level of modification of plant cell wall xylan acetylation;

the acetylation of the xylan is the acetylation of the 0-2 position and/or the 0-3 position of the xylan.

5. Use of a protein according to claim 1 or a related biomaterial according to claim 2 or 3 to modulate the level of xylan acetylation modification;

or the protein of claim 1 or the related biomaterial of claim 2 or 3 for use in the preparation of a product for modulating the level of xylan acetylation modification;

6. Use according to claim 4 or 5, characterized in that:

the regulation and control of the plant cell wall xylan acetylation modification level is to catalyze the plant cell wall xylan deacetylation reaction.

7. A product for regulating deacetylation of xylan, the active ingredient of which is the protein of claim 1 or a fusion protein thereof.

8. A method for modulating deacetylation of xylan comprising the step of treating xylan with the protein of claim 1 or a fusion protein thereof.