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

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 regulating plant cell wall xylan deacetylation

technical field

The invention belongs to the field of biotechnology, in particular to the application of BS1-CT protein in regulating the deacetylation reaction of plant cell wall xylan.

Background technique

O-acetylation is a common modification in plant cell wall polysaccharides. With the exception of cellulose, β-1,3-1,4-linked glucans and some glycoproteins, O-acetylation modifications are present on nearly all other cell wall polysaccharides.

O-acetylation modification may affect cell wall structure by affecting the physiological and biochemical properties of cell wall polysaccharides. The study found that acetylation affects the interaction of polysaccharides with polar molecules. The pattern of acetylation modification on xylan affects how xylan binds to cellulose, which in turn affects cell wall structure. The degree of O-acetylation modification of plant cell wall polysaccharides shows dynamic changes with plant tissues and organs and growth and development stages, indicating that O-acetylation modification is closely related to plant growth and development status and is strictly regulated in plants. Therefore, the regulation mechanism of O-acetylation modification of plant cell wall polysaccharides is of great significance for plants to maintain cell wall structure and normal growth.

Studies have found that there are acetyltransferases in plants, which are involved in the acetylation modification in the process of plant cell wall polysaccharide synthesis, but whether there is a mechanism of plant deacetylation modification in this process has not been reported yet. Analyzing the mechanism of deacetylation 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 groups released from plant cell wall polysaccharides lead to acidification of the environment, affecting the fermentation process, and optimizing conditions increases 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.

SUMMARY OF THE INVENTION

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

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

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

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

c) A protein with the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in positions 25-382 of SEQ ID NO: 1.

In order to facilitate purification of the protein in a), a tag as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein shown in the sequence 1 at positions 25-382 in the sequence listing.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

For the protein in the above c), the substitution and/or deletion and/or addition of one or several amino acid residues are substitutions and/or deletions and/or additions of no more than 10 amino acid residues.

The protein in the above c) can be obtained by artificial synthesis, or by first synthesizing its encoding gene and then biologically expressing it.

The coding gene of the protein in the above c) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown at positions 73-1149 of SEQ ID NO: 2, and/or making one or several base pair errors. A sense mutation, and/or the coding sequence of the tag shown in Table 1 is attached to its 5' end and/or 3' end.

The amino acid sequence of the fusion protein in the above b) is shown in sequence 3.

The second object of the present invention is to provide biomaterials related to the BS1-CT protein.

The biological material related to the BS1-CT protein provided by the present invention is any one of the following A1) to A12):

A1) nucleic acid molecule encoding BS1-CT protein;

A2) an expression cassette containing the nucleic acid molecule of A1);

A3) a recombinant vector containing the nucleic acid molecule of A1);

A4) a recombinant vector containing the expression cassette described in A2);

A5) a recombinant microorganism containing the nucleic acid molecule of A1);

A6) a recombinant microorganism containing the expression cassette described in A2);

A7) a recombinant microorganism containing the recombinant vector described in A3);

A8) a recombinant microorganism containing the recombinant vector described in A4);

A9) a transgenic plant cell line containing the nucleic acid molecule of A1);

A10) a transgenic plant cell line containing the expression cassette of A2);

A11) a transgenic plant cell line containing the recombinant vector described in A3);

A12) A transgenic plant cell line containing the recombinant vector described in A4).

In the above-mentioned biological material, the nucleic acid molecule of A1) is the gene shown in the following 1) or 2) or 3):

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

2) A cDNA molecule or a genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the BS1-CT protein;

3) A cDNA molecule or a genomic DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and encodes the BS1-CT protein.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

One of ordinary skill in the art can easily mutate the nucleotide sequence encoding BS1-CT of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence encoding 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 sequences of the present 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 the protein consisting of the amino acid sequence shown in the coding sequence 1 of the present invention. , or a nucleotide sequence of 95% or greater identity. Identity can be assessed with the naked eye or with 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 75% or more identity may be 80%, 85%, 90% or more than 95% identity.

In the above biological materials, the stringent conditions are hybridization in a solution of 2×SSC, 0.1% SDS at 68° C. and washing the membrane twice for 5 min each time, and then in a solution of 0.5×SSC, 0.1% SDS, Hybridize and wash the membrane twice at 68°C for 15 min each; or, in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS, hybridize and wash the membrane at 65°C.

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

In the above biological materials, the microorganisms can be yeast, bacteria, algae or fungi, such as Agrobacterium.

In the above biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

The third object of the present invention is to provide new uses of the BS1-CT protein.

The present invention provides the application of BS1-CT protein as acetyl esterase.

The fourth object of the present invention is to provide a new use of BS1-CT protein or biomaterials related to BS1-CT protein.

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

The invention also provides the application of the BS1-CT protein or the biological material related to the BS1-CT protein in the preparation of a product for regulating the acetylation modification level of plant cell wall xylan.

In the above application, the regulation of the acetylation modification level of plant cell wall xylan is to catalyze the deacetylation reaction of plant cell wall xylan.

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

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

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

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

The 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 BS1-CT protein or a fusion protein of BS1-CT protein.

The last object of the present invention is to provide a method for regulating the deacetylation of xylan.

The method for regulating deacetylation of xylan provided by the present invention includes the step of treating xylan with BS1-CT protein or a fusion protein of BS1-CT protein.

In the above product or method, the regulation of xylan deacetylation is regulation of plant cell wall xylan deacetylation; the regulation of plant cell wall xylan deacetylation is to catalyze the deacetylation of plant cell wall xylan.

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

It is proved by experiments that the BS1-CT protein of the present invention has the function of catalyzing the deacetylation reaction of plant cell wall xylan, can be used to regulate the acetylation modification level of plant cell wall xylan, and will be used in the process of converting biomass resources into energy. Play a major role, help reduce the production cost of bioenergy, and have important economic value.

Description of drawings

Figure 1 is a schematic diagram of the structure of the BS1 protein.

Figure 2 is an electrophoresis image of the fusion protein BS1-CT-His solution.

Figure 3 is a comparison of the activity of the fusion protein BS1-CT-His on different commercial acetylated monosaccharides detected by the acetic acid assay kit and the quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) method, respectively. The left graph takes the five acetylated monosaccharides as the abscissa and the acetic acid release rate (μmol min ^-1 mg ^-1 ) as the ordinate. The right graph takes 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 the experimental group with the fusion protein BS1-CT-His added, and Mock is the control group without the fusion protein BS1-CT-His added.

Fig. 4 is a kinetic curve of the reaction of fusion protein BS1-CT-His to acetylated xylose measured with an acetic acid assay kit. Take the gradient concentration of triacetylmethylxylose (mM) as the abscissa and the acetic acid release rate (μmol min ^-1 mg ^-1 ) as the ordinate.

Figure 5 is a comparison of the activity of fusion protein BS1-CT-His on acetyl xylan extracted from rice and acetyl oligosaccharide produced by endo-xylanase. The left graph takes 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. On the right, the abscissa is the acetyl xylo-oligosaccharide produced by endo-xylanase, and the ordinate is the amount of acetic acid released by the reaction (μmol mg ^-1 acetyl xylo-oligosaccharide). BS1 is the experimental group with the fusion protein BS1-CT-His added, and Mock is the control group without the fusion protein BS1-CT-His added.

Figure 6 is the detection of the deacetylation activity of fusion protein BS1-CT-His on rice aceto-oligosaccharide by LC-QTOF-MS method. Taking four xylose aceto-oligosaccharides (respectively, DP3, DP4, DP5, DP5 and DP6) as abscissas, the relative abundance of AO is Y-axis. - represents the control group, + represents the experimental group added with fusion protein BS1-CT-His.

Figure 7 is the kinetic curve of the deacetylation reaction of fusion protein BS1-CT-His on rice xylo-oligosaccharides. Take the gradient concentration of xylo-acetyl-oligosaccharide (mg mL ^-1 ) as the abscissa, and take the release rate of acetic acid (μmol min ^-1 mg ^-1 ) as the ordinate.

Fig. 8 is the detection of the deacetylation activity of the fusion protein BS1-CT-His on the mutant bs1 acetyl-oligosaccharide by NMR method.

Detailed ways

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

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Quantitative experiments in the following examples are all set up to repeat the experiments three times, and the results are averaged.

The japonica rice variety "Huangjinqing" (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, Australia.

Agrobacterium EHA105 in the following examples was purchased from CAMIA, 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 example was purchased from Invitrogen Company, and the vector has the coding sequence of the His tag, and expresses the exogenous protein with the His tag.

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 acetyl esterase specifically acts on the acetylated modified cell wall polysaccharide to deacetylate it.

Example 1. Obtainment of rice brittle sheath mutant and BS1-CT protein

1. The acquisition of rice brittle sheath mutants and their BS1 protein sequence analysis

1. Phenotype of rice brittle sheath mutants

The brittle sheath1 mutant of rice (bs1 or mutant bs1 for short) is a spontaneous mutation material of the japonica rice variety "Golden Qing". Compared with the japonica rice variety "Huangjinqing", the mutant bs1 mainly showed as follows: (1) the leaf sheath became brittle (the mechanical strength of the leaf sheath was significantly reduced, and the structure of the xylem vessel was abnormal); (2) the plant became shorter.

2. BS1 protein sequence analysis of rice brittle sheath mutants

Sequencing showed that compared with wild-type rice plants (Jinjinqing), the rice brittle sheath mutant only mutated the first base from the 5' end of the second intron of the BS1 gene from G to A, and the rest of the genome The sequences are all identical to wild-type rice plants. This base mutation caused the second intron of the BS1 gene to not be spliced normally, resulting in premature termination of the translation of the BS1 protein.

The protein sequence of the BS1 protein is shown in Sequence 1 of the Sequence Listing, and the nucleotide sequence of the BS1 gene is shown in Sequence 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 main 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

The mutant bs1 and wild-type plants (japonica rice variety "Golden Qing") grown for 4 weeks were taken respectively, and 6 plants were randomly selected from each line, mixed and powdered, and the acetyl groups on the acetyl xylan in the cell walls of the plants to be tested were detected. content. The specific operations are as follows:

1. Extraction of acetyl xylan

Four-week-old plant seedlings were freeze-dried until their weight did not change, ground to a powder, and sieved with a 200-mesh sieve to remove coarse particles. Wash three times with 70% ethanol aqueous solution and three times with an equal volume of mixed chloroform-methanol mixture, and collect the precipitate by centrifugation at 12000 rpm for 10 min after each rinsing. The obtained precipitate was washed with acetone and dried to obtain an alcohol insoluble residue (Alcohol insoluble residue/AIR for short) mainly composed of cell wall components. Weigh 400 mg of plant stem alcohol insoluble matter (AIR), add 40 mL of 1% (mass fraction) ammonium oxalate solution, and react at 37° C. overnight to remove pectin components. After the sample was centrifuged, the precipitate was collected, rinsed with 5 mL of 11% (volume fraction) peracetic acid solution, and the supernatant was discarded. Then, 20 mL of 11% (volume fraction) peracetic acid solution was added, and the reaction was carried out at 85° C. for 30 minutes. After centrifugation at 2500 rpm for 15 minutes, the supernatant was discarded to obtain a delignified sample. The pellet 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, reacted at 97 °C for 35 min, and then transferred to a 60 °C water bath for 1 h to remove starch. After centrifugation at 2500 rpm for 15 minutes, the supernatant was discarded, and 5 mL of acetone was added to rinse the precipitate three times, and vacuum-dried. The extraction was performed with 20 mL of DMSO overnight at 70°C, and after two repetitions, the supernatant was transferred to a new tube. Add 5 times the volume of ethanol:methanol:water (7:2:1, v/v, pH 2-3), and precipitate at 4°C for 3 days to obtain acetylxylan precipitation. The precipitate collected by centrifugation at 2500 rpm for 15 minutes was rinsed three times with absolute ethanol and dried in vacuum to obtain the acetyl xylan fraction.

2. Acetyl content detection

Dissolve 1 mg of acetylxylan in 100 μL of 1N NaOH, and react at 28°C and 200 rpm for 1 h. Then add 100 μL of 1N HCl to neutralize, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant for testing. Acetic acid assay kit (Megazyme, K-ACET) was used to measure the amount of acetic acid released by the reaction in the supernatant (the acetic acid released by the reaction came from the acetyl group in the cell wall, representing a part of the acetyl group in the cell wall participating in the reaction). Take 10 μL of the supernatant in a UV capable 96-well flat plate and add 94 μL of water. Subsequently, 42 μL (2.5:1) of a mixture of solution 1 and solution 2, solution 3 and solution 4 were added in sequence. Read the corresponding absorbance values A0, A1 and A2 at 340 nm, respectively. Using solution 5 to draw a standard curve, 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 = (A2-A0)-(A1-A0)(A1-A0)/(A2-A0).

the result shows. The content of acetyl groups on acetylxylan (the amount of released acetic acid) in the cell walls of mutant bs1 was significantly increased compared to wild-type plants.

Third, the acquisition of BS1-CT protein

The gene shown at positions 73-1149 of sequence 2 is named BS1-CT gene, the amino acid sequence of the protein encoded by BS1-CT gene is shown as position 25-382 of sequence 1, and the amino acid sequence of position 25-382 of sequence 1 is shown The protein was named BS1-CT protein.

Example 2. Preparation of BS1-CT protein

1. Construction of recombinant plasmids

1. The total RNA of the japonica rice variety "Golden Qing" was extracted and reverse transcribed into cDNA.

2. Using the cDNA extracted in step 1 as a template, perform PCR amplification with a primer pair composed of F2 and R2, recover the PCR amplification product, and connect the T vector.

F2: 5'-TCTCGAGAAGAGAGAGGCTGAAGCAGAGGGGAAGGTGAACGGGA-3';

R2: 5'-TTCTAGACCTGAAGATTGGAAGATCGGTTGG-3'.

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

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

5. Connect the enzyme-digested product of step 3 with the vector backbone of step 4 to obtain a recombinant plasmid pPICZαC-BS1-CT. According to the sequencing results, the structure of the recombinant plasmid pPICZαC-BS1-CT is described as follows: The sequence 2 of the sequence listing is inserted between the XhoI and XbaI restriction sites of the pPICZαC vector. The double-stranded DNA molecule shown. In the recombinant plasmid pPICZαC-BS1-CT, the exogenous insertion sequence and some nucleotides on the vector backbone form the encoding 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 sequence 3 of the sequence listing.

2. 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.

3. Inducible expression and purification of fusion protein BS1-CT-His

1. Induction and expression of fusion protein BS1-CT-His

Pick a single clone and culture it in 25mL BMGY medium. 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 bacteria with 100-200mL BMMY medium. The concentration of bacterial cells, so that its OD600 value is about 1.0. Induction of protein expression was initiated and sampling was performed 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. Compared with the results of monoclonal expression, the strain with the highest expression level and the optimal expression time were selected for mass induction. The medium used in the experiment and the detailed operating procedure can be found in EasySelect ^™ Pichia Expression Kit (Invitrogen). The electropherogram of the fusion protein BS1-CT-His solution is shown in Figure 2, and the size of the fusion protein BS1-CT-His is about 60 kDa. However, there is no target band in the control strain.

2. Purification of fusion protein BS1-CT-His

Pure系统对融合蛋白BS1-CT-His进行纯化。具体步骤如下：添加硫酸铵至大量诱导的蛋白溶液中，使硫酸铵的终浓度为1M，12000rpm离心10min，取上清，使其流穿已被缓冲液(1M硫酸铵，50mM Tris-HCl，pH 7.0)平衡的HiTrap phenyl FF(HS)柱，并以(1-0)M梯度浓度硫酸铵溶液洗脱，用HiTrap脱盐柱进行脱盐处理，得到纯化后的融合蛋白BS1-CT-His。use

The Pure system purifies the fusion protein BS1-CT-His. The specific steps are as follows: add ammonium sulfate to a large amount of induced protein solution, make the final concentration of ammonium sulfate 1M, centrifuge at 12000rpm for 10min, take the supernatant, make it flow through the buffer solution (1M ammonium sulfate, 50mM Tris-HCl, pH 7.0) equilibrated HiTrap phenyl FF(HS) column, eluted with (1-0) M gradient concentration ammonium sulfate solution, and used HiTrap desalting column for desalting treatment to obtain the purified fusion protein BS1-CT-His.

Example 3. Application of fusion protein BS1-CT-His in regulating xylan deacetylation in vitro

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

1. Acetylated monosaccharide samples

The acetylated monosaccharide samples are as follows: 1,2,3,4,6-5-O-acetyl-β-D-glucose (Glc) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 604-69-3); 1 ,2,3,4,6-5-O-acetyl-β-D-galactopyranose (Gal) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 4163-60-4); 1,2,3 ,5-4-O-Acetyl-α-L-arabinofuranosose (Ara) (Tianjin Xiensi 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 -Xylopyranose (Xyl) (Beijing Kaisenlai Pharmaceutical Technology Co., Ltd., 13007-37-9) (referred to as triacetylmethyl xylose).

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

Acetic acid assay kit (Megazyme, K-ACET) was used to determine the activity of fusion protein BS1-CT-His on acetylated monosaccharide, and the control group (Mock) was used without fusion protein BS1-CT-His. The specific steps are as follows: take 2 mM acetylated monosaccharide as the reaction substrate, use 50 mM Tris-HCl as the reaction buffer, add 2 μg of the purified fusion protein BS1-CT-His, catalyze the reaction at 37 °C for 2 h, and then use acetic acid to determine the reagent The box detects the amount of acetic acid released in the reaction (the acetic acid released by the reaction comes from the acetyl group in the cell wall, representing a part of the acetyl group in the cell wall participating in the reaction), and calculates the release rate of acetic acid according to the amount of acetic acid released (μmol min ^-1 mg ^-1 ) . The specific steps are as follows: take 10 μL of the supernatant into a UV capable 96-well flat plate, and add 94 μL of water. Subsequently, 42 μL (2.5:1) of a mixture of solution 1 and solution 2, solution 3 and solution 4 were added in sequence. Read the corresponding absorbance values A0, A1 and A2 at 340 nm, respectively. Using solution 5 to draw a standard curve, 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 = (A2-A0)-(A1-A0)(A1-A0)/(A2-A0).

The results are shown in Figure 3A. The results showed that compared with the control group, the release rate of acetic acid (μmol min ^-1 mg ^-1 ) in the experimental group added with the fusion protein BS1-CT-His was significantly improved, indicating that the fusion protein BS1-CT-His of the present invention has a significant effect on Glc, Gal, Ara, Man and Xyl all have higher deacetylation activities.

2. Deacetylation activity of fusion protein BS1-CT-His on acetylated xylose and determination of kinetic curve of enzyme activity

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

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), and the fusion protein BS1-CT-His was used as the control group without the addition of the fusion protein. (Mock). 2 mM triacetylmethyl xylose (Xyl) 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 time was 16 h. The protein in the reaction system was filtered using a 10KDa concentrated centrifuge tube. The reaction solution was transferred to a liquid chromatography injection bottle and detected on the machine. The analysis of experimental data adopts Aglient Mass HunterQualitative Analysis B.07.00.

The results are shown in Figure 3B. The results show that: compared with the control group, the fusion protein BS1-CT-His of the present invention can specifically catalyze the removal of two acetyl groups from triacetylmethylxylose to generate diacetylmethylxylose and monoacetylmethylxylose respectively. Xylose.

2. Determination of the enzymatic kinetic curve of fusion protein BS1-CT-His to triacetylmethylxylose (Xyl)

The kinetic curve of the enzymatic activity of fusion protein BS1-CT-His to triacetylmethylxylose (Xyl) was determined by acetate assay kit. The specific steps are as follows: take triacetylmethylxylose (Xyl) as the substrate, and set a series of concentration gradients for the substrate: 0.5mM, 1mM, 1.5mM, 2.0mM, 4.0mM, 6mM, 8mM, 10mM, 14mM, 20mM, 25mM, the enzymatic reaction system is the same as step 1, and then the amount of acetic acid released by the fusion protein BS1-CT-His catalyzed by different concentrations of substrates was measured with an acetic acid assay kit, and finally the amount of acetic acid released by triacetylmethylxylose (Xyl) was used. The concentration is the abscissa, and the release rate of acetic acid is the ordinate, and the kinetic curve of the enzyme activity of the fusion protein BS1-CT-His to triacetylmethylxylose (Xyl) is obtained. Data analysis was performed with Origin v8.0 software, and the calculated Km value was 4.4±0.78mM. The kinetic curve of enzyme activity is shown in Figure 4.

3. Deacetylation activity of fusion protein BS1-CT-His on acetyl xylan and acetyl oligosaccharide extracted from rice

1. Preparation of acetyl xylan and acetyl oligosaccharide

Acetylxylan was extracted from wild-type rice plants (Huangjinqing) and mutant bs1, respectively. Same as 1 in step 2 of Example 1.

The acetyl xylan obtained by extraction is prepared into acetyl oligosaccharide. The specific steps are as follows: Weigh 1 mg of the acetyl xylan extracted by the above method, take 200 μL of 50 mM NaAc as buffer (pH 6.0), add 8Uβ-Xylanase M6 endo-xylanase (Megazyme, E-XYRU6) The acetyl xylo-oligosaccharide was obtained by treatment, centrifuged at 12,000 rpm for 10 min, the supernatant was taken, and lyophilized to obtain the acetyl xylo-oligosaccharide.

2. Deacetylation activity of fusion protein BS1-CT-His on acetyl xylan and acetyl oligosaccharide

Acetic acid assay kit was used to determine the deacetylation activity of fusion protein BS1-CT-His on acetyl xylan and acetyl oligosaccharide, while the control group (Mock) was used without fusion protein BS1-CT-His. The specific steps are as follows: Weigh 1 mg of wild-type rice plants (Jinjinqing) and 4 copies of mutant bs1 acetyl xylan and acetyl oligosaccharide respectively, 50 mM Tris-HCl (pH 7.0) is the reaction buffer, add 2 μg The purified fusion protein BS1-CT-His, catalyzed the reaction at 37 °C for 2 h, and the amount of acetic acid released in the reaction was determined by using an acetic acid assay kit (the acetic acid released in the reaction came from the acetyl group in the cell wall, representing a part of the acetyl group in the cell wall participating in the reaction. base). The specific steps are the same as 2 in step 1.

The results are shown in Figure 5. Since the content of acetyl groups on the acetyl xylan in the cell wall of the mutant bs1 is higher than that of the wild type, the amount of acetic acid released from the acetyl xylan in the mutant bs1 is also higher than that of the wild type after the reaction (left panel of Figure 5). However, the acetyl xylan in the same amount of 1 mg of wild type and mutant bs1 was treated with the same amount of β-Xylanase M6 endo-xylanase, and then the acetyl xylan was obtained under the same enzymatic reaction system mentioned above. The amount of acetic acid released after the detection reaction was higher than that of acetyl xylan (right panel of Figure 5). It shows that the fusion protein BS1-CT-His has higher activity with acetyl-oligosaccharide as the reaction substrate.

3. Deacetylation activity of fusion protein BS1-CT-His on different acetyl-oligosaccharides

Quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) method was used to determine the effect of fusion protein BS1-CT-His on different acetyl-oligosaccharides (tri-xylose DP3, tetra-xylose DP4, penta-xylose DP5 The deacetylation activity of xylan and hexaxylose DP6), and the control group (Mock) without the fusion protein BS1-CT-His. 2 mM acetyl-xylo-oligosaccharide 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 time was 16 h. The protein in the reaction system was filtered using a 10KDa concentrated centrifuge tube. The reaction solution was transferred to a liquid chromatography injection bottle and detected on the machine. The analysis of experimental data used Aglient 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 on trimeric acetyl xylan (trimeric xylan), and is more inclined to act on short-chain acetyl oligosaccharides.

4. Determination of the enzymatic kinetic curve of the mutant bs1 acetyl-oligosaccharide by fusion protein BS1-CT-His

The kinetic curve of the enzyme activity of the fusion protein BS1-CT-His on the mutant bs1 acetyl-oligosaccharide was determined by the acetate assay kit. The determination method is the same as 4 in step 2. The acetyl-xylo-oligosaccharide extracted from mutant bs1 was used as the substrate, and a series of concentration gradients were set for the substrate: 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, 0.8 mg/mL, 1.0 mg/mL mL, 1.5mg/mL, 2.0mg/mL, 3.6mg/mL, 7.2mg/mL, and then the amount of acetic acid released by the fusion protein BS1-CT-His catalyzed by different concentrations of substrates was determined by the acetic acid assay kit. The xylo-oligosaccharide concentration is taken as the abscissa, and the acetic acid release rate is taken as the ordinate to obtain the enzymatic kinetic curve of the fusion protein BS1-CT-His on acetyl-xylo-oligosaccharide. Data analysis was performed with Origin v8.0 software, and the calculated Km value was 1.58 ± 0.52 mg/mL. The results of the enzymatic kinetic curves are shown in Figure 7.

4. Detection of the effect of fusion protein BS1-CT-His on the acetylation sites of plant xylan by nuclear magnetic resonance technique NMR

1. Preparation of acetyl-xylo-oligosaccharides in mutant bs1

The acetyl xylan in the mutant bs1 is extracted, and the acetyl xylo-oligosaccharide is prepared, and the extraction and preparation method is the same as that in step 3.

2. Determination of Acetylation Modification Sites of Aceto-Oligosaccharides by NMR Experiments

Take 1 mg of acetyl xylo-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, and at the same time with no The fusion protein was added as a control group, heated for 15 min to inactivate the protein, 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 set to 599.90MHz, the experimental temperature of 1H-NMR and HSQC-NMR was set to 298K, and the probe used was a 5-mmHCN triple resonance cryoprobe. Agilent's standard pulse sequence gHSQCAD was used to detect 13C-1H-related single bonds in cell walls. The acquisition range of all 1H-13C HSQC spectra was 10 ppm in F2 (1H) and 200 ppm in F1 (13C). The data matrix of 2048×512 (F2×F1) is obtained by collecting, and the sampling parameters are: the receiving gain is 30, the number of scans is 64 times/FID, and the Interscan delay (d1) is 1s. 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 results are shown in Figure 8. The results showed that the O-2 and O-3 acetylation modifications of acetyl xylan in the mutant bs1 were significantly lower than those in the control group, indicating that the fusion protein BS1-CT-His was specifically involved in xylan O-2 and O-3. Deacetylation of O-3.

sequence listing

<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

Application of <120> BS1-CT protein in regulating the deacetylation of plant cell wall xylan

<160> 3

<210> 1

<211> 382

<212> PRT

<213> Artificial sequences

<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 sequences

<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 sequences

<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

210 215 220

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

370 375 380

Claims (8)

1. A protein, which is a protein of the following a) or b): a) The amino acid sequence is the protein shown at positions 25-382 of SEQ ID NO: 1; b) A fusion protein obtained by linking a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID NO: 25-382. 2. The biological material relevant to the protein of claim 1 is any one of the following A1) to A8): A1) a nucleic acid molecule encoding the protein of claim 1; A2) an expression cassette containing the nucleic acid molecule of A1); A3) a recombinant vector containing the nucleic acid molecule of A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule of A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) a recombinant microorganism containing the recombinant vector described in A3); A8) A recombinant microorganism containing the recombinant vector described in A4). 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 positions 73-1149 of sequence 2. 4 . 4. the application in the protein described in claim 1 or the relevant biological material described in claim 2 or 3 in the regulation of plant cell wall xylan acetylation modification level; Or the application of the protein described in claim 1 or the related biological material described in claim 2 or 3 in the preparation of a product that regulates the level of xylan acetylation modification of plant cell walls; The xylan acetylation is the acetylation of the 0-2 and/or 0-3 positions of xylan. 5. the application in the protein described in claim 1 or the relevant biological material described in claim 2 or 3 in regulating xylan acetylation modification level; Or the application of the protein described in claim 1 or the related biological material described in claim 2 or 3 in the preparation of a product that regulates the level of xylan acetylation modification; The xylan acetylation is the acetylation of the 0-2 and/or 0-3 positions of xylan. 6. The application according to claim 4 or 5, characterized in that: The regulation of the acetylation modification level of plant cell wall xylan is to catalyze the deacetylation reaction of plant cell wall xylan. 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 regulating deacetylation of xylan, comprising the step of treating xylan with the protein of claim 1 or a fusion protein thereof.

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