A xyloglucanase nfxyg12a with the function of exciting plant immune activity, mutants and application thereof

Abstract

The application discloses a xyloglucanase NfXYG12A with plant immune activity, a mutant and application thereof. The xyloglucanase NfXYG12A disclosed by the application is a protein shown in positions 94-313 of SEQ ID NO. 1, and the mutant is a protein shown in positions 94-313 of SEQ ID NO. 3, positions 94-313 of SEQ ID NO. 5 and positions 94-313 of SEQ ID NO. 7. Experiments prove that the NfXYG12A of the application is a high-temperature acid xyloglucanase, the enzyme has excellent pH stability and catalytic activity, and can be used as a plant immune elicitor, and has high application value in enhancing plant immunity. The NfXYG12A of the application has wide application prospect.

Description

Technical Field

[0001] The present invention relates to the fields of microbial enzyme engineering technology and biochemistry and molecular biology technology, and in particular to a xyloglucanase NfXYG12A capable of stimulating plant immunity, a mutant thereof and applications thereof. Background Art

[0002] Global population growth poses significant challenges to agricultural production and quality. Ensuring the yield and quality of staple crops is crucial for maintaining national security. Given the public's pursuit of green, high-quality agricultural products, microbial-derived apoplastic immune elicitors have attracted significant attention in the past decade due to their potential for broad-spectrum plant resistance.

[0003] Xyloglucanase is an extracellular enzyme that is widely distributed in the CAZy (http: / / www.cazy.org / ) database and exists in the GH5, GH12, GH16, GH44 and GH74 families. Among them, the glycoside hydrolases of the GH12 family mainly include cellulase (EC 3.2.1.4) and xyloglucanase (EC 3.2.1.151) ( www.cazy.org).

[0004] Xyloglucanase specifically degrades xyloglucan and has the ability to hydrolyze polysaccharides with a β-1,4 glycosidic backbone. Xyloglucan, a key component of hemicellulose polysaccharides in plant cell walls, is present in the primary cell walls of most plants and plays a crucial role in regulating cell wall structure and growth. Cellulose and hemicellulose are renewable resources. my country, a major agricultural country, produces a large amount of crop waste straw each year that can be recycled and degraded. Xyloglucanase, a key enzyme in the degradation of cellulose and hemicellulose, can collaborate with other cellulases to efficiently degrade plant cellulose. Furthermore, xyloglucanase is generally used in various industries due to its good temperature stability and excellent catalytic effect under alkaline conditions. For example: in the bioethanol industry, the synergistic effect of xyloglucanase and cellulase can increase the hydrolysis of wood cellulose, thereby improving the economic benefits of biomass raw material conversion; in the food industry, on the one hand, xyloglucan can be used as a food thickener, and on the other hand, xyloglucanase can make turbidity in juice clear and improve the quality of juice; in the textile and papermaking industry, since most xyloglucanases are alkaline and have good stability in alkaline environments, they have great application potential in the papermaking and textile industries.

[0005] Most processes for enzymatic degradation of xyloglucan and cellulose and conversion of their degradation products need to be carried out under acidic conditions. However, xyloglucanases with high hydrolysis activity under acidic conditions are extremely rare. Summary of the Invention

[0006] The present invention provides a highly efficient xyloglucanase NfXYG12A derived from Aspergillus fischeri and exhibiting plant immune activity, and its mutants. NfXYG12A exhibits high-temperature and slightly acidic hydrolysis, and its mutants exhibit excellent catalytic activity under pH stability conditions, potentially unlocking the potential of xyloglucanase in feed fermentation production and the food additive industry. This invention utilizes its plant immune activity for the first time, spraying it on plant foliage to induce plant resistance.

[0007] The xyloglucanase NfXYG12A with plant immune activity provided by the present invention is any one of the following A1)-A6):

[0008] A1) a protein whose amino acid sequence is positions 94-313 of SEQ ID NO. 1;

[0009] A2) a protein whose amino acid sequence is positions 94-313 of SEQ ID NO. 3;

[0010] A3) a protein whose amino acid sequence is positions 94-313 of SEQ ID NO. 5;

[0011] A4) a protein whose amino acid sequence is positions 94-313 of SEQ ID NO. 7;

[0012] A5) a protein having the same function as the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, or SEQ ID NO. 7, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0013] A6) A fusion protein obtained by linking a tag to the N-terminus or / and C-terminus of A1) or A2) or A3) or A4) or A5).

[0014] To facilitate purification of the proteins in A1) to A4), a tag as shown in the following table can be attached to the amino or carboxyl terminus.

[0015] Table: Sequence of tags

[0016] Label residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0017] The NfXYG12A protein in A5) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0018] The gene encoding the NfXYG12A protein in the above A5) can be obtained by deleting one or several codons for amino acid residues from the DNA sequence shown in positions 280-939 of SEQ ID NO.2 or positions 280-939 of SEQ ID NO.4 or positions 280-939 of SEQ ID NO.4, and / or performing a missense mutation of one or several base pairs, and / or linking the coding sequence of the tag shown in the above table to its 5′ and / or 3′ end. Among them, the DNA molecule shown at positions 280-939 of SEQ ID NO.2 encodes the protein shown at positions 94-313 of SEQ ID NO.1; the DNA molecule shown at positions 280-939 of SEQ ID NO.4 encodes the protein shown at positions 94-313 of SEQ ID NO.3; the DNA molecule shown at positions 280-939 of SEQ ID NO.6 encodes the protein shown at positions 94-313 of SEQ ID NO.5; and the DNA molecule shown at positions 280-939 of SEQ ID NO.8 encodes the protein shown at positions 94-313 of SEQ ID NO.7.

[0019] In an embodiment of the present invention, the protein described in A6) is shown as SEQ ID NO.1 or SEQ ID NO.3 or SEQ ID NO.5 or SEQ ID NO.7.

[0020] The present invention also provides a biomaterial related to NfXYG12A, wherein the biomaterial is any one of the following B1) to B7):

[0021] B1) a nucleic acid molecule encoding NfXYG12A;

[0022] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0023] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0024] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0025] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

[0026] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0027] B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2).

[0028] In the above-mentioned biological material, the nucleic acid molecule in B1) may be any one of the following b1) to b14):

[0029] b1) the coding sequence is the cDNA molecule or DNA molecule at positions 280-939 of SEQ ID NO. 2 in the sequence listing;

[0030] b2) the DNA molecule shown in positions 280-939 of SEQ ID NO. 2 in the sequence listing;

[0031] b3) the DNA molecule shown in SEQ ID NO.2 in the sequence listing;

[0032] b4) the coding sequence is the cDNA molecule or DNA molecule at positions 280-939 of SEQ ID NO. 4 in the sequence listing;

[0033] b5) the DNA molecule shown in positions 280-939 of SEQ ID NO. 4 in the sequence listing;

[0034] b6) the DNA molecule shown in SEQ ID NO.4 in the sequence listing;

[0035] b7) the coding sequence is the cDNA molecule or DNA molecule at positions 280-939 of SEQ ID NO.6 in the sequence listing;

[0036] b8) the DNA molecule shown in positions 280-939 of SEQ ID NO. 6 in the sequence listing;

[0037] b9) the DNA molecule shown in SEQ ID NO.6 in the sequence listing;

[0038] b10) the coding sequence is the cDNA molecule or DNA molecule at positions 280-939 of SEQ ID NO. 8 in the sequence listing;

[0039] b11) the DNA molecule shown in positions 280-939 of SEQ ID NO. 8 in the sequence listing;

[0040] b12) the DNA molecule shown in SEQ ID NO.8 in the sequence listing;

[0041] b13) a cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined in any one of b1) to b12) and encodes the protein of claim 1;

[0042] b14) A cDNA molecule or DNA molecule that hybridizes with the nucleotide sequence defined in any one of b1) to b12) under stringent conditions and encodes the protein of claim 1.

[0043] 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.

[0044] Those skilled in the art can readily mutate the nucleotide sequence encoding the NfXYG12A protein of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequence of the isolated NfXYG12A protein of the present invention are derived from and are equivalent to the nucleotide sequences of the present invention, as long as they encode the NfXYG12A protein and possess the function of the NfXYG12A protein.

[0045] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that are 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identical to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in positions 94-313 of SEQ ID NO.1, positions 94-313 of SEQ ID NO.3, positions 94-313 of SEQ ID NO.5, or positions 94-313 of SEQ ID NO.7. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0046] In the above biological materials, the stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 2×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 1×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 0.5×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 0.1×SSC, 0.1% Alternatively, the hybridization step can be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and then washing at 65°C in 0.1×SSC, 0.1% SDS; or: 6×SSC, 0.5% SDS solution, at 65 o The membranes were hybridized at 400 °C and then washed once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS. Alternatively, the membranes were hybridized and washed twice at 68°C for 5 min each in a 2×SSC, 0.1% SDS solution, and then hybridized and washed twice at 68°C for 15 min each in a 0.5×SSC, 0.1% SDS solution. Alternatively, the membranes were hybridized and washed at 65°C in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution.

[0047] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0048] In the above-mentioned biological materials, the expression cassette containing the nucleic acid molecule encoding the NfXYG12A protein (NfXYG12A gene expression cassette) described in B2) refers to DNA capable of expressing the NfXYG12A protein in a host cell. This DNA may include not only a promoter for initiating transcription of the NfXYG12A gene, but also a terminator for terminating transcription of the NfXYG12A gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0049] Existing expression vectors can be used to construct a recombinant vector containing the NfXYG12A gene expression cassette.

[0050] In the above biological materials, the vector may be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid may be pPIC9.

[0051] B3) The recombinant vector may specifically be pPIC9-NfXYG12A, pPIC9-N106S / S173A, pPIC9-N106S or pPIC9-S173A.

[0052] pPIC9-NfXYG12A is a recombinant plasmid obtained by replacing the DNA fragment between the EcoR I and Not I recognition sequences of the pPIC9 plasmid with the NfXYG12A gene, represented by positions 280-942 of SEQ ID No. 2 in the sequence listing. pPIC9-NfXYG12A contains the DNA fragment represented by SEQ ID No. 2 in the sequence listing and expresses NfXYG12A, represented by SEQ ID No. 1.

[0053] pPIC9-N106S / S173A is a recombinant plasmid obtained by replacing the gene represented by SEQ ID NO. 2 in pPIC9-NfXYG12A with the gene represented by SEQ ID NO. 4.

[0054] pPIC9-N106S is a recombinant plasmid obtained by replacing the gene shown in SEQ ID NO. 2 in pPIC9-NfXYG12A with the gene shown in SEQ ID NO. 6.

[0055] pPIC9-S173A is a recombinant plasmid obtained by replacing the gene shown in SEQ ID NO. 2 in pPIC9-NfXYG12A with the gene shown in SEQ ID NO. 8.

[0056] In the above-mentioned biological material, the microorganism can be yeast, bacteria, algae or fungi. Among them, the yeast can be Pichia pastoris GS115.

[0057] Among the above-mentioned biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include reproductive materials.

[0058] The use of NfXYG12A as xyloglucanase also falls within the protection scope of the present invention.

[0059] The present invention also provides NfXYG12A, or any of the following uses of the biomaterial:

[0060] M1, preparation of xyloglucanase;

[0061] M2, hydrolyzed xyloglucan;

[0062] M3, preparing hydrolyzed xyloglucan product;

[0063] M4, production of glucose;

[0064] M5. Preparation and production of glucose products;

[0065] M6, hydrolysis of cellulose or hemicellulose;

[0066] M7. Preparation of hydrolyzed cellulose or hemicellulose products;

[0067] M8, improve plant resistance to stress;

[0068] M9. Preparation of products for improving plant stress resistance;

[0069] M10, stimulate plant immunity to diseases and insect pests;

[0070] M11. Prepare products that stimulate plant immunity against diseases and insect pests.

[0071] The improvement of plant stress resistance can be reflected in the germination of seeds in a salt stress environment. The salt stress environment can be a growth environment with a salt concentration higher than that in a normal plant growth environment (such as NaCl with a mass percentage concentration of 0.5%).

[0072] The stimulation of plant immunity to pests and diseases can be manifested in the induction of plant cell necrosis, the induction of expression of defense-related genes (such as PTI and ETI pathway-related genes), and / or the induction of callose precipitation. The PTI and ETI pathway-related genes can be Pti5, Acre31, H1N1, and / or HSR203J genes.

[0073] In the above application, the adverse environment may be a salt stress environment.

[0074] The plant may be P1) or P2) or P3):

[0075] P1) dicots or monocots;

[0076] P2) Solanaceae;

[0077] P3) Tobacco.

[0078] The present invention also provides a method for hydrolyzing xyloglucan, which comprises: placing NfXYG12A in a reaction system containing xyloglucan to carry out a reaction, thereby achieving the hydrolysis of the xyloglucan.

[0079] In the above method, the pH of the reaction system may be 2-8. The reaction may be carried out at 30-80°C.

[0080] The pH of the reaction system may be 2-7 or 2-6 or 2-5 or 2-4.5 or 3-4.5 or 3.5-4.5.

[0081] The pH of the reaction system can specifically be 2, 3, 3.5, 4, 4.5, 5, 6, 7 or 8.

[0082] The reaction may be carried out at 40-70°C, or 50-70°C, or 55-70°C, or 60-70°C.

[0083] The reaction can be carried out at 30, 40, 50, 55, 60, 65, 70, or 80°C.

[0084] The present invention also provides a method for promoting seed germination under a salt stress environment, the method comprising: treating plant seeds with NfXYG12A to promote seed germination under a salt stress environment.

[0085] In the above method, the plant may be P1) or P2) or P3):

[0086] P1) dicots or monocots;

[0087] P2) Solanaceae;

[0088] P3) Tobacco.

[0089] Experiments have shown that the NfXYG12A of the present invention is a thermophilic, acidic xyloglucanase with excellent pH stability and catalytic activity. Compared with the original xyloglucanase NfXYG12A, the catalytic efficiency of the mutant is significantly increased. The xyloglucanase mutant of the present invention can be used for the hydrolysis of lignocellulose and improve the economic benefits of biomass raw material conversion. The NfXYG12A of the present invention can also be used as a plant immune elicitor and has high application value in enhancing plant immunity. The NfXYG12A of the present invention has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 Shown are the results of the optimal pH test for xyloglucanase rNfXYG12A and its mutants. WT represents rNfXYG12A, M2 represents rNfXYG12A-N106S / S173A, N106S represents rNfXYG12A-N106S, and S173A represents rNfXYG12A-S173A.

[0091] Figure 2 The results show the pH stability of xyloglucanase rNfXYG12A and its mutants. WT represents rNfXYG12A, M2 represents rNfXYG12A-N106S / S173A, N106S represents rNfXYG12A-N106S, and S173A represents rNfXYG12A-S173A.

[0092] Figure 3The results show the optimal temperature detection results of xyloglucanase rNfXYG12A and its mutants. WT represents rNfXYG12A, M2 represents rNfXYG12A-N106S / S173A, N106S represents rNfXYG12A-N106S, and S173A represents rNfXYG12A-S173A.

[0093] Figure 4 Shown are the temperature stability test results of xyloglucanase rNfXYG12A and its mutants at 65°C and 70°C. WT represents rNfXYG12A, M2 represents rNfXYG12A-N106S / S173A, N106S represents rNfXYG12A-N106S, and S173A represents rNfXYG12A-S173A.

[0094] Figure 5 Shown are the Tm and catalytic efficiency measurements of xyloglucanase rNfXYG12A and its mutants. A represents rNfXYG12A, B represents rNfXYG12A-N106S / S173A, C represents rNfXYG12A-N106S, and D represents rNfXYG12A-S173A.

[0095] Figure 6 Figure 2 shows varying degrees of cell necrosis after injection of different concentrations of the plant immune elicitor protein NfXYG1 into tobacco plants. NfXYG1-E116A is an enzyme-active mutant. The buffer is PBS containing MES, MgCl2, and AS.

[0096] Figure 7 This figure shows that the plant immunity elicitor protein NfXYG1 significantly stimulates the expression of PTI- and ETI-related marker genes. Panels A and B show tobacco leaves immersed in a protein solution; Panels C and D show tobacco leaves injected with the protein solution. EV denotes empty vector protein; NbPti5, NbAcre31, NbH1N1, and NbHSR203J are PTI- and ETI-related marker genes. * indicates a significant difference, p < 0.05; *** indicates a significant difference, p < 0.001.

[0097] Figure 8 Shown: Plant immune elicitor NfXYG1 protein induces callose precipitation in plant leaves. The top image shows injection of PBS buffer, while the bottom image shows injection of NfXYG1 protein solution. DETAILED DESCRIPTION

[0098] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0099] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. The quantitative tests in the following examples were all repeated three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0100] The Aspergillus fischeri P1 in the following examples was collected on September 3, 2013 at the General Microbiology Center of the China Culture Collection Administration Committee (referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101). The registration number of the collection center is CGMCC No. 3.15369. Since the date of collection, the public can obtain the strain from the General Microbiology Center of the China Culture Collection Administration Committee. The website address of CGMCC is: https: / / cgmcc.net / , and the public can directly order strains online. The website address of Aspergillus fischeri P1 is

[0101] https: / / cgmcc.net / directory / detail?cgmccid=3.15369&number=3.15369&genus=&specifics=&yiming=&page=1.

[0102] Both pPIC9 plasmid and Pichia pastoris GS115 were products of Invitrogen.

[0103] Example 1. Construction of NfXYG12A gene expression vector

[0104] Using the cDNA of Aspergillus fischeri P1 as template, the primers EG5-F (5'-GCC G AATTC GCCACCCAGTTCTGTGACCAATG-3') and EG5-R(5'-GCC GCGGCCGCTTAGGCAACACTGACAGAGTAG-3') was used to amplify the NfXYG12A gene to obtain a PCR product.

[0105] The obtained PCR product was double-digested with EcoR I and Not I to recover the large fragment; the obtained large fragment and the pPIC9 plasmid were double-digested with EcoR I and Not I, the digested PCR product and plasmid were ligated with T4 ligase, and the recombinant plasmid with the correct sequence was recorded as pPIC9-NfXYG12A.

[0106] pPIC9-NfXYG12A is a recombinant plasmid obtained by replacing the DNA fragment between the EcoR I and Not I recognition sequences of the pPIC9 plasmid with the NfXYG12A gene shown at positions 280-942 of SEQ ID No. 2 in the sequence listing. pPIC9-NfXYG12A contains the DNA fragment shown in SEQ ID No. 2 in the sequence listing (denoted as the rNfXYG12A fusion gene). Positions 1-279 of SEQ ID No. 2 represent the fragment on the pPIC9 plasmid, and positions 280-942 represent the NfXYG12A gene. pPIC9-NfXYG12A can express the rNfXYG12A fusion protein shown in SEQ ID No. 1.

[0107] The rNfXYG12A fusion gene encodes the rNfXYG12A fusion protein shown in SEQ ID No.1, positions 1-93 of SEQ ID No.1 are encoded by positions 1-279 of SEQ ID No.2, and positions 94-313 of SEQ ID No.1 are the NfXYG12A protein encoded by the NfXYG12A gene.

[0108] Example 2: Construction of gene mutation vector

[0109] Using pPIC9-NfXYG12A obtained in Example 1 as a template, a first round of PCR was performed using primers N106S-F and N106S-R, and S173A-F and S173A-R, respectively, to obtain two PCR products. A second round of PCR was then performed using these two PCR products as primers and pPIC9-NfXYG12A as a template to obtain recombinant plasmids of each mutant (N106S / S173A).

[0110] The mutation primers are as follows:

[0111] N106S-F: 5'-GACTCGACTTGGACGTGGAGCTACTCTGGCTCC-3';

[0112] N106S-R: 5'-GCTCCACGTCCAAGTCGAGTCAATGCTCTTGAC-3';

[0113] S173A-F: 5'-CCTCGGCCCGAACGGCGCGATGCAGGTGTAC-3';

[0114] S173A-R: 5'-CGCCGTTCGGGCCGAGGTACAGGTTAAAC-3'.

[0115] The second-round PCR products were electrophoresed on a 1% agarose gel. The target band was excised and purified, dissolved in 25 μl of ddH₂O, treated with DMT enzyme at 37°C for 2 h, and transformed into DMT-competent cells (Beijing Zhuangmeng International Biogene Technology Co., Ltd.). Positive transformants were screened on solid LB medium plates containing 100 μg / ml ampicillin. Single clones were selected and cultured overnight on 600 μl of liquid LB medium containing 100 μg / ml ampicillin. Sequencing confirmed the presence of recombinant bacteria containing the recombinant plasmids pPIC9-N106S / S173A, pPIC9-N106S, and pPIC9-S173A. Plasmids were extracted from each recombinant strain to obtain the recombinant plasmids pPIC9-N106S / S173A, pPIC9-N106S, and pPIC9-S173A, respectively.

[0116] Among them, the recombinant plasmid pPIC9-N106S / S173A is a recombinant plasmid obtained by replacing the rNfXYG12A gene in pPIC9-NfXYG12A with the rNfXYG12A-N106S / S173A gene (SEQ ID NO.4), the rNfXYG12A-N106S / S173A gene is a DNA fragment obtained by mutating aac at positions 541-543 of the rNfXYG12A fusion gene to agc and mutating tcg at positions 742-744 to GCG, and the rNfXYG12A-N106S / S173A gene encodes the rNfXYG12A-N106S / S173A protein shown in SEQ ID NO.3.

[0117] The recombinant plasmid pPIC9-N106S is a recombinant plasmid obtained by replacing the rNfXYG12A gene in pPIC9-NfXYG12A with the rNfXYG12A-N106S gene (SEQ ID NO.6). The rNfXYG12A-N106S gene is a DNA fragment obtained by mutating aac at positions 541-543 of the rNfXYG12A fusion gene to agc. The rNfXYG12A-N106S gene encodes the rNfXYG12A-N106S protein shown in SEQ ID NO.5.

[0118] The recombinant plasmid pPIC9-S173A is a recombinant plasmid obtained by replacing the rNfXYG12A gene in pPIC9-NfXYG12A with the rNfXYG12A-S173A gene (SEQ ID NO.8). The rNfXYG12A-S173A gene is a DNA fragment obtained by mutating the tcg at positions 742-744 of the rNfXYG12A fusion gene to gcg. The rNfXYG12A-S173A gene encodes the rNfXYG12A-S173A protein shown in SEQ ID NO.7.

[0119] Example 3. Preparation of recombinant bacteria

[0120] pPIC9-NfXYG12A, pPIC9-N106S / S173A, pPIC9-N106S and pPIC9-S173A obtained in Examples 1 and 2 were linearized using the restriction endonuclease SacI (ThermoFisher Scientific FD1133) and then operated according to the following steps: the linearized plasmid was dissolved in 10 μL of sterile water, 80 μL of Pichia pastoris GS115 competent cells were mixed therewith, electroporated, spread on MD plates, and cultured in an incubator at 30°C for 2 days. Transformants with xyloglucanase activity (i.e., recombinant bacteria) were screened from all transformants.

[0121] The recombinant bacteria obtained from pPIC9-NfXYG12A, pPIC9-N106S / S173A, pPIC9-N106S and pPIC9-S173A were named GS115 / pPIC9-NfXYG12A, GS115 / pPIC9-N106S / S173A, GS115 / pPIC9-N106S and GS115 / pPIC9-S173A, respectively.

[0122] The pPIC9 plasmid was used as an empty vector control, and the recombinant bacteria obtained from the pPIC9 plasmid were designated as GS115 / pPIC9.

[0123] Example 4: Cultivation of recombinant bacteria and protein purification

[0124] The five recombinant bacteria obtained in Example 3 were used as test bacteria, and the xyloglucanase activity was detected according to the following steps:

[0125] Cultivation of the test strain: Cultivate the recombinant strain in 100 ml YPD medium in a 100 ml shake flask at 30°C, 220 rpm for 16 h. Inoculate the recombinant strain into 400 ml of BMGY medium in a 1 L Erlenmeyer flask at a ratio of 1:100. Incubate the culture at 30°C, 220 rpm, and shake for 48 h. Centrifuge the culture (6000 rpm, 10 min), discard the supernatant, and resuspend the cells in 200 ml of BMMY medium. Incubate the culture at 30°C, 220 rpm, and shake for 48 h. Add 2 ml of methanol every 12 h.

[0126] After the incubation period, the supernatant was collected and protein purification was performed as follows: ion exchange chromatography was used. The supernatant was centrifuged at 12,000 rpm for 10 minutes, and the cells were discarded to obtain a crude enzyme solution. The enzyme solution was then concentrated by ultrafiltration using a 30 kDa membrane. The concentrated enzyme solution was then dialyzed overnight against phosphate-citrate buffer for desalination. The treated enzyme solution was then applied to an equilibrated HiTrap QXY anion column and eluted using a 0-1 mol / L NaCl gradient. The protein corresponding to the peak was collected. GS115 / pPIC9-NfXYG12A, GS115 / pPIC9-N106S / S173A, GS115 / pPIC9-N106S and GS115 / pPIC9-S173A obtained rNfXYG12A fusion protein, rNfXYG12A-N106S / S173A protein, rNfXYG12A-N106S protein and rNfXYG12A-S173A protein, respectively; GS115 / pPIC9 obtained empty vector protein (EV).

[0127] Example 5. Detection of protein xyloglucanase properties

[0128] The activity unit (U) of xyloglucanase is defined as the amount of enzyme required to decompose xyloglucan to produce 1 μm old-(+)-glucose per minute under given conditions.

[0129] The rNfXYG12A fusion protein, rNfXYG12A-N106S / S173A fusion protein, rNfXYG12A-N106S fusion protein and rNfXYG12A-S173A fusion protein purified in Example 4 were used as test samples to detect the optimal pH, pH stability, optimal temperature and temperature stability of the proteins.

[0130] (1) Optimum pH

[0131] The xyloglucanase activity of the four purified proteins at pH 2–8 was tested to determine the optimal pH for each protein. The reaction system for enzyme activity was set to 500 μL:

[0132] Add 400 μL of 0.5% xyloglucanase substrate (i.e. xyloglucan, Megazyme, Ireland) solution (diluted with buffer of corresponding pH) to a glass test tube and preheat at 75°C for 2 min. Add 100 μL of citric acid-disodium hydrogen phosphate (McIlvaine) buffer of different pH to dilute the pure enzyme in appropriate multiples. Set up 3 parallels and 1 control for each pH treatment (DNS was added to the control first and then the enzyme solution was added). React at 75°C for 10 min. After removing the test tube, add 1.5 mL of DNS reagent to terminate the reaction. Add 100 μL of buffer of corresponding pH to the control and boil in water for 5 min. After cooling, measure the OD value with the increase of glucan as the indicator. 540 The absorbance value at nm wavelength was calculated. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated as shown in Table 1. The relative enzyme activity curves of the four enzymes were plotted as shown in Table 1. Figure 1 .

[0133] Citric acid-sodium hydrogen phosphate buffers of different pH values ​​were prepared by mixing 0.1 M citric acid solution and 0.2 M sodium hydrogen phosphate in different volumes.

[0134] Table 1. Optimum pH test results

[0135] pH rNfXYG12A rNfXYG12A-N106S / S173A rNfXYG12A-N106S rNfXYG12A-S173A 2.0 11.82％ 12.34％ 14.49％ 16.64％ 3.0 67.23％ 58.95％ 62.77％ 71.95％ 3.5 96.66％ 88.77％ 85.84％ 100.00％ 4.0 100.00％ 95.02％ 94.16％ 95.75％ 4.5 99.09％ 100.00％ 100％ 82.34％ 5.0 91.37％ 98.21％ 86.56％ 14.52％ 6.0 31.26％ 27.21％ 27.78％ 6.33％ 7.0 8.57％ 8.21％ 11.57％ 4.99％ 8.0 7.86％ 8.05％ 11.13％ 16.64％

[0136] The results showed that the optimal pH values ​​of rNfXYG12A fusion protein, rNfXYG12A-N106S / S173A fusion protein, rNfXYG12A-N106S fusion protein and rNfXYG12A-S173A fusion protein were 4.0, 4.5, 4.5 and 3.5, respectively.

[0137] (2) pH stability

[0138] The xyloglucanase activity of the four proteins obtained in step 4 was tested after incubation at pH 2–10 to determine the pH stability of each protein. The reaction system for enzyme activity was set to 500 μL:

[0139] The buffers used were: McIlvaine buffers at pH 2, 3, 3.5, 4, 4.5, 5, 6, 7, and 8, 0.1 mol / L Tris-HCl buffer at pH 9, and Gly-NaOH buffer at pH 10. The four pure enzymes were diluted 10-fold with different pH buffers and incubated in a 37°C water bath for 1 hour to obtain the enzyme solution to be tested. The enzyme solution to be tested was diluted to an appropriate multiple, and the enzyme activity was determined by referring to the enzyme activity detection method in (1). The enzyme activity of the unincubated pure enzyme at 75°C for 10 minutes was set as 100%, and the relative enzyme activity was calculated, as shown in Table 2. The relative enzyme activity curve was drawn, as shown in Table 2. Figure 2 .

[0140] Table 2. pH stability test results

[0141]

[0142] The results showed that the pH stability of rNfXYG12A fusion protein, rNfXYG12A-N106S / S173A fusion protein, rNfXYG12A-N106S fusion protein and rNfXYG12A-S173A fusion protein remained basically unchanged, and they all had good stability in a wide range of pH 2-8. When the pH was greater than 9, the pH stability dropped sharply. Compared with the rNfXYG12A fusion protein, the enzymatic activity of the rNfXYG12A-N106S protein under acidic conditions (pH = 4.5) was improved.

[0143] (3) Optimum temperature

[0144] The xyloglucanase activity of the four purified proteins was tested in the temperature range of 30-80°C to determine the optimal temperature for each protein. The reaction system for enzyme activity was set to 500 μL:

[0145] The four pure enzymes were diluted to appropriate multiples using McIlvaine buffer at pH 4.0. The xyloglucanase activity was detected at 30-80°C according to the enzyme activity detection method in (1). The relative enzyme activity was calculated with the highest enzyme activity as 100%, as shown in Table 3, and the relative enzyme activity curve was plotted as shown in Table 3. Figure 3 .

[0146] Table 3. Optimum temperature test results

[0147]

[0148] The results showed that the optimum temperature of rNfXYG12A fusion protein, rNfXYG12A-N106S / S173A fusion protein, rNfXYG12A-N106S fusion protein and rNfXYG12A-S173A fusion protein was 65℃. At 60℃, the enzyme activity of rNfXYG12A-S173A protein was significantly increased compared with that of rNfXYG12A fusion protein.

[0149] (4) Temperature stability

[0150] Take 1 mL of each of the four enzyme solutions (4 pure enzymes diluted with McIlvaine buffer at pH 4.0) and place them in 1.5 mL centrifuge tubes. Incubate 7 tubes of each enzyme at 65°C and 70°C for 5 min, 10 min, 20 min, 30 min, and 60 min, respectively. Use the untreated enzyme solution (treatment time is 0) as a control. After the incubation, refer to the enzyme activity detection method in (1) to determine the xyloglucanase activity of the enzyme solutions with different treatments. The enzyme activity of the four untreated enzyme solutions was taken as 100%, and the relative enzyme activity of each treatment was calculated, as shown in Table 4. Draw a relative enzyme activity curve, and the results are shown in Table 4. Figure 4 .

[0151] Table 4. Test results of temperature stability

[0152]

[0153]

[0154] In Table 4, “ / ” indicates not detected.

[0155] The results showed that the wild-type (i.e., rNfXYG12A fusion protein) maintained a relative residual enzyme activity of approximately 80% after treatment at 65°C for 90 minutes, and approximately 55% after treatment at 70°C for 10 minutes. The rNfXYG12A-N106S fusion protein and rNfXYG12A-S173A fusion protein maintained a relative residual enzyme activity of approximately 45% after treatment at 65°C for 90 minutes, and essentially lost their enzyme activity after treatment at 70°C for 60 minutes. The results of the different treatment times indicate the following thermal stability effects:

[0156] rNfXYG12A>rNfXYG12A-S173A>rNfXYG12A-N106S>rNfXYG12A-N106S / S173A.

[0157] Example 6: Determination of enzyme kinetic parameters

[0158] The proteins to be tested were the rNfXYG12A fusion protein, rNfXYG12A-N106S / S173A fusion protein, rNfXYG12A-N106S fusion protein and rNfXYG12A-S173A fusion protein purified in Example 4.

[0159] The first-order reaction time of the reaction was determined, and the reaction time of Km (Km value is the substrate concentration at half the maximum speed of the enzymatic reaction) and Vmax was determined to be 5 min. Xyloglucan solutions of 5 mg / ml, 4 mg / ml, 3 mg / ml, 2.5 mg / ml, 2 mg / ml, 1.25 mg / ml, and 1 mg / ml were prepared using the optimal pH citric acid-sodium hydrogen phosphate buffer for each enzyme. After incubation at the optimal temperature for 5 min, the enzyme activities of the four fusion proteins were detected under the optimal pH buffer conditions according to the enzyme activity determination method in Example 5 (1), and the corresponding reaction speeds were calculated. The Km value and Vmax were calculated using the double reciprocal plotting method using GraphPad Prism5 software. The catalytic efficiency determination data showed that the catalytic efficiency of each protein was rNfXYG12A-N106S / S173A>rNfXYG12A-N106S>rNfXYG12A>rNfXYG12A-S173A.

[0160] Table 4. Enzyme kinetic parameters

[0161]

[0162] A standard curve was constructed using the Bio-Rad kit protocol. The assay method: First, the target protein content was calculated using the standard curve. Next, the recombinant enzyme activity was measured under optimal conditions. The specific activity of the enzyme was calculated by dividing the activity by the protein concentration. Specific activity is defined as the number of units of enzyme activity per milligram of enzyme protein.

[0163] The specific activities of the rNfXYG12A fusion protein, rNfXYG12A-N106S / S173A fusion protein, rNfXYG12A-N106S fusion protein, and rNfXYG12A-S173A fusion protein were 217.00, 754.85, 389.75g, and 401.12 U / mg, respectively. This indicates that compared with the rNfXYG12A fusion protein, the enzyme activities of the three mutants (rNfXYG12A-N106S / S173A fusion protein, rNfXYG12A-N106S fusion protein, and rNfXYG12A-S173A fusion protein) were significantly improved.

[0164] Example 7, Tm and catalytic efficiency determination

[0165] The Tm value of each protein was determined using differential scanning calorimetry (DSC). The test proteins were the rNfXYG12A fusion protein, rNfXYG12A-N106S / S173A fusion protein, rNfXYG12A-N106S fusion protein, and rNfXYG12A-S173A fusion protein purified in Example 4.

[0166] The thermal denaturation process of the four fusion proteins was analyzed using a GE Microcal Capillary DSC instrument. The measured data were processed by subtracting the calorimetric values ​​of the corresponding buffer and normalizing the protein samples to molar concentrations.

[0167] The results are as follows Figure 5 The Tm values ​​of rNfXYG12A fusion protein, rNfXYG12A-N106S / S173A fusion protein, rNfXYG12A-N106S fusion protein, and rNfXYG12A-S173A fusion protein were 57.22, 55.09, 55.77, and 54.88°C, respectively. The Tm value of the NfXYG12A protein decreased by 2 to 3°C after mutation, and the thermal stability of these mutant proteins was lower than that of the wild-type enzyme.

[0168] Example 8: Plant immune elicitor rNfXYG12A can induce tobacco necrosis

[0169] The purified protein rNfXYG12A obtained in Example 4 was named plant immune elicitor protein NfXYG1, and 5 different concentrations of 500nM, 1μM, 5μM, 10μM, and 20μM were set. 20μM of the purified enzyme-active mutant protein NfXYG1-E116A was used as a positive control. Each protein was dissolved to the corresponding concentration using PBS buffer containing MES, MgCl2, and AS, and a PBS buffer containing MES, MgCl2, and AS (acetosyringone) was used as a blank control. The PBS buffer containing MES, MgCl2, and AS consists of a solvent and a solute, the solvent being PBS buffer, and the solute and its concentration in the resulting solution being MES1mol / L, MgCl21mol / L, and AS1mol / L, respectively.

[0170] Select the middle portion of a 5- to 6-leaf tobacco leaf, which is actively growing. Use a 1 mL syringe without a needle to inject 20 μL of the protein at the indicated concentration into the underside of the leaf. After 36 hours, observe the leaf for cell necrosis and take photos.

[0171] Results: As Figure 6As shown in the results, NfXYG1 protein at a concentration of 500nM can still induce necrosis in tobacco cells, and the enzyme activity mutant NfXYG1-E116A also triggers necrosis in tobacco cells, indicating that high enzyme activity has no effect on inducing necrosis in tobacco cells, and low concentrations of NfXYG1 protein can cause typical plant cell necrosis.

[0172] Example 9: Plant immunity elicitor NfXYG1 increases the expression level of tobacco defense response-related genes

[0173] The purified protein rNfXYG12A obtained in Example 4 was named plant immune elicitor protein NfXYG1, and was dissolved in PBS buffer to obtain a protein solution with a concentration of 10 μM; a 1 mL needleless syringe was used to select the middle leaves of tobacco in the 5-6 leaf stage with vigorous growth, and 20 μL of protein solution was injected from the back or immersed in the protein solution. PBS buffer was used as a blank control, and the empty vector protein (EV) obtained in Example 4 with the same concentration was used as a negative control. 12 hours after the injection, the leaves in the injection area were collected, RNA was extracted, reverse transcribed into cDNA, and the expression levels of defense response-related genes were detected by RT-PCR. The internal references were NbPti5, NbAcre31, NbH1N1, and NbHSR203J.

[0174] Results: In tobacco leaves injected with the plant immunity elicitor NfXYG1, the expression of PTI and ETI pathway-related genes Pt i5, Acre31, H1N1, and HSR203J were induced to a certain extent. The above results indicate that NfXYG1 can effectively increase the expression of tobacco defense-related genes. Figure 7 shown.

[0175] Using a 1 mL needleless syringe, 20 μL of the protein solution obtained above was injected from the back into the middle of a 5- to 6-leaf tobacco leaf, which was actively growing. PBS buffer was used as a blank control. Two days after injection, the injected leaves were washed with deionized water, decolorized with 95% ethanol, and the residual ethanol was rinsed with deionized water. The leaves were then placed in 150 mM phosphate buffer (pH 9.5) containing 0.1% (w / v) aniline blue for 2 hours in the dark. The leaves were then removed and observed and photographed using a fluorescence microscope.

[0176] Results: In tobacco leaves injected with plant immune elicitor NfXYG1, a large number of blue spots appeared in the injected area; in tobacco leaves injected with PBS buffer, only a few blue spots appeared in the injected area. The above results indicate that plant immune elicitor NfXYG1 can induce callose precipitation in tobacco leaves. Figure 8 shown.

[0177] Example 10: Plant immunity elicitor NfXYG1 can promote early germination rate of seeds

[0178] To further verify the feasibility of the application of NfXYG1 protein, rice seeds were treated with different concentrations of NfXYG1 protein as follows:

[0179] Rice seeds were soaked. For each treatment, 100 plump, healthy seeds were randomly selected and disinfected in a 1% sodium hypochlorite solution for 30 minutes. The seeds were then rinsed several times with distilled water and dried. A Petri dish filter paper method was used to test rice seed germination. Three different concentrations of NfXYG1 protein (0.15 mg / L, 0.30 mg / L, and 0.60 mg / L) were added to the treated rice seedlings in an isotropic NaCl solution (0.5% NaCl by mass, water as the solvent). Three different concentrations of Alternaria tenuis protein (Beijing Zhongbao Green Agriculture Technology Group Co., Ltd.) (0.15 mg / L, 0.30 mg / L, and 0.60 mg / L) were used as negative controls, while an isotropic saline solution was used as a positive control. Germination rates were observed 48 and 96 hours after treatment.

[0180] The results are shown in Table 5.

[0181] Table 5. Plant immunity elicitor NfXYG1 improves seed germination rate under salt stress

[0182]

[0183] Note: In Table 5, in the same column of data, the differences between the data marked with the same lowercase letters did not reach the significant level (p≥0.05), and the differences between the data marked with different lowercase letters reached the significant level (p<0.05).

[0184] The results showed that 48h and 96h after treatment, the germination rate of seeds was significantly increased when the NfXYG1 protein was treated at a concentration of 0.60mg / L, indicating that the plant immune elicitor NfXYG1 has significant stress resistance and can promote the early germination rate of seeds under salt stress.

[0185] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0186] The present invention relates to the following sequence:

[0187] The amino acid sequence of rNfXYG12A is shown in SEQ ID NO.1:

[0188]

[0189] The gene sequence of rNfXYG12A is shown in SEQ ID NO.2

[0190] The amino acid sequence of rNfXYG12A-N106S / S173A is shown in SEQ ID NO.3:

[0191] The gene sequence of rNfXYG12A-N106S / S173A is shown in SEQ ID NO.4:

[0192] The amino acid sequence of rNfXYG12A-N106S is shown in SEQ ID NO.5:

[0193] The gene sequence of rNfXYG12A-N106S is shown in SEQ ID NO.6:

[0194]

[0195]

[0196] The amino acid sequence of rNfXYG12A-S173A is shown in SEQ ID NO.7:

[0197] The gene sequence of rNfXYG12A-S173A is shown in SEQ ID NO.8:

Claims

1. Protein, any one of the following A1) to A4): A1) a protein having an amino acid sequence of positions 94-313 of SEQ ID NO. 3; A2) a protein whose amino acid sequence is positions 94-313 of SEQ ID NO. 5; A3) a protein whose amino acid sequence is positions 94-313 of SEQ ID NO. 7; A4) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of A1), A2), or A3).

2. The biological material related to the protein according to claim 1, which is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule is any one of the following b1) to b9): b1) The coding sequence is the cDNA molecule or DNA molecule at positions 280-939 of SEQ ID NO. 4 in the sequence listing; b2) the DNA molecule shown in positions 280-939 of SEQ ID NO. 4 in the sequence listing; b3) the DNA molecule shown in SEQ ID NO.4 in the sequence listing; b4) The coding sequence is the cDNA molecule or DNA molecule at positions 280-939 of SEQ ID NO. 6 in the sequence listing; b5) the DNA molecule shown in positions 280-939 of SEQ ID NO. 6 in the sequence listing; b6) the DNA molecule shown in SEQ ID NO.6 in the sequence listing; b7) The coding sequence is the cDNA molecule or DNA molecule at positions 280-939 of SEQ ID NO. 8 in the sequence listing; b8) the DNA molecule shown in positions 280-939 of SEQ ID NO. 8 in the sequence listing; b9) The DNA molecule shown in SEQ ID NO. 8 in the sequence listing.

4. Use of the protein according to claim 1 as xyloglucanase.

5. Any of the following uses of the protein according to claim 1 or the biomaterial according to claim 2 or 3: M1, preparation of xyloglucanase; M2, hydrolyzed xyloglucan; M3, preparing hydrolyzed xyloglucan product; M4, production of glucose; M5. Preparation and production of glucose products; M6, hydrolysis of cellulose or hemicellulose; M7. Preparation of hydrolyzed cellulose or hemicellulose products.

6. Any of the following uses of a protein or a biological material related to the protein: M8, improve rice salt tolerance; M9, preparing rice salt tolerance products; M10, stimulates the immune activity of tobacco; M11. Preparation of products that stimulate tobacco immune activity; The protein is as follows (C1) or (C2): A1) a protein having an amino acid sequence of positions 94-313 of SEQ ID NO. 1; C2) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of C1); The biological material related to the protein is any one of the following D1) to D4): D1) a nucleic acid molecule encoding the protein; D2) an expression cassette containing the nucleic acid molecule described in D1); D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3).

7. The use according to claim 6, characterized in that: D1) The nucleic acid molecule is any one of the following d1) to d3): b1) The coding sequence is the cDNA molecule or DNA molecule at positions 280-939 of SEQ ID NO. 2 in the sequence listing; b2) the DNA molecule shown in positions 280-939 of SEQ ID NO. 2 in the sequence listing; b3) The DNA molecule shown in SEQ ID NO. 2 in the sequence listing.

8. A method for hydrolyzing xyloglucan, comprising: The protein according to claim 1 is placed in a reaction system containing xyloglucan to carry out a reaction, thereby achieving hydrolysis of the xyloglucan.

9. The method according to claim 8, characterized in that: The pH of the reaction system is 2-8; and / or the reaction is carried out at 30-80°C.

10. A method for promoting rice seed germination under salt stress, comprising: Treating rice seeds with protein to promote germination under salt stress; The protein is as follows (C1) or (C2): A1) a protein having an amino acid sequence of positions 94-313 of SEQ ID NO. 1; C2) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of C1).

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