Application of substance for inhibiting BC17 protein in cultivation of brittle-stalk plant material

Abstract

The invention discloses application of a substance for inhibiting BC17 protein in cultivation of a brittle-stalk plant material. The invention further discloses application of the BC17 protein to regulation and control of plant stalk brittleness and / or mechanical strength and / or breaking force and regulation and control of plant cell wall thickness and cell Young modulus. By knocking out BC17 in rice, the invention finds that the stalk breaking force of the BC17 mutant is reduced and the three-point bending resistance is reduced. The further analysis on the cellular level shows that the function loss of the BC17 can cause the thinning of the cell wall of the fiber cell, the increase of Young modulus and the enhancement of the brittleness of the stem. In addition, the yield is not obviously influenced while the stalks become brittle due to function deficiency of the BC17. The invention discovers that BC17-mediated acetylation modification regulation plays an important role in stalk brittleness regulation for the first time, provides a gene resource and an innovative approach for cultivating grain-stalk dual-purpose high-quality rice varieties, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of a substance that inhibits BC17 protein in cultivating brittle-stalk plant materials. Background Art

[0002] How to realize the high value and resource utilization of straw, a major byproduct of crops, is an important theme in today's agriculture. Traditional rice stalks are tough and not easy to break or degrade, which is a key constraint on the high value utilization of straw. Most materials that improve the brittleness of straw have also been affected in terms of yield and quality. Therefore, exploring rice materials that can be used for both rice and straw has important production and ecological significance.

[0003] Straw is mainly composed of cell walls. As the basic structural unit of straw, the cell wall is mainly composed of cellulose, hemicellulose and other polysaccharides cross-linked to form a network structure, which constitutes the structural basis of plant load-bearing. Therefore, genetic improvement of cell wall polysaccharides is an important way to optimize the mechanical properties of straw.

[0004] Xylan, as the main hemicellulose of grasses, is the core of polysaccharide cross-linking and plays a bridging role in the cross-linking of cellulose and lignin. It has important regulatory significance for the control of the higher-order structure and mechanical properties of the cell wall. Therefore, it is an important polysaccharide target for genetic improvement of the mechanical properties of the cell wall. The side chain structure of xylan is complex, among which acetylation modification plays an important regulatory role in its conformation and polysaccharide cross-linking. However, there have been no reports on controlling the brittleness of stems by modifying xylan and its acetylation modification. Discovering the key xylan acetylation genes that control the mechanical properties of stems will provide important molecular targets and innovative approaches for obtaining brittle stalk genetic materials that can be used for both cereal and grain purposes through genetic modification. Summary of the invention

[0005] The purpose of the invention is to improve the brittleness of plant stems and cultivate brittle-stalk plant materials.

[0006] In a first aspect, the present invention claims new uses of BC17 protein or related biological materials.

[0007] The present invention claims the use of BC17 protein or its related biological materials in any of the following 1)-7):

[0008] 1) Regulate the fragility of plant stems;

[0009] 2) Regulate the mechanical strength of plant stems;

[0010] 3) Regulate the breaking force of plant stems;

[0011] 4) Regulate the thickness of plant cell walls;

[0012] 5) Regulate the Young's modulus of plant cells;

[0013] 6) Cultivating transgenic plants with reduced stem brittleness and / or increased stem mechanical strength and / or increased stem breaking force and / or increased cell wall thickness and / or reduced cell Young's modulus;

[0014] 7) Plant breeding;

[0015] The BC17 protein is any one of the following proteins (a1)-(a4):

[0016] (a1) the protein shown in SEQ ID No. 2;

[0017] (a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (a1);

[0018] (a3) a protein related to plant stem fragility and / or mechanical strength and / or breaking force obtained by substituting and / or deleting and / or adding one or more amino acid residues in (a1);

[0019] (a4) A protein having 98% or more identity with (a1) and associated with plant stem brittleness and / or mechanical strength and / or breaking force.

[0020] In the protein described in (a2) above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, 6His tag protein, MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0021] In the protein described in (a3) ​​above, the substitution and / or deletion and / or addition of one or several amino acid residues is substitution and / or deletion and / or addition of no more than 10 amino acid residues, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or substitution and / or deletion and / or addition of no more than 2 amino acid residues, or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0022] In the protein described in (a4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be calculated.

[0023] In the above application, the relevant biological material is a nucleic acid molecule encoding the BC17 protein or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule.

[0024] Furthermore, the nucleic acid molecule encoding the BC17 protein may be a DNA molecule as described in any one of (A1) or (A2):

[0025] (A1) a DNA molecule represented by SEQ ID No. 1 or SEQ ID No. 3;

[0026] (A2) A DNA molecule that has 75% or more identity with (A1) and encodes the BC17 protein.

[0027] Those skilled in the art can easily mutate the nucleotide sequence encoding the BC17 protein of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the BC17 nucleotide sequence isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the BC17 protein and have the same function.

[0028] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No.2. Identity can be evaluated by the naked eye or by 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.

[0029] The expression cassette refers to a DNA capable of expressing BC17 protein in a host cell, and the DNA may include not only a promoter for initiating BC17 transcription, but also a terminator for terminating BC17 transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0030] The vector may be a plasmid, a phage, a cosmid, a Ti plasmid or a viral vector.

[0031] In practical applications, existing plant expression vectors can be used to construct a recombinant expression vector containing the BC17 gene. The plant expression vectors include but are not limited to binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. The plant expression vectors may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression.

[0032] When the BC17 gene is used to construct a recombinant plant expression vector, any enhancing promoter or constitutive promoter can be added before its transcription start nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the ubiquitin promoter of corn. They can be used alone or in combination with other plant promoters. In addition, when the gene of the present invention is used to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence.

[0033] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0034] By using any vector that can guide the expression of foreign genes in plants, the BC17 gene or gene fragment provided by the present invention is introduced into plant cells or recipient plants, and transgenic cell lines and transgenic plants with altered stem brittleness can be obtained. The expression vector carrying the BC17 gene can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues can be cultivated into plants.

[0035] The microorganism may be yeast, bacteria, algae or fungi; the bacteria may be Agrobacterium (such as Agrobacterium EHA105).

[0036] In the above application, the regulating the brittleness of the plant stems is to reduce the brittleness of the plant stems.

[0037] The regulating the mechanical strength of the plant stems is to increase the mechanical strength of the plant stems.

[0038] The regulating the breaking force of the plant stem is to increase the breaking force of the plant stem.

[0039] The regulating the thickness of the plant cell wall is to increase the thickness of the plant cell wall, specifically to increase the thickness of the cell wall of the plant fiber cells.

[0040] The regulating the Young's modulus of the plant cells is to reduce the Young's modulus of the plant cells, specifically to reduce the Young's modulus of the plant fiber cells.

[0041] In a second aspect, the present invention claims a new use of a substance that inhibits the above-mentioned BC17 protein.

[0042] The present invention claims the use of a substance that inhibits the above-mentioned BC17 protein in any one of the following (d1)-(d7):

[0043] (d1) increasing the brittleness of plant stems;

[0044] (d2) reduce the mechanical strength of plant stems;

[0045] (d3) reducing the breaking force of plant stems;

[0046] (d4) reducing the thickness of plant cell walls;

[0047] (d5) increasing the Young's modulus of plant cells;

[0048] (d6) cultivating transgenic plants with increased stem fragility and / or reduced stem mechanical strength and / or reduced stem breaking force and / or reduced cell wall thickness and / or increased cell Young's modulus;

[0049] (d7) Plant breeding.

[0050] Furthermore, the substance that inhibits the above-mentioned BC17 protein may be a substance that inhibits the activity of the above-mentioned BC17 protein, or a substance that inhibits the expression of the gene encoding the above-mentioned BC17 protein, or a substance that knocks out the gene encoding the above-mentioned BC17 protein.

[0051] The substance that inhibits the activity of the above-mentioned BC17 protein can be any substance that can cause the loss of the activity of the above-mentioned BC17 protein in plants, such as proteins, polypeptides or small molecule compounds (such as protein activity inhibitors) that inhibit the synthesis of the above-mentioned BC17 protein or promote the degradation of the above-mentioned BC17 protein or inhibit the function of the above-mentioned BC17 protein.

[0052] The substance that inhibits the expression of the gene encoding the above-mentioned BC17 protein can be any substance that can prevent the gene encoding the above-mentioned BC17 protein from being expressed in plants, such as a substance that silences the gene encoding the above-mentioned BC17 protein in plants (such as miRNA, siRNA, dsRNA, shRNA, etc.).

[0053] The substance for knocking out the gene encoding the BC17 protein can be a substance that achieves the host cell not producing the functional protein product of the BC17 gene in any way, such as removing all or part of the coding gene sequence, introducing a frameshift mutation so that no functional protein is produced, removing or changing the regulatory component (such as promoter editing) so that the coding gene sequence is not transcribed, and preventing translation by binding to mRNA. Usually, the knockout is performed at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout. Furthermore, the substance for knocking out the gene encoding the BC17 protein can be any substance that can cause the gene encoding the BC17 protein in the plant to mutate (the mutation form can be a deletion mutation and / or an insertion mutation and / or a base substitution) and thereby lose activity, such as a zinc finger protein ZFN gene editing system or a TALENs gene editing system or a CRISPR / Cas9 gene editing system, etc.

[0054] Furthermore, the substance for knocking out the gene encoding the BC17 protein is the CRISPR / Cas9 gene editing system.

[0055] In any of the above applications, reducing the thickness of plant cell walls is reducing the thickness of plant fiber cell walls.

[0056] The method of increasing the Young's modulus of plant cells is to increase the Young's modulus of plant fiber cells.

[0057] In a third aspect, the present invention claims a method for cultivating transgenic plants with reduced stem brittleness and / or increased stem mechanical strength and / or increased stem breaking force and / or increased cell wall thickness and / or reduced cell Young's modulus.

[0058] The method for cultivating transgenic plants with reduced stem brittleness and / or increased stem mechanical strength and / or increased stem breaking force and / or increased cell wall thickness and / or reduced cell Young's modulus claimed in the present invention includes the steps of increasing the activity and / or content of the above-mentioned BC17 protein in the recipient plant to obtain the transgenic plants.

[0059] Furthermore, the method for increasing the activity and / or content of the above-mentioned BC17 protein in the recipient plant is to overexpress the above-mentioned BC17 protein in the recipient plant.

[0060] The overexpression method may be to introduce the coding gene of the BC17 protein into a recipient plant.

[0061] Furthermore, the gene encoding the BC17 protein may be a DNA molecule shown by SEQ ID No.1 or SEQ ID No.3.

[0062] In a fourth aspect, the present invention claims a method for cultivating transgenic plants with increased stem brittleness and / or reduced stem mechanical strength and / or reduced stem breaking force and / or reduced cell wall thickness and / or increased cell Young's modulus.

[0063] The method for cultivating transgenic plants with enhanced stem brittleness and / or reduced stem mechanical strength and / or reduced stem breaking force and / or reduced cell wall thickness and / or increased cell Young's modulus claimed in the present invention is the following method 1 or method 2:

[0064] The method 1 comprises the step of reducing the activity and / or content of the above-mentioned BC17 protein in the recipient plant to obtain a transgenic plant.

[0065] The second method comprises the step of replacing the BC17 gene (SEQ ID No. 3) in the recipient plant with the DNA molecule shown in SEQ ID No. 4 or SEQ ID No. 5 to obtain a transgenic plant.

[0066] Furthermore, in the method 1, the method for reducing the activity and / or content of the above-mentioned BC17 protein in the recipient plant is to introduce a substance that knocks out the gene encoding the above-mentioned BC17 protein into the recipient plant.

[0067] Furthermore, the substance for knocking out the gene encoding the BC17 protein is the CRISPR / Cas9 gene editing system.

[0068] The CRISPR / Cas9 gene editing system includes Cas9 nuclease and sgRNA targeting the bc17 gene; the target sequence of the sgRNA is ACGGCGATGGCGGCCGCCGCCGG.

[0069] In a specific embodiment of the present invention, the substance for knocking out the gene encoding the BC17 protein is the recombinant vector pYLCRISPR / Cas9-MH-BC17.

[0070] In the second method, the replacement is a homozygous replacement, that is, the same replacement occurs in the homologous chromosomes.

[0071] In any of the above applications or methods, the plant is any of the following plants:

[0072] N1) monocots or dicots;

[0073] N2) Gramineae;

[0074] N3) Gramineae;

[0075] N4) Oryza plants;

[0076] N5) Rice (such as Nipponbare).

[0077] The present invention first constructs bc17 mutants and transgenic BC17 rice and analyzes their phenotypes and xylan acetylation modification levels to find that the BC17 mutation can lead to a decrease in the acetylation level of xylan in the stems, and the most significant phenotype is an increase in the brittleness of the stems. Further observations and mechanical analysis at the cellular level show that the BC17 mutation can lead to thinning of the cell walls of fiber cells, a significant increase in Young's modulus, and an increase in the rigidity of the fiber cell walls, indicating that the acetylation modification mediated by BC17 plays an important role in regulating the mechanical properties of the cell walls and the brittleness of the stems. BC17 can control the mechanical properties of the cell walls and the brittleness of the stems by regulating the acetylation modification level of xylan. The present invention provides genetic resources for cultivating brittle stalk genetic materials for both grain and straw. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1The phenotype, mutation site, protein diagram, acetylation level and yield analysis of BC17 related genetic materials. A is the phenotypic diagram of BC17 related genetic materials, with a scale of 12 cm; B is the diagram of the mutation site and mutant protein of the bc17 mutant; C is the acetylation modification level of xylan in BC17 related genetic materials; Figure 1 D is the yield of 15 strains of BC17 related genetic materials.

[0079] Figure 2 The bending load and breaking force analysis of the stem of BC17 related genetic materials. A is the three-point bending load analysis of the second section of the stem under the mature ear of BC17 related genetic materials; B is the breaking force analysis of the second section of the stem under the mature ear of BC17 related genetic materials.

[0080] Figure 3 The cell wall thickness and mechanical characterization of BC17-related genetic material fiber cells. A is a display diagram of the cell wall thickness of BC17-related genetic material fiber cells, with a scale of 5μm; B is a statistical diagram of the cell wall thickness of BC17-related genetic material fiber cells; C is a stress-strain curve diagram of the Young's modulus of the cell wall of BC17-related genetic material fiber cells; and D is a statistical diagram of the Young's modulus of the cell wall of BC17-related genetic material fiber cells. DETAILED DESCRIPTION

[0081] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0082] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0083] The quantitative experiments in the following examples were performed three times unless otherwise specified, and the results were averaged.

[0084] The following examples used SPSS19.0 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. Tukey's test was used. Different lowercase letters in the results indicate significant differences at the 0.05 level, and * and ** represent significant differences at the 0.05 and 0.01 levels, respectively.

[0085] The pYLsgRNA-OsU3 and pYLCRISPR / Cas9-MH vectors in the following examples were kindly provided by the laboratory of Professor Liu Yaoguang, State Key Laboratory of Conservation and Utilization of Subtropical Agricultural Biological Resources, School of Life Sciences, South China Agricultural University, and are both recorded in the following literature: Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. 2015. Arobust CRISPR / Cas9 system for convenient high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant. 8: 1274-1284. The public can obtain them from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0086] The pCAMBIA1300 vector in the following examples is a product of CAMIA (Australia).

[0087] The pCAMBIA1300-ccdB vector in the following examples is recorded in the following document: Zhang, D., Xu, Z., Cao, S., Chen, K., Li, S., Liu, X., Gao, C., Zhang, B., and Zhou, Y. 2018. An uncanonicalCCCH-tandem zinc-finger protein represses secondary wall synthesis and controls mechanical strength in rice. Molecular Plant. 11: 163-174, which is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0088] The pDNOR207 vector in the following examples is a product of Invitrogen (USA).

[0089] The wild japonica rice variety “Nipponbare” in the following examples is a product of the China National Rice Research Institute.

[0090] Agrobacterium EHA105 in the following examples is a product of CAMIA (Australia).

[0091] The E. coli strain DH5α in the following examples is a product of Beijing Qingke Biotechnology Co., Ltd.

[0092] The amino acid sequence of the BC17 protein in the following examples is shown in SEQ ID No.2, and the encoding gene sequence is shown in SEQ ID No.1.

[0093] Example 1. Construction of BC17-related genetic materials, phenotypic observation and acetylation analysis

[0094] I. Construction of bc17-1 mutant

[0095] The bc17-1 mutant was obtained by EMS mutagenesis. The specific steps of EMS mutagenesis are as follows:

[0096] 1. Rinse: Take a portion of wild rice Nipponbare seeds (about 1,200 seeds, packed in a mesh bag), put them in a fruit and vegetable basket, wash them, and remove surface dust.

[0097] 2. Soaking seeds: Soak the seeds in tap water (25℃, temperature controlled in a thermostat) for 36 hours.

[0098] 3. Prepare phosphate buffer (3750 mL of phosphate buffer): 1) Prepare Na 2 HPO 4 Mother liquor 2250mL (2000mL + 250mL): weigh Na 2 HPO 4 12H 2 O 47.75g dilute to 2L, mix well; weigh Na 2 HPO 4 12H 2 O 5.97g dilute to 250mL. 2) Weigh KH 2 PO 4 18.15g dilute to 2L, prepare KH 2 PO 4 2000mL mother liquor. 3) Press Na 2 HPO 4 : KH 2 PO 4 =3:2 to prepare phosphate buffer, that is, 3750mL of buffer requires Na 2 HPO 4 2250mL mother liquor and KH 2 PO 4 Pour 1500mL of the mother liquor into a 5L reagent bottle (shielded from light) and mix well (pH was measured to be about 7.3), and store at 4°C.

[0099] 4. Prepare EMS working solution (1% EMS solution): first add phosphate buffer to the volumetric flask, then use a pipette to transfer 5 mL of EMS mother solution to the volumetric flask, then make up to 500 mL, and finally shake well (prepare 2 hours in advance).

[0100] 5. Preparation of Na 2 S 2 O 3 Solution (5% Na 2 S 2 O 3 Solution): weigh 26.3 g of solid Na 2 S 2 O 3 , stir with a glass rod until melted, then dilute to 500 mL. The main function is to neutralize the EMS solution.

[0101] 6. EMS treatment: Before EMS treatment, label the bottle and the bottle cap, put the seeds (use absorbent paper to absorb excess water) into a 250mL bottle (the bottle is wrapped in a black plastic bag and shielded from light), then add 100mL (pipette) of 0.82% EMS solution to each bottle (the time for adding EMS reagent to each batch should be controlled within 20min), tighten the bottle cap, put it in a shaker, and treat it for 22h at a speed of 125r / min, a temperature of 22°C, and dark conditions.

[0102] 7. Cleaning: After EMS treatment, pour out the EMS solution in the bottle and immediately rinse it with tap water for 3 times. Collect the water used for cleaning and use Na 2 S 2 O 3 Neutralize. Then wash with tap water for 3 times, add water to the bottleneck each time, shake up and down about 30 times (cleaning time is controlled within 20 minutes), and after washing, pour the seeds into a mesh bag (mark it well) and rinse with running water for 1 hour.

[0103] 8. Germination: Spread the seeds evenly and wrap them with a towel, put them in a light incubator, and germinate them for 3 days under dark conditions at 35℃. Rinse the seeds with water twice a day and soak the towel at the same time to prevent the sprouts from burning.

[0104] 9. Sequencing of mutants: After germination, sowing was carried out according to conventional methods, and mutagenic plants with brittle stalk phenotype were selected to extract genomic DNA. PCR amplification was performed using primers BC17-test-F and BC17-test-R to obtain a product of about 400 bp. The mutation was confirmed by sequencing, and finally a homozygous mutant of the bc17 gene was obtained and recorded as bc17-1. The primer sequences are as follows:

[0105] BC17-test-F: 5'-GGCACGGCGATGGCGGCCCGCCGC-3';

[0106] BC17-test-R: 5'-AAACGCGGCGGCCGCCATCGCCG-3'.

[0107] The sequencing results showed that compared with the wild-type rice Nipponbare, the only difference between the bc17-1 mutant and the wild-type rice Nipponbare is that a base mutation occurred in the gene sequence encoding the BC17 protein (SEQ ID No.1). The base mutation was that the base G at position 738 of SEQ ID No.1 was mutated to base A, which caused the translation of the BC17 protein to terminate prematurely. The schematic diagram of the BC17 protein structure in the bc17-1 mutant is shown in Figure 1 As shown in B.

[0108] 2. Construction of bc17-2 mutant

[0109] 1. Construction of CRISPR / Cas9 knockout vector for BC17 gene

[0110] 1) Design of gRNA target sequence

[0111] In the BC17 gene sequence, a specific nucleotide sequence 19-20 bp upstream of NGG was searched as the target of gRNA. The final gRNA target sequence screened was as follows: 5'-ACGGCGATGGCGGCCGCCGCCGG-3'.

[0112] 2) Design of primer sequences

[0113] The following primer sequences were designed according to the target sequence: BC17-target-F: 5'-GGCACGGCGATGGCGGCCGCCGC-3'; BC17-target-R: 5'-AAACGCGGCGGCCGCCATCGCCG-3'.

[0114] 3) Preparation of target linker

[0115] The above target primers were mixed in equal amounts and diluted to 1 μM, reacted at 90°C for 30 seconds, and moved to room temperature to complete annealing.

[0116] 4) Preparation of gRNA expression cassette pYLsgRNA-OsU3 containing target

[0117] The target linker was connected to the gRNA expression cassette pYLsgRNA-OsU3 to obtain the gRNA expression cassette pYLsgRNA-OsU3 containing the target. The specific steps are as follows:

[0118] 4-1) Prepare the following reaction system: 20ng pYLsgRNA-OsU3, 0.5μL target linker, 2×T4 DNA ligation buffer, 1×Cutsmart digestion buffer, 40U T4 DNA ligase, 5U BsaI endonuclease, and make up to 10μL with ultrapure water. Cut and ligate at the same time to obtain the ligation product.

[0119] 4-2) The gRNA expression cassette was amplified by two rounds of PCR using the above ligation product.

[0120] First round of PCR amplification: The ligation product was used as a template and the primer pair U3F / BC17-target-R and the primer pair BC17-target-F / gRNA-R were used for PCR amplification to obtain the first round of amplification products. PCR amplification system (total volume 20 μL): 1 μL of the above ligation product, 0.2 μM of each primer, 0.2 U KOD-Plus. PCR amplification system program: 95°C 10s, 60°C 15s, 68°C 20s amplification for 30 cycles. The primer sequences are as follows:

[0121] U3F: 5'-CTCCGTTTTACCTGTGGAATCG-3':

[0122] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3'.

[0123] Second round of PCR amplification: PCR amplification was performed using the first round amplification product as a template and primer pair F / R to obtain the gRNA expression cassette pYLsgRNA-OsU3 containing the target. PCR amplification system (total volume 20 μL): 0.2 μL of the first round amplification product, 0.15 μM each of homologous recombination primers F and R, 0.2 U KOD-Plus. PCR amplification system program: 95°C 10s, 58°C 15s, 68°C 20s amplification for 30 cycles. The primer sequences are as follows:

[0124] F: 5'-TTCAGAGGTCTCTCTCGACTAGTGGAATCGGCAGCAAAGG-3';

[0125] R: 5'-AGCGTGGGTCTCGACCGACGCGTCCATCCACTCCAAGCTC-3'.

[0126] 5) Homologously recombinantly connect the gRNA expression cassette pYLsgRNA-OsU3 containing the target to the binary vector pYLCRISPR / Cas9-MH to obtain the recombinant vector pYLCRISPR / Cas9-MH-BC17. Transform the recombinant vector pYLCRISPR / Cas9-MH-BC17 into competent cells of Escherichia coli strain DH5α. Pick a single clone, shake the bacteria, extract the plasmid and sequence it, and keep the plasmid with correct sequencing.

[0127] 2. Obtaining bc17 mutant plants

[0128] The recombinant vector pYLCRISPR / Cas9-MH-BC17 was transformed into wild-type rice Nipponbare using Agrobacterium-mediated genetic transformation to obtain bc17 mutant plants. The specific steps are as follows:

[0129] 1) Rice embryo culture

[0130] The immature embryos of wild-type rice Nipponbare seeds were dehulled and sterilized as follows: 70% ethanol for 2 minutes, 0.1% mercuric chloride solution for 5 minutes, 10% sodium hypochlorite solution for 20 minutes, and sterile distilled water for 4-5 times. The sterilized seeds were spread on NB medium, and callus tissue grew from the mature embryo scutellum in 20 days. It was subcultured on NB medium and then subcultured every two weeks.

[0131] 2) Preparation of recombinant Agrobacterium suspension

[0132] The recombinant plasmid pYLCRISPR / Cas9-MH-BC17 was introduced into Agrobacterium tumefaciens EHA105 competent cells by electroporation and cultured in YEP liquid medium until the bacterial concentration OD 600nm is 0.8-1.0, and the recombinant Agrobacterium EHA105 / pYLCRISPR / Cas9-MH-BC17 is obtained.

[0133] 3) Co-cultivation of rice materials and Agrobacterium stalks

[0134] The wild-type embryonic callus was infected with the recombinant Agrobacterium suspension EHA105 / pYLCRISPR / Cas9-MH-BC17 and immersed in water at room temperature for 20 minutes. The callus was transferred to a solid NB medium containing 20 μM acetosyringone covered with a layer of sterile filter paper and cultured in the dark at 26°C for 2-3 days.

[0135] 4) Screening of resistant callus and plant regeneration

[0136] The callus tissue was spread on a selection medium containing 50 mg / L hygromycin for screening and culture for 2 weeks, and then transferred to the second round of selection medium for further screening for 2 weeks. The resistant callus tissue with vigorous growth was selected and transferred to the differentiation medium for differentiation. After it grew into a seedling, it was transferred to a 1 / 2MS solid medium for rooting and seedling growth, and then moved into a greenhouse, which is the T0 generation plant. The T0 generation plant was self-pollinated, and the seeds were harvested and cultivated into plants, which is the T1 generation plant.

[0137] 3. Identification of bc17 mutants

[0138] The genomic DNA of the T1 generation plants was extracted, and PCR amplification was performed using primers BC17-test-F and BC17-test-R to obtain a product of about 400 bp. The mutation of the bc17 gene was determined by sequencing. At the same time, PCR amplification was performed using primers Hyg-F and Hyg-R. Plants without screening markers were screened by electrophoresis (plants that amplified a target band of 1001 bp contained screening markers, and plants that could not amplify bands did not contain screening markers). Finally, a homozygous mutant of the bc17 gene without screening markers was obtained and recorded as bc17-2. The primer sequences are as follows:

[0139] Hyg-F: 5'-TTCTTTGCCCTCGGACGAGT-3';

[0140] Hyg-R: 5'-GCGACGTCTGTCGAGAAGTT-3'.

[0141] The sequencing results showed that compared with the wild-type rice Nipponbare, the only difference between the bc17-2 mutant and the wild-type rice Nipponbare is that there is a base deletion in the gene sequence encoding the BC17 protein (SEQ ID No. 1). The missing base is the base C at position 344 of SEQ ID No. 1. This mutation causes the translation of the BC17 protein to terminate prematurely. The schematic diagram of the BC17 protein structure in the bc17-2 mutant is shown in the figure below. Figure 1 As shown in B.

[0142] 3. Construction of Transgenic BC17 Rice

[0143] 1. Construction of BC17 overexpression vector

[0144] 1) Amplification of the BC17 gene coding region

[0145] Using the cDNA reverse transcribed from total RNA extracted from the stems of wild-type rice Nipponbare as a template, PCR amplification was performed using forward primers and reverse primers to obtain the PCR amplification product (i.e., the coding region of the BC17 gene). The primer sequence is as follows (the primer pair contains the sequence of the attB site):

[0146] Forward primer: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTACATGCAGCAGCGGCGGAAGTC-3′;

[0147] Reverse primer: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTACTGGTCGGATGACCATGGGG-3′.

[0148] The PCR reaction system was 25 μL, and the components included: KOD-FX (2×) 12.5 μL, upstream and downstream primers (10 μmmol / L) 1 μL each, cDNA 2 μL, H 2 2.5 μL of HO.

[0149] The PCR reaction program was: pre-denaturation at 98°C for 3 min, 35 cycles of 98°C for 10 s, 55°C for 15 s and 68°C for 30 s, and 68°C for 5 min.

[0150] 2) Obtaining plasmid pDONR207-BC17

[0151] The PCR amplification product was subjected to 1% agarose gel electrophoresis and gel recovery, and then connected to the vector pDONR207 through BP reaction. The reaction system is as follows: gel recovery product 100ng, pDONR207 150ng, BP enzyme 1μL, 1×TE buffer (pH8.0) to make up to 5μl. After 6h of reaction at 25℃, transform the competent cells of Escherichia coli strain DH5α. Pick a single clone, shake the bacteria, extract the plasmid and sequence it, and the plasmid with correct sequencing is named pDONR207-BC 17.

[0152] 3) The pDONR207-BC17 plasmid was subjected to LR reaction to connect the coding region of the BC17 gene to the pCAMBIA1300 vector. The reaction system is as follows: pDONR207-BC17 150ng, pCAMBIA1300-ccdB 150ng, LR enzyme 1μl, 1×TE buffer (pH 8.0) to 5μl. After 6h of reaction at 25℃, the competent cells of E. coli strain DH5α were transformed. Single clones were picked, shaken, plasmids were extracted and sequenced, and the plasmid with correct sequencing was named pCAMBIA 1300-BC17.

[0153] The structure of the recombinant vector pCAMBIA1300-BC17 is described as follows: the recombinant vector pCAMBIA1300-BC17 is obtained by replacing the fragment between the attR1 site and the attR2 site of the pCAMBIA1300-ccdB vector with attL1-BC17-attL2, and keeping the other sequences of the pCAMBIA1300-ccdB vector unchanged. pCAMBIA1300-BC17 contains the CDS sequence of the BC17 gene shown in SEQ ID No. 1, the Actin promoter and the NOS terminator, and can express the BC17 protein, and the expression of the protein is driven by the Actin promoter.

[0154] 2. Obtaining BC17 Transgenic Rice

[0155] The recombinant vector pCAMBIA1300-BC17 was transformed into wild-type rice Nipponbare using Agrobacterium-mediated genetic transformation. The specific steps were the same as 2 in step 2. The transgenic plant transformed with plasmid pCAMBIA1300-BC17 was named BC17-Ox.

[0156] IV. Observation of brittle culm phenotype of BC17 related genetic materials

[0157] T1 generation bc17-1 mutant, bc17-2 mutant, BC17-Ox overexpression material and wild-type rice Nipponbare were sown in the field, with 24 plants in each treatment. After four months of growth, the brittle stalk phenotype was observed by hand-breaking the second node of the fresh stalk and taking pictures.

[0158] The results are as follows Figure 1 As shown in A, the results showed that compared with the wild type, there was no significant difference in plant height between the mutants bc17-1 and bc17-2. By hand-breaking the second section of the fresh stems and observing the brittle stalk phenotype, it was found that the bc17 mutant had obvious broken sections, while the wild type did not break.

[0159] 5. Analysis of acetylation modification of BC17-related genetic materials

[0160] The xylan acetylation modification levels of the T1 generation bc17-1, bc17-2 mutants and wild-type rice Nipponbare plants were detected respectively. The specific steps are as follows:

[0161] 1. Take T1 generation bc17-1, bc17-2 mutants and wild-type rice Nipponbare plants, freeze-dry until the weight does not change, mix and grind into powder, and sieve out coarse particles with a 200-mesh sieve to obtain powder.

[0162] 2. Weigh 400 mg of powder, wash three times with 20 mL of 70% ethanol solution, and three times with 20 mL of an equal volume of chloroform-methanol mixture. Centrifuge at 12000 rpm for 10 min after each rinse to collect the precipitate. Wash the precipitate with acetone and dry it to obtain the alcohol insoluble fraction (Alcohol insoluble residue, referred to as AIR) mainly composed of cell wall components.

[0163] 3. Rinse the precipitate with 5 mL of MES / Tris buffer (Tris, Sigma, 77-86-1; MES, Sigma, 1266615-59-1) and discard the supernatant. Then add 20 mL of MES / Tris buffer and 40 U of amylase (Megazyme, K-TDFR-100A), react at 97°C for 35 min, then transfer to a 60°C water bath for 1 h to remove starch. Centrifuge at 2500 rpm for 15 minutes, discard the supernatant, add 5 mL of acetone to rinse the precipitate three times, and vacuum dry to obtain the plant seedling alcohol insoluble matter (AIR) with starch removed.

[0164] 4. Weigh 400 mg of plant seedling alcohol insoluble matter (AIR) after removing starch, add 40 mL of 1% (mass fraction) ammonium oxalate solution, react at 37°C overnight to remove the pectin component. Then centrifuge the sample, collect the precipitate, rinse with 5 mL of 11% (volume fraction) peracetic acid solution, and discard the supernatant.

[0165] 5. Add 20 mL of 11% (volume fraction) peracetic acid solution to the precipitate and react at 85°C for 30 minutes. Centrifuge at 2500 rpm for 15 minutes and discard the supernatant.

[0166] 6. Add 5 mL of acetone to the precipitate to rinse it three times and vacuum dry it. Extract it with 20 mL of DMSO at 70°C overnight, repeat twice, and transfer the supernatant to a new tube. Then add 5 times the volume of ethanol: methanol: water (7:2:1, v / v, pH 2-3) and precipitate it at 4°C for 3 days to obtain xylan precipitate.

[0167] 7. Centrifuge the xylan precipitate at 2500 rpm for 15 minutes, collect the precipitate, rinse it three times with anhydrous ethanol, and vacuum dry it to obtain the xylan component.

[0168] 8. Dissolve 1 mg of xylan in 100 μL 1N NaOH, repeat five times, react at 28°C, 200 rpm for 1 hour. Then add 100 μL 1N HCl to neutralize, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant for testing.

[0169] 9. Use the acetic acid assay kit (Megazyme, K-ACET) to determine the acetic acid content released by the reaction in the supernatant (the acetic acid released by the reaction comes from the acetyl groups in the cell wall, representing part of the acetyl groups in the cell wall that participate in the reaction). The specific steps are as follows: Take 10 μL of the supernatant in a UV capable 96-well flat plate and add 94 μL of water. Then add 42 μL (2.5:1) of the mixture of solution 1 and solution 2 in the acetic acid assay kit, solution 3 and solution 4 in turn. Read the corresponding absorbance values ​​A0, A1 and A2 at 340 nm respectively. Use solution 5 (acetic acid standard) to draw a standard curve: Take 5, 10, 15, 30, 50 μL of solution 5, corresponding to 0.5 μg, 1 μg, 1.5 μg, 3 μg, 5 μg of acetic acid, respectively, fill with double distilled water to 104 μL, and then add solution 1, solution 2, solution 3 and solution 4 according to the above method to prepare a standard curve of the corresponding absorbance value of acetic acid content. The acetic acid content (μg / mg xylan) in the sample was calculated using the following formula: Sample = (A2-A0)-(A1-A0)(A1-A0) / (A2-A0)-Blank; Blank = (A2-A0)-(A1-A0)(A1-A0) / (A2-A0).

[0170] The results are as follows Figure 1 As shown in C, the results show that compared with the wild-type rice Nipponbare, the acetyl content of xylan in the mutant bc17 (the amount of acetic acid released) is significantly reduced, indicating that the BC17 protein has the ability to regulate the acetylation modification level of plant cell wall xylan.

[0171] 6. Yield statistics of BC17 related genetic materials

[0172] The yield of T1 generation bc17-1 mutant and wild-type rice Nipponbare plants were counted, and 15 plants of each material were selected.

[0173] The results are as follows Figure 1 As shown in D, the results showed that the yield of 15 wild-type rice Nipponbare plants was 322.08±7.52g, and the yield of 15 bc17-1 mutant plants was 353.66±18.81g. Compared with the wild-type rice Nipponbare, the yield of the bc17-1 mutant did not show significant changes.

[0174] Example 2: Analysis of mechanical properties of BC17 related genetic materials

[0175] 1. Determination of mechanical strength of stems of BC17 related genetic materials

[0176] Test materials: T1 generation bc17-1 mutant, BC17-Ox overexpression material and wild-type rice Nipponbare plants.

[0177] The main panicle of mature rice plants was measured using a three-point bending tester (DZ-107), and the bending load of the second internode under the main panicle (without the leaf sheath) was measured. The second internode of about 15 cm was cut and fixed with a three-point bending tester, and the bending displacement of the sample when the load was applied was recorded. The obtained data was used to calculate the stress-strain curve. The main stems of at least five individual plants were tested for each test material.

[0178] The results are as follows Figure 2 As shown in A, the results showed that the maximum bending load of the mature stems of wild-type rice Nipponbare was 2.88 N, the maximum bending load of the mature stems of the bc17-1 mutant was 1.97 N, and the maximum bending load of the mature stems of the BC17-Ox overexpression material was 3.33 N. Compared with the wild-type rice Nipponbare, the mechanical strength of the stems of the bc17 mutant was reduced, and the mechanical strength of the stems of the BC17-Ox overexpression material was increased.

[0179] 2. Determination of stem breaking force of BC17 related genetic materials

[0180] Test materials: T1 generation bc17-1, bc17-2 mutants and wild-type rice Nipponbare plants.

[0181] The second internode below the panicle of mature fresh rice stalks was selected, the leaf sheaths were peeled off, and the stalk breaking force was measured using a stalk digital force gauge (NK-200).

[0182] The results are as follows Figure 2 As shown in B, the results showed that the mature stem breaking force of wild-type rice Nipponbare was 94.44±9.25 N, the mature stem breaking force of bc17-1 mutant was 14.98±6.27 N, and the mature stem breaking force of bc17-2 mutant was 12.86±3.72 N. Compared with wild-type rice Nipponbare, the stem breaking force of bc17 mutant was significantly reduced.

[0183] The above results indicate that BC17 protein has the ability to regulate the mechanical strength and breaking force of plant stems.

[0184] Example 3. Cell wall thickness and mechanical characterization of BC17-related genetic material fiber cells

[0185] 1. Observation and determination of cell wall thickness of BC17-related genetic material fiber cells

[0186] Test materials: T1 generation bc17-1 mutant and wild-type rice Nipponbare plants.

[0187] Select the second internode under the panicle of fresh rice stalks at maturity and fix them overnight with 4% paraformaldehyde fixative. Use a blade to cut the stalks crosswise (for observing the thickness of the fiber cell wall) or longitudinally (for observing the morphology of the pit vessels), and the positions of the wild type and mutants are consistent. Wash twice with PBS, 30 minutes each time. Dehydrate with gradient ethanol, 30%, 50%, 75%, 90% and 100% ethanol in sequence, and change each gradient twice, 30 minutes each time. After dehydration, dry the sample with a critical point dryer (LeicaEM CPD300). After the sample is dried, take out the sample sticky stage, spray gold, and then observe it with a scanning electron microscope (HITACHIS-3000N).

[0188] The results are as follows Figure 3 A and Figure 3 As shown in B, the results show that the fiber cell wall thickness of wild-type rice Nipponbare is 2.47±0.02μm, and the fiber cell wall thickness of bc17-1 mutant is 2.07±0.02μm. Compared with wild-type rice Nipponbare, the cell wall thickness of fiber cells of bc17-1 mutant is significantly reduced.

[0189] 2. Determination of Young's modulus of BC17-related genetic material fiber cells

[0190] Test materials: T1 generation bc17-1 mutant and wild-type rice Nipponbare plants.

[0191] Select the mature part of the second internode under the panicle of fresh rice stalks at maturity, cut into about 1cm*1mm*1cm size, put into a 2mL screw-capped tube, then add 1mL 11% peracetic acid, high temperature treatment at 80℃ for 12h to remove lignin, and wash repeatedly with ultrapure water several times. Ultrasonicate the sample in an ultrasonic cleaner to loosen and separate the cells. Separate the fiber cells under a stereo microscope, spread the fiber cells evenly on a slide, and after drying naturally, use an atomic force microscope (NanoWizard3) and a probe RTESPA-525 (Bruker) for nanoindentation analysis, and calculate the Young's modulus using JPK Data Processing software.

[0192] The results are as follows Figure 3 As shown in C and 3D, the results show that the Young's modulus of wild-type rice Nipponbare is 61.61±0.85, and the Young's modulus of the bc17-1 mutant is 90.47±2.65. Compared with the wild-type rice Nipponbare, the Young's modulus of the bc17-1 mutant is increased, and the rigidity (brittleness) is significantly enhanced.

[0193] The above results indicate that BC17 protein has the ability to regulate the thickness and Young's modulus of plant cell walls.

[0194] In summary, the BC17 mutation can reduce the acetylation modification level of xylan in rice stalks and present a brittle stalk phenotype. Further analysis of the phenotype and mechanical properties at the cellular level revealed that the secondary wall of stalk fiber cells became thinner, the Young's modulus increased, and the rigidity was enhanced. The present invention provides an innovative way to achieve the dual use of rice stalks.

[0195] 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of BC17 protein or related biological materials in any of the following 1)-7): 1) Regulate the fragility of plant stems; 2) Regulate the mechanical strength of plant stems; 3) Regulate the breaking force of plant stems; 4) Regulate the thickness of plant cell walls; 5) Regulate the Young's modulus of plant cells; 6) Cultivating transgenic plants with reduced stem brittleness and / or increased stem mechanical strength and / or increased stem breaking force and / or increased cell wall thickness and / or reduced cell Young's modulus; 7) Plant breeding; The BC17 protein is any one of the following proteins (a1)-(a4): (a1) protein shown in SEQ ID No. 2; (a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) a protein related to plant stem fragility and / or mechanical strength and / or breaking force obtained by substituting and / or deleting and / or adding one or more amino acid residues in (a1); (a4) A protein having 98% or more identity with (a1) and associated with plant stem brittleness and / or mechanical strength and / or breaking force.

2. The use according to claim 1, Features: The related biological material is a nucleic acid molecule encoding the BC17 protein or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule.

3. The nucleic acid molecule according to claim 2, Features: The nucleic acid molecule encoding the BC17 protein is a DNA molecule as described in any one of (A1) or (A2): (A1) a DNA molecule represented by SEQ ID No. 1 or SEQ ID No. 3; (A2) A DNA molecule that has 75% or more identity with (A1) and encodes the BC17 protein.

4. The use according to any one of claims 1 to 3, Features: The regulating the thickness of the plant cell wall is regulating the thickness of the cell wall of the plant fiber cells; Alternatively, the regulating the Young's modulus of plant cells is regulating the Young's modulus of plant fiber cells.

5. Use of a substance that inhibits the BC17 protein of claim 1 in any one of the following (d1) to (d7): (d1) increasing the brittleness of plant stems; (d2) reduce the mechanical strength of plant stems; (d3) reduce the breaking force of plant stems; (d4) reducing the thickness of plant cell walls; (d5) increasing the Young's modulus of plant cells; (d6) cultivating transgenic plants with increased stem fragility and / or reduced mechanical strength and / or reduced breaking force and / or reduced cell wall thickness and / or increased cell Young's modulus; (d7) Plant breeding.

6. The use according to claim 5, Features: The reducing the thickness of the plant cell wall is reducing the thickness of the cell wall of the plant fiber cells; Alternatively, the method of increasing the Young's modulus of plant cells is increasing the Young's modulus of plant fiber cells.

7. A method for cultivating transgenic plants with reduced stem brittleness and / or improved stem mechanical strength and / or improved stem breaking force and / or increased cell wall thickness and / or reduced cell Young's modulus, comprising the steps of increasing the activity and / or content of the BC17 protein described in claim 1 in a recipient plant to obtain a transgenic plant.

8. A method for cultivating transgenic plants with enhanced stem brittleness and / or reduced stem mechanical strength and / or reduced stem breaking force and / or reduced cell wall thickness and / or increased cell Young's modulus, comprising the steps of reducing the activity and / or content of the BC17 protein described in claim 1 in a recipient plant to obtain a transgenic plant.

9. A method for cultivating transgenic plants with enhanced stem brittleness and / or reduced stem mechanical strength and / or reduced stem breaking force and / or reduced cell wall thickness and / or increased cell Young's modulus, comprising the step of replacing the BC17 gene in a recipient plant with a DNA molecule shown in SEQ ID No.4 or SEQ ID No.5 to obtain a transgenic plant.

10. The use according to any one of claims 1 to 6 or the method according to any one of claims 7 to 9, Features: The plant is any of the following plants: N1) monocots or dicots; N2) Gramineae; N3) Gramineae; N4) Oryza plants; N5) Rice.

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