Method for regulating pit fine structure on plant vessels and use thereof

Abstract

This invention discloses the application of the BS1 protein in regulating plant vessel and pit structure and transport capacity. BS1 mediates acetylation modification of xylan at the pit edge, affecting the arrangement of nanofibers at the pit edge and the formation of pit structure, thereby influencing plant transport capacity. This invention is the first to discover that BS1-mediated acetylation modification at the pit edge plays an important role in pit nanostructure and morphology, as well as vessel transport capacity. Furthermore, BS1 It also mediates changes in pit structure under low-nitrogen treatment. Therefore, it can be controlled by regulating... BS1 The expression level or activity of rice under low nitrogen conditions, and the cultivation of rice with high transport capacity under low nitrogen conditions, have broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to methods and applications for regulating the fine structure of plant vessel pits. Background Technology

[0002] The vascular system plays a crucial role in plants' adaptation to terrestrial life. Primarily composed of the xylem and phloem, the vascular system provides mechanical support and channels for the transport of water and nutrients. The xylem transports water and minerals from the soil to various tissues within the plant. The xylem vessel walls possess various ornamental structures, such as spirals, reticulations, step patterns, and pits, with pits exhibiting a higher degree of development. These ornamental structures play a vital role in balancing the transport capacity and safety of the vessels.

[0003] The formation of ornamentation involves a series of biological processes, including vessel cell elongation, cell wall deposition, and intracellular lysate exudation. The deposition mode of the cell wall is a crucial regulatory process, requiring the participation of polymers such as cellulose, hemicellulose, and lignin. However, current research remains unclear regarding the properties of these polymers at pits, how they regulate pit morphology, and their physiological significance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for regulating the fine structure of pits, thereby improving the plant's transport capacity.

[0005] In a first aspect, the present invention claims protection for novel uses of the BS1 protein or related biological materials thereof.

[0006] This invention claims protection for the use of BS1 protein or related biological materials in any of the following 1)-7): 1) Regulating the size of plant vessel pits; 2) Regulating the fine structure of plant vessel pits; 3) Regulating xylan deposition at pit edges; 4) Regulating the arrangement of nanofibers at the edge of pits; 5) Regulate the water transport capacity of the ducts; 6) Regulate nitrogen transport capacity; The BS1 protein is any one of the proteins described in (a1)-(a4) below: (a1) The proteins shown in SEQ ID No. 3 and SEQ ID No. 4; (a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) Proteins obtained by substituting and / or deleting and / or adding one or more amino acid residues of (a1) that are related to plant vessel pit size and / or pit fine structure and / or pit xylan deposition and nanofibril arrangement and / or nitrogen transport capacity. (a4) is a protein that shares more than 98% identity with (a1) and is associated with plant vessel pit size and / or pit fine structure and / or pit xylan deposition and nanofiber arrangement and / or nitrogen transport capacity.

[0007] In the protein described in (a2) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag includes, but is not limited to: GST (glutathione thiotransferase) 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 (monomer red fluorescent protein), or AviTag tag protein.

[0008] In the protein described in (a3) ​​above, the substitution and / or deletion and / or addition of one or more amino acid residues is as follows: 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.

[0009] 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 homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can be obtained.

[0010] In the above applications, the relevant biological material is a nucleic acid molecule encoding the BS1 protein or an expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule.

[0011] Furthermore, the nucleic acid molecule encoding the BS1 protein may be a DNA molecule as described in either (A1) or (A2): (A1) The DNA molecule shown in SEQ ID No. 1 or SEQ ID No. 2; (A2) is a DNA molecule that shares more than 75% identity with (A1) and encodes the BS1 protein.

[0012] Those skilled in the art can readily mutate the nucleotide sequence encoding the BS1 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides having 75% or higher identity with the BS1 nucleotide sequence isolated according to this invention, as long as they encode BS1, can also be used. Hap2 Proteins that have the same function are all derived from the nucleotide sequence of this invention and are equivalent to the sequence of this invention.

[0013] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2 of this invention. 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.

[0014] The expression cassette refers to DNA capable of expressing the BS1 protein in host cells, and this DNA may include not only the promoter but also... BS1The promoter of transcription may also include a terminator. BS1 A transcription terminator. Furthermore, the expression cassette may also include an enhancer sequence.

[0015] The vector may be a plasmid, bacteriophage, granule, Ti plasmid, or viral vector.

[0016] In practical applications, existing plant expression vectors can be used to construct structures containing... BS1 Hap2 Recombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing the polyadenylated nucleotide signal and any other DNA fragments involved in mRNA processing or gene expression.

[0017] use BS1 When constructing recombinant plant expression vectors, any type of enhancing promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. In addition, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but they must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence.

[0018] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0019] Using any vector capable of guiding the expression of exogenous genes in plants, the present invention can be used to... BS1 Introducing genes or gene fragments into plant cells or recipient plants can yield transgenic cell lines and transgenic plants with altered stem xylan acetylation, pit structure, and transport capabilities. BS1 Hap2 Gene expression vectors can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0020] The microorganism may be yeast, bacteria, algae or fungi; the bacteria may be Agrobacterium (such as Agrobacterium EHA105).

[0021] In the above applications, regulating the size of plant vessel pits means reducing the pit size.

[0022] The regulation of the fine structure of plant vascular bundles and pits involves reducing the pore area, primarily based on the pit diameter.

[0023] The regulation of xylan deposition at the pit edge is to improve the deposition of deacetylated xylan.

[0024] The arrangement of nanofibers at the edge of the controlled pits is specifically characterized by the nanofibers being neat and orderly.

[0025] The regulation of nitrogen transport capacity specifically refers to improving nitrogen transport capacity.

[0026] Secondly, the present invention claims protection against the above-mentioned... BS1 Hap2 New uses for proteins.

[0027] This invention claims protection against the above-mentioned suppression BS1 The application of protein substances in any of the following (d1)-(d7): (d1) Increase pit size; (d2) Increase the area of ​​pit diameter; (d3) Increases the deposition of acetylated xylan; (d4) Disordered arrangement of porous nanofibers; (d5) Nitrogen transport capacity decreased; Furthermore, the substance that inhibits the BS1 protein may be a substance that inhibits the activity of the BS1 protein, a substance that inhibits the expression of the gene encoding the BS1 protein, or a substance that knocks out the gene encoding the BS1 protein.

[0028] The substance that inhibits the activity of the BS1 protein can be any substance that can cause the loss of the activity of the BS1 protein in plants, such as inhibiting the synthesis of the BS1 protein or promoting the synthesis of the BS1 protein. BS1 Proteins, peptides, or small molecule compounds (such as protein activity inhibitors) that degrade or inhibit the function of the aforementioned BS1 proteins.

[0029] The substance that inhibits the expression of the gene encoding the BS1 protein can be any substance that prevents the gene encoding the BS1 protein in the plant from being expressed, such as substances that silence the gene encoding the BS1 protein in plants (e.g., miRNA, siRNA, dsRNA, shRNA, etc.).

[0030] The substance that knocks out the gene encoding the BS1 protein can be any substance that prevents the host cell from producing the functional protein product of the BS1 gene. Specific methods include removing all or part of the coding gene sequence, introducing frameshift mutations to prevent the production of the functional protein, removing or altering regulatory components (e.g., promoter editing) to prevent transcription of the coding gene sequence, or blocking translation by binding to mRNA. Typically, knockout is performed at the genomic DNA level, so that the cell's offspring permanently carry the knockout. Furthermore, the substance that knocks out the gene encoding the BS1 protein can be any substance capable of causing mutations in the gene encoding the BS1 protein in plants (the mutations can be deletion mutations and / or insertion mutations and / or base substitutions) thereby rendering it inactive, such as zinc finger protein (ZFN) gene editing systems, TALENs gene editing systems, or CRISPR / Cas9 gene editing systems.

[0031] Furthermore, the substance used to knock out the gene encoding the BS1 protein is the CRISPR / Cas9 gene editing system.

[0032] Thirdly, the present invention claims a method for cultivating plants with smaller pits and / or smaller pit diameter and / or pit deacetylated xylan deposition and / or orderly arrangement of pit nanofibers and / or improved nitrogen transport capacity.

[0033] The method for cultivating plants with smaller pits and / or smaller pit diameter and / or deacetylated xylan deposition and / or orderly arrangement of nanofibers surrounding the pits and / or improved nitrogen transport capacity, as claimed in this invention, includes increasing the above-mentioned BS1 in the recipient plant. Hap2 The steps to obtain transgenic plants include determining the activity and / or content of proteins.

[0034] Furthermore, the enhancement of the above-mentioned receptor plants BS1 Methods for determining protein activity and / or content include expressing BS1 in recipient plants. Hap2 Form protein or related to BS1 Hap1 Compared to BS1 homologs with higher deacetylation activity, or by overexpressing the aforementioned BS1 protein.

[0035] The overexpression method may involve introducing the gene encoding the BS1 protein into a recipient plant.

[0036] Furthermore, the gene encoding the BS1 protein may be a DNA molecule as shown in SEQ ID No. 1 or SEQ ID No. 2.

[0037] Fourthly, the present invention claims a method for cultivating plants with enlarged pits and / or enlarged pit diameter and / or pit acetylated xylan deposition and / or disordered arrangement of pit nanofibers and / or reduced nitrogen transport capacity.

[0038] The method for cultivating plants with enlarged pits and / or enlarged pit pore area and / or pit acetylated xylan deposition and / or disordered arrangement of pit nanofibers and / or reduced nitrogen transport capacity, as claimed in this invention, is as follows: Method 1 includes the step of reducing the activity and / or content of the aforementioned BS1 protein in the recipient plant to obtain a transgenic plant.

[0039] In any of the above applications or methods, the plant is any of the following: N1) Monocotyledonous or dicotyledonous plants; N2) Plants of the order Poales; N3) Gramineae plants; N4) Rice plants; N5) Rice (such as Nipponbare).

[0040] This invention discovers a gene that regulates pit size through population genetics analysis. BS1 The enzyme BS1 encodes xylan acetylesterase. BS1 mediates xylan acetylation modification at the pit site. A high degree of xylan deacetylation results in orderly arrangement of nanofibers at the pit edge, smaller pits, and a significant increase in plant transport capacity; conversely, a high degree of acetylation leads to disordered arrangement of nanofibers at the pit edge, larger pits, and a significant decrease in plant transport capacity. BS1 The acetylation modification mediated by BS1 at the pit edge plays an important role in pit nanostructure, pit morphology, and vessel transport capacity. Furthermore, low-nitrogen-induced expression of BS1 leads to smaller plant vessel pits, while... bs1 The fact that the pits of the mutant were not affected by low nitrogen indicates that... BS1 Specific response to low-nitrogen induction modulates changes in pit structure. Therefore, it is possible to regulate... BS1 This invention provides a method for regulating the fine structure of pits on xylem vessels and its application in plant transport capacity, thereby cultivating rice with high transport capacity under low nitrogen conditions by controlling expression levels or activity levels under low nitrogen. Attached Figure Description

[0041] Figure 1 for BS1 Observation, statistics, and comparison of pit size and transport capacity of related genetic materials. A represents NP observed under a scanning electron microscope. bs1 BS1 Hap1 and BS1 Hap2 The pit morphology of the vessels is shown in Figure A, with a scale bar of 2 μm; B represents the statistical results of pit size in Figure A; C represents the NP... bs1BS1 Hap1 and BS1 Hap2 A comparison of the transport capacity of Rhodamine. Figure 2 for BS1 The three-dimensional structure of pits in related genetic materials and a comparison of pit diameter area and pit width are presented. Where A represents NP, bs1 BS1 Hap1 and BS1 Hap2 The three-dimensional structure is displayed, with a scale bar of 0.5 μm; B represents NP, bs1 BS1 Hap1 and BS1 Hap2 Comparison of pit diameter area and pit width.

[0042] Figure 3 This section compares and observes the deposition patterns of pitted xylan. A shows the distribution pattern of xylan at the pit edges using immunofluorescence, with a scale bar of 2 μm; B shows the quantitative statistics of xylan signal at the pit edges in Figure A; C shows BS1... Hap1 and BS1 Hap2 Comparison of the activities of the two proteins.

[0043] Figure 4 The arrangement of nanofibers at the pit edges was observed using atomic force microscopy. A represents the morphological observation with a scale bar of 1 μm; B represents the statistical distribution of the nanofibers.

[0044] Figure 5 For observation BS1 The responses of relevant genetic materials to low nitrogen and comparisons of nitrogen transport and transpiration capacity were analyzed. Figure A shows the pit morphology and size of the NP and bs1 mutants observed by scanning electron microscopy (scale bar: 2 μm); Figure B presents the statistical results of Figure A; Figure C compares the transport capacity of stable isotopic nitrogen; and Figure D shows field measurements. BS1 Transpiration potential of relevant genetic materials. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0048] The following examples use Graphpad Prism 8.0 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The graph test is used. Different lowercase letters in the results indicate that the difference is significant at the 0.05 level, while * and ** represent significant differences at the 0.05 and 0.01 levels, respectively.

[0049] The pCAMBIA1300 vector used in the following examples is a product of CAMBIA Corporation (Australia), which was subsequently modified for use in the laboratory. The vector is described in the following literature: Zhang, B., Zhang, L., Li, F., Zhang, D., Liu, X., Wang, H., Xu, Z., Chu, C., and Zhou, Y. (2017). Control of secondary cellwall patterning involves xylan deacetylation by a GDSL esterase. Nat Plants 3, 17017. It is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to replicate the relevant experiments of this invention and should not be used for other purposes.

[0050] The wild-type japonica rice varieties “Nipponbare” and 93-11 mentioned in the following examples are products of the China National Rice Research Institute.

[0051] The Agrobacterium EHA105 used in the following examples is a product of CAMBIA (Australia).

[0052] The Escherichia coli strain DH5α in the following examples is a product of Beijing Qingke Biotechnology Co., Ltd.

[0053] Example 1 BS1 Construction of relevant genetic materials, observation of pit morphology, and comparison of transport capacity one, bs1 Creation of mutants bs1 The mutant was obtained through EMS mutagenesis. The specific steps of EMS mutagenesis are as follows: 1. Rinse: Take one portion of wild-type rice Nipponbare seeds (about 1200 seeds, packed in a mesh bag), rinse with tap water to remove surface dust.

[0054] 2. Soaking seeds: Soak seeds in tap water (25℃, temperature controlled in an incubator) for 36 hours.

[0055] 3. Preparation of phosphate buffer (3750 mL): 1) Prepare 2250 mL of Na2HPO4 stock solution (2000 mL + 250 mL) in two batches as follows: Weigh 47.75 g of Na2HPO4·12H2O and bring the volume to 2 L, mix well; weigh 5.97 g of Na2HPO4·12H2O and bring the volume to 250 mL. 2) Weigh 18.15 g of KH2PO4 and bring the volume to 2 L to prepare 2000 mL of KH2PO4 stock solution. 3) Prepare phosphate buffer solution according to the ratio of Na2HPO4:KH2PO4 = 3:2, that is, 3750 mL of buffer solution requires 2250 mL of Na2HPO4 stock solution and 1500 mL of KH2PO4 stock solution. Pour both into a 5 L reagent bottle (protected from light) and mix well (the pH was measured to be approximately 7.3). Store at 4℃.

[0056] 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 stock solution into the volumetric flask, then make up to 500 mL, and finally shake well (prepare 2 hours in advance).

[0057] 5. Prepare Na2S2O3 solution (5% Na2S2O3 solution): Weigh 26.3 g of solid Na2S2O3, stir with a glass rod until melted, and then make up to 500 mL. The main purpose is to neutralize the EMS solution.

[0058] 6. EMS treatment: Before EMS treatment, label the bottle and cap. Put the seeds (with excess water absorbed with absorbent paper) into a 250 mL bottle (wrap the bottle in a black plastic bag and protect it from light). Then add 100 mL (pipette transfer) of 0.82% EMS solution to each bottle (the time for adding EMS reagent for each batch should be controlled within 20 min). Tighten the cap and place the bottle on a shaker. Treat for 22 h at a speed of 125 r / min, a temperature of 22℃ and in the dark.

[0059] 7. Cleaning: After EMS treatment, first pour out the EMS solution from the bottle and immediately rinse three times with tap water. Collect all the water used for rinsing and neutralize it with Na2S2O3. Then rinse three times with tap water, adding water to the neck of the bottle each time and shaking it up and down about 30 times (the rinsing time should be controlled within 20 minutes). After rinsing, pour the seeds into a mesh bag (mark it) and rinse with running water for 1 hour.

[0060] 8. Germination: Spread the seeds evenly, wrap them in a towel, and place them in a light incubator. Germinate them at 35℃ in the dark for 3 days. Rinse the seeds with water twice a day and wet the towel at the same time to prevent scorching of the sprouts.

[0061] 9. Mutant Sequencing: After germination, sow the seeds using standard methods. Select mutagenic plants exhibiting the brittle sheath phenotype, extract genomic DNA, and perform PCR amplification using BS1-test-F and BS1-test-R primers to obtain a product of approximately 1 kb. Sequencing is then used to determine the mutation status, ultimately yielding the desired mutant. bs1 Homozygous mutant of the gene. Primer sequences are as follows: BS1-test-F: 5'-TGTTCAACTTCGGGGACTCG-3'; BS1-test-R: 5'-TGATCCTCACATTGGGGTGC-3'.

[0062] Sequencing results show that, compared with wild-type rice Nipponbare, bs1 The mutant's genomic base mutation involves changing base G to base A at position 1019 of the genomic sequence of the BS1 protein from Nipponbare (as shown in SEQ ID No. 5 in the sequence listing). After the mutation, bs1 The introns cannot be properly cleaved, leading to bs1 The translation of the corresponding BS1 protein terminates prematurely in the mutant. bs1 mutant bs1 The amino acid sequence of the protein is shown in SEQ ID No. 7, and its coding sequence is shown in SEQ ID No. 6.

[0063] II. Transfer BS1 The construction of rice 1. BS1 Overexpression vector BS1 Hap1 Construction 1) BS1 Hap1 Amplification of gene coding regions Using cDNA derived from the reverse transcription of total RNA extracted from the young stems of wild-type rice Nipponbare as a template, PCR amplification was performed using forward and reverse primers to obtain the PCR amplification product (i.e., the sequence corresponding to the coding region of the BS1 gene in the sequence listing). The primer sequences are as follows: Forward primer: 5'-GGGCCCTCACAAGTTTGTACAAAATGGGGGCAGTTCGGGGGA-3'; Reverse primer: 5'-ATTCACTAGTTCACACCACTTTGTACAATGAAGATTGGAAGATCGGTT-3'. The PCR reaction system consisted of 25 μL of the following components: 12.5 μL of KOD-FX (2×), 1 μL each of upstream and downstream primers (10 μmmol / L), 2 μL of cDNA, and 8.5 μL of H2O.

[0064] The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min, followed by 35 cycles of 98℃ for 10 s, 58℃ for 15 s, and 68℃ for 30 s, and 68℃ for 5 min.

[0065] The PCR amplification products were subjected to 1% agarose gel electrophoresis and then recovered from the gel.

[0066] 2) Enzyme digestion of pCAMBIA1300-ccdB vector The pCAMBIA1300-ccdB vector was digested with BsrGI-HF restriction enzyme, and the product was recovered by gel electrophoresis on a 1% agarose gel. The enzyme digestion reaction system was as follows: 1 μg DNA (pCAMBIA1300-ccdB vector), 10 μL 10x Cutsmart buffer, 1 μL BsrGI-HF restriction enzyme, and double-distilled water to a final volume of 100 μL. The enzyme digestion reaction was carried out at 37°C for 2 h.

[0067] 3) Obtaining the recombinant vector pCAMBIA1300-BS1 The PCR product recovered in step 1) and the vector recovered in step 2) were subjected to homologous recombination using a homologous recombinase to obtain the reaction product. The homologous recombination reaction system was as follows: 200 ng of gel-recovered PCR product, 100 ng of gel-recovered enzyme-digested vector, 5 μL of homologous recombinase, and double-distilled water to a final volume of 10 μL. The homologous recombination reaction conditions were as follows: 40℃ for 15 min.

[0068] The reaction product was transformed into E. coli strain DH5α competent cells. Single clones were picked, cultured, plasmids were extracted and sequenced. Clones with correct sequencing were selected, plasmids were extracted, and double digested with KpnI and ApaI.

[0069] 4) BS1 Amplification of gene promoter regions Using cDNA derived from the reverse transcription of total RNA extracted from the young stems of wild-type rice Nipponbare as a template, PCR amplification was performed using forward and reverse primers to obtain the PCR amplification product (i.e., the promoter region of the BS1 gene). The primer sequences are as follows: Forward primer: 5'-ACGGCCAGTGCCAAGCTTGGTACCGCTGCAGTGAGTAGGAAGGTG-3'; Reverse primer: 5'-GAACGATCGGGGAAATTCACTAGTCGCCGCCCGCCCTATCCCTC-3'. The PCR reaction system consisted of 25 μL of the following components: 12.5 μL of KOD-FX (2×), 1 μL each of upstream and downstream primers (10 μmmol / L), 2 μL of cDNA, and 8.5 μL of H2O.

[0070] The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min, followed by 35 cycles of 98℃ for 10 s, 58℃ for 15 s, and 68℃ for 30 s, and 68℃ for 5 min.

[0071] 5) Recombinant vector BS1 Hap1 The acquisition The PCR product recovered in step 4) and the vector recovered in step 3) were subjected to homologous recombination using a homologous recombinase to obtain the reaction product. The homologous recombination reaction system was as follows: 200 ng of gel-recovered PCR product, 100 ng of gel-recovered enzyme-digested vector, 5 μL of homologous recombinase, and double-distilled water to a final volume of 10 μL. The homologous recombination reaction conditions were as follows: 40℃ for 15 min.

[0072] The reaction product was transformed into *E. coli* strain DH5α competent cells. Single clones were picked, cultured, plasmids were extracted and sequenced. Clones with correct sequencing were selected, and the plasmid with correct sequencing was named BS1. Hap1 .

[0073] Recombinant vector BS1 Hap1 The structure is described as follows: Recombinant vector BS1 Hap1 To deliver the DNA molecule (BS1) shown in SEQ ID No. 1 Hap1 The coding region sequence of the gene was ligated into the BsrGI site of the modified pCAMBIA1300 vector. The BS1 promoter shown in SEQ ID No. 8 was then ligated into the KpnI and ApaI sites of the pCAMBIA1300-ccdB vector, while keeping the other sequences of the pCAMBIA1300 vector unchanged. BS1 Hap1 Contains ingredients from Japan. BS1 A gene expression cassette, comprising, in sequence, the BS1 promoter and the gene shown in SEQ ID No. 1. BS1 The gene's CDS sequence and NOS terminator enable the expression of BS1. Hap1 protein.

[0074] 2. BS1 Overexpression vector BS1 Hap2 Construction 1) BS1 Hap2 Amplification of gene coding regions Using cDNA derived from the reverse transcription of total RNA extracted from the young stems of rice 93-11 as a template, PCR amplification was performed using forward and reverse primers to obtain the PCR amplification product (i.e., the coding region of the BS1 gene). The primer sequences are as follows: Forward primer: 5'-GGGCCCTCACAAGTTTGTACAAAATGGGGGCAGTTCGGGGGA-3'; Reverse primer: 5'-ATTCACTAGTTCACACCACTTTGTACAATGAAGATTGGAAGATCGGTT-3'.

[0075] The PCR reaction system consisted of 25 μL of the following components: 12.5 μL of KOD-FX (2×), 1 μL each of upstream and downstream primers (10 μmmol / L), 2 μL of cDNA, and 8.5 μL of H2O.

[0076] The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min, followed by 35 cycles of 98℃ for 10 s, 58℃ for 15 s, and 68℃ for 30 s, and 68℃ for 5 min.

[0077] The PCR amplification products were subjected to 1% agarose gel electrophoresis and then recovered from the gel.

[0078] 2) Enzyme digestion of pCAMBIA1300-ccdB vector The pCAMBIA1300-ccdB vector was digested with BsrGI-HF restriction enzyme, and the product was recovered by gel electrophoresis on a 1% agarose gel. The enzyme digestion reaction system was as follows: 1 μg DNA (pCAMBIA1300-ccdB vector), 10 μL 10x Cutsmart buffer, 1 μL BsrGI-HF restriction enzyme, and double-distilled water to a final volume of 100 μL. The enzyme digestion reaction was carried out at 37°C for 2 h.

[0079] 3) Obtaining the recombinant vector pCAMBIA1300-BS1 The PCR product recovered in step 1) and the vector recovered in step 2) were subjected to homologous recombination using a homologous recombinase to obtain the reaction product. The homologous recombination reaction system was as follows: 200 ng of gel-recovered PCR product, 100 ng of gel-recovered enzyme-digested vector, 5 μL of homologous recombinase, and double-distilled water to a final volume of 10 μL. The homologous recombination reaction conditions were as follows: 40℃ for 15 min.

[0080] The reaction product was transformed into E. coli strain DH5α competent cells. Single clones were picked, cultured, plasmids were extracted and sequenced. Clones with correct sequencing were selected, plasmids were extracted, and double digested with KpnI and ApaI.

[0081] 4) BS1 Amplification of gene promoter regions Using cDNA derived from the reverse transcription of total RNA extracted from the young stems of wild-type rice Nipponbare as a template, PCR amplification was performed using forward and reverse primers to obtain the PCR amplification product (i.e., the promoter region of the BS1 gene). The primer sequences are as follows: Forward primer: 5'-ACGGCCAGTGCCAAGCTTGGTACCGCTGCAGTGAGTAGGAAGGTG-3'; Reverse primer: 5'-GAACGATCGGGGAAATTCACTAGTCGCCGCCCGCCCTATCCCTC-3'. The PCR reaction system consisted of 25 μL of the following components: 12.5 μL of KOD-FX (2×), 1 μL each of upstream and downstream primers (10 μmmol / L), 2 μL of cDNA, and 8.5 μL of H2O.

[0082] The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min, followed by 35 cycles of 98℃ for 10 s, 58℃ for 15 s, and 68℃ for 30 s, and 68℃ for 5 min.

[0083] 5) Recombinant vector BS1 Hap2 The acquisition The PCR product recovered in step 4) and the vector recovered in step 3) were subjected to homologous recombination using a homologous recombinase to obtain the reaction product. The homologous recombination reaction system was as follows: 200 ng of gel-recovered PCR product, 100 ng of gel-recovered enzyme-digested vector, 5 μL of homologous recombinase, and double-distilled water to a final volume of 10 μL. The homologous recombination reaction conditions were as follows: 40℃ for 15 min.

[0084] The reaction product was transformed into *E. coli* strain DH5α competent cells. Single clones were picked, cultured, plasmids were extracted and sequenced. Clones with correct sequencing were selected, and the plasmid with correct sequencing was named BS1. Hap2 .

[0085] Recombinant vector BS1 Hap1 The structure is described as follows: Recombinant vector BS1 Hap2 To deliver the DNA molecule (BS1) shown in SEQ ID No. 2 Hap2 The coding region sequence of the gene was ligated into the BsrGI site of the pCAMBIA1300-ccdB vector. The BS1 promoter shown in SEQ ID No. 9 was then ligated into the KpnI and ApaI sites of the pCAMBIA1300-ccdB vector while maintaining the other sequences of the pCAMBIA1300-ccdB vector unchanged. BS1Hap2 Contains from 93-11 BS1 A gene expression cassette, comprising, in sequence, the BS1 promoter and the gene shown in SEQ ID No. 2. BS1 The gene's CDS sequence and NOS terminator enable the expression of BS1. Hap2 protein.

[0086] BS1 Hap2 With BS1 Hap1 The two protein sequences differ by only one amino acid, at position 163 (Val (BS1)). Hap1 ) becomes Ala (BS1) Hap2 ), BS1 Hap2 It is better than BS1 Hap1 Alleles with higher activity.

[0087] 3. Turn BS1 Rice harvest The recombinant vector BS1 was transformed using Agrobacterium-mediated genetic transformation. Hap1 and BS1 Hap2 Transform separately bs1 Mutant.

[0088] 1) Rice embryo culture Will bs1 The young embryos of the mutant seeds were dehulled and sterilized as follows: sterilized with 70% ethanol for 2 minutes, soaked in 10% sodium hypochlorite solution for 20 minutes, and washed 4-5 times with sterile distilled water. The sterilized seeds were then spread on NB medium. Callus tissue grew from the mature embryo scutellum after 20 days. This callus tissue was then subcultured on NB medium, and subcultured every two weeks thereafter.

[0089] 2) Preparation of recombinant Agrobacterium suspension Recombinant vector BS1 Hap1 and BS1 Hap2 The bacteria were introduced into *Agrobacterium tumefaciens* EHA105 competent cells via electroporation and cultured in YEP liquid medium until the bacterial cell concentration reached OD500. 600nm The concentration was 0.8-1.0, resulting in recombinant Agrobacterium tumefaciens EHA105 / BS1. Hap1 and BS1 Hap2 .

[0090] 3) Co-culture of rice materials and Agrobacterium tumefaciens Recombinant Agrobacterium tumefaciens suspension EHA105 / BS1 Hap1 and EHA105 / BS1 Hap2 Infection bs1 The mutant embryonic callus was soaked in a shaking bath at room temperature for 20 minutes. The callus was then transferred to NB solid medium containing 20 μM acetylsyl syringone and lined with a layer of sterile filter paper. The medium was then incubated in the dark at 26°C for 2-3 days.

[0091] 4) Screening of resistant callus and plant regeneration Callus tissue was cultured on selective medium containing 50 mg / L hygromycin for 2 weeks, then transferred to a second selective medium for another 2 weeks. Vigorous, resistant callus tissue was selected and transferred to differentiation medium for differentiation. After seedling development, the seedlings were transferred to 1 / 2 MS solid medium for rooting and then transplanted into a greenhouse; these were the T0 generation plants. The T0 generation plants were self-pollinated, and the seeds were harvested and cultured into plants, which were the T1 generation plants.

[0092] 5) Identification of genetically modified plants: DNA was extracted from transgenic plants and used as a template for PCR amplification using Hyg-F and Hyg-R primers. Plants containing the hygromycin marker were screened by electrophoresis (plants that amplified a target band of 1001 bp contained the selection marker, while plants that did not amplify a band did not contain the selection marker). Positive BS1 transgenic plants were finally obtained. Hap1 and BS1 Hap2 Rice, and designated it as BS1 Hap1 and BS1 Hap2 The primer sequences are as follows: Hyg-F: 5'-TTCTTTGCCCTCGGACGAGT-3'; Hyg-R: 5'-GCGACGTCTGTCGAGAAGTT-3'.

[0093] Transform plasmid BS1 Hap1 and BS1 Hap2 The transgenic plant was named BS1. Hap1 and BS1 Hap2 .

[0094] three, BS1 Observation of pit phenotype in relevant genetic materials Rice Nipponbare, bs1 T2 generation BS1 Hap1 and BS1 Hap2Twenty-four plants were sown in the field for each treatment. After four months of growth, the second node of mature stems was harvested and fixed overnight with 4% paraformaldehyde fixative. The stems were longitudinally cut with a blade to expose the vascular bundles, with the same location used for wild-type and mutant plants. The plants were washed twice with PBS (Phosphate buffered solution) for 30 min each time. Dehydration was performed using a gradient of ethanol: 30%, 50%, 75%, 90%, and 100% ethanol, with two ethanol changes per gradient for 30 min each time. After dehydration, the samples were dried using a critical point dryer (Leica EM CPD300). The samples were then mounted on a stage, sputter-coated with gold, and observed using a scanning electron microscope (HITACHI S-3000N). Pits were analyzed and statistically analyzed using ImageJ.

[0095] The results are as follows Figure 1 As shown in A and 1B: Compared to the wild type, the mutant... bs1 The pits were significantly enlarged, with an area of ​​1.95 ± 0.05 μm. 2 BS1 Hap1 The pit size and NP were not significantly different, with areas of 0.52±0.02 μm. 2 0.58±0.02 μm 2 , and BS1 Hap2 The pits were significantly smaller than those of NP and BS1. Hap1 The area is 0.35±0.01 μm. 2 The results showed that BS1 negatively regulated pit size.

[0096] IV. Rhodamine Transport Experiment Rice Nipponbare, bs1 T2 generation BS1 Hap1 and BS1 Hap2 After two weeks of culture in the culture medium until the four-leaf stage, the cells were transferred to a solution containing 0.5 mM Rhodamine B for 3 hours for transport experiments. The base 2 cm of the second leaf was powdered and extracted with ethanol. The Rhodamine content in the extract was analyzed using a microplate reader. The results are shown in Figure 1C; the Rhodamine content of wild-type NP was 40.22 ± 10.09 nmol.g -1 , bs1 The mutant concentration was 9.55 ± 4.04 nmol.g -1 BS1 Hap1 and BS1 Hap2 The values ​​were 27.1 ± 3.92 nmol·g. -1 and 57.37±3.92 nmol.g -1 The results indicate that BS1 positively regulates water transport capacity. Example 2 BS1 Regulation of the three-dimensional structure of duct pits I. Reconstruction of the Three-Dimensional Structure of Vessel Pits Stem samples from plants grown for four months were fixed with a fixative containing 2.5% (v / v) glutaraldehyde and 2% (v / v) paraformaldehyde, then washed twice with phosphate buffer (0.1 M, pH 7.4) and twice with deionized water. Next, the samples were immersed at 4°C in a 1% (w / v) osmium tetroxide and 1.5% (w / v) potassium ferricyanide aqueous solution for 2 hours. After washing, the tissues were dehydrated sequentially by a gradient of ethanol (30%, 50%, 70%, 80%, 90%, 100%, 100%, 100%, 10 min each time) and finally transferred to pure acetone (2 × 10 min). The samples were then infiltrated in a gradient mixture of acetone and Spurr resin (5:1, 3:1, 1:1, 1:3), then replaced with pure resin. Finally, the tissues were embedded in pure resin and polymerized at 45°C for 12 hours, followed by polymerization at 70°C for 48 hours. For the study of three-dimensional ultrastructures, a dual-beam scanning electron microscope (Helios Nanolab 600i, FEI) was used to sequentially cut and image the samples via FIB. After the images were aligned using ImageJ software, all datasets were analyzed using Imaris software.

[0097] II. Measurement of Three-Dimensional Structure Indicators of Vessel Pits The area and width of pits were measured using Imaris software, and statistical analysis was performed.

[0098] The results are as follows Figure 2 As shown in Figures A and 2B, the results indicate that the pore size of the Nipponbare mineral is 0.92 ± 0.05 μm. 2 , bs1 The mutant pit diameter is 1.59 ± 0.08 μm. 2 BS1 Hap1 and BS1 Hap2 The pore sizes were 1.07 ± 0.07 μm. 2 and 0.54±0.03 μm 2 Compared to wild-type Nipponbare rice, the mutant... bs1 The pits are significantly enlarged, BS1 Hap1 The pit size and NP showed no significant difference, while BS1 Hap2 The pits were significantly smaller than those of NP and BS1. Hap1 The pit width of the Nipponbare specimen is 0.43 ± 0.01 μm. bs1 The mutant pit width was 0.53 ± 0.01 μm, BS1 Hap1 and BS1 Hap2The pit widths were 0.47±0.02 μm and 0.33±0.02 μm, respectively. Compared with wild-type rice Nipponbare, the mutant... bs1 The width has increased significantly, BS1 Hap1 The pit width and size of BS1 showed no significant difference from those of NP, while BS1... Hap2 The pit width is significantly smaller than that of NP and BS1. Hap1 .

[0099] The above results indicate that the BS1 protein has the function of regulating the fine structure of pits.

[0100] Example 3: Deposition Analysis of Acetylated Xylan Mature stem material was treated with 10% peracetic acid solution at 80℃ for 12 hours. Vessel cells were isolated under a stereomicroscope using micromanipulation. The cells were then incubated with xylan-specific antibody M150 at a 1:50 ratio for 2 hours, followed by incubation with AF488-labeled secondary antibody for another 2 hours. The cell walls were stained with Direct Red 23 dye, and finally observed using an LSM 980 laser scanning confocal microscope.

[0101] The results are as follows Figure 3 As shown in A and 3B, wild-type rice Nipponbare and BS1 Hap1 The deacetylated xylan signals were comparable, at 49.31 ± 7.39 and 48.49 ± 5.24, respectively, for BS1. Hap2 The deacetylation signal was higher in wild-type rice Nipponbare and BS1. Hap1 The value was 59.33±9.11, indicating that BS1 mediated the deposition of low-acetylated xylan at the pit edges. Hap2 The degree of deacetylation modification is greater than that of BS1 Hap1 High. Xylan, xylan. Merge, integration of xylan signal and Direct Red 23 signal.

[0102] Example 4: Atomic force microscopy observation of nanofiber arrangement at pit edges Mature stem material was treated with 10% peracetic acid solution at 80℃ for 12 hours. Vessel cells were then isolated under a stereomicroscope using micromanipulation. The vessel cells were evenly spread on mica sheets, dried, and the morphology of cellulose microfilaments on the surface of the stem vessel cells was observed under an atomic force microscope (MM-SPM; Bruker). The width of the cellulose microfilaments was statistically analyzed using ImageJ, and the arrangement of the cellulose microfilaments was analyzed using SOAX 3.6.1 (https: / / omictools.com / soax-tool).

[0103] The results are as follows Figure 4 As shown, bs1 The medium-sized nanofibers are arranged in a disordered manner, while NP and BS1 Hap1 The layout is quite similar, BS1 Hap2 The more concentrated arrangement of the nanofibers indicates that BS1 regulates the arrangement of the nanofibers.

[0104] Example 5: Comparison of the response of BS1-related genetic materials to low nitrogen and their nitrogen transport and transpiration capabilities. I. Observation of pit structure under low nitrogen treatment Material processing and pit observation are described in Section 3 of Example 1, where the pit morphology was observed using a scanning electron microscope. Statistics were calculated using ImageJ. The results are shown below. Figure 5 A. In Nipponbare materials, the pitting size gradually decreases with decreasing nitrogen concentration, reaching 0.81 ± 0.03 μm at high nitrogen concentrations. 2 The nitrogen content was 0.58 ± 0.02 μm. 2 The value under low nitrogen conditions is 0.42 ± 0.01 μm. 2 ,and bs1 The mutant showed little change in pit size under nitrogen treatment, with values ​​of 1.95 ± 0.05 μm under high, medium, and low nitrogen conditions. 2 1.91±0.03 μm 2 1.93±0.03 μm 2 This indicates that BS1 mediates changes in pit structure under low-nitrogen treatment.

[0105] II. Detection of transport efficiency of isotopic nitrogen Using stable isotopes 15 The nitrogen tracer detection method was used to determine nitrogen transport efficiency. Rice seedlings were first cultured in normal nutrient solution for 10 days, then transferred to a mononitrogen hydroponic solution containing only ammonium nitrogen for 3 days of acclimatization. Afterwards, the seedlings were transferred to a solution containing 2 mM... 15 N (98% atom 15 After treating the plants in a hydroponic solution containing N-NH4Cl (Sigma-Aldrich) for 5 hours, the roots were then immersed in a 0.1 mM CaSO4 solution for 60 seconds to rinse (to remove adsorbed substances from the root surface). 15 The root and aboveground parts were then rinsed in pure water for 30 seconds, dried with absorbent paper, and freeze-dried separately. The resulting powders were then ground into a uniform powder using a ball mill. The content of N in the samples was determined using an isotope ratio mass spectrometer (Thermo Finnigan Delta Plus XP). 15 Nitrogen stable isotope content. Used from the above-ground parts and roots. 15 The N ratio indicates the nitrogen transport activity of a plant. The results are as follows Figure 5 B, aboveground and root parts of wild-type rice Nipponbare material 15The ratio of N is 0.41 ± 0.04. bs1 The mean value was 0.27 ± 0.01, significantly lower than that of wild-type rice Nipponbare, BS1 Hap1 The value was 0.56±0.02, which was not significantly different from that of wild-type rice Nipponbare, while BS1 Hap2 The mean value was 0.75 ± 0.01, significantly higher than that of Nipponbare and BS1. Hap1 This indicates that BS1 regulates the transport efficiency of nitrogen.

[0106] III. Measurement of transpiration potential Wild-type Japanese Haru BS1 The relevant genetic materials were cultured in the field for 3 months, and the plant transpiration potential was measured at three time points throughout the day using a photosynthesis instrument.

[0107] The results are as follows Figure 5 As shown in Figure C, the transpiration potential of the leaves of each plant fluctuated over time, reaching its highest point at 13:00 (noon). During this period, the transpiration potential of the leaves of *Nipponbare* was 7.55 ± 0.32 mmol / L. -2 .s -1 ; bs1 The leaf transpiration potential of the mutant was 4.34 ± 0.95 mmol·m⁻². -2 .s -1 BS1 Hap1 The leaf transpiration potential was 9.50 ± 0.62 mmol·m⁻². -2 .s -1 BS1 Hap1 The leaf transpiration potential was 11.90 ± 0.35 mmol·m⁻². -2 .s -1 The results showed that BS1 positively regulated the water transport capacity of the duct.

[0108] In summary, BS1-mediated acetylation modification of xylan at the pit edge leads to a high degree of xylan deacetylation, resulting in orderly arrangement of nanofibers at the pit edge, smaller pits, and a significant increase in plant transport capacity. Conversely, a high degree of acetylation modification at the pit edge leads to disordered nanofiber arrangement, larger pits, and a significant decrease in plant transport capacity. This indicates that BS1-mediated acetylation modification at the pit edge plays an important role in pit nanostructure, pit morphology, and vessel transport capacity. Furthermore, BS1-induced reduction in plant vessel pit size in response to low nitrogen levels... bs1 The mutant's pits were unaffected by low nitrogen, indicating that BS1 specifically responds to low nitrogen and regulates changes in pit structure. Therefore, by regulating the expression level or activity of BS1 under low nitrogen conditions, rice with high transport capacity under low nitrogen can be bred.

[0109] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of BS1 protein or related biological materials in any of the following 1)-7): 1) Regulating the size of plant vessel pits; 2) Regulating the size of plant vessel pits; 3) Regulating xylan deposition at pit edges; 4) Regulating the arrangement of nanofibers at the edge of pits; 5) Regulate the water transport capacity of the ducts; 6) Regulate nitrogen transport capacity; 7) Plant breeding; The BS1 protein is any one of the proteins described in A1)-A8): A1) The protein shown in SEQ ID No. 4; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in A1); A3) Proteins related to plant vessel pit structure and transport capacity obtained by substituting and / or deleting and / or adding one or more amino acid residues of A1). A4) is a protein that shares more than 98% identity with A1) and is related to the regulation of plant pits; A5) The protein shown in SEQ ID No. 3; A6) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in A5); A7) Proteins related to plant vessel pit structure and transport capacity obtained by substituting and / or deleting and / or adding one or more amino acid residues of A5. A8) and A5) are proteins that share more than 98% identity and are related to the regulation of plant pits.

2. The application according to claim 1, characterized in that: The relevant biological material is any one of B1) to B6) below: B1) A nucleic acid molecule encoding the protein of 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) Reduce the amount of nucleic acid molecules expressed by the protein of claim 1; B6) Expression cassettes, recombinant vectors, or recombinant microorganisms containing the nucleic acid molecules described in B5).

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule encoding the protein of claim 1 is any one of the following DNA molecules (b1)-b4): b1) The DNA molecule shown in SEQ ID No. 1; b2) A DNA molecule that shares more than 75% identity with b1) and encodes the BS1 protein; b3) The DNA molecule shown in SEQ ID No. 2; b4) and b3) are DNA molecules that share more than 75% identity and encode the BS1 protein.

4. A method for cultivating any of the following plants, characterized in that, Seeds overexpressing the protein of claim 1 are selected for breeding, wherein the plant is... 1) Plants with smaller pits; 2) Plants with smaller pit diameters; 3) Plants with deacetylated xylan deposits at the pit edges; 4) Plants with neatly arranged nanofibers at the edges of pits; 5) Plants that enhance nitrogen transport capacity.

5. A method for regulating the fine structure of plant vessel pits, characterized in that, Regulate the expression level of the protein described in claim 1 in a plant or alter the activity of the protein described in claim 1 in a plant.

6. The method according to claim 5, characterized in that, The fine structure of plant vessel pits includes the size of plant vessel pits, pit diameter area, plant with deacetylated xylan deposited at the pit edge, and plant with orderly arranged nanofibers at the pit edge.

7. A method for regulating the transport capacity of plant vascular bundles, characterized in that, This includes regulating the expression level of the protein described in claim 1 in plants or altering the activity of the protein described in claim 1 in plants.

8. The method according to claim 7, characterized in that, The plant's vascular transport capacity includes its water transport capacity and nitrogen transport capacity.

9. The application according to any one of claims 1-3 or the method according to any one of claims 4-8, characterized in that: The plant is any of the following: N1) Monocotyledonous or dicotyledonous plants; N2) Plants of the order Poales; N3) Gramineae plants; N4) Rice plants; N5) rice.

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