PmNST3 gene of plum blossom and its alternative splice variant and its application

Abstract

This invention relates to the field of genetic engineering technology, and discloses plum blossom PmNST3 Genes and their alternative splice variants and their applications. This invention reveals the plum blossom... PmNST3 Genes and their alternative splice variants PmNST3‑E1、 PmNST3‑E2 Related to the lignin pathway, by increasing PmNST3 The expression level of this substance can increase the lignin content in plants, causing them to exhibit an upright phenotype; by increasing... PmNST3‑E1 or PmNST3‑E2 The expression level of this substance can reduce the lignin content in plants, causing the branches to droop. The plum blossom provided by this invention... PmNST3 or PmNST‑E1 or PmNST3‑E2 Genes and the proteins they encode can be applied to plant architecture breeding in various plants, and have broad application prospects in the field of molecular breeding of plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to plum blossoms. PmNST3 Genes and their alternative splice variants and their applications. Background Technology

[0002] plum bossom( Prunus mume Sieb. et Zucc.) is a member of the genus Prunus in the family Rosaceae. Prunus Plum blossoms are plants with extremely high ornamental value. Currently, there are over 400 varieties. They are beloved for their unique tree shapes, diverse flower colors, and fragrant aromas, holding a vital position in landscaping. Besides the common upright-branch type, there are also weeping and dragon-shaped varieties; however, the weeping and dragon-shaped varieties are relatively rare, making it difficult to meet the diversity requirements of landscaping.

[0003] Currently, plant type breeding is one of the important directions in plum blossom breeding. Traditional plum blossom breeding mainly uses methods such as seedling selection, intervarietal hybridization, and distant hybridization. Both hybridization techniques and seedling selection suffer from problems such as long breeding cycles, low efficiency, and the inability to achieve targeted breeding. With the development of genetic engineering and molecular biotechnology, molecular breeding has become a shortcut to quickly obtain varieties with ideal plant types. By analyzing the molecular mechanisms of special plant type traits and identifying key genes, targeted breeding can be achieved. Drooping branches are caused by a variety of factors, with lignin content in the branches being a significant one. When the lignin content in the branches is low, the branches will exhibit a drooping phenotype under the influence of gravity. Currently, drooping plum blossom varieties are scarce, and their numbers are far from meeting the needs of landscaping applications. Therefore, it is urgent to accelerate the cultivation of drooping plum blossoms through genetic engineering methods to fundamentally solve the problem of insufficient drooping plum blossom resources from the perspective of germplasm genetic improvement. Summary of the Invention

[0004] The purpose of this invention is to provide plum blossom PmNST3 Genes and their alternative splice variants and their applications.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides an isolated protein, said protein being NST3, NST3-E1, or NST3-E2; The NST3 protein is: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:1; or, (b) A protein derived from (a) with the sequence shown in SEQ ID NO:1 substituted, deleted or added with one or more amino acids and having the same function; NST3-E1 protein is: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO:3; or, (b1) A protein derived from (a1) with one or more amino acids substituted, deleted or added to the sequence shown in SEQ ID NO:3 and having the same function; The NST3-E2 protein is: (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO:5; or, (b2) Proteins derived from (a2) with the sequence shown in SEQ ID NO:5 substituted, deleted or added with one or more amino acids and having the same function.

[0006] Secondly, the present invention provides an isolated nucleic acid molecule, said nucleic acid molecule being a plum blossom PmNST3 Genes, plum blossoms PmNST3-E1 Genes or plum blossoms PmNST3-E2 Gene; Among them, plum blossom PmNST3-E1 , PmNST3-E2 Genes of plum blossom PmNST3 Different variants of genes; plum bossom PmNST3 The genes are: A) The nucleotide sequence shown in SEQ ID NO:2; B) A nucleotide sequence of SEQ ID NO:2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; C) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:2 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the solution. D) A nucleotide sequence that has more than 90% homology with the nucleotide sequences of A), B), or C) and expresses the same functional protein; or, E) A nucleotide sequence that is completely complementary to the nucleotide sequence of A), B), C), or D); plum bossom PmNST3-E1 The genes are: A1), the nucleotide sequence shown in SEQ ID NO:4; B1) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:4 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; C1) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:4 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS, followed by washing the membrane with the solution. A nucleotide sequence (D1) that has more than 90% homology with the nucleotide sequences of A1), B1), or C1) and expresses the same functional protein; or, A nucleotide sequence that is completely complementary to the nucleotide sequence of E1), A1), B1), C1), or D1); plum bossom PmNST3-E2 The genes are: A2), the nucleotide sequence shown in SEQ ID NO:6; B2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:6 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; C2) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:6 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS, followed by washing the membrane with the solution. D2) nucleotide sequences that have more than 90% homology with the nucleotide sequences of A2), B2), or C2) and express the same functional protein; or, A nucleotide sequence that is completely complementary to the nucleotide sequences of E2, A2, B2, C2, or D2.

[0007] Thirdly, the present invention provides biological materials containing the nucleic acid molecules, including but not limited to expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or non-renewable plant parts.

[0008] Fourthly, the present invention provides any of the following applications of the described protein, nucleic acid molecule, or biological material: (1) Regulating plant lignin content; (2) Regulating plant architecture; (3) Preparation of transgenic plants; (4) Prepare kits for regulating plant type, lignin content or preparing transgenic plants.

[0009] Furthermore, utilizing plum blossoms PmNST3 Genes or their expression products promote upright plant growth; or... Using plum blossoms PmNST3-E1 Genes or their expression products, and / or plum blossoms PmNST3-E2Genes or their expression products promote drooping of plant branches.

[0010] Furthermore, improve plum blossom PmNST3 Gene expression levels increase lignin content; or, Improve plum blossom PmNST3-E1 and / or PmNST3-E2 Gene expression levels are reduced to decrease lignin content.

[0011] The plants described in this invention include, but are not limited to, plum blossoms or Arabidopsis thaliana.

[0012] Furthermore, methods to increase gene expression levels can be selected from the following 1) to 5), or any combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on plant chromosomes; 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes; 4) By operatively linking a strong promoter to the gene; 5) By introducing enhancers.

[0013] Fifthly, the present invention provides a method for cultivating plants with a specific plant type, comprising the step of introducing the nucleic acid molecule into the plant and causing it to be expressed; When cultivating upright plants, the introduced nucleic acid molecules are plum blossoms. PmNST3 Genes; when it is necessary to cultivate weeping plants, the introduced nucleic acid molecules are plum blossoms. PmNST3-E1 Genes and / or plum blossoms PmNST3-E2 Gene.

[0014] In a sixth aspect, the present invention provides a method for regulating the lignin content of plants, comprising the step of introducing the nucleic acid molecule into the plant and causing it to be expressed; When it is necessary to increase the lignin content of plants, the nucleic acid molecules introduced are plum blossoms. PmNST3 Genes; when it is necessary to reduce the lignin content of plants, the introduced nucleic acid molecules are plum blossoms. PmNST3-E1 Genes and / or plum blossoms PmNST3-E2 Gene.

[0015] Expression vectors carrying target genes can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2). nd Edition).

[0016] In a seventh aspect, the present invention provides the application of transgenic plants obtained according to the method in plant breeding.

[0017] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0018] Eighthly, the present invention provides a kit comprising the protein NST3 or NST3-E1 or NST3-E2, or the plum blossom [protein name missing]. PmNST3 or PmNST3-E1 or PmNST3-E2 Genes, or the biological material mentioned above.

[0019] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention provides a protein NST3 or NST3-E1 or NST3-E2 and its encoding gene. PmNST3 or PmNST-E1 or PmNST3-E2 Studies show that plum blossoms PmNST3 or PmNST3-E1 or PmNST3-E2 Genes, associated with the lignin pathway, by increasing PmNST3 The expression level of this substance can increase the lignin content in plants, causing them to exhibit an upright phenotype; by increasing... PmNST3-E1 or PmNST3-E2 The expression level of this substance can reduce the lignin content in plants, causing the branches to droop. The plum blossom provided by this invention... PmNST3 or PmNST-E1 or PmNST3-E2 Genes and the proteins they encode can be applied to plant architecture breeding in various plants, and have broad application prospects in the field of molecular breeding of plants. Attached Figure Description

[0020] Figure 1 This is the clone provided in Embodiment 1 of the present invention. PmNST3 or PmNST3-E1 or PmNST3-E2 Electrophoresis image.

[0021] Figure 2 The results are the alignment results of three bands amplified using the same pair of primers, as provided in Example 1 of this invention.

[0022] Figure 3 This is an evolutionary tree of the plum blossom NST3 protein provided in Example 1 of the present invention, and the poplar and Arabidopsis NST proteins.

[0023] Figure 4 The sequence alignment results are those of the encoded sequences of PmNST3, PmNST3-E1 and PmNST3-E2 provided in Embodiment 2 of the present invention.

[0024] Figure 5 This is provided in Embodiment 2 of the present invention. PmNST3 , PmNST3-E1 and PmNST3-E2 Schematic diagrams of gene and protein structures. A is the DNA structure of PmNST3, B is a schematic diagram of the full-length mRNA and protein structure of PmNST3, C is a schematic diagram of the mRNA and protein structure of PmNST3-E2, and D is a schematic diagram of the mRNA and protein structure of PmNST3-E1.

[0025] Figure 6 This is the protein tertiary structure model of PmNST3, PmNST3-E1 and PmNST3-E2 provided in Example 2 of the present invention.

[0026] Figure 7 This is provided in Embodiment 3 of the present invention. PmNST3 Expression levels in different tissues of plum blossom.

[0027] Figure 8 This is provided in Embodiment 3 of the present invention. PmNST3 or PmNST3-E1 or PmNST3-E2 Expression levels in different developmental stages of upright and weeping plum branches, where Z represents upright branches, C represents weeping branches, 1 represents non-lignified, 2 represents semi-lignified, and 3 represents fully lignified. Different lowercase letters indicate significant differences.

[0028] These are phenotypic diagrams of wild-type and transgenic Arabidopsis thaliana provided in Example 4 of this invention.

[0029] Figure 9 This is a qRT-PCR result diagram of the transgenic Arabidopsis thaliana provided in Example 4 of the present invention.

[0030] Figure 10 This is a graph showing the lignin content in the stems of wild-type and transgenic Arabidopsis thaliana provided in Example 4 of this invention, where OE1, OE3, and OE13 are different numbers. Figure 11The transgenic lines OE11, OE23, and OE24 are different numbering systems. PmNST3 Transgenic lines, OE4, OE18, and OE19 are PmNST3-E1 Different transgenic lines with different numbers. Different lowercase letters indicate significant differences. Detailed Implementation

[0031] The present invention adopts the following technical solution: This invention aims to provide plum blossom PmNST3-E2 This study focuses on genes and their alternative splice variants, particularly their application in regulating plant architecture. Specifically, it examines three alternative splice variants of the NST homolog in plum blossom, each with distinct functions in lignin synthesis.

[0032] In a first aspect, the present invention provides a protein NST3 (NAC secondary wall thickening promoting factor), comprising: an amino acid sequence as shown in SEQ ID NO:1.

[0033] The present invention provides a protein NST3-E1, comprising the amino acid sequence shown in SEQ ID NO:3.

[0034] The present invention provides a protein NST3-E2, comprising the amino acid sequence shown in SEQ ID NO:5.

[0035] Further, the coding gene for protein NST3 includes the nucleotide sequence shown in SEQ ID NO:2. The coding gene for protein NST3-E1 includes the nucleotide sequence shown in SEQ ID NO:4. The coding gene for protein NST3-E2 includes the nucleotide sequence shown in SEQ ID NO:6.

[0036] Secondly, the present invention provides a plum blossom PmNST3 Genes, the plum blossom PmNST3 The gene includes a nucleotide sequence as shown in SEQ ID NO:2.

[0037] This invention provides a plum blossom PmNST3 Genes, the plum blossom PmNST3-E1 The gene includes a nucleotide sequence as shown in SEQ ID NO:4.

[0038] This invention provides a plum blossom Genes, the plum blossom PmNST3-E1 The gene includes a nucleotide sequence as shown in SEQ ID NO:6.

[0039] The present invention further provides a biomaterial, the biomaterial comprising the plum blossom described above. or PmNST3-E2 or Gene; the biological material is an expression cassette, vector, or transgenic cell.

[0040] The present invention further provides a kit comprising the aforementioned protein NST3 or NST3-E1 or NST3-E2, or the aforementioned plum blossom [protein]. PmNST3-E2 or or PmNST3 Genes, or the biological material mentioned above.

[0041] The present invention further provides the protein NST3 or NST3-E1 or NST3-E2, or the plum blossom [protein name missing]. PmNST3-E1 or PmNST3-E2 or PmNST3 The application of the gene, or the biological material, or the kit described herein, in regulating plant architecture, or in the preparation of transgenic plants, or in plant genetic breeding.

[0042] Furthermore, by increasing the expression level of the gene encoding the protein NST3 or NST3-E1 or NST3-E2, the lignin content in the plant can be increased or decreased, or the plant can be made upright or drooping; or by crossing a line that overexpresses the gene encoding the protein NST3 or NST3-E1 or NST3-E2 with other plants, upright or drooping plant lines can be cultivated.

[0043] Furthermore, the genes encoding the proteins NST3, NST3-E1, or NST3-E2 regulate lignin synthesis, causing the plant to stand upright or the branches to droop.

[0044] Furthermore, the plant is either plum blossom or Arabidopsis thaliana.

[0045] The present invention provides proteins NST3, NST3-E1, or NST3-E2 and their encoding genes. These encoding genes can regulate the expression levels of genes involved in the lignin synthesis pathway in plants, participating in the regulation of lignin synthesis. Increasing the expression level of the encoding gene of this protein can cause plants to grow upright or drooping. The proteins NST3, NST3-E1, or NST3-E2 and their encoding genes provided by the present invention can be applied to the cultivation of various upright or drooping plant types, which is of great significance to the field of plant molecular breeding.

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0047] Example 1: Plum Blossom PmNST3-E1 or PmNST3-E2 orPmNST3 Gene cloning This embodiment provides PmNST3-E1 or or PmNST3-E2 The specific process of gene cloning is as follows: According to the Longyou plum reference genome (https: / / www.rosaceae.org / Analysis / 13114608) Specific primers (SEQ ID NO: 7-8) were designed at the beginning and end of the gene coding region. Using 'Fentai Chuizhi' stem cDNA as a template, PCR amplification was performed using novizan high-fidelity enzyme. Electrophoresis on a 2% agarose gel revealed that the same primer pair amplified three bands. PmNST3 The three target fragments obtained by PCR were recovered by gel extraction and ligated into pTOPO vector (purchased from Sino-American Taihe Biotechnology (Beijing) Co., Ltd.), transformed into E. coli DH5α competent cells, single clones were picked for expansion culture, and the resulting bacterial solutions were identified by PCR. Positive bacterial solutions were sent for testing, and the sequencing results were compared.

[0048] PmNST3-E1 Gene-specific primers (SEQ ID NO:7-8): PmNST3-F: 5'- ATGGCTGAGGATCATATGAATCTATC-3' PmNST3-R: 5'-TTATACCGACAAGTGGCACAGTG-3' 1. Synthesis of the first strand of cDNA Total RNA from intact plum blossom stems was reverse transcribed into cDNA using the TaKaRa PrimeScript RT reagent Kit. The experimental procedure was performed according to the kit instructions, as follows: 2.0 μL 5× PrimeScript® buffer, 0.5 μL PrimeScript® reverse transcriptase mixture, 0.5 μL oligo-dT primer (50 μM), 0.5 μL random hexamer primer (50 μM), 0.5 μg total RNA from plum blossom stems, and 6 μL RNase-free ddH2O.

[0049] Reaction conditions: 37℃ for 20 min; 80℃ for 5 sec.

[0050] 2. Gene amplification Using reverse-transcribed stem cDNA as a template and PmNST3-F / R as primers, the target gene was cloned using novizan high-fidelity enzyme PCR with the sequence shown in SEQ ID NO:7-8. The PCR reaction system consisted of: 25 μL of rapid high-fidelity enzyme premix (2 × Phanta Flash Master Mix, Dye Plus), 2.0 μL of PmNST3-F (10 μM), 2.0 μL of PmNST3-R (10 μM), 2 μL of cDNA template, and 19 μL of dd H2O.

[0051] PCR program: 95℃ for 3 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 15 s, 30 cycles; 72℃ for 5 min, 16℃ to infinity. After the PCR reaction, 2 μL was taken for 1% agarose gel electrophoresis.

[0052] 3. Subcarrier construction PCR products containing a single target band were purified using the Novizan gel extraction kit. The purified products were collected, and the recovery quality was assessed using agarose gel electrophoresis. The purified products were stored at -20°C. The three purified fragments were then ligated using the Zero BackgroundBlunt TOPO Cloning Kit from Sino-American Pharmaceutical Group.

[0053] Ligation system: 1.0 μL pTOPO cloning vector, 2.0 μL PmNST3 PCR product, 1.0 μL 10× enhancer, and 6.0 μL dd H2O. After mixing, the ligation reaction was performed at 25℃ for 5 min. Immediately after ligation, the mixture was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing ampicillin, and incubated overnight.

[0054] 4. Sequencing and identification of single colonies Several single-clone plaques were randomly selected from the antibiotic-resistant plate and cultured in liquid LB medium. An appropriate amount of the bacterial culture was sent to Shanghai Sangon Biotech Co., Ltd. for bidirectional sequencing.

[0055] The results are as follows , PmNST3-E2 and As shown: PmNST3 This invention demonstrates that it utilizes specific primers for PCR amplification, resulting in three fragments of different lengths. PmNST3-E1Phylogenetic analysis showed that the brightest band encoding a protein closely related to Arabidopsis thaliana AtNST3 / SND1 and poplar PtrWND1 was named PmNST3, with a CDS length of 1185 bp. Sequence alignment revealed that the three fragments were transcripts formed from the same pre-RNA through alternative splicing. The other two bands retained introns of the PmNST3 DNA fragment and were named PmNST3-E1 and PmNST3-E2, respectively. PmNST3-E1 retained two introns; the first intron contained an early stop codon, causing premature termination of transcription at that site. Its coding sequence was 192 bp long and encoded 65 amino acids. PmNST3-E2 retained the second intron; the presence of the early stop codon also caused premature termination of transcription. Its coding sequence was 465 bp long and encoded 156 amino acids.

[0056] Example 2: Structural analysis of proteins NST3, NST3-E1, or NST3-E2 Further analysis results are as follows and PmNST3-E2 As shown: Encoding 394 amino acids, PmNST3 and Figure 1 E2The encoded amino acid numbers are 65 aa and 156 aa, respectively. Sequence alignment revealed that the first 62 amino acids of the three proteins are identical, but the last few amino acids differ. In PmNST3-E1, positions 63, 64, and 65 are glycine, threonine, and histidine, respectively. In PmNST3 and PmNST3-E2, positions 63, 64, and 65 are glutamic acid, lysine, and cysteine, respectively. The 155th and 156th amino acids in PmNST-E2 are serine and histidine, respectively, while in PmNST3 they are cysteine ​​and asparagine. The relative molecular masses of PmNST3, PmNST3-E2, and PmNST3-E1 are 45.44 kDa, 18.4 kDa, and 7.72 kDa, respectively, and their isoelectric points are 5.94, 9.14, and 5.16, respectively. The protein instability coefficients were 53.32, 36.96, and 50.99, respectively, and the average hydrophilicity coefficients were -0.889, -0.881, and -0.652, respectively. Conserved domain analysis revealed that all three transcripts contained the NAM conserved domain specific to the NAC family. The NAM conserved domains of PmNST3 and PmNST3-E2 both contained four subdomains: A, B, C, and D. Furthermore, PmNST3 also contained the E subdomain of NAC transcription factors. Among the NAC family subdomains, the A domain is thought to be potentially involved in protein dimer formation, while the C and D domains are thought to be involved in DNA binding. Based on this, it is speculated that PmNST3-E1 may not possess a DNA-binding domain. PmNST-E1 and PmNST-E2 also lack the variable transcriptional regulatory region contained in the C-terminus of the NAC family. PmNST3, PmNST-E1, and PmNST-E2 all lack signal peptides and transmembrane domains. SOPMA was used to predict the secondary structure of proteins. The results showed that the PmNST3 protein had 43 α-helices (10.91%), 307 random coils (77.92%), 38 extended strands (9.64%), and 6 β-turns (1.52%). The PmNST3-E2 protein had 26 α-helices (16.67%), 91 random coils (58.33%), 31 extended strands (19.87%), and 8 β-turns (5.13%). The PmNST3-E1 protein had 17 α-helices (26.15%), 38 random coils (58.46%), 8 extended strands (12.31%), and 2 β-turns (3.08%). A tertiary structure model of the protein was constructed using SWISS-MODEL, and the results are as follows: PmNST3 As shown.

[0057] Example 3 Plum Blossom Figure 1 , Figure 2 and Figure 3 Gene expression pattern analysis This embodiment is... Figure 1 , Figure 3 andFigure 4 The specific process for analyzing expression patterns is as follows: 1. Using transcriptome analysis Figure 5 Expression levels in different tissues of plum blossom Using *Prunus cerasifera* as material, a total of 22 tissues were sequenced for transcriptome sequencing, including different parts and developmental stages of the roots and stems (bark, xylem, cambium, phloem, young stems, semi-lignified stems, and fully lignified stems), leaves (young leaves, old leaves, and mature leaves), different floral organs (buds, sepals, petals, anthers, filaments, styles, and ovaries), seeds, and fruits (young fruits, mature fruits, and semi-mature fruits). The transcriptome data were then analyzed. PmNST3 The level of gene expression in tissues. PmNST3-E1 The results show that PmNST3- It is mainly expressed in the stems of plum blossoms, with higher expression levels in semi-lignified and fully lignified stems than in young stems, roots, flowers, fruits, and leaves. In different stem tissues, Figure 6 The expression level is highest in the cambium, and higher in the phloem than in the xylem.

[0058] 2. PmNST3 , PmNST3-E1 and PmNST3-E2 Gene expression analysis In the F1 generation of 'six-petaled' (straight branch) and 'pink-platform weeping' (weeping branch) varieties, extremely drooping and extremely upright individuals were selected. One-year-old branches were used as material, harvested when the stem length was approximately 25 cm. At this stage, the stem tip was in the elongation stage and not yet lignified (labeled 1); the middle branches were in the lignification process (semi-lignified, labeled 2); and the basal branches were fully lignified (labeled 3). RNA was extracted and reverse transcribed into cDNA. Total RNA extraction from the plum blossoms was performed using the RNAsimple Total RNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd., following the kit's instructions. Quantitative specific primers were designed using the Integrated DNA Technologies online platform (https: / / sg.idtdna.com / page). PmNST3 (NCBI ID: XM_008244977) was used as an internal reference gene. cDNA synthesis was performed using the Novizuma Hiscript IV All-in-One Ultra RT SuperMix for qPCR kit, and real-time quantitative PCR was performed using the Novizuma Taq Pro UniversalSYBR qPCR Master Mix kit. The experiments were conducted according to the kit instructions. A control was set up, and the expression level of the target gene was calculated using the ΔΔCt method.

[0059] Quantitative primers (SEQ ID NO:9-16): PmNST3-RT-F: 5'- AAGCTTGCTACCAGGCCATT-3' PmNST3-RT-R: 5'-AGTTGACCCAGCTGAGATGC-3' PmNST3-E1-RT-F: 5'-CTCAAGTTCCTCCGGGCTTTC-3' PmNST3-E1-RT-R: 5'- AATGCGTACCTTGAATATCCCAT-3' PmNST3-E2-RT-F: 5'- ACCGGAACTCGAACCAATCG-3' PmNST3-E2-RT-R: 5'-TGGTGTCATGGGTGTTGCTT-3' PmPP2A-RT-F: 5'- AGGGTTTCGGCTCGCAATAATAGA-3' PmPP2A-RT-R: 5'-AGCAGCAGCATCACGAATTGAGTAG-3' Using qPCR method to PmNST3-E1 , PmNST3-E2 and P PmNST3 Gene quantification was performed using qPCR primers as shown in SEQ ID NO:9-16. Results are as follows: PmNST3 As shown, Figure 7 , PmNST3 and PmNST3 Expression levels were lowest at the shoot apex, with no significant difference between straight and drooping branches. In both semi-lignified and fully lignified branches, expression levels were highest in straight branches. PmNST3 The expression level was higher in the drooping branches, but PmNST3-E1 and PmNST3-E2 The expression level in straight branches was significantly lower than that in drooping branches. PmPP2A The expression patterns of different variable shearing variants differed in upright and drooping branches.

[0060] Example 4 Plum Blossom PmNST3 , PmNST3-E1 and mNST3-E2 Gene function verification This embodiment performs Figure 8 , PmNST3 and PmNST3-E1 The functional verification process in Arabidopsis thaliana is as follows: 1. Construction of plant expression vectors Amplification primers (SEQ ID NO:17-18): PmNST3-E1-R: 5'-TTAATGCGTACCTTGAATATCCCA-3' PmNST3-E2-R: 5'-TTAATGGCTTACGGTGGTGTCA-3' Designed with the stop codon as the end. PmNST3-E2 and PmNST3 The reverse primer (SEQ ID NO:17-18) was used, and the forward primer was PmNST3-F (SEQ ID NO:7). Homologous arms of the NcoI and BstEII restriction sites of pCAMBIA1304 were added to the 5' end of the primers. pTOPO- PmNST3-E1 pTOPO- PmNST3-E2 pTOPO- PmNST3 PCR amplification was performed using the plasmid as a template. pCAMBIA1304 was double-digested with NcoI and BstEII. The recovered plasmid was then... PmNST3 , PmNST3-E1 and PmNST3-E2 The fragment from the pCAMBIA1304 vector (preserved in the laboratory) was ligated using the ClonExpress Ultra OneStep Cloning Kit V3 ligase from Novizan to obtain the recombinant plasmid. After the ligation reaction, the plasmid was transformed into E. coli DH5α competent cells, plated on LB agar plates containing kanamycin, and incubated overnight at 37°C. Single colonies were picked from the plates, expanded, and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0061] 2. Genetic transformation of Agrobacterium Add 1 μL of recombinant plasmid to 50 μL of freshly thawed Agrobacterium competent cells, tap the centrifuge tube to mix, and incubate sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and on ice for 5 min. Add 700 μL of liquid LB to the centrifuge tube and incubate at 200 rpm for 2-3 h at 28℃. Centrifuge at 5000 rpm to remove excess LB, leaving 100 μL, and spread it on LB solid medium containing antibiotics (kanamycin + rifampin). Incubate at 28℃ for 48 h, pick single colonies and expand them in liquid LB containing kanamycin and rifampin. Then perform PCR to identify whether the transformants are positive.

[0062] 3. Preparation of infiltration solution pCAMBIA1304- PmNST3 pCAMBIA1304- PmNST3-E1 pCAMBIA1304- PmNST3-E2Agrobacterium GV3101 was streaked onto LB agar containing 50 mg / L kanamycin and 25 mg / L rifampin and incubated at 28°C for 48 h. Single colonies were picked and expanded into LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampin. Colony PCR was performed again to confirm the positive result. The bacterial culture was then expanded at a 1:100 ratio and incubated at 28°C and 200 rpm for 14 h until the OD of the bacterial culture was reached. 600 Centrifuge at 0.6-0.8 °C, 4 °C, 5000 rpm for 5 min, and discard the supernatant. Resuspend the bacterial cells in staining buffer (50 g / L sucrose + 0.02% Silwet-77) to OD200. 600 =0.6-0.8. Pour the bacterial solution into a wide-mouthed beaker for later use.

[0063] 4. Genetic transformation and resistance selection in Arabidopsis thaliana When wild-type Arabidopsis thaliana enters its vigorous growth period and the inflorescence reaches approximately 20 cm, infection is performed. The inflorescence is collected and immersed in the infection solution, gently agitated for 30 seconds to 1 minute, and excess solution is absorbed with absorbent paper. The infected Arabidopsis is then covered with a black plastic bag and incubated in the dark in a climate chamber for 24 hours before being transferred to normal light conditions. Mature seeds are collected, dried, and placed in 2 ml centrifuge tubes with silica gel added, then stored at 4°C. After sterilization, Arabidopsis seeds are spread on 1 / 2 MS solid medium containing 30 mg / L hygromycin. The plates are sealed and refrigerated at 4°C for 2-3 days, then transferred to a tissue culture room. After 10 days, resistant plants with two green true leaves are transferred to soil-based culture medium for continued growth.

[0064] 5. Molecular detection of transgenic plants Total RNA was extracted from leaves of resistant transgenic Arabidopsis thaliana using the Tiangen Plant Total RNA Extraction Kit. Using the extracted transgenic Arabidopsis thaliana cDNA as a template, qRT-PCR was performed using quantitative primers for the target gene (SEQ ID NO: 9-14). A control was set up, and the expression level of the target gene was calculated using the ΔΔCt method.

[0065] 6. Phenotypic determination of transgenic Arabidopsis thaliana After sterilization, seeds of different transgenic lines and wild-type Arabidopsis thaliana were spread on 1 / 2 MS medium and refrigerated at 4 ℃ for 3 days. They were then transferred to a tissue culture chamber for 10 days, after which they were transplanted into a substrate and placed in an artificial climate chamber for further culture (16 hours light / 8 hours darkness). When the transgenic and overexpressing Arabidopsis thaliana bolted, the pots were tilted at 90° for continued culture.

[0066] The results are as follows PmNST3-E1 As shown: This invention screens out positive lines through resistance screening, and selects lines with high expression levels for subsequent experiments using qRT-PCR. PmNST3-E2 The relative expression levels of the target gene in the selected strains are shown. PmNST3 PmNST3-E1 PmNST3-E2 PmNST3 PmNST3-E1 PmNSTE-E2 PmNST3 PmNST3-E1 PmNST3-E2 Figures 9 - 11 Figure 10 Figure 11 This study demonstrates the phenotypic differences between wild-type Arabidopsis and those overexpressing the three genes. When different strains of Arabidopsis were cultured horizontally, in the early growth stages, the overexpressing strains showed no significant difference in growth compared to the wild-type. However, as the stems elongated,… PmNST3-E2 and PmNST3-E1 Stems overexpressing Arabidopsis thaliana showed improvement compared to wild-type and PmNST3 The stem is slightly tilted, and around the 50th day, PmNST3-E2 The stems of Arabidopsis thaliana exhibit a drooping phenotype.

[0067] This invention further analyzes whether the lignin content in the stem is a factor in drooping. Stems from wild-type and three gene-overexpressing Arabidopsis thaliana plants were harvested, and the lignin content was determined using the Huayueyang Lignin Detection Kit (UV spectrophotometry). Three plants from each line were pooled, and three biological replicates were set up. Figure 11 As shown, the lignin content in the stems of wild-type Arabidopsis thaliana is approximately 55.62 mg / g. PmNST3 The lignin contents of the three overexpression lines OE1, OE3, and OE13 were 82.12, 83.19, and 76.20 mg / g, respectively, which were significantly higher than those of wild stems. PmNST3-E1 The lignin contents of the overexpression lines OE11, OE23, and OE24 were 36.01, 46.59, and 35.72 mg / g, respectively. PmNST3-E2 The lignin contents of the stems of the positive lines OE14, OE18, and OE19 were 27.57, 32.52, and 23.20 mg / g, respectively, all lower than those in the stems of wild-type Arabidopsis thaliana. It is speculated that... PmNST3 It can promote lignin synthesis in Arabidopsis stems, and PmNST3-E1 and PmNST3-E2 It has the function of delaying the lignification of Arabidopsis thaliana, and PmNST3-E2 The delay function compared to PmNST3-E1 Stronger.

[0068] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An isolated protein, characterized in that, The protein is NST3, NST3-E1, or NST3-E2; The NST3 protein is: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:1; or, (b) A protein derived from (a) with the sequence shown in SEQ ID NO:1 substituted, deleted or added with one or more amino acids and having the same function; NST3-E1 protein is: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO:3; or, (b1) A protein derived from (a1) with one or more amino acids substituted, deleted or added to the sequence shown in SEQ ID NO:3 and having the same function; NST3-E2 protein is: (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO:5; or, (b2) Proteins derived from (a2) with the sequence shown in SEQ ID NO:5 substituted, deleted or added with one or more amino acids and having the same function.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule is a plum blossom PmNST3 Genes, plum blossoms PmNST3- E1 Genes or plum blossoms PmNST3-E2 Gene; Among them, plum blossom PmNST3-E1 , PmNST3-E2 Genes of plum blossom PmNST3 Different variants of genes; plum bossom PmNST3 The genes are: A) The nucleotide sequence shown in SEQ ID NO:2; B) A nucleotide sequence of SEQ ID NO:2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; C) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:2 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the solution. D) A nucleotide sequence that has more than 90% homology with the nucleotide sequences of A), B), or C) and expresses the same functional protein; or, E) A nucleotide sequence that is completely complementary to the nucleotide sequence of A), B), C), or D); plum bossom PmNST3-E1 The genes are: A1), the nucleotide sequence shown in SEQ ID NO:4; B1) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:4 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; C1) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:4 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS, followed by washing the membrane with the solution. A nucleotide sequence (D1) that has more than 90% homology with the nucleotide sequences of A1), B1), or C1) and expresses the same functional protein; or, A nucleotide sequence that is completely complementary to the nucleotide sequence of E1), A1), B1), C1), or D1); plum bossom PmNST3-E2 The genes are: A2), the nucleotide sequence shown in SEQ ID NO:6; B2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:6 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; C2) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:6 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS, followed by washing the membrane with the solution. D2) nucleotide sequences that have more than 90% homology with the nucleotide sequences of A2), B2), or C2) and express the same functional protein; or, A nucleotide sequence that is completely complementary to the nucleotide sequences of E2, A2, B2, C2, or D2.

3. A biomaterial containing the nucleic acid molecule of claim 2, characterized in that, The biological material is an expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

4. Any of the following applications of the protein of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3: (1) Regulating plant lignin content; (2) Regulating plant architecture; (3) Preparation of transgenic plants; (4) Prepare kits for regulating plant type, lignin content or preparing transgenic plants.

5. The application according to claim 4, characterized in that, Using plum blossoms PmNST3 Genes or their expression products promote upright plant growth; or... Using plum blossoms PmNST3-E1 Genes or their expression products, and / or plum blossoms PmNST3-E2 Genes or their expression products promote drooping of plant branches.

6. The application according to claim 4, characterized in that, Improve plum blossom PmNST3 Gene expression levels increase lignin content; or, Improve plum blossom PmNST3-E1 and / or PmNST3-E2 Gene expression levels are reduced to decrease lignin content.

7. The application according to any one of claims 4-6, characterized in that, The plant in question is either plum blossom or Arabidopsis thaliana.

8. A method for cultivating plants with a specific plant type, characterized in that, Includes the step of introducing the nucleic acid molecule of claim 2 into a plant and causing it to be expressed; When cultivating upright plants, the introduced nucleic acid molecules are plum blossoms. PmNST3 Genes; when it is necessary to cultivate weeping plants, the introduced nucleic acid molecules are plum blossoms. PmNST3-E1 Genes and / or plum blossoms PmNST3-E2 Gene; The plant in question is either plum blossom or Arabidopsis thaliana.

9. A method for regulating the lignin content of plants, characterized in that, Includes the step of introducing the nucleic acid molecule of claim 2 into a plant and causing it to be expressed; When it is necessary to increase the lignin content of plants, the nucleic acid molecules introduced are plum blossoms. PmNST3 Genes; when it is necessary to reduce the lignin content of plants, the introduced nucleic acid molecules are plum blossoms. PmNST3-E1 Genes and / or plum blossoms PmNST3-E2 Gene; The plant in question is either plum blossom or Arabidopsis thaliana.

10. The application of the transgenic plant obtained according to the method of claim 8 or 9 in plant breeding; Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

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