5D006949-1D001661-3C003541 gene module in regulating the application of oat crude protein content

Abstract

The application discloses application of a 5D006949-1D001661-3C003541 gene module in regulating oat crude protein content and belongs to the technical field of plant molecular biology. The nucleotide sequence of 5D006949 is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2; the nucleotide sequence of 1D001661 is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4; and the nucleotide sequence of 3C003541 is shown in SEQ ID NO. 5, and the amino acid sequence is shown in SEQ ID NO. 6. By analyzing the coding sequences of the genes in the module, VIGS strains and overexpression transgenic oat strains are constructed to evaluate the functions of the genes in the gene module in regulating the crude protein content of oats. The results show that the gene module can regulate the crude protein content of oats, thereby providing a reference basis for genetically improving cultivated oats by using genetic engineering technology and providing a new target for cultivating new oat varieties with high yield and high quality.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and in particular to the application of the 5D006949-1D001661-3C003541 gene module in regulating the crude protein content of oats. Background Technology

[0002] Increasing the crude protein content of leaves in forage crops, especially oat grass (Avena sativa L.), is crucial for enhancing their nutritional value as high-quality forage. Current technologies primarily improve nitrogen use efficiency or increase grain protein content by regulating nitrogen absorption, assimilation-related genes, or transcription factors; however, their application in oats has significant limitations.

[0003] (1) Manipulation of nitrogen transporters: In model plants or cereals, overexpression of high-affinity nitrate transporter genes can increase root nitrogen uptake. However, the application of this strategy in oat grass has not been reported. Simply enhancing root nitrogen uptake often involves two problems: ① the absorption of inorganic nitrogen (NO3-) - NH4 + ① It fails to effectively assimilate nitrogen into organic nitrogen (amino acids, proteins) in leaves (the main nitrogen assimilation and storage organs), resulting in inefficient nitrogen accumulation; ② Non-specific expression leads to energy waste or excessive growth of the aboveground parts, and the increase in crude protein concentration per unit dry matter of leaves is limited or unstable.

[0004] (2) Regulation of key nitrogen assimilation enzymes: Overexpression of glutamine synthase (GS) or glutamate synthase (GOGAT) genes (such as GS2, which is predominantly expressed in leaves) aims to enhance the assimilation capacity of leaves for inorganic nitrogen. For example, overexpression of GS2 in tobacco increases the dry weight of the aboveground parts. However, such strategies have not yet been achieved in oat grass, and may face the following challenges: ① The enhancement of GS / GOGAT activity may be limited by the substrate (NH4+). + ① The nitrogen source (inorganic nitrogen, amino acids) was not supplied (such as glutamate, α-ketoglutarate) or energy (ATP, reducing power); ② The nitrogen source (inorganic nitrogen, amino acids) transport efficiency to the leaves was not simultaneously enhanced; ③ The nitrogen assimilation products were not specifically driven to flow towards the synthesis of storage proteins.

[0005] (3) Utilizing transcription factors to regulate nitrogen metabolism: Some studies have explored the use of Dof (DNA-binding with one finger) family transcription factors to regulate nitrogen response gene networks. For example, introducing the maize ZmDof1 gene into rice can enhance its carbon and nitrogen assimilation capacity under low nitrogen conditions. However: ① No specific functions of genes related to nitrogen response regulation and their target gene networks have been reported in oats; ② Existing studies mostly focus on overall nitrogen utilization or root nodule symbiosis, lacking reports on transcription factors and their synergistic target genes that directly target increasing leaf crude protein content.

[0006] Currently, improving the quality of oats still faces significant challenges:

[0007] (1) Traditional methods to increase the crude protein content of oat grass rely on breeding high-protein varieties or optimizing fertilization management (such as applying nitrogen fertilizer in the later stage). The application of molecular methods in oats is relatively lagging behind and is concentrated on the discovery of molecular markers such as grain β-glucan or oil improvement. Research on genetic improvement of crude protein content of vegetative organs (leaves) is extremely scarce.

[0008] (2) In oats, the core transcription factor (Master Regulator) that can sense nitrogen signals (such as nitrate) and simultaneously activate the expression of genes related to nitrogen transport, nitrogen assimilation and storage protein synthesis, and its synergistic mechanism have not been elucidated.

[0009] (3) Lack of modular design: There is a lack of gene modules that can integrate nitrogen signal sensing, nitrogen uptake / transport gene expression activation, nitrogen assimilation gene expression activation, and storage protein synthesis-related gene expression activation (such as NLP-regulated targets).

[0010] (4) Weak research foundation: There is very little understanding of the molecular mechanisms of nitrogen metabolism in oat leaves, especially the regulation of storage protein synthesis.

[0011] (5) Low genetic transformation efficiency: Oat grass is difficult to genetically transform, which limits gene function verification and molecular breeding applications.

[0012] The main reason for the above problems is:

[0013] (1) Insufficient analysis of the core regulatory network: In oats, little is known about the core transcription factor network that regulates nitrogen uptake, transport, assimilation, and storage protein synthesis in leaves. There is a lack of knowledge to identify the "major" regulatory modules that can efficiently coordinate nitrogen uptake and assimilation.

[0014] (2) Molecular design for improving forage quality is lagging behind: Traditional forage breeding focuses more on biomass (hay yield) and resistance. Molecular design breeding for nutritional quality (such as crude protein) started late and lacks efficient gene modules and targeted expression strategies (such as leaf-specific promoters) suitable for improving the quality of vegetative organs. Summary of the Invention

[0015] The purpose of this invention is to provide the application of the 5D006949-1D001661-3C003541 gene module in regulating the crude protein content of oats, so as to provide a new target for the genetic improvement of crude protein content of oats and lay the foundation for the breeding of high-yield and high-quality oat varieties.

[0016] To achieve the above objectives, on the one hand, the present invention provides the application of the 5D006949-1D001661-3C003541 gene module in regulating the crude protein content of oats. The nucleotide sequence of 5D006949 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the nucleotide sequence of 1D001661 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of 3C003541 is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0017] Preferably, 5D006949 is an upstream negative regulator of 1D001661. 5D006949 inhibits the expression of 1D001661 by specifically binding to the promoter region of 1D001661, thereby reducing the crude protein content of oats. Knocking down 5D006949 can significantly upregulate the expression level of 1D001661.

[0018] Preferably, 1D001661 is a positive regulator of crude protein content in oats, and overexpression of 1D001661 can significantly increase the crude protein content of oat leaves.

[0019] Preferably, 3C003541 and 1D001661 have protein-level interactions and the expression of 3C003541 is induced by nitrate signaling.

[0020] On the other hand, the present invention provides a method for regulating the crude protein content of oats, which regulates the expression of at least one gene in the above-mentioned gene module by means of genetic engineering, specifically including at least one of the following (1)-(3):

[0021] (1) Overexpression of the 1D001661 gene;

[0022] (2) Knock down or silence the 5D006949 gene;

[0023] (3) Synergistically regulate the expression levels of 1D001661 and 3C003541.

[0024] Preferably, the vector overexpressing 1D001661 is constructed using homologous recombination, and the primer sequences are shown in SEQ ID NO.23 and SEQ ID NO.24.

[0025] Preferably, the gene knockdown method employs viral-induced gene silencing technology, including: constructing a TRV2::Gene recombinant vector and achieving gene knockdown through vacuum infection of germinating buds.

[0026] On the other hand, the present invention provides an application of the 5D006949-1D001661-3C003541 gene module in oat breeding, using the 5D006949-1D001661-3C003541 gene module as a molecular marker or target gene, and cultivating oat varieties with high crude protein content through genetic transformation or molecular design breeding.

[0027] Preferably, genetic transformation includes Agrobacterium-mediated callus transformation, with specific steps including: embryogenic callus culture, Agrobacterium infection, resistance screening, and regenerated plant culture.

[0028] Therefore, the application of the gene module 5D006949-1D001661-3C003541 of this invention in regulating the crude protein content of oats has the following beneficial effects:

[0029] (1) For the first time, a cascade regulatory network was discovered to form between 5D006949, 1D001661 and 3C003541: EMSA experiments verified that 5D006949 inhibits the expression of 1D001661 by binding to the promoter, and BIFC experiments verified that 3C003541 and 1D001661 interact at the protein level and are specifically induced by nitrate signals;

[0030] (2) The crude protein content of leaves of the 1D001661 overexpression line was significantly higher than that of the wild type (WT), and there was no decrease in biomass or abnormal growth; the expression level of 1D001661 in the 5D006949 knockdown line was upregulated by 1.8 times, and the crude protein content was significantly higher than that of the control group.

[0031] (3) The 5D006949-1D001661-3C003541 gene module can regulate the crude protein content of oats, providing a reference for genetic improvement of cultivated oats using genetic engineering technology, and providing a new target for cultivating high-yield and high-quality oat varieties.

[0032] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1The results are for the 1D001661 knockdown strains. In the results, a represents the phenotype of the 1D001661 knockdown strains, and b represents the crude protein content of the 1D001661 knockdown strains.

[0035] Figure 2 The results are for the 5D006949 knockdown strains. In the image, a represents the phenotype of the 5D006949 knockdown strains, and b represents the crude protein content of the 5D006949 knockdown strains.

[0036] Figure 3 The results are for the 3C003541 knockdown strain. In the image, a represents the phenotype of the 3C003541 knockdown strain, and b represents the crude protein content of the 3C003541 knockdown strain.

[0037] Figure 4 The results show the crude protein content of the 1D001661 overexpression transgenic line. In the figure, a represents the DNA detection and Western blot results, b represents the relative gene expression level of 1D001661, and c represents the crude protein content.

[0038] Figure 5 Results of Electrophoretic Mobility Variation Experiment (EMSA);

[0039] Figure 6 Results from the dual-luciferase reporter system (dual-LUC);

[0040] Figure 7 The results show the upregulation of 1D001661 expression in the 5D006949 gene knockdown line;

[0041] Figure 8 The results of the interaction test between 1D001661 and 3C003541 are shown. Among them, a is the relative expression level of 3C003541 under low nitrogen (LN) level, b is the relative expression level of 3C003541 under high nitrogen (HN) level, and c is the result of bimolecular fluorescence complementarity (BIFC) experiment. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0043] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0044] 5D006949, 1D001661, and 3C003541 are newly discovered oat gene molecular modules in a gene co-expression network constructed based on genome-wide association analysis and transcriptome data from 270 oat samples.

[0045] The nucleotide sequence of 5D006949 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the nucleotide sequence of 1D001661 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of 3C003541 is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0046] SEQ ID NO.1:ATGGCTCCAGCTTCCGCATCCCTCTTCCCCGCCGCCAACGCCG.

[0047] SEQ ID NO.2:MAPASASLFPAANAGAKRPAPAAAVFDADHPLPQDGVAAGKN NQSQQQQLECPRCQSSNTKFCYYNNYSTTQPRHFCRACRRYWTHGGTLRKVPVGGA CRRAGNKRRRSADPSPSSAASPSAEAKEAHWPDDMAPATPFFPFLFPFLQLDFLPQDGVAAGKN PPGFPWTAASATTDLYDGLAAPWSGSDGNLTGGAWDDFTTGLDLTWPPTPAGN*.

[0048]

[0049] SEQ ID NO.4:*.

[0050]

[0051]

[0052] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0053] Example 1

[0054] Using cDNA from the oat variety Marvellous as a template, the VIGS target sequences of 5D006949, 1D001661, and 3C003541 were amplified, and TRV2::5D006949, TRV2::1D001661, and TRV2::3C003541 vectors were constructed using the NC cloning method. The TRV2 vector was a commercially available product from Nixing Biotechnology Co., Ltd.

[0055] The VIGS primer sequences for 5D006949, 1D001661, and 3C003541 are shown in Table 1 below. Among them, the reverse target primer design requires the addition of adapter sequences F: cagggtctctgtccagtcct, as shown in SEQ ID NO.7, and R: cggtctcagcagaccacaagt, as shown in SEQ ID NO.8.

[0056] Table 1 VIGS primer sequence design

[0057]

[0058] Example 2

[0059] Gene knockdown (VIGS) lines of 5D006949, 1D001661, or 3C003541 were obtained by vacuum infection of germinating shoots, as follows:

[0060] S1. Wash with 75% alcohol for 10 minutes, rinse twice with sterile water, wash with 10% NaClO for 20 minutes, and rinse five times with sterile water. Place two round filter papers in a petri dish, moisten them with sterile water, and place the sterilized seeds with the ventral groove facing down on the moistened filter paper. Germinate in the dark for 24 hours.

[0061] S2. After activating positive single colonies by streaking, incubate at 28°C in YEP medium (containing antibiotics Kan and Rif) at 200 rpm for 24 hours. Simultaneously activate and incubate Agrobacterium carrying empty TRV1 and TRV2 vectors.

[0062] S3. Dilute the shaken bacterial culture with YEP+Kan+Rif liquid medium to OD=1.2.

[0063] S4. Mix the TRV1 bacterial solution with adjusted OD value with TRV2 bacterial solution and TRV2::Gene (5D006949, 1D001661 or 3C003541) bacterial solution at a volume ratio of 1:1.

[0064] S5. Add a permeation solution to the mixed bacterial culture. The permeation solution includes 400 mg / L cysteine, 5 mL / L Tween-20, and 19.62 mg / L acetylsuccinone.

[0065] S6. Place Marvellous oat seeds that have germinated to a length of 3 mm in the dark together with the bacterial solution in a small glass bottle, vacuum at 20 kPa for 5 min, and then culture at 28°C and 180 rpm for 16 h.

[0066] S7. After rinsing twice with sterilized water, plant it in the soil. The planting conditions are 15℃ at night and 22℃ during the day, with a photoperiod of 16 hours of light and 8 hours of darkness.

[0067] Ultimately, three 1D001661 knockdown lines (V-1D001661-1, V-1D001661-2, and V-1D001661-3), three 5D006949 knockdown lines (V-5D006949-1, V-5D006949-2, and V-5D006949-3), one 3C003541 knockdown line (V-3C003541-1), and the control group V1::V2 line were obtained.

[0068] Example 3

[0069] Using cDNA from the oat variety Marvellous as a template, the complete coding sequence of 1D001661 was amplified, and the pUBI::1D001661-flag overexpression vector was constructed by homologous recombination according to the instructions (Norwayzan, C112).

[0070] The primers for vector construction are:

[0071] pUBI::1D001661-flag-F:gtttggtgttacttctgcagaATGGCCTCGGTGCTGCCGGA, SEQ ID NO.23;

[0072] pUBI::1D001661-flag-R:gtctttgtagtccatggatcATGGCCGACAATCATGGCCT, SEQ ID NO. 24.

[0073] Example 4

[0074] The 1D001661 overexpressing transgenic line was obtained using an oat-based genetic transformation system. The method for establishing the 1D001661 overexpressing transgenic line includes:

[0075] S1. Cultivating callus tissue: Mature embryos of oat variety 'c918' were cultured on L3-M medium (4.6 g / L L3 base salt, 30 g / L maltose, 4 g / L plant gel, 2 mL / L 2,4-D, 1 mL / L dicamba, with H2O added to 1 L, pH = 5.8) until embryogenic callus tissue was produced.

[0076] S2. Preparation of Infection Solution: Agrobacterium strain GV3101 containing pUBI::1D001661 was cultured overnight at 28°C in YEP medium containing kanamycin and rifampin. After centrifugation, the precipitate was resuspended to OD=0.5 with Wls solution (4.6 g / L L3 basic salt, 10 g / L glucose, 0.5 g / L MES, H2O added to 1 L, pH=5.8), and then mixed in an equal proportion with Agrobacterium strain GV3101 carrying pUBI::taWOX5.

[0077] S3. Callus Infection: Selected embryogenic callus tissues were immersed in a mixed Agrobacterium tumefaciens strain for 30 minutes. Residual bacterial liquid was absorbed using filter paper, and the tissues were air-dried on the filter paper for 5 minutes. The embryogenic callus tissues were then placed on filter paper containing 0.15 mM As and cultured in the dark for 3 days. The embryogenic callus tissues were then cultured on Wils-Res medium for 5 days. Viable callus tissues were screened using Wils-P5 medium (Wils-Res medium containing 100 μL / L Basta).

[0078] S4. Callus regeneration: Transfer the callus to a regeneration medium (4.6 g / L L3 basic salt, 20 g / L sucrose, 0.5 g / L MES, 200 μL / L 12.5 g / L CuSO4·5H2O, 4 g / L plant gel, add H2O to 1 L, pH=5.8, sterilize and add 1 mL / L termethin, 100 μL / L Basta, 5 mL / L zeatin) and culture until leaves of 1-2 cm differentiate.

[0079] S5. Rooting culture is carried out on rooting medium. The rooting medium formula is: 4.6 g / L L3 basic salt, 15 g / L sucrose, 0.5 g / L MES, 4 g / L plant gel, add H2O to 1L, pH=5.8, sterilize and add 1 mL / L termethin, 100 uL / L Basta, and 20 uL / L IBA.

[0080] The 1D001661 overexpression transgenic line 1D001661OE was finally obtained.

[0081] Test Example 1

[0082] Using 5 mM KNO3 as a high-concentration nitrogen source and 0.25 mM KNO3 as a low-concentration nitrogen source, the phenotypic characteristics of oat seedlings were observed by soaking the roots, and the changes in crude protein content before and after treatment were detected by a crude protein analyzer.

[0083] like Figures 1-3 As shown, Figure 1 In the figure, a represents the phenotype of the 1D001661 gene knockdown line, and b represents the crude protein content detection in the 1D001661 gene knockdown line. The results show that the crude protein content of oats is significantly reduced in the 1D001661 gene knockdown line, indicating that the 1D001661 gene positively regulates the accumulation of crude protein in oats.

[0084] Figure 2 In the table, a represents the phenotype of the 5D006949 gene knockdown line, and b represents the crude protein content detection in the 5D006949 gene knockdown line. The results show that the crude protein content of oats is significantly upregulated in the 5D006949 gene knockdown line, indicating that the 5D006949 gene negatively regulates the accumulation of crude protein in oats. The 1D001661 gene and the 5D006949 gene show a negatively correlated regulatory relationship in their effects on crude protein content in oats.

[0085] Figure 3 In the figure, a represents the expression level of the 3C003541 gene in the 3C003541 gene knockdown line, and b represents the crude protein content in the 3C003541 gene knockdown line. The results show that the crude protein content of oats is significantly downregulated in the 3C003541 gene knockdown line, indicating that the 3C003541 gene positively regulates the accumulation of crude protein in oats.

[0086] Test Example 2

[0087] The crude protein content of the 1D001661 overexpression transgenic line was detected, and the results are as follows: Figure 4 As shown, a represents the results of DNA detection and Western blot analysis, b represents the relative gene expression level of 1D001661, and c represents the crude protein content.

[0088] Depend on Figure 4 As shown in Figure a, no obvious band was detected in the WT group during DNA testing, while a clear band was observed in the 1D001661OE group, indicating that the DNA of 1D001661 inserted in the overexpression group was successfully expressed.

[0089] The α-flag detection results showed that there was no band in the WT group, while there was a specific band in the 1D001661OE group, indicating that the overexpressed 1D001661 protein carried a flag tag and was successfully expressed, and the transgenic lines were positive seedlings.

[0090] The α-TUB (α-tubulin) assay results showed that both the WT group and the 1D001661OE group had clear bands with similar brightness, which, as internal reference proteins, verified that the sample loading amount was consistent.

[0091] Depend on Figure 4 As shown in Figure b, the gene expression level of the 1D001661OE group was significantly higher than that of the WT group (P<0.01), indicating the successful construction of 1D001661 overexpression plants.

[0092] Depend on Figure 4 As shown in Figure c, the crude protein content of the 1D001661OE group was significantly higher than that of the WT group (P<0.01), indicating that overexpression of 1D001661 can significantly increase the crude protein content.

[0093] Test Example 3

[0094] 5D006949 negative regulation 1D001661 verification:

[0095] ① Electrophoretic mobility variation experiment (EMSA), the steps are as follows:

[0096] S1. Vector construction: The PGEX4T-1-5D006949 protein expression vector was constructed by homologous recombination according to the instructions (Novizan, C112).

[0097] The primers are:

[0098] PGEX4T-1-5D006949-F: CTGGTTCCGCGTGGATCATGGCTCCAGCTTCCGCATC, SEQ IDNO.25;

[0099] PGEX4T-1-5D006949-R: TCGAGTCGACCCGGGGTTGCCGGCCGGCGTTGGCG, SEQ ID NO. 26.

[0100] S2, Protein Expression:

[0101] 1) The constructed expression vectors PGEX4T-1-5D006949-GST and the empty vector PGEX4T-1-GST were transformed into BL21 strain. Single colonies were inoculated into 10 mL of LB medium containing Amp and antibiotics and cultured overnight. After culture, the culture was diluted 1:100 and inoculated into LB medium containing antibiotics. The culture was incubated at 37°C for 2 h until the OD of the bacterial culture was reached. 600 Reach 0.6. Add 0.5 mM IPTG and incubate at 28°C for 3 hours. Collect the cultured bacterial solution into a centrifuge tube and centrifuge at 8000 rpm for 20 minutes at 4°C. Discard the supernatant and collect the precipitate.

[0102] 2) Take the precipitate after induced expression and add 40 mL of non-denaturing lysis buffer to fully suspend the cells. The non-denaturing lysis buffer formula is: 50 mM Tris-HCl (pH = 8.0), 1 mM DTT, 200 mM NaCl, and add 1 cOmplete EDTA-free protease inhibitor tablet (Roche) to every 50 mL of lysis buffer.

[0103] 3) Add lysozyme to a final concentration of 1 mg / mL, mix well, and place in an ice bath for 30 minutes; then perform high-pressure sterilization on the bacteria.

[0104] 4) Centrifuge at 4℃, 10000g for 20 minutes, collect the supernatant and place it on ice.

[0105] 5) Take 300 μL of GST tag magnetic beads, wash three times with non-denaturing lysis buffer, and then incubate them in the collected supernatant.

[0106] 6) The magnetic beads were washed four times with GST-rinsing solution, each time rotating and rinsing for 10 minutes at 4°C.

[0107] GST-rinse solution formula: 154mM NaCl, 50mM Tris-HCl, pH=8.0.

[0108] 7) Use GST-elution buffer to elute the magnetic beads 3 times, each time rotating and eluting for 10 minutes at 4°C.

[0109] GST-elution buffer formulation: 50mM Tris-HCl (pH=8.0), 1mM DTT, 200mM NaCl, 10mM GSH.

[0110] 8) Collect the eluent from each elution into a clean centrifuge tube; this is the final purified protein. Protein purity is analyzed by Coomassie Brilliant Blue staining.

[0111] S3, EMSA probe preparation:

[0112] 1) Prepare probe dilution solution: 10mM Tris-HCl (pH=8.0), 1mM EDTA (pH=8.0), 50mM NaCl.

[0113] 2) Dilute the primers to 10 μmol using probe dilution buffer.

[0114] 3) Anneal the probe to obtain a probe concentration of 1 pmol / μL. Store at -20℃ after annealing, or at 4℃ for 1-2 weeks.

[0115] Annealing conditions are as follows:

[0116] Step 1: React at 95℃ for 5 minutes.

[0117] Step 2: React at 95℃ (-1℃ / cycle) for 1 minute, for 70 cycles.

[0118] Step 3: 4℃, ∞.

[0119] S4. Prepare 6% non-modified adhesive, the formulation of which is shown in Table 2 below:

[0120] Table 2 Non-modified adhesive formulations

[0121] Reagent Name Dosage 5×TBE 1.5mL 30% Acrylamide 3mL 10% AP 105μL TEMED 6μL <![CDATA[ddH2O]]> 10.35mL

[0122] S5, Electrophoresis buffer preparation:

[0123] 5×TBE electrophoresis buffer: 54g Trisbase (tris(hydroxymethyl)aminomethane), 27.5g boric acid, 20mL 0.5MEDTA (pH=8.0, ethylenediaminetetraacetic acid).

[0124] Place the prepared gel stably in the electrophoresis tank, set the voltage to 100V, run it dry for 30-60 minutes, and use 0.5×TBE as the electrophoresis solution.

[0125] S6. Binding reaction: The binding reaction is carried out in 200 μL of an octet. The reagents for the binding reaction should be added slowly in the order shown in Table 3. The binding reaction system is as follows:

[0126] Table 3 Combined Reaction System

[0127]

[0128]

[0129] After incubating at room temperature for 20 minutes, add 5 μL of 5× loading buffer to each binding reaction and mix by aspirating and swirling several times.

[0130] S7. Electrophoresis: After rinsing the gel cavity, load 10 μL of each sample and perform electrophoresis on ice at 100V until the bromophenol blue dye reaches 2 / 3 or 3 / 4 of the gel.

[0131] S8, Transfer:

[0132] 1) N + The membrane was immersed in 0.5×TBE for 10 minutes.

[0133] 2) Gel, N + The membrane and absorbent paper were sandwiched in a clean electrophoresis transfer apparatus. 0.5×TBE was used for transfer on ice.

[0134] 3) Transfer the membrane at 380mA for 30 minutes.

[0135] 4) After the transfer is complete, place the membrane with the bromophenol blue side up on a dry paper towel to allow the buffer solution on the membrane surface to be absorbed into the membrane.

[0136] 5) Use a UV-crosslinking instrument equipped with a 254nm bulb at 120MJ / cm 2 The degree of cross-linking is automatically cross-linked for 60 seconds.

[0137] S9. Detection of biotin-labeled DNA by chemiluminescence:

[0138] 1) Heat the blocking buffer and 4× washing buffer to 50°C until all particles dissolve.

[0139] 2) Add 20 mL of blocking buffer and gently shake to incubate for 15 minutes.

[0140] 3) Prepare the conjugate / blocking buffer by adding 66.7 μL of streptavidin-horseradish peroxidase conjugate to 20 mL of blocking buffer.

[0141] 4) Pour off the blocking buffer from the membrane and replace it with conjugate / blocking buffer. Gently shake and incubate for 15 minutes.

[0142] 5) Add 40 mL of 4× washing buffer to 120 mL of ultrapure water to prepare 1× washing solution.

[0143] 6) Transfer the membrane to a new container and rinse briefly with 20 mL of 1× washing solution.

[0144] 7) Gently shake and wash the membrane 4 times in 20 mL of 1× washing solution, 5 minutes each time.

[0145] 8) Transfer the membrane to a new container, add 30 mL of substrate equilibration buffer, and gently shake the membrane to incubate for 5 minutes.

[0146] 9) Expose using a luminescent liquid.

[0147] Experimental results of electrophoretic mobility variation are as follows Figure 5As shown in the experimental results, the first column (negative control) contained only the control protein GST and the biotin-labeled target DNA probe in the reaction system. The results showed only a free probe signal, indicating that the negative control protein did not bind to the target DNA probe (a segment of the 1D001661 promoter region). The second column (experimental group) included the 5D006949 protein fused with the GST tag. 5D006949 bound to the biotin-labeled target DNA probe, showing a shift band, indicating that 5D006949 can bind to the target DNA fragment. The third column (competitive experimental group) contained 5D006949 protein in the reaction system, along with the biotin-labeled target DNA probe and the unlabeled probe (150 times stronger than the biotin-labeled probe; one "+" represents 50 times). The results showed that the binding band in the third column was weaker than in the second column, further demonstrating that 5D006949 can bind to the target DNA fragment. The fourth column shows the mutation experiment. Point mutations were performed on the DNA site in the biotin-tagged probe that might bind to the 5D006949 protein. The results showed that after the mutation, the probe did not bind to the 5D006949 protein, indicating that the binding site of the 5D006949 protein to the promoter region of 1D001661 is correct. The experiment confirms that 5D006949 can specifically bind to the promoter region of 1D001661.

[0148] ②LUC activity detection test:

[0149] (1) The empty vector plasmid pNC-Cam3304-MCS35S, the recombinant plasmids pNC-Cam3304-MCS35S-5D006949, pNC-Green-Luc-1D001661-pro, and pNC-Green-Luc-1D001661-promu were transformed into GV3101 (pSoup-p19) competent cells, respectively, and heat shock transformation was performed according to the reagent instructions (Weidi Bio; AC1002L). The transformed Agrobacterium was cultured at 28°C for 2 days on LB solid medium (25 μg / mL Rif and 50 μg / mL Kana). Single colonies were picked and dissolved in LB liquid medium containing 25 μg / mL Rif and 50 μg / mL Kana and cultured with shaking until OD. 600 =0.8. Resuspend the bacterial cells in an equal volume of tobacco infection solution, and then let them stand in the dark for 2-3 hours.

[0150] (2) Mix the following different combinations of Agrobacterium suspensions (pNC-Cam3304-MCS35S+pNC-Green-Luc-1D001661-pro, pNC-Cam3304-MCS35S-5D006949+pNC-Green-Luc-1D001661-pro and pNC-Cam3304-MCS35S-5D006949+pNC-Gree n-Luc-1D001661-promu) in equal proportions. Inject the different Agrobacterium mixtures into tobacco leaves.

[0151] (3) After 2 days of light incubation, approximately 100 mg of leaf tissue from the injection site was placed in a 2 mL EP tube containing two 3 mm steel beads. After quick-freezing in liquid nitrogen, the tissue was ground into powder. The activities of firefly luciferase (Fluc) and kidney luciferase (Rluc) were measured according to the manufacturer's instructions (Lambolid; DR 075).

[0152] like Figure 6 As shown, the dual-luciferase reporter system (dual-LUC) showed that 5D006949 significantly inhibited the promoter activity of 1D001661 (P<0.0001).

[0153] At the same time, such as Figure 7 As shown, upregulation of 1D001661 was detected in the 5D006949 gene knockdown line.

[0154] The above results clarify that 5D006949 is an upstream negative regulator of 1D001661, providing new clues for elucidating the accumulation regulatory network of oat crude protein content.

[0155] Test Example 4

[0156] Interaction test between 1D001661 and 3C003541:

[0157] ① Bimolecular fluorescence complementary (BIFC) experiment, the steps are as follows:

[0158] S1. Vector construction: The vector was constructed according to the instructions (Nixing Bio, NC001). The primers for vector construction were pNC-BiFC-Enn-1D001661 and pNC-BiFC-Enc-3C003541.

[0159] 1D001661-F: ATGGCCTCGGTGCTGCCGGA, SEQ ID NO. 27.

[0160] 1D001661-R: ATGGCCGACAATCATGGCCT, SEQ ID NO. 28.

[0161] 3C003541-F: ATGTGTGTGAGTGATTCCTT, SEQ ID NO. 29.

[0162] 3C003541-R:ACCGGAGCTTCCACAAGAAC, SEQ ID NO. 30.

[0163] The constructed vector was transformed into Agrobacterium according to the instructions (Weidi Bio, AC1002).

[0164] S2. Bacterial culture treatment: Pour the turbid bacterial culture into a 2mL centrifuge tube, centrifuge at 4000rpm for 10min, discard the supernatant, add an equal volume of buffer to the precipitate, and measure the OD. 600 Add the corresponding bacterial solution according to the required protein-interacting pairs, adjust the bacterial solution concentration with buffer, mix well, and let stand for 3 hours before injecting tobacco.

[0165] S3. Tobacco Injection: Select fresh, thick upper leaves of robust tobacco plants for injection with Agrobacterium-mediated bacterial suspension. Use a 1mL syringe (without a needle) to draw up the suspension, support the underside of the tobacco leaf, and inject into the smooth leaf tissue, avoiding the veins. Gently push the syringe with your fingertip, being careful not to puncture the leaf. The extent of infection can be observed by looking for water stains on the leaves; most leaves should be infected. You can select 3-4 leaves from different plants for injection as replicates, and mark the leaves. After infection, incubate the tobacco plants in an incubator for 2 days to allow for sufficient protein expression, then observe the fluorescence signal under a laser confocal microscope.

[0166] The results are as follows Figure 8 As shown, a represents the relative expression level of 3C003541 under low nitrogen (LN) levels, and b represents the relative expression level of 3C003541 under high nitrogen (HN) levels. It can be seen that KNO3 treatment significantly upregulated 3C003541 expression under both LN and HN conditions, while KCl treatment showed no significant change, indicating that induction depends on nitrate signaling rather than chloride ion levels. Under HN conditions, KNO3 induction was more intense and showed significance earlier, suggesting that high nitrogen levels enhance the inductive effect of nitrate on 3C003541.

[0167] Figure 8 Part c presents the results of bimolecular fluorescence complementarity (BIFC) experiments, demonstrating the protein-level interaction between 3C003541 and 1D00168.

[0168] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. 5D006949 or 1D001661 The application of genes in increasing the crude protein content of oat leaves is characterized by: 5D006949 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; by knocking down 5D006949 This increases the crude protein content of oat leaves; 1D001661 The nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; through overexpression 1D001661 This increases the crude protein content of oat leaves.

2. A method for increasing the crude protein content of oat leaves, characterized in that, Regulating the expression of at least one gene in claim 1 by means of genetic engineering means specifically includes at least one of the following (1)-(2): (1) Overexpression 1D001661 Gene; (2) Knock down 5D006949 Gene.

3. The method for increasing the crude protein content of oat leaves according to claim 2, characterized in that: overexpression 1D001661 The vector was constructed using homologous recombination, and the primer sequences are shown in SEQ ID NO.23 and SEQ ID NO.

24.

4. The method for increasing the crude protein content of oat leaves according to claim 2, characterized in that, The gene knockdown method employs viral-induced gene silencing technology, including: constructing a TRV2::Gene recombinant vector and achieving gene knockdown through vacuum infection of germinating buds.

5. The application of the method for increasing the crude protein content of oat leaves as described in any one of claims 2-4 in oat breeding, characterized in that: Will 5D006949 or 1D001661 Genes are used as molecular markers or target genes to cultivate oat varieties with high crude protein content in leaves through genetic transformation or molecular design breeding.

6. The application according to claim 5, characterized in that, Genetic transformation includes Agrobacterium-mediated callus transformation, which involves the following steps: embryogenic callus culture, Agrobacterium infection, resistance screening, and regenerated plant culture.

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