Application of medicago sativa cFBP protein in regulation and control of plant type

Abstract

The invention belongs to the field of plant genetic engineering, and provides application of alfalfa cFBP protein in regulation and control of plant types, and the nucleotide sequence of an alfalfa cFBP protein coding gene is SEQ ID NO.1 or SEQ ID NO.3. The invention further provides a preparation method of the alfalfa cFBP protein. The alfalfa cFBP protein coding gene can positively regulate and control the plant height, the branch number and the biomass of alfalfa, and the plant height, the branch number and the biomass of the plant after overexpression of the MscFBP gene are remarkably improved compared with those of a control material. According to the invention, the MscFBP gene is overexpressed through a genetic engineering technology, so that the transgenic alfalfa with remarkably improved biological yield is successfully obtained, and important normal forms and gene resources are provided for cultivating high-yield plant varieties.

Description

Technical Field

[0001] This application belongs to the field of plant genetic engineering. Specifically, this application provides the application of alfalfa cFBP protein in regulating plant architecture. Background Technology

[0002] Plant architecture is a crucial agronomic trait determining crop yield, encompassing plant height, number of branches / tillers, and branch / tiller angles, among other aspects. It directly impacts yield by regulating the plant's efficiency in utilizing resources such as light, water, and fertilizer. The concept of "ideal plant architecture" was first proposed in 1968, referring to the form in which competition among individual crop plants is minimized while the accumulation of grain assimilates is maximized—essentially achieving the "greatest common denominator" of crop yield and quality through efficient use of light and soil fertility. "Ideal plant architecture" has become an important breeding target for crops such as rice, corn, wheat, soybeans, and rapeseed. However, a clear concept has not yet been established for most forage crops. Therefore, identifying genes related to crop plant architecture regulation and elucidating their molecular mechanisms is of great significance for obtaining higher-yielding varieties.

[0003] Alfalfa (Medicago sativa L.), known as the "King of Forage," is widely cultivated worldwide and is a representative of high-quality perennial forage. Plant type is a key factor affecting alfalfa yield, and developing alfalfa varieties with upright plant type, rapid growth, and high biomass is an urgent need for the industry. Summary of the Invention

[0004] On the one hand, this application provides the application of alfalfa cFBP protein in regulating plant architecture, wherein the amino acid sequence of the alfalfa cFBP protein is SEQ ID NO.2 or SEQ ID NO.4.

[0005] Furthermore, in the application, the expression level of alfalfa cFBP protein or its encoding gene in the plant is increased to achieve one or more effects selected from (A1)-(A3);

[0006] (A1) Increase plant height;

[0007] (A2) Increase the number of branches in the plant;

[0008] (A3) Increase plant biomass.

[0009] Furthermore, the expression level of alfalfa cFBP protein or its encoding gene in the plant is achieved by one or more tools selected from (B1)-(B2):

[0010] (B1) A tool for overexpressing the gene encoding alfalfa cFBP protein;

[0011] (B2) Tools to enhance the activity of alfalfa cFBP protein.

[0012] Further, (B1) is a nucleic acid molecule encoding the alfalfa cFBP protein gene, or a vector, expression cassette, recombinant microorganism, plant cell, plant tissue, or plant organ containing the alfalfa cFBP protein encoding gene; preferably, an overexpression vector containing the alfalfa cFBP protein encoding gene; more preferably, the overexpression vector contains a constitutive promoter; and even more preferably, a pCAMBIA1307 vector containing the alfalfa cFBP protein encoding gene.

[0013] In addition, known techniques such as CRISPRa activation or saRNA stimulation can also be used.

[0014] Furthermore, the plant in question is alfalfa.

[0015] Furthermore, the application is used for one or more of (C1)-(C3):

[0016] (C1) Improves plant shape;

[0017] (C2) Provide plant propagation materials for improving plant structure, preferably seeds, tissue culture materials, and organs that can be used for propagation;

[0018] (C3) Provides plant strains with improved plant type.

[0019] On the other hand, this application provides alfalfa cFBP protein, wherein the amino acid sequence of the alfalfa cFBP protein is SEQ ID NO.2 or SEQ ID NO.4.

[0020] On the other hand, this application provides the encoding gene of the above-mentioned alfalfa cFBP protein.

[0021] Furthermore, the nucleotide sequence of the encoding gene is SEQ ID NO.1 or SEQ ID NO.3.

[0022] On the other hand, this application provides the application of any one of the tools (D1)-(D3) for detecting the expression level of alfalfa cFBP protein and the expression level of the alfalfa cFBP protein encoding gene:

[0023] (D1) Determine the plant type of alfalfa;

[0024] (D2) Predicting plant architecture of alfalfa propagation material;

[0025] (D3) Develop alfalfa varieties with improved plant type.

[0026] The tools used to detect the expression levels of alfalfa cFBP protein and the expression levels of the alfalfa cFBP protein encoding gene are one or more of the following:

[0027] (E1) Primer;

[0028] (E2) Probe;

[0029] (E3) PCR reagents;

[0030] (E4) chip;

[0031] (E5) Antibody;

[0032] (E6) ELISA reagent.

[0033] The nucleotide sequence of the alfalfa cFBP protein encoding gene is SEQ ID NO.1 or SEQ ID NO.3; the amino acid sequence of the alfalfa cFBP protein is SEQ ID NO.2 or SEQ ID NO.4.

[0034] The codons of proteins can be varied by those skilled in the art based on the host requirements or expression needs, and on known codon preferences and other factors.

[0035] This application identifies two MscFBP gene haplotypes, MscFBPHap1 and MscFBP Hap2, that positively regulate alfalfa plant height, branch number, and biomass. Knocking out MscFBP using gene editing technology resulted in mutant plants with reduced plant height, fewer branches, and lower aboveground dry weight compared to the control. Overexpression of MscFBP Hap1 or MscFBP Hap2 via constitutive promoters significantly increased transgenic plant height, branch number, and biomass compared to the control, with the MscFBP Hap2 overexpression material showing significantly higher values ​​for all indicators than the MscFBP Hap1 overexpression material. This invention successfully obtained transgenic alfalfa with significantly increased biomass through overexpression of MscFBP using genetic engineering technology, providing an important paradigm and genetic resource for breeding high-yielding plant varieties. Attached Figure Description

[0036] Figure 1 Genome-wide association analysis (GWAS) for growth habit index and plant height phenotype, and mining of the major gene MScFBP. Part A is a Manhattan plot of the GWAS analysis for the growth habit index phenotype, n=132. Part B is a Manhattan plot of the GWAS analysis for the plant height phenotype, n=132. The y-axis represents the t-test correlation test result - log. 10 (P) value, x-axis is marked with chromosome number.

[0037] Figure 2This section describes the assay of the FBP enzyme activities of MsFBP Hap1 and Hap2. Parts A and B show the protein purification assays for His-MscFBP Hap1 and Hap2. The purification efficiency of His-MscFBP Hap1 and Hap2 was assessed by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining. M represents the protein molecular weight marker; precipitation refers to cell precipitation; supernatant refers to lysis supernatant; and flow-through refers to the flow-through buffer. Lanes 0, 20, 50, 80, and 100 correspond to elution fractions with imidazole concentrations of 0, 20, 50, 80, and 100 mM, respectively. Part C presents the time-matter curves of NADPH generation catalyzed by MsFBPHap1 and MsFBP Hap2, with the absorbance at 340 nm reflecting the FBP enzymatic reaction rate. Part D shows the FBP enzyme activity assay results for MsFBP Hap1 and MsFBP Hap2. The difference was analyzed by t-test, and the result showed that p < 0.0001, indicating that the FBP enzyme activity of MsFBP Hap2 was significantly higher than that of Hap1.

[0038] Figure 3 This is a schematic diagram of the structure of the alfalfa expression vector p6401-MscFBP.

[0039] Figure 4 The results show the identification of the Mscfbp mutant. The results of transgenic plants obtained by transforming the p6401-MscFBP vector into alfalfa via Agrobacterium EHA105 were also presented. In the diagram, M represents the standard molecular weight of DNA, CK-1 represents the control material, and the positive control "+" represents the plasmid p6401-MscFBP. The results indicate that samples Mscfbp-61, Mscfbp-78, and Mscfbp-89 all showed amplified bands, and these were all positive transgenic plants obtained by transforming the p6401-MscFBP vector into alfalfa via Agrobacterium EHA105.

[0040] Figure 5 The results show the mutational patterns of the Mscfbp mutant. Part A analyzes the DNA-level mutational patterns of the Mscfbp mutant. Horizontal lines indicate target regions, bold letters represent PAM sequences, "-" indicates deleted bases, and lowercase letters indicate inserted bases. Part B analyzes the protein-level mutational patterns of the Mscfbp mutant.

[0041] Figure 6 The growth status of alfalfa CK-1 and Mscfbp mutant materials was observed 3 weeks after mowing. The overall morphology is shown in Part A. Parts B and C show the plant height, number of branches, aboveground fresh weight, and aboveground dry weight, respectively. The results showed that the plant height, number of branches, aboveground dry weight, and aboveground dry weight of the Mscfbp mutant were all significantly lower than those of the control material CK-1.

[0042] Figure 7 This is a schematic diagram of the structures of the overexpression vectors pCAMBIA1307-MscFBP Hap1 and pCAMBIA1307-MscFBP Hap2.

[0043] Figure 8 This section presents the transgenic identification results of plants overexpressing MscFBP Hap1 and MscFBP Hap2. Part A shows the identification results of transgenic plants obtained by transforming the pCAMBIA1307-MscFBP Hap1 vector into Alfalfa 'Zhongmu No. 1' via Agrobacterium EHA105. M represents the standard molecular weight of DNA, positive control "+" indicates plasmid pCAMBIA1307-MscFBPHap1, and CK-2 represents the control plants. MscFBP-F and GFP-R represent primers for transgenic identification, with 1163 bp being the molecular weight of the target fragment. MscFBP Hap1-OE-1 and MscFBP Hap1-OE-2 represent genetically transformed and regenerated plants. Part B represents the identification results of transgenic plants obtained by transforming the pCAMBIA1307-MscFBP Hap2 vector into Alfalfa 'Zhongmu No. 1' via Agrobacterium EHA105. Here, M represents the standard molecular weight of DNA, the positive control "+" represents the plasmid pCAMBIA1307-MscFBP Hap2, and CK-2 represents the control plant. MscFBP-F and GFP-R represent primers for transgenic identification, with 1163 bp being the molecular weight of the target fragment. MscFBP Hap2-OE-1 and MscFBP Hap2-OE-2 represent genetically transformed and regenerated plants.

[0044] Figure 9 RT-qPCR detection was performed on transgenic plants overexpressing MscFBP Hap1 and MscFBP Hap2. CK-2 was the control plant.

[0045] Figure 10 To detect the MscFBP protein in transgenic plants overexpressing MscFBP Hap1 and MscFBP Hap2 using Western blotting. CK-2 served as the control plant.

[0046] Figure 11 The growth status of MscFBP Hap1 and MscFBP Hap2 overexpressing plants was observed 3 weeks after cutting. The overall morphology is shown in Part A, while Parts B and C represent the measurements of plant height, number of branches, aboveground fresh weight, and aboveground dry weight, respectively. The results showed that the plant height, number of branches, aboveground fresh weight, and aboveground dry weight of the MscFBP overexpressing plants were significantly higher than those of CK-2, and the growth of the MscFBP Hap2 overexpressing plants was superior to that of the MscFBP Hap1 overexpressing plants. Detailed Implementation

[0047] The culture media and their formulations involved in the examples are as follows:

[0048] a. The formulation of SM4 liquid medium (1L) is as follows: MURASHIGE&SKOOG(MS) BASAL MEDIUM (M519) (Phyto Technology Laboratories™) 4.43g, 2,4-D stock solution (10mg / mL) 0.4mL, 6-BAP stock solution (1mg / mL) 0.2mL, sucrose 30g, and pH adjusted to 5.85 using KOH. Add 3.2g of plant gel to the solid medium.

[0049] b. The formulation of MSBK liquid medium (1L) is as follows: MURASHIGE&SKOOG(MS) BASAL MEDIUM (M519) (Phyto Technology Laboratories™) 4.43g, kinetin (1mg / mL) 1mL, 6-BAP stock solution (1mg / mL) 0.5mL, sucrose 30g, and pH adjusted to 5.85 using KOH. Add 3.2g of plant gel to the solid medium.

[0050] c. The formulation of SH9 liquid culture medium (1L) is as follows: 100mL of 10×N6 large volume stock solution, 1mL of 1000×SH micro volume stock solution, 1mL of 1000×SH organic stock solution, 20mL of 50×EDFS iron salt stock solution, 100mg of inositol, and 20g of sucrose. The pH is adjusted to 5.85 using KOH. 8g of agar is added to the solid culture medium.

[0051] d. The formulation of 1 / 2 MS liquid medium (1L) is as follows: 2.22g MURASHIGE & SKOOG (MS) BASAL MEDIUM (M519) (Phyto Technology Laboratories™), 12g sucrose, and pH adjusted to 5.85 using KOH. 8g agar is added to the solid medium.

[0052] e. 10×N6 large-volume mother liquor (1L): MgSO4·7H2O 1.85g, KNO3 28.3g, (NH4)2SO4 4.63g, CaCl2·2H2O 1.66g, KH2PO4 4g, H2O to a final volume of 1L.

[0053] f. 1000×SH micro-volume stock solution (1L): MnSO4·H2O 10g, H3BO3 5g, ZnSO4·7H2O 1g, KI 1g, Na2MoO4·2H2O 100mg, CuSO4·5H2O 200mg, CoCl2·6H2O 100mg, H2O to a final volume of 1L.

[0054] g, 1000×SH organic mother liquor (1L): 5g nicotinic acid, 5g pyridoxine hydrochloride, 5g thiamine hydrochloride, H2O to a final volume of 1L.

[0055] h, 50×EDFS iron salt mother liquor (1L): NaFe·EDTA 6.974g, H2O to a final volume of 1L.

[0056] Example 1: GWAS revealed a significant association between the major gene MScFBP and plant architecture-related traits.

[0057] GWAS analysis of plant type-related traits: Plant type-related phenotypes were investigated in May 2022 using 137 core alfalfa germplasm accessions grown at the Shangzhuang Experimental Station in Haidian District, Beijing (40 N, 116°E). Growth habit index phenotypes were determined by visual grading, classifying the plants into very upright, upright, semi-upright, intermediate, semi-prostrate, and prostrate types based on branching angle, and denoted as indices 8, 7, 6, 5, 4, and 3, respectively. Plant height phenotype was defined as the natural height from the ground to the top of the plant. GWAS analysis of the collected data showed that the Manhattan plots of both the growth habit index and plant height phenotypes located a signal peak on chromosome 6, with the peak SNP located near the candidate gene MScFBP. Figure 1 ).

[0058] Cloning and haplotype analysis of MscFBP: Two extreme types of germplasm resources, erect and prostrate, were selected. RNA was extracted and reverse transcribed into cDNA. Using cDNA as a template, primers synthesized by BGI Genomics were used for amplification: MscFBP-F: 5′-ATGGACCACAGCGCAGATGCAC-3′ (SEQ ID NO.5); MscFBP-R: 5′-TTATTTAGCCTCTGCAGCATAAAGAGC-3′ (SEQ ID NO.6).

[0059] The amplification product was 1020 bp in size. After DNA gel recovery, it was sequenced, and two different haplotypes of the cFBP gene were obtained. The haplotype cloned from the upright extremophile material C68 was named MscFBP Hap1, and the haplotype cloned from the creeping extremophile material C11 was named MscFBP Hap2.

[0060] Two haplotypes, Hap1 and Hap2, were cloned from germplasm resources, with only 5 amino acid differences between them.

[0061] MscFBP Hap1 cds (SEQ ID NO.1)

[0062]

[0063] MscFBP Hap1 protein(SEQ ID NO.2)

[0064] MDHSADAHRTDLMTITRFVLNEQSKHPESRGDFTILLNNIVLGCKFVCSAVNKAGLAKLIGLAGETNVQGEEQKKLDVLSNEVFCKALISSGRTCILVSEEDEDAIFVEPTQRGKYCVVFDPLDGSSNIDCGVSIGTIFGIYMMKDNHEPIIEDVLQPGKNMLAAGYCMYGSSCTFVITTGSGVNGFTLDPSLGEFILTHPDIKIPKKGKIYSVNEGNAKNWDGPTAAYVEKCKFPTDGSPAKSLRYIGSMVADVHRTLLYGGTFLYPADKKSPNGKLRVLYEVFPMSFLMEQAGGQAFTGKERALDLVPTKLHERSPIFLGSYDDIEEIKALYAAEAK

[0065] MscFBP Hap2 cds(SEQ ID NO.3)

[0066]

[0067] MscFBP Hap2 protein (SEQ ID NO.4)

[0068] MDHSADAHRTDLMTITRFVLNEQSKHPESRGDFTILLNNIVLGCKFVCSAVNKAGLAKLIGLAGETNVQGEEQKKLDVLSNEVFCKALISSGRTCILVSEEDEDAIFVDPKQRGKYCVVFDPLDGSSNIDCGVSIGTIFGIYMMKDNHEPVIDDVLQPGKNMLAAGYCM YGSSCTFVITTGSGVNGFTLDPSLGEFILTHPDIKIPKKGKIYSVNEGNAKNWDGPTAAYVEKCKFPTDGSPAKSLRYIGSMVADVHRTLLYGGTFLYPADKKSPNGKLRVLYEVFPMSFLMEQAGGQAFTGKERALDLIPTKLHERSPIFLGSYDDIEEIKALYAAEAK

[0069] Detection of two haplotype protease activities:

[0070] Construction of vectors pET30a-MscFBP Hap1 and pET30a-MscFBP Hap2

[0071] a. Using pLB-MscFBP Hap1 and pLB-MscFBP Hap2 as templates, primers were synthesized by BGI Genomics and PCR amplification was performed; MsFBP-pET30a-F: 5′-GGCTGATATCGGATCCGAATTCATGGACCACAGCGCAGATGC-3′ (SEQ ID NO.7); MsFBP-pET30a-R: 5′-GTGCGGCCGCAAGCTTGTCGACGTTTAGCCTCTGCAGCAT-3′ (SEQ ID NO.8).

[0072] b. The product was detected by 1% agarose gel electrophoresis. The product fragment size was 1062 bp. The gel block containing the target fragment was excised and the fragment was recovered using a gel extraction kit. After homologous recombination ligation, the pET30a-MscFBP Hap1 and pET30a-MscFBP Hap2 protein expression vectors were obtained.

[0073] Prokaryotic proteins His-MscFBP Hap1 and His-MscFBP Hap2 expression and purification

[0074] a. Recombinant plasmids pET30a-MscFBP Hap1 and pET30a-MscFBP Hap2 were transformed into BL21 protein expression strain using the heat shock transformation method. 50 μL of the bacterial culture was plated onto LB agar plates containing Kan resistance, incubated overnight, and single clones were picked for bacterial PCR detection.

[0075] b. Transfer the positive bacterial culture to 5 mL of LB liquid medium containing Kan resistance and incubate at 37°C and 230 rpm for 4 h. Then transfer the bacterial culture to 1 L of LB liquid medium containing Kan resistance and incubate again at 37°C and 230 rpm for about 4 h until OD (Organic Discharge) is reached. 600 It is 0.6-0.8;

[0076] c. Add 1 mL of IPTG to the culture medium and induce protein expression at 16℃ for 16 h;

[0077] d. Collect bacterial cells using a 250 mL centrifuge bottle and discard the supernatant. Resuspend the bacterial cells in 30 mL MCAC-0 and add 300 μL of 1% Triton X-100 and 300 μL of 100× PMSF to the resuspending solution. Sonicate the cells on ice at 400 W for 2 seconds, followed by a 4-second interval, until the sample is clear.

[0078] e. Transfer the ultrasonicated bacterial culture into a 50 mL centrifuge bottle, centrifuge at 12000 g for 15 min at 4℃, retain the precipitate, filter the supernatant into a 50 mL beaker using a 0.8 μm filter, pump the sample into the Ni-NTA column at a flow rate of 15 mL / h and collect the eluent.

[0079] f. Prepare MCAC-0, MCAC-20, MCAC-50, MCAC-80, MCAC-100, MCAC-200 and MCAC-500 gradient eluents, 5 mL for each gradient, and collect the eluents stepwise.

[0080] g. Take 40 μL of precipitate, supernatant, percolation buffer and protein samples eluted at each gradient, add 10 μL of 5× SDS PAGE loading buffer, boil for 10 min to fully denature the protein, and then cool on ice.

[0081] Detection of the content and purity of prokaryotic proteins His-MscFBP Hap1 and His-MscFBP Hap2

[0082] a. Take 20 μL of the protein sample prepared above into the SDS PAGE gel wells, separate by electrophoresis at 80 V for 30 min, and then separate by electrophoresis at 120 V for 1 h;

[0083] b. Place the SDS PAGE gel in a 15 cm diameter round glass dish, add Coomassie Brilliant Blue staining solution to cover the gel, and stain at 80 rpm for 1 h at room temperature.

[0084] c. Recover the Coomassie Brilliant Blue staining solution and pour destaining solution into the round dish to cover the gel. Destain at room temperature and 80 rpm until the background color of the gel disappears and the protein bands are clear.

[0085] d. Observe and take photos;

[0086] e. Use ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa to concentrate the protein by centrifugation at 4°C.

[0087] Assay of prokaryotic protein His-MscFBP Hap1 and His-MscFBP Hap2 FBP enzyme activities

[0088] The FBPase activity assay was performed according to the instructions of the Fructose-1,6-bisphosphatase Activity Assay Kit (catalog number BC0925) manufactured by Solarbio Science & Technology Co., Ltd.

[0089] a. Dilute the two proteins His-MscFBP Hap1 and His-MscFBP Hap2 with the extraction buffer to a total protein amount of 40 μg each (Note that MCAC reagent contains imidazole, which will inhibit FBPase activity, so the protein dissolution medium needs to be replaced with PBS (pH 7.4) before measuring enzyme activity).

[0090] b. Preparation of the reaction system shown in Table 1:

[0091] Table 1 Reaction system for enzyme activity assay

[0092]

[0093] c. Take 100 μL from the reaction system and immediately measure the absorbance at a wavelength of 340 nm, which is recorded as A1. After 4 min, measure the absorbance at a wavelength of 340 nm and record it as A2. ΔA measurement tube = A2 measurement tube - A1 measurement tube, ΔA blank tube = A2 blank tube - A1 blank tube, ΔA = ΔA measurement tube - ΔA blank tube.

[0094] Enzyme activity calculation: FBP (U / g) = ΔA ÷ (ε × d) × 10 9 ×V 反总 ÷(Cpr×V 样 ) ÷T=321.5×ΔA÷Cpr

[0095] ε: NADPH molar extinction coefficient, 6.22 × 10⁻⁶ 3L / mol / cm; d: optical path of the cuvette, 1 cm; V 反总 : Total volume of the reaction system, 500 μL; W: Sample mass, g; V 样 Add 50 μL to the total sample volume; V 样总 : Total volume of extraction solution added: 500 μL; T: Reaction time; Cpr: Sample protein concentration, μg / μL; Unit conversion factor: 1 mol = 10 9 nmol.

[0096] The results are as follows Figure 2 As shown, the enzyme activity of the MScFBP Hap2 haplotype was significantly higher than that of Hap1.

[0097] Example 2: Cultivation of Mscfbp mutant plants and phenotypic identification

[0098] Cultivation of Mscfbp mutant plants

[0099] Construction of vector p6401-MscFBP:

[0100] a. Gene editing target design: A target list was generated using an online target prediction website (http: / / crispor.tefor.net / ). Target 1 was selected as 5′-TGTTGGAGGAGCCGTCTAA-3′ (SEQ ID NO.9), and target 2 was selected as 5′-TCGCCAAGAGAAGGGTCAA-3′ (SEQ ID NO.10).

[0101] b. Construction of sgRNA module:

[0102] Primers MscFBP-BsF: 5′- synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0103] ATATATGGTCTCGCTTGTGTTGGAGGAGCCGTCTAAGTT-3′ (SEQ ID NO.11), where nucleotides 18 to 36 are the designed target 1 sequence; MscFBP-F0: 5′-GTGTTGGAGGAGCCGTCTAAGTTTTAGAGCTAGAAATAGC-3′ (SEQ ID NO.12), where nucleotides 2 to 20 are the designed target 1 sequence; MscFBP-R0: 5′-AACTTGACCCTTCTCTTGGCGACAATTTAATGGTTCGCTTGTA-3′ (SEQ ID NO.13), where nucleotides 4 to 22 are the reverse complementary sequence of the designed target 2; MscFBP-BsR: 5′-ATTATTGGTCTCGAAACTTGACCCTTCTCTTGGCGAC-3′ (SEQ ID NO.11). NO.14), where positions 18 to 36 are the reverse complementary sequences of the designed target 2; the sgRNA module MscFBP-5CBC with the target sequence was obtained by PCR amplification.

[0104] c. Construction of a binary gene editing vector: Golden Gate digestion and ligation reaction: The MScFBP-5CBC fragment carrying the target sequence and the p6401 vector were digested with Bsa I and ligated with T4 DNA Ligase to form p6401-MscFBP. Figure 3 After the reaction was completed, the product was added to E. coli competent TOP10 cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, and then added to LB liquid medium without antibiotics. The cells were incubated at 37℃ and 150 rpm for 45 min. Subsequently, the bacterial culture was spread on LB solid medium containing kanamycin (50 mg / L) and incubated in the dark at 37℃ for 12 h for selection. Single clones were picked and cultured in 400 μL of LB liquid medium containing kanamycin (50 mg / L) at 37°C and 230 rpm for 6–8 h. Subsequently, bacterial colony-linked PCR was performed for identification. Amplification was performed using primers MScFBP-BsF: 5′-ATATATGGTCTCGCTTG TGTTGGAGGAGCCGTCTAAGTT-3′ (SEQ ID NO.15) and MScFBP-BsR: 5′-ATTATTGGTCTCGAAACTTGACCCTTCTCTTGGCGAC-3′ (SEQ ID NO.16). Positive clones exhibited a specific band of 841 bp. Plasmids of positive clones were extracted and verified by sequencing.

[0105] Obtaining Mscfbp mutant plants of alfalfa

[0106] a) Plant transformation vector p6401-MscFBP was transformed into Agrobacterium tumefaciens EHA105:

[0107] Add 50 ng of p6401-MscFBP plasmid to competent Agrobacterium tumefaciens cells EHA105 and incubate on ice for 5 min. Add the mixture to a pre-chilled electroporation vessel and electroporate at 2100 V. Then add 500 μL of antibiotic-free YEP liquid medium and mix thoroughly by pipetting. Transfer the bacterial culture to centrifuge tubes and incubate at 28℃ and 200 rpm for 1 h. Spread 30 μL of the bacterial culture onto YEP solid medium containing rifampicin (75 mg / L) and kanamycin (50 mg / L), and incubate in the dark at 28℃ for 48 h. Select clones for colony PCR identification using the primers MscFBP-BsF and MscFBP-BsR. Positive clones exhibit a specific band of 841 bp. Select the positive clone (the recombinant Agrobacterium tumefaciens obtained by introducing p6401-MscFBP into Agrobacterium tumefaciens EHA105, named EHA105 / p6401-MscFBP) will then proceed to the next stage of infection.

[0108] b. Agrobacterium tumefaciens infection of alfalfa with EHA105 / pCAMBIA1307-MScFBP:

[0109] Agrobacterium tumefaciens (EHA105 / pCAMBIA1307-MScFBP) was inoculated at a ratio of 1:1000 into 200 mL of YEP medium (75 mg / L rifampin and 50 mg / L kanamycin), and cultured at 28°C and 230 rpm until OD500 was reached. 600 The value was 0.3. The EHA105 / pCAMBIA1307-MscFBP bacterial culture was transferred to a sterile 250 mL centrifuge bottle, centrifuged at 24℃, 4000 g for 10 min, the supernatant was discarded in a laminar flow hood, and the bacterial cells were resuspended in 200 mL SM4-0 (containing 100 µM acetylsyl syringone) liquid medium as the infection solution.

[0110] The 35 compound leaves collected the previous day were soaked in 0.1% Tween 20 solution for 5 min, then transferred to a sterile empty glass bottle and rinsed 3 times with deionized water in a laminar flow hood. The leaves were then transferred to a blue-capped bottle, 30% bleach was added, and the solution was treated for 10 min. The leaves were then rinsed 3 times with deionized water.

[0111] After disinfection, the leaves were transferred to sterile empty glass bottles and inoculated with the infection solution. Vacuuming was performed for 5 minutes, followed by sonication for 3 minutes, and then vacuuming again for 5 minutes. The infection solution was removed in a laminar flow hood. Excess liquid was removed from the leaves using sterile filter paper. After drying, the leaves were cut into small pieces and laid flat on SM4 solid medium. After 3 days of incubation in the dark at room temperature, the leaves were transferred to SM4 solid medium (containing 200 mg / L cephalosporin and 10 mg / L hygromycin) and cultured under light at 24°C. Every 2 weeks, the leaves were transferred to fresh SM4 solid medium (containing 200 mg / L cephalosporin and 10 mg / L hygromycin). Once callus formed, the callus tissue was transferred to MSBK regeneration solid medium (containing 200 mg / L termethin and 10 mg / L hygromycin) and grown for 3 weeks. Then, the callus was transferred to SH9 solid medium (containing 200 mg / L cephalosporin and 5 mg / L hygromycin). The resulting plantlets were transferred to 1 / 2 MS solid medium. After rooting, the plantlets were transferred to a greenhouse for further cultivation, and positive seedlings were identified.

[0112] Identification of Mscfbp mutant in alfalfa

[0113] a. Transgenic identification: Genomic DNA was extracted from regenerated seedlings for PCR detection, using plasmid p6401-MscFBP as a positive control and genomic DNA as a negative control. The primers were the aforementioned MscFBP-BsF and MscFBP-BsR. After the reaction, the product was detected by 1% agarose gel electrophoresis. If a specific band of 841 bp was present, the sample was considered transgenic positive.

[0114] Figure 4 Part A represents the transgenic identification results of alfalfa regenerated plants obtained by Agrobacterium tumefaciens-mediated transformation into the p6401-MscFBP vector. M indicates the standard molecular weight of DNA. The positive control "+" represents the p6401-MscFBP vector plasmid, and the negative control "-" represents the control material CK-1. The results show that Mscfbp-61, Mscfbp-78, and Mscfbp-89 are transgenic positive plants.

[0115] b. Mutation mode identification: The genotype of the transgenic positive plants Mscfbp was detected. Genomic DNA fragments containing the target region were amplified by PCR using primers MscFBP-TF: 5′-CTAGGGAGAAGTTTAACTGTGAT-3′ (SEQ ID NO.17) and MscFBP-TR: 5′-GAGCAGGCAGGTAATCATATC-3′ (SEQ ID NO.18). The results were detected by 1% agarose gel electrophoresis. A fragment of 855 bp was amplified from the positive transgenic plants. After the reaction, 5 μL of the product was analyzed by 1% agarose gel electrophoresis. Figure 4 Part B of the document. Figure 4 In part B, M represents the standard molecular weight of DNA, and CK-1 is the control material. The results show that samples Mscfbp-61, Mscfbp-78, and Mscfbp-89 all have amplified bands, which need to be further verified by Sanger sequencing.

[0116] The remaining PCR products were directly recovered and ligated into the pLB vector. Multiple single clones were then selected for sequencing verification. Examples of mutation methods are analyzed below. Figure 5 Part A of the diagram shows the target region (black solid line), PAM sequences (bold letters), deleted bases (-), and inserted bases (lowercase letters). The Mscfbp-61, Mscfbp-78, and Mscfbp-89 plants all show mutations in all four MscFBP alleles. Figure 5 Part B contains the amino acid coding of the four alleles of MscFBP in Mscfbp-61, Mscfbp-78 and Mscfbp-89 plants.

[0117] Phenotypic analysis of alfalfa Mscfbp mutant

[0118] Three weeks after mowing, alfalfa CK-1 and Mscfbp mutant plants were observed to measure plant height, number of branches, and aboveground biomass. The Mscfbp mutant plants ( Figure 6 The plant height of Part A was significantly reduced ( Figure 6 Part B), the number of branches is significantly reduced ( Figure 6 Part C), fresh weight of the above-ground parts ( Figure 6 Part D) and dry weight ( Figure 6 All components (part E) were significantly reduced. The average plant height of the wild type was 37.2 cm, while that of the Mscfbp mutant was 25.8 cm, a decrease of 30.6%. The average number of branches of the wild type was 6.0, while that of the Mscfbp mutant was 3.7, a decrease of 38.3%. The average fresh weight of the aboveground parts of the wild type was 7.2 g, while that of the Mscfbp mutant was 4.3 g, a decrease of 40.3%. The average dry weight of the aboveground parts of the wild type was 1.9 g, while that of the Mscfbp mutant was 1.1 g, a decrease of 42.1%.

[0119] Example 3: Cultivation of MscFBP Hap1 and MscFBP Hap2 overexpressing plants and their phenotypic identification

[0120] Cultivating MscFBP Hap1 and MscFBP Hap2 overexpression plants

[0121] Construction of vectors 1307-MscFBP Hap1 and 1307-MscFBP Hap2

[0122] Primers synthesized by BGI Genomics: 1307-MscFBP-F: 5′-GCGTCGACGGATGGACCACAGCGCAGATGCAC-3′ (SEQ ID NO.19), where positions 1-2 are protective bases and positions 3-8 are Sal I restriction sites. 1307-MscFBP-R: 5′-GCTCTAGATTTAGCCTCTGCAGCATAAAGAGC-3′ (SEQ ID NO.20), where positions 1-2 are protective bases and positions 3-8 are Xba I restriction sites.

[0123] First, amplification was performed using primers 1307-MscFBP-F and 1307-MscFBP-R with pLB-MscFBP Hap1 and pLB-MscFBPHap2 as templates. After DNA gel recovery, the amplification products underwent enzyme digestion and ligation to obtain pCAMBIA1307-MscFBP Hap1 (… Figure 7 Part A) and pCAMBIA1307-MScFBP Hap2 vector ( Figure 7 Part B).

[0124] Obtaining alfalfa plants overexpressing MScFBP Hap1 and MScFBP Hap2

[0125] a. Transformation of plant transformation vectors pCAMBIA1307-MscFBP Hap1 and pCAMBIA1307-MscFBP Hap2 into Agrobacterium EHA105: 50 ng of each plasmid (pCAMBIA1307-MscFBP Hap1 and pCAMBIA1307-MscFBP Hap2) was added to Agrobacterium EHA105 competent cells and incubated on ice for 5 min. The mixture was then added to a pre-chilled electroporation vessel and electroporated at 2100 V. Following this, 500 μL of antibiotic-free YEP liquid medium was added and mixed thoroughly by pipetting. The bacterial culture was transferred to centrifuge tubes and incubated at 28℃ and 200 rpm for 1 h. 50 μL of the bacterial culture was then plated onto YEP solid medium containing rifampicin (75 mg / L) and kanamycin (50 mg / L) and cultured in the dark at 28℃ for 48 hours. h. Clones were selected for colony PCR identification. Amplification was performed using the primers 1307-MscFBP-F and 1307-MscFBP-R. Positive clones showed a specific band of 1035 bp. The recombinant Agrobacterium tumefaciens was named EHA105 / pCAMBIA1307-MscFBP Hap1 and EHA105 / pCAMBIA1307-MscFBPHap2 for the next step of infection.

[0126] b. Infection of alfalfa with Agrobacterium tumefaciens EHA105 / pCAMBIA1307-MscFBP Hap1 and EHA105 / pCAMBIA1307-MscFBP Hap2: Inoculate Agrobacterium tumefaciens EHA105 / pCAMBIA1307-MscFBP Hap1 and EHA105 / pCAMBIA1307-MscFBP Hap2 into 200 mL YEP medium (75 mg / L rifampin and 50 mg / L kanamycin) at a ratio of 1:1000, and incubate at 28°C and 230 rpm until OD. 600 The value was 0.3. The bacterial cultures of EHA105 / pCAMBIA1307-MscFBP Hap1 and EHA105 / pCAMBIA1307-MscFBP Hap2 were transferred to sterile 250 mL centrifuge bottles, centrifuged at 24℃, 4000 g for 10 min, and the supernatant was discarded in a laminar flow hood. The bacterial cells were resuspended in 200 mL of SM4-0 (containing 100 µM acetylsyleugenol) liquid medium as the infection solution.

[0127] The 35 compound leaves collected the previous day were soaked in 0.1% Tween 20 solution for 5 min, then transferred to a sterile empty glass bottle and rinsed 3 times with deionized water in a laminar flow hood. The leaves were then transferred to a blue-capped bottle, 30% bleach was added, and the solution was treated for 10 min. The leaves were then rinsed 3 times with deionized water.

[0128] After disinfection, the leaves were transferred to sterile empty glass bottles, and the inoculation solution was added. Vacuuming was performed for 5 minutes, followed by sonication for 3 minutes, and then vacuuming again for 5 minutes. The inoculation solution was removed in a clean bench, and excess liquid was removed from the leaves using sterile filter paper. After drying the leaves, they were cut into small pieces and laid flat on SM4 solid medium. After 3 days of incubation in the dark at room temperature, the leaves were transferred to SM4 solid medium (containing 200 mg / L cephalosporin and 10 mg / L hygromycin) and cultured under light at 24°C. Every 2 weeks, the leaves were transferred to fresh SM4 solid medium (containing 200 mg / L cephalosporin and 10 mg / L hygromycin). After callus formation, the callus tissue was transferred to MSBK regeneration solid medium (containing 200 mg / L termethin and 10 mg / L hygromycin) and grown for 3 weeks. Then, it was transferred to SH9 solid medium (containing 200 mg / L cephalosporin and 5 mg / L hygromycin). The resulting plantlets were transferred to 1 / 2 MS solid medium. After rooting, they were transferred to a greenhouse for further cultivation, and positive seedlings were identified.

[0129] Detection of alfalfa plants overexpressing MScFBP Hap1 and MScFBP Hap2

[0130] a. DNA level detection of 1307-MscFBP Hap1 and 1307-MscFBP Hap2 transgenic plants: Genomic DNA was extracted from 1307-MscFBP Hap1 and 1307-MscFBP Hap2 transgenic plants, respectively. Using these as templates, MscFBP-F: 5′-CGGAATTCATGGACCACAGCGCAGATGC-3′ (SEQ ID NO.21); GFP-R: 5′-GTAAGTTTTCCGTATGTTGCATCAC-3′ (SEQ ID NO.22) were used as primers for amplification. Plasmids pCAMBIA1307-MscFBP Hap1 and pCAMBIA1307-MscFBP Hap2 were used as positive controls, and genomic DNA from the control plant CK-2 was used as a negative control. A fragment of 1163 bp in size was amplified from the positive transgenic plants (…). Figure 8 ).

[0131] b. RNA Level Detection of Alfalfa 1307-MscFBP Hap1 and 1307-MscFBP Hap2 Overexpression Plants: To detect whether the MscFBP Hap1 and MscFBP Hap2 genes were transcribed in the PCR-positive plants from step 3.a, RT-qPCR was further performed. Total RNA was extracted from the PCR-positive plants from the previous step using the TRIzol method. Using intact, uncontaminated RNA as a template, reverse transcription was performed using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R312, Vazyme). The RT-qPCR amplification product of MsActin4A was used as an internal standard for total RNA template quantification. The primers used were MsActin4A-F: 5′-CCAAAGGCCAACAGAGAAAA-3′ (SEQ ID NO.23) and MsActin4A-R: 5′-ACGACCAGCAAGATCCAAAC-3′ (SEQ ID NO.24). The primers for amplifying the MscFBP gene were MscFBP-qF: 5′-TGGAAGCATGGTAGCTGATG-3′ (SEQ ID NO.25) and MscFBP-qR: 5′-CACTGACAGAAAATTTGTGCCCA-3′ (SEQ ID NO.26). The results were as follows: Figure 9 As shown.

[0132] c. Protein Level Detection in Alfalfa 1307-MscFBP Hap1 and 1307-MscFBP Hap2 Overexpressing Plants: Total protein was extracted from transgenic plants, separated by SDS-PAGE gel electrophoresis, and transferred to cellulose acetate membranes for immunoblotting hybridization using an electroporation device. After blocking, specific antibody GFP-tag was added, along with Rabbit pAb (31002ES60, YEASEN) polyclonal antibody against GFP-tag. For the secondary antibody reaction, alkaline phosphatase-labeled goat anti-rabbit IgG diluted with blocking buffer was added. After washing the hybridization membrane three times, a colorimetric reaction was performed. Positive plants showed a specific band of approximately 70 kDa. Figure 10 ).

[0133] Phenotypic identification of alfalfa plants overexpressing MScFBP Hap1 and MScFBP Hap2

[0134] Three weeks after mowing, alfalfa plants overexpressing CK-2, MscFBP Hap1, and MscFBP Hap2 were observed and their growth indicators, including plant height, number of branches, and aboveground biomass, were recorded. The number of plants overexpressing MscFBP Hap1 and MscFBP Hap2 was [not specified]. Figure 11The plant height of Part A was significantly increased ( Figure 11 Part B), with a significantly increased number of branches ( Figure 11 Part C), fresh weight of the above-ground parts ( Figure 11 Part D) and dry weight ( Figure 11 The E portion of both were significantly increased; further comparison revealed that the overall growth of MscFBP Hap2 overexpressing plants was better than that of MscFBP Hap1 overexpressing plants. The average plant height of wild-type plants was 46.1 cm, while that of MscFBPHap1-overexpressing plants was 54.7 cm, an increase of 18.7%, and that of MscFBP Hap2-overexpressing plants was 65.8 cm, an increase of 42.7%. The average number of branches was 3.7 for wild-type plants, 5.6 for MscFBP Hap1-overexpressing plants (an increase of 51.4%), and 7.1 for MscFBP Hap2-overexpressing plants (an increase of 91.9%). The average fresh weight of the aboveground parts was 3.8 g for wild-type plants, 6.1 g for MscFBP Hap1-overexpressing plants (1.6 times that of wild-type plants), and 7.1 g for MscFBP Hap2-overexpressing plants (1.9 times that of wild-type plants). The average dry weight of the aboveground parts was 0.8 g for wild-type plants. The average fresh weight of the aboveground parts of Hap1-overexpressing plants was 1.4 g, which was 1.75 times that of wild-type plants, while the average fresh weight of the aboveground parts of MscFBP Hap2-overexpressing plants was 2.1 g, which was 2.6 times that of wild-type plants.

Claims

1. The application of alfalfa cFBP protein in regulating plant architecture, characterized by, The amino acid sequence of the alfalfa cFBP protein is SEQ ID NO.2 or SEQ ID NO.

4.

2. The application according to claim 1, wherein the application increases the expression level of alfalfa cFBP protein or its encoding gene in the plant to achieve one or more effects selected from (A1)-(A3): (A1) Increase plant height; (A2) Increase the number of plant branches; (A3) Increase plant biomass.

3. In the application according to claim 1 or 2, increasing the expression level of alfalfa cFBP protein or its encoding gene in the plant is achieved by one or more tools selected from (B1)-(B2): (B1) A tool for overexpressing the gene encoding alfalfa cFBP protein; (B2) Tools to enhance the activity of alfalfa cFBP protein.

4. In the application according to claim 3, (B1) is a nucleic acid molecule encoding the alfalfa cFBP protein gene, or a vector, expression cassette, recombinant microorganism, plant cell, plant tissue, or plant organ containing the alfalfa cFBP protein encoding gene; preferably, an overexpression vector containing the alfalfa cFBP protein encoding gene; more preferably, the overexpression vector contains a constitutive promoter; and even more preferably, a pCAMBIA1307 vector containing the alfalfa cFBP protein encoding gene.

5. The application according to any one of claims 1-4, wherein the plant is alfalfa.

6. The application according to any one of claims 1-5, wherein the application is used for one or more of (C1)-(C3): (C1) Improves plant shape; (C2) Provide plant propagation materials for improving plant structure, preferably seeds, tissue culture materials, and organs that can be used for propagation; (C3) Provides plant strains with improved plant type.

7. Alfalfa cFBP protein, characterized in that, The amino acid sequence of the alfalfa cFBP protein is SEQ ID NO.2 or SEQ ID NO.

4.

8. The gene encoding the alfalfa cFBP protein according to claim 7.

9. The coding gene according to claim 8, wherein the nucleotide sequence of the coding gene is SEQ ID NO.1 or SEQ ID NO.

3.

10. Application of any one of the tools (D1)-(D3) for detecting the expression level of alfalfa cFBP protein and the expression level of the alfalfa cFBP protein encoding gene: (D1) Determine the plant type of alfalfa; (D2) Predicting plant architecture of alfalfa propagation material; (D3) Develop alfalfa varieties with improved plant type; The tools used to detect the expression levels of alfalfa cFBP protein and the expression levels of the alfalfa cFBP protein encoding gene are one or more of the following: (E1) Primer; (E2) Probe; (E3) PCR reagents; (E4) chip; (E5) Antibody; (E6) ELISA reagent; The nucleotide sequence of the alfalfa cFBP protein encoding gene is SEQ ID NO.1 or SEQ ID NO.3; the amino acid sequence of the alfalfa cFBP protein is SEQ ID NO.2 or SEQ ID NO.4.

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