Method for improving characters of alfalfa by using MsDELLA1 gene

Abstract

The invention discloses a method for improving characters of alfalfa, which is characterized in that DELLA1 genes in the alfalfa are highly expressed by an agrobacterium-mediated method; the method for improving the characters of the alfalfa comprises the following steps: increasing branches of alfalfa plants; the fresh weight of alfalfa plants is increased; the leaf number of alfalfa plants is increased; the content of starch in alfalfa leaves is increased; the content of soluble protein in alfalfa leaves is increased; the content of soluble sugar in alfalfa leaves is increased. The method has application and popularization prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to a method for improving alfalfa traits using the MsDELLA1 gene. Background Technology

[0002] Alfalfa (scientific name: *Medicago sativa* L.) is a perennial herbaceous plant belonging to the legume family and the *Medicago* genus. It is an important economic crop with value in forage, ecology, food, and medicine, and is known as the "King of Forage." It is highly adaptable, exhibiting outstanding cold resistance, drought resistance, salt and alkali tolerance, and tolerance to poor soil. It has a long growth cycle and strong regeneration ability, allowing for multiple harvests and utilization, making it suitable for large-scale cultivation in various ecological environments.

[0003] Branching number is one of the core agronomic traits affecting alfalfa plant architecture and biomass. More branches result in more robust plant growth, stronger regeneration ability after harvesting, increased biomass, and enhanced utilization value and economic benefits, thus meeting the large-scale demand for high-quality forage in livestock farming. However, the molecular regulatory mechanisms influencing alfalfa branching remain unclear, severely impacting the breeding of high-quality alfalfa varieties.

[0004] Gibberellins (GAs) regulate numerous developmental processes, including seed germination, cell elongation, leaf expansion, flowering, and fruit development. DELLA protein is a key component of the GA signaling pathway. There are no reports of the function of the DELLA gene in regulating alfalfa branching. There are also no reports of genetic modifications to alfalfa that simultaneously increase biomass by increasing branching and leaf number while also increasing protein, starch, and sugar content. Summary of the Invention

[0005] To address the problems existing in the prior art, the first aspect of the present invention provides a method for improving alfalfa traits, the method being: to achieve high expression of the DELLA1 gene in alfalfa through genetic engineering.

[0006] The improvement of alfalfa traits includes:

[0007] Increase the number of branches in alfalfa plants;

[0008] Increase the fresh weight of alfalfa plants;

[0009] Increase the number of leaves in alfalfa plants;

[0010] Increase the starch content of alfalfa leaves;

[0011] Increase the soluble protein content in alfalfa leaves;

[0012] Increase the soluble sugar content in alfalfa leaves.

[0013] In some embodiments, the alfalfa is alfalfa.

[0014] In some embodiments, the alfalfa variety is Zhongmu No. 3.

[0015] In some embodiments, the amino acid sequence of the protein encoded by the DELLA1 gene is shown in SEQ ID NO.4.

[0016] In some embodiments, the method includes the following steps:

[0017] S1: The coding sequence of the DELLA1 gene is transferred into an expression vector to obtain a recombinant vector containing the DELLA1 gene;

[0018] S2: Transform Agrobacterium with the recombinant vector to obtain a recombinant Agrobacterium bacterial suspension containing the DELLA1 gene;

[0019] S3: Infect alfalfa leaves with the recombinant Agrobacterium bacterial solution to obtain infected alfalfa leaves;

[0020] S4: The infected alfalfa leaves are placed on a co-culture medium and cultured to obtain co-cultured alfalfa leaves.

[0021] S5: The co-cultured alfalfa leaves are placed on a selective culture medium to obtain preliminary alfalfa callus tissue;

[0022] S6: The alfalfa preliminary callus tissue is placed on a callus culture medium and cultured to obtain alfalfa callus tissue;

[0023] S7: The alfalfa callus tissue is cultured on a differentiation medium to obtain alfalfa plants with roots and buds.

[0024] In some embodiments, in S1, the backbone of the expression vector is a pFGC-eYFP vector;

[0025] In S2, the Agrobacterium is Agrobacterium GV3101;

[0026] In S3, the alfalfa leaf is a sterilized leaf from an alfalfa tissue culture seedling that has grown for 4-6 weeks.

[0027] In S4, the co-culture medium is based on SH medium, which also contains 15-25 g / L sucrose, 3.0-3.5 g / L plant gel, 0.8-1.2 mg / mL 2,4-D, 0.8-1.2 mg / mL 6-BAP, and pH 5.5-6.0;

[0028] In S5, the selective medium is based on SH medium, which also contains 16-24 g / L sucrose, 3.0-3.5 g / L plant gel, 0.8-1.2 mg / mL 2,4-D, 0.8-1.2 mg / mL 6-BAP, 150-250 mg / L cephalosporin, 150-250 mg / L termethin, 6.0-9.0 mg / L glufosinate, and pH 5.5-6.0;

[0029] In S6, the callus culture medium is based on MS medium, which also contains 25-35 g / L sucrose, 3.0-3.5 g / L phytogel, 0.8-1.2 mg / mL kinetin, 0.8-1.2 mg / mL 6-BAP, 150-250 mg / L cephalosporin, 150-250 mg / L termethin, 6.0-9.0 mg / L glufosinate, and pH 5.5-6.0; or

[0030] In S7, the differentiation medium is based on SH medium, which also contains 8-12 g / L sucrose, 3.0-3.5 g / L plant gel, 0.8-1.2 mg / mL 6-BAP, 150-250 mg / L termethin, 1.5-2.5 mg / L glufosinate, and pH 5.5-6.0.

[0031] In some implementations, in S3, the infection step is as follows:

[0032] The alfalfa leaves were placed into the recombinant Agrobacterium bacterial solution;

[0033] Evacuate for 4-12 minutes under a vacuum level of 0.06-0.12 MPa.

[0034] Ultrasonic treatment for 6-10 seconds at 0-4℃, ultrasonic power 80-120 W, and ultrasonic frequency 30-50 kHz.

[0035] Evacuate for 2-6 minutes under a vacuum level of 0.06-0.12 MPa;

[0036] In S4, the culture conditions are 22-26℃, cultured in the dark for 0.5-1.5 days;

[0037] In S5, the culture conditions are 22-26℃, cultured in the dark for 50-70 days;

[0038] In S6, the cultivation conditions are 22-26℃, 6-10h light per day, light intensity 1000-2000 Lux, and cultivation for 25-35 days; or

[0039] In S7, the culture conditions are 22-26℃, 6-10h light per day, light intensity of 1000-2000 Lux, and culture for 55-65 days.

[0040] A second aspect of the present invention provides the use of a biomaterial in the preparation of a formulation for improving alfalfa traits in alfalfa breeding;

[0041] The improvement of alfalfa traits includes:

[0042] Increase the number of branches in alfalfa plants;

[0043] Increase the fresh weight of alfalfa plants;

[0044] Increase the number of leaves in alfalfa plants;

[0045] Increase the starch content of alfalfa leaves;

[0046] Increase the soluble protein content in alfalfa leaves;

[0047] Increase the soluble sugar content in alfalfa leaves;

[0048] The biological material is selected from any one of the following: P1, P2, P3, P4, P5, P6, P7, and P8:

[0049] P1: Protein

[0050] The protein in question is the DELLA1 protein.

[0051] P2: Fusion protein

[0052] The amino acid sequence of the fusion protein contains the amino acid sequence of the DELLA1 protein described in P1 and the amino acid sequence of the tag peptide and / or signal peptide used for protein isolation and purification.

[0053] P3: RNA

[0054] The RNA can be translated into the DELLA1 protein described in P1 or the fusion protein described in P2;

[0055] P4: Genes

[0056] The coding sequence of the gene can encode the DELLA1 protein described in P1 or the fusion protein described in P2;

[0057] P5: Gene Expression Kit

[0058] The gene expression product in the gene expression cassette is the RNA described in P3;

[0059] P6: Gene Engineering Vector

[0060] The genetic engineering vector contains the gene expression cassette described in P5;

[0061] P7: Cells

[0062] The cells contain the gene engineering vector described in P6;

[0063] Constitutive expression, tissue-specific expression, or artificially induced expression of the encoded protein in the gene expression cassette of the genetic engineering vector; and

[0064] P8: Composition

[0065] The composition contains the RNA described in P3, the gene engineering vector described in P6, or the cell described in P7.

[0066] In some embodiments, the alfalfa is alfalfa;

[0067] The amino acid sequence of the DELLA1 protein is shown in SEQ ID NO.4;

[0068] The backbone of the genetic engineering vector is a pFGC-eYFP vector; and / or

[0069] The cells were selected from Agrobacterium GV3101 cells and Escherichia coli cells.

[0070] In some embodiments, the alfalfa variety is Zhongmu No. 3. Attached Figure Description

[0071] Figure 1 This is a schematic diagram of the target knockout.

[0072] Figure 2 This is a sequence analysis diagram of the knockout sequence.

[0073] Figure 3 These are comparison photos of plant morphology.

[0074] Figure 4 This is a statistical chart of the number of branches of the plant.

[0075] Figure 5 This is a statistical chart of plant height.

[0076] Figure 6 This is a chart showing the fresh weight of the plants at ground level.

[0077] Figure 7 This is a chart showing the number of blades.

[0078] Figure 8 This is a statistical chart showing the soluble protein content in leaves.

[0079] Figure 9 This is a statistical chart showing the soluble sugar content in leaves.

[0080] Figure 10 This is a statistical chart of starch content in leaves. Detailed Implementation

[0081] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0082] Example 1: Preparation of DELLA1 gene knockout plants from Zhongmu No. 3

[0083] I. Amplification and Sequencing of the Target Gene

[0084] The alfalfa variety used in this invention is Zhongmu No. 3 (also called Zhongmu No. 3). Primers F1 (SEQ ID NO. 1) and R1 (SEQ ID NO. 2) were designed to amplify the coding sequence of the DELLA1 gene in Zhongmu No. 3.

[0085] F1:ATGGACGAGTTGTTAGCAGCA

[0086] R1:TCACTTGGACTCATTTTGTGGAAG

[0087] RNA was extracted from *Agropyron cristatum* var. *mongolica* using the TRIZOL method and then reverse transcribed into cDNA for gene cloning. PCR amplification was performed using primers F1 and R1 and the high-fidelity enzyme Phanta Max Master Mix. The amplification program in the PCR thermal cycler was set as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 58°C annealing for 30 s, and 72°C extension for 2 min, with 29 cycles of denaturation-annealing-extension, followed by a final 72°C complete extension for 10 min. The PCR product was purified and recovered, ligated into the pMD18-T cloning vector (purchased from Sangon Biotech), and the insert was sequenced using the Sanger assay. The recombinant vector (named pMD18-T-DELLA1) was used for subsequent gene expression vector construction.

[0088] Sequencing results show that the coding sequence of the DELLA1 gene in alfalfa variety Zhongmu 3 is as follows (SEQ ID NO.3):

[0089]

[0090] The protein sequence of the DELLA1 gene in alfalfa variety Zhongmu 3 is as follows (SEQ ID NO.4):

[0091] MDELLAALGYKVRSSDMADVAQKLEQLEMVMGSAQEEGINHLSSDTVHYDPTDLYSWVQTMLTELNPDSSQINDPLASLGSSSSSSILNNTFNDDSEYDLSAIPGMAAYPPQEENITAKRMKTWSEPEPAVAM SPPAVVENTRPVVLVDTQETGVRLVHTLMACAEAIQQENLKLAEALVKHISLLASLQTGAMRKVASYFAQALARRIYGNPEETIDSSFSEILHMHFYESSPYLKFAHFTANQAILEAFAGAGRVHVIDFGLKQG MQWPALMQALALRPGGPPTFRLTGIGPPQADNTDALQQVGWKLAQLAQTIGVQFEFRGFVCNSIADLDPNMLEIRPGEAVAVNSVFELHTMLARPGSVEKVLNTVKKINPKIVTIVEQEANHNGPVFVDRFTEA LHYYSSLFDSLEGSNSSSNNSNLNSTGLGSPSQDLLMSEIYLGRQICNVVAYEGVDRVERHETLTQWRSRMGSAGFEPVHLGSNAFKQASTLLALFAGGDGYRVEENNGCLMLGWHTRSLIATSAWKLPQNESK

[0092] II. Gene Target Selection

[0093] Based on the actual DNA sequence of the DELLA1 gene obtained through amplification and sequencing, two high-quality target sites, sgRNA1 (SEQ ID NO.5) and sgRNA2 (SEQ ID NO.6), were designed in the exon region of the target gene using an online design website (https: / / www.sciencedirect.com / science / article / abs / pii / S1360138502022513) following the design rules of low off-target probability and absence of secondary structures larger than 7 nt. See the knockout diagram below. Figure 1 .

[0094] sgRNA1: GACGAGTTGTTAGCAGCATT

[0095] sgRNA2: ATGGGTAGTGCTCAAGAAGA

[0096] Gene editing fragments were synthesized and constructed into gene editing vectors. Reference: Wolabu TW, Cong L, Park JJ, Bao Q, Chen M, Sun J, Xu B, Ge Y, Chai M, Liu Z, Wang ZY. Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa). Front Plant Sci. 2020 Jul17;11:1063. doi: 10.3389 / fpls.2020.01063.

[0097] The CRISPR / Cas9 system used in this invention (for usage instructions, please refer to the literature Wolabu TW, CongL, Park JJ, Bao Q, Chen M, Sun J, Xu B, Ge Y, Chai M, Liu Z, Wang ZY. Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa). Front Plant Sci. 2020, 17;11:1063.) was donated by the Samuel Nobel Foundation. Its polycistronic tRNA-gRNA (PTG) gene was artificially synthesized. The target vector pRGEB31 has a Cas9 protein-coding sequence and an sgRNA expression element. After the synthesized target sequence gene is inserted into the target vector through homologous recombination, the target receptor is transformed by Agrobacterium.

[0098] Polycistronic tRNA-gRNA (PTG) gene sequence (SEQ ID NO.7): ATTGAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGACGAGTTGTTAGCAGCATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAATGGGTAGTGCTCAAGAAGACGTTT

[0099] Homologous recombination primer-F (SEQ ID NO.8): TCGAAGTAGTGATTG AACAAAGCACCAGTGGTCTAG

[0100] Homologous recombination primer-R (SEQ ID NO.9): TTCTAGCTCTAAAAC TCTTCTTGAGCACTACCCAT

[0101] The pRGEB31 vector was linearized by restriction endonuclease Bsa I. The linearized vector was verified by electrophoresis and purified. The recovered product was ligated with double-stranded PTG using homologous recombinase. The recombinant vector was transformed into *E. coli* DH5α competent cells and cultured on LB solid medium containing 50 mg / L kanamycin. Single colonies were picked and PCR was performed using sgRNA1_F (SEQ ID NO.10) and M13_R (SEQ ID NO.11) as primers. After confirming the successful recombinant positive clones by electrophoresis, the correctness of the recombinant sequence was verified by Sanger sequencing using CRISPR_F primers (SEQ ID NO.12). Positive recombinant plasmids were selected and transformed into *Agrobacterium* GV3101 competent cells. The cells were cultured on YEP solid medium containing 50 mg / L kanamycin and 50 mg / L rifamycin. PCR was performed using CRISPR_F and M13_R as primers. Successfully transformed single colonies were confirmed by electrophoresis. Recombinant Agrobacterium tumefaciens bacterial culture was obtained by expanding positive monoclonal cultures.

[0102] sgRNA1_F:GACGAGTTGTTAGCAGCATT

[0103] M13_R:GTAAAACGACGGCCAGT

[0104] Crispr_F:TTCAAAAGTCCCACATCGC

[0105] III. Alfalfa leaf disc inoculation and transformation

[0106] (a) Explant treatment: Tender leaves from tissue culture seedlings of Amyda cum Euphratica var. 3, which have grown for 4-6 weeks after transplanting, were used as explant transformation materials. After collection, the entire leaf was first washed with clean water, then disinfected in a clean bench with a 20% (w / v) sodium hypochlorite solution for 20 minutes, and rinsed 5 times with sterile distilled water to ensure that there was no sodium hypochlorite residue on the surface of the material. Excess distilled water was absorbed with sterile filter paper.

[0107] (II) Agrobacterium culture: Recombinant Agrobacterium culture solution 1 was cultured overnight at 28°C and 180 rpm in 200 mL of YEP liquid medium containing 100 mg / L kanamycin and 50 mg / L rifamycin. After amplification in the logarithmic phase, the cells were centrifuged at 3600 rpm and 4°C for 10-5 min. The cells were resuspended in sterile 1 / 2 MS solution (containing 30 g / L sucrose) to OD. 600 The concentration was approximately 0.4, resulting in bacterial culture solution 1 for later use.

[0108] (III) Receptor infection: Place the previously disinfected explant leaves in a tissue culture bottle containing infection bacterial solution 1, and evacuate for 5-10 min to a vacuum degree of 0.08 MPa. Then, place the tissue culture bottle in an ultrasonic instrument containing an ice-water mixture for treatment. The ultrasonic power is 100W, 40 kHz, and the treatment time is 10s. Evacuate again for 3-5 min to a vacuum degree of 0.08 MPa. In a clean bench, spread the treated leaves on filter paper, press gently, and absorb the bacterial solution on the leaf surface to avoid bacterial growth in subsequent experiments.

[0109] (iv) Co-culture: After infection, the leaves were blotted dry with filter paper and spread evenly on the co-culture medium. They were then placed in the dark for 1 day at a temperature of 24℃. The co-culture medium (SH3a solid medium) was based on SH medium and also contained 20 g / L sucrose, 3.3 g / L Phytagel (purchased from Beijing Cooler Master Technology Co., Ltd.), 1 mg / mL 2,4-D, 1 mg / mL 6-BAP (6-benzylaminopurine), and pH 5.8.

[0110] (V) Selective Culture: After co-culturing on SH3a solid medium for 1 day, the leaves were transferred to SH3a selective medium and placed in an incubator at 24℃ in the dark. The medium was changed every 15 days until about 60 days of culture, at which point pale yellow callus tissue with a diameter of about 0.5 cm was produced. SH3a selective medium is based on SH and also contains 20 g / L sucrose, 3.3 g / L plant gel, 1 mg / mL 2,4-D, 1 mg / mL 6-BAP, 200 mg / L cephalosporin, 200 mg / L termethin, 7.5 mg / L glufosinate, and pH 5.8.

[0111] (vi) Callus induction culture: After growing on SH3a selective medium for 2-3 months, healthy callus tissue was transferred to MSBK medium and cultured in an incubator with alternating light and dark cycles of 24℃ and 16 h / 8 h. During the light culture phase, the light intensity was 1500 Lux. The medium was changed approximately every 15 days. Pale green callus tissue with a diameter of about 1 cm was cultured. The callus medium (MSBK medium) was based on MS medium and also contained 30 g / L sucrose, 3.3 g / L plant gel, 1 mg / mL kinetin, 1 mg / mL 6-BAP, 200 mg / L cephalosporin, 200 mg / L termethin, 7.5 mg / L glufosinate, and pH 5.8.

[0112] (VII) Differentiation Culture: After approximately 30 days of growth on MSBK medium, the callus tissue began to turn green. It was then promptly transferred to SH9a medium and placed in an incubator at 24℃ with alternating light and dark conditions (16 h / 8 h). During the light culture phase, the light intensity was 1500 Lux, and the medium was changed approximately every 15 days. When roots and shoots emerged from the callus tissue (approximately 60 days of differentiation culture), it was transferred to tissue culture bottles containing SH9a medium for further cultivation under the same conditions. Seedlings with robust root systems and upright growth were then transferred to the culture medium (soil) for further growth. The differentiation medium (SH9a medium) was based on SH medium and also contained 10 g / L sucrose, 3.3 g / L plant gel, 1 mg / mL 6-BAP, 200 mg / L termethin, 3 mg / L glufosinate, and pH 5.8.

[0113] (viii) Positive detection: Two weeks after rooting, DNA was extracted from the leaves and PCR was performed using specific primers F2 (SEQ ID NO.13) and R2 (SEQ ID NO.14) to obtain four positive transgenic plants for subsequent analysis.

[0114] F2: ATGAAAAAGCCTGAACTCACCG

[0115] R2:CTATTTCTTTGCCCTCGGACG

[0116] IV. Mutation Identification in Transgenic Plants

[0117] Genomic DNA was extracted from positive transgenic poplar trees and amplified by PCR using target-specific primers Crispr_F and M13_R. The amplified products were then ligated into a cloning vector. Single colonies were obtained by transforming *E. coli* DH5α competent cells and used for Sanger sequencing of target site amplicon sequences to analyze mutation events. For a comparison of the corresponding target sequences of plants #1 and #2 with the wild-type control (CK), please refer to [reference needed]. Figure 2 Each plant's four sequences represent four alleles. This indicates that all sgRNA1 alleles in this plant underwent base mutations, successfully editing the target gene. The two edited lines were named crispr-MsDELLA1#1 (KO-1) and crispr-MsDELLA1#2 (KO-2), respectively.

[0118] Example 2: Preparation of plants with high DELLA1 gene expression in Zhongmu No. 3

[0119] I. Construction of MsDELLA1 gene expression vector

[0120] The pFGC-eYFP vector (purchased from Sangon Biotech) was double-digested with BamHI and NcoI restriction endonucleases (purchased from NEB). The target band was detected by agarose gel electrophoresis, and the linearized vector fragment was obtained by gel recovery.

[0121] Design the target gene recombination primers F3 (SEQ ID NO.15) and R3 (SEQ ID NO.16), and design the universal primer eYFP-R (SEQ ID NO.17) downstream of the pFGC-eYFP vector insertion sequence.

[0122] F3: TACATTTACAATTAC GGATCC ATGGACGAGTTGTTAGCAGCA;

[0123] R3: GCTCCTCGCCCTTGC CCATGG TCACTTGGACTCATTTTGTGGAAG

[0124] eYFP-R: GGACACGCTGAACTTGTGGC

[0125] Example 2 and Example 1 are parallel operations, and the tissue culture of genetically engineered plants was completed simultaneously.

[0126] Using the recombinant cloning vector pMD18-T-DELLA1 containing the MsDELLA1 gene coding sequence prepared in Example 1 as a template, PCR amplification was performed. The PCR amplification product was ligated to the linearized vector fragment using the homologous recombinase Exnase II (purchased from Nanjing Vazyme), and cultured at 37°C for 30 min, followed by 5 min on ice. The resulting culture was transformed into *E. coli* DH5α competent cells and screened using LB solid medium containing 50 mg / L kanamycin. The extracted recombinant pFGC-eYFP vector was amplified using primers F3 and eYFP-R, yielding a target band of 1770 bp, indicating successful insertion of the DELLA1 gene coding sequence. The amplified product was then verified by Sanger sequencing using primers F3 and R3, showing no mutation in the DELLA1 gene coding sequence. Sequencing confirmed the transformation of the recombinant pFGC-eYFP vector containing the MsDELLA1 gene coding sequence into *Agrobacterium* GV3101 competent cells. Single-clonal detection PCR was performed using F3 and eYFP-R primers, and successfully transformed single clones were identified by electrophoresis. Positive single clones were expanded to obtain recombinant Agrobacterium tumefaciens containing the MsDELLA1 gene (culture 2), which was stored at -80℃ for later use.

[0127] II. Preparation of alfalfa with high expression of the DELLA1 gene in Zhongmu No. 3

[0128] (a) Explant treatment: Tender leaves from tissue culture seedlings of Amyda cum Euphratica var. 3, which have grown for 4-6 weeks after transplanting, were used as explant transformation materials. After collection, the entire leaf was first washed with clean water, then disinfected in a clean bench with a 20% (w / v) sodium hypochlorite solution for 20 minutes, and rinsed 5 times with sterile distilled water to ensure that there was no sodium hypochlorite residue on the surface of the material. Excess distilled water was absorbed with sterile filter paper.

[0129] (II) Agrobacterium culture: Recombinant Agrobacterium culture solution 2 was cultured overnight at 28°C and 180 rpm in YEP liquid medium (200 mL) containing 100 mg / L kanamycin and 50 mg / L rifamycin. After amplification in the logarithmic phase, the cells were centrifuged at 3600 rpm and 4°C for 10-15 min. The cells were resuspended in sterile 1 / 2 MS solution (containing 30 g / L sucrose) to OD. 600 The concentration was approximately 0.4, resulting in 2 infecting bacterial solutions for later use.

[0130] (III) Receptor infection: Place the previously disinfected explant leaves in a tissue culture bottle containing infection bacterial solution 2, and evacuate for 5-10 min to a vacuum degree of 0.08 MPa. Then, place the tissue culture bottle in an ultrasonic instrument containing an ice-water mixture for treatment. The ultrasonic power is 100W, 40 kHz, and the treatment time is 8 seconds. Evacuate again for 3-5 min to a vacuum degree of 0.08 MPa. In a clean bench, spread the treated leaves on filter paper, press gently, and absorb the bacterial solution on the leaf surface to avoid bacterial growth in subsequent experiments.

[0131] (iv) Co-culture: After infection, the leaves were blotted dry with filter paper and spread evenly on the co-culture medium. They were then placed in the dark for 1 day at a temperature of 24℃. The co-culture medium (SH3a solid medium) was based on SH medium and also contained 20 g / L sucrose, 3.3 g / L Phytagel (purchased from Beijing Cooler Master Technology Co., Ltd.), 1 mg / mL 2,4-D, 1 mg / mL 6-BAP, and pH 5.8.

[0132] (V) Selective Culture: After co-culturing on SH3a solid medium for 1 day, the leaves were transferred to SH3a selective medium and placed in an incubator at 24℃ in the dark. The medium was changed approximately every 15 days until pale yellow callus tissue with a diameter of about 0.5 cm was cultured for 60 days. SH3a selective medium is based on SH and also contains 20 g / L sucrose, 3.3 g / L plant gel, 1 mg / mL 2,4-D, 1 mg / mL 6-BAP, 200 mg / L cephalosporin, 200 mg / L termethin, 7.5 mg / L glufosinate, and pH 5.8.

[0133] (vi) Callus induction culture: After growing on SH3a selective medium for 2-3 months, healthy callus tissue was transferred to MSBK medium and cultured in an incubator with alternating light and dark cycles of 24℃ and 16 h / 8 h. During the light culture phase, the light intensity was 1500 Lux. The medium was changed approximately every 15 days. Pale green callus tissue with a diameter of about 1 cm was cultured. The callus medium (MSBK medium) was based on MS medium and also contained 30 g / L sucrose, 3.3 g / L plant gel, 1 mg / mL kinetin, 1 mg / mL 6-BAP, 200 mg / L cephalosporin, 200 mg / L termethin, 7.5 mg / L glufosinate, and pH 5.8.

[0134] (VII) Differentiation Culture: After approximately 30 days of growth on MSBK medium, the callus tissue began to turn green. It was then promptly transferred to SH9a medium and placed in an incubator at 24℃ with alternating light and dark conditions (16 h / 8 h). During the light culture phase, the light intensity was 1500 Lux, and the medium was changed approximately every 15 days. When roots and shoots emerged from the callus tissue (approximately 60 days of differentiation culture), it was transferred to tissue culture bottles containing SH9a medium for further cultivation under the same conditions. Seedlings with robust root systems and upright growth were then transferred to the culture medium (soil) for further growth. The differentiation medium (SH9a medium) was based on SH medium and also contained 10 g / L sucrose, 3.3 g / L plant gel, 1 mg / mL 6-BAP, 200 mg / L termethin, 3 mg / L glufosinate, and pH 5.8.

[0135] III. Identification of Genetically Modified Alfalfa

[0136] DNA was extracted from leaves of 15 plants to be tested, and PCR detection was performed using specific primers F3 and eYFP-R. Ten transgenic plants (MsDELLA1#1, #4, #5, #6, #7, #8, #9, #13, #14, #15) showed positive amplification results, indicating that the MsDELLA1 gene had been transferred into the recipient genome, resulting in positive transgenic plants (ox MsDELLA1). Plants designated OE-5 and OE-14 were selected for further research.

[0137] At the same time as the aforementioned tissue culture seedlings, Zhongmu No. 3 tissue culture seedlings were prepared as wild-type controls.

[0138] Example 3: Characterization of traits

[0139] Ten one-month-old seedlings (one month after conventional tissue culture and transplanting, and one month after harvesting) of five alfalfa plant species (referred to as five alfalfa germplasms) were taken from each parental line WT, OE-5 and OE-14 high-expression plants, and KO-1 and KO-2 knockout plants, and the following parallel test experiments were conducted.

[0140] I. Morphological Observation

[0141] One month after transplanting from tissue culture plants, the plants were cut on the same day to compare their morphology immediately after cutting and one month later. See also Figure 3 The scale bar in the diagram represents 4cm.

[0142] II. Statistics on the number of branches

[0143] For five alfalfa varieties, the number of branches per plant was counted one month after harvesting. The results are shown in [link to results]. Figure 4 Therefore, knockout of the MsDELLA1 gene can reduce the number of branches, while high expression of the MsDELLA1 gene can increase the number of branches.

[0144] III. Plant height statistics

[0145] For five alfalfa varieties, the plant height was measured one month after harvesting. The results are shown in [link to results]. Figure 5 .

[0146] IV. Statistics on Fresh Weight

[0147] For five alfalfa varieties, the fresh weight of the above-ground parts of the plants was calculated one month after harvesting. The results are shown in [link to results]. Figure 6 Therefore, knockout of the MsDELLA1 gene can reduce plant fresh weight, while high expression of the MsDELLA1 gene can increase plant fresh weight.

[0148] V. Statistics on the number of leaves

[0149] For five alfalfa varieties, the number of leaves per plant was counted one month after harvesting. The results are shown in [link to results]. Figure 7 Therefore, knockout of the MsDELLA1 gene can reduce the number of leaves, while overexpression of the MsDELLA1 gene can increase the number of leaves.

[0150] VI. Determination of soluble protein in leaves

[0151] Leaves were taken from alfalfa plants of five different genotypes one month after harvesting, and the soluble protein content was determined using the Coomassie brilliant blue staining method, following the method described in Reference 1.

[0152] Reference 1: Li Hesheng. 2002. Principles and Techniques of Plant Physiological and Biochemical Experiments. Beijing: Higher Education Press.

[0153] Soluble proteins refer to a class of small-molecule proteins, polypeptides, and nitrogen-containing compounds that can dissolve in water or other solvents.

[0154] For statistics on soluble protein content, please refer to [link / reference]. Figure 8 Therefore, knockout of the MsDELLA1 gene can reduce the content of soluble protein, while overexpression of the MsDELLA1 gene can increase the content of soluble protein.

[0155] VII. Determination of Soluble Sugar and Starch Content in Leaves

[0156] Leaves were taken from alfalfa plants of five different genotypes one month after harvesting, and the soluble sugar and starch contents were determined using the sulfuric acid anthrone colorimetric method according to the technical route described in the literature.

[0157] Reference 2: Wang Yimin. 2019. Study on the physiological mechanism of carbon metabolism response to potassium concentration in Fritillaria thunbergii leaves. [Master's Thesis]. Hangzhou: Zhejiang University of Traditional Chinese Medicine.

[0158] Soluble sugars refer to monosaccharides, disaccharides, and some oligosaccharides (such as glucose, fructose, sucrose, etc.) that are soluble in water.

[0159] Starch refers to the polysaccharide storage carbohydrates stored in plant tissues, which are composed of a large number of glucose molecules and include amylose and amylopectin.

[0160] For statistics on soluble sugar content, please refer to [link / reference]. Figure 9 Therefore, knockout of the MsDELLA1 gene can reduce soluble sugar content, while overexpression of the MsDELLA1 gene can increase soluble sugar content.

[0161] For statistics on starch content, please refer to [link / reference]. Figure 10 Therefore, knockout of the MsDELLA1 gene can reduce starch content, while overexpression of the MsDELLA1 gene can increase starch content.

[0162] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A method for improving alfalfa traits, the method comprising: using genetic engineering techniques to achieve high expression of the DELLA1 gene in alfalfa; The improvement of alfalfa traits includes: Increase the number of branches in alfalfa plants; Increase the fresh weight of alfalfa plants; Increase the number of leaves in alfalfa plants; Increase the starch content of alfalfa leaves; Increase the soluble protein content in alfalfa leaves; Increase the soluble sugar content in alfalfa leaves.

2. The method as described in claim 1, characterized in that, The alfalfa in question is alfalfa.

3. The method as described in claim 2, characterized in that, The alfalfa variety mentioned is Zhongmu No.

3.

4. The method according to any one of claims 1-3, characterized in that, The amino acid sequence of the protein encoded by the DELLA1 gene is shown in SEQ ID NO.

4.

5. The method according to any one of claims 1-4, characterized in that, The method includes the following steps: S1: The coding sequence of the DELLA1 gene is transferred into an expression vector to obtain a recombinant vector containing the DELLA1 gene; S2: Transform Agrobacterium with the recombinant vector to obtain a recombinant Agrobacterium bacterial suspension containing the DELLA1 gene; S3: Infect alfalfa leaves with the recombinant Agrobacterium bacterial solution to obtain infected alfalfa leaves; S4: The infected alfalfa leaves are placed on a co-culture medium and cultured to obtain co-cultured alfalfa leaves. S5: The co-cultured alfalfa leaves are placed on a selective culture medium to obtain preliminary alfalfa callus tissue; S6: The alfalfa preliminary callus tissue is placed on a callus culture medium and cultured to obtain alfalfa callus tissue; S7: The alfalfa callus tissue is cultured on a differentiation medium to obtain alfalfa plants with roots and buds.

6. The method as described in claim 5, characterized in that, In S1, the backbone of the expression vector is the pFGC-eYFP vector; In S2, the Agrobacterium is Agrobacterium GV3101; In S3, the alfalfa leaf is a sterilized leaf from an alfalfa tissue culture seedling that has grown for 4-6 weeks. In S4, the co-culture medium is based on SH medium, which also contains 15-25 g / L sucrose, 3.0-3.5 g / L plant gel, 0.8-1.2 mg / mL 2,4-D, 0.8-1.2 mg / mL 6-BAP, and pH 5.5-6.0; In S5, the selective medium is based on SH medium, which also contains 16-24 g / L sucrose, 3.0-3.5 g / L plant gel, 0.8-1.2 mg / mL 2,4-D, 0.8-1.2 mg / mL 6-BAP, 150-250 mg / L cephalosporin, 150-250 mg / L termethin, 6.0-9.0 mg / L glufosinate, and pH 5.5-6.0; In S6, the callus culture medium is based on MS medium, which also contains 25-35 g / L sucrose, 3.0-3.5 g / L phytogel, 0.8-1.2 mg / mL kinetin, 0.8-1.2 mg / mL 6-BAP, 150-250 mg / L cephalosporin, 150-250 mg / L termethin, 6.0-9.0 mg / L glufosinate, and pH 5.5-6.0; or In S7, the differentiation medium is based on SH medium, which also contains 8-12 g / L sucrose, 3.0-3.5 g / L plant gel, 0.8-1.2 mg / mL 6-BAP, 150-250 mg / L termethin, 1.5-2.5 mg / L glufosinate, and pH 5.5-6.

0.

7. The method as described in claim 5 or 6, characterized in that, In S3, the infection steps are as follows: The alfalfa leaves were placed into the recombinant Agrobacterium bacterial solution; Evacuate for 4-12 minutes under a vacuum level of 0.06-0.12 MPa. Ultrasonic treatment for 6-10 seconds at 0-4℃, ultrasonic power 80-120 W, and ultrasonic frequency 30-50 kHz. Evacuate for 2-6 minutes under a vacuum level of 0.06-0.12 MPa; In S4, the culture conditions are 22-26℃, cultured in the dark for 0.5-1.5 days; In S5, the culture conditions are 22-26℃, cultured in the dark for 50-70 days; In S6, the cultivation conditions are 22-26℃, 6-10h light per day, light intensity 1000-2000 Lux, and cultivation for 25-35 days; or In S7, the culture conditions are 22-26℃, 6-10h light per day, light intensity of 1000-2000 Lux, and culture for 55-65 days.

8. Use of a biomaterial in the preparation of a formulation for improving alfalfa traits in alfalfa breeding; The improvement of alfalfa traits includes: Increase the number of branches in alfalfa plants; Increase the fresh weight of alfalfa plants; Increase the number of leaves in alfalfa plants; Increase the starch content of alfalfa leaves; Increase the soluble protein content in alfalfa leaves; Increase the soluble sugar content in alfalfa leaves; The biological material is selected from any one of the following: P1, P2, P3, P4, P5, P6, P7, and P8: P1: Protein The protein in question is the DELLA1 protein. P2: Fusion protein The amino acid sequence of the fusion protein contains the amino acid sequence of the DELLA1 protein described in P1 and the amino acid sequence of the tag peptide and / or signal peptide used for protein isolation and purification. P3: RNA The RNA can be translated into the DELLA1 protein described in P1 or the fusion protein described in P2; P4: Genes The coding sequence of the gene can encode the DELLA1 protein described in P1 or the fusion protein described in P2; P5: Gene Expression Kit The gene expression product in the gene expression cassette is the RNA described in P3; P6: Gene Engineering Vector The genetic engineering vector contains the gene expression cassette described in P5; P7: Cells The cells contain the gene engineering vector described in P6; Constitutive expression, tissue-specific expression, or artificially induced expression of the encoded protein in the gene expression cassette of the genetic engineering vector; and P8: Composition The composition contains the RNA described in P3, the gene engineering vector described in P6, or the cell described in P7.

9. The use as described in claim 8, characterized in that, The alfalfa mentioned is purple alfalfa; The amino acid sequence of the DELLA1 protein is shown in SEQ ID NO.4; The backbone of the genetic engineering vector is a pFGC-eYFP vector; and / or The cells were selected from Agrobacterium GV3101 cells and Escherichia coli cells.

10. The use as described in claim 9, characterized in that, The alfalfa variety mentioned is Zhongmu No. 3.

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