An alfalfa MsCAL gene and its application

Abstract

The present invention relates to the field of biotechnology and relates to a kind of alfalfa MsCAL Gene, and its application, MsCAL The nucleotide sequence of the gene is shown in SEQ ID No.29. MsCAL The gene is specifically expressed in the plant stem apex, with the highest expression level in the floral organs during the reproductive growth stage, indicating that it is specifically involved in the plant flowering process. MsCAL Gene overexpression caused transgenic Arabidopsis to flower early, become dwarfed, and have fewer rosette leaves. In addition, the transgenic Arabidopsis changed from an indeterminate inflorescence to a finite inflorescence. Quantitative results of known genes regulating flowering time and floral organ development in Arabidopsis showed that MsCAL It can regulate the expression of downstream flowering and flower organ development related genes. MsCAL The expression delayed flowering, reduced crude fiber and lignin content, and increased forage digestibility and crude protein content.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kind of alfalfa MsCAL Genes and their applications. Background Art

[0002] Alfalfa is widely cultivated for its high yield, excellent quality, and wide adaptability. High in protein and rich in various vitamins and minerals, it not only improves livestock milk production but also boosts immunity. As a forage legume, alfalfa also plays a vital role in improving soil fertility, fixing nitrogen, and promoting ecological balance. Alfalfa holds an irreplaceable position and economic value in modern animal husbandry and agricultural production.

[0003] The flowering time of alfalfa significantly influences its yield and quality. When alfalfa flowers, nutrients are redistributed toward flower and seed development, while nutrients and energy invested in the vegetative phase decrease, leading to lower yield. Furthermore, after flowering, the cellulose and lignin content of alfalfa increases significantly, resulting in poor palatability and reduced digestibility, thus affecting forage quality. Flowering time is also closely linked to seed yield. Early flowering leads to incomplete development of the vegetative phase, resulting in less accumulated nutrients and fewer flowers, lower seed yield, and lower quality. Alfalfa is a forage grass that requires multiple mowing cycles to obtain its vegetative phase, and flowering is one of the indicators of mowing time. Mowing before flowering maintains quality but reduces yield, while mowing after flowering maintains yield but reduces quality. Therefore, delaying the flowering time of alfalfa and extending its vegetative phase are currently urgent challenges.

[0004] Therefore, the purpose of the present invention is to use transcriptome technology to study the expression differences of regulatory genes in the process of alfalfa flower bud differentiation, to explore the key genes in the alfalfa flowering regulatory pathway, and to analyze the functions and mechanisms of action of key regulatory genes in alfalfa flowering through molecular biology and genetics methods, so as to provide theoretical support for the genetic improvement of forage and the cultivation of high-yield and high-quality alfalfa varieties. Summary of the Invention

[0005] In order to achieve the above object, the present invention first provides a kind of alfalfa MsCAL gene, the MsCAL The nucleotide sequence of the gene is shown in SEQ ID No.29.

[0006] The present invention also provides a kind of alfalfa MsCAL gene, the MsCAL The amino acid sequence encoded by the gene is shown in SEQ ID No.30.

[0007] The present invention also provides MsCALThe relevant biological material is any one of the following B1) to B5):

[0008] B1) contains the above MsCAL expression cassette;

[0009] B2) contains the above MsCAL The recombinant vector

[0010] B3) contains the above MsCAL recombinant microorganisms;

[0011] B4) a recombinant vector containing the expression cassette described in B1);

[0012] B5) A recombinant microorganism containing the recombinant vector described in B2).

[0013] The present invention also provides a MsCAL Application in alfalfa breeding.

[0014] In certain embodiments, the use is to produce high-quality alfalfa.

[0015] In certain embodiments, the application is achieved by regulating MsCAL expression realization.

[0016] The present invention also provides an application of the above-mentioned biological material in alfalfa breeding.

[0017] In certain embodiments, the use is to produce high-quality alfalfa.

[0018] In certain embodiments, the application is achieved by regulating MsCAL expression realization.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] The present invention discovered MsCAL The N-terminus of the gene encodes a conserved MADS domain and belongs to the plant MADS transcription factor family. MsCAL Genes and Medicago truncatula MtCAL The gene shares a high degree of homology (93.9%) and is specifically expressed in the stem apex, with peak expression in floral organs during the reproductive growth phase, indicating its specific involvement in flowering. The MsCAL-GFP protein is primarily localized to the nucleus and exhibits in vitro transcriptional activation activity, consistent with the characteristics of a transcription factor. MsCAL Gene overexpression caused transgenic Arabidopsis to flower early, become dwarfed, and have fewer rosette leaves. In addition, the transgenic Arabidopsis changed from an indeterminate inflorescence to a finite inflorescence. Quantitative results of known genes regulating flowering time and floral organ development in Arabidopsis showed that MsCALIt can regulate the expression of downstream flowering and flower organ development related genes. MsCAL The expression delayed flowering, reduced crude fiber and lignin content, and increased forage digestibility and crude protein content. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Medicago sativa total RNA and MsCAL Agarose gel electrophoresis of PCR products of gene coding regions; a. Quality testing of total RNA extracted from alfalfa stem tips; b. MsCAL Amplification of gene CDS sequence; M: represents DNA molecular weight standard;

[0022] Figure 2 Alfalfa MsCAL Gene coding sequence;

[0023] Figure 3 MsCAL Bioinformatics analysis of genes; a. MsCAL Analysis of conserved gene domains. b. Analysis of the secondary structure of MsCAL protein. c. Analysis of the hydrophilicity of MsCAL protein. d. MsCAL Gene transmembrane domain analysis;

[0024] Figure 4 MsCAL Differential expression of genes in different tissues; n = 3, significance was calculated using one-way ANOVA analysis of variance, where different letters indicate significant differences between the two groups, P < 0.01;

[0025] Figure 5 MsCAL In vitro transcriptional activation assay for genes; a. Schematic diagram of the yeast reporter system. GAL4 BD represents the GAL4 binding domain, and GAL4 AD represents the activation domain. HIS3 represents the histidine reporter gene. b. Detection in yeast cells MsCAL Transcriptional activation activity assay. SC / -Trp: yeast growth medium lacking tryptophan; SC-Trp / His: yeast growth medium lacking both tryptophan and histidine;

[0026] Figure 6 MsCAL Gene subcellular localization; DAPI: cell nucleus specific dye;

[0027] Figure 7 MsCAL Transformation of Arabidopsis thaliana with the gene; a. Schematic diagram of the overexpression vector and PCR primer sites. b. Agarose gel electrophoresis of the PCR product. c. MsCALGene expression analysis. WT: wild-type Arabidopsis thaliana (Columbia-0). 1#–5#: represent five independent 35S:MsCAL-GFP overexpressing lines. Each line had three biological replicates. Results were analyzed using one-way ANOVA. Different letters indicate significant differences between the two groups, P < 0.01.

[0028] Figure 8 Overexpression MsCAL Genes promote early flowering in Arabidopsis; a. Flowering phenotypes of Arabidopsis under long-day conditions. b. Statistical analysis of flowering time in Arabidopsis under long-day conditions, n = 12. c. Statistical analysis of rosette leaf number at bolting in Arabidopsis under long-day conditions, n = 12. d. Expression analysis of genes regulating flowering time, n = 3. WT: wild-type Arabidopsis; 1#-4# represent four independent 35S:MsCAL-GFP overexpressing lines. Differences between transgenic lines and controls were analyzed using t-tests. ** indicates significant difference (P < 0.01).

[0029] Figure 9 Overexpression MsCAL The gene causes abnormal floral organ development in Arabidopsis; a. Terminal flower; b. Primary inflorescence; c. Secondary inflorescence; d. Rosette inflorescence; e. Mature pod; f. RT-qPCR analysis of genes related to floral organ development in transgenic Arabidopsis. WT: wild-type Arabidopsis. 1#-3# represent two independent 35S:MsCAL-GFP overexpressing lines. Student's t-test was used to analyze differences between transgenic lines and controls. Represents significant difference analysis P < 0.01;

[0030] Figure 10 Alfalfa MsCAL Analysis of flowering time of interference strains; a. Phenotype of alfalfa grown under long-day conditions, bar = 7 cm. b. MsCAL Gene expression analysis, n = 3. c. Statistical analysis of flowering time of transgenic alfalfa under long-day conditions. The dotted line represents the experimental cutoff time. d. Number of alfalfa nodes at the end of the experiment. The differences between the six transgenic cutting-regenerated lines and the control group were analyzed using t-tests. Represents significant difference analysis P < 0.01;

[0031] Figure 11 Downward adjustment MsCAL Effects on alfalfa quality; bar = ±SD, differences between the six transgenic lines regenerated by cuttings and the control group were analyzed using t-test. Represents significant difference analysis P < 0.01;

[0032] Figure 12 Transcriptome analysis of alfalfa stem tissue; a. Differential gene enrichment analysis; b. Transcriptome analysis of alfalfa lignin biosynthesis genes; c. Transcriptome analysis of alfalfa cellulose synthesis-related genes. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0034] The materials and equipment involved in the present invention are:

[0035] Plant materials: Medicago sativa cv zhongmu NO.1; Nicotiana tabacum L.; Arabidopsis thaliana Columbia-0;

[0036] Bacterial species: Escherichia coli (DH5α, Bioengineering (Shanghai) Co., Ltd.); Agrobacterium tumefaciens (EHA105, Beijing Huayueyang Biotechnology);

[0037] Plasmid vector

[0038] Cloning vector: TA / Blunt-Zero Cloning Kit (Nanjing Novozymes Biotech Co., Ltd., Nanjing);

[0039] pGBKT7 yeast expression vector (purchased from Takara);

[0040] Plant expression vector: pSmRYCE-GFP (stored in our laboratory);

[0041] Reagents and drugs are shown in Table 1.

[0042] Table 1 Chemical reagents and consumables

[0043]

[0044] The instruments and equipment are shown in Table 2;

[0045] Table 2 Summary of instruments and equipment

[0046]

[0047] MsCAL The nucleotide sequence is shown in SEQ ID No. 29, and the amino acid sequence encoded by the gene is shown in SEQ ID No. 30.

[0048] SEQ ID No. 29:

[0049] ;

[0050] SEQ ID No .30: MGRSKVQLKRIDNKINRQVTFSKRTGLLKKAKEISVLCDAEVALIIFSDNGKLFDYSTDSCMEQILERHERYGYAERLLVGNDTNTQENWTMEYTRLKAKIELLQRNHRHYMGE DLDSMSLKELQCLEQQLDTGLKHIRTRRTQVMYEAISELQKKEKGIQEQNNMLSKEIKEKEKVVAQEAACQWEQPNYRVDTSFLLQNPLPGLNMGGNYGEGAQELGRNGLDLTLEPLYY;

[0051] Example 1

[0052] Based on the preliminary research of the present invention, it was found that MsCAL The gene may play an important role in the transition to flowering. MsCAL The MsCAL gene was cloned and bioinformatics analysis was performed to analyze the expression pattern and biological function of the MsCAL gene.

[0053] Here’s how:

[0054] MsCAL gene cloning

[0055] (1) Primer design

[0056] Based on the published XinJiangDaYe Medicago sativa reference genome (https: / / figshare.com / s / fb4ba8e0b871007a9e6c) MsCAL The full-length CDS sequence of the gene was used as the reference sequence, and specific primers MsCAL-f and MsCAL-r were designed in the 5' and 3' noncoding regions for PCR amplification. The sequences are shown in Table 3.

[0057] Table 3 Summary of primers and sequences

[0058]

[0059] Note: Lowercase letters represent homology arm sequences of seamless cloning vectors

[0060] (2) Total RNA extraction and reverse transcription of alfalfa: Total RNA from alfalfa stem tips was extracted according to the operation method provided by the Promega Plant Total RNA Extraction Kit, and then reverse transcribed into cDNA using the Novozymes Fluorescence Quantitation Kit.

[0061] (3) MsCALPCR amplification of the full-length CDS sequence: Using primers MsCAL-f / r (Table 3), total cDNA obtained by reverse transcription of total RNA from the stem tip was used as a template, and PCR amplification was performed using high-fidelity Taq enzyme. The system is as follows:

[0062] Table 4 PCR amplification reaction system

[0063]

[0064] The PCR program settings are as follows:

[0065] Step 1: Melting (98°C, 5 min); Step 2 (27 cycles): 98°C (30 s), 56.5°C (30 s), 72°C (1 min); Step 3: Extension at 72°C for 5 min; Step 4: Reduce to 4°C for storage.

[0066] Cloning of target gene:

[0067] (1) After mixing the PCR product with DNA loading buffer, run electrophoresis on a 1% agarose gel with the electrophoresis parameters set at 121V, 30W, and the electrophoresis time set to 15min.

[0068] (2) Separate the target band into a nuclease-free centrifuge tube, add the melting solution and heat it at 56°C to melt the gel, and use a gel recovery kit to recover the target PCR product.

[0069] (3) Recover and purify the PCR product and connect it to the T vector according to the instructions of the cloning vector kit, which is named T-MsCAL. Heat shock the product into Escherichia coli DH5α competent cells at 42°C, and spread it onto LB solid medium containing ampicillin resistance. Screen for positive single clones at 37°C overnight.

[0070] (4) Send the monoclonal clone to Beijing Qingke Biotechnology Co., Ltd. for sequencing and obtain MsCAL Gene nucleic acid sequence.

[0071] Results: Total RNA was extracted from the shoot tip using an RNA extraction kit. The quality of the total RNA was tested by 1% agarose gel electrophoresis. The electrophoresis bands were clear and had no tailing, indicating high-quality RNA without degradation. Total cDNA was obtained by reverse transcription using RNA as a template. Using cDNA as a template, the MsCAL band of alfalfa was cloned. The gel results are shown in Figure 2. Figure 1 As shown in b, the size is approximately 800 bp, which is consistent with the CDS size of the genome annotation.

[0072] MsCAL bioinformatics analysis: The bioinformatics software DNAMAN 7 was used to predict the MsCAL open reading frame, translate the nucleic acid sequence into a protein sequence according to the codons, and calculate the protein molecular weight and isoelectric point of the gene.

[0073] The sequencing results are displayed after removing the T-linker sequence (see Figure 2 ), MsCAL is 830 bp long and contains a maximum open reading frame of 705 bp, which encodes 234 amino acid residues, with a molecular weight of 27.24 KDa and a theoretical isoelectric point of pI=7.44.

[0074] Protein hydrophilicity analysis was performed using Protscale online software (https: / / web.expasy.org / protscale / ); the transmembrane domain of the MsCAL gene was analyzed using TMHMM2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ); and the secondary structure of the gene was analyzed using SOPMA (https: / / npsa-pbil.ibcp.fr / cgi-bin / secpred_sopma.pl). Conserved domains of the MsCAL gene were analyzed using NCBI online software CD-Search; multiple sequence alignment was performed using DNAMAN software; and a phylogenetic tree of the MsCAL gene was constructed using MEGA (version 6.0). To investigate the structure of MsCAL, the encoded protein sequence was aligned with the CDD database using the online conserved domain identification software CD-Search (Conserved Domains-Search). The gene contains a conserved Type II MADS domain (1-71 aa) at its N-terminus, which belongs to the MADS-box family. Following this domain, there is a conserved K-box domain (83-173 aa), which is responsible for interacting with other proteins ( Figure 3 a). In addition to the MADS-box and K-box domains, MsCAL also contains a conserved AP1 motif at its C-terminus, which is thought to play a role in transcriptional activation. Interestingly, the MsCAL protein sequences from alfalfa and Medicago truncatula are shorter, lacking the LGCFA motif at the C-terminus, resulting in a partial loss of the putative AP1 domain. The MsCAL gene protein sequence shows high similarity to homologous genes from legumes, such as soybean (94.8%) and Medicago truncatula (99.1%). Sequence identity with homologous proteins from other selected species is lower, suggesting that the MsCAL gene may share or have similar biological functions with its Medicago truncatula and soybean homologs.

[0075] The secondary structure analysis of the protein sequence showed that the MADS domain is mainly composed of α-helix and β-sheet, and the K-box region is mainly composed of α-helix (see Figure 3 b). Analysis of the physicochemical properties of MsCAL protein showed that there is a hydrophobic peak in the MADS region at the N-terminus of MsCAL protein, which may be related to its biological function of binding to DNA (see Figure 3 c). In addition, the analysis of the transmembrane domain showed that there was no transmembrane domain in MsCAL protein (see Figure 3 d), which indicates that MsCAL transcription factor mainly plays a transcriptional regulatory role in the cell nucleus.

[0076] Example 2 Determination of MsCAL gene expression

[0077] The specific experimental steps are as follows:

[0078] (1) Alfalfa seeds were disinfected with 10% sodium hypochlorite solution for 12 minutes, then sown in a wide-mouth glass bottle containing 1 / 2 MS solid culture medium and placed in a light incubator for germination (about 5 days).

[0079] (2) Select seedlings with fully expanded cotyledons and relatively uniform size and plant them in flower pots (nutrient soil: vermiculite: perlite = 3:2:1), and transfer them to an artificial climate culture room for further growth.

[0080] (3) Roots, stems, and leaf tissues of alfalfa during the vegetative growth stage (day 20) and roots, stems, leaves, flower buds, and flower tissues during the reproductive growth stage (day 35) were cut and wrapped in tin foil. The tissues were quickly frozen in liquid nitrogen for 10 min and immediately transferred to a -80°C freezer for subsequent total RNA extraction.

[0081] (4) The sample was crushed using a high-throughput tissue grinder with a frequency of 50 Hz and a time of 30 s. Total RNA extraction and reverse transcription of alfalfa: Total RNA from alfalfa stem tips was extracted according to the operation method provided by the Promega Plant Total RNA Extraction Kit, and reverse transcribed into cDNA using the Novozymes Fluorescence Quantification Kit.

[0082] The expression levels of the genes in roots, stems, leaves, buds, and flowers of alfalfa at the vegetative growth stage (day 20) and reproductive growth and development stage (day 45) were analyzed by RT-qPCR. Figure 4). The MsCAL gene showed a high abundance expression level specifically in the reproductive organs during the reproductive growth stage, especially in the sepals, where the expression level was the highest, followed by the petals, and similar expression levels in the buds and pistils. In addition, significant expression was also detected in the pods. The MsCAL gene was expressed in the stem segment tissues during the reproductive growth stage, but the expression level was low. No MsCAL gene expression was detected in the leaf and root tissues. Compared with the reproductive growth stage, the expression of the MsCAL gene was only detected in the stem apex during the vegetative growth stage, which was 5.6 times that of the stem tissues during the mature stage. No expression of the gene was detected in other tissues including stems, leaves, and roots. In general, the MsCAL gene is highly expressed specifically in the floral organs, suggesting that it may be involved in regulating the biological function of the floral transition and the development of floral organs in alfalfa.

[0083] Example 3 In vitro transcription activation ability experiment

[0084] Construction of pGBKT7-MsCAL vector:

[0085] (1) Using the BD-CAL-f and BD-CAL-r primers (sequences shown in Table 3), and the T-MsCAL plasmid as a template, amplify the full-length coding sequence of MsCAL with homology arms.

[0086] (2) Linearize the pGBKT7 plasmid using EcoRI restriction endonuclease. The system is shown in Table 5.

[0087] Table 5 Restriction enzyme reaction system

[0088]

[0089] (3) Use a seamless cloning kit to connect the PCR product to the linearized pGBKT7 vector. The system is shown in the table below. Keep it in a constant temperature metal bath at 50°C for 15 minutes.

[0090] Table 6 Seamless cloning reaction system

[0091]

[0092] (4) After the seamless cloning reaction is completed, the entire reaction solution is added to 50 μL of E. coli (DH5α) competent cells and the pGBKT7-MsCAL fusion plasmid is transformed into the E. coli competent cells using the heat shock transformation method. After screening, the positive single clone plasmid is sequenced to ensure that no amino acid residue mutations have occurred. The single clone is propagated in LB liquid medium (containing 50 mg / L kanamycin) to an OD = 2.0. The plasmid is extracted using an E. coli plasmid extraction kit for subsequent transformation into yeast.

[0093] Yeast cell culture and electroporation:

[0094] (1) Thaw the yeast (Y2H) glycerol culture stored in the ultra-low temperature refrigerator at room temperature, dip the bacterial solution with a sterilized inoculation loop, streak on YPDA, and resuscitate and culture for 3 days until the diameter of the bacterial plaque is about 2 mm.

[0095] (2) Use a sterilized yellow pipette tip to pick up the Y2H monoclonal plaque and transfer it to 50 ml of liquid YPDA medium. Add 50 mg / L of kanamycin and place it in a shaker at 30°C, set the speed to 220 rpm, and shake for about 20 hours until the OD = 0.8.

[0096] (3) Transfer the bacterial solution to a 50 ml sterilized centrifuge tube and place the tube in an ice box for 5 min. Centrifuge at 4°C and 2500 g for 5 min, discard the supernatant and collect the bacteria.

[0097] (4) Resuspend the yeast pellet in 50 mL of ice-cold deionized water and vortex to mix thoroughly. Repeat the previous centrifugation step on the resuspended bacterial solution and wash the collected bacteria again with ice-cold deionized water.

[0098] (5) Add 20 mL of pre-cooled 1 mol / L sorbitol solution and vortex to mix. Centrifuge again to collect the yeast cells, resuspend the pellet in 500 μL of sorbitol solution, and place in an ice box for electroporation.

[0099] (6) Pipette salmon sperm DNA into a 200 μL centrifuge tube and melt it at 95°C for 5 min using a PCR instrument. Remove the centrifuge tube and immediately insert it into an ice box to cool. Repeat once and use it immediately for transformation.

[0100] (7) Mix the plasmid to be transformed and 5 μL of melted salmon sperm DNA on ice, immediately add 150 μL of yeast competent cells, flick to mix, and place in an ice box for 3 minutes.

[0101] (8) Add all the yeast competent cells from the previous step to the pre-cooled electroporation cup, and then perform electroporation transformation at the parameters of 1.5KV / 200Ω. Add 1ml of sorbitol solution to the yeast competent cells and incubate in a 30℃ water bath for 1 hour.

[0102] (9) The incubated bacterial liquid was concentrated by high-speed instant centrifugation, spread on SC medium lacking tryptophan (SC / -Trp) containing 50 mg / L, and incubated in an incubator at 30°C for 3-5 days to screen for transformants.

[0103] Yeast plasmid extraction and identification

[0104] The specific method for extracting plasmids using the Coolable glass bead yeast crushing method is as follows:

[0105] (1) Pick transformants from the plate and culture them in 5 mL SC / -Trp liquid medium (supplemented with 50 mg / L kan) overnight.

[0106] (2) Pipette 2 mL of yeast solution into a centrifuge tube and centrifuge at 13,000 rpm for 30 seconds at room temperature. Discard the supernatant and collect the bacterial precipitate.

[0107] (3) Add 200 μL of solution A to the centrifuge tube and vortex to suspend the bacteria. Add 0.2 g of glass beads and vortex thoroughly for 5 minutes to break up the bacteria.

[0108] (4) Heat in a boiling water bath for 3 minutes, then immediately cool in an ice box and centrifuge at 13,000 rpm for 10 minutes.

[0109] (5) Transfer 100 μL of supernatant to a new sterilized centrifuge tube and immediately add 50 μL of solution B. Place the centrifuge tube in a -40°C refrigerator for 1 hour.

[0110] (6) Centrifuge the tube at 13,000 rpm for 10 min, transfer 100 μL of the supernatant to a new sterilized centrifuge tube. Then add 200 μL of pre-cooled solution C, pipette and mix immediately, and place on ice for 15 min.

[0111] (7) Centrifuge and discard the supernatant. Add 1 ml of solution D to the centrifuge tube to wash once. Centrifuge again and aspirate and discard the supernatant. After drying the precipitate in an open container, add 30 μL of sterile water to dissolve the plasmid.

[0112] (8) Use gene-specific primers BD-CAL-f and BD-CAL-r (sequences in Table 3) to determine whether the plasmid has been successfully transformed into yeast by PCR.

[0113] Yeast in vitro transcription activation activity assay:

[0114] (1) Inoculate the yeast carrying pGBKT7-MsCAL into SC-Trp liquid medium and culture in a shaker at 30°C and 220 rpm to OD600 = 0.5.

[0115] (2) After centrifugation, resuspend the bacterial solution in sterile water to OD600 = 0.1, and then dilute it to 10 times and 100 times the bacterial solution in sequence.

[0116] (3) Pipette 10 μL of yeast solution with different dilution concentrations and spot it on the selection medium lacking tryptophan and histidine (SC / -Trp-His) containing 15 mM 3-AT. Invert it in a constant temperature incubator at 30°C for 3 days. Evaluate the transcriptional activation activity of the gene based on the growth status of the yeast.

[0117] like Figure 5 As shown in a, the yeast system HIS3 gene is used as a reporter gene, and the empty vector (pGBKT7) and GAL4 (pGBKT7-GAL4) are negative and positive controls, respectively. On the control medium lacking tryptophan (SC / -Trp), yeast cells carrying pGBKT7-GAL4, pGBKT7-MsCAL and pGBKT7 empty vectors can grow healthily, indicating that the fusion plasmid has been successfully transformed into yeast cells and has successfully expressed Trp resistance, allowing yeast cells to grow normally on tryptophan-deficient plates. In contrast, on tryptophan and histidine-deficient medium (SC / -Trp-His), yeast cells transformed with the pGBKT7 empty vector cannot grow normally, while yeast cells expressing the pGBKT7-MsCAL fusion plasmid grow normally in a manner similar to the positive control, indicating that MsCAL induces the expression of the HIS3 reporter gene ( Figure 5 b). In summary, the experimental results show that MsCAL This gene can activate the expression of a reporter gene in vitro and exhibits transcriptional activation activity. This study is consistent with previous results on CAL homologous genes, which function as transcription factors in regulating floral organ formation in plants.

[0118] Example 4 Subcellular localization experiment

[0119] (1) Construction of pSmRYCE-MsCAL-GFP vector: Use a pair of primers GFP-CAL-f and GFP-CAL-r (Table 3) with GFP-tagged vector homology arms to clone the MsCAL gene. Cut the vector with restriction endonuclease Spe I and construct the pSmRYCE-MsCAL-GFP vector using the seamless cloning method, as described above.

[0120] (2) Tobacco culture: Add 1 ml of sodium hypochlorite solution (10%, v / v) to a centrifuge tube and vortex the tube thoroughly to allow the tobacco seeds to fully contact the sodium hypochlorite solution. Sterilize the centrifuge tube by inverting it for 10 minutes. After disinfection, discard the disinfectant in a sterile environment and rinse the seeds several times with sterile distilled water to remove the disinfectant. Sow the seeds on 1 / 2 MS solid culture medium and culture them for 5 days. Transplant the germinated seedlings into nutrient soil and continue to culture them in an artificial climate chamber. When 4 to 5 leaves grow, use them for subcellular localization experiments.

[0121] (3) The constructed pSmRYCE-MsCAL-GFP plasmid and the pSmRYCE-GFP empty vector were transformed into Agrobacterium (EHA105) competent cells and spread on LB solid medium supplemented with kanamycin and rifampicin (50 mg / L) to isolate Agrobacterium carrying the target plasmid.

[0122] (4) Streak the Agrobacterium carrying the plasmid on an LB plate to isolate a single colony. Seal the plate and culture it in a constant temperature incubator at 28°C for 3-4 days until a circular monoclonal colony grows.

[0123] (5) In a sterile environment, pick a single colony and transfer it to 50 ml of LB liquid culture medium. Seal the culture medium and place it in a shaker at 220 rpm and 28°C in the dark for about 18 h until the OD = 0.8.

[0124] (6) Transfer the bacterial solution to a 50 mL sterile centrifuge tube and centrifuge at 4700 rpm for 10 min at room temperature. Discard the supernatant LB medium, collect the bacterial pellet, and invert the centrifuge tube on sterile absorbent paper to drain the liquid medium.

[0125] (7) Resuspend the pellet with 1 / 2 MS suspension (pH = 6.0) containing 150 mM / L acetosyringone, adjust OD = 1.0, and incubate at room temperature in the dark for 2 h.

[0126] (8) The suspension was injected into the tobacco leaves through the stomata of the lower epidermis and transferred to a dark and humid environment for 24 h. Then, the leaves were transferred to a greenhouse with normal light and continued to grow for about 2 days.

[0127] (9) Prepare the nucleus-specific dye DAPI with PBS buffer to a working concentration of 10 μg / mL. DAPI is easily decomposed by light and should be prepared immediately before use. Inject it into the leaves through the original injection hole and move the tobacco to a dark place and leave it at room temperature for 5-10 minutes to stain the nuclei of the tobacco leaves.

[0128] (10) Avoid the injection hole and the surrounding area. Use a blade to cut a 0.3 cm square leaf (do not cut into the main vein, as protruding veins are not conducive to sealing with a coverslip). Immerse the leaf in sterile distilled water, cover with a coverslip, absorb the excess distilled water with absorbent paper, and place the slide under a fluorescence microscope for observation. The excitation wavelength of DAPI is 360 nm, and the emission wavelength is 460 nm; the excitation wavelength of GFP is 488 nm, and the emission wavelength is 507 nm.

[0129] MsCAL with green fluorescent protein (GFP) tag was transiently transformed into tobacco leaf cells using CaMV35S promoter. The green fluorescence signal of the empty vector control (35S:GFP) was detected, and green fluorescence was detected in both the cell membrane and the cell nucleus ( Figure 6 The fluorescent signal of the GFP-tagged MsCAL protein was primarily localized in the cell nucleus, with trace amounts also expressed on the cell membrane. These results indicate that MsCAL is primarily localized in the cell nucleus, consistent with its typical nuclear localization as a MADS transcription factor. The presence of some fluorescent signal on the cell membrane may be due to the unique mechanism of action of this gene, which warrants further investigation.

[0130] Example 5 RNAi vector construction and host bacterial transformation, Arabidopsis thaliana and alfalfa genetic transformation

[0131] MsCAL-RNAi vector construction:

[0132] (1) Using T-MsCAL as template and CALi-F1 / R1 as primers (Table 3), the PCR amplification system is shown in Table 7.

[0133] Table 7 PCR reaction system

[0134]

[0135] (2) PCR program is shown in Table 8.

[0136] Table 8 PCR reaction procedure

[0137]

[0138] After PCR amplification, the target band was purified from gel. The psmRYCE-Ri vector was linearized with BamH I restriction endonuclease. The target band and the vector were ligated using seamless cloning.

[0139] (3) Repeat the cloning of the fragment using CALi-F2 and CALi-R2 and recover the fragment. Extract the plasmid connected in the previous step, cut the vector with Spe I restriction enzyme to linearize it, mix the PCR product with the linearized vector, and use the seamless cloning method to connect the fragment to the vector again, using the same method as the previous step.

[0140] Agrobacterium competent cell preparation and plasmid transformation:

[0141] (1) Thaw the Agrobacterium EHA105 glycerol stock stored in an ultra-low temperature freezer at room temperature, then streak it onto LB solid medium (50 mg / L rifampicin), and incubate it upside down at 28°C for 3 days to isolate the monoclonal strain.

[0142] (2) Pick a single colony plaque and place it in 500 μL LB liquid culture medium. Use a pipette to pipette several times to break up the plaque. Transfer all the bacterial liquid into 20 ml LB liquid culture medium containing 50 mg / L rifampicin. Rotate and culture in a shaker until OD600 = 0.8.

[0143] (3) Transfer the entire bacterial solution to a 50ml sterilized centrifuge tube. Centrifuge at 4700rpm for 5 minutes at room temperature. Discard the upper culture medium and keep the bacterial pellet.

[0144] (4) Suspend the Agrobacterium tumefaciens precipitate in 10 ml of pre-cooled 0.1 M calcium chloride solution and place it in an ice box for 20 minutes.

[0145] (5) After the ice bath, centrifuge at 4700 rpm for 5 min at 4°C and discard the supernatant. Add 2 ml of pre-chilled calcium chloride solution containing 15% glycerol, aliquot, and take 100 μL of competent cells for plasmid transformation.

[0146] (6) Add 200 ng of pSmRYCE-MsCAL plasmid to 100 μL of Agrobacterium competent cells, flick to mix, place in an ice box and let stand for 20 min; quick freeze in liquid nitrogen for 5 min; and keep warm at 37°C for 2 min.

[0147] (7) Add 1 ml of sterile LB liquid medium to the centrifuge tube and resuscitate at 28°C for 1 hour. Centrifuge the culture at 8000 rpm, discard the supernatant, and resuspend the cells by pipetting several times. Spread the suspension on an LB plate (containing kanamycin and rifampicin at a final concentration of 50 mg / L). Incubate the plate upside down at 28°C for 3 days. Identify the Agrobacterium carrying the pSmRYCE-MsCAL plasmid by colony PCR.

[0148] Genetic transformation of transgenic Arabidopsis thaliana:

[0149] (1) Arabidopsis seeds were sterilized with a sodium hypochlorite solution containing 1% Tween for 10 min and washed 5-6 times with sterile water.

[0150] (2) Sow the seeds on 1 / 2MS solid culture medium and culture them in an artificial climate incubator for 10 days until the seeds germinate and grow two true leaves. Select single plants with consistent growth and plant them in flower pots (vermiculite: nutrient soil = 1:1). Then transfer them to a greenhouse and grow them until they bolt. The photoperiod conditions are 16h light / 8h dark and 30%-40% humidity.

[0151] (3) Suspend Agrobacterium cells in 1 / 2 MS liquid medium (containing 0.2% Silweet-77) to an OD600 of 1.0. Remove the opened flowers and leave only the unopened buds of the Arabidopsis inflorescences. Immerse the inflorescences in the suspension for 5 minutes, shaking them at intervals.

[0152] (4) Transfer the Arabidopsis thaliana to a foam box, add water to keep it moist, and seal it for 24 hours. Remove the Arabidopsis thaliana and return it to the culture room to continue culture until pods grow.

[0153] (5) Collect the pods, dry them, and separate the seeds. Sterilize the seeds as in step (1) and spread them on 1 / 2 MS plates containing 4 mg / L glufosinate ammonium to screen for transgenic Arabidopsis. Plant the positive Arabidopsis plants for three consecutive generations to obtain homozygous plants whose offspring no longer separate.

[0154] Alfalfa genetic transformation:

[0155] (1) Alfalfa seeds were germinated on moist filter paper, then transplanted into pots and grown in an artificial climate chamber. Healthy leaves were used as explants for Agrobacterium infection.

[0156] (2) Streak the Agrobacterium tumefaciens EHA105 glycerol culture containing the target gene on an LB agar plate containing 50 mg / L kanamycin and rifampicin and culture at 28°C for 2 days. Pick a single colony from the plate with a sterile pipette tip, add 4 ml of LB medium, and culture overnight in a shaker at 28°C at 200 rpm. The next afternoon, take 1 ml of the overnight culture and inoculate it into 30 ml of liquid LB medium containing antibiotics (50 mg / L kanamycin and rifampicin) and continue to culture in a shaker at 28°C at 200 rpm. The next day, monitor the culture liquid. When the OD600 = 0.8, centrifuge the Agrobacterium culture liquid at 4700 rpm for 15 minutes at room temperature to collect the precipitate, and use SH3a liquid medium to suspend the bacteria to OD600 = 0.3.

[0157] (3) Sterilize the alfalfa leaves by shaking them continuously in a 10% sodium hypochlorite solution for 10 min. Then wash the alfalfa leaves five times with sterile distilled water, dry them with absorbent paper, and scratch the leaves with a sterile scalpel.

[0158] (4) Then add the Agrobacterium suspension to the alfalfa leaves, seal them with parafilm, and use a vacuum pump to evacuate for 10 minutes to allow the leaves to fully absorb the suspension.

[0159] (5) Drain the bacterial liquid on the surface of alfalfa leaves and spread them on SH3a solid culture medium, then culture them in a 24℃ incubator in the dark for 24 hours.

[0160] (6) The next day, the explants were transferred to SH3a selection medium (supplemented with 4 mg / L glufosinate) and cultured in a dark incubator at 24°C.

[0161] (7) Transfer the explants to fresh SH3a selection medium every 2 weeks. During the first transfer, use forceps to separate the explants and place them on the selection medium. Callus spots will be visible after about 3 weeks, but it will take 5-6 weeks for significant callus proliferation to occur.

[0162] (8) When the resistant callus is large enough (about 0.5 cm in diameter), transfer the callus to MSBK regeneration medium. Continue to culture in a light incubator (16 h light / 8 h dark, 24°C / 20°C) for 10-14 days until green embryoids are visible.

[0163] (9) Transfer the callus with embryos to SH9 medium to regenerate buds, subculture every 20 days until the embryos grow.

[0164] (10) When the embryo develops into 2-3 fully expanded tripterygium leaves, it is transferred to 1 / 2 MS medium for rooting culture. The rooted transgenic plants are transplanted into pots and continue to grow in the greenhouse.

[0165] Determination of flowering time:

[0166] To measure flowering time in Arabidopsis, homozygous T3 seeds were sterilized, sown on 1 / 2 MS solid medium, and cultured in a light incubator until four true leaves developed (approximately 10 days). The seeds were then transplanted into pots and transferred to long-day conditions (16 hours of light / 8 hours of darkness) to induce flowering. Flowering time was assessed using two methods: the growth time from germination to bolting and the number of rosette leaves at bolting.

[0167] Because the flowering time of tissue culture-derived original lines may be affected by tissue culture effects or mosaic mutations, to accurately assess the flowering time of transgenic lines, artificially propagated plants using stem cuttings were analyzed. The transgenic lines underwent at least two rounds of mowing in the greenhouse to maintain the consistency of their growth state as much as possible. Then, to minimize the variation in flowering time that may be caused by environmental factors, cuttings from stem segments in the same position were used to propagate at least six plants per line. After uniform mowing, the plants were grown in the greenhouse for one month, and long-day conditions (16 hours of light / 8 hours of dark) were used to induce flowering of alfalfa.

[0168] Flowering-related gene expression: Total RNA was extracted from aerial parts of Arabidopsis thaliana according to the instructions of the Plant Total RNA Isolation Kit and evaluated by NanoDrop. First-strand cDNA synthesis was performed using 1 μg of total RNA using the cDNA Synthesis Kit. RT-qPCR analysis was performed using the SYBR Premix Ex Taq fluorescence quantitative PCR system. AtACIN2 (At3g18780) was used as an internal reference gene for Arabidopsis thaliana. Relative expression levels were calculated using the 2-ΔΔCT method.

[0169] result:

[0170] (1) Identification of MsCAL-overexpressing Arabidopsis

[0171] To reveal the biological function of MsCAL, a 35S promoter overexpression vector was constructed ( Figure 7 a) Arabidopsis thaliana was genetically transformed using the inflorescence infection method. Five independent MsCAL Arabidopsis transgenic lines were screened for glufosinate resistance. To determine whether the MsCAL gene had integrated into the Arabidopsis genome, genomic DNA from homozygous T2 lines was used as a template for PCR amplification. Specific primer sites were designed as follows: Figure 7 As shown in a, the positive control was the pSmRYCE-MsCAL-GFP plasmid, and the negative control was wild-type Arabidopsis genomic DNA. The PCR amplification band positions of the five homozygous transgenic Arabidopsis lines were consistent with those of the positive control plasmid, while no band was amplified at the corresponding position in wild-type Arabidopsis ( Figure 7 b) shows that MsCAL is integrated into the Arabidopsis genome.

[0172] To further identify the mRNA expression level of MsCAL in transgenic Arabidopsis, the MsCAL gene in five T2 generation Arabidopsis plants was quantitatively analyzed by RT-qPCR ( Figure 7c). No fluorescent signal (No Signal) was detected in the wild-type (WT) line, while clear expression signals were detected in the transgenic lines, indicating that the MsCAL gene is expressed in Arabidopsis. The 35S:MsCAL-GFP 5# line had the lowest expression level, while the 35S:MsCAL-GFP 1# line had the highest expression level, 9.33-fold higher. This was followed by 35S:MsCAL-GFP 4# (7.09-fold higher), followed by 35S:MsCAL-GFP 2# and 35S:MsCAL-GFP 3#, with expression levels of 4.97 and 5.47-fold higher, respectively.

[0173] (2) Phenotypic analysis of Arabidopsis overexpressing MsCAL

[0174] To reveal the biological function of MsCAL, four homozygous overexpressing Arabidopsis lines with high expression levels were selected, and 12 individuals from each line were used for phenotypic analysis. Under long-day conditions, the four Arabidopsis lines overexpressing MsCAL all flowered earlier than the wild type ( Figure 8 a). The average flowering time of each transgenic Arabidopsis line is 14.7-16.3 days, which is 10.5-12.1 days earlier than the wild-type control (26.8 days). Figure 8 b); The average number of rosette leaves in the overexpression lines at flowering was 4.9-5.6 (1# and 2#), which was significantly less than that in the wild type (8.1), indicating that overexpression of MsCAL promoted flowering in Arabidopsis ( Figure 8 c).

[0175] To investigate the molecular mechanism of flowering regulation by the MsCAL gene, RT-qPCR analysis was performed on genes regulating flowering time in two transgenic Arabidopsis lines overexpressing 35S:MsCAL-GFP 1# and 35S:MsCAL-GFP 3#. Figure 8As shown in Figure d, AtAGL6 promotes flowering in Arabidopsis, with its expression levels significantly increased in overexpression lines. Expression of both AtLFY and AtSOC1 significantly promoted flowering in Arabidopsis, indicating a role in the flowering process. In both 35S:MsCAL-GFP 1# and 35S:MsCAL-GFP 3# transgenic lines, AtLFY expression levels were significantly increased, reaching 5.72- and 12.05-fold compared to the WT, respectively. However, AtSOC1 expression levels were significantly decreased, reaching 0.24- and 0.19-fold compared to the control. MsCAL genes may mediate flowering in Arabidopsis through the AtAGL6 and AtLFY pathways. In addition to regulating flowering time, overexpression of MsCAL has also been observed to result in a dwarf, thin, and weak phenotype in Arabidopsis plants, suggesting that MsCAL genes also regulate growth and development in Arabidopsis.

[0176] (3) Analysis of floral organ phenotypes in Arabidopsis overexpressing MsCAL

[0177] Observation of the flowers of four homozygous Arabidopsis lines overexpressing MsCAL revealed that MsCAL not only regulates the flowering time of Arabidopsis, but also affects the normal development of floral organs, inflorescences, and pods. Wild-type Arabidopsis petals are cross-shaped, while the petals of the terminal flowers of transgenic Arabidopsis overexpressing the MsCAL gene are irregularly curved inward ( Figure 9 a); In addition, the sepals thicken and become concave inward to form a spoon shape, and multiple stamens and pistils develop at the same time ( Figure 9 a and b). The overexpressing Arabidopsis lines showed the emergence of terminal flowers at the top of the primary inflorescence, resulting in the transformation of the indeterminate inflorescence into a determinate inflorescence. The stems of the cauline inflorescence and the rosette inflorescence stems also transformed into flowers ( Figure 9 c and d). Observation of pods showed that the pods of Arabidopsis overexpressing thaliana exhibited abnormal curved growth, reduced pod length and fewer seeds ( Figure 9 e). RT-qPCR analysis showed that the expression of class A genes (AtAP1), class B genes (PI, AP3), and class C genes (AG) that control petal and sepal development were significantly increased in 35S::MsCAL-GFP 1# and 35S::MsCAL-GFP 3# transgenic Arabidopsis ( Figure 9 f). For the overexpression of TFL gene that regulates inflorescence development in Arabidopsis, the expression level was significantly reduced, which was 0.04 times and 0.22 times that of the control group respectively ( Figure 9 f).

[0178] (4) Flowering phenotype analysis of MsCAL-RNAi alfalfa strains

[0179] To investigate the effect of MsCAL on flowering in alfalfa, 10 independent alfalfa RNAi transgenic lines were generated through genetic transformation. Three lines with lower expression levels (RNAi-1#, RNAi-2#, and RNAi-3#) were analyzed by measuring the expression of the MsCAL gene. The transcript levels of the MsCAL gene in RNAi-interference plants were reduced by 0.66-0.32 times compared to the control group ( Figure 10 b). These transgenic lines were propagated by cuttings and analyzed for flowering time under long-day conditions. Compared with the regeneration control, the apical meristems of all transgenic lines showed no signs of imminent flowering at the end of the experiment (see Figure 10 a), indicating that flowering time was delayed. In addition, the growth and development of the transgenic lines were normal, and the total number of internodes per plant was the same as that of the wild-type plants at the end of the experiment ( Figure 10 d).

[0180] Example 6 MsCAL-RNAi Alfalfa Quality Analysis

[0181] The digestibility of alfalfa forage is mainly affected by non-digestible tissues such as crude fiber and lignin. To investigate the quality of late-flowering alfalfa strains, this study used a near-infrared analysis system to measure the forage quality of late-flowering plants at the initial flowering stage and a control.

[0182] Method for determining the quality of alfalfa forage:

[0183] When 20% of the control plants were in bloom, the aerial parts of the plants were harvested. The fresh weight was immediately measured using a scale and placed in a kraft paper bag. The plants were then dried in a constant-temperature forced-air drying oven at 55 ± 5°C to constant weight (approximately 5 days). After thorough grinding, the following alfalfa quality parameters were measured using near-infrared reflectance spectroscopy: crude protein (on a dry matter basis), acid detergent fiber (ADF), neutral detergent fiber (NDF), lignin content, and in vitro dry matter digestibility.

[0184] Results: Compared with the control, there were no significant differences in fresh weight, plant height and branch number of the RNAi-interference transgenic lines ( Figure 11 a, b and c), indicating that reducing MsCAL expression does not affect the normal growth of alfalfa. The neutral detergent fiber content of the alfalfa aboveground part of the control group was 51.08%, which was reduced by about 2% (48.68%-48.79%) compared with the control group ( Figure 11 e). The acid detergent fiber content (32.16%-31.66%) was also significantly reduced, down 4.44%-4.94% compared to the control group (36.6%). Figure 11f). At the same time, reducing MsCAL expression also reduced lignin content by approximately 1.39%. Compared with the control group, the in vitro dry matter digestibility of transgenic alfalfa increased by 3.75%-3.19%, and the crude protein content increased by 3%-5% ( Figure 11 d and h). The above results indicate that reducing the expression of MsCAL gene improves the quality of alfalfa.

[0185] Example 7 MsCAL-RNAi alfalfa transcriptome analysis

[0186] Since lignin and cellulose are the main components of stems, transcriptome analysis was performed on the stem tissues of MsCAL-RNAi 3# and control at the early flowering stage. GO enrichment analysis of differentially expressed genes showed that among the top 10 GO terms, a large number of genes related to secondary metabolic pathways were significantly enriched ( Figure 12 a), including phenylpropanoid synthesis and metabolism pathways, lipid synthesis and metabolism pathways, and fatty acid synthesis pathways. In particular, genes in the lignin synthesis pathway and cell wall organization and synthesis pathway, which are related to alfalfa quality, were significantly enriched. Analysis of genes in the lignin synthesis pathway showed that genes related to lignin synthesis, such as MsPAL, Ms4CL, MsHCT, MsCOMT, MsCCoAOMT, and MsCAD, were significantly reduced in RNAi plants ( Figure 12 b). In addition, the expression levels of a large number of oxidases that catalyze the synthesis of lignin monomers were also reduced. This indicates that reducing MsCAL expression leads to reduced expression of lignin synthesis genes in alfalfa stems. This supports the results of reduced lignin content in RNAi lines. Analysis of cell wall organization and synthesis pathway genes showed that most cellulose and hemicellulose synthesis genes (CESAs, CSLs, and IRXs) were downregulated in RNAi transgenic lines ( Figure 12 c). Several XTH genes involved in catalyzing the breakdown and rearrangement of xyloglucan polymers in primary cell walls and in cell wall construction of growing tissues were also found to be significantly down-regulated in the RNAi lines.

[0187] In summary, MsCAL The N-terminus of the gene encodes a conserved MADS domain and belongs to the plant MADS transcription factor family. MsCAL Genes and Medicago truncatula MtCAL The gene shares a high degree of homology (93.9%) and is specifically expressed in the stem apex, with peak expression in floral organs during the reproductive growth phase, indicating its specific involvement in flowering. The MsCAL-GFP protein is primarily localized to the nucleus and exhibits in vitro transcriptional activation activity, consistent with the characteristics of a transcription factor. MsCALGene overexpression caused transgenic Arabidopsis to flower early, become dwarfed, and have fewer rosette leaves. In addition, the transgenic Arabidopsis changed from an indeterminate inflorescence to a finite inflorescence. Quantitative results of known genes regulating flowering time and floral organ development in Arabidopsis showed that MsCAL It can regulate the expression of downstream flowering and flower organ development related genes. MsCAL The expression delayed flowering, reduced crude fiber and lignin content, and increased forage digestibility and crude protein content.

[0188] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. MsCAL The application of genes in the construction of high-quality alfalfa MsCAL The nucleotide sequence of the gene is shown in SEQ ID No. 29; MsCAL The amino acid sequence encoded by the gene is shown in SEQ ID No. 30, which is characterized in that: The high quality is to reduce the crude fiber and lignin content and increase the digestibility and crude protein content of alfalfa; The application is down-regulated by RNAi MsCAL expression realization.

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