Alfalfa mspl19 protein and gene in regulating alfalfa plant height, fresh weight or biological yield

Abstract

The present application relates to the technical field of plant genetic engineering, and particularly relates to an application of Medicago sativa MsPL19 protein and gene in regulating plant height, fresh weight or biological yield of Medicago sativa. The present application provides the MsPL19 protein, gene and application thereof, which can significantly improve the plant height, biological yield and fresh weight of Medicago sativa, and further improve the quality of Medicago sativa. Meanwhile, it is found that the MsPL19 protein and gene can respond to low temperature, drought, salt, hormone and other pathways, the proteins interacting with the MsPL19 are obtained through immunoprecipitation-mass spectrometry analysis, the corresponding molecular module can be enriched, the molecular mechanism of Medicago sativa MsPL19 responding to low temperature environment is analyzed, and a new cold-resistant variety can be selected in an alpine region, thereby providing a new breeding direction for improving the yield of Medicago sativa.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of alfalfa MsPL19 protein and its gene in regulating alfalfa plant height, fresh weight or biomass yield. Background Technology

[0002] alfalfa ( Medicago sativa Alfalfa (L.) is a perennial, cut-tolerant legume forage grass, renowned for its high yield and rich nutritional value, earning it the title of "King of Forages." Alfalfa is a long-day crop with typical fall dormancy characteristics. Its regeneration rate, number of cuts, and forage yield are directly related to its fall dormancy. Furthermore, research indicates a significant correlation between alfalfa regeneration and overwintering rate. Alfalfa varieties with good spring regeneration performance green up early, extending the vegetative growth period, increasing forage yield, and suppressing weed growth, effectively mitigating weed damage. Alfalfa regeneration significantly impacts its overwintering ability, biomass, and quality. Identifying the genes regulating alfalfa regeneration and biomass in alfalfa, and subsequently studying their regulatory networks, is of significant practical value and guiding importance for creating stress-resistant, high-yielding new alfalfa germplasm and breeding new alfalfa varieties.

[0003] Pectate lyase (PL) is a class of enzymes that catalyzes the degradation of α-1,4-glycosidic bonds in the smooth region of pectin through β-trans elimination. This degradation produces oligosaccharides containing unsaturated galacturonic acid residues at the non-reducing ends, thereby cleaving pectin molecules. PL is involved in various plant functions, playing a crucial role in stress resistance, including plant growth and development, fruit ripening and softening, and interactions between plants and pathogens.

[0004] Low temperatures significantly affect the growth, development, productivity, and geographical distribution of alfalfa, especially in high-altitude and cold regions such as Hulunbuir. Frequent frosts and cold snaps in late autumn and early spring severely impact alfalfa harvesting, regeneration, and yield.

[0005] Currently, alfalfa PL The function of genes is still poorly understood; therefore, research on alfalfa... PL The function of the gene and the regulatory mechanism of its molecular modules are of great significance for improving alfalfa yield, enhancing its mowing regeneration ability and overwintering ability. Summary of the Invention

[0006] This invention has discovered a new alfalfa gene. MsPL19This method can significantly increase the biomass and fresh weight of alfalfa, thereby improving its quality. Furthermore, it can respond to low temperature and light conditions and is regulated by genes involved in hormone synthesis and signal transduction, cell wall synthesis, and metabolism. Based on this, the following technical solution is proposed.

[0007] In a first aspect, the present invention provides alfalfa MsPL19 protein, the amino acid sequence of which is shown in (1) or (2) below:

[0008] (1) As shown in SEQ ID NO.1;

[0009] (2) An amino acid sequence derived from (1) with the amino acid sequence shown in SEQ ID NO.1 having one or more amino acids replaced, deleted or added and having the same function.

[0010] Secondly, the present invention provides a gene encoding the MsPL19 protein of alfalfa.

[0011] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0012] Thirdly, the present invention provides biological materials containing the said gene, wherein the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable plant cells or tissues.

[0013] Fourthly, this invention provides alfalfa MsPL19 protein and alfalfa... MsPL19 The application of genes or the aforementioned biological materials in regulating alfalfa plant height, fresh weight, or biomass yield.

[0014] Preferably, increasing the expression level or activity of the MsPL19 protein or gene in alfalfa can improve plant height, fresh weight, or biomass yield.

[0015] Fifthly, this invention provides alfalfa MsPL19 protein and alfalfa... MsPL19 The application of genes or the aforementioned biological materials in alfalfa variety improvement.

[0016] In the specific implementation process, variety improvement is carried out through methods including but not limited to genetic modification, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0017] Sixthly, the present invention provides alfalfa MsPL19 protein and alfalfa... MsPL19 The application of genes or the aforementioned biological materials in the resistance of alfalfa to abiotic stress; preferably, the abiotic stress includes drought stress, low temperature stress, or salt stress.

[0018] Preferably, alfalfa MsPL19 protein, alfalfa MsPL19 Genes or the biological materials described herein regulate alfalfa plant height, fresh weight, or biomass yield by modulating hormone signaling pathway genes and cell wall development genes.

[0019] Preferably, the hormone signal includes IAA, GA, or CK.

[0020] Preferably, the expression levels of hormone signaling pathway genes and cell wall development genes are increased by increasing the expression level or activity of alfalfa MsPL19 protein or gene.

[0021] Preferably, the activity or expression level of the alfalfa MsPL19 protein or gene is regulated by photoperiod or temperature.

[0022] In a seventh aspect, the present invention provides a method for increasing the plant height, fresh weight or biomass yield of alfalfa, comprising: overexpressing the alfalfa MsPL19 protein or the gene in alfalfa;

[0023] Preferably, the overexpression mode is selected from one or more combinations of the following (1) to (5):

[0024] (1) By introducing a plasmid containing the gene;

[0025] (2) By increasing the copy number of the genes described on the chromosome;

[0026] (3) By altering the promoter sequence of the genes described on the chromosome;

[0027] (4) By operatively linking a strong promoter (such as the CaMV35S promoter) to the gene;

[0028] (5) By introducing enhancers.

[0029] The CaMV35S promoter can be efficiently expressed in dicotyledonous plant tissues, enabling... MsPL19 Overexpression in alfalfa plants allows for full utilization of its function.

[0030] The beneficial effects of this invention are as follows:

[0031] This invention provides the MsPL19 protein, gene, and their applications, which can significantly improve the plant height, biomass yield, and fresh weight of alfalfa, thereby improving alfalfa quality. Simultaneously, this invention discovers that the MsPL19 protein and gene can respond to low temperature, drought, salt, and hormones, further providing direction for studying the biological function of the alfalfa MsPL19 protein, thus enriching the molecular breeding pathways for alfalfa. Furthermore, this invention uses immunoprecipitation-mass spectrometry (IP-MS) to obtain proteins interacting with MsPL19, enriching the corresponding molecular modules and elucidating the molecular mechanism of alfalfa MsPL19's response to low-temperature environments. This provides a new breeding direction for selecting new cold-resistant varieties in high-altitude and cold regions, thereby increasing alfalfa yield. Attached Figure Description

[0032] Figure 1 yes MsPL19 Structure diagram of the PC-GW-Hyg-eGFP gene vector.

[0033] Figure 2 yes MsPL19 Identification of RNA overexpression levels of genes in alfalfa.

[0034] Figure 3 These are the results of pectinase content determination in OE7, OE-9, and OE-16.

[0035] Figure 4 yes MsPL19 The phenotypes of transgenic alfalfa include plant height (A), leaf shape (B), and root fresh weight (C).

[0036] Figure 5 yes MsPL19 Analysis of gene expression levels in different tissues of alfalfa.

[0037] Figure 6 yes MsPL19 Promoter analysis.

[0038] Figure 7 Under different treatment conditions MsPL19 Gene expression status.

[0039] Figure 8 It is overexpression MsPL19 Expression of genes related to hormone synthesis, signal transduction, and cell wall development in transgenic alfalfa materials.

[0040] Figure 9 This describes the phenotypic characteristics of wild-type alfalfa No. 3 under short-day and low-temperature treatment.

[0041] Figure 10Transcriptome analysis of wild-type Alfalfa No. 3 under short-day and low-temperature conditions PL Significant enrichment of genes.

[0042] Figure 11 These are the results of immunoprecipitation-mass spectrometry (IP-MS) analysis. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0044] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0045] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. Furthermore, depending on the experimental purpose, those skilled in the art complete the corresponding experiments under the guidance of the operating manuals accompanying various commercial kits or entrust them to specialized companies, such as gene sequencing, plasmid sequencing, and molecular weight determination.

[0046] Example 1: Alfalfa MsPL19 Cloning of genes

[0047] 1.1 Extraction of total RNA from alfalfa: mainly using... TransZol The Up method, with the following specific steps:

[0048] (1) Using alfalfa leaves as material, grind them rapidly into powder in liquid nitrogen, and then immediately put them into centrifuge tubes (the sample volume is about 1 / 5 of the centrifuge tube). Add 1 mL of alfalfa leaves to the centrifuge tubes. TransZol Add 200 μL of RNA Exaction Agent to the extract, vortex thoroughly for 2 min, and then centrifuge at 12000 rpm and 4 ℃ for 15 min.

[0049] (2) Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol and mix well. Incubate at room temperature for 10 min. Centrifuge at 12000 rpm and 4 ℃ for 10 min, discard the supernatant and retain the precipitate.

[0050] (3) Add 1 mL of pre-cooled 75% alcohol to the centrifuge tube, wash the precipitate, and then centrifuge at 12000 rpm and 4 ℃ for 10 min. Repeat the above steps twice.

[0051] (4) Pour off 75% of the alcohol, dry the remaining alcohol in a fume hood, then add 20 μL of DEPC water to dissolve it, measure the concentration of the extracted RNA, and place the RNA solution at -80 ℃ for later use.

[0052] 1.2 cDNA Synthesis

[0053] Using the Total Gold Reverse Transcription Kit, 1 μg of total RNA was added sequentially to 4 μL of 5×EasyScript. ® All-in-One One-Step SuperMix, 1 μL gDNA Remover Mix, and the remaining solution were brought to a total of 20 μL with sterile ddH2O. The mixture was then placed in a PCR instrument and reverse transcribed according to the following program: 42 ℃ for 15 min; 85 ℃ for 5 s. The reaction was stopped immediately on ice after the reaction was completed, and then stored at -20 ℃ for later use.

[0054] 1.3 MsPL19 Cloning of genes

[0055] Design specific primers and use the following primers for... MsPL19 Gene cloning:

[0056] Upstream primer: 5'-ATGGCTGCAATTGCAATGAAGA-3' (SEQ ID NO.3)

[0057] Downstream primer: 5'-GCATGGCCTACCCACAACACA-3' (SEQ ID NO.4)

[0058] Using the reverse-transcribed cDNA as a template, the total volume was 50 μL, consisting of 25 μL of 2×Phanta Max Master Mix (DyePlus); 2 μL each of forward and reverse primers; 2 μL of cDNA; and 19 μL of ddH2O. PCR amplification was performed according to the following program: 95 ℃, 5 min; 98 ℃, 15 s; 57 ℃, 15 s; 72 ℃, 1 min for 40 cycles; 72 ℃, 5 min; and termination at 16 ℃. The PCR products were detected by electrophoresis and recovered using a gel extraction kit. The product was then ligated into the pEASy-Blunt Zero vector and transformed into DH5α *E. coli*. Batch PCR was used to screen for banded colonies, which were sent to the company for sequencing. Positive colonies were preserved for later use, and plasmids were extracted from these colonies. The specific steps for gel extraction (using reagents from the Novizan FastPure Gel DNA purification and recovery kit) are as follows:

[0059] (1) Cut a single target gene band from the agarose gel and place it in a 2 mL centrifuge tube. Add an appropriate amount of GDP solution (if the weight of the recovered band is 0.1 g, add 100 μL of GDP solution). Then place it in a 65°C water bath until the gel block is completely dissolved.

[0060] (2) Add the dissolved solution to the adsorption column, let it stand at room temperature for 1 min, then centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back on the collection tube.

[0061] (3) Add 300 μL of GDP solution back into the adsorption column and centrifuge at 12000 rpm for 1 min.

[0062] (4) Add 700 μL of washing solution PW to the adsorption column (note whether anhydrous ethanol is added before use), then centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back on the collection tube.

[0063] (5) Repeat step (4).

[0064] (6) Place the adsorption column on the collection tube and centrifuge at 12,000 rpm for 2 min. Discard the waste liquid in the collection tube. Let the adsorption column stand at room temperature for a few minutes to remove residual rinsing liquid.

[0065] (7) Place the adsorption column on a new centrifuge tube, add 20-30 μL of elution buffer, let stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to collect the target gene solution. If a higher concentration of DNA solution is required, the collected solution can be added to the adsorption column, let stand at room temperature for 2 min, and then centrifuged under the same conditions for 2 min.

[0066] The specific steps for homologous recombination are as follows:

[0067] 0.5-4 μL of PCR amplification product

[0068] pEASy-Blunt Zero Cloning Vector 1 μL

[0069] Mix gently and react at 37 °C for 5 min. After the reaction is complete, place the centrifuge tube on ice.

[0070] The specific steps for E. coli transformation are as follows:

[0071] (1) Add the above ligation product to Escherichia coli competent cells DH5α (thaw the competent cells gently by hand, do not shake violently), mix evenly by lightly tapping with your fingers, and then incubate on ice for 30 min.

[0072] (2) Heat shock in a 42 ℃ water bath for 45 s, then immediately place in ice for 2 min.

[0073] (3) Add 300 μL of non-resistant LB solution to the transformed solution and incubate at 37 °C and 200 rpm for more than 1 h.

[0074] (4) Centrifuge the bacterial culture at 6000 rpm for 30 s, then retain 100 μL to resuspend the bacterial cells, spread them on a plate, and incubate overnight.

[0075] (5) Following a 15 μL system, i.e., 0.7 μL of forward and reverse primers; 1 μL of bacterial culture; 7.5 μL of enzyme; and 5.1 μL of ddH2O, batch PCR was performed using the following program: 95 ℃ for 5 min; 30 cycles: 98 ℃ for 15 s; 57 ℃ for 15 s; 72 ℃ for 1 min; and 72 ℃ for 5 min. Subsequently, bacterial cultures with bands were screened by agarose gel electrophoresis and sent to the company for sequencing.

[0076] (6) Store the correctly sequenced bacterial culture at -80 ℃ using 70% glycerol.

[0077] Plasmid extraction (the following reagents are from Tiangen Biotech Co., Ltd. plasmid mini-prep kit) The specific steps are as follows:

[0078] (1) Add 500 μL of BL equilibration solution to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back on the collection tube.

[0079] (2) Take 4-8 mL of overnight cultured Escherichia coli and centrifuge at 12000 rpm for 1 min to retain the bacterial cells and remove as much supernatant as possible. If there are many bacterial cells, centrifuge multiple times to ensure that more Escherichia coli are obtained.

[0080] (3) Add 500 μL of P1 solution to the centrifuge tube (check whether RNase A has been added before adding), and completely suspend the bacterial culture with a pipette.

[0081] (4) Add 500 μL of P2 solution to the centrifuge tube and gently invert it 6-8 times to fully lyse the cells.

[0082] (5) Add 700 μL of P3 solution to the centrifuge tube and immediately gently invert it 6-8 times to mix thoroughly. A white flocculent precipitate will appear at this time. Centrifuge at 12000 rpm for 15 min.

[0083] (6) Take the supernatant into the adsorption column, being careful not to take the white precipitate. Centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back onto the collection tube.

[0084] (7) Optional steps: Add 500 μL of protein removal solution PD to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back on the collection tube.

[0085] (8) Add 700 μL of washing solution PW to the adsorption column (note whether anhydrous ethanol is added before use), then centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back on the collection tube.

[0086] (9) Repeat step (8).

[0087] (10) Place the adsorption column on the collection tube and centrifuge at 12,000 rpm for 2 min. Discard the waste liquid in the collection tube. Let the adsorption column stand at room temperature for a few minutes to remove residual rinsing liquid.

[0088] (11) Place the adsorption column on a new centrifuge tube, add 30-50 μL of elution buffer, let stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to collect the plasmid solution. If a higher concentration of plasmid solution is required, the collected solution can be added to the adsorption column, let stand at room temperature for 2 min, and then centrifuged under the same conditions for 2 min.

[0089] The cloned gene is 1152 bp in length, as shown in SEQ ID NO.2, and the protein sequence encoded by this gene is shown in SEQ ID NO.1. This gene is named... MsPL19 .

[0090] Example 2 Construction of plant expression vectors

[0091] The vector (purchased from New England Biolabs) was digested using Xbal I single enzyme. The digestion system consisted of 40 μL of Xbal I, which included 1.5 μL of rapid digestion enzyme, 5 μL of 10× buffer, and 1000 ng of vector plasmid. The remaining volume was made up with ddH2O. The mixture was incubated at 37 °C for 3 h. After detection by agarose gel electrophoresis, the gel was excised and recovered using a gel recovery kit. The gel was stored at -20 °C for later use.

[0092] PCR was performed using the following forward and reverse primers, with the plasmids described above as templates: F: 5'-GGCTGCAGGAAAGACGGATGGCTGCAATTGCAATGAAGA-3' (SEQ ID NO.5); R: 5'-CTCGCCCTTGCTACCATGCATGGCCTACCCACAACACA-3' (SEQ ID NO.6). The total volume was 50 μL, consisting of 25 μL of 2×Phanta Max Master Mix (Dye Plus); 2 μL each of the forward and reverse primers; 2 μL of cDNA; and 19 μL of ddH2O. The amplification program was as follows: 95 ℃ for 5 min; 95 ℃ for 15 s; 60 ℃ for 15 s; 72 ℃ for 1 min for 40 cycles; 72 ℃ for 5 min; and 16 ℃ to terminate the reaction. The PCR products were detected by agarose gel electrophoresis, and the gel was excised and recovered using a gel extraction kit. The products were stored at -20 ℃ for later use.

[0093] The homologous recombination system is as follows:

[0094] PCR product: 200-300 ng; PC-GW-Hyg-eGFP vector (provided by the Institute of Botany, Chinese Academy of Sciences): 100-200 ng; homologous recombinase: 5 μL; reaction was carried out at 50 ℃ for 30 min in a PCR instrument. Subsequently, the homologous recombinant transformation product was transformed into E. coli according to the above-described E. coli transformation method, and sequencing was performed using the above-described gene primers.

[0095] Retain the bacterial culture with correct sequencing, extract plasmids, and transform Agrobacterium EHA105 competent cells. This expression vector ( MsPL19 The structure of the PC-GW-Hyg-eGFP gene vector is shown below. Figure 1 It can be directly used for the conversion of plants such as alfalfa, Arabidopsis thaliana, and tobacco.

[0096] The specific steps for Agrobacterium EHA105 transformation are as follows:

[0097] (1) After gently thawing Agrobacterium EHA105 competent cells by hand, add 10 μL of the target plasmid to them, gently tap them with your fingers to mix them thoroughly, and incubate on ice for 5 min.

[0098] (2) Quickly place the centrifuge tube in liquid nitrogen and freeze for 5 min.

[0099] (3) Quickly transfer to a 37 ℃ water bath and let stand for 5 min. After the reaction is complete, quickly place it in an ice bath and let stand for 5 min.

[0100] (4) Add 700 μL of non-resistant LB solution to it and incubate on a shaker at 200 rpm and 28 ℃ for 2-3 h to allow the cells to recover and express resistance.

[0101] (5) Centrifuge at 6000 rpm for 1 min to collect the bacteria, retain about 100 μL of supernatant, gently resuspend the bacteria by pipetting, and spread evenly on LB solid medium plates containing the corresponding antibiotics. After the liquid in the plate is completely absorbed, invert the plate and incubate at 28℃ for 2-3 days.

[0102] (6) Using its own primers and a negative control, the 15 μL system consisted of 0.7 μL of forward and reverse primers, 1 μL of bacterial culture, 7.5 μL of enzyme, and 5.1 μL of ddH2O. Batch PCR was performed using the following program: 95 ℃ for 5 min; 30 cycles: 98 ℃ for 15 s; 60 ℃ for 15 s; 72 ℃ for 1 min; 72 ℃ for 5 min. Subsequently, bacterial cultures showing bands were selected by agarose gel electrophoresis, indicating successfully transformed Agrobacterium EHA105, which can be used for subsequent genetic transformation.

[0103] Example 3: Alfalfa MsPL19 Genetic transformation

[0104] Agrobacterium-mediated transformation was used to transform alfalfa tissue culture seedlings. MsPL19 The genetic transformation process involves the following steps:

[0105] (1) Leaf disinfection: Select the third leaf from the top of the alfalfa 3 variety with good growth. After washing the leaf in water, soak it in 75% alcohol and shake and rinse for 5 seconds. Disinfect it with 10% NaClO solution for 15 minutes, and then rinse it with sterile water 5-8 times.

[0106] (2) Preparation of bacterial culture: Take an appropriate amount of Agrobacterium and add it to 5 mL of YEP liquid medium with the corresponding resistance and incubate overnight. Then, take 200 μL of the cultured Agrobacterium culture and add it to 50-60 mL of YEP liquid medium with the corresponding resistance, and shake until OD. 600 =0.6-0.8, centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend the bacteria in resuspending solution to OD. 600 =0.5-0.6, let stand for 2-3 hours before use.

[0107] (3) Explant preparation: Use a sterile scalpel to cut a wound in the leaf.

[0108] (4) Infection and co-culture: Cut the explant leaves into small pieces and put them into a 50 mL centrifuge tube. Add about 10 mL of resuspended bacterial solution (containing 100 μM AS) to the tube, sonicate for 30 s, then vacuum pump the air to 0.8 MPa for 10 min, let it stand for 15 min, aspirate the bacterial solution, and then place it on a co-culture medium in the dark for 2-3 days.

[0109] (5) Selection and culture: The co-cultured explants were inoculated onto selective medium (hygromycin 5 mg / L) and cultured for two weeks. The hygromycin concentration was increased to 10 mg / L, and cultured for another two weeks. The callus tissue was then placed under a photoperiod of 16 h light / 8 h darkness for 10 days to continue growth. Callus tissue was successfully induced. The green, sticky callus tissue was inoculated onto differentiation medium and subcultured every two weeks.

[0110] (6) Somatic cell culture: Embryoids are inoculated onto rooting medium for rooting culture.

[0111] (7) Culture medium components:

[0112] ① YEP medium: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar (pH=7) on solids. Add rifampicin 50 mg / L and kanamycin 100 mg / L when culturing Agrobacterium.

[0113] ② Co-culture medium: Modified N6 medium, 3.0 mg / L 2,4-D, 0.05 mg / L KT, 0.6 g / L MES, 150 μM AS (pH=5.4).

[0114] ③ Selected culture medium: Modified N6 medium, 2.0 mg / L 2,4-D, 0.05 mg / L KT, 0.6 g / L LMES, 200 mg / L termethin, 5 mg / L or 10 mg / L hygromycin (pH=5.8).

[0115] ④ Differentiation medium: Modified N6 medium, 0.4 mg / L KT, 0.6 g / L MES, 200 mg / L termethin, 5 mg / L hygromycin (pH=5.8).

[0116] ⑤ Rooting medium: MS medium (15 g / L sucrose), 100 mg / L inositol, 100 mg / L termethin, 1 mg / L hygromycin (pH=5.8).

[0117] Among them, 2,4-D, KT, and MES are 2,4-dichlorophenoxyacetic acid, kinetin, and 2-(N-morpholino)ethanesulfonic acid, respectively.

[0118] This embodiment provides a method for cloning alfalfa genes and an expression vector, which will pave the way for subsequent... MsPL19 The transformation of alfalfa into gene vectors laid the foundation for obtaining transgenic plants, which plays an important role in the breeding, production and promotion of new varieties.

[0119] Example 4: Overexpression in alfalfa MsPL19 Gene

[0120] According to Example 3, it was obtained in alfalfa MsPL19 Plants with overexpressed genes were identified based on RNA levels. Figure 2 Three strains, OE-7, OE-9, and OE-16, were selected, and their pectin lyase activity was significantly increased. Figure 3 ).

[0121] MsPL19 Phenotypic results for transgenic alfalfa are shown below. Figure 4 The results showed that overexpression MsPL19 After gene modification, its plant height and biomass per plant were significantly higher than those of the wild type. MsPL19The regenerated plants of the gene overexpression material reached a height of 52 cm, 1.79 times that of the control plant, Zhongmu 3; the fresh weight of a single plant was 27.63 g, 4.40 times that of the control. Furthermore, the leaf shape of the transgenic material differed significantly from the control, with a leaf area 0.55 times that of Zhongmu 3. Finally, root development analysis showed a significant increase in root biomass compared to the control, with a fresh weight 5.52 times that of the control. These data indicate that gene overexpression can significantly improve the biomass and fresh weight of alfalfa; overexpression or interference with gene expression can improve the forage quality of alfalfa.

[0122] Example 5 MsPL19 Gene functional analysis

[0123] right MsPL19 Tissue-specific expression analysis, results as follows Figure 5 As shown, it indicates MsPL19 The gene is expressed in alfalfa roots, root nodules, root caps, stems, and leaves, with significantly increased expression levels in root nodules, young leaves, and mature leaves.

[0124] Will MsPL19 Functional analysis of gene promoters yielded the following results: Figure 6 As shown, the results indicate that MsPL19 The promoter contains light-responsive elements, methyl jasmonic acid-related binding elements, auxin-responsive elements, abscisic acid-responsive elements, and drought stress-inducing elements.

[0125] Cold treatment (4 °C), drought (PEG) treatment (15% PEG-6000 treatment), salt treatment (10 mg / L), ABA treatment (50 mg / L), IAA treatment (20 mg / L), and GA treatment (50 mg / L) all induced [the condition]. MsPL19 The expression, the result is as follows Figure 7 As shown, this illustrates MsPL19 It plays an important role under these adverse stresses and is related to hormone-related pathways.

[0126] The expression of genes related to hormone synthesis and signal transduction, as well as cell wall development, was determined in transgenic lines OE-7, OE-9, and OE-16. IAA1 The primers are shown in SEQ ID NO.7 and SEQ ID NO.8. ARF The primers are shown in SEQ ID NO. 9 and SEQ ID NO. 10. GH3 The primers are shown in SEQ ID NO.11 and SEQ ID NO.12. KO The primers are shown in SEQ ID NO.13 and SEQ ID NO.14. DELLAThe primers are shown in SEQ ID NO.15 and SEQ ID NO.16. AHK2 The primers are shown in SEQ ID NO.17 and SEQ ID NO.18. BARR The primers are shown in SEQ ID NO.19 and SEQ ID NO.20. EXPANSIN The primers are shown in SEQ ID NO.21 and SEQ ID NO.22. XET The primers are shown in SEQ ID NO.23 and SEQ ID NO.24.

[0127] The results are as follows Figure 8 As shown, the results revealed the auxin inhibitor gene. IAA1 Auxin response factor ARF Auxin-binding gene amide synthase GH3 Gibberellin synthesis gene Kaurene oxidase ( KO , ent-kaurene pxidase (Gibberellin signaling pathway negatively regulated genes DELLA protein and cytokinin receptor) AHK2 Cytokinin B response factor BARR Cell wall expansin EXPANSIN and XET ( xyloglucan endotransglucosylase The expression level was significantly upregulated, indicating that MsPL19 Increased biomass yield in transgenic materials is regulated by hormone signaling pathway genes and cell wall development genes.

[0128] Example 6 MsPL Gene expression response to short-day and low-temperature treatment

[0129] The PL gene was analyzed under the following conditions: control (12 h light / 8 h dark, 22 °C), short-day treatment (8 h light / 12 h dark, 22 °C), and low-temperature treatment (12 h light / 8 h dark, 10 °C). The short-day and low-temperature phenotypes of wild-type alfalfa are shown below. Figure 9 As shown, under short-day and low-temperature treatments, the phenotypes of the treatment groups were weaker than those of the control.

[0130] Further analysis was conducted using transcriptome analysis. PL Gene expression under different temperatures and photoperiods, such as Figure 10 As shown, the results indicate that under the control experimental conditions (12 h light / 8 h darkness, 22 ℃) and the short-day treatment conditions (8 h light / 12 h darkness, 22 ℃), the six [unclear text - possibly related to experimental conditions or parameters]... PL Genes were significantly enriched and their expression levels were significantly upregulated; under low-temperature treatment (12 h light / 8 h dark, 10 °C), 8 genes were significantly enriched and their expression levels were significantly upregulated. PL The genes were significantly enriched, with 5 genes upregulated and 3 genes downregulated, indicating that... PL Gene expression is regulated by photoperiod and temperature.

[0131] Furthermore, this embodiment successfully constructed an alfalfa yeast cDNA library, and obtained 540 MsPL19 interacting proteins using immunoprecipitation-mass spectrometry (IP-MS) technology. Among them, low-temperature responsive proteins such as Aspartate aminotransferase, Peroxidase, Glutathione S-transferase, F-box / FBD-like domain protein, and E3 ubiquitin-protein ligase were significantly enriched. Figure 11 The above results further illustrate... MsPL19 It responds to low temperatures and plays an important role under low-temperature conditions.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of alfalfa MsPL19 protein, the gene encoding said protein, or biological material containing said gene in improving alfalfa plant height, fresh weight, or biomass yield; The amino acid sequence of the alfalfa MsPL19 protein is shown in SEQ ID NO.1; The biological material is an expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria containing a gene encoding the alfalfa MsPL19 protein.

2. Application of alfalfa MsPL19 protein, the gene encoding the protein, or biological material containing the gene in alfalfa variety improvement; The amino acid sequence of the alfalfa MsPL19 protein is shown in SEQ ID NO.1; The biological material is an expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria containing a gene encoding the alfalfa MsPL19 protein.

3. A method for increasing the plant height, fresh weight, or biomass yield of alfalfa, characterized in that, include: Overexpression of the alfalfa MsPL19 protein or the gene encoding the protein in alfalfa; The amino acid sequence of the alfalfa MsPL19 protein is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that, The overexpression mode is selected from one or more combinations of the following (1) to (5): (1) By introducing a plasmid containing the gene; (2) By increasing the copy number of the genes described on the chromosome; (3) By altering the promoter sequence of the genes described on the chromosome; (4) By operatively linking a strong promoter to the gene; (5) By introducing enhancers.

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