Application of medicago sativa MsPL19 protein and gene thereof in regulating and controlling plant height, fresh weight or biological yield of medicago sativa

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to an application of an alfalfa MsPL19 protein and a gene thereof in regulating and controlling the plant height, fresh weight or biological yield of alfalfa. The invention provides an MsPL19 protein, a gene and application thereof, which can obviously improve the plant height, the biological yield and the fresh weight of medicago sativa and further improve the quality of the medicago sativa. Meanwhile, it is found that the MsPL19 protein and gene can respond to low temperature, drought, salt, hormone and other pathways, the protein interacting with MsPL19 is obtained through immunoprecipitation-mass spectrometry analysis, corresponding molecular modules can be enriched, the molecular mechanism of alfalfa MsPL19 responding to the low-temperature environment is analyzed, new cold-resistant varieties can be bred for alpine regions, and the application prospect is broad. And a new breeding direction is provided for increasing the yield of alfalfa.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to application of alfalfa MsPL19 protein and a gene thereof in regulating alfalfa plant height, fresh weight or biological yield. Background Art

[0002] Alfalfa ( Medicago sativa L.) is a perennial, mowing-tolerant leguminous forage grass with the advantages of high grass yield and rich nutritional value. It is known as the "king of forage grass". Alfalfa is a long-day crop with typical fall dormancy characteristics. Its biological yield indicators such as regeneration rate, mowing times and grass yield are directly related to the fall dormancy of alfalfa. In addition, studies have shown that the mowing regeneration and wintering rate of alfalfa are significantly correlated. Alfalfa varieties with good spring regeneration performance turn green early, which can not only extend the vegetative growth period and increase alfalfa grass yield, but also inhibit the growth of weeds and effectively resist weed damage. The mowing regeneration of alfalfa has an important influence on its wintering ability, biological yield and alfalfa quality. The discovery of genes that regulate the mowing regeneration and biological yield of alfalfa in purple alfalfa, and the subsequent study of the regulatory network of this gene, has important application value and guiding significance for the creation of new alfalfa germplasm with stress resistance and high yield, and the cultivation of new alfalfa varieties.

[0003] Pectate lyase (PL) is a type of enzyme that catalyzes the degradation of α-1,4-glycosidic bonds located in the smooth region of pectin through β-trans elimination, producing oligosaccharides containing unsaturated galacturonic acid residues at the non-reducing end, thereby cleaving pectin molecules. PL is related to various functions of plants and plays an important role in plant growth and development, fruit ripening and softening, and plant-pathogenic microorganism interactions.

[0004] Low temperature significantly affects the growth and development ability, productivity and geographical distribution of alfalfa, especially in high-altitude cold areas such as Hulunbuir. The sudden temperature changes such as frost and late spring cold that often occur in late autumn and early spring seriously affect the mowing, regeneration and grass yield of alfalfa.

[0005] Currently, alfalfa PL The function of genes is still less studied, so it is necessary to study PL The function of the gene and the regulatory mechanism of the molecular module of the gene are of great significance to improving alfalfa yield, enhancing its cutting regeneration ability and wintering ability. Summary of the invention

[0006] The present invention discovered a new alfalfa gene MsPL19, which can significantly increase the biological yield and fresh weight of alfalfa, thereby improving the quality of alfalfa. In addition, it can respond to low temperature and light environment, and is regulated by genes such as 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 an alfalfa MsPL19 protein, the amino acid sequence of which is shown in (1) or (2): (1) As shown in SEQ ID NO.1; (2) An amino acid sequence derived from (1) with equivalent function, wherein one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention provides a gene encoding the alfalfa MsPL19 protein.

[0009] Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0010] In a third aspect, the present invention provides a biological material containing the gene, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium, or a non-renewable plant cell or tissue.

[0011] In a fourth aspect, the present invention provides alfalfa MsPL19 protein, alfalfa MsPL19 Application of the gene or the biological material in regulating plant height, fresh weight or biological yield of alfalfa.

[0012] Preferably, the plant height, fresh weight or biological yield of alfalfa is increased by increasing the expression level or activity of the alfalfa MsPL19 protein or gene.

[0013] In a fifth aspect, the present invention provides alfalfa MsPL19 protein, alfalfa MsPL19 Application of the gene or the biological material in alfalfa variety improvement.

[0014] In the specific implementation process, it includes but is not limited to variety improvement through genetic modification, hybridization, backcrossing, self-pollination or asexual reproduction.

[0015] In a sixth aspect, the present invention provides alfalfa MsPL19 protein, alfalfa MsPL19 The application of the gene or the biological material in alfalfa's resistance to adverse stress; preferably, the adverse stress includes drought stress, low temperature stress or salt stress.

[0016] Preferably, the alfalfa MsPL19 protein, MsPL19The gene or the biological material regulates the plant height, fresh weight or biological yield of alfalfa by regulating hormone signal pathway genes and cell wall development genes.

[0017] Preferably, the hormone signal comprises IAA, GA or CK.

[0018] Preferably, the expression level of hormone signaling pathway genes and cell wall development genes is increased by increasing the expression amount or activity of alfalfa MsPL19 protein or gene.

[0019] Preferably, the activity or expression level of the alfalfa MsPL19 protein or gene is regulated by photoperiod or temperature.

[0020] In a seventh aspect, the present invention provides a method for increasing plant height, fresh weight or biological yield of alfalfa, comprising: overexpressing the alfalfa MsPL19 protein or the gene in alfalfa; Preferably, the overexpression method 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 gene on the chromosome; (3) by changing the promoter sequence of the gene on the chromosome; (4) by operably linking a strong promoter (such as CaMV35S promoter) to the gene; (5) By introducing enhancers.

[0021] The CaMV35S promoter can be efficiently expressed in dicotyledonous plant tissues, making MsPL19 Overexpressed in alfalfa plants to fully exert its function.

[0022] The beneficial effects of the present invention are: The present invention provides MsPL19 protein, gene and application thereof, which can significantly improve the plant height, biological yield and fresh weight of alfalfa, thereby improving the quality of alfalfa. At the same time, the present invention finds that MsPL19 protein and gene can respond to low temperature, drought, salt, hormone and other pathways. The present invention further provides a direction for studying the biological function of alfalfa MsPL19 protein, thereby enriching the molecular breeding approach of alfalfa. In addition, the present invention obtains proteins that interact with MsPL19 through immunoprecipitation-mass spectrometry (IP-MS) technology, which can enrich the corresponding molecular modules and analyze the molecular mechanism of alfalfa MsPL19 responding to low temperature environment, which can provide a new breeding direction for breeding new cold-resistant varieties in high-cold areas and thus improve alfalfa yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 yes MsPL19 Gene PC-GW-Hyg-eGFP vector structure diagram.

[0024] Figure 2 yes MsPL19 Identification of genes overexpressed at the RNA level in alfalfa.

[0025] Figure 3 These are the results of determination of pectinase content in OE7, OE-9, and OE-16.

[0026] Figure 4 yes MsPL19 Transgenic alfalfa phenotypes, including plant height (A), leaf shape (B), and root fresh weight (C).

[0027] Figure 5 yes MsPL19 Analysis of gene expression in different tissues of alfalfa.

[0028] Figure 6 yes MsPL19 Promoter analysis.

[0029] Figure 7 Under different treatment conditions MsPL19 Gene expression status.

[0030] Figure 8 Overexpression MsPL19 The expression of genes related to hormone synthesis, signal transduction and cell wall development in transgenic alfalfa materials.

[0031] Fig. 9 This is the phenotype of wild-type alfalfa Zhongmu 3 under short-day and low-temperature treatments.

[0032] Fig.10 The transcriptome analysis showed that the wild type Zhongmu 3 alfalfa had a low temperature and short-day light condition. PL Significantly enriched genes.

[0033] Fig.11 is the result of immunoprecipitation-mass spectrometry (IP-MS) analysis. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0036] The present invention relates to molecular biology experiments. If not otherwise specified, reference can be made to the book Molecular Cloning (J. Sambrook, EF Fritsch, T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used reference books for technicians in the field of molecular biology when conducting experimental operations related to molecular biology. In addition, according to different experimental purposes, technicians in the field of molecular biology complete corresponding experiments under the guidance of the operating manuals attached to various commercial kits or entrust specialized companies to conduct experiments, such as gene sequencing, plasmid sequencing, and molecular weight determination.

[0037] Example 1 Alfalfa MsPL19 Gene cloning 1.1 Extraction of total RNA from alfalfa: TransZol Up method, the specific steps are as follows: (1) Use alfalfa leaves as the material, quickly grind them into powder in liquid nitrogen, and then immediately put them into a centrifuge tube (the sample volume is about 1 / 5 of the centrifuge tube). Add 1 mL TransZol Up extract, add 200 μL of RNA Exaction Agent, shake thoroughly for 2 min, and then centrifuge at 12000 rpm and 4 °C for 15 min.

[0038] (2) Pipette the supernatant into a new centrifuge tube, add an equal volume of isopropanol and mix well, incubate at room temperature for 10 min. Centrifuge at 12,000 rpm, 4 °C for 10 min, discard the supernatant, and retain the precipitate.

[0039] (3) Add 1 mL of pre-cooled 75% alcohol to the centrifuge tube to wash the precipitate, then centrifuge at 12,000 rpm and 4°C for 10 min. Repeat the above steps twice.

[0040] (4) Pour off 75% of the alcohol and dry the remaining alcohol in a fume hood. Then add 20 μL of DEPC water to dissolve the RNA. Measure the concentration of the extracted RNA and place the RNA solution at -80 °C for later use.

[0041] 1.2 cDNA synthesis Using the full-strand gold reverse transcription kit, 1 μg of total RNA was added to 4 μL of 5×EasyScript ®All-in-One One-Step SuperMix, 1 μL gDNA Remover Mix, the remaining solution was made up with sterile ddH2O, a total of 20 μL, and then placed in a PCR instrument, according to the following program: 42 ℃, 15 min; 85 ℃, 5 s for reverse transcription, immediately placed on ice after the reaction is completed to terminate the reaction, and then stored at -20 ℃ for use.

[0042] 1.3 MsPL19 Gene cloning Design specific primers and use the following primers for MsPL19 Gene cloning: Upstream primer: 5'-ATGGCTGCAATTGCAATGAAGA-3' (SEQ ID NO. 3) Downstream primer: 5'-GCATGGCCTACCCACAACACA-3' (SEQ ID NO. 4) The above reverse transcribed cDNA was used as a template, and the total system was 50 μL, namely 2×Phanta Max Master Mix (DyePlus) 25 μL; 2 μL of each forward and reverse primer; 2 μL of cDNA; 19 μL of ddH2O. PCR amplification was performed according to the following amplification program. The amplification program was: 95 ℃, 5 min; 98 ℃, 15 s; 57 ℃, 15 s; 72 ℃, 1 min for 40 cycles of amplification; 72 ℃, 5 min; 16 ℃ to terminate the reaction. The PCR product was detected by electrophoresis and recovered using a gel recovery kit, and then the pEASy-Blunt Zero vector was connected and transformed into DH5α Escherichia coli. The bacterial solution with bands was screened by bacterial batch PCR and sent to the company for sequencing. The positive colonies were saved for standby use, and the plasmid was extracted using the bacterial solution. The specific steps of gel recovery (the following reagents are from the Novozymes FastPure Gel DNA Purification and Recovery Kit) are as follows: (1) Cut the 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 gel recovery 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.

[0043] (2) Add the dissolved solution to the adsorption column, let it stand at room temperature for 1 min, then centrifuge it at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column on the collection tube again.

[0044] (3) Add 300 μL of GDP solution to the adsorption column and centrifuge at 12,000 rpm for 1 min.

[0045] (4) Add 700 μL of rinse solution PW to the adsorption column (pay attention to whether anhydrous ethanol is added before use), then centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column on the collection tube again.

[0046] (5) Repeat step (4).

[0047] (6) Place the adsorption column on the collection tube and continue centrifugation at 12,000 rpm for 2 min. Discard the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to remove the residual rinse solution.

[0048] (7) Place the adsorption column in a new centrifuge tube, add 20-30 μL of elution buffer, let stand at room temperature for 2 min, and then centrifuge at 12,000 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 centrifuge for 2 min under the same conditions.

[0049] The specific steps of homologous recombination operation are as follows: PCR amplification product 0.5-4 μL pEASy-Blunt Zero Cloning Vector 1 μL Mix gently and react at 37°C for 5 min. After the reaction is complete, place the centrifuge tube on ice.

[0050] The specific steps of E. coli transformation are as follows: (1) Add the above ligation product to the competent E. coli DH5α (thaw the competent cell gently by hand, do not shake it violently), flick it with your fingers to mix evenly, and place it on ice for 30 min.

[0051] (2) Heat shock in a 42°C water bath for 45 seconds and immediately place on ice for 2 minutes.

[0052] (3) Add 300 μL of LB solution without resistance to the transformed solution and incubate at 37 °C in a shaker at 200 rpm for more than 1 h.

[0053] (4) Centrifuge the bacterial solution at 6000 rpm for 30 s, then retain 100 μL of the resuspended bacteria, spread it on a plate, and culture overnight.

[0054] (5) According to the 15 μL system, i.e. 0.7 μL of forward and reverse primers; 1 μL of bacterial solution; 7.5 μL of enzyme; 5.1 μL of ddH2O. Batch PCR was performed using the following procedure, i.e. 95 ℃, 5 min; 30 cycles: 98 ℃, 15 s; 57 ℃, 15 s; 72 ℃, 1 min; 72 ℃, 5 min. The bacterial solution with bands was then screened by agarose gel electrophoresis and sent to the company for sequencing.

[0055] (6) The correctly sequenced bacterial solution was stored in 70% glycerol at -80°C.

[0056] Plasmid extraction (the following reagents are from the plasmid extraction kit of Tiangen Biochemical Co., Ltd.) The specific steps are as follows: (1) Add 500 μL of BL equilibrium solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column on the collection tube again.

[0057] (2) Take 4-8 mL of overnight cultured E. coli and centrifuge at 12,000 rpm for 1 min. Keep the cells and remove as much supernatant as possible. If there are many cells, centrifuge several times to ensure that more E. coli are obtained.

[0058] (3) Add 500 μL of P1 solution to the centrifuge tube (check whether RNase A has been added before adding) and use a pipette to thoroughly suspend the bacterial solution.

[0059] (4) Add 500 μL of P2 solution to the centrifuge tube and gently invert it upside down 6-8 times to fully lyse the bacteria.

[0060] (5) Add 700 μL of P3 solution to the centrifuge tube and gently invert it 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 15 min.

[0061] (6) Pipette the supernatant into the above adsorption column, being careful not to absorb the white precipitate. Centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column on the collection tube again.

[0062] (7) Optional step: Add 500 μL of deproteinized solution PD to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column on the collection tube again.

[0063] (8) Add 700 μL of rinse solution PW to the adsorption column (pay attention to whether anhydrous ethanol is added before use), then centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column on the collection tube again.

[0064] (9) Repeat step (8).

[0065] (10) Place the adsorption column on the collection tube and continue centrifugation at 12,000 rpm for 2 min. Discard the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to remove the residual rinse solution.

[0066] (11) Place the adsorption column in a new centrifuge tube, add 30-50 μL of elution buffer, let stand at room temperature for 2 min, and then centrifuge at 12,000 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 centrifuge for 2 min under the same conditions.

[0067] The full length of the cloned gene is 1152 bp, as shown in SEQ ID NO.2, and the protein sequence encoded by the gene is shown in SEQ ID NO.1. The gene was named MsPL19 .

[0068] Example 2 Construction of plant expression vector The vector was cut with Xbal I (purchased from New England Biolabs) and the enzyme cutting system was 40 μL, i.e. 1.5 μL of fast cutting enzyme, 5 μL of 10× buffer, and 1000 ng of vector plasmid. The remaining volume was filled with ddH2O and the mixture was incubated in a 37°C water bath for 3 h. After agarose gel electrophoresis, the gel was cut and recovered using a gel recovery kit and stored at -20°C for later use.

[0069] PCR reaction was carried out with the following forward and reverse primers, and the above plasmid was used as template, F: 5'-GGCTGCAGGAAAGACGGATGGCTGCAATTGCAATGAAGA-3' (SEQ ID NO.5); R: 5'-CTCGCCCTTGCTACCATGCATGGCCTACCCACAACACA-3' (SEQ ID NO.6), according to the total system of 50 μL, i.e. 2×Phanta Max Master Mix (Dye Plus) 25 μL; 2 μL of forward and reverse primers respectively; 2 μL of cDNA; 19 μL of ddH2O, the amplification program was: 95 ℃, 5 min; 95 ℃, 15 s; 60 ℃, 15 s; 72 ℃, 1 min for 40 cycles of amplification; 72 ℃, 5 min; 16 ℃ to terminate the reaction, and PCR reaction was carried out. The PCR product was detected by agarose gel electrophoresis, and then the gel was cut and recovered using a gel recovery kit and stored at -20 ℃ for use.

[0070] The homologous recombination system is as follows, PCR product: 200-300 ng; PC-GW-Hyg-eGFP vector (provided by the Institute of Botany, Chinese Academy of Sciences): 100-200 ng; homologous recombination enzyme: 5 μL; react in a PCR instrument at 50 °C for 30 min. Then, the homologous recombination transformation product was transformed into Escherichia coli according to the above Escherichia coli transformation method, and sequenced with the above gene self primers.

[0071] The bacterial solution with correct sequencing was retained, and the plasmid was extracted and transformed into Agrobacterium EHA105 competent cells. MsPL19 The gene PC-GW-Hyg-eGFP vector structure is shown in Figure 1 ) can be directly used for the transformation of plants such as alfalfa, Arabidopsis, and tobacco.

[0072] The specific steps of Agrobacterium EHA105 transformation are as follows: (1) Gently thaw the competent cells of Agrobacterium tumefaciens EHA105 by hand, add 10 μL of the target plasmid, flick the cells gently to mix thoroughly, and place on ice for 5 min.

[0073] (2) Quickly place the centrifuge tube in liquid nitrogen and freeze it for 5 min.

[0074] (3) Quickly transfer to a 37 ℃ water bath and let stand for 5 min. After the reaction is complete, quickly place in an ice bath and let stand for 5 min.

[0075] (4) Add 700 μL of LB solution without resistance and incubate the culture on a shaker at 200 rpm and 28 °C for 2-3 h to allow the bacteria to revive and express resistance.

[0076] (5) Collect the bacteria by centrifugation at 6000 rpm for 1 min. Keep about 100 μL of supernatant and gently blow to resuspend the bacteria. Spread the bacteria evenly on an LB solid culture medium plate containing the corresponding antibiotics. After the liquid in the plate is completely absorbed, invert the plate and culture at 28°C for 2-3 days.

[0077] (6) Use self-primers and add negative control, according to the 15 μL system, that is, 0.7 μL of forward and reverse primers; 1 μL of bacterial solution; 7.5 μL of enzyme; 5.1 μL of ddH2O. Use the following program to perform batch PCR, that is, 95 ℃, 5 min; 30 cycles: 98 ℃, 15 s; 60 ℃, 15 s; 72 ℃, 1 min; 72 ℃, 5 min. Then, screen the bacterial solution with bands by agarose gel electrophoresis, which is the successfully transformed Agrobacterium EHA105 and can be used for subsequent genetic transformation.

[0078] Example 3 Alfalfa MsPL19 Genetic transformation Transformation of alfalfa seedlings using Agrobacterium tumefaciens MsPL19 The specific steps for genetic transformation are as follows: (1) Leaf disinfection: Select the third leaf of Zhongmu No. 3 with good growth condition from the top, wash the leaf in water, soak it in 75% alcohol and rinse it with shaking for 5 seconds, disinfect it with 10% NaClO solution for 15 minutes, and then rinse it with sterile water for 5-8 times.

[0079] (2) Preparation of bacterial solution: Take an appropriate amount of Agrobacterium and add it to 5 mL of YEP liquid medium with corresponding resistance for overnight culture. Then take 200 μL of the cultured Agrobacterium solution and add it to 50-60 mL of YEP liquid medium with corresponding resistance. Shake until OD 600 =0.6-0.8, centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend the cells in resuspension buffer to OD 600 =0.5-0.6, let stand for 2-3 hours before use.

[0080] (3) Explant preparation: Use a sterilized scalpel to cut a wound on the leaf.

[0081] (4) Infection and co-cultivation: Cut the explant leaves into small pieces and place them in a 50 mL centrifuge tube. Add about 10 mL of resuspended bacterial solution (containing 100 μM AS) and sonicate for 30 s. Then, use a vacuum pump to evacuate the air to 0.8 MPa for 10 min and leave it for 15 min. After the bacterial solution is dried, place it on the co-culture medium and culture it in the dark for 2-3 d.

[0082] (5) Screening culture: The co-cultured explants were inoculated on a selection medium (hygromycin 5 mg / L) and cultured for two weeks. The hygromycin concentration was increased to 10 mg / L and cultured for two weeks. The callus was then placed under a photoperiod of 16 h light / 8 h dark and continued to grow for 10 days. Callus was successfully induced. The green, sticky callus was inoculated on a differentiation medium and subcultured every two weeks.

[0083] (6) Somatic cell culture: The embryoids are inoculated onto rooting medium for rooting culture.

[0084] (7) Culture medium composition: ① YEP medium: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, solid plus 15 g / L agar (pH=7). When culturing Agrobacterium, add 50 mg / L rifampicin and 100 mg / L kanamycin.

[0085] 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).

[0086] ③ Select culture medium: modified N6 medium, 2.0 mg / L 2,4-D, 0.05 mg / L KT, 0.6 g / LMES, 200 mg / L timentin, 5 mg / L or 10 mg / L hygromycin (pH=5.8).

[0087] ④ Differentiation medium: modified N6 medium, 0.4 mg / L KT, 0.6 g / L MES, 200 mg / L timentin, 5 mg / L hygromycin (pH = 5.8).

[0088] ⑤ Rooting medium: MS medium (15 g / L sucrose), 100 mg / L inositol, 100 mg / L timentin, 1 mg / L hygromycin (pH=5.8).

[0089] Among them, 2,4-D, KT and MES are 2,4-dichlorophenoxyacetic acid, kinetin and 2-(N-morpholine)ethanesulfonic acid respectively.

[0090] This example provides a cloning method and expression vector of alfalfa gene, which is for subsequent MsPL19 The use of gene vectors to transform Medicago truncatula has laid the foundation for obtaining transgenic plants, and plays an important role in the breeding, production and promotion of new varieties.

[0091] Example 4 Overexpression in Alfalfa MsPL19 Gene According to Example 3, alfalfa was obtained. MsPL19 Gene overexpression plants 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 ).

[0092] MsPL19 The phenotype of transgenic alfalfa is shown in Figure 4 The results showed that overexpression MsPL19 After the gene was added, the plant height and single plant biomass yield were significantly higher than those of the wild type. MsPL19 The height of the regenerated plant of the gene overexpression material is 52 cm, which is 1.79 times that of the control Zhongmu 3 plant; the fresh weight of the single plant biological yield is 27.63 grams, which is 4.40 times that of the control. In addition, the leaf shape of the transgenic material is significantly different from that of the control, and the leaf area is 0.55 times that of Zhongmu 3; finally, the root development is analyzed. Compared with the control, the root biological yield of the transgenic material is significantly increased, and the fresh weight is 5.52 times that of the control. The above data show that the overexpression of this gene can significantly increase the biological yield and fresh weight of alfalfa; overexpression or interference with the expression of this gene can improve the forage quality of alfalfa.

[0093] Example 5 MsPL19 Functional analysis of genes right MsPL19 The tissue-specific expression analysis of Figure 5 Shown, indicating MsPL19 The gene was expressed in roots, nodules, root caps, stems and leaves of alfalfa, with the expression level significantly increased in nodules, young leaves and mature leaves.

[0094] Will MsPL19 The promoter of the gene was functionally analyzed. Figure 6 As shown, the results show that MsPL19 The promoter contains light-responsive elements, methyl jasmonate-related binding elements, auxin-responsive elements, abscisic acid-responsive elements, and drought-stress-induced elements.

[0095] The induced growth of β-actin was observed under cold treatment (4 ℃), 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). MsPL19 The expression of Figure 7 As shown, the description MsPL19 It plays an important role under these adverse stresses and is associated with hormone-related pathways.

[0096] The expression of genes related to hormone synthesis and signal transduction, as well as cell wall development, was measured 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, DELLA The 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 as SEQ ID NO.23 and SEQ ID NO.24.

[0097] The results are as follows Figure 8 As shown in the results, it was found that the somatostatin gene IAA1 , auxin response factor ARF , auxin binding gene amide synthase GH3 、Gibberellin biosynthesis gene Kaurene oxidase ( KO , ent-kaurene pxidase )、Gibberellin signaling pathway negative regulatory gene DELLA protein, cytokinin receptor AHK2 , cytokinin type B response factor BARR , cell wall expansins EXPANSIN and XET ( xyloglucan endotransglucosylase ) expression level was significantly upregulated, indicating MsPL19 The increase in biomass yield of transgenic materials is regulated by hormone signaling pathway genes and cell wall development genes.

[0098] Example 6 MsPL Gene expression in response to short-day and low-temperature treatments The PL gene was analyzed under the control experimental conditions (12 h light / 8 h dark, temperature 22 ℃), short-day treatment conditions (8 h light / 12 h dark, temperature 22 ℃), and low temperature treatment conditions (12 h light / 8 h dark, temperature 10 ℃). Fig. 9As shown. Under short-day and low-temperature treatments, the phenotypes of the treatment groups were weaker than those of the control.

[0099] Transcriptome analysis was further performed to analyze PL The expression of genes under different temperatures and photoperiods, such as Fig.10 The results showed that under the control experimental conditions (12 h light / 8 h dark, temperature 22 ℃) and the short-day treatment conditions (8 h light / 12 h dark, temperature 22 ℃), the six PL The genes were significantly enriched and their expression levels were significantly upregulated; under low temperature treatment (12 h light / 8 h dark, temperature 10 ℃), 8 PL The genes were significantly enriched, among which 5 genes were up-regulated and 3 genes were down-regulated, indicating PL Gene expression is regulated by photoperiod and temperature.

[0100] In addition, this example successfully constructed a cDNA library of alfalfa yeast, and obtained 540 MsPL19-interacting proteins through immunoprecipitation-mass spectrometry (IP-MS) technology, among which Aspartate aminotransferase, Peroxidase, Glutathione S-transferase, F-box / FBD-like domain protein, E3 ubiquitin-proteinligase and other low-temperature responsive proteins were significantly enriched ( Fig.11 The above results further illustrate that MsPL19 Responds to low temperatures and plays an important role under low temperature conditions.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Alfalfa MsPL19 protein, characterized in that Its amino acid sequence is shown as follows (1) or (2): (1) As shown in SEQ ID NO.1; (2) An amino acid sequence derived from (1) with equivalent function, wherein one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO.

1.

2. A gene encoding the alfalfa MsPL19 protein according to claim 1.

3. The gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

2.

4. A biological material containing the gene according to claim 2 or 3, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or non-renewable plant cells or tissues.

5. Use of the protein according to claim 1, the gene according to claim 2 or 3, or the biological material according to claim 4 in regulating plant height, fresh weight or biological yield of alfalfa.

6. Use of the protein according to claim 1, the gene according to claim 2 or 3, or the biological material according to claim 4 in improving alfalfa varieties.

7. Use of the protein according to claim 1, the gene according to claim 2 or 3, or the biological material according to claim 4 in alfalfa in resisting adverse stress; preferably, the adverse stress includes drought stress, low temperature stress or salt stress.

8. The use according to any one of claims 5 to 7, characterized in that: The protein, the gene or the biological material regulates the plant height, fresh weight or biological yield of alfalfa by regulating hormone signal pathway genes and cell wall development genes.

9. The use according to any one of claims 5 to 7, characterized in that: The activity or expression level of the alfalfa MsPL19 protein or gene is regulated by photoperiod or temperature.

10. A method for increasing plant height, fresh weight or biomass yield of alfalfa, characterized in that: include: Overexpressing the alfalfa MsPL19 protein according to claim 1 or the gene according to claim 2 or 3 in alfalfa; Preferably, the overexpression method 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 gene on the chromosome; (3) by changing the promoter sequence of the gene on the chromosome; (4) by operably linking a strong promoter to the gene; (5) By introducing enhancers.

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