Medicago sativa MsSANT gene and application thereof in regulation and control of plant height and biological yield

Abstract

The invention discloses a medicago sativa MsSANT gene and application of the medicago sativa MsSANT gene in regulation and control of plant height and biological yield. It is found for the first time that overexpressed MsSANT gene can improve the plant height and the biological yield of medicago sativa, and the method has important guiding significance on cultivation of high-yield and high-quality medicago sativa varieties; meanwhile, the molecular mechanism of the MsSANT gene in regulating and controlling the alfalfa plant height is defined, a molecular module for regulating and controlling the alfalfa plant height and the biological yield is further analyzed, the corresponding molecular mechanism can be enriched, and then a new breeding direction is provided for breeding high-yield alfalfa varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, in particular, to Medicago sativa MsSANT gene and its application in regulating plant height and biomass. BACKGROUND

[0002] Medicago sativa Medicago Sativa L. is a high-quality perennial legume forage, which has high protein content, high digestibility, fast growth, strong environmental adaptability and other excellent characteristics. It plays an important role in agriculture in 80 countries and regions including China, and is often used for hay and silage production, which is a high-quality feed for livestock. In terms of ecology, Medicago sativa can convert atmospheric nitrogen into soil nitrogen, thereby improving soil fertility. Its root system penetrates deep into the soil, and in areas with serious wind and water erosion, it can reduce water and soil loss, improve soil desertification, and promote land greening and ecological restoration. In addition, planting Medicago sativa promotes the aggregation of beneficial insects in the farmland ecosystem, which helps to protect and improve the local ecological balance. At the same time, terminal foods based on Medicago sativa have high economic potential, and these products provide rich nutrients for hundreds of millions of consumers, so Medicago sativa is figuratively referred to as the "engine of food production".

[0003] The SANT domain is a conserved protein module widely present in eukaryotes, which is involved in chromatin remodeling and gene expression regulation. It can interact with the histone tail to regulate chromatin structure and play a role in DNA repair by promoting the binding of repair proteins through affecting chromatin state. The SANT domain has similarity with the Myb-type DNA binding domain, but usually has an acidic isoelectric point and a negative electrostatic surface potential, making it more likely to interact with the basic region of the histone tail or other proteins, rather than directly binding to DNA. It plays a key role in multiple chromatin remodeling complexes and serves as a bridge connecting histones and enzymes.

[0004] By comparing and analyzing the differential transcripts in overexpression plants and wild type, the transcriptional regulation molecular mechanism of biological processes can be revealed, thereby providing an important basis for in-depth understanding of related biological processes. At the same time, the SANT function of the Medicago sativa gene in regulating plant height is less studied, and its mechanism in regulating plant growth and development is unclear. SUMMARY

[0005] The purpose of the present application is to provide Medicago sativa MsSANT gene and its application in regulating plant height and biomass.

[0006] The present application selects wild type and overexpression MsSANT Medicago sativa, performs phenotype and transcriptome analysis, and excavates the MsSANTResponse pathways and genes related to plant height in order to reveal MsSANT Molecular mechanisms regulating plant height and biomass yield in alfalfa.

[0007] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides alfalfa MsSANT Genes encoding proteins such as: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; or (b) A protein derived from (a) with equivalent function, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO: 2.

[0008] Furthermore, the MsSANT gene is: i) the nucleotide sequence shown in SEQ ID NO: 1; ii) a nucleotide sequence in which one or more nucleotides are substituted, deleted, and / or added to the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence expresses a protein with the same function; iii) a nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO: 1 under stringent conditions, wherein the hybridization is carried out in 0.1× SSPE containing 0.1% SDS or in 0.1× SSC containing 0.1% SDS at 65°C, and the membrane is washed with the solution; or iv) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses a protein with the same function.

[0009] In a second aspect, the present invention provides a MsSANT Genetic biological materials include but are not limited to expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.

[0010] In a third aspect, the present invention provides MsSANT Gene or contains MsSANT Application of genetic biomaterials in regulating plant height and biomass yield.

[0011] Furthermore, the regulation is positive regulation, which increases the internode distance of the plant, thereby increasing its plant height.

[0012] The plant includes herbaceous plants of the genus Medicago of the family Leguminosae, preferably alfalfa.

[0013] In a fourth aspect, the present invention provides a method for increasing the internode distance, plant height and biomass yield of alfalfa, the method comprising: overexpressing MsSANT Gene.

[0014] Further, the overexpression mode can be selected from 1) to 5) below, or optional combinations: 1) by introducing a plasmid having the gene; 2) by increasing the copy number of the gene on the plant chromosome; 3) by changing the promoter sequence of the gene on the plant chromosome; 4) by operably linking a strong promoter (such as CaMV 35S promoter) to the gene; 5) by introducing an enhancer.

[0015] Gene MsSANT The PEG100-eGFP vector can be successfully connected, and the Flag tag on the vector does not affect the function, property of the target protein, does not interact with the target protein, only serves as a detection function, and the downstream of the fusion protein can be studied.

[0016] The CaMV 35S promoter can be efficiently expressed in the tissues of dicotyledonous plants, so that MsSANT The gene is overexpressed in the alfalfa plant, and its function is fully exerted.

[0017] In a fifth aspect, the present application provides the use of the above method in cultivating high-yield and high-quality alfalfa varieties.

[0018] In a sixth aspect, the present application provides the use of the transgenic alfalfa obtained according to the method in plant breeding.

[0019] Further, the breeding method includes but is not limited to transgenesis, crossing, backcrossing, selfing or vegetative reproduction.

[0020] Further, the expression vector carrying the target gene can be introduced into plant cells by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation and other conventional biological technology methods (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pages 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2 nd Edition).

[0021] By the above technical solution, the present application has at least the following advantages and beneficial effects: (1) The present application first clones a gene capable of promoting the increase of the internode distance and the plant height of alfalfa MsSANT , and the application of the gene can increase the internode distance of alfalfa, thereby increasing the plant height and the biological yield, and has important guiding significance for cultivating alfalfa high-yield varieties.

[0022] (II) The present application discloses MsSANT The molecular mechanism of the gene in regulating the plant height and biological yield of alfalfa, further analysis of the molecular module of regulating the plant height of alfalfa, can enrich the corresponding molecular mechanism, and then provide a new breeding direction for breeding alfalfa high-yield varieties. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 For the preferred embodiment of the present application MsSANT Gene vector structure diagram.

[0024] Figure 2 For the preferred embodiment of the present application MsSANT Gene expression in transgenic alfalfa MsSANT

[0025] Figure 3 For the preferred embodiment of the present application MsSANT Transgenic alfalfa phenotype

[0026] Figure 4 For the preferred embodiment of the present application MsSANT Transgenic alfalfa plant height analysis

[0027] Figure 5 For the preferred embodiment of the present application MsSANT Transgenic alfalfa internode distance analysis

[0028] Figure 6 For the preferred embodiment of the present application MsSANT Transgenic alfalfa biological yield analysis

[0029] Figure 7 For the preferred embodiment of the present application MsSANT Transgenic alfalfa mesophyll cell statistical analysis

[0030] Figure 8 For the preferred embodiment of the present application MsSANT Transgenic alfalfa stem cell statistical analysis

[0031] Figure 9 For the preferred embodiment of the present application MsSANT Gene expression amount analysis in different tissues of alfalfa

[0032] Figure 10 For the preferred embodiment of the present application MsSANT Transgenic alfalfa hormone synthesis and signal transduction, and cell wall development related gene expression amount analysis

[0033] Figure 11 For the preferred embodiment of the present application MsSANT Transgenic alfalfa transcriptome differential expression gene statistical analysis​

[0034] Figure 12 overexpressed in the preferred embodiments of the present application MsSANT Transgenic alfalfa transcriptome differential expression gene regulation pathway enrichment chart.

[0035] Figure 13 overexpressed in the preferred embodiments of the present application MsSANT Transgenic alfalfa hormone signal pathway gene expression analysis. DETAILED DESCRIPTION

[0036] The present application provides a new alfalfa gene MsSANT which can increase the internode distance and plant height of alfalfa, thereby regulating the biological yield of alfalfa.

[0037] The present application also provides a method for cloning and constructing an expression vector of an alfalfa MsSANT gene, further obtaining an overexpression transgenic line of alfalfa MsSANT , so as to study the function of the alfalfa MsSANT gene. Through transcriptome data analysis, the transcriptional regulation mechanism affecting the plant height of alfalfa is further understood. Through the application of the gene, it is beneficial to improve the yield of alfalfa.

[0038] The present application also provides the application of the alfalfa MsSANT gene in regulating the internode distance and plant height. Through the application of the gene, it is beneficial to improve the yield of alfalfa.

[0039] The present application adopts the following technical solutions: The present application provides an alfalfa MsSANT gene, the nucleotide sequence of the gene is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2. MsSANT The mRNA sequence of the gene has 1695 bases, and can encode a protein with 565 amino acids.

[0040] The present application also provides an expression vector, wherein the expression vector comprises the above-mentioned alfalfa MsSANT gene.

[0041] The application also provides a method for constructing the expression vector. First, total RNA of Medicago sativa is extracted, and then the total RNA is rapidly reverse transcribed into cDNA for standby. Specific primers are designed, and a full-length gene of 1695 bp is obtained by using a PCR instrument for amplification. After agarose gel electrophoresis, the PCR product is cut and recovered, the recovered product is connected with a pEASy-Blunt Zero cloning vector (Beijing Quanshijin Biotechnology Co., Ltd.), and after transformation, plasmid extraction is performed. The extracted plasmid is subjected to bacterial batch PCR verification, and the bacterial liquid with a band is sent to the company for sequencing. The bacterial liquid with correct sequencing is preserved to extract the plasmid. The PEG100-eGFP vector (Wuhan Boyuan Biotechnology Co., Ltd.) is double-enzyme cut by using restriction endonuclease Spe I and Noc I, the target plasmid is extracted after gel recovery, the plasmid is connected to the plant expression vector, and after transformation, PCR bacterial batch verification is performed. The bacterial liquid with a band is sent to the company for sequencing again, the bacterial liquid with correct sequencing is preserved, and the extracted plasmid is the recombinant vector containing the target gene.

[0042] The application provides transcriptome data analysis, which analyzes potential gene networks and key genes that regulate MsSANT Specific steps are as follows: 1. After extracting RNA, a library is constructed, and a Qubit 3.0 fluorescence quantifier is used for preliminary quantification (concentration > 1 ng / μL), then a Qsep400 high-throughput analysis system is used to detect the insert fragments of the library, after the insert fragments meet the expectation, a Q-PCR method is used to accurately quantify the effective concentration of the library (library effective concentration > 2 nM) to ensure the quality of the library.

[0043] 2. After the library quality inspection is qualified, an Illumina NovaSeq6000 sequencing platform is used for PE150 mode sequencing. The cDNA library is sequenced by using an Illumina high-throughput sequencing platform, and a large amount of high-quality data, called raw data, is output. The transcript splicing and merging are performed by using StringTie v2.2.1 software. After the raw sequencing amount, effective sequencing amount and the like are counted, comprehensive evaluation is performed, and subsequent high-quality analysis based on clean data is performed.

[0044] 3. The alfalfa genome website (https: / / modms.lzu.edu.cn / ) downloads the annotation file of the reference genome and gene model of 'Zhongmo 4'. The sequenced Clean Reads are compared with the reference genome sequence using HISAT2 v2.0.5 software. Gene function is annotated based on the following databases through sequence alignment: Nr (NCBI non-redundant protein sequences); Pfam (Protein family); KOG / COG (Clusters of Orthologous Groups of proteins); Swiss-Prot (A manually annotated and reviewed protein sequence database); KO (KEGG Ortholog database); GO (Gene Ontology).

[0045] The application provides MsSANT The gene and the application thereof can increase the plant height of alfalfa, lay a foundation for subsequent construction of a gene vector and further obtaining a high-yield transgenic plant, and the gene can also affect the biological yield of alfalfa.

[0046] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are conventional means familiar to those skilled in the art, and the raw materials used are commercially available goods.

[0047] The present application relates to molecular biology experiments, and if not specifically indicated, reference can be made to the book Molecular Cloning (J. Sambrook, E.F. Fritsch, T. Maniatis, Science Press, 1994). In addition, according to different experimental purposes, those skilled in the art can complete the corresponding experiments under the guidance of the operation manual attached to various commercial kits (Kit) or entrust a professional company, such as gene sequencing, plasmid sequencing and determination of molecular weight.

[0048] Experimental materials: The alfalfa 'Zhongmo 3' is used as the background material.

[0049] Example 1 Alfalfa MsSANT Gene cloning 1.1 The total RNA of alfalfa is extracted by the TransZol Up (Beijing Zoonking Biotech Co., Ltd.) method, and the specific operation process is as follows: (1) Take the alfalfa leaf as the experimental sample, and quickly place it in a liquid nitrogen environment for grinding until the sample is in powder form. After grinding is complete, immediately transfer the powder to a centrifuge tube. Then, add 1 mL of TransZol Up extraction solution and 200 μL of RNA extraction solution to the centrifuge tube, mix thoroughly for 2 minutes, and fully dissociate the sample. Then, centrifuge at 4°C and 12000 rpm for 15 minutes.

[0050] (2) Precipitation acquisition: After centrifugation is complete, carefully transfer the supernatant to a new centrifuge tube. Then, add an equal volume of isopropanol to the supernatant, mix gently, and incubate at room temperature for 10 minutes. Then, centrifuge again at 4°C and 12000 rpm for 10 minutes, and discard the supernatant after centrifugation, leaving the precipitate at the bottom of the tube.

[0051] (3) Precipitate washing: Inject 1 mL of pre-cooled 75% alcohol into the centrifuge tube containing the precipitate to wash the precipitate. After washing, centrifuge at 4°C and 12000 rpm for 10 minutes. This washing step needs to be repeated 2 times to ensure the purity of the precipitate.

[0052] (4) RNA dissolution and preservation: After washing is complete, pour out the 75% alcohol in the centrifuge tube and blow dry the remaining alcohol in a fume hood. Then, add 20 μL of DEPC water to the centrifuge tube to fully dissolve the precipitate. After dissolution, determine the concentration of the extracted RNA. Finally, store the RNA solution at -80°C for subsequent use.

[0053] 1.2 Synthesis of cDNA The synthesis of cDNA was performed using the All-in-One One-Step SuperMix kit. First, measure 1 μg of total RNA, and then add 4 μL of 5×EasyScript® All-in-One One-Step SuperMix and 1 μL of gDNA removal agent mixture to it. Then, use sterile ddH2O to make up the reaction system to 20 μL. After completing the system preparation, place the reaction tube in the PCR instrument, and perform the reverse transcription reaction according to the preset program, which is: first react at 42°C for 15 minutes, then raise the temperature to 85°C and hold for 5 seconds. After the reverse transcription reaction is completed, immediately place the reaction tube on ice to terminate the reaction. Finally, store the treated cDNA product at -20°C for subsequent experimental use.

[0054] 1.3 MsSANT Gene cloning Specific primers were designed, and the following primers were used MsSANT Cloning of the gene: Upstream primer: 5'-ATGGATGTGAAAAATATCCTTGGAGG-3' Downstream primer: 5'-CTAACCACAATCAGAACTGCCAGG-3' A PCR reaction system with a total volume of 50 μL was constructed using the cDNA obtained by reverse transcription as the template. The system specifically contained: 25 μL of 2x Phanta Max Master Mix; 2 μL of each of the upstream and downstream primers; 2 μL of the cDNA template; and 19 μL of ddH2O. The PCR reaction was carried out according to the following amplification program: first, pre-denaturation at 95 ℃ for 5 minutes; then, the cycling stage, denaturation at 98 ℃ for 15 seconds, annealing at 57 ℃ for 15 seconds, extension at 72 ℃ for 1 minute, and so on for 40 cycles; after the cycling stage, extension at 72 ℃ for 5 minutes; finally, the temperature was reduced to 16 ℃ to terminate the reaction. After the completion of the PCR amplification, the obtained product was subjected to electrophoresis detection, and the target band was recovered using a gel recovery kit (the reagents below are from the Novizen FastPure Gel DNA Purification Recovery Kit). The specific steps of gel recovery are as follows: (1) The single target gene band was cut from the agarose gel and placed in a 2 mL centrifuge tube, and an appropriate amount of DNA binding liquid solution was added (if the weight of the recovered band is 0.1 g, 100 μL of DNA binding liquid is added) to release the DNA. It was placed in a 65 ℃ metal bath, and every 3 min, it was inverted once until the agarose gel in the centrifuge tube completely melted.

[0055] (2) The above melted gel liquid was transferred to the adsorption column with a pipette gun, and centrifuged at a speed of 12000 rpm for 1 min at room temperature.

[0056] (3) The filtrate in the collection column was discarded, 300 μL of DNA binding liquid was added to the adsorption column, and centrifuged at a speed of 12000 rpm for 1 min at room temperature.

[0057] (4) The filtrate in the collection column was discarded, 700 μL of rinse liquid was added to the adsorption column, and centrifuged at a speed of 12000 rpm for 1 min at room temperature, so as to wash away the impurities and purify the DNA sample.

[0058] (5) Discard the filtrate in the collection column, and add 700 μL of the rinse solution to the adsorption column again. Centrifuge at 12000 rpm for 1 min to further remove impurities and purify the DNA sample.

[0059] (6) Discard the filtrate in the collection column, and place the adsorption column and the collection tube in the centrifuge at 12000 rpm for 2 min.

[0060] (7) Replace the adsorption column in a new 1.5 ml centrifuge tube, and place it at room temperature to dry for 5-10 min. After the alcohol evaporates completely, add 30-40 μl of the elution solution or ddH2O.

[0061] (8) Place it at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min to collect the target DNA in the centrifuge tube.

[0062] Subsequently, the recovered product is connected with a pEASy-Blunt Zero (Beijing Quanshi Gold Biotechnology Co., Ltd.) vector, and transformed into DH5α E. coli competent cells. The bacterial liquid containing the target band is screened by colony PCR, and these bacterial liquids are sent to a professional company for sequencing analysis. For the colonies with positive sequencing results, preservation is performed for subsequent use, and plasmids are extracted using these bacterial liquids.

[0063] The specific steps of the homologous recombination operation are as follows: PCR amplification product 0.5-4 μL pEASy-Blunt Zero cloning vector 1 μL Gently mix, and react at 37 ℃ for 5 min. After the reaction is completed, place the centrifuge tube on ice.

[0064] The specific steps of the E. coli transformation operation are as follows: (1) Take the E. coli strain DH5α frozen in the -80 ℃ ultra-low temperature refrigerator, and place it on ice to thaw, so as to activate the strain. Add the above connection product to the E. coli competent DH5α, and cool on ice for 30 min.

[0065] (2) After heat shocking the competent cells in a metal bath at 42 ℃ for 85 s, place the competent cells on ice to cool for 3 min.

[0066] (3) Super-clean bench operation, 500 μL of the above-mentioned LB liquid medium was added to the competent cells, and then placed on a 37 ℃ shaker for 1-2 h. After centrifugation at a speed of 5000 rpm for 1 min, the supernatant was discarded, and the cells were mixed and evenly spread on LB solid culture dishes with the addition of the corresponding antibiotics.

[0067] (4) The solid culture dishes were inverted and cultured overnight in a 37 ℃ incubator. 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; and 5.1 μL of ddH2O. Bacterial batch PCR was performed using the following program: 94 ℃, 3 min; 94 ℃, 30 s; 63 ℃, 30 s; 72 ℃, 1 min, 30 cycles; and 72 ℃, 5 min. Subsequently, the bacterial solution with bands was screened by agarose gel electrophoresis and sent to the company for sequencing.

[0068] (6) The bacterial solution with correct sequencing was stored at -80 ℃ using 60% glycerol.

[0069] Plasmid extraction (the following reagents are from the plasmid extraction kit of Tiangen Biochemical Co., Ltd.) The specific steps are as follows: 1) 500 μL of BL balancing solution was added to the adsorption column, and centrifuged at 12000 rpm for 1 min. The waste liquid in the collection tube was discarded, and the adsorption column was placed again in the collection tube.

[0070] 2) 4-8 ml of overnight cultured bacterial solution was taken in a centrifuge tube, and centrifuged at a speed of 12000 rpm for 1 min. The supernatant was discarded, and if the bacterial mass was large, it could be centrifuged multiple times.

[0071] 3) 500 μL of solution P1 with RNase was added to the centrifuge tube to suspend the precipitated bacterial mass.

[0072] 4) 500 μL of solution P2 was added to the centrifuge tube, and gently inverted 6-8 times.

[0073] 5) 350 μL of solution P3 was added and immediately mixed thoroughly, and gently inverted 6-8 times until a white flocculent precipitate appeared. The supernatant was aspirated using a pipette gun and placed in an adsorption column, which was centrifuged at 12000 rpm for 1 min to remove the waste liquid.

[0074] 6) 700 μL of rinse solution PW was added to the adsorption column, which was centrifuged at a speed of 12000 rpm for 1 min at room temperature, and the waste liquid was discarded. This step was repeated once.

[0075] 7) The adsorption column and the collection tube are placed in the centrifuge at room temperature for 2 min to remove the residual rinse solution in the adsorption column.

[0076] 8) The adsorption column is changed to a new 1.5 ml centrifuge tube, and is placed at room temperature for 5-10 min to dry, and after the alcohol is completely volatilized, 30-40 μL of the eluent is added. It is placed at room temperature for 2 min, and is centrifuged at a speed of 12000 rpm for 2 min, and the plasmid DNA is collected in the centrifuge tube and is stored at -20℃.

[0077] The full length of the gene is 1695 bp (SEQ ID NO: 1), and the amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO: 2. The gene is named MsSANT .

[0078] Example 2 MsSANT Construction of a plant expression vector The Bar single enzyme digestion vector is used, the enzyme digestion system is 50 μL, the enzyme digestion system is 40 μL, that is, fast-cut enzyme 1.5 μL; 10×buffer 5 μL; vector plasmid 1000 ng, the remaining volume is supplemented with ddH2O, and is placed in a 37℃ water bath for 3 h. After detection by agarose gel electrophoresis, the gel is cut and recovered using a gel recovery kit, and is stored at -20℃ for later use.

[0079] The following forward and reverse primers are used for PCR reaction, and the above plasmid is used as a template (forward primer: 5'-ACTACCTGAGCACCCAGTCC-3'; reverse primer: 5'-CTTGGCAAAAGAATAGTCCCCTGT-3'), and the total system is 50 μL, that is, 2×Phanta Max Master Mix 25 μL; 2 μL of each of the forward and reverse primers; cDNA 2 μL; ddH2O 19 μL, and the amplification program is: 95℃, 5 min; 95℃, 15 s; 59℃, 15 s; 72℃, 1 min, 40 cycles; 72℃, 5 min; 16℃, termination reaction, and PCR reaction is performed. After detection by agarose gel electrophoresis, the gel is cut and recovered using a gel recovery kit, and is stored at -20℃ for later use.

[0080] The homologous recombination is operated as follows: the solution (PCR product, 3.5 μL; homologous recombination enzyme, 5 μL; vector, 1.5 μL) is reacted at 50℃ in PCR for 30 min. Subsequently, the homologous recombination product is transformed into E. coli according to the above E. coli transformation method, and sequencing is performed using a universal primer of the vector.

[0081] The bacterial liquid with correct sequencing is reserved, and the plasmid is extracted to transform the Agrobacterium EHA105 competent cells. The expression vector can be directly used for transformation of plants such as alfalfa, Arabidopsis, tobacco, etc. MsSANT The gene vector structure is as follows Figure 1 The expression vector can be directly used for transformation of plants such as alfalfa, Arabidopsis, tobacco, etc.

[0082] The bacterial liquid with correct sequencing is reserved, and the plasmid is extracted to transform the Agrobacterium EHA105 competent cells. The expression vector can be directly used for transformation of plants such as alfalfa, Arabidopsis, tobacco, etc.

[0083] The specific steps of Agrobacterium EHA105 transformation are as follows: The GV3101 Agrobacterium competent cells are taken out from the low-temperature frozen environment at -80°C, and are placed on the ice surface to be naturally thawed. When the cells are thawed to a part of liquid state, the expression vector plasmid with completed recombination is added, and then the bottom of the test tube is shaken gently. Subsequently, the Agrobacterium competent cells containing the mixture are placed on ice and stand for 5 minutes. Then, they are quickly transferred to liquid nitrogen for 5 minutes of treatment. After the treatment is completed, they are immediately placed in a water bath environment at 37°C for 5 minutes of heat shock treatment, and then are placed back on the ice for 5 minutes of cooling.

[0084] After the above steps are completed, 700 μL of LB medium without antibiotics is added to the test tube containing the competent cells, and then the test tube is placed on a shaker for 3 hours of culture at 28°C and 200 rpm to promote cell recovery. After the recovery is completed, the cells are precipitated by centrifugation at a speed of 5000 rpm for 5 minutes. Most of the supernatant is discarded, and only 200 μL of supernatant is reserved, and the Agrobacterium competent cells are resuspended with the 200 μL of supernatant.

[0085] Then, the resuspended cells are uniformly coated on the agar plate containing kanamycin (concentration of 50 mg / L) and rifampicin (concentration of 50 mg / L) using a pipette, and the plate surface is waited until there is no obvious liquid residue. Then, the plate is sealed with Parafilm M sealing film and placed in a 28°C incubator for 2 days of continuous culture.

[0086] After the colonies grow on the plate, 5 single colonies are selected from each plate for gene transformation, and PCR positive identification is performed using the expression vector homologous recombination primers. The PCR reaction system and reaction program refer to the method of positive identification of E. coli in Example 3. The clones identified as positive are expanded into bacterial liquid and properly stored for subsequent test use.

[0087] Example 3 MsSANT Genetic transformation of Medicago sativa L. The tissue culture seedlings of Medicago sativa L. were transformed by Agrobacterium transformation method, and the specific steps were as follows: MsSANT (1) Leaf disinfection: select the third leaf from top to bottom of well-grown Zhongmou No. 3, wash the leaf in water, then soak in 75% alcohol for 5 s, disinfect with 10% NaClO solution for 15 min, and then wash with sterile water for 5-8 times. (2) Preparation of bacterial solution: take appropriate amount of Agrobacterium and add it into 5 mL of corresponding resistant YEP liquid medium for overnight culture, then take 200 μL of the cultured Agrobacterium solution and add it into 50-60 mL of corresponding resistant YEP liquid medium, shake to OD 600 =0.6-0.8, centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend the bacterial body to OD 600 =0.5-0.6 using resuspension solution, and stand for 2-3 h.

[0088] (3) Preparation of explants, use a sterile scalpel to cut the leaf to make wounds.

[0089] (4) Infection and co-culture: cut the explant leaf into small pieces and put them into a 50 mL centrifuge tube, add about 10 mL of resuspended bacterial solution (containing 100 μM acetyl-syringone) into the tube, ultrasonic for 30 s, then vacuum pump to 0.8 MPa for 10 min, stand for 15 min, dry the bacterial solution, and then place it on the co-culture medium in the dark for 2-3 d.

[0090] (5) Selection culture: inoculate the co-cultured explants on the selection medium (hygromycin 5 mg / L) for two weeks. Increase the concentration of hygromycin to 10 mg / L and culture for two weeks. Then place the callus in a light cycle of 16 h light / 8 h dark for continuous growth for 10 d. Successfully induce callus. Inoculate the green and sticky callus on the differentiation medium, and subculture every two weeks.

[0091] (6) Somatic culture: inoculate the embryoid on the rooting medium for rooting culture.

[0092] (7) Medium composition:

[0093] ① YEP medium: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar (pH=7) for solidification. When culturing Agrobacterium, add rifampicin 50 mg / L and kanamycin 100 mg / L.

[0094] ​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 acetosyringone (pH=5.4).

[0095] ③ Selection medium: modified N6 medium, 2.0 mg / L 2,4-D, 0.05 mg / L KT, 0.6 g / L MES, 200 mg / L timentin, 5 mg / L or 10 mg / L hygromycin (pH=5.8).

[0096] 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).

[0097] ⑤ Rooting medium: MS medium (15 g / L sucrose), 100 mg / L myo-inositol, 100 mg / L timentin, 1 mg / L hygromycin (pH=5.8).

[0098] Wherein, 2,4-D, KT, MES are 2,4-dichlorophenoxyacetic acid, kinetin and 2-(N-morpholino)-ethanesulfonic acid, respectively.

[0099] The present embodiment provides a cloning method of alfalfa gene and an expression vector, which lays a foundation for subsequent transformation of alfalfa by a gene vector and obtaining a transgenic plant, and plays an important role in new variety breeding and production promotion. MsSANT

[0100] Example 4 Overexpression of MsSANT alfalfa transgenic material According to Example 3, three strains of alfalfa transgenic material were obtained MsSANT According to the RNA level identification Figure 2 ), three strains (phenotype see Figure 3 ) were screened out OEMsSANT-4 , OEMsSANT-7 , OEMsSANT-10 After being cultured for one month under indoor potting conditions, MsSANT the plant height of the gene overexpression material was 42, 52 and 46 cm, respectively, which was 1.43, 1.70 and 1.20 times that of the control Zhongmu 3 (control) (see Figure 4 ); and the internode distance was also significantly higher than that of the wild type, and the internode distance of the three strains was 13, 15 and 12 cm, respectively, which was 1.21, 1.35 and 1.15 times that of the control Zhongmu 3 (control) (see Figure 5 ​); the biological yield of the overexpression plants was also significantly higher than that of the wild type, and the biological yield of the three lines was 4.38, 4.46, and 4.05 g, which was 2.01, 2.21, and 2.45 times that of Zhongmian No. 3 (control) Figure 6 , A and B). In addition, the stem and leaf cells of the transgenic material were significantly different from the control, and the leaf cell area was 0.74 times that of Zhongmian No. 3 (control) Figure 7 , A and B); the stem cell area was 1.32 times that of Zhongmian No. 3 (control) Figure 8 , A and B), indicating that gene editing MsSANT and gene overexpression can obtain materials with increased plant height and internode distance.

[0101] Example 5 Medicago sativa MsSANT Gene function analysis and molecular module analysis The tissue-specific expression analysis of MsSANT is shown in Figure 9 , indicating that MsSANT the gene is expressed in the roots, stems, leaves, and flowers of Medicago sativa, and the expression amount in the roots and flowers is significantly increased.

[0102] The expression of genes related to hormone synthesis and signal transduction, and cell wall development was determined in the transgenic lines OEMsSANT-4 , OEMsSANT-7 , OEMsSANT-10 , which were used to amplify IAA1, ARF, GH3, KO, DELLA, AHK2, BARR, XTR. The primers for XTR are as follows: IAA1 : Forward primer: 5'-AGAGTTCTTCTGCTGCCCCT-3' Reverse primer: 5'-GGCACCATCCATGCTCACT-3' ARF : Forward primer: 5'-CTCCTCCAGCAAACTCCCAG-3' Reverse primer: 5'-CCATGTTGAGGGTCGCGATA-3' GH3 : Forward primer: 5'-CAGAACACACGACCCTTGGA-3' Reverse primer: 5'-GGCGCCTAGCCTTAGAACTT-3' KO : Forward primer: 5'-GCAATTGAGGTGGATTGGCG-3' Reverse primer: 5'-CCCTGAAGCTAACCGCTTCT-3' DELLA : Upstream primer: 5'-TGGGATCTATGGCTAGTGCTTC-3' Downstream primer: 5'-GAAGTCCACTGTCTTGTTCCTG-3' AHK2 : Upstream primer: 5'-ATTTTGCGAGCAAGGGCATC-3' Downstream primer: 5'-TGTTCAGGCGTAGCATCTGG-3' BARR : Upstream primer: 5'-CCCTGTCAGAAGTGGTTCCTC-3' Downstream primer: 5'-ACAGCAACGATGTCTGTCGAA-3' XTR: Upstream primer: 5'-TCTTGGTTGACAACATCCCAA-3' Downstream primer: 5'-ATCATCAGCGTTCCAGAGAC-3' The results are shown in Table 1. Figure 10 It was found that the expression level of auxin inhibitor protein gene was significantly up-regulated, and the expression levels of auxin response factor IAA1 , ARF , Auxin Response Factor , GH3 , Gretchen Hagen 3 , KO , ent-kaurene pxidase , AHK2 , Arabidopsis Histidine Kinase 2 , XTR , Xyloglucan Endotransglucosylase / Hydrolase , BARR , Type-B Arabidopsis Response Regulators , MsSANT The expression levels of DELLA protein, a negative regulator of gibberellin signaling pathway, a cytokinin receptor ,

[0103] , MsSANT , , SANT , Figure 11 The expression of genes in the transgenic material and wild-type genes was analyzed by transcriptome analysis, and the statistical analysis of differentially expressed genes is shown in Table 1.The number of differentially expressed genes was 7057, of which 3037 were up-regulated and 4020 were down-regulated. Through the functional enrichment of KEGG ( Figure 12 ), the significantly up-regulated genes were enriched in photosynthesis-antenna proteins, starch and sucrose metabolism, carotenoid biosynthesis, etc. In the photosynthesis-antenna protein pathway, the up-regulated genes may be related to chlorophyll-binding proteins in chloroplasts, enhancing the efficiency of light energy capture and conversion, and promoting photosynthesis; the up-regulated genes in the starch and sucrose metabolism pathway may regulate the synthesis and decomposition of carbohydrates, affecting the energy metabolism and growth and development of plants; the up-regulation of genes in the carotenoid biosynthesis pathway may change the composition and content of carotenoids, affecting the light protection mechanism and color of plants.

[0104] The significantly down-regulated genes were enriched in ribosomes, ubiquinone and other terpene-quinone biosynthesis, amino acid biosynthesis, caffeine metabolism, etc. The down-regulated genes in the ribosome pathway may slow down the rate of protein synthesis; the down-regulation in the ubiquinone and other terpene-quinone biosynthesis pathway may affect the redox state and energy metabolism of cells; the down-regulation in the amino acid biosynthesis pathway may limit the raw materials for protein synthesis; the down-regulation in the caffeine metabolism pathway may change the composition of secondary metabolites in plants. The enrichment of these differentially expressed genes reveals that in transgenic plants, the phenotype and quality of overexpression lines may be affected by regulating photosynthesis, metabolic processes, and circadian rhythms, etc.

[0105] Through correlation analysis, a co-expression network was constructed, and some key differentially expressed genes ( Figure 13 ) were found: auxin response protein (Myb-like DNA-binding domain, Msa0224840), gibberellin-regulated protein (Msa1006930), AUX / IAA family protein (Msa0841820), auxin response factor (Msa1236630), membrane transport protein (Msa1383860), transcriptional coactivator p15 (Msa1362670), etc. The genes in this module are related to plant hormone signal transduction and cell elongation, etc., and by regulating the changes of hormones in plants, the MsSANT overexpression of alfalfa is higher. The above analysis shows MsSANT may regulate these related pathways, thereby affecting the plant height and biomass of alfalfa.

[0106] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.

Claims

1. Alfalfa MsSANT A gene characterized by It is a gene that encodes the following protein: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; or (b) A protein derived from (a) with equivalent function, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO:

2.

2. The biological material containing the gene according to claim 1, characterized in that: The biological material is an expression cassette, a transposon, a plasmid vector, a virus vector or an engineered bacterium.

3. Use of the gene according to claim 1 or the biological material according to claim 2 in regulating plant height and biomass yield.

4. The use according to claim 3, characterized in that The regulation is positive regulation, which increases the distance between plant nodes, thereby increasing the plant height.

5. The use according to claim 3 or 4, characterized in that The plant includes herbaceous plants of the genus Medicago of the family Leguminosae, preferably alfalfa.

6. A method for increasing internode distance, plant height and biomass yield of alfalfa, characterized in that: The method comprises: using genetic engineering means to overexpress the gene of claim 1 in alfalfa MsSANT Gene.

7. The method according to claim 6, characterized in that The overexpression method is selected from the following 1) to 5), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the copy number of the gene on the plant chromosome; 3) by changing the promoter sequence of the gene on the plant chromosome; 4) by operably linking a strong promoter to the gene; 5) by introducing enhancers; Preferably, the promoter is the CaMV35S promoter.

8. Use of the method according to claim 6 or 7 in breeding high-yield and high-quality alfalfa varieties.

9. Use of the transgenic alfalfa obtained according to the method of claim 6 or 7 in plant breeding.

10. The use according to claim 9, characterized in that Breeding methods include transgenics, hybridization, backcrossing, selfing or asexual reproduction.

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