Medicago sativa MsPPR1 gene and application thereof in regulation and control of plant photosynthetic carbon metabolism and biological yield

Abstract

The invention discloses a medicago sativa MsPPR1 gene and application of the medicago sativa MsPPR1 gene in regulation and control of photosynthetic carbon metabolism and biological yield of plants. It is found for the first time that the MsPPR1 gene can improve the chlorophyll content, enhance the photosynthetic efficiency and optimize energy metabolism, then the biological yield of medicago sativa is improved, and the MsPPR1 gene has important guiding significance on cultivation of medicago sativa high-yield and high-quality varieties; by analyzing the molecular mechanism of the MsPPR1 gene for regulating and controlling the chloroplast function and energy metabolism, a new target gene and theoretical support are provided for the genetic improvement of forage grass, and a new breeding direction is provided for cultivating a new variety of high-yield and high-quality medicago sativa.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to alfalfa MsPPR1 Genes and their applications in regulating plant photosynthetic carbon metabolism and biomass production. Background Art

[0002] Alfalfa ( Medicago sativa Alfalfa (Medicago sativa L.) is a perennial, high-quality forage grass in the genus Medicago in the Leguminosae family. It is known as the "King of Forage Grasses" for its high protein content and excellent palatability. As one of the world's most important forage crops, the efficient cultivation of alfalfa is of great significance to the sustainable development of animal husbandry. Alfalfa is not only rich in protein but also contains a variety of essential amino acids, vitamins, and minerals. Its high-quality production is crucial to the sustainable development of modern animal husbandry and the dairy industry. However, in actual production processes, insufficient photosynthetic carbon assimilation efficiency leads to unstable biomass yields, severely limiting the full realization of alfalfa's production potential. Therefore, in-depth analysis of the regulatory mechanisms of alfalfa's photosynthetic characteristics is of great scientific significance and application value for the breeding of new high-yield varieties.

[0003] PPR (Pentatricopeptide repeat) proteins are a family of RNA-binding proteins that are widely present in plants. They are characterized by containing 2-30 tandemly repeated 35-amino acid sequence units. These proteins are mainly located in mitochondria and chloroplasts, and recognize target RNA molecules through sequence specificity, participating in post-transcriptional processing such as RNA editing, splicing, stability and translation regulation. Studies have shown that PPR proteins play a key role in the establishment of plant photosynthetic systems, chloroplast development and energy metabolism regulation. For example, PPR proteins in Arabidopsis thaliana regulate rbcL The stability of gene transcripts affects the synthesis of Rubisco enzyme; maize PPR10 protein can protect atpH mRNA from degradation and activate its translation process.

[0004] By comparing and analyzing the differential transcripts in samples before and after treatment, we can accurately reveal MsPPR1 Although some progress has been made in the functional research of PPR proteins, the current research on the molecular mechanism of photosynthetic carbon metabolism in alfalfa is still unclear. PPR There is still a lack of research on how genes affect biomass production by regulating photosynthetic carbon assimilation pathways. The lack of this research area has seriously limited PPR The potential of genes in breeding high-yield alfalfa. Summary of the Invention

[0005] The present invention aims to provide alfalfa MsPPR1 Genes and their applications in regulating plant photosynthetic carbon metabolism and biomass production.

[0006] The application selects wild type and overexpression MsPPR1 Medicago sativa L. genes, and performs phenotype and transcriptome analysis under different environmental conditions, excavates the related response pathways and genes related to photosynthetic characteristics, growth and development and biological yield changes of overexpression MsPPR1 Strains, so as to reveal MsPPR1 The molecular mechanism of regulating photosynthetic efficiency and biological yield of Medicago sativa L..

[0007] In order to achieve the purpose of the application, in the first aspect, the application provides a Medicago sativa L. MsPPR1 Gene, which is a gene encoding 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) by substitution, deletion or addition of one or more amino acids in the sequence shown in SEQ ID NO: 2 and having equivalent function.

[0008] Further, the MsPPR1 gene is: i) the nucleotide sequence shown in SEQ ID NO: 1; ii) a nucleotide sequence shown in SEQ ID NO: 1 by substitution, deletion and / or addition of one or more nucleotides and expressing the same functional protein; iii) a nucleotide sequence hybridizing to the sequence shown in SEQ ID NO: 1 under stringent conditions, which are hybridization in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS at 65°C, and washing the membrane with the solution; or iv) a nucleotide sequence having more than 90% homology with the nucleotide sequence of i), ii) or iii) and expressing the same functional protein.

[0009] In the second aspect, the application provides a biological material containing MsPPR1 The gene, which includes but is not limited to an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

[0010] In the third aspect, the application provides MsPPR1 The gene or the biological material containing MsPPR1 The gene in regulating plant photosynthetic carbon metabolism, growth and development and biological yield.

[0011] Further, the regulation is positive regulation, which improves the chlorophyll content, photosynthetic carbon metabolism (photosynthetic efficiency) and biological yield of the plant.

[0012] The plant includes legume Medicago sativa L. herb, preferably Medicago sativa L..

[0013] In a fourth aspect, the present invention provides a method for improving photosynthetic carbon metabolism of alfalfa, the method comprising: using genetic engineering means to overexpress MsPPR1 Gene.

[0014] In a fifth aspect, the present invention provides a method for cultivating high-yield alfalfa varieties or improving the quality of alfalfa forage, the method comprising: using genetic engineering means to overexpress MsPPR1 Gene.

[0015] Furthermore, the overexpression method can be 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 (such as the CaMV35S promoter) to the gene; 5) By introducing enhancers.

[0016] Gene MsPPR1 It can be successfully connected to the PEG100-eGFP vector, and the Flag tag on the vector will not affect the function and properties of the target protein, will not interact with the target protein, and is only used as a detection function to study the downstream of the fusion protein.

[0017] The CaMV35S promoter can be expressed efficiently in dicotyledonous plant tissues, MsPPR1 Overexpression in alfalfa plants allows it to fully function.

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

[0019] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, selfing, or asexual reproduction.

[0020] Furthermore, expression vectors carrying target genes can be introduced into plant cells by conventional biotechnology methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 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: (I) The present application first clones a gene capable of improving the photosynthetic carbon metabolism efficiency and biological yield of alfalfa MsPPR1 The gene can improve biological yield by regulating the expression of key pathway genes of photosynthetic carbon metabolism, and has important guiding significance for cultivating high-yield and high-quality alfalfa varieties.

[0022] (II) The present application clarifies MsPPR1 The molecular mechanism of the gene for improving photosynthetic carbon assimilation efficiency by regulating chloroplast RNA metabolism, further analyzes the molecular module for regulating the photosynthetic efficiency and biological yield of alfalfa, can enrich the corresponding molecular mechanism, and further provides a new molecular module and breeding direction for breeding high-yield and high-quality alfalfa varieties, and has important application value for promoting the sustainable development of modern animal husbandry. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 For the gene vector structure in the preferred embodiment of the present application MsPPR1 .

[0024] Figure 2 For the overexpression MsPPR1 strain in the preferred embodiment of the present application MsPPR1 Relative expression amount of gene.

[0025] Figure 3 For the phenotype of transgenic alfalfa in the preferred embodiment of the present application MsPPR1 .

[0026] Figure 4 For the overexpression MsPPR1 Strain height measurement in the preferred embodiment of the present application. Among them, Indicates P <0.05, Indicates P <0.01, Indicates P <0.001.

[0027] Figure 5 For the overexpression MsPPR1 Strain internode number measurement in the preferred embodiment of the present application. Among them, Indicates P <0.05, Indicates P <0.01, Indicates P <0.001.

[0028] Figure 6 For the overexpression of the gene in the preferred embodiments of the present application MsPPR1 Strain single plant fresh weight analysis. Among them, <0.05, P <0.01, <0.001. <0.01, P <0.001. <0.05, <0.01, <0.001. P <0.05, <0.01,

[0029] <0.001. Figure 7 For the overexpression of the gene in the preferred embodiments of the present application MsPPR1 Strain stem cell analysis. Among them, <0.05, P <0.01, <0.001. <0.01, P <0.001. <0.05, <0.01, <0.001. P <0.05, <0.01,

[0030] <0.001. Figure 8 For the overexpression of the gene in the preferred embodiments of the present application MsPPR1 Strain leaf cell analysis. Among them, <0.05, P <0.01, <0.001. <0.01, P <0.001. <0.05, <0.01, <0.001. P <0.05, <0.01,

[0031] <0.001. Figure 9 For the overexpression of the gene in the preferred embodiments of the present application MsPPR1 Strain chlorophyll content analysis. Among them, <0.05, P <0.01, <0.001. <0.01, P <0.001. <0.05, <0.01, <0.001. P <0.05, <0.01,

[0032] <0.001. Figure 10 For the overexpression of the gene in the preferred embodiments of the present application MsPPR1 Gene expression analysis in different tissues of Medicago sativa. <0.05, <0.01, <0.001.

[0033] <0.05, <0.01, <0.001. Figure 11In a preferred embodiment of the present invention, overexpression MsPPR1 Analysis of gene expression levels in plant hormone signaling pathways.

[0034] Figure 12 In a preferred embodiment of the present invention, overexpression MsPPR1 Statistical analysis of differentially expressed genes in plant transcriptomes.

[0035] Figure 13 In a preferred embodiment of the present invention, overexpression MsPPR1 Enrichment map of regulatory pathways of differentially expressed genes in plant transcriptomes.

[0036] Figure 14 In a preferred embodiment of the present invention, overexpression MsPPR1 Diagram of photosynthesis-related differential gene expression analysis in plants. DETAILED DESCRIPTION

[0037] The present invention provides a new alfalfa gene MsPPR1 This gene significantly improves the photosynthetic efficiency and biomass yield of alfalfa by regulating the expression of genes in key pathways of photosynthetic carbon metabolism.

[0038] The present invention also provides a kind of alfalfa MsPPR1 Gene cloning and expression vector construction method, further obtain alfalfa MsPPR1 Overexpression transgenic lines to study alfalfa MsPPR1 The function of genes. Transcriptome data analysis revealed MsPPR1 The molecular mechanism of regulating photosynthetic carbon assimilation efficiency. The application of this gene provides an effective means for breeding new high-yield and high-quality alfalfa varieties.

[0039] The present invention adopts the following technical solutions: The present invention provides alfalfa MsPPR1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2. MsPPR1 The mRNA sequence of the gene has 1776 bases and can encode a protein with 592 amino acids.

[0040] The present invention also provides an expression vector, which comprises the alfalfa MsPPR1 Gene.

[0041] The present invention also provides a method for constructing the expression vector. First, total RNA of alfalfa is extracted and then reverse transcribed into cDNA for standby use. Specific primers are designed and PCR amplified to obtain a 1776 bp long cDNA. MsPPR1After the gel electrophoresis of the genes, the PCR product was cut and recovered, the recovered product was connected with pEASy-Blunt Zero (Beijing Quanshi Gold Biotechnology Co., Ltd.) cloning vector, the plasmid was extracted after transformation, and the positive clone was sent to the company for sequencing, and the correct bacterial liquid was preserved to extract the plasmid. The PEG100-eGFP (Wuhan Boyuan Biotechnology Co., Ltd.) vector was double-digested with restriction endonuclease NcoI and XmaI, the target plasmid was extracted by gel recovery, the plasmid was connected to the plant expression vector, and after transformation, the positive clone was verified by PCR, and the correct plasmid was confirmed by sequencing, which was the recombinant vector containing the target gene.

[0042] The present application provides transcriptome data analysis, which analyzes the potential genes of regulation MsPPR1 The specific steps are as follows: 1. After extracting RNA, construct a library, use Qubit 3.0 fluorescence quantitative instrument to determine the RNA concentration (concentration > 1 ng / μL), use Qsep400 high-throughput analysis system to detect library insert fragments, and use Q-PCR method 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, use Illumina NovaSeq6000 sequencing platform to perform PE150 mode sequencing. Use Illumina high-throughput sequencing platform to sequence cDNA library, and output a large amount of high-quality Data, called raw data. Use StringTie v2.2.1 software to splice and merge transcripts. After the raw sequencing amount, effective sequencing amount and the like are counted, comprehensive evaluation is carried out, and subsequent high-quality analysis based on clean data is carried out.

[0044] 3. The alfalfa genome website (https: / / modms.lzu.edu.cn / ) was used to download the annotation files of the reference genome and gene model of 'Zhongmo 4'. The sequencing Clean Reads were compared with the reference genome sequence using HISAT2 v2.0.5 software. Gene function was 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 MsPPR1 The gene and the application thereof can significantly improve the chlorophyll content and photosynthetic efficiency of alfalfa, and provide an important basis for constructing a gene expression vector and creating a high photosynthetic efficiency transgenic plant. Meanwhile, the gene involved in the application can improve the biological yield of alfalfa and improve the forage quality, and provides an effective molecular breeding method for cultivating a new alfalfa variety with high yield and high quality.

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

[0047] The 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 corresponding experiments under the guidance of the operation manuals attached to various commercial kits or entrust professional companies, such as gene sequencing, plasmid sequencing and determination of molecular weight.

[0048] Experimental material: 'Zhongmo 3' alfalfa was used as the background material.

[0049] Example 1 MsPPR1 Cloning of the gene 1.1 Extraction of total RNA of alfalfa: mainly using the TransZol Up method (1) 0.1 g of alfalfa leaves was used as the material, which was rapidly ground into powder in liquid nitrogen, 1 mL of TransZol Up reagent was added into a centrifugal tube, and the mixture was mixed by vortexingTransZol Up (Beijing Quanjing Gold Biotechnology Co., Ltd.) extraction solution, 200 μL of RNA extraction solution was added, shaken and mixed thoroughly for 2 min, and then centrifuged at 12000 rpm, 4 ℃ for 15 min.

[0050] (2) The supernatant was taken into a new centrifuge tube, an equal volume of isopropanol was added and mixed, and incubated at room temperature for 10 min. Centrifugation was performed at 12000 rpm, 4 ℃ for 10 min, the supernatant was discarded, and the precipitate was retained.

[0051] (3) 1 mL of pre-cooled 75% alcohol was added to the centrifuge tube, the precipitate was washed, and then centrifuged at 12000 rpm, 4 ℃ for 10 min. The above step was repeated 2 times.

[0052] (4) The 75% alcohol was poured out, the remaining alcohol was blown dry in a fume hood, then 20 μL of DEPC water was added for dissolution, the concentration of the extracted RNA was determined, and the RNA solution was stored at -80 ℃ for use.

[0053] 1.2 Synthesis of cDNA Using the Quanjing reverse transcription kit, 1 μg of total RNA was added to 4 μL of 5x EasyScript® All-in-One One-Step SuperMix, 1 μL of gDNA removal agent mixture, and the remaining solution was supplemented with sterile ddH2O to a total of 20 μL, then placed in a PCR instrument, and the following program was performed: 42 ℃, 15 min; 85 ℃, 5 s for reverse transcription, and immediately after the reaction was terminated, the obtained product was placed in a -20 ℃ refrigerator for storage.

[0054] 1.3 MsPPR1 Cloning of genes Specific primers were designed, and the primer sequences with NcoI and XmaI enzyme digestion sites were as follows: Upstream primer: 5'-ATGACAACCTTTTCAACCGAATTCCTC-3' Downstream primer: 5'-TCATTCTGATGATAAGGCAAGCTCT-3' The above reverse transcription cDNA was used as a template, and the total system was 50 μL, that is, 2x Phanta Max Master Mix 25 μL; 2 μL of upstream and downstream primers; 2 μL of cDNA; and 19 μL of ddH2O. PCR amplification was performed according to the following amplification program. The amplification program was as follows: 95 ℃, 3 min; 95 ℃, 20 s; 56 ℃, 20 s; 72 ℃, 1 min, 40 cycles of amplification; 72 ℃, 10 min; 12 ℃, termination reaction. The PCR product was subjected to electrophoresis detection and gel recovery. Subsequently, the pEASy-Blunt Zero vector (Beijing Quanshijin Biotechnology Co., Ltd.) was connected and transformed into DH5α E. coli, and the bacterial liquid with a band was screened by bacterial PCR and sent to the company for sequencing. The positive colonies were saved for use, and the plasmid was extracted using the bacterial liquid.

[0055] Gel recovery uses Novizen FastPure Gel DNA purification and recovery kit, and the specific steps are as follows: (1) Cut the single target DNA band from the agarose gel (try to remove the excess part) into a 2 mL centrifuge tube, add an appropriate amount of DNA binding liquid solution (if the gel weight is 0.1 g, its volume can be considered as 100 ul, then add 100 μL of DNA binding liquid), then place it in a 65 ℃ water bath pot until the gel block is completely dissolved.

[0056] (2) Add the dissolved solution to the adsorption column, stand at room temperature for 1 min, then centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column on the collection tube again.

[0057] (3) Add 300 μL of DNA binding liquid to the adsorption column, and centrifuge at 12000 rpm for 1 min.

[0058] (4) Add 700 μL of rinse solution (pay attention to whether anhydrous ethanol is added before use) to the adsorption column, then centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column on the collection tube again.

[0059] (5) Repeat step (4).

[0060] (6) Place the adsorption column on the collection tube and continue to centrifuge at 12000 rpm for 2 min, discard the waste liquid in the collection tube. Place the adsorption column at room temperature for a few minutes to remove the residual rinse solution.

[0061] (7) Put the adsorption column on a new centrifuge tube, add 20-30 μL elution buffer, stand for 2 min at room temperature, then centrifuge at 12000 rpm for 2 min to collect the target gene solution. If a DNA solution with higher concentration is needed, the collected solution can be added to the adsorption column again, stand for 2 min at room temperature, and then centrifuge at the same condition for 2 min.

[0062] The specific steps of homologous recombination operation are as follows: PCR amplification product 0.5-4 μL pEASy-Blunt Zero cloning vector 1 μL Gently mix and blow with a pipette, centrifuge briefly to collect at the bottom of the tube, and react at 37 °C for 5 min. After the reaction is completed, place the centrifuge tube on ice.

[0063] The specific steps of E. coli transformation operation are as follows: (1) Melt the DH5a competent cells on ice. After the competent cells are completely melted, add the product of the previous step to the competent cells, mix gently by tapping the wall of the tube, and stand on ice for 30 min.

[0064] (2) Heat shock at 42 °C in a metal bath for 90 s, and immediately place on ice for 2-3 min.

[0065] (3) Add 300 μL of antibiotic-free LB solution to the transformed solution, and incubate at 37 °C in a shaker at 200 rpm for more than 1 h.

[0066] (4) Centrifuge the recovered bacterial solution at 5000 rpm for 1 min, resuspend 100 μL of the bacterial solution, and then plate it. After the plate has no obvious liquid accumulation, invert and culture in a 37 °C incubator for 12-16 h.

[0067] (5) According to the 15 μL system, i.e. 1 μL of each of the upstream and downstream primers; 1 μL of bacterial solution; 7.5 μL of enzyme; and 4.5 μL of ddH2O. Perform bacterial batch PCR using the following program: 95 °C for 3 min; 30 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min; and 72 °C for 5 min. Then select the bacterial solution with a band by agarose gel electrophoresis, and send it to the company for sequencing.

[0068] (6) Store the bacterial solution with correct sequencing in 70% glycerol at -80 °C.

[0069] The specific steps of plasmid extraction (the reagents are from the plasmid mini-prep kit of Tian Gen Biochemical Co., Ltd.) are as follows: 1) Column equilibration: Add 500 μL of equilibration buffer BL to the adsorption column CP3, centrifuge at 12000 rpm for 1 min, discard the waste in the collection tube, and place the adsorption column back into the collection tube.

[0070] (1) Take 5 mL of the overnight culture of E. coli, and divide it into 2 mL centrifuge tubes. Centrifuge at 12000 rpm for 1 min, discard the supernatant, and if there are many bacteria, centrifuge several times to ensure that more E. coli is obtained.

[0071] 3) Add 250 μL of P1 solution to the centrifuge tube (check whether RNase A has been added before adding), and mix well using a vortex machine to suspend the bacterial solution completely.

[0072] 4) Add 250 μL of P2 solution to the centrifuge tube, and gently invert 6-8 times to lyse the bacteria completely. At this time, the bacterial solution should be clear and viscous.

[0073] 5) Add 350 μL of P3 solution to the centrifuge tube, and gently invert 6-8 times to mix well. At this time, a white flocculent precipitate should appear. Centrifuge at 12000 rpm for 10 min.

[0074] 6) Take the supernatant and place it into the adsorption column CP3, taking care not to take the white precipitate. Centrifuge at 12000 rpm for 1 min. 7) Discard the waste in the collection tube, and place the adsorption column back into the collection tube. Add 700 μL of rinse buffer PW (check whether anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste in the collection tube, and place the adsorption column back into the collection tube.

[0075] 8) Repeat step (7).

[0076] 9) Place the adsorption column in the collection tube, and continue centrifuging at 12000 rpm for 2 min, and discard the waste in the collection tube. Place the adsorption column CP3 in a place with good ventilation, and air-dry at room temperature for 5 min to ensure that the ethanol in the column evaporates completely.

[0077] 10) Place the adsorption column CP3 in a new centrifuge tube, and add 50 μL of elution buffer to it. Let it stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to collect the plasmid solution. If a plasmid solution with a higher concentration is needed, the collected solution can be added to the adsorption column CP3 again, let it stand at room temperature for 2 min, and then centrifuge under the same conditions for 2 min.

[0078] The full length of the cloned gene is 1776 bp, and the sequence is shown as SEQ ID NO: 1. The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO: 2. The gene is named as MsPPR1 .

[0079] Example 2 Construction of plant expression vector The Bar single enzyme-digested vector (purchased from New England Biolabs) was used, and the enzyme digestion system was 40 μL, i.e. fast enzyme 1.5 μL; 10x buffer 5 μL; vector plasmid 1000 ng, and the remaining volume was supplemented with ddH2O. The enzyme digestion was performed in a 37 ℃ water bath for 3 h. The gel was recovered after agarose gel electrophoresis detection, and stored at -20 ℃ for use.

[0080] The following forward and reverse primers were used for PCR reaction, and the above plasmid was used as the template (forward primer: 5'-actacctgagcacccagtcc-3', reverse primer: 5'-GTCTTGTTTCATTGACGCGGATT-3'), and the total system was 50 μL, i.e. 2x Phanta Max Master Mix (Dye Plus) 25 μL; 2 μL of each of the forward and reverse primers; 2 μL of cDNA; 19 μL of ddH2O. The amplification program was as follows: 95 ℃, 5 min; 95 ℃, 15 s; 59 ℃, 15 s; 72 ℃, 1 min, 40 cycles of amplification; 72 ℃, 5 min; 16 ℃, termination reaction. The PCR product was recovered using a gel recovery kit after agarose gel electrophoresis detection, and stored at -20 ℃ for use.

[0081] The homologous recombination was performed as follows. The solution (PCR product, 3.5 μL; homologous recombination enzyme, 5 μL; vector, 1.5 μL) was reacted at 50 ℃ in PCR for 30 min. Subsequently, the homologous recombination product was transformed into E. coli according to the above E. coli transformation method, and sequencing was performed using the above gene self primers.

[0082] The bacterial liquid with correct sequencing was reserved, and the plasmid was extracted to transform the Agrobacterium EHA105 competent cells. After screening the positive clones, extracting the plasmid and performing restriction enzyme digestion verification, it was proved that the Agrobacterium was successfully transformed. The expression vector (pBI121-PEG100-eGFP) MsPPR1 The structure of the PEG100-eGFP vector is shown in Figure 1 ) can be directly used for transformation of plants such as alfalfa, Arabidopsis thaliana, and tobacco.

[0083] The specific steps of Agrobacterium EHA105 transformation are as follows: (1) Agrobacterium EHA105 competent cells were gently thawed by hand, and the recombinant expression vector plasmid was added. The mixture was mixed thoroughly by flicking with fingers, and the centrifuge tube was placed in liquid nitrogen for 5 min.

[0084] (2) Quickly transfer to a 37 °C water bath for 5 min, and then quickly place on ice for 5 min.

[0085] (3) Add 700 μL of LB solution without resistance to the mixture, and incubate at 28 °C on a shaker at 200 rpm for 3 h to recover the bacterial cells.

[0086] (4) After recovery, centrifuge the cells at 6000 rpm for 1 min to precipitate the cells. Retain about 100 μL of supernatant, resuspend the bacterial cells by gently flicking, and evenly spread them on LB solid medium plates containing the corresponding antibiotics. After the liquid in the plates is completely absorbed, invert the plates and place them in a 28 °C incubator for further incubation for 2-3 d.

[0087] (5) After the colonies grow, pick single colonies from each gene-transformed plate for PCR positive identification. The identification primers are the same as the homologous recombination primers of the expression vector. The system is 15 μL, i.e. 1 μL of each of the upstream and downstream primers; 1 μL of bacterial solution; 7.5 μL of enzyme; and 4.5 μL of ddH2O. Perform bacterial batch PCR using the following program: 95 °C for 5 min; 95 °C for 10 s; 56 °C for 15 s; 72 °C for 40 s, for 30 cycles; and 72 °C for 5 min. Then select the bacterial solution with bands by agarose gel electrophoresis, which is the successfully transformed Agrobacterium EHA105.

[0088] Example 3 Medicago sativa MsPPR1 Genetic transformation of the gene Agrobacterium-mediated transformation of Medicago sativa tissue culture seedlings was performed, and the specific steps were as follows: MsPPR1 (1) Leaf disinfection: Select the third leaf from the top of Medicago sativa No. 3 with good growth, wash the leaf in water, and then soak it in 75% alcohol for 5 s. Disinfect with 10% NaClO solution for 15 min, and then rinse with sterile water for 5-8 times.

[0089] ​(2) Bacterial liquid preparation: take appropriate amount of Agrobacterium and add into 5 mL corresponding resistant YEP liquid medium for overnight culture, then take 200 μL of the cultured Agrobacterium liquid and add into 50-60 mL of corresponding resistant YEP liquid medium, shake to OD600=0.6-0.8, centrifuge at 5000 rpm for 5 min, discard the supernatant, resuspend the bacterial body to OD600=0.5-0.6 using resuspension liquid, and stand for 2-3 h for standby use.

[0090] (3) Explant preparation, use a sterilized scalpel to cut the leaf to form a wound.

[0091] (4) Infection and co-culture: cut the explant leaf into small pieces and put into a 50 mL centrifuge tube, add about 10 mL of resuspended bacterial liquid (containing 100 μM acetosyringone) into the tube, ultrasonic for 30 s, then pump to 0.8 MPa under vacuum, 10 min, stand for 15 min, dry the bacterial liquid, and place on the co-culture medium in the dark for 2-3 d.

[0092] (5) Selection culture: inoculate the co-cultured explants on the selection medium (hygromycin 5 mg / L) for two weeks. Increase the hygromycin to 10 mg / L and culture for two weeks. Continue to grow the callus under a light cycle of 16 h light / 8 h dark for 10 d. Successfully induce callus. Inoculate the green and sticky callus on the differentiation medium, subculture every two weeks.

[0093] (6) Somatic cell culture: inoculate the embryoid on the rooting medium for rooting culture.

[0094] (7) Medium composition: ① YEP medium: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar (pH=7) for solid. Add rifampicin 50 mg / L and kanamycin 100 mg / L when culturing Agrobacterium.

[0095] 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).

[0096] 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).

[0097] (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).

[0098] (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).

[0099] Among them, 2,4-D, KT, MES are 2,4-dichlorophenoxyacetic acid, kinetin and 2-(N-morpholino) ethanesulfonic acid, respectively.

[0100] The application provides a cloning method and an expression vector of a Medicago sativa L. gene, and lays a foundation for subsequent MsPPR1 transformation of Medicago truncatula by the gene vector, which is of great significance for the cultivation, production and popularization of new varieties.

[0101] Example 4 Overexpression of Medicago sativa L. in Medicago truncatula MsPPR1 According to Example 3, three strains of OEMsPPR1-4, OEMsPPR1-9 and OEMsPPR1-14 are screened out from the Medicago sativa L. overexpression plants, and the phenotypes are shown in MsPPR1 According to the RNA level identification (OEMsPPR1-4, OEMsPPR1-9 and OEMsPPR1-14 are screened out from the Medicago sativa L. overexpression plants, and the phenotypes are shown in Figure 2 ). Figure 3 After overexpression of the gene, the plant height is obviously higher than that of the wild type (OEMsPPR1-4, OEMsPPR1-9 and OEMsPPR1-14 are screened out from the Medicago sativa L. overexpression plants, and the phenotypes are shown in MsPPR1 ), Figure 4 The plant height of the gene overexpression material is 44 cm, 42 cm and 40 cm respectively, which is 1.29 times, 1.21 times and 1.15 times of the plant height of the control Medicago sativa L. (OEMsPPR1-4, OEMsPPR1-9 and OEMsPPR1-14 are screened out from the Medicago sativa L. overexpression plants, and the phenotypes are shown in MsPPR1 ), Figure 5 The internode number of the three strains is 15, 13 and 12 respectively, which is 1.5 times, 1.3 times and 1.2 times of the internode number of the control Medicago sativa L. (OEMsPPR1-4, OEMsPPR1-9 and OEMsPPR1-14 are screened out from the Medicago sativa L. overexpression plants, and the phenotypes are shown in Figure 6 ), Figure 7 The single plant fresh weight of the OEMsPPR1 strain is 4.85 g, 5.92 g and 4.03 g respectively, which is 2.23 times, 2.92 times and 2.44 times of the single plant fresh weight of the control Medicago sativa L. (OEMsPPR1-4, OEMsPPR1-9 and OEMsPPR1-14 are screened out from the Medicago sativa L. overexpression plants, and the phenotypes are shown in Figure 8 ),

[0102] The above results show that, MsPPR1Gene overexpression not only promotes the growth of alfalfa plant height and internode development, but also directly improves yield potential by increasing single plant fresh weight, while regulating the morphological development of stem and leaf cells, providing a theoretical basis for further analyzing the molecular mechanism of the gene in alfalfa high-yield breeding.

[0103] Example 5 Overexpression MsPPR1 Transgenic plant chlorophyll content analysis Compared with Zhongmuyu No. 3, the overexpression MsPPR1 The chlorophyll content of the transgenic plants showed a significant increase Figure 9 ), wherein the chlorophyll a content reached 1.78 times that of the control group, the chlorophyll b content increased to 1.76 times that of the control group, the carotenoid content increased to 2.43 times that of the control group, and the total chlorophyll content also increased to 1.77 times that of the control group. This finding provides important experimental evidence for the core innovation point of the present application.

[0104] In terms of specific strain performance, the OEMsPPR1-4 strain showed the most significant chlorophyll accumulation ability. The chlorophyll a content reached 2962.62 mg·g⁻¹ FW, which was 1.86 times that of the control group. The chlorophyll b content reached 1328.10 mg·g⁻¹ FW, which was 2.11 times that of the control group. The carotenoid content and total chlorophyll content also reached 2.74 times and 1.92 times that of the control group, respectively. These data fully confirm MsPPR1 The regulatory effect of the gene on chlorophyll synthesis.

[0105] The present application discloses MsPPR1 The key role of the gene in regulating the photosynthesis of alfalfa. By significantly increasing the chlorophyll content, especially the accumulation of chlorophyll a, the gene can effectively enhance the photosynthesis efficiency of the plant. This finding not only provides a new perspective for understanding the regulatory mechanism of plant photosynthetic carbon metabolism.

[0106] Example 6 MsPPR1 Gene function analysis The tissue-specific expression analysis of MsPPR1 The results are shown in Figure 10 , indicating that MsPPR The gene is expressed in the roots, stems, leaves and flowers of alfalfa, with significantly increased expression in roots, stems and leaves.

[0107] In transgenic strains OEMsPPR1-4, OEMsPPR1-9 and OEMsPPR1-14, the expression of genes related to hormone synthesis and signal transduction, and cell wall development was determined to detect ARF , XTR , DELLA , AHK2 , BARR , IAA1, GH3 The primers are as follows: ARF : Upstream primer: 5'-CTCCTCCAGCAAACTCCCAG-3' Downstream primer: 5'-CCATGTTGAGGGTCGCGATA-3' XTR : Upstream primer: 5'-TCTTGGTTGACAACATCCCAA-3' Downstream primer: 5'-ATCATCAGCGTTCCAGAGAC-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' IAA1 : Upstream primer: 5'-GCAATTGAGGTGGATTGGCG-3' Downstream primer: 5'-CCCTGAAGCTAACCGCTTCT-3' GH3 : Upstream primer: 5'-CAGAACACACGACCCTTGGA-3' Downstream primer: 5'-GGCGCCTAGCCTTAGAACTT-3' The results are shown in Table 1, and the auxin response factor (ARF), auxin binding gene (ABP1), and cell wall loosening protein gene (XYL1) were detected. Figure 11 ARF Auxin Response Factor GH3 Gretchen Hagen 3 XTR Xyloglucan Endotransglucosylase / Hydrolase ​​​​​​expression of auxin signaling inhibitor IAA1 ( Indole-3-Acetic Acid Inducible 1 ) was significantly down-regulated. Meanwhile, the expression of cytokinin signaling pathway related genes, receptor AHK2 ( Arabidopsis Histidine Kinase 2 ) and B-type response regulator BARR ( Type-B Arabidopsis Response Regulators ) were also significantly enhanced. In addition, the expression of DELLA protein, a negative regulator of gibberellin signaling pathway, was inhibited. These molecular changes were consistent with the significant increase of chlorophyll content observed in previous studies, suggesting that the plants might be in an active energy metabolism and growth development state.

[0108] These results suggest MsPPR1 that the gene promotes the expression of key chlorophyll biosynthesis enzyme genes by coordinating hormone signal transduction and cell development processes, regulates chloroplast development, and builds a regulatory network for chlorophyll synthesis and photosynthetic efficiency improvement through multiple mechanisms.

[0109] Example 7 Overexpression MsPPR1 Transcriptome analysis of transgenic plants Statistical results of differentially expressed genes show that Figure 12 ), OEMsPPR1 there are 5126 differentially expressed genes compared with WT, of which 2272 are up-regulated and 2854 are down-regulated. This result shows MsPPR1 that the gene may affect the phenotype by regulating specific functional gene networks, and also confirms the extensive regulatory effect of the gene on alfalfa gene expression at the transcriptome level. MsPPR1

[0110] KEGG pathway enrichment analysis shows that differentially expressed genes are significantly enriched in multiple key pathways related to photosynthesis and carbon metabolism Figure 13 . Among them, the “carbon fixation photosynthetic organisms” and “photosynthesis-antenna protein” pathways are particularly prominent, which is highly consistent with the previous finding of the present application that the chlorophyll content is increased. In addition, the significant enrichment of energy metabolism related pathways such as “carbon metabolism”, “starch and sucrose metabolism” and “pentose phosphate pathway” further supports the core role of the gene in regulating photosynthetic carbon assimilation and energy metabolism. MsPPR1 Further analysis of differentially expressed genes found that

[0111] , Figure 14 MsPPR1 ​​In the overexpression plants, multiple significantly differentially expressed genes (|log2FC| ≥ 1, FDR<0.05) were identified, involving photosynthesis, RNA editing and hormone response pathways. Among them, the expression levels of pentapeptide repeat proteins (Msa1412080 and Msa1432640) were up-regulated by 50.23-fold and 49.85-fold, respectively. These two genes belong to the PPR family, which can affect the metabolic process of plants by regulating chloroplast or mitochondrial RNA editing. In addition, the expression level of photosystem II reaction center W protein (Msa0168360) was up-regulated by 35.76-fold, which directly participates in photosynthesis, suggesting that PPR The overexpression may significantly enhance the light energy utilization efficiency of the plants.

[0112] On the other hand, the expression level of photosynthetic NDH subunit B1 (Msa0625820) was down-regulated by 60.11-fold, which is related to the function of NAD(P)H dehydrogenase complex. Its significant down-regulation may affect the energy metabolism balance. The expression level of photosystem I chlorophyll a / b binding protein (Msa0323790) was down-regulated by 17.27-fold, which may interfere with light signal transmission and photosynthetic efficiency. In addition, the expression level of constitutive ROP interacting protein ICR2 (Msa0116680) was down-regulated by 4.31-fold, which is involved in cytoskeleton regulation and may be related to auxin transport or cell morphogenesis.

[0113] The above results show that, MsPPR1 The genes can optimize the efficiency of light energy capture, electron transfer and carbon assimilation by coordinating the expression network of photosynthesis-related genes, thereby significantly improving the photosynthetic performance of alfalfa. This has important guiding significance for breeding high-yield and high-quality alfalfa varieties.

[0114] Although the present application has been described in detail in the foregoing description with general principles and specific embodiments thereof, it should be further appreciated that modifications and improvements can be made to the present application which will be apparent to those skilled in the art. Therefore, these modifications and improvements are intended to be included within the scope of the present application. The scope of the present application should be construed based on the appended claims.

Claims

1. Medicago truncatula MsPPR1 gene characterized in that, 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. Biomaterials 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 biomaterial according to claim 2 in regulating plant photosynthetic carbon metabolism, growth and development, and biomass yield.

4. Use according to claim 3, characterized in that, The regulation is positive regulation, which increases the chlorophyll content, photosynthetic carbon metabolism and biological yield of plants.

5. 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 of increasing photosynthetic carbon metabolism in alfalfa, comprising, The method comprises: overexpressing the gene of claim 1 in alfalfa by genetic engineering means. MsPPR1 The gene.

7. A method of breeding alfalfa cultivars with high yield or improving the quality of alfalfa forage, characterized by, The method comprises: overexpressing the gene of claim 1 in alfalfa by genetic engineering means. MsPPR1 The gene.

8. The method according to claim 6 or 7, 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.

9. Use of the transgenic alfalfa obtained according to the method according to any one of claims 6 to 8 in plant breeding.

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

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