Application of gene MtPrx38 in regulation and control of growth, development and quality of forage grass

Abstract

The invention discloses application of a gene MtPrx38 in regulation and control of growth, development and quality of forage grass. The method comprises the following steps: carrying out genome PCR (Polymerase Chain Reaction) identification to obtain homozygotes MtPrx38-1 and MtPrx38-2 of the medicago truncatula MtPrx38 gene mutant; the expression quantity of the MtPrx38 gene detected by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) is obviously reduced, and compared with wild medicago truncatula, the peroxidase activity of the mutant homozygotes MtPrx38-1 and MtPrx38-2 is obviously reduced. Compared with wild medicago truncatula, the MtPrx38 gene mutant has the advantages that the root length, the lateral root number, the plant height, the branch number, the leaf area and the secondary branch number are obviously increased; the flowering time is advanced; the lignin content and the cellulose content are obviously reduced, which indicates that the gene MtPrx38 has the functions of regulating and controlling the growth, development and quality of forage grass. The method has application prospects in the aspects of promoting growth and development of forage grass, improving forage grass quality and the like.

Description

Technical Field

[0001] The present invention relates to Leguminosae ) plants, particularly Medicago truncatula ( Medicago truncatula ) MtPrx38 Use of the encoded protein in regulating the growth and development or quality of forage, belonging to gene MtPrx38 and application areas of the proteins encoded therein. Background Art

[0002] Peroxidase (PRX) is an important antioxidant enzyme. As a superfamily, this family plays an important role in various physiological activities of plants. According to different functions, peroxidases are divided into three categories: Class I, Class II, and Class III. Class I includes ascorbate peroxidase, cytochrome c peroxidase, and hydrogen peroxidase (a fusion protein with two catalytic activities); Class II includes secreted fungal peroxidases (such as manganese peroxidase and lignin peroxidase); Class III is a plant secreted peroxidase (Class III Plant Peroxidase, Prx), which has very high thermal stability and is widely involved in various life activities of plants, such as cell wall formation and lignification, signal transduction, ROS (reactive oxygen species) metabolism, stress response, IAA (indole-3-acetic acid) decomposition and plant growth and development; Due to the complexity of peroxidase family genes and functional redundancy between genes, the physiological functions of most proteins in this family are not yet clear.

[0003] There are 73 and 138 Class III gene family members in Arabidopsis and rice, respectively. However, to date, only a few biological functions of Class III Prx genes have been revealed, and they are mainly concentrated in the model plant Arabidopsis. Most of the functions have not been reported, especially in alfalfa, there are no reports of Class III plant peroxidases involved in regulating growth, development or quality.

[0004] Medicago truncatula Medicago truncatula ) is a typical annual herbaceous plant of the genus Medicago in the Leguminosae family. Its natural population types are diverse, with rich mutant and ecotype resources. In addition, it has a short growth cycle, a small genome, and stable and efficient genetic transformation. It is also very representative in the study of genetics of leguminous plants. It is similar to Alfalfa ( Medicago sativa ) are closely related and have become an important model plant for studying legumes. Summary of the invention

[0005] The main purpose of the present invention is to MtPrx38 Gene, MtPrx38 protein 、 contain MtPrx38 Gene expression cassette or containing MtPrx38 The recombinant plant expression vector of the gene is used to regulate the growth, development or quality of forage.

[0006] In order to achieve the above object, the main technical solutions adopted by the present invention include: One aspect of the present invention is to MtPrx38 Gene, MtPrx38 protein 、 contain MtPrx38 Gene expression cassette or containing MtPrx38 The recombinant plant expression vector of the gene is used to regulate the growth and development of forage.

[0007] In a preferred specific embodiment of the present invention, the regulating forage growth and development is promoting forage growth and development.

[0008] In a preferred specific embodiment of the present invention, the promoting the growth and development of forage grass includes: increasing the root length, the number of lateral roots, the plant height, the number of branches, the leaf area or the number of secondary branches of the forage grass; and advancing the flowering time.

[0009] Another aspect of the present invention is to MtPrx38 Gene, MtPrx38 protein 、 contain MtPrx38 Gene expression cassette or containing MtPrx38 The recombinant plant expression vector of the gene is used to regulate forage quality.

[0010] In a preferred specific embodiment of the present invention, the regulating forage quality is to improve the forage quality.

[0011] In a preferred specific embodiment of the present invention, the improving the quality of forage includes: reducing the lignin content and cellulose content of the forage.

[0012] In a preferred embodiment of the present invention, the forage is a legume plant; wherein the legume plant is Medicago truncatula.

[0013] As a reference, the present invention provides an implementation method. MtPrx38 Gene mutation or silencing MtPrx38 The gene expression level is reduced or the normal function of MtPrx38 protein is defective, thereby promoting the growth and development of forage or improving the quality of forage, thereby increasing the root length, lateral root number, leaf area, plant height, lateral branch number or secondary branch number of the forage; flowering time is advanced; and the lignin content and cellulose content of the forage are reduced.

[0014] A preferred specific embodiment of the present invention is a method for promoting the growth and development of forage grass or improving the quality of forage grass, comprising: MtPrx38 Gene mutation or silencing MtPrx38 Reduced gene expression may cause defects in the normal function of the MtPrx38 protein.

[0015] The mutations include MtPrx38 One or more nucleotides are substituted, deleted and / or added to the nucleotide sequence of a gene or its promoter. Preferably, the mutation can be obtained by physical mutagenesis, chemical mutagenesis, or gene editing. Physical mutagenesis includes, but is not limited to, radiation mutagenesis, space breeding, etc.; chemical mutagenesis includes mutagenesis caused by treatment with mutagens such as EMS; the gene editing method includes, but is not limited to, ZFN, TALE and / or CRISPR / Cas, etc.

[0016] Those skilled in the art can use conventional methods such as conventional gene knockout or gene editing technology to modify the gene in plants. MtPrx38 Gene knockout mutation, such as constructing MtPrx38 Gene knockout vectors or constructed using gene editing technology MtPrx38 CRISPR / Cas9 gene editing vectors, etc., can be used to edit genes in plants MtPrx38 Gene knockout or mutation are methods that are well known to those skilled in the art.

[0017] Those skilled in the art know that the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the location of gene editing in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, to target the DNA sequence, and then cut the DNA through the Cas protein. In this application, the Cas protein includes but is not limited to Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13 and / or Cas14.

[0018] The interference MtPrx38 The normal expression or normal function of the gene or promoter can be interfered with by RNA interference technology (RNAi) to interfere with the normal expression of the gene encoding the MtPrx38 protein or its promoter or to cause defects in its normal function. RNA interference technology is a conventional technology in the art. It specifically binds to the homologous region of the mRNA expressed by the target gene through a 21-23bp short double-stranded RNA (siRNA) or a long double-stranded RNA (dsRNA; double-strand RNA), thereby degrading the mRNA and achieving the effect of inhibiting gene expression.

[0019] Another aspect of the present invention is to provide a Medicago truncatula-derived material capable of regulating the growth and development or quality of forage. MtPrx38 gene or its encoded protein.

[0020] The present invention MtPrx38 The nucleotide sequence of the CDS of the gene is selected from any one of the nucleotide sequences described in (a) to (e) below: (a) the polynucleotide sequence shown in SEQ ID No. 1; (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2; (c) a polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and the polynucleotide sequence still has the function of regulating the growth, development or quality of forage; (d) a polynucleotide sequence having at least 95% or more identity with any of the polynucleotide sequences shown in (a) to (c), and the polynucleotide sequence still has the function of regulating the growth, development or quality of forage; (e) A polynucleotide sequence that is complementary to the polynucleotide sequence described in any one of (a) to (d), and the polynucleotide sequence still has the function of regulating the growth, development or quality of forage.

[0021] The percentage of sequence identity described in the present invention can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0022] Those skilled in the art can easily adopt known methods, such as directed evolution or point mutation, to MtPrx38 The nucleotide sequence of the gene is mutated. MtPrx38 Nucleotides with 75% or higher identity to the nucleotide sequence of a gene are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the encoded protein has the function of regulating the growth or quality of forage.

[0023] In addition, the nucleotide sequence described in the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA.

[0024] The amino acid sequence of the MtPrx38 protein of the present invention is selected from any one of the following amino acid sequences (a) to (d): (a) the amino acid sequence shown in SEQ ID NO.2; (b) a protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2, wherein the protein variant still has the function or activity of regulating the growth and development or quality of forage; (c) a protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.2, wherein the protein variant still has the function or activity of regulating the growth, development or quality of forage; (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 2, wherein the protein still has the function or activity of regulating the growth or quality of forage.

[0025] The MtPrx38 protein of the present invention can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed.

[0026] The main beneficial technical effects of the present invention are: (1) The present invention obtains Medicago truncatula by genome PCR and RT-PCR identification. MtPrx38 Homozygous gene mutant MtPrx38 -1, MtPrx38 -2; compared with wild-type Medicago truncatula, the mutant homozygous MtPrx38 -1, MtPrx38 -2 had a significant increase in the mass fraction of H2O2 and a significant decrease in the activity of peroxidase.

[0027] (2) Phenotypic observations and related data statistics show that compared with wild-type Medicago truncatula, MtPrx38 The root length, lateral root number, plant height, branch number, leaf area, and secondary branch number of the gene mutant were significantly increased; the flowering time was advanced, that is, MtPrx38 The growth performance of the gene mutant was improved; the lignin content and cellulose content were significantly reduced. MtPrx38 The forage quality of the gene mutant was improved; it proved that the gene MtPrx38 It has the function of regulating the growth or quality of forage such as Medicago truncatula.

[0028] (3) The present invention can be used to cultivate forage varieties with fast growth and development or high quality, and has important application prospects in promoting forage growth and development, improving forage quality, and cultivating high-yield forage varieties.

[0029] Definitions of terms used in this invention Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0030] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (phosphorothioates, phosphoramidates, etc.) used in antisense technology. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0031] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions directed to polypeptides apply equally to describing peptides and describing proteins, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0032] The "stringent hybridization conditions" described in the present invention refer to the conditions of low ionic strength and high temperature known in the art. Generally, under stringent conditions, the detectable degree of hybridization of the probe to its target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times higher than the background). Stringent hybridization conditions are sequence-dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or washing conditions. For detailed guidance on nucleic acid hybridization, reference can be made to the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are usually selected to be lower than the thermal melting point (T ) of the specific sequence at a specified ionic strength pH. m ) about 5-10℃. T mThe temperature (under specified ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, the temperature at T m Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60°C for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions may also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least two times the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubated at 65°C, washed in 0.2×SSC and washed in 0.1% SDS at 65°C. The washing may be performed for 5, 15, 30, 60, 120 minutes or longer. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Medicago truncatula MtPrx38 Gene CDS and amino acid sequence details; Figure 2 for MtPrx38 The nucleotide sequence alignment result of the CDS of the gene and the CDS of the homologous gene in Arabidopsis thaliana; Figure 2 A in MtPrx38 Arabidopsis thaliana AT5G67400 The nucleotide sequence alignment results of the CDS of the gene; Figure 2 B is MtPrx38 Arabidopsis thaliana AT3G49960 The nucleotide sequence alignment results of the CDS of the gene; Figure 3 is the result of comparing the amino acid sequence of MtPrx38 protein with the amino acid sequence of the homologous protein of Arabidopsis thaliana; wherein, Figure 3 A in the middle is the amino acid sequence alignment result of MtPrx38 protein and Arabidopsis AT5G67400 protein; Figure 3 Middle B is the amino acid sequence alignment result of MtPrx38 protein and Arabidopsis AT3G49960 protein; Figure 4 Medicago truncatula MtPrx38 Gene homozygous mutant screening and verification diagram; among them, Figure 4 A is Medicago truncatula MtPrx38Screening diagram of homozygous mutants with gene insertion into Tnt1; Figure 4 B is Medicago truncatula MtPrx38 Gene expression graph; Figure 4 C is wild type Medicago truncatula and MtPrx38 In gene mutants MtPrx38 Statistical graph of relative gene expression; Figure 5 Wild-type Medicago truncatula and MtPrx38 Statistical graph of H2O2 mass fraction and peroxidase activity of gene mutants; Figure 5 A in the figure is wild type Medicago truncatula and MtPrx38 Statistical graph of H2O2 mass fraction of gene mutants; Figure 5 B is wild type Medicago truncatula and MtPrx38 Statistical graph of peroxidase activity of gene mutants; Figure 6 Wild-type Medicago truncatula and MtPrx38 Root growth of gene mutants; Figure 6 A in the figure is wild type Medicago truncatula and MtPrx38 Root phenotypes of gene mutants; Figure 6 B is wild type Medicago truncatula and MtPrx38 Root length statistics of gene mutants; Figure 6 C is wild type Medicago truncatula and MtPrx38 Statistics of lateral root numbers of gene mutants; Figure 7 Wild-type Medicago truncatula and MtPrx38 The plant height and branch number of gene mutants; Figure 7 A in the figure is wild type Medicago truncatula and MtPrx38 Phenotypes of plant height and branch number of gene mutants; Figure 7 B is wild type Medicago truncatula and MtPrx38 Statistical graph of plant height of gene mutants; Figure 7 C is wild type Medicago truncatula and MtPrx38 Statistical diagram of the number of branches of gene mutants; Figure 8 Wild-type Medicago truncatula and MtPrx38 Leaf area of ​​gene mutants; Figure 8 A in the figure is wild type Medicago truncatula and MtPrx38 Leaf area phenotype of gene mutants; Figure 8 B is wild type Medicago truncatula and MtPrx38 Leaf area statistics of gene mutants; Fig. 9 Wild-type Medicago truncatula and MtPrx38 Secondary branching of gene mutants; among them, Fig. 9 A in the figure is wild type Medicago truncatula and MtPrx38Secondary branching phenotype diagram of gene mutants; Fig. 9 B is wild type Medicago truncatula and MtPrx38 Statistical graph of the growth rate of secondary branches of gene mutants; Fig.10 Wild-type Medicago truncatula and MtPrx38 Statistical graph of lignin content in gene mutants. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the scope of protection of the present invention.

[0035] Medicago truncatula MtPrx38 Gene cloning and comparative analysis Screening genes from the Medicago truncatula genome by multi-omics analysis MtPrx38 (GeneID: LOC11405719, MtrunA17_Chr4g0053871), designed primers MtPrx38-F / MtPrx38-R for PCR amplification of the full-length gene CDS sequence, PCR reaction enzyme selected Novozymes Biotech's high-fidelity enzyme 2×Phanta Flash MasterMix with catalog number P520, after electrophoresis detection, the product was sent to the company for sequencing, and the sequencing results were compared with the downloaded sequence information to obtain the correct CDS sequence and amino acid sequence, Medicago truncatula MtPrx38 The detailed information of the gene CDS sequence and amino acid sequence is as follows Figure 1 shown.

[0036] MtPrx38 The nucleotide sequence of the CDS of the gene is close to that in Arabidopsis AT5G67400 , AT3G49960 The nucleotide sequences of the CDS of the genes were subjected to multiple sequence alignment. The alignment results of the nucleotide sequences are shown in Figure 2 As shown, Figure 2 A and Figure 2 The consistency of B in MtPrx38 protein was 64.72% and 64.55% respectively. The amino acid sequence of MtPrx38 protein was compared with that of AT5G67400 protein and AT3G49960 protein. The comparison results of amino acid sequence are shown in Figure 3 As shown, Figure 3 A and Figure 3 The consistency of B is 66.57%. MtPrx38 Arabidopsis thaliana AT5G67400 , AT3G49960 The nucleotide sequence of the CDS of the gene, the amino acid sequence of the MtPrx38 protein, and the Arabidopsis AT5G67400 protein and AT3G49960 protein are significantly different. MtPrx38 The gene is a new gene that has not been reported.

[0037] MtPrx38The nucleotide sequence of the CDS of the gene is shown below: ATGGGTCGTTATAATGTTATACTTGTGTGGTCACTAGCATTAACGTTGTGTCTTATTCCATATACAACTTTTGCTCAACTTAGTCCAAACCATTATGCTAACATTTGTCCTAATGTTCAAAGCATTGTAAGATCAGCAGTTCAAAAGAAATTTCAACAAACTTTTGTTACTGTTCCTGCTACCCTTCGTCTCTTCTTTCATGATTGTTTTGTTCAGGGTTGTGATGCTTCAGTTTTGGTGGCATCAAGTGGAAATAACAAAGCTGAAAAGGATCATCCTGAAAATTTGTCATTGGCTGGTGATGGTTTTGACACAGTGATTAAAGCAAAAGCTGCATTAGATGCTGTTCCACAGTGCAGAAACAAAGTTTCTTGTGCTGATATTCTTGCTTTGGCTACTCGAGATGTTATTAATCTGGCTGGTGGTCCATCCTATACAGTAGAATTGGGAAGGTTTGATGGTCTAGTTTCAAGATCTTCAGATGTAAATGGAAGGCTCCCTCAGCCAAGTTTCAATTTGAATCAGTTGAATACTCTCTTTGCAAACAATGGACTCACTCAAACAGACATGATTGCCCTATCAGGTGCACACACTCTTGGATTCTCTCACTGTGACAGATTCTCCAATAGAATTCAAACTCCAGTGGACCCCACATTGAACAAACAATATGCTGCCCAGTTACAACAAATGTGCCCTAGGAATGTTGATCCAAGAATAGCCATCAACATGGACCCAACAACTCCTAGAACATTTGACAATGTTTACTACAAAAATCTTCAACAAGGAAAAGGTTTATTTACTTCTGATCAAATCCTCTTCACTGACACAAGATCAAGGAACACTGTTAACTCTTTCGCGACTAACGGTAACGTTTTCAATGCCAATTTCATCACTGCCATGACCAAGTTGGGTCGTGTTGGAGTTAAAAATGCGAGAAATGGGAAAATTCGTACCGATTGTTCTGTGCTTTAA (SEQ ID NO.1). .

[0038] The amino acid sequence of the MtPrx38 protein is as follows: MGRYNVILVWSLALTLCLIPYTTFAQLSPNHYANICPNVQSIVRSAVQKKFQQTFVTVPATLRLFFHDCFVQGCDASVLVASSGNNKAEKDHPENLSLAGDGFDTVIKAKAALDAVPQCRNKVSCADILALATRDVINLAGGPSYTVELGR FDGLVSRSSDVNGRLPQPSFNLNQLNTLFANGLTQTDMIALSGAHTLGFSHCDRFSNRIQTPVDPTLNKQYAAQLQQMCPRNVDPRIAINMDPTTPRTFDNVYYKNLQQGKGLFTSDQILFTDTRSRNTVNSFATNGNVFNANFITAMTKLGRVGVKNARNGKIRTDCSVL (SEQ ID NO. 2).

[0039] The nucleotide sequence of primers MtPrx38-F / MtPrx38-R is shown below: MtPrx38-F:GGGTCGTTATAATGTTATACTTGTGTGG (SEQ ID NO.3); MtPrx38-R: CAGAACAATCGGTACGAATTTTCCC (SEQ ID NO. 4).

[0040] Test Example 1 Medicago truncatula MtPrx38 Screening and verification test of homozygous mutants of genes Purchased from the Medicago truncatula mutant library (https: / / medicago-mutant.dasnr.okstate.edu / ) MtPrx38 Gene mutant NF14058 ( MtPrx38 -1) and NF3872 ( MtPrx38 -2). Primers NF14058-F / NF14058-R and NF3872-F / NF3872-R were designed 200 bp before the insertion site. Homozygotes were identified using Tnt1 flanking primers LTR6 / LTR31 and gene-specific primers. The identification results were as follows: Figure 4 As shown in A. The homozygous mutant material was propagated and the harvested seeds were stored at 4°C.

[0041] The nucleotide sequences of primers NF14058-F / NF14058-R, NF3872-F / NF3872-R, LTR6 / LTR31 are as follows: NF14058-F: CCCAACAACTCCTAGAACATTTGAC (SEQ ID NO.5); NF14058-R: GGGGACACTGAATTATTTGGAAC (SEQ ID NO. 6).

[0042] NF3872-F: CAGCCAAGTTTCAATTTGAATCAGCTG (SEQ ID NO.7); NF3872-R: CAACTTGGTCATGGCAGTGATG (SEQ ID NO. 8).

[0043] LTR6: CTCCTCTCGGGGTCGTGGTT (SEQ ID NO.9); LTR31: GCTACCAACCAAACCAAGTCAA (SEQ ID NO. 10).

[0044] Wild-type Medicago truncatula and MtPrx38 After disinfection, the mutant seeds were spread on 1 / 2MS plates and vernalized at 4°C for 48 h. They were transferred to an incubator at 24°C, 16 hours of light and 8 hours of darkness, and cultured for 7 days. Three strains with healthy and consistent growth conditions were selected and mixed for RNA extraction. Refer to the instructions of the RNAprep Pure Plant Total RNA Extraction Kit with the catalog number DP432 of Tiangen Biochemical Company. The integrity of the extracted RNA was detected by gel electrophoresis. Specific primers MtPrx38-F / MtPrx38-R and internal reference primers MtActin7-F / MtActin7-R were designed to detect genes MtPrx38 The first strand of cDNA was synthesized by reverse synthesis according to the instructions of HiScript IV 1st Strand cDNA Synthesis Kit (+gDNAwiper) with catalog number R412-01 from Novozymes Biotech. qPCR experiments were performed using Taq Pro Universal SYBR QPCR Master Mix with cDNA as template. The results of qPCR experiments are shown in the figure. Figure 4 Middle B, Figure 4 As shown in C, the Medicago truncatula mutant MtPrx38 The expression level of the gene was significantly lower than that of wild type Medicago truncatula MtPrx38 The expression level of the gene, MtPrx38 The gene expression level was significantly reduced in the mutant.

[0045] The nucleotide sequences of primers MtPrx38-F / MtPrx38-R and MtActin7-F / MtActin7-R are as follows: MtPrx38-F: CCAGTGGACCCCACATTGAAC (SEQ ID NO. 11); MtPrx38-R: CACGACCCAACTTGGTCATGG (SEQ ID NO. 12).

[0046] MtActin7-F: CCGAGGAGCACCCAGTACTTC (SEQ ID NO. 13); MtActin7-R:CGAAGGATGGCATGTGGGAGTG (SEQ ID NO. 14).

[0047] Test Example 2 MtPrx38 Gene mutant physiological index determination test 1 Test methods 1.1 MtPrx38 Determination of H2O2 Mass Fraction in Gene Mutants Wild-type Medicago truncatula and MtPrx38 Fresh tissues of gene mutants were fully ground in liquid nitrogen, 9 volumes of physiological saline were added, mechanically homogenized in an ice-water bath, centrifuged at 12000 r / min for 10 min, and the supernatant was taken for testing. Three replicates were set for each sample, and the H2O2 mass fraction was determined using the H2O2 kit with the catalog number A064-1-1 of Nanjing Jiancheng Bioengineering Institute.

[0048] 1.2 MtPrx38 Determination of peroxidase activity of gene mutants 0.5 g of fresh leaves from 2-month-old Medicago truncatula seedlings were ground into powder in liquid nitrogen, mixed with 1 mL of 0.05 mol / L PBS, and quickly thawed in a 2 mL centrifuge tube. The mixture was centrifuged at 12000 r / min and 4°C for 15 min, and the supernatant containing POD was collected. Guaiacol was used as a hydrogen donor, and the OD value was measured at 405 nm using a spectrophotometer, and the peroxidase activity was calculated according to the formula.

[0049] 2 Test results 2.1 MtPrx38 Determination results of H2O2 mass fraction in gene mutants The results of the determination of H2O2 mass fraction are as follows Figure 5 As shown in A, compared with wild-type Medicago truncatula, MtPrx38 The H2O2 mass fraction of the gene mutant increased significantly.

[0050] 2.2 MtPrx38 Results of peroxidase activity assay of gene mutants The results of peroxidase activity assay were as follows Figure 5 As shown in B, compared with the wild type Medicago truncatula, MtPrx38 The POD content of the gene mutant was significantly reduced.

[0051] Test Example 3 MtPrx38 Phenotypic identification of gene mutants and lignin content determination test 1 MtPrx38 Phenotypic identification of gene mutants Observation of the wild type Medicago truncatula and MtPrx38 Root phenotypes of mutant seedlings. Wild-type Medicago truncatula and MtPrx38 Root growth of gene mutants Figure 6 As shown, Figure 6 A is wild type Medicago truncatula and MtPrx38 Root phenotypes of gene mutants; Figure 6 B is wild type Medicago truncatula and MtPrx38 Root length statistics of gene mutants; Figure 6 C is wild type Medicago truncatula and MtPrx38 Statistical diagram of lateral root number of gene mutants. The results showed that compared with wild-type Medicago truncatula, MtPrx38 The root length and lateral root number of the gene mutant were significantly higher than those of the wild type Medicago truncatula.

[0052] The seedlings were transplanted into soil and cultured under the same conditions for 1 month, and the phenotypes of the 1-month-old plants were observed. MtPrx38 The plant height and branch number of the gene mutants are as follows Figure 7 As shown, Figure 7 A in the figure is wild type Medicago truncatula and MtPrx38 Phenotypes of plant height and branch number of gene mutants; Figure 7 B is wild type Medicago truncatula and MtPrx38 Statistical graph of plant height of gene mutants; Figure 7 C is wild type Medicago truncatula and MtPrx38 Statistical diagram of the number of branches of gene mutants. The results show that compared with wild-type Medicago truncatula, MtPrx38 The plant height and branch number of the mutant were significantly higher than those of wild-type Medicago truncatula. MtPrx38 Leaf area of ​​gene mutants Figure 8 As shown, Figure 8 A in the figure is wild type Medicago truncatula and MtPrx38 Leaf area phenotype of gene mutants; Figure 8 B is wild type Medicago truncatula and MtPrx38 Leaf area statistics of gene mutants. The results show that compared with wild-type Medicago truncatula, MtPrx38 The leaf area of ​​the mutant was significantly larger than that of wild-type Medicago truncatula. MtPrx38 Secondary branching of gene mutants Fig. 9 As shown, Fig. 9 A in the figure is wild type Medicago truncatula and MtPrx38 Secondary branching phenotype diagram of gene mutants; Fig. 9 B is wild type Medicago truncatula and MtPrx38 The statistical diagram of the growth rate of secondary branches of gene mutants. The results show that compared with wild-type Medicago truncatula, each primary branch has MtPrx38 The growth rate of secondary branches of the gene mutant was significantly higher than that of wild-type Medicago truncatula.

[0053] The above results can prove that MtPrx38 The gene negatively regulates the growth and development of forage grasses such as Medicago truncatula.

[0054] 2 MtPrx38 Determination of lignin content in gene mutants The stem base, middle part and top of 2-month-old Medicago truncatula were dried at 80°C to constant weight, ground and weighed to 3 mg into a centrifuge tube, and the lignin content of wild-type Medicago truncatula and MtPrx38 Lignin content of mutants. Wild-type Medicago truncatula and MtPrx38 The results of lignin content of gene mutants are as follows Fig.10 As shown, the results showed that compared with wild-type Medicago truncatula, MtPrx38 The lignin content in the base, middle and top of the stem of the gene mutant was significantly reduced.

Claims

1. MtPrx38 Gene, MtPrx38 protein 、 contain MtPrx38 Gene expression cassette or containing MtPrx38 The invention discloses the use of a recombinant plant expression vector of a gene in regulating the growth and development of forage grass.

2. The use according to claim 1, characterized in that The regulating forage growth and development is to promote the growth and development of forage.

3. The use according to claim 2, characterized in that: The forage grass growth and development promotion includes: increasing the root length, the number of lateral roots, the plant height, the number of branches, the leaf area or the number of secondary branches of the forage grass; and advancing the flowering time.

4. MtPrx38 Gene, MtPrx38 protein 、 contain MtPrx38 Gene expression cassette or containing MtPrx38 The invention discloses use of a recombinant plant expression vector of a gene in regulating forage quality.

5. The use according to claim 4, characterized in that The forage quality regulation is to improve the forage quality.

6. The use according to claim 5, characterized in that The improving of forage quality includes: reducing the lignin content and cellulose content of the forage.

7. The use according to claim 2 or 5, characterized in that: include: The forage MtPrx38 Gene mutation or silencing MtPrx38 Reduced gene expression may cause defects in the normal function of the MtPrx38 protein.

8. The use according to claim 1 or 4, characterized in that: The forage grass is a legume plant; wherein the legume plant is Medicago truncatula.

9. The use according to claim 1 or 4, characterized in that: The MtPrx38 The nucleotide sequence of the CDS of the gene is selected from the polynucleotide sequences described in (a) or (b) below: (a) the polynucleotide sequence shown in SEQ ID NO.1; (b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

10. The use according to claim 1 or 4, characterized in that: The amino acid sequence of the MtPrx38 protein is the amino acid sequence shown in SEQ ID NO.2.

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