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 Prx38-1 and Prx38-2 of the medicago truncatula MtPrx38 gene mutant; rT-PCR (reverse transcription-polymerase chain reaction) detection shows that the expression quantity of the MtPrx38 gene is obviously reduced, and compared with wild medicago truncatula, the peroxidase activity of the mutant homozygotes Prx38-1 and Prx38-2 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 application relates to a new use of a gene isolated from Leguminosae plants, in particular to the use of a gene MtPrx38 isolated from Medicago truncatula and a protein encoded by the gene in regulating the growth and development or quality of forage grasses, and belongs to the field of application of the gene MtPrx38 and the protein encoded by the gene. BACKGROUND

[0002] Peroxidase (PRX) is an important antioxidant enzyme, and as a superfamily, the family plays an important role in various physiological activities of plants. According to different functions, peroxidases are divided into Class I, Class II and Class III. Class I includes ascorbate peroxidase, cytochrome c peroxidase and hydrogen peroxide peroxidase (a fusion protein with two catalytic activities); Class II includes secretory fungal peroxidases (such as manganese peroxidase and lignin peroxidase); Class III is a plant secretory 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 the functional redundancy between genes, the physiological functions of most proteins in the family are not clear.

[0003] There are 73 and 138 gene Class III family members in Arabidopsis and rice, respectively, but so far only a few Class III Prx genes have been revealed, and most of the functions have not been reported, especially in alfalfa, there is no report of Class III plant peroxidase participating in the regulation of growth and development or quality.

[0004] Medicago truncatula is a typical annual legume herb, and its natural population type is diverse, with rich mutant and ecotype resources. In addition, it has a short growth cycle, a small genome, stable and efficient genetic transformation, and is highly representative in legume genetics research. It is closely related to Medicago sativa, which is known as the "king of forage grasses". At present, it has become an important model plant for studying legumes. SUMMARY

[0005] The main purpose of the present application is to apply the MtPrx38 gene, the MtPrx38 protein, the expression cassette containing the MtPrx38 gene or the recombinant plant expression vector containing the MtPrx38 gene to regulate the growth and development or the quality of forage grass.

[0006] In order to achieve the above-mentioned purpose, the main technical solutions adopted by the present application include: One aspect of the present application is to apply the MtPrx38 gene, the MtPrx38 protein, the expression cassette containing the MtPrx38 gene or the recombinant plant expression vector containing the MtPrx38 gene to regulate the growth and development of forage grass.

[0007] In a preferred embodiment of the present application, the regulation of the growth and development of forage grass is to promote the growth and development of forage grass.

[0008] In a preferred embodiment of the present application, the promotion of 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 forage grass; and advancing the flowering time.

[0009] Another aspect of the present application is to apply the MtPrx38 gene, the MtPrx38 protein, the expression cassette containing the MtPrx38 gene or the recombinant plant expression vector containing the MtPrx38 gene to regulate the quality of forage grass.

[0010] In a preferred embodiment of the present application, the regulation of the quality of forage grass is to improve the quality of forage grass.

[0011] In a preferred embodiment of the present application, the improvement of the quality of forage grass includes: reducing the lignin content and the cellulose content of forage grass.

[0012] In a preferred embodiment of the present application, the forage grass is a legume; and the legume is Medicago truncatula.

[0013] For reference, the present application provides an implementation method. That is, by mutating or silencing the MtPrx38 gene in plants, the expression amount of the MtPrx38 gene is reduced or the normal function of the MtPrx38 protein is defective, so as to promote the growth and development of forage grass or improve the quality of forage grass, thereby increasing the root length, the number of lateral roots, the leaf area, the plant height, the number of lateral branches or the number of secondary branches of forage grass; advancing the flowering time; and reducing the lignin content and the cellulose content of forage grass.

[0014] In a preferred embodiment of the present application, a method for promoting the growth and development of forage grass or improving the quality of forage grass includes: mutating or silencing the MtPrx38 gene in plants, so as to reduce the expression amount of the MtPrx38 gene or cause the normal function of the MtPrx38 protein to be defective.

[0015] The mutation includes substitution, deletion and / or addition of one or more nucleotides on the nucleotide sequence of the MtPrx38 gene or its promoter. Preferably, the mutation can be obtained by physical mutagenesis, chemical mutagenesis, gene editing. The physical mutagenesis includes but is not limited to radiation mutagenesis, space breeding, etc.; the method of chemical mutagenesis includes mutagenesis caused by treatment with mutagenic agents such as EMS; the method of gene editing includes but is not limited to ZFN, TALE and / or CRISPR / Cas, etc.

[0016] The skilled person in the art can use conventional gene knockout or gene editing techniques to knockout the MtPrx38 gene in the plant, for example, constructing a MtPrx38 gene knockout vector or using a gene editing technique to construct a CRISPR / Cas9 gene editing vector of the MtPrx38 gene, etc., to knockout or mutate the MtPrx38 gene in the plant, which are all familiar to the skilled person in the art.

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

[0018] The interference with the normal expression or normal function of the MtPrx38 gene or its promoter can use RNA interference technology (RNAi) to interfere with the normal expression of the coding gene of the MtPrx38 protein or its promoter or make its normal function defective. The RNA interference technology is a conventional technology in the art, which specifically binds to the homologous region of the mRNA expressed by the target gene through 21-23 bp short-chain double-stranded RNA (siRNA) or long-chain double-stranded RNA (dsRNA; double-strand RNA), degrades the mRNA, and achieves the effect of inhibiting gene expression.

[0019] Another aspect of the present application is to provide a MtPrx38 gene or its encoded protein from Medicago truncatula, which can regulate the growth and development or quality of forage grass.

[0020] The nucleotide sequence of the CDS of the MtPrx38 gene in the present application is selected from any one of the nucleotide sequences in (a)-(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 capable of hybridizing to the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, which still has the function of regulating the growth and development or quality of forage grasses; (d) a polynucleotide sequence having at least 95% or more identity to the polynucleotide sequence described in any one of (a) to (c), which still has the function of regulating the growth and development or quality of forage grasses; (e) a polynucleotide sequence complementary to the polynucleotide sequence described in any one of (a) to (d), which still has the function of regulating the growth and development or quality of forage grasses.

[0021] The percentage of sequence identity described in the present application can be obtained by 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's algorithm, which are well known to those skilled in the art.

[0022] A person of ordinary skill in the art can easily mutate the nucleotide sequence of the MtPrx38 gene using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the MtPrx38 gene, as long as the encoded protein has the function of regulating the growth and development or quality of forage grasses, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

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

[0024] The amino acid sequence of the MtPrx38 protein described in the present application is selected from any one of the following (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, which still has the function or activity of regulating the growth and development or quality of forage grasses; (c) a protein variant obtained by inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2, which still has the function or activity of regulating the growth and development or quality of forage grasses; (d) a protein having 80% or more identity to the amino acid sequence shown in SEQ ID NO. 2, which protein still has the function or activity of regulating the growth and development or quality of forage grass.

[0025] The MtPrx38 protein described in the present application can be artificially synthesized, or the encoding gene thereof can be synthesized first and then expressed biologically.

[0026] The main beneficial technical effects of the present application are: (1) The present application obtains the pure homozygote MtPrx38-1 and MtPrx38-2 of Medicago truncatula MtPrx38 gene mutant through genome PCR and RT-PCR identification. Compared with the wild type of Medicago truncatula, the mass percentage content of H2O2 in the pure homozygote MtPrx38-1 and MtPrx38-2 is significantly increased, and the peroxidase activity is significantly reduced.

[0027] (2) Through phenotype observation and related data statistics, it can be seen that compared with the wild type of Medicago truncatula, the root length, lateral root number, plant height, branch number, leaf area, and secondary branch number of the MtPrx38 gene mutant are all significantly increased; the flowering time is advanced, that is, the growth performance of the MtPrx38 gene mutant is improved; the lignin content and cellulose content are significantly reduced, that is, the forage quality of the MtPrx38 gene mutant is improved; it is proved that the gene MtPrx38 has the function of regulating the growth and development or quality of forage grass such as Medicago truncatula.

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

[0029] Definitions of terms involved in the present invention Unless otherwise defined, 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 application belongs.

[0030] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides in either single- or double-stranded form, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically indicated otherwise, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, simplifying degenerate codon substitutions) and

[0031] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and to a description of a protein, and vice versa. The terms apply to naturally-occurring amino acid polymers as well as to amino acid polymers in which one or more of the 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 connected by covalent peptide bonds.

[0032] "stringent hybridization conditions" means conditions known in the art to be low in ionic strength and high in temperature. Typically, under stringent conditions, a detectable degree of hybridization of a probe to its target sequence is higher than a detectable degree of hybridization to other sequences (e.g., at least 2-fold over background). Stringent hybridization conditions are sequence dependent, and will be different in varying environmental conditions, with longer sequences being specifically hybridized at higher temperatures. By controlling the stringency or washing conditions of the hybridization, one can identify target sequences that are 100% complementary to the probe. For a detailed discussion of nucleic acid hybridization, see 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 typically chosen to be around 5-10°C lower than the thermal melting point (T m ) for the specific sequence at the specified ionic strength pH. T mThe temperature at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium at 50% occupancy (Tm) under a designated ionic strength, pH, and nucleic acid concentration (as the target sequences are present in excess, at Tm50% of the probes will be occupied at equilibrium). Stringent conditions can 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 can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least twice background hybridization, and in some cases 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 x SSC, and 1% SDS, at 42°C, or 5 x SSC, 1% SDS, at 65°C, with wash in 0.2 x SSC and 0.1% SDS at 65°C. The washes can be for 5, 15, 30, 60, 120 minutes or longer. m The temperature at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium at 50% occupancy (Tm) under a designated ionic strength, pH, and nucleic acid concentration (as the target sequences are present in excess, at Tm50% of the probes will be occupied at equilibrium). Stringent conditions can 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 can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least twice background hybridization, and in some cases 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 x SSC, and 1% SDS, at 42°C, or 5 x SSC, 1% SDS, at 65°C, with wash in 0.2 x SSC and 0.1% SDS at 65°C. The washes can be for 5, 15, 30, 60, 120 minutes or longer. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure 1 is a detailed information of CDS and amino acid sequence of Medicago truncatula MtPrx38 gene; Figure 2 Figure 2 is a nucleotide sequence alignment result of CDS of MtPrx38 gene and CDS of Arabidopsis homologous gene; wherein, Figure 2 Figure 2A is a nucleotide sequence alignment result of CDS of MtPrx38 gene and CDS of Arabidopsis AT5G67400 gene; Figure 2 Figure 2B is a nucleotide sequence alignment result of CDS of MtPrx38 gene and CDS of Arabidopsis AT3G49960 gene; Figure 3 Figure 3 is an amino acid sequence alignment result of MtPrx38 protein and Arabidopsis homologous protein; wherein, Figure 3 Figure 3A is an amino acid sequence alignment result of MtPrx38 protein and Arabidopsis AT5G67400 protein; Figure 3 Figure 3B is an amino acid sequence alignment result of MtPrx38 protein and Arabidopsis AT3G49960 protein; Figure 4 Figure 4 is a screening verification diagram of homozygous mutant of Medicago truncatula MtPrx38 gene; wherein, Figure 4 Figure 4A is a screening diagram of homozygous mutant of Medicago truncatula MtPrx38 gene with Tnt1 insertion; Figure 4 Figure 4B is an expression amount diagram of Medicago truncatula MtPrx38 gene; Figure 4Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 5 Figure 7 is a graph showing the H2O2 mass percentage content and peroxidase activity of wild type Medicago truncatula and MtPrx38 gene mutants; wherein, Figure 5 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 5 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 6 Figure 8 is a graph showing the root growth of wild type Medicago truncatula and MtPrx38 gene mutants; wherein, Figure 6 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 6 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 6 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 7 Figure 9 is a graph showing the plant height and branch number of wild type Medicago truncatula and MtPrx38 gene mutants; wherein, Figure 7 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 7 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 7 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 8 Figure 10 is a graph showing the leaf area of wild type Medicago truncatula and MtPrx38 gene mutants; wherein, Figure 8 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 8 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 9 Figure 11 is a graph showing the secondary branch of wild type Medicago truncatula and MtPrx38 gene mutants; wherein, Figure 9 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 9 Figure 6 is a graph showing the relative expression of MtPrx38 gene in wild type Medicago truncatula and MtPrx38 gene mutants; Figure 10 Figure 12 is a graph showing the lignin content of wild type Medicago truncatula and MtPrx38 gene mutants. DETAILED DESCRIPTION

[0034] The application will be further described in connection with the specific embodiments. The advantages and features of the application will become apparent from such description. It is to be understood that the embodiments are merely exemplary and that no limitations are implied by describing the application in the context of the embodiments. Those skilled in the art will understand that modifications or substitutions can be made to the details and forms of the application without departing from the spirit and scope of the application.

[0035] Cloning and comparative analysis of Medicago truncatula MtPrx38 gene MtPrx38 gene (Gene ID: LOC11405719, MtrunA17_Chr4g0053871) was screened from Medicago truncatula genome by multi-omics analysis. Primers MtPrx38-F / MtPrx38-R were designed for PCR amplification of the full-length sequence of the CDS of the gene. The PCR reaction enzyme was selected as 2x Phanta Flash MasterMix from Novietek Biotech Co., Ltd. (P520). After electrophoresis detection, the product was sent to the company for sequencing. The correct CDS sequence and amino acid sequence were obtained by comparing the sequencing results with the downloaded sequence information. The CDS sequence and amino acid sequence of Medicago truncatula MtPrx38 gene are shown in detail in Figure 1 .

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

[0037] The nucleotide sequence of the CDS of the MtPrx38 gene is set forth below: ATGGGTCGTTATAATGTTATACTTGTGTGGTCACTAGCATTAACGTTGTGTCTTATTCCATATACAACTTTTGCTCAACTTAGTCCAAACCATTATGCTAACATTTGTCCTAATGTTCAAAGCATTGTAAGATCAGCAGTTCAAAAGAAATTTCAACAAACTTTTGTTACTGTTCCTGCTACCCTTCGTCTCTTCTTTCATGATTGTTTTGTTCAGGGTTGTGATGCTTCAGTTTTGGTGGCATCAAGTGGAAATAACAAAGCTGAAAAGGATCATCCTGAAAATTTGTCATTGGCTGGTGATGGTTTTGACACAGTGATTAAAGCAAAAGCTGCATTAGATGCTGTTCCACAGTGCAGAAACAAAGTTTCTTGTGCTGATATTCTTGCTTTGGCTACTCGAGATGTTATTAATCTGGCTGGTGGTCCATCCTATACAGTAGAATTGGGAAGGTTTGATGGTCTAGTTTCAAGATCTTCAGATGTAAATGGAAGGCTCCCTCAGCCAAGTTTCAATTTGAATCAGTTGAATACTCTCTTTGCAAACAATGGACTCACTCAAACAGACATGATTGCCCTATCAGGTGCACACACTCTTGGATTCTCTCACTGTGACAGATTCTCCAATAGAATTCAAACTCCAGTGGACCCCACATTGAACAAACAATATGCTGCCCAGTTACAACAAATGTGCCCTAGGAATGTTGATCCAAGAATAGCCATCAACATGGACCCAACAACTCCTAGAACATTTGACAATGTTTACTACAAAAATCTTCAACAAGGAAAAGGTTTATTTACTTCTGATCAAATCCTCTTCACTGACACAAGATCAAGGAACACTGTTAACTCTTTCGCGACTAACGGTAACGTTTTCAATGCCAATTTCATCACTGCCATGACCAAGTTGGGTCGTGTTGGAGTTAAAAATGCGAGAAATGGGAAAATTCGTACCGATTGTTCTGTGCTTTAA (SEQ ID NO: 1)ID NO.1)

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

[0039] The nucleotide sequences of the primers MtPrx38-F / MtPrx38-R are shown as follows: MtPrx38-F: GGGTCGTTATAATGTTATACTTGTGTGG (SEQ ID NO. 3); MtPrx38-R: CAGAACAATCGGTACGAATTTTCCC (SEQ ID NO. 4).

[0040] Test Example 2 Screening and verification test of homozygous mutant of Medicago truncatula MtPrx38 gene MtPrx38 gene mutants NF14058 (MtPrx38-1) and NF3872 (MtPrx38-2) were purchased from Medicago truncatula mutant library (https: / / medicago-mutant.dasnr.okstate.edu / ). Primers NF14058-F / NF14058-R, NF3872-F / NF3872-R were designed respectively 200 bp before the insertion site. Using Tnt1 flanking primers LTR6 / LTR31 and gene-specific primers, homozygotes were identified, and the identification results are shown in Table A. Figure 4 The obtained homozygous mutant materials were 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] After the wild type Medicago truncatula and the mutant seed of MtPrx38 gene were sterilized, they were placed on 1 / 2MS plates and placed at 4°C for 48 h. Then they were transferred to a culture box and cultured at 24°C for 7 days under the condition of 16 hours light and 8 hours darkness. Three strains with healthy and consistent growth state were selected and mixed to extract RNA. The RNAprep Pure Plant Total RNA Extraction Kit (DP432) of Tiangen Biochemical Technology Co., Ltd. was referred to for the extraction of RNA. 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 the expression of the gene MtPrx38. The cDNA first strand was synthesized by referring to the instructions of HiScript IV 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R412-01) of Novoprotein Biological Technology Co., Ltd. The qPCR experiment was performed by using Taq Pro Universal SYBR Qpcr Master Mix with cDNA as the template. The results of the qPCR experiment are shown in Tables 1-3 and Figs. 1-3. Figure 4 Table 1 Figure 4 Table 2

[0045] The nucleotide sequences of primers MtPrx38-F / MtPrx38-R, 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 3 Transcriptional sequencing analysis of expression of MtPrx38 gene mutant The Mtprx38 gene mutants NF14058 and NF3872 purchased from the Medicago truncatula mutant website are Tnt1 sequence inserted in the MtPrx38 gene, which causes the transcriptional expression amount of the MtPrx38 gene to be significantly reduced compared with wild-type Medicago truncatula. In the mutants NF14058 (MtPrx38-1) and NF3872 (MtPrx38-2), although there are Tnt1 sequence insertions in other genes, the transcriptional expression amount of these inserted genes does not change significantly compared with wild-type Medicago truncatula, and some Tnt1 insertions are in intron positions; only the Tnt1 sequence insertion in the MtPrx38 gene is inserted in the non-intron positions such as exons and 3'-UTR of the MtPrx38 gene and causes the transcriptional expression amount of the MtPrx38 gene in the two mutants to be significantly reduced compared with wild-type. Therefore, the phenotype of the mutants NF14058 and NF3872 is only the phenotype produced by the mutation of the MtPrx38 gene (genotype), and the specific verification method is as follows: Fresh young tissues of Mtprx38 gene mutant and wild type Medicago truncatula R108 were taken respectively, immediately frozen in liquid nitrogen, RNA was extracted, cDNA was reverse transcribed, sequencing library was constructed, sequencing was performed, bioinformatics analysis was performed, and differentially expressed genes of the mutant relative to the wild type were obtained. Input the number of MtPrx38-1 mutant in the Medicago mutant website (medicago-mutant.dasnr.okstate.edu), that is: NF14058, and all flanking sequences of the mutant can be obtained or displayed (https: / / medicago-mutant.dasnr.okstate.edu / mutant / lineseq.php?id=NF14058&hlvalue=high). The following is the full flanking sequence of Tnt1 insertion of Medicago truncatula mutant NF14058 (MtPrx38-1): >NF14058_high_1 signature: GAGATGGACATCATCA 5' end >NF14058_high_10 signature: AATGAGATGGACATCATCA 5' end >NF14058_high_11 signature: AATGAGATGGACATCATCA 5' end >NF14058_high_12 signature: TGAGATGGACATCATCA 5' end >NF14058_high_13 signature: AGATGGACATCATCA 5' end >NF14058_high_14 signature: GAGATGGACATCATCA 5' end >NF14058_high_15 signature: TGAGATGGACATCATCA 5' end >NF14058_high_16 signature: GAGATGGACATCATCA 5' end > NF14058_high_17 signature: TGAGATGGACATCATCA 5' end > NF14058_high_18 signature: No signature > NF14058_high_19 signature: AATGAGATGGACATCATCA 5' end > NF14058_high_2 signature: ATGAGATGGACATCATCA 5' end > NF14058_high_20 signature: AATGAGATGGACATCATCA 5' end > NF14058_high_21 signature: TGAGATGGACATCATCA 5' end > NF14058_high_22 signature: GAGATGGACATCATCA 5' end > NF14058_high_23 signature: AATGAGATGGACATCATCA 5' end > NF14058_high_24 signature: TGAGATGGACATCATCA 5' end > NF14058_high_3 signature: GAGATGGACATCATCA 5' end > NF14058_high_4 signature: GAGATGGACATCATCA 5' end >NF14058_high_5 signature: ATGAGATGGACATCATCA 5' end >NF14058_high_6 signature: ACATCATCA 5' end >NF14058_high_7 signature: AATGAGATGGACATCATCAA 5' end NF14058_high_8 signature: TGAGATGGACATCATCA 5' end >NF14058_high_9 signature: GATGGACATCATCA 5' end All the above flanking sequences were blasted on Medicago genome website (https: / / medicago.toulouse.inra.fr / MtrunA17r5.0-ANR / ), and 122 sequences (these 122 sequences belong to 112 genes) were obtained. The Tntl insertion sites of these genes and their expression in the transcriptome sequencing data of mutant NF14058 and wild type Medicago truncatula were analyzed. Table 1 is the Tntl insertion sites of these 122 sequences and their expression in the transcriptome sequencing data of mutant NF14058 and wild type Medicago truncatula. According to Table 1, the MtrunA17_Chr4g0053871 gene (which is the MtPrx38 gene described in the application) in the transcriptome sequencing data of the mutant NF14058 has a gene expression difference fold of 0.30 compared with the wild type, is significantly down-regulated, the expression amount of the MtPrx38 gene of the Medicago truncatula mutant is significantly lower than that of the wild type Medicago truncatula MtPrx38 gene, and the expression amount of the MtPrx38 gene in the mutant is significantly reduced. Among the remaining 111 genes, 18 genes have no flanking sequence tags, and no Tnt1 sequence insertion, 79 genes have Tnt1 sequence insertion at the intron position, the intron is a non-coding DNA fragment, which does not cause the change of the expression amount, and in the transcriptome sequencing data, the expression fold of the 97 genes in the mutant NF14058 compared with the wild type Medicago truncatula is not significantly different from that of the wild type Medicago truncatula; in addition, the Tnt1 sequence of 14 genes is inserted at the exon position, but in the transcriptome data, the expression fold of the 14 genes in the mutant NF14058 compared with the wild type Medicago truncatula is not significantly different from that of the wild type Medicago truncatula; the above results show that although the Tnt1 sequence in the mutant NF14058 described in the application is inserted, it does not cause significant change of the expression amount of the 111 genes, therefore, the mutant NF14058 (MtPrx38-1) does not have mutation of the above 111 genes, and only has mutation of the target gene MtPrx38. Similarly, by inputting the number of the MtPrx38-2 mutant, i.e. NF3872, in the Medicago truncatula mutant website (medicago-mutant.dasnr.okstate.edu), all flanking sequences of the mutant can be obtained or displayed (https: / / medicago-mutant.dasnr.okstate.edu / mutant / lineseq.php?id=NF3872&hlvalue=high). The complete flanking sequence of the Tnt1 insertion of the Medicago truncatula mutant NF3872 (MtPrx38-2 mutant) is as follows: >NF3872_high_1 signature: GAGATGGACATCATCA 5' end NF3872_high_10 signature: GAGATGGACATCATCA 5' end NF3872_high_11 signature: ATGAGATGGACATCATCA 5' end NF3872_high_12 signature: ATGAGATGGACATCATCA 5' end NF3872_high_13 signature: GAGATGGACATCATCA 5' end NF3872_high_14 signature: TGAGATGGACATCATCA 5' end NF3872_high_15 signature: GAGATGGACATCATCA 5' end NF3872_high_16 signature: No signature NF3872_high_17 signature: AATGAGATGGACATCATCA 5' end NF3872_high_18 signature: GAGATGGACATCATCA 5' end NF3872_high_19 signature: GATGGACATCATCA 5' end NF3872_high_9 signature: TGAGATGGACATCATCA 5' end NF3872_high_2 signature: AGATGGACATCATCA 5' end > NF3872_high_20 signature: GAGATGGACATCATCA 5' end > NF3872_high_21 signature: GAGATGGACATCATCA 5' end > NF3872_high_22 signature: No signature > NF3872_high_23 signature: AATGAGATGGACATCATCAA 5' end > NF3872_high_24 signature: No signature > NF3872_high_25 signature: AATGAGATGGACATCATCA 5' end > NF3872_high_26 signature: GATGGACATCATCA 5' end > NF3872_high_27 signature: AGATGGACATCATCA 5' end > NF3872_high_28 signature: TGAGATGGACATCATCA 5' end > NF3872_high_29 signature: GAGATGGACATCATCA 5' end > NF3872_high_3 signature: ATGAGATGGACATCATCA 5' end > NF3872_high_30 signature: No signature NF3872_high_31 signature: No signature NF3872_high_32 signature: No signature NF3872_high_33 signature: No signature NF3872_high_34 signature: GAGATGGACATCATCA 5' end NF3872_high_35 signature: No signature NF3872_high_36 signature: AGATGGACATCATCA 5' end NF3872_high_37 signature: AGATGGACATCATCA 5' end NF3872_high_38 signature: No signature NF3872_high_39 signature: GATGGACATCATCA 5' end NF3872_high_4 signature: ATGAGATGGACATCATCA 5' end NF3872_high_40 signature: No signature NF3872_high_41 signature: TGAGATGGACATCATCA 5' end >NF3872_high_4 signature: ATGAGATGGACATCATCA 5' end >NF3872_high_5 signature: GAGATGGACATCATCA 5' end >NF3872_high_6 signature: ATGGACATCATCA 5' end >NF3872_high_7 signature: AGATGGACATCATCA 5' end >NF3872_high_8 signature: ATGAGATGGACATCATCA 5' end All the above flanking sequences were blasted against the Medicago truncatula genome website (https: / / medicago.toulouse.inra.fr / MtrunA17r5.0-ANR / ), and a total of 151 sequences (these 151 sequences belong to 128 genes) were obtained. The Tnt1 insertion sites of these genes and their expression in the transcriptome sequencing data of mutant NF14058 and wild-type Medicago truncatula were analyzed. According to Table 2, the MtrunA17_Chr4g0053871 gene (which is the MtPrx38 gene described in the application) in the transcriptome sequencing data of the mutant NF3872 has a gene expression difference fold of 0.23 compared with the wild type, is significantly down-regulated, the expression amount of the MtPrx38 gene of the mutant Medicago polymorpha is significantly lower than that of the wild type Medicago polymorpha, and it is indicated that the expression amount of the MtPrx38 gene in the mutant is significantly reduced. Among the remaining 127 genes, 31 have no flanking sequence tags, and no Tnt1 sequence insertion, and the Tnt1 sequence of the other 71 genes is inserted in the intron position. The intron is a non-coding DNA fragment, which does not cause a change in the expression amount, and in the transcriptome sequencing data, the expression fold of the above 102 genes in the mutant NF3872 compared with the wild type Medicago polymorpha is not significantly different from that of the wild type Medicago polymorpha; the Tnt1 sequence of the other 25 genes is inserted in the exon position, but in the transcriptome data, the expression fold of the 25 genes in the mutant NF3872 compared with the wild type Medicago polymorpha is not significantly different from that of the wild type Medicago polymorpha; the above results show that although the Tnt1 sequence in the mutant NF3872 described in the application is inserted, it does not cause a significant change in the expression amount of the 127 genes, and therefore, the mutant NF3872 does not have mutations in the above 127 genes, and only has a mutation in the target gene MtPrx38.

[0048] Test Example 4: Determination of physiological indexes of the MtPrx38 gene mutant 1. Test method 1.1 Determination of H2O2 mass percentage content of the MtPrx38 gene mutant The wild type Medicago polymorpha and the fresh tissues of the MtPrx38 gene mutant were ground in liquid nitrogen, 9 times the volume of physiological saline was added, and mechanical homogenization was carried out under ice water bath conditions, centrifuged at 12000 r / min for 10 min, and the supernatant was taken for determination. Three replicates were set for each sample, and the H2O2 reagent kit with the product number A064-1-1 of Nanjing Jiancheng Biological Engineering Institute was used to determine the H2O2 mass percentage content.

[0049] 1.2 Determination of peroxidase activity of the MtPrx38 gene mutant 0.5 g of fresh leaves of 2-month-old Medicago polymorpha seedlings were ground into powder in liquid nitrogen, mixed with 1 mL of 0.05 mol / L PBS in a 2 mL centrifuge tube, quickly thawed, and then centrifuged at 12000 r / min at 4℃ for 15 min, and the supernatant containing POD was collected. With guaiacol as the hydrogen donor, the OD value was determined at 405 nm by spectrophotometry, and the peroxidase activity was calculated according to the formula.

[0050] 2. Test results 2.1 MtPrx38 gene mutant H2O2 mass percentage content determination results H2O2 mass percentage content determination results are shown in FIG. 2.1, wherein, Figure 5 compared with wild type Medicago polymorpha, the H2O2 mass percentage content of the MtPrx38 gene mutant was significantly increased.

[0051] 2.2 MtPrx38 gene mutant peroxidase activity determination results Peroxidase activity determination results are shown in FIG. 2.2, wherein, Figure 5 compared with wild type Medicago polymorpha, the POD content of the MtPrx38 gene mutant was significantly reduced.

[0052] Test Example 5 Phenotype identification and lignin content determination test of MtPrx38 gene mutant 1 Phenotype identification of MtPrx38 gene mutant The root phenotypes of wild type Medicago polymorpha and MtPrx38 gene mutant seedlings vertically cultured on 1 / 2MS plates for 7 days were observed. The root growth of wild type Medicago polymorpha and MtPrx38 gene mutant Figure 6 are shown in FIG. 3.1, wherein, Figure 6 FIG. 3.1A is a root phenotype diagram of wild type Medicago polymorpha and MtPrx38 gene mutant; Figure 6 FIG. 3.1B is a root length statistical diagram of wild type Medicago polymorpha and MtPrx38 gene mutant; Figure 6 FIG. 3.1C is a lateral root number statistical diagram of wild type Medicago polymorpha and MtPrx38 gene mutant. The results show that compared with wild type Medicago polymorpha, the root length and lateral root number of the MtPrx38 gene mutant are significantly higher than those of wild type Medicago polymorpha.

[0053] The seedlings were transplanted into soil and cultured for 1 month under the same conditions, and the 1-month-old plant phenotypes were observed. The plant height and branch number of wild type Medicago polymorpha and MtPrx38 gene mutant are shown in FIG. 4.1, wherein, Figure 7 Figure 7 FIG. 4.1A is a plant height and branch number phenotype diagram of wild type Medicago polymorpha and MtPrx38 gene mutant; Figure 7 FIG. 4.1B is a plant height statistical diagram of wild type Medicago polymorpha and MtPrx38 gene mutant; Figure 7 FIG. 4.1C is a branch number statistical diagram of wild type Medicago polymorpha and MtPrx38 gene mutant. The results show that compared with wild type Medicago polymorpha, the plant height and branch number of the MtPrx38 gene mutant are significantly higher than those of wild type Medicago polymorpha. The leaf area of wild type Medicago polymorpha and MtPrx38 gene mutant is shown in FIG. 4.2, wherein, Figure 8 Figure 8 FIG. 4.2A is a leaf area phenotype diagram of wild type Medicago polymorpha and MtPrx38 gene mutant; Figure 8 ​​Figure 6B is a chart showing leaf area of wild type Medicago truncatula and MtPrx38 gene mutant. The results show that the leaf area of the MtPrx38 gene mutant is significantly higher than that of the wild type Medicago truncatula. Figure 9 Figure 6A is a chart showing secondary branch of wild type Medicago truncatula and MtPrx38 gene mutant. The results show that the secondary branch of the MtPrx38 gene mutant is significantly higher than that of the wild type Medicago truncatula. Figure 9 Figure 6A is a chart showing secondary branch of wild type Medicago truncatula and MtPrx38 gene mutant. The results show that the secondary branch of the MtPrx38 gene mutant is significantly higher than that of the wild type Medicago truncatula. Figure 9 Figure 6B is a chart showing leaf area of wild type Medicago truncatula and MtPrx38 gene mutant. The results show that the leaf area of the MtPrx38 gene mutant is significantly higher than that of the wild type Medicago truncatula.

[0054] The above results can prove that the MtPrx38 gene negatively regulates the growth and development of Medicago truncatula and other forage grasses.

[0055] 2 Lignin content determination of MtPrx38 gene mutant The stem base, middle and top of 2-month-old Medicago truncatula were dried at 80°C to constant weight, ground and weighed 3 mg into a centrifuge tube. The lignin content of wild type Medicago truncatula and MtPrx38 gene mutant was determined using a lignin content detection kit with product number BC4205 from Solabio. The results of lignin content of wild type Medicago truncatula and MtPrx38 gene mutant are shown in Figure 10 The results show that the lignin content of the stem base, middle and top of the MtPrx38 gene mutant is significantly lower than that of the wild type Medicago truncatula.

Claims

1. The use of the MtPrx38 gene, MtPrx38 protein, expression cassettes containing the MtPrx38 gene, or recombinant plant expression vectors containing the MtPrx38 gene in regulating the growth and development of forage crops.

2. The use according to claim 1, characterized in that, The regulation of forage growth and development mentioned above is to promote forage growth and development.

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

4. The use of the MtPrx38 gene, MtPrx38 protein, expression cassettes containing the MtPrx38 gene, or recombinant plant expression vectors containing the MtPrx38 gene in regulating forage quality.

5. The use according to claim 4, characterized in that, The aforementioned regulation of forage quality refers to improving forage quality.

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

7. The use according to claim 2 or 5, characterized in that, include: Mutating or silencing the MtPrx38 gene in forage can reduce the expression level of the MtPrx38 gene or impair the normal function of the MtPrx38 protein.

8. The use according to claim 1 or 4, characterized in that, The forage is a legume; specifically, alfalfa.

9. The use according to claim 1 or 4, characterized in that, The nucleotide sequence of the CDS of the MtPrx38 gene is selected from the polynucleotide sequence 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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