MtPRP4 gene isolated from Medicago truncatula and its encoded protein and application
By isolating and verifying the MtPRP4 gene and its encoded protein from Medicago truncatula, the problem of unclear function of the PRP gene in Medicago truncatula was solved, growth and development regulation and enhancement of abiotic stress resistance were achieved, and genetic resources for improving alfalfa yield and quality were provided.
Patent Information
- Application Number
- CN202510368422.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-27
- Publication Date
- 2025-09-05
- Estimated Expiration
- 2045-03-27
Smart Images
Figure CN119899850B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of genetic engineering, and particularly relates to an MtPRP4 gene separated from Medicago truncatula, a coded protein and an application thereof. Background Art
[0002] Plant proline-rich proteins (PRPs) are a class of structural proteins rich in proline and hydroxyproline, widely present in plant cell walls. They participate in cell wall construction and plant stress responses. PRPs often contain repetitive sequences within the proline-rich polypeptide region, which play a key role in the specific structure and function of the cell wall. Studies have shown that PRP gene expression can respond to a variety of external stimuli, such as hormones, injury, low temperature, and salt stress. Furthermore, in response to fungal inducers and mechanical damage, PRPs can rapidly oxidatively cross-link with other cell wall components (such as lignin and sugars) to form a protective layer, enhancing the plant's defense capabilities.
[0003] Although the importance of PRP in plant growth, development, and stress response has been widely recognized, there are still many deficiencies in the specific functions of PRP genes and their regulatory mechanisms:
[0004] First, the diversity of PRP gene functions remains largely unexplored. While studies have shown that PRP genes are involved in responses to abiotic stresses (such as drought, high salinity, and low temperature) in plants like Arabidopsis and pigeon pea, significant differences exist in the functions of PRP genes across different plant species. For example, knockout of the EARLI1 gene in Arabidopsis makes plants more susceptible to cold stress, while the CcHyPRP gene in pigeon pea improves the resistance of transgenic Arabidopsis to drought, high salinity, and high temperature. However, research on the specific functions of PRP genes in legumes, such as Medicago truncatula, particularly their roles in growth and development and in responses to abiotic stresses, remains limited.
[0005] Second, research gaps exist regarding PRP genes in Medicago truncatula. As a model legume, Medicago truncatula offers advantages such as a small genome, ease of genetic transformation, and close genetic relationship to various economic crops (such as alfalfa, pea, and broad bean). This makes it an ideal material for studying symbiotic nitrogen fixation, compound leaf development, and stress tolerance genes in legumes. However, while 23 PRP genes have been identified in Medicago truncatula, current research has primarily focused on nodule formation, and the specific roles of these genes in plant growth and development and responses to abiotic stresses have yet to be systematically investigated. In particular, the function of the MtPRP4 gene and its role in regulating growth and stress tolerance in alfalfa remain largely unknown.
[0006] Third, there is a lack of genetic resources to improve alfalfa yield and quality: Alfalfa is an important forage crop, and its yield and quality directly affect the development of animal husbandry. However, there is currently a lack of high-quality genetic resources that can significantly improve alfalfa yield and quality. As a potential stress resistance gene, the functional research of the PRP gene in alfalfa may provide new ideas for solving this problem. However, existing technologies have not yet fully utilized the potential of the PRP gene, especially in the regulation of resistance under abiotic stress, and there is still a large research gap. Summary of the Invention
[0007] Existing research on the functions of PRP genes still has many deficiencies. In particular, in Medicago truncatula, the specific roles of PRP genes in growth and development and abiotic stress responses have not been fully revealed. Therefore, in-depth research on the functions of the MtPRP4 gene will not only help fill the research gap in this field, but also provide new technical approaches for developing genetic resources that improve alfalfa yield and quality. Based on this, the present invention provides an MtPRP4 gene isolated from Medicago truncatula, its encoded protein, and applications, as follows:
[0008] A MtPRP4 gene isolated from Medicago truncatula, characterized in that the MtPRP4 gene is isolated from Medicago truncatula, the nucleotide sequence of the MtPRP4 gene is shown in SEQ ID NO: 1, and the MtPRP4 gene belongs to a proline protein (PRP) gene; the MtPRP4 gene is used to regulate the growth and development of Medicago truncatula and enhance the resistance of Medicago truncatula to abiotic stress.
[0009] Furthermore, the CDS sequence of the MtPRP4 gene is shown in SEQ ID NO: 2.
[0010] Furthermore, the amino acid sequence of the protein encoded by the MtPRP4 gene is shown in SEQ ID NO: 3, and the amino acid sequence is obtained by translating the CDS sequence shown in SEQ ID NO: 2.
[0011] Furthermore, the CDS sequence of the MtPRP4 gene shown in SEQ ID NO: 2 has a sequence identity of 32.77% with the CDS sequence of AT2G21140 in Arabidopsis thaliana, and has a sequence identity of 47.14% with the CDS sequence of AT4G38770 in Arabidopsis thaliana.
[0012] Furthermore, the amino acid sequence of the MtPRP4 gene shown in SEQ ID NO: 3 is 31.00% identical to the amino acid sequence of AT2G21140 in Arabidopsis thaliana, and is 44.75% identical to the amino acid sequence of AT4G38770 in Arabidopsis thaliana.
[0013] In a second aspect, the present invention provides a protein encoded by the MtPRP4 gene. The protein is encoded by the MtPRP4 gene, and the amino acid sequence of the protein is shown in SEQ ID NO: 3.
[0014] Furthermore, the encoded protein is rich in proline and / or hydroxyproline and has the characteristics of a cell wall structural protein.
[0015] In a third aspect, the present invention provides an application of the MtPRP4 gene isolated from Medicago truncatula for regulating the growth and development of Medicago truncatula and enhancing the resistance of Medicago truncatula to abiotic stress.
[0016] Furthermore, the abiotic stress is salt stress.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] 1. This invention isolated the MtPRP4 gene from Medicago truncatula for the first time and verified its function in regulating the growth and development of Medicago truncatula and enhancing resistance to abiotic stress through experiments, filling the gap in the research of PRP genes in Medicago truncatula and providing a new perspective for understanding the function of PRP genes in legumes.
[0019] 2. By studying the function of the MtPRP4 gene, the present invention found that its mutants showed advantages in growth and development, such as enlarged leaves, increased plant height and number of branches. These phenotypic changes indicate that the MtPRP4 gene can be used as a genetic resource to improve alfalfa yield and quality, providing a new target for the genetic improvement of alfalfa.
[0020] 3. During the experiment, it was found that under salt stress treatment, although the MtPRP4 mutant was more sensitive to stress, it proved the important role of the MtPRP4 gene in alfalfa's response to abiotic stress. The MtPRP4 gene or its related genes can be used to improve alfalfa's resistance to abiotic stress, thereby enhancing its survival ability and yield stability under adverse conditions. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 for MtPRP4 Sequence alignment results of the gene with the homologous gene ATG21140 in Arabidopsis thaliana; 1 (A) is Medicago truncatula MtPRP4 Arabidopsis thaliana ATG21140 CDS sequence alignment results; 1 (B) is Medicago truncatula MtPRP4 Arabidopsis thaliana ATG21140 Amino acid sequence alignment results.
[0022] Figure 2 MtPRP4 Comparison of the amino acid sequence of the gene with that of the homologous gene in Arabidopsis; 2 (A) is Medicago truncatula MtPRP4 Arabidopsis thaliana AT4G38770 CDS sequence alignment results; 2 (B) is Medicago truncatula MtPRP4 Arabidopsis thaliana AT4G38770 Amino acid sequence alignment results.
[0023] Figure 3 for MtPRP4-1, MtPRP4-2 Schematic diagram of the mutant insertion site.
[0024] Figure 4 Medicago truncatula MtPRP4 Homozygous material of gene Tnt1 mutant; 4 (A) is Medicago truncatula MtPRP4 Results of homozygous screening of Tnt1 insertion mutants; 4 (B) is a mutant MtPRP4 Gene RT-PCR results.
[0025] Figure 5 Medicago truncatula MtPRP4 Root phenotypes of mutants; 5 (A) is Medicago truncatula MtPRP4 Root phenotype of mutants, 5 (B) is Medicago truncatula MtPRP4 Root length statistics of mutants.
[0026] Figure 6 Medicago truncatula MtPRP4 Phenotypic results of plant height and branch number of mutants at 60 days; 6 (A) is Medicago truncatula MtPRP4 Figure 6 (B) shows the plant height and branching phenotype of the mutant; Figure 6 (B) shows Medicago truncatula MtPRP4 Statistical diagram of plant height of mutants; 6 (C) is Medicago truncatula MtPRP4 Statistics of branch numbers of mutants.
[0027] Figure 7 Medicago truncatula MtPRP4 Leaf area phenotype of mutants; 7 (A) is Medicago truncatula MtPRP4 Leaf area phenotype of mutants; 7 (B) is Medicago truncatula MtPRP4 Leaf area statistics of mutants.
[0028] Figure 8 The phenotypic results of Medicago truncatula under 150mmol / L NaCl stress in hydroponic culture; 8 (A) is the phenotypic results of Medicago truncatula under 150mmol / L NaCl stress MtPRP4 Mutant phenotype; 8 (B) is Medicago truncatula under 150mmol / L NaCl MtPRP4 Superoxide dismutase content of mutants; 8 (C) is Medicago truncatula under 150mmol / L NaCl MtPRP4 Proline content. DETAILED DESCRIPTION
[0029] Example 1 Medicago truncatula MtPRP4Gene cloning
[0030] The NCBI website tool Blast was used to search for closely homologous sequences in Arabidopsis thaliana. AT2G21140、AT4G38770 The gene sequence and amino acid sequence were compared to find the Medicago truncatula A17 ecotype MtPRP4 (GeneID: LOC25492843, MtrunA17_Chr4g0038461) full-length sequence and CDS sequence, design PCR amplification upstream primers MtPRP4 -F (SEQ ID NO: 4) and downstream primers MtPRP4 -R (SEQ ID NO: 5). The PCR reaction enzyme used was Novozymes Biotech's high-fidelity 2xPhanta Flash Master Mix (Cat. No. P520) to ensure the fidelity of the amplified product and reduce sequence mutations caused by manipulation. After electrophoresis, the product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the electronic sequence to obtain the correct nucleotide sequence (SEQ ID NO: 1), CDS sequence (SEQ ID NO: 2), and amino acid sequence (SEQ ID NO: 3).
[0031] MtPRP4 The CDS sequence of the gene AT2G21140 、 AT4G38770 The multiple sequence alignment showed that the identities were 32.77% and 47.14%, respectively. Figure 1 (A), Figure 2 (A). MtPRP4 The amino acid sequence of the gene AT2G21140 、 AT4G38770 Multiple sequence alignment was performed, and the identities were 31.00% and 44.75%, see Figure 1 (B), Figure 2 (B).
[0032] The results showed that MtPRP4 Arabidopsis thaliana AT2G21140 、 AT4G38770 The gene CDS and amino acid sequences are significantly different. MtPRP4 This gene is a new gene that has not been reported before.
[0033] Example 2 MtPRP4 Screening of Tnt1 insertion homozygous mutants
[0034] Purchased from the Medicago truncatula mutant library (https: / / medicago-mutant.dasnr.okstate.edu / ) MtPRP4 Mutant NF14275 ( MtPRP4-1 ) and NF16150 ( MtPRP4-2), the insertion sites of the two mutants are as follows Figure 3 As shown. Upstream primers NF14275-F (SEQ ID NO: 6) and NF16150-F (SEQ ID NO: 7) were designed 200 bp before the insertion site, and downstream primers NF14275-R (SEQ ID NO: 8) and NF16150-R (SEQ ID NO: 9) were designed 200 bp after the insertion site. Homozygotes were identified using Tnt1 flanking primers LTR6 (SEQ ID NO: 10) and LTR31 (SEQ ID NO: 11) and gene-specific primers. Figure 4 (A). The homozygous mutant material was propagated and the harvested seeds were stored at 4°C. MtPRP4 After disinfecting the mutant seeds, they were spread on 1 / 2MS plates and vernalized at 4°C for 48 hours. They were transferred to an incubator at 24°C with 16 hours of light and 8 hours of darkness and cultured for 7 days. Three plants with healthy and consistent growth conditions were selected and mixed for RNA extraction. The integrity of the extracted RNA was checked by gel electrophoresis according to the instructions of Tiangen Biochemical's RNAprep Pure Plant Total RNA Extraction Kit (Cat. No. DP432). Specific primers were designed. MtPRP4 -F ， (SEQ ID NO: 12) and MtPRP4 -R ， (SEQ ID NO: 13), internal reference gene primers MtActin7 -F (SEQ ID NO: 14:) and MtActin7 -R (SEQ ID NO: 15) to detect gene expression. st The first strand of cDNA was synthesized by reverse synthesis using the Strand cDNA Synthesis Kit (+gDNAwiper) (Cat. No. R412-01). RT-PCR was performed using the cDNA as a template using Taq Pro Universal SYBR qPCR Master Mix. Figure 4 (B).
[0035] The results showed that the gene MtPRP4 In mutant NF14275 ( MtPRP4-1 ) and NF16150 ( MtPRP4-2 ) is not expressed.
[0036] Example 3 MtPRP4 Mutant phenotype identification
[0037] Observation of the wild type Medicago truncatula and MtPRP4 The root phenotypes of mutant seedlings are shown in Figure 5 , Figure 5 Medicago truncatula MtPRP4 Root phenotype of mutants, 5 (A) is Medicago truncatula MtPRP4 Root phenotype of mutants, 5 (B) is Medicago truncatula MtPRP4 Root length statistics of mutants. The results show that the root length of mutants is significantly longer than that of wild type.
[0038] Use sandpaper to gently break the seed coat to allow it to absorb water and swell. Then place the seeds in a petri dish lined with moist filter paper and move them to a 4°C refrigerator for vernalization for 3 days. Move them to a light incubator for normal cultivation. When the seed roots are about 1-2 cm long, move the seedlings to a 10 cm diameter square pot covered with nutrient soil and place them in the incubator for cultivation. Greenhouse growth conditions are: 16 hours of light, 8 hours of darkness, temperature 22-24°C, humidity 60%-70%, and light intensity 150 μmol / m -2 s -1 The phenotypes of 2-month-old plants were observed under the same conditions. Figure 6 、 Figure 7 .
[0039] Figure 6 Medicago truncatula MtPRP4 Phenotypes of plant height and branch number of mutants at 60 days old. 6 (A) is Medicago truncatula MtPRP4 Figure 6 (B) shows the plant height and branching phenotype of the mutant; Figure 6 (B) shows Medicago truncatula MtPRP4 Statistical diagram of plant height of mutants; 6 (C) is Medicago truncatula MtPRP4 Statistics of branch numbers of mutants.
[0040] Figure 7 Medicago truncatula MtPRP4 Leaf area phenotype of mutants; 7 (A) is Medicago truncatula MtPRP4 Leaf area phenotype of mutants; 7 (B) is Medicago truncatula MtPRP4 Leaf area statistics of mutants.
[0041] The results showed that the plant height, branch number and leaf area of the mutant material increased compared with the wild type. The number of secondary branches on each primary branch of the wild type and mutant was counted separately. The results showed that the number of secondary branches on each primary branch of the mutant was higher than that of the wild type. MtPRP4 Genes affecting growth and development of Medicago truncatula.
[0042] Example 4 MtPRP4 Determination of physiological indicators of mutants
[0043] Combine wild type and MtPRP4The mutants were hydroponically cultured under the following conditions: seedlings with roots 1-2 cm long were placed in a planting sponge containing 1 / 2 Hoagland nutrient solution, with the roots in contact with the liquid surface. After 14 days of growth, 150 mmol / L NaCl was applied, and the phenotypes and physiological parameters were observed ( Figure 8 ), phenotype diagram see Figure 8 (A).
[0044] 0.1 g of fresh plant tissue was thoroughly ground in liquid nitrogen, 1 ml of extract was added, and the mixture was homogenized in an ice bath. Centrifuged at 8000 g for 10 min at 4°C, the supernatant was placed on ice, and the absorbance OD value was recorded at a wavelength of 560 nm. Three replicates were set for each sample. The SOD kit (Cat. No. BC0170) from Beijing Solebaugh Technology Co., Ltd. was used to calculate the superoxide dismutase (SOD) content using the formula. The results are shown in the table. Figure 8 (B).
[0045] Weigh approximately 0.1 g of fresh plant sample, add 1 mL of extract solution, and homogenize in an ice bath. Then, place in a boiling water bath and shake for 10 minutes. Centrifuge at 10,000 g for 10 minutes at room temperature. Collect the supernatant, cool, and measure the OD value at 520 nm using a spectrophotometer. Set up three replicates for each sample. Proline (Pro) activity was determined using the Pro kit (Cat. No. BC0290) from Beijing Solebau Technology Co., Ltd. according to the formula. The results are shown in the table. Figure 8 (C).
[0046] The results showed that under 150mmol / L NaCl treatment, the roots produced more superoxide ions to resist salt stress, and the proline content of the mutant material was significantly lower than that of the control, indicating that the mutant material was more sensitive to salt stress.
Claims
1. A MtPRP4 gene isolated from Medicago truncatula, characterized in that: The MtPRP4 gene is isolated from Medicago truncatula, and the nucleotide sequence of the MtPRP4 gene is shown in SEQ ID NO:
1. The MtPRP4 gene belongs to a PRP gene. The MtPRP4 gene is used to regulate the growth and development of Medicago truncatula and enhance the resistance of Medicago truncatula to abiotic stress.
2. The MtPRP4 gene isolated from Medicago truncatula according to claim 1, wherein The CDS sequence of the MtPRP4 gene is shown in SEQ ID NO:
2.
3. The use of the MtPRP4 gene isolated from Medicago truncatula according to claim 1 or 2, characterized in that: The invention is used for regulating the growth and development of Medicago truncatula and enhancing the resistance of Medicago truncatula to abiotic stress; the abiotic stress is salt stress.