MtPRP4 gene separated from medicago truncatula as well as encoding protein and application of MtPRP4 gene

Abstract

The invention provides an MtPRP4 gene separated from medicago truncatula and an encoding protein and application thereof, and belongs to the field of genetic engineering, the MtPRP4 gene is separated from medicago truncatula, the nucleotide sequence of the MtPRP4 gene is shown as SEQ ID NO: 1, and the MtPRP4 gene belongs to a PRP gene; the CDS sequence of the MtPRP4 gene is as shown in SEQ ID NO: 2; the amino acid sequence of the protein coded by the MtPRP4 gene is as shown in SEQ ID NO: 3; according to the invention, the MtPRP4 gene is separated from medicago truncatula for the first time, and the function of the MtPRP4 gene in regulating the growth and development of medicago truncatula and enhancing abiotic stress resistance is verified through experiments, so that the blank of research on the PRP gene in medicago truncatula is filled, and a new perspective is provided for understanding the function of the PRP gene in leguminous plants.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a MtPRP4 gene separated from Medicago truncatula and a coded protein and application thereof. Background Art

[0002] Plant proline-rich proteins (PRPs) are a class of structural proteins rich in proline and hydroxyproline. They are widely present in plant cell walls and are involved in the construction of cell walls and plant stress responses. PRPs usually contain repetitive sequences in the proline-rich polypeptide region, which play a key role in the specific structure and function of the cell wall. Studies have shown that the expression of PRP genes can respond to a variety of external stimuli, such as hormones, injury, low temperature, salt stress, etc., and under fungal inducers and mechanical damage, PRP can form a protective layer with other cell wall components (such as lignin and sugars) through rapid oxidative cross-linking, thereby enhancing the defense capabilities of plants.

[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: First, the diversity of PRP gene functions has not been fully elucidated: Although studies have shown that PRP genes are involved in the response to abiotic stresses (such as drought, high salt, and low temperature) in plants such as Arabidopsis and pigeon pea, there are significant differences in the functions of PRP genes in different plants. For example, after the EARLI1 gene in Arabidopsis was knocked out, the plants were more vulnerable to cold stress, while the CcHyPRP gene in pigeon pea can improve the resistance of transgenic Arabidopsis to drought, high salt, and high temperature. However, research on the specific functions of PRP genes in legumes (such as Medicago truncatula), especially their role in growth and development and abiotic stress responses, is still very limited.

[0004] Second, the research gap of PRP genes in Medicago truncatula: Medicago truncatula, as a model plant of the Leguminosae, has the advantages of small genome, convenient genetic transformation, and close relationship with a variety of economic crops (such as alfalfa, pea, and broad bean). It is an ideal material for studying symbiotic nitrogen fixation, compound leaf development, and stress resistance genes in leguminous plants. However, although 23 PRP genes have been identified in Medicago truncatula, current research mainly focuses on nodule formation, and there has been no systematic study on the specific role of these genes in plant growth and development and abiotic stress response. In particular, the function of the MtPRP4 gene and its role in regulating alfalfa growth and development and stress resistance are still unknown.

[0005] 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 PRP gene in alfalfa may provide new ideas for solving this problem. However, existing technologies have not yet fully utilized the potential of PRP gene, especially in the regulation of resistance under abiotic stress, and there is still a large research gap. Summary of the invention

[0006] There are still many deficiencies in the existing research on the function of PRP genes, especially in Medicago truncatula, the specific role of PRP genes in growth and development and abiotic stress response has not been fully revealed. Therefore, in-depth research on the function of MtPRP4 gene will not only help fill the research gap in this field, but also provide new technical approaches for the development of gene resources to improve alfalfa yield and quality. Based on this, the present invention provides a MtPRP4 gene isolated from Medicago truncatula, its encoded protein and application, as follows: 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.

[0007] Furthermore, the CDS sequence of the MtPRP4 gene is shown in SEQ ID NO:2.

[0008] 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.

[0009] Furthermore, the CDS sequence of the MtPRP4 gene shown in SEQ ID NO: 2 has a 32.77% identity with the CDS sequence of AT2G21140 in Arabidopsis thaliana, and has a 47.14% identity with the CDS sequence of AT4G38770 in Arabidopsis thaliana.

[0010] 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.

[0011] In a second aspect, the present invention provides a protein encoded by the MtPRP4 gene, wherein the encoded protein is encoded by the MtPRP4 gene, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 3.

[0012] Furthermore, the encoded protein is rich in proline and / or hydroxyproline and has the characteristics of a cell wall structural protein.

[0013] 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.

[0014] Furthermore, the abiotic stress is salt stress.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present 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 leguminous plants.

[0016] 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, etc. These phenotypic changes indicate that the MtPRP4 gene can be used as a genetic resource for improving alfalfa yield and quality, providing a new target for the genetic improvement of alfalfa.

[0017] 3. During the experiment, it was found that under salt stress, 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

[0018] Figure 1 for MtPRP4 Sequence alignment 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.

[0019] Figure 2 MtPRP4 Comparison results of the amino acid sequence of the gene with that of the homologous gene in Arabidopsis; 2 (A) is Medicago truncatula MtPRP4Arabidopsis thaliana AT4G38770 CDS sequence alignment results; 2 (B) is Medicago truncatula MtPRP4 Arabidopsis thaliana AT4G38770 Amino acid sequence alignment results.

[0020] Figure 3 for MtPRP4-1, MtPRP4-2 Schematic diagram of the mutant insertion site.

[0021] Figure 4 Medicago truncatula MtPRP4 Homozygous material of gene Tnt1 mutant; 4 (A) is Medicago truncatula MtPRP4 Results of homozygous screening of Tnt1 gene insertion mutants; 4 (B) is a mutant MtPRP4 Gene RT-PCR results.

[0022] Figure 5 Medicago truncatula MtPRP4 Root phenotypes of mutants; 5 (A) is Medicago truncatula MtPRP4 Root phenotypes of mutants, 5 (B) is Medicago truncatula MtPRP4 Root length statistics of mutants.

[0023] 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 mutants; 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.

[0024] 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.

[0025] Figure 8 The phenotypic results of Medicago truncatula hydroponically cultured under 150mmol / L NaCl stress; 8 (A) is the phenotypic results of Medicago truncatula hydroponically cultured 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) Medicago truncatula under 150mmol / L NaCl MtPRP4 Proline content. DETAILED DESCRIPTION

[0026] Example 1 Medicago truncatula MtPRP4 Gene cloning The NCBI website tool Blast was used to search for similar homologous genes in Arabidopsis AT2G21140、AT4G38770 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 selected was the high-fidelity enzyme 2xPhanta Flash Master Mix (Cat. No.: P520) from Novazon Biotech to ensure the fidelity of the amplified product and reduce sequence mutations caused by human manipulation. After electrophoresis detection, the product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and 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).

[0027] MtPRP4 The CDS sequence of the gene AT2G21140 , AT4G38770 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).

[0028] The results show that MtPRP4 Arabidopsis thaliana AT2G21140 , AT4G38770 The gene CDS and amino acid sequences are significantly different. MtPRP4 The gene is a new gene that has not been reported.

[0029] Example 2 MtPRP4 Screening of Tnt1 insertion homozygous mutant materials 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 3As 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 (SEQID 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, see Figure 4 (A). The homozygous mutant material was propagated and the harvested seeds were stored at 4°C. MtPRP4 After the mutant seeds were sterilized, they were spread on 1 / 2MS plates and vernalized at 4°C for 48 hours. They were transferred to an incubator at 24°C, 16 hours of light / 8 hours of darkness, and cultured for 7 days. Three strains with healthy and consistent growth conditions were selected and mixed for RNA extraction. The integrity of the extracted RNA was detected by gel electrophoresis according to the instructions of the RNAprep Pure Plant Total RNA Extraction Kit (Cat. No.: DP432) of Tiangen Biochemical Company. Specific primers were designed. MtPRP4 -F ， (SEQ ID NO: 12) and MtPRP4 -R ， (SEQ ID NO: 13), internal reference gene primer MtActin7 -F (SEQ ID NO: 14:) and MtActin7 -R (SEQ ID NO: 15) to detect gene expression. st Strand cDNA Synthesis Kit (+gDNAwiper) (Cat. No.: R412-01) Instructions Reverse synthesis of the first strand of cDNA. RT-PCR experiments were performed using cDNA as a template using Taq Pro Universal SYBR QPCR Master Mix, see Figure 4 (B).

[0030] The results showed that the gene MtPRP4 In mutant NF14275 ( MtPRP4-1 ) and NF16150 ( MtPRP4-2 ) is not expressed.

[0031] Example 3 MtPRP4 Mutant phenotype identification Observation of the growth of wild-type Medicago truncatula and MtPRP4 The root phenotypes of mutant seedlings are shown in Figure 5 , Figure 5 Medicago truncatula MtPRP4 Root phenotypes of mutants, 5 (A) is Medicago truncatula MtPRP4 Root phenotypes of mutants, 5 (B) is Medicago truncatula MtPRP4 Statistical diagram of mutant root length. The results showed that the root length of the mutant was significantly longer than that of the wild type.

[0032] Use sandpaper to gently break the seed epidermis so that it can absorb water and swell. Then place the seeds in a culture dish covered with moist filter paper and move them to a 4°C refrigerator for vernalization for 3 days. Move them to a light culture box for normal culture. 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 culture room for culture. The greenhouse growth conditions are: 16 hours of light, 8 hours of darkness, temperature 22-24°C, humidity 60%-70%, and light intensity of 150umolm -2 s -1 The phenotypes of the 2-month-old plants were observed under the same conditions. Figure 6 , Figure 7 .

[0033] Figure 6 Medicago truncatula MtPRP4 Plant height and branch number phenotypes of mutants at 60 days. 6 (A) is Medicago truncatula MtPRP4 Figure 6 (B) shows the plant height and branching phenotype of mutants; 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.

[0034] 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.

[0035] The results showed that compared with the wild type, the mutant material had an increased plant height, branch number and leaf area. The number of secondary branches on each primary branch of the wild type and mutant was counted separately, and 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.

[0036] Example 4 MtPRP4 Determination of physiological indicators of mutants Combine wild type and MtPRP4 The 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 treated to observe the phenotype and measure physiological indicators ( Figure 8 ), phenotype diagram see Figure 8 (A).

[0037] 0.1g of fresh plant tissue was fully ground in liquid nitrogen, 1ml of extract was added, ice bath homogenized, centrifuged at 8000g for 10min at 4℃, the supernatant was placed on ice, and the absorbance OD value was recorded at a wavelength of 560nm. 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 by the formula. The results are shown in Figure 8 (B).

[0038] Weigh about 0.1g of fresh plant sample, add 1mL of extract solution and homogenize in ice bath; then place in boiling water bath for 10min, centrifuge at 10000g for 10min at room temperature, take the supernatant, and measure the OD value at 520nm with a spectrophotometer after cooling. Set 3 replicates for each sample. Use the Pro kit (Cat. No.: BC0290) of Beijing Solebow Technology Co., Ltd. to measure the proline (Pro) activity according to the formula. The results are shown in Figure 8 (C).

[0039] 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, the nucleotide sequence of the MtPRP4 gene is shown in SEQ ID NO: 1, and 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, characterized in that: The CDS sequence of the MtPRP4 gene is shown in SEQ ID NO:

2.

3. The MtPRP4 gene isolated from Medicago truncatula according to claim 2, characterized in that: The amino acid sequence of the protein encoded by the MtPRP4 gene is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by the MtPRP4 gene is obtained by translating the CDS sequence shown in SEQ ID NO:

2.

4. The MtPRP4 gene isolated from Medicago truncatula according to claim 2, characterized in that: The CDS sequence of the MtPRP4 gene shown in SEQ ID NO: 2 has a 32.77% identity with the CDS sequence of AT2G21140 in Arabidopsis thaliana, and has a 47.14% identity with the CDS sequence of AT4G38770 in Arabidopsis thaliana.

5. The MtPRP4 gene isolated from Medicago truncatula according to claim 3, characterized in that: 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.

6. A protein encoded by the MtPRP4 gene, characterized in that: The encoded protein is encoded by the MtPRP4 gene described in claim 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:

3.

7. The protein encoded by the MtPRP4 gene according to claim 6, characterized in that: The encoded protein is rich in proline and / or hydroxyproline and has the characteristics of a cell wall structural protein.

8. The use of the MtPRP4 gene isolated from Medicago truncatula according to any one of claims 1 to 5, characterized in that: Used to regulate the growth and development of Medicago truncatula and enhance the resistance of Medicago truncatula to abiotic stress.

9. The use of the MtPRP4 gene isolated from Medicago truncatula according to claim 8, characterized in that: The abiotic stress is salt stress.

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