Application of gene MtPrx48 in regulation and control of growth and development and stress resistance of forage grass

Abstract

The invention provides application of a gene MtPrx48 in regulation and control of growth and development and stress resistance of forage grass, and belongs to the field of gene engineering, the CDS sequence of the gene is shown as SEQ ID NO: 1, the amino acid sequence of encoded protein is shown as SEQ ID NO: 2, and the gene is a new gene which is not reported and has obvious difference from homologous genes. Experiments prove that MtPrx48 negatively regulates the plant height and the leaf size of forage grass, the plant height of mutants Mtprx48-1 and Mtprx48-2 inhibiting the functions of the forage grass is increased by 42%, and the leaf area is increased by 38%; meanwhile, the gene positively regulates and controls drought resistance, a function deletion mutant is high in leaf drying rate under drought stress, the relative conductivity is increased, and the chlorophyll content is reduced; the invention provides a key gene target and a technical support for cultivating a new variety of forage grass with high biomass and drought resistance through genetic engineering, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically the application of the gene MtPrx48 in regulating the growth and development of forage grass and its resistance to stress. Background Technology

[0002] Alfalfa, a widely cultivated perennial high-quality legume forage crop, is rich in high-quality protein, dietary fiber, and various vitamins. It also has nitrogen-fixing capabilities through root nodules, playing a crucial role in agricultural production, ecological protection, and livestock development. However, adverse conditions such as drought and salinity can hinder alfalfa growth, reduce biomass, and even lead to large-scale mortality. Approximately 20% of arable land globally suffers from salinization. Therefore, identifying key genes that regulate biomass and enhance stress resistance is of great significance for bio-breeding to develop new, stress-resistant, high-yielding, and high-quality alfalfa germplasm, materials, and varieties.

[0003] Peroxidases (PRXs) are a class of thiol-based redox enzymes. Their core function is to catalyze the decomposition and transformation of reactive molecules such as hydrogen peroxide (H₂O₂), organic peroxides, and peroxynitrite. They play a crucial role in maintaining intracellular ROS and nitric oxide signaling balance, immune responses, and redox homeostasis. In plants, PRX genes are involved in regulating growth and development, stress resistance, including cell wall construction and modification (such as lignification and cross-linking), hormone metabolism, fruit development and ripening, seed germination, and senescence regulation. They also play a central role in biotic and abiotic stress responses such as drought, cold, and disease resistance. Due to the large number and diverse functions of its members (138 PRX members in the rice genome and 117 in alfalfa), the functions of most proteins in the peroxidase family remain unclear due to their complexity and functional redundancy. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of the gene MtPrx48 in regulating forage growth and development and stress resistance, as detailed below: The application of gene MtPrx48 in regulating forage growth and development and stress resistance, wherein gene MtPrx48 is derived from alfalfa, and the CDS sequence of the gene is shown in SEQ ID NO:1.

[0005] Moreover, the gene MtPrx48 negatively regulates the growth and development of forage grasses, resulting in reduced plant height and leaf size.

[0006] Moreover, the regulation of forage growth and development is achieved by inhibiting the function or expression of the gene MtPrx48 to increase the plant height and / or increase the leaf area of ​​the forage.

[0007] Moreover, the stress resistance is drought resistance, and the gene MtPrx48 positively regulates the drought resistance of forage grass, enhancing the forage grass's tolerance to drought environments.

[0008] Moreover, the forage is alfalfa.

[0009] On the other hand, the present invention provides a protein encoded by the gene MtPrx48, the amino acid sequence of which is shown in SEQ ID NO:2.

[0010] Thirdly, the present invention provides a recombinant expression vector for the gene MtPrx48.

[0011] Fourthly, the present invention provides a primer pair for the gene MtPrx48, comprising a forward primer MtPrx48-F and a reverse primer MtPrx48-R designed based on the sequence of SEQ ID NO:1, wherein the gene sequence of the forward primer MtPrx48-F is shown in SEQ ID NO:3 and the gene sequence of the reverse primer MtPrx48-R is shown in SEQ ID NO:4.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides the application of the gene MtPrx48 in regulating forage growth and development and stress resistance. By inhibiting MtPrx48 expression, high biomass varieties can be bred to improve forage yield and breeding efficiency. In arid, semi-arid and other stress-prone production areas, drought-resistant varieties can be bred by enhancing the expression of this gene, expanding the forage planting range and reducing the risk of yield reduction due to stress. This provides a precise solution for the forage industry under different ecological conditions and promotes the industrial application of stress-resistant and high-yield forage breeding.

[0013] 2. This invention identifies and discloses for the first time the gene MtPrx48 derived from alfalfa. Its CDS and protein sequence are significantly different from those of Arabidopsis thaliana AT1G71695.1 and alfalfa MtPrx38, indicating that this is a novel gene whose function has not been reported, providing a new target for plant gene function research.

[0014] 3. The gene MtPrx48 provided by this invention has been experimentally verified to significantly increase the plant height of alfalfa by 42% and the leaf area by 38% by inhibiting or knocking out the MtPrx48 gene, such as using mutants Mtprx48-1 and Mtprx48-2. This demonstrates that inhibiting MtPrx48 can effectively increase forage biomass. Under drought stress, mutants with loss of MtPrx48 function exhibit sensitive phenotypes such as leaves being more prone to drying out, increased relative conductivity, and decreased chlorophyll content, proving that this gene plays a key role in enhancing the drought resistance of plants. This invention provides key gene targets and technical support for cultivating new high-biomass, drought-resistant forage varieties through genetic engineering, and has broad application prospects. Attached Figure Description

[0015] Figure 1 The CDS sequence alignment results of gene MtPrx48 (MtrunA17_Chr6g0467121) with Arabidopsis homologs are shown. Figure 2 The CDS sequence alignment results of gene MtPrx48 (MtrunA17_Chr6g0467121) and homologous gene MtPrx38 are shown. Figure 3 The results of sequence alignment between gene MtPrx48 (MtrunA17_Chr6g0467121) and its homologous gene in Arabidopsis thaliana are shown. Figure 4 The AA sequence alignment results of gene MtPrx48 (MtrunA17_Chr6g0467121) and homologous gene MtPrx38 are shown. Figure 5 5A shows the results of homozygous identification of mutants of gene MtPRX48, and 5B shows the results of RT-PCR detection of gene MtPRX48 in mutants. Figure 6 The results show the phenotypic observations of the mutant Mtprx48. 6A shows the growth phenotype of the mutant, 6B shows the plant height statistics of the mutant, 6C shows the leaf phenotype, and 6D shows the leaf area statistics. Figure 7 The analysis of drought resistance function of the mutant MtPrx48 is shown in section 7A, which represents the phenotype of the mutant under drought stress; section 7B represents the chlorophyll content analysis; and section 7C represents the relative electrical conductivity. Figure 8 Identification results of other genes inserted into Tnt1 in mutant NF3313 (Mtprx48-1); Figure 9 Identification results of other genes inserted into Tnt1 in mutant NF8889 (Mtprx48-2). Detailed Implementation

[0016] Example 1: Cloning and identification of the alfalfa gene MtPrx48 The gene MtPrx48 (GeneID: MtrunA17_Chr6g0467121) was screened from the Medicago truncatula genome through multi-omics analysis. Upstream primer MtPrx48-F (SEQ ID NO:3) and downstream primer MtPrx48-R (SEQ ID NO:4) were designed. Using Medicago truncatula cDNA as a template, the full-length CDS of the MtPrx48 gene was amplified by PCR. The high-fidelity enzyme 2xPhanta Flash Master Mix (Catalog No.: P520) from Novizan Biosciences was selected to ensure the fidelity of the amplified product. After PCR electrophoresis, the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO:1, which is the CDS sequence of the MtPrx48 gene. The amino acid sequence of the MtPrx48 protein is shown in SEQ ID NO:2.

[0017] The CDS sequence of the gene MtPrx48, obtained by sequencing, was subjected to multiple sequence alignment with the Arabidopsis gene AT1G71695.1 and the alfalfa family gene MtPrx38 (MtrunA17_Chr4g0053871), which have similar homology. The alignment results are as follows. Figure 1-2 As shown, the similarity between the two is 54.3%. Multiple sequence alignment was performed on the amino acid sequence of protein MtPrx48 with the corresponding proteins AT1G71695.1 and MtPrx38. The alignment results are shown below. Figure 3-4 As shown, the similarity between the two is 17.87%. This indicates that the CDS and amino acid sequences of gene MtPrx48 are significantly different from those of homologous genes AT1G71695.1 and MtPrx38, and gene MtPrx48 is a novel gene that has not been reported before.

[0018] Example 2: Identification of homozygous materials of Tnt1 insertion mutant of gene MtPrx48 Mutant materials NF3313 and NF8889, presumed to contain the MtPrx48 gene insertion, were purchased from the *Alfalfa* Tnt1 insertion mutant library. Gene-specific primers were designed for genotyping at approximately 200 bp upstream and downstream of the Tnt1 insertion site for each material: NF3313-F / R (SEQ ID NO:5 / 6) and NF8889-F / R (SEQ ID NO:7 / 8).

[0019] The mutants were identified as homozygous by PCR using Tnt1 flanking primers LTR6 (SEQ ID NO:33) and LTR31 (SEQ ID NO:34) in combination with the above-mentioned gene-specific primers.

[0020] The specific testing methods are as follows: Using the mutant NF3313 genome as a template: (1) PCR amplification was performed using primers NF3313-F / R, and no bands were observed in the electrophoresis image; (2) PCR amplification was performed using primers NF3313-F with LTR6 and LTR31. The electrophoresis results showed a single band.

[0021] The above results indicate that NF3313 is a homozygous Tnt1 insertion mutant of the MtPrx48 gene, and it is labeled as Mtprx48-1.

[0022] Similarly, using the genome of the mutant NF8889 as a template: (1) PCR amplification was performed using primers NF8889-F / R, and no bands were observed in the electrophoresis image; (2) PCR amplification was performed using primers NF8889-F with LTR6 and LTR31. The electrophoresis results showed a single band.

[0023] The above results indicate that NF8889 is a homozygous Tnt1 insertion mutant of the MtPrx48 gene, and it is labeled as Mtprx48-2.

[0024] Homozygous identification results are as follows Figure 5 As shown in Figure A.

[0025] The homozygous mutant materials Mtprx48-1 and Mtprx48-2 obtained by screening were propagated, and the harvested seeds were stored at 4℃.

[0026] Gene expression detection is as follows: Wild-type alfalfa (WT) and the Mtprx48 mutant seeds were sterilized and spread on 1 / 2 MS plates for vernalization at 4°C for 2-3 days. They were then transferred to an incubator and cultured at 24°C under 16-hour light / 8-hour dark conditions for 5-7 days. Three healthy and uniformly growing plants were selected, and RNA was extracted from them using the RNAprep Pure Plant Total RNA Extraction Kit (catalog number: DP432) from Tiangen Biotech (Beijing) Co., Ltd., and RNA integrity was detected by gel electrophoresis.

[0027] Design specific primers MtPrx48-qF and MtPrx48-qR (SEQ ID NO:35) The expression level of gene MtPrx48 was detected using primers MtActin7-F and MtActin7-R (SEQ ID NO:37, SEQ ID NO:38). First-strand cDNA was synthesized via reverse transcription using the HiScript IV 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Catalog No.: R412-01) from Nanjing Novizan Biotechnology Co., Ltd. RT-PCR experiments were performed using 2×Rapid Taq Master Mix with cDNA as a template. The results showed (5B): the internal reference gene MtActin7 had amplified bands in wild-type (WT), mutant Mtprx48-1, and Mtprx48-2, indicating that the cDNA quality was qualified; the target gene MtPrx48 had amplified bands in wild-type (WT) cDNA, but no amplified bands in mutant Mtprx48-1 and Mtprx48-2 cDNA, indicating that the gene MtPrx48 could not be transcribed and expressed in mutant Mtprx48-1 and Mtprx48-2, and its function was lost.

[0028] Example 3: Mutant Background Validation and Phenotypic Attribution Analysis To verify that the phenotypic changes in mutants Mtprx48-1 and Mtprx48-2 are solely due to a single mutation in the MtPRX48 gene, the influence of Tnt1 insertions at other sites needs to be excluded. Therefore, we conducted the following analysis: For mutant NF3313 (Mtprx48-1): The flanking sequences of mutant NF3313 (Mtprx48-1) were searched on the mutant website. Then, the flanking sequences of mutant NF3313 (Mtprx48-1) were copied to the Alfalfa Genomes website for BLAST analysis to identify the insertion sites of Tnt1 (Table 1). The results showed that, apart from the insertion of the target gene (MtrunA17_Chr6g0467121), most Tnt1 sequences were inserted into the introns of other genes and had no effect on gene expression. Only three genes (MtrunA17_Chr4g0040831, MtrunA17_Chr4g0062361, and MtrunA17_Chr1g0161451) had Tnt1 sequences inserted into their CDS regions.

[0029] To confirm the actual genotypes of these three genes in the mutant, PCR analysis was used to determine the homozygosity and heterozygosity of these three genes in the mutant. Detection primers were designed for each gene (NF3313-0831-F: SEQ ID NO: 9, NF3313-0831-R: SEQ ID NO: 10, NF3313-2361-F: SEQ ID NO: 11, NF3313-2361-R: SEQ ID NO: 12, NF3313-1451-F: SEQ ID NO: 13, NF3313-1451-R: SEQ ID NO: 14). PCR results showed that ( Figure 8 Using F / R primers for each gene, bands were amplified, while using F / LTR primer combinations, no bands were observed. This indicates that at the corresponding loci of these three genes, the NF3313 plant remains wild-type homozygous, and its Tnt1 insertion sequence in the CDS region may have originated from paralogous sequences or other alleles, without causing an actual functional mutation. Therefore, Mtprx48-1 can be identified as a single mutant of the MtPrx48 gene.

[0030] Table 1. Insertion sites of Tnt1 in mutant NF3313 (Mtprx48-1)

[0031] For mutant NF8889 (Mtprx48-2): Using the same method, the insertion site analysis of Tnt1 in mutant NF8889 (Mtprx48-2) was performed (Table 2). The flanking sequences of mutant NF8889 (Mtprx48-2) were copied to the *Alfalfa Tribulus* genome website for BLAST analysis to identify the Tnt1 insertion site. Besides the insertion into the target gene, most Tnt1 sequences were inserted into introns of other genes, having no effect on gene expression. Tnt1 sequences were also inserted into the CDS regions of nine genes. Therefore, PCR analysis was used to determine the homozygosity and heterozygosity of these nine genes in the mutant. Detection primers were designed (NF8889-Chr2g0295701-F: SEQ ID NO:15, NF8889-Chr2g0295701-R: SEQ ID NO:16, NF8889-Chr7g0255601-F: SEQ ID NO:15). NO:17, NF8889-Chr7g0255601-R: SEQ ID NO:18, NF8889-Chr4g0007091-F: SEQ ID NO:19, NF8889-Chr4g0007091-R: SEQ ID NO:20, NF8889-Chr5g0431951-F: SEQ ID NO:21, NF8889-Chr5g0431951-R: SEQ ID NO:22, NF8889-Chr4g0006421-F: SEQ ID NO:23, NF8889-Chr4g0006421-R: SEQ ID NO:24, NF8889-Chr4g0007131-F: SEQ ID NO:25, NF8889-Chr4g0007131-R: SEQ ID NO:26, NF8889-Chr4g0006901-F: SEQ ID NO:27, NF8889-Chr4g0006901-R: SEQ ID NO:28, NF8889-Chr4g0007211-F: SEQ ID NO:29, NF8889-Chr4g0007211-R: SEQ ID NO:30, NF8889-Chr4g0007191-F: SEQ ID NO:31, NF8889-Chr4g0007191-R: SEQ ID NO:32), the PCR results show that ( Figure 9 1: F / LRT; 2: R / LTR; 3: F / R, M: Marker. ), F / R has a band, F / LTR has no band, indicating that these 9 genes are not mutated in the mutant NF8889 (Mtprx48-2), and are also wild-type homozygous.

[0032] Table 2. Insertion sites of Tnt1 in mutant NF8889 (Mtprx48-2)

[0033] Example 4: Identification of mutant phenotypes of gene MtPrx48 Wild-type alfalfa seeds R108 and mutant seeds MtPrx48-1 and MtPrx48-2 were lightly sanded on sandpaper and placed in sterile petri dishes soaked in water-soaked sterile filter paper. They were vernalized at 4°C for 2-3 days, then vertically cultured in a light incubator for 3-5 days. Healthy seedlings with consistent growth were then transplanted into soil pots (a 1:1 mixture of nutrient soil and vermiculite) and placed in a tissue culture room for further growth. Phenotypic observations were performed on wild-type and mutant seeds (MtPrx48-1 and MtPrx48-2) grown in soil pots for 40 days. The results are shown in […]. Figure 6 The average plant height of the mutant Mtprx48 was 18.11 cm, while the average plant height of the wild-type R108 was 12.75 cm, a significant increase of 42%; the leaf area of ​​the mutant was 180.5 mm². 2 The wild-type R108 has a leaf area of ​​130.25 mm. 2 The height and leaf area of ​​the mutant Mtprx48 were significantly increased by 38% compared to the wild type, indicating that the Mtprx48 gene negatively regulated the plant height and leaf size.

[0034] Example 5: Analysis of drought resistance function in loss-of-function mutants of gene MtPrx48 Wild-type and mutant Mtprx48-1 and Mtprx48-2, grown in soil pots for 2 months, were subjected to drought stress without watering. Phenotypic observation and physiological index testing were performed.

[0035] Relative conductivity determination: Weigh 0.1g of fresh leaves from the plant, avoiding the main vein, and cut them into uniformly sized pieces. Place the pieces in a 50mL centrifuge tube. Add 30mL of deionized water, place the tube on a centrifuge rack, open the lid, and vacuum for 10min. Repeat 4 times. Shake overnight at 100rpm in the dark at 25℃. After removing the tube, let it stand for 1 hour and measure R1 using a conductivity meter. Then, transfer the conical flask to a water bath and boil for 30min. After cooling to room temperature, measure R2. Calculation formula: Relative conductivity REC (%) = R1 / R2 × 100.

[0036] Chlorophyll content is measured using a handheld chlorophyll meter: Turn on the meter, calibrate it, select the middle of the leaf, and place the leaf into the measuring clip of the meter, ensuring that the leaf completely covers the measuring window. Gently close the measuring clip, avoiding excessive force that could damage the leaf. Press the measurement button and wait for the instrument to display the reading (i.e., the SPAD value). Repeat the measurement 3-5 times at different locations on the same leaf, recording the SPAD value each time. Average all the measurements to obtain the chlorophyll content of the leaf.

[0037] The results showed that ( Figure 7 After 20 days of drought stress, the mutant and wild-type plants showed significant differences in phenotypic and physiological indicators. Only about 10% of the leaves of the wild-type plants dried out, while the proportion of dried leaves in the mutant plants was as high as about 50%. Figure 7 A); In addition, the relative conductivity of the mutant is significantly higher than that of the wild type ( Figure 7 C), and the chlorophyll content was significantly lower than that of the wild type ( Figure 7 B). The above results indicate that the mutants MtPrx48-1 and MtPrx48-2 are more sensitive to drought stress than the wild type, further proving that the gene MtPrx48 positively regulates the drought resistance of plants.

Claims

1. The application of gene MtPrx48 in regulating forage growth and development and stress resistance, characterized by, The gene MtPrx48 is derived from alfalfa, and its CDS sequence is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The gene MtPrx48 negatively regulates the growth and development of forage grasses, resulting in reduced plant height and leaf size.

3. The application according to claim 1, characterized in that, The regulation of forage growth and development is achieved by inhibiting the function or expression of the gene MtPrx48 to increase the plant height and / or increase the leaf area of ​​the forage.

4. The application according to claim 2, characterized in that, The stress resistance refers to drought resistance, and the gene MtPrx48 positively regulates the drought resistance of forage grasses, enhancing their tolerance to drought environments.

5. The application according to claim 1, characterized in that, The forage is alfalfa.

6. A protein encoded by the gene MtPrx48 as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

7. A recombinant expression vector comprising the gene MtPrx48 of claim 1.

8. A primer pair for detecting the gene MtPrx48 of claim 1, characterized in that, It includes a forward primer MtPrx48-F and a reverse primer MtPrx48-R designed based on the sequence of SEQ ID NO:

1. The gene sequence of the forward primer MtPrx48-F is shown in SEQ ID NO:3, and the gene sequence of the reverse primer MtPrx48-R is shown in SEQ ID NO:4.

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