Salt-alkali tolerance RpMAPK3 gene of Rhododendron splendidum, its encoded protein and application

Abstract

The present invention discloses a salt-alkali-tolerant RpMAPK3 gene of Rhododendron splendidum, its encoded protein and application, and relates to the field of genetic engineering technology. The nucleotide sequence of the salt-alkali-tolerant RpMAPK3 gene of Rhododendron splendidum is shown in SEQ ID NO.1. The amino acid sequence of the RpMAPK3 protein encoded by the salt-alkali-tolerant RpMAPK3 gene of Rhododendron splendidum is shown in SEQ ID NO.2. The present invention adopts the above-mentioned salt-alkali-tolerant RpMAPK3 gene of Rhododendron splendidum, its encoded protein and application. The RpMAPK3 gene can improve the adaptability and growth conditions of azalea in alkaline soil, not only expand the planting range of azalea, but also improve its growth conditions under adverse soil conditions, provide more options for ornamental gardening and ecological environment protection, and can also be used for the cultivation of new varieties of azalea, providing effective gene resources for the cultivation of new varieties of salt-alkali-tolerant azalea.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a Rhododendron splendidum salt-alkali resistant RpMAPK3 gene, its encoded protein and application. Background Art

[0002] Salt-alkali stress is a common abiotic stress that significantly affects plant growth, development, and yield. Salt-alkali stress primarily manifests as ionic, osmotic, and oxidative stress, while alkaline stress, which adds high pH stress to salt stress, can cause more severe and complex damage to plants. Plants respond and adapt to these stresses through a variety of physiological and molecular mechanisms.

[0003] Azalea is an evergreen shrub belonging to the genus Rhododendron in the Ericaceae family. Its large, colorful flowers remain green year-round, making it highly ornamental and economically valuable. This genus has strict ecological requirements, preferring an acidic environment. Suitable soil pH for growth is between 4.5 and 6, and its alkalinity tolerance is poor. Salt-alkali stress is the main limiting factor for its growth, development, ex situ conservation, and landscape applications. Rhododendron pulchrum, belonging to the subgenus Tsutsusi of the genus Ericaceae, is a commonly cultivated azalea in my country. Due to its strong stress resistance, it is widely used in green space cultivation in the Yangtze River Delta region, as well as in Fujian, Hunan, and Guangdong. However, there are few varieties of Rhododendron pulchrum, and the purple-flowered varieties used in green spaces are primarily those with the most common flowers, resulting in a monotonous ornamental appearance.

[0004] Mitogen-activated protein kinases (MAPKs) are a class of highly conserved serine / threonine (Ser / Thr) protein kinases ubiquitous in eukaryotes. The MAPK signaling cascade comprises MAPK kinase kinase (MEKK), MAPK kinase (MEK), and MAPK. These three kinases are sequentially activated to regulate a variety of important cellular physiological and pathological processes, playing a crucial role in normal plant growth and development and responses to environmental stress. As the terminal protein kinase, MAPK is a key protein kinase in cellular signal transduction and amplification, acting as a direct link between the cascade signal and downstream proteins. Numerous MAPK family members have been identified from diverse plant species. Generally, the MAPK family can be divided into two subfamilies: TEY and TDY, based on the conserved TXY amino acid residue sequence phosphorylated by MEK. The TEY subfamily is further divided into three categories: A, B, and C, based on the characteristics of their conserved regions. The MAPK3 gene belongs to the TEY subfamily A, which is involved in various environmental stress responses and hormone-regulated physiological processes. Studies have shown that in response to salt stress, MPK3 is a direct downstream activator of MKK4 and mediates the resistance of overexpressing plants to high salt. Summary of the Invention

[0005] The present invention aims to provide a salt-alkali tolerant RpMAPK3 gene of Rhododendron splendidum, its encoded protein and application. The RpMAPK3 gene can improve the adaptability and growth condition of azalea in alkaline soil, not only expanding the planting range of azalea, but also improving its growth condition under unfavorable soil conditions, providing more options for ornamental gardening and ecological environment protection. At the same time, it can also be used for the cultivation of new varieties of azalea, providing an effective gene resource for the cultivation of new varieties of salt-alkali tolerant azalea.

[0006] To achieve the above object, the present invention provides a salt-alkali-tolerant RpMAPK3 gene of Rhododendron splendidum. The nucleotide sequence of the salt-alkali-tolerant RpMAPK3 gene of Rhododendron splendidum is shown in SEQ ID NO.1.

[0007] The present invention also provides the RpMAPK3 protein encoded by the salt-alkali tolerant RpMAPK3 gene of Rhododendron splendidum. The amino acid sequence of the RpMAPK3 protein is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector comprising the salt-alkali tolerant RpMAPK3 gene of Rhododendron splendidum. The recombinant vector comprises the nucleotide sequence shown as SEQ ID NO.1.

[0009] Furthermore, the recombinant vector includes a cloning vector or a plant overexpression vector.

[0010] Furthermore, the recombinant vector is a CaMV35S-RpMAPK3-GFP plant expression vector.

[0011] The present invention also provides the use of the salt-alkali tolerance RpMAPK3 gene of Rhododendron splendidum in improving the salt-alkali tolerance of Rhododendron splendidum.

[0012] The present invention also provides application of the recombinant vector in improving the salt-alkali resistance of Rhododendron splendidum.

[0013] The advantages and positive effects of the salt-alkali tolerant RpMAPK3 gene of Rhododendron splendidum, its encoded protein and application of the present invention are:

[0014] 1. The RpMAPK3 gene can improve the adaptability and growth of azalea in alkaline soils, which not only expands the planting range of azalea, but also improves its growth conditions under adverse soil conditions, providing more options for ornamental gardening and ecological environment protection, and has important practical application value.

[0015] 2. Overexpression of the RpMAPK3 gene can significantly improve the salt-alkali tolerance of transgenic Arabidopsis thaliana, which can be used to breed new azalea varieties in the future, providing effective genetic resources for the breeding of new salt-alkali-tolerant azalea varieties.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 1 shows the effect of RpMAPK3 gene overexpression on germination rate in plants according to the present invention, wherein A shows the germination rate phenotype of RpMAPK3 gene transgenic plants under control and saline-alkali stress; B shows the statistical results of germination rate of RpMAPK3 gene transgenic plants under control and saline-alkali stress, Col is the wild-type control, and 35S:RpMAPK3 is the transgenic line;

[0018] Figure 2 Root phenotypes of Arabidopsis thaliana overexpressing the RpMAPK3 gene under saline-alkali stress in the present invention, wherein A is the root phenotype of the wild type and overexpressing plants under pH 5.8 (0 mM NaHCO3); B is the root phenotype of the wild type and overexpressing plants under pH 8.2 (5 mM NaHCO3), Col is the wild type control, and 35S:RpMAPK3 is the transgenic line;

[0019] Figure 3Figure 2 shows the effect of overexpressing the RpMAPK3 gene on the root system of Arabidopsis thaliana under saline-alkali stress in the examples of the present invention, wherein A shows the root length, root surface area, and root tip number of the wild type and overexpressing plants under pH 5.8 (0 mM NaHCO3); B shows the root length, root surface area, and root tip number of the wild type and overexpressing plants under pH 8.2 (5 mM NaHCO3), Col is the wild type control, and 35S:RpMAPK3 is the transgenic line;

[0020] Figure 4 These are the quantitative PCR results of the RpMAPK3 gene in the examples of the present invention, where A represents the expression of the RpMAPK3 gene in the wild-type and overexpressing plants under pH 5.8 (0 mM NaHCO3), B represents the expression of the RpMAPK3 gene in the wild-type and overexpressing plants under pH 8.2 (5 mM NaHCO3), Col represents the wild-type control, and OE represents the transgenic line. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0023] Unless otherwise defined, the instruments, equipment, and reagents used in the present invention are all commercially available.

[0024] The invention cloned the Rhododendron splendidum RpMAPK3 gene and constructed a plant expression vector CaMV35S-RpMAPK3-GFP. Then, transgenic Arabidopsis thaliana was obtained by Agrobacterium infection, and the function of RpMAPK3 was verified in Arabidopsis thaliana.

[0025] The nucleotide sequence of the Rhododendron splendidum MAPK3 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the Rhododendron splendidum MAPK3 gene is shown in SEQ ID NO.2.

[0026] Example 1 Experimental method

[0027] 1.1 Cloning and vector construction of Rhododendron splendidum RpMAPK3 gene:

[0028] Based on the CDS sequence of the RpMAPK3 gene, primers were designed to amplify the MAPK3 gene (Table 1). The PCR reaction system was as follows: 10 μL of 5× PCR mix, 4 μL of 2.5 mM dNTPs, 1.5 μL of Prime F (10 μM / L), 1.5 μL of Prime R (10 μM / L), 2 μL of template (cDNA), 0.2 μL of Prime Star, and ddH2O to 50 μL. The PCR amplification program was as follows: 95°C for 5 min; 94°C for 30 s, 54°C for 30 s, 72°C for 2 min, 38 cycles; 72°C for 2 min, 4°C for 10 min. The PCR product was recovered using a gel extraction kit from Generic. The plasmid digestion system was as follows: 5 μL of 10× Q-cut buffer, 2 μL of endonuclease, 1 μg of plasmid, and ddH2O to 50 μL. Enzyme digestion was performed at 37°C for 2–3 hours. The digested product was recovered using the Tiangen Gel Extraction Kit. The ligation system was as follows: 6 μL of PCR product, 2 μL of plasmid, 1 μL of 10× T4 ligase buffer, 0.5 μL of T4 ligase, and ddH2O to 10 μL. Ligation was performed overnight at 4°C. The ligation product was added to 50 μL of TOP10 competent cells, incubated on ice for 30 minutes, heat-shocked at 37°C for 30 seconds, and then incubated on ice for 2 minutes. 1 mL of antibiotic-free LB medium was added and allowed to recover at 37°C for 45 minutes. The cells were then plated on LB plates containing the appropriate resistance and incubated at 37°C overnight. Positive clones were identified by colony PCR.

[0029] Table 1 MAPK3 gene primers

[0030]

[0031] 1.2 Agrobacterium transformation:

[0032] Add 0.5 μl of plasmid vector to 50 μl of GV3101 competent Agrobacterium cells and mix thoroughly. Transfer the mixture to an electroporation cuvette and place on ice for 1 minute. Electroporate at 2.2 kV for 5.0 ms. Immediately after the electroporation, add 1 mL of antibiotic-free liquid LB. Let the cells recover on a shaker at 30°C for 1 hour. Then, spread the cells onto solid LB selective medium containing Kan+Gen and incubate at 30°C for 48 hours.

[0033] 1.3 Arabidopsis transformation and transgenic plant screening:

[0034] Arabidopsis transformation was performed using the inflorescence infection method, using Arabidopsis plants in good growth and early flowering. Agrobacterium GV3101 containing the target vector was shaken at 30°C for 12 hours. After shaking vigorously until the OD reached 0.8-1.0, the cells were centrifuged for 10 minutes and resuspended in freshly prepared transformation solution. For transformation, all flowers were immersed in the transformation solution containing Agrobacterium for 45 seconds. The transformed flowers were then wrapped in plastic wrap and protected from light for 24 hours before removal.

[0035] Transformed T0 generation Arabidopsis seeds were dried in a 30°C incubator for 3-7 days, sterilized, and plated onto MS plates containing a selection antibiotic. Positive Arabidopsis were identified and individual T1 generation Arabidopsis plants were harvested. T1 generation seeds were screened on a medium containing a selection antibiotic. Lines with a 3:1 segregation ratio, i.e., lines with a single-copy insertion, were selected for passage and subsequent experiments.

[0036] 1.4 Phenotypic experiments of overexpression of RpMAPK3 gene:

[0037] Prepare 1 / 2 MS medium with 30% sucrose. Adjust the pH of the medium to 5.8 and 8.2 with 1 M / L KOH solution, respectively. Add 7 g of agar powder and sterilize by autoclaving. After sterilization, cool to approximately 40°C. Add 0.8 M / L NaHCO3 to prepare an alkaline stress medium. Pour the medium into a Petri dish and allow it to cool and solidify before subsequent experiments. The stress medium recipe is shown in Table 2.

[0038] Arabidopsis wild type and T2 generation seeds overexpressing RpMAPK3 gene were selected and spotted on alkaline stress culture medium plates, with 10 seeds spotted on each plate. The experiment was repeated 3 times and placed in a light incubator for 14 days to observe their phenotypes.

[0039] Table 2 Stress culture medium formula (1L)

[0040]

[0041] 1.5 Verification of RpMAPK3 gene expression in transgenic plants by real-time fluorescence quantitative PCR

[0042] Quantitative primers for the RpMAPK3 gene were designed using SnapGene (Table 3). After 14 days of culture, T2 transgenic Arabidopsis seeds were cultured on 1 / 2 MS medium. RNA was extracted from the entire plant and reverse-transcribed into cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMIX for qPCR Kit. Real-time PCR reagents used were SYBY Green Master Mix (Takara). The reaction system was as follows: 10 μL 2× SYBR buffer, 0.5 μL Forward Primer (10 μM), 0.5 μL Reverse Primer (10 μM), 1 μL cDNA, and 8 μL HO. The PCR program was: 94°C for 30 s; 94°C for 5 s, 60°C for 30 s, for 45 cycles.

[0043] Table 3 RT-PCR primers for RpMAPK3 gene

[0044]

[0045]

[0046] Example 2 Experimental results

[0047] 2.1 Effect of overexpression of RpMAPK3 gene on germination rate of Arabidopsis thaliana under saline-alkali stress conditions:

[0048] Wild-type Arabidopsis thaliana and T2 seeds overexpressing the RpMAPK3 gene were seeded on pH 5.8 (0 mM NaHCO3) and pH 8.2 (5 mM NaHCO3) media, respectively. Three overexpressing plants were selected as three biological replicates. Ten seeds from each plant were seeded on plates, with three replicates forming three technical replicates. Germination rates were calculated after 7 days.

[0049] The results are as follows Figure 1 As shown, the germination rate of wild-type Arabidopsis thaliana on a medium with pH 5.8 (0 mM NaHCO₃) was 93.33%, while that on a medium with pH 8.2 (5 mM NaHCO₃) was 36.67%. Compared to the two, the germination rate was significantly reduced under saline-alkali stress. The germination rate of transgenic RpMAPK3 seeds at pH 5.8 (0 mM NaHCO₃) and pH 8.2 (5 mM NaHCO₃) was not significantly different, reaching 92.21% and 95.57%, respectively. This suggests that overexpressing RpMAPK3 can improve the germination rate of plants under saline-alkali stress.

[0050] 2.2 Effects of overexpression of RpMAPK3 gene on Arabidopsis roots under saline-alkali stress conditions:

[0051] T2 seeds of wild-type Arabidopsis and transgenic RpMAPK3 plants were seeded on media at pH 5.8 (0 mM NaHCO₃) and pH 8.2 (3 mM NaHCO₃, 5 mM NaHCO₃), respectively. Three overexpressing plants were selected as three biological replicates. Ten seeds from each plant were seeded on plates, with three plates replicated as three technical replicates. Root phenotypes were observed 14 days later.

[0052] The results are as follows Figure 2 and Figure 3 As shown, on a medium without saline-alkali stress (pH 5.8, 0 mM NaHCO₃), there were no significant differences in root length and surface area between wild-type and overexpressing plants. However, the wild-type had 9.09% more root tips than the overexpressing plants. On a saline-alkali stress medium (pH 8.2, 5 mM NaHCO₃), the transgenic plants exhibited higher root length, surface area, and root tip numbers than the wild-type, by 27.27%, 47.71%, and 36.21%, respectively. This suggests that under saline-alkali stress conditions, root development in the wild-type is significantly inhibited, while overexpressing the RpMAPK3 gene can enhance the plant's resistance to saline-alkali stress.

[0053] 2.3 Quantitative PCR detection of RpMAPK3 gene expression:

[0054] To verify the phenotype of transgenic plants with improved plant resistance under saline-alkali stress, the screened T2 generation seeds were sown on pH 5.8 (0mM NaHCO3) and pH 8.2 (5mM NaHCO3) culture media, respectively. After 14 days of culture, RNA was extracted from the whole plant, reverse transcribed into cDNA, and then quantitative PCR detection was carried out.

[0055] The results are as follows Figure 4 As shown in the results, under no salinity or alkali stress, the expression level of the RpMAPK3 gene in the overexpressing plants was 30 times higher than that in the wild type. Under high salinity and high alkali conditions, the expression level of the RpMAPK3 gene in the overexpressing plants was significantly higher than that in the wild type by 70,000 times. This indicates that overexpressing the RpMAPK3 gene can significantly improve the salt and alkali tolerance of plants.

[0056] Therefore, the present invention adopts the above-mentioned salt-alkali tolerance RpMAPK3 gene of Rhododendron splendidum, its encoded protein and application. The RpMAPK3 gene can improve the adaptability and growth conditions of azalea in alkaline soil, not only expanding the planting range of azalea, but also improving its growth conditions under adverse soil conditions, providing more options for ornamental gardening and ecological environment protection. At the same time, it can also be used for the cultivation of new varieties of azalea, providing an effective gene resource for the cultivation of new varieties of salt-alkali tolerance azalea.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Rhododendron splendidum is salt- and alkali-tolerant RpMAPK3 The application of the gene in improving the salt-alkali tolerance of Arabidopsis thaliana is characterized by: Rhododendron splendidum is salt-alkali tolerant RpMAPK3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; Overexpression of Rhododendron splendidum RpMAPK3 Genes that can improve the salt and alkali tolerance of Arabidopsis thaliana and Rhododendron splendidum RpMAPK3 Genetically encoded RpMAPK3 The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. Application of a recombinant vector in improving salt and alkali tolerance in Arabidopsis thaliana, characterized by: The recombinant vector contains the salt-alkali resistance of Rhododendron splendidum shown in SEQ ID NO.1 RpMAPK3 Gene, recombinant vector overexpressed by Rhododendron splendidum salt and alkali tolerance RpMAPK3 Gene that can improve salt-alkali tolerance in Arabidopsis thaliana.

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