Rhododendron simsii saline-alkaline tolerant RpMAPK3 gene as well as encoding protein and application thereof

Abstract

The invention discloses a rhododendron simsii saline-alkaline tolerant RpMAPK3 gene as well as an encoding protein and application thereof, and relates to the technical field of gene engineering. The nucleotide sequence of the saline-alkaline tolerant RpMAPK3 gene of the rhododendron simsii is as shown in SEQ ID NO. 1. The amino acid sequence of the RpMAPK3 protein encoded by the Rhododendron simsii saline-alkaline tolerant RpMAPK3 gene is as shown in SEQ ID NO. 2. According to the RpMAPK3 gene, the encoding protein and the application of the RpMAPK3 gene, the RpMAPK3 gene can improve the adaptability and the growth condition of rhododendron in alkaline soil, the planting range of the rhododendron is enlarged, the growth condition of the rhododendron under adverse soil conditions is improved, more choices are provided for ornamental horticulture and ecological environment protection, and the application of the RpMAPK3 gene is promoted. Meanwhile, the gene can also be used for cultivating new varieties of azalea, and an effective gene resource is provided for cultivating new varieties of saline-alkaline tolerant azalea.

Description

Technical Field

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

[0002] Salt-alkali stress is one of the common abiotic stresses, which has a significant impact on plant growth, development and yield. Salt-alkali stress mainly includes two forms: salt stress and alkali stress, which have different characteristics of damage to plants. Salt stress is mainly manifested as ion stress, osmotic stress and oxidative stress, while alkali stress adds high pH stress on the basis of salt stress, which causes more serious and complex damage to plants. Plants respond and adapt to these stresses through a variety of physiological and molecular mechanisms.

[0003] Rhododendron is an evergreen shrub belonging to the genus Rhododendron in the family Ericaceae. It has large, colorful flowers that are evergreen all year round and has extremely high ornamental and economic value. This genus of plants has strict requirements on the ecological environment and prefers an acidic growth environment. The pH of the soil suitable for its growth is 4.5 to 6. It has poor alkalinity tolerance, and salinity stress is the main limiting factor for its growth and development, ex situ conservation, and garden application. Rhododendron pulchrum belongs to the subgenus Rhododendron Tsutsusi in the genus Ericaceae. It is a commonly cultivated rhododendron group in my country. Due to its strong stress resistance, it is widely used in green space cultivation in the Yangtze River Delta region and Fujian, Hunan, and Guangdong. However, there are few varieties of Rhododendron pulchrum, and the purple-flowered varieties used in green space are basically the purple-flowered varieties, and the monotonous ornamental characteristics are extremely prominent.

[0004] Mitogen-activated protein kinases (MAPK) are a class of serine / threonine (Ser / Thr) protein kinases that are ubiquitous and highly conserved in eukaryotes. The MAPK cascade signaling system includes MAPK kinase kinase (MAP kinase kinase kinase, MEKK), MAPK kinase (MAP kinase kinase, MEK) and MAPK. These three kinases can be activated in sequence and jointly regulate a variety of important cellular physiological / pathological processes, playing an important role in the normal growth and development of plants and in responding to external environmental stress. As the last protein kinase, MAPK is a key protein kinase in the process of cell signal conversion and amplification, and is a direct factor that connects cascade signals with downstream proteins. At present, a large number of MAPK family members have been identified from different plants. Generally, according to the conserved TXY amino acid residue sequence phosphorylated by MEK, the MAPKs family can be divided into two subfamilies: TEY and TDY. The TEY subfamily is further divided into three categories: A, B, and C according to the characteristics of its conserved region. The MAPK3 gene belongs to the TEY subfamily A class, 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 activation substrate of MKK4 and mediates the resistance of overexpressed plants to high salt. Summary of the invention

[0005] The 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 expand the planting range of azalea, but also improve its growth condition under unfavorable soil conditions, provide more choices 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.

[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 resistant RpMAPK3 gene of Rhododendron splendidum, and the recombinant vector contains 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 soil, which not only expands the planting range of azalea, but also improves its growth 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, 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 : The effect of the RpMAPK3 gene overexpression plant on the germination rate in the embodiment of the present invention, wherein A is the phenotype of the germination rate of the RpMAPK3 gene transgenic plant under the control and saline-alkali stress; B is the statistical result of the germination rate of the RpMAPK3 gene transgenic plant under the control and saline-alkali stress, Col is the wild type control, and 35S:RpMAPK3 is the transgenic strain;

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

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

[0020] Figure 4 : The quantitative PCR results of the RpMAPK3 gene in the example of the present invention, wherein A is the expression of the RpMAPK3 gene in the wild type and overexpressing plants under pH 5.8 (0 mM NaHCO3), B is the expression of the RpMAPK3 gene in the wild type and overexpressing plants under pH 8.2 (5 mM NaHCO3), Col is the wild type control, and OE is the transgenic line. DETAILED DESCRIPTION

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

[0022] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

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

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

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

[0026] Example 1 Experimental method

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

[0028] According to the CDS sequence of RpMAPK3 gene, primers for amplifying MAPK3 gene were designed (Table 1). The PCR reaction system was as follows: 5×PCR mix 10μL, dNTP (2.5mM) 4μL, Prime F (10uM / L) 1.5μL, Prime R (10μM / L) 1.5μL, template (cDNA) 2μL, Prime star 0.2μL, ddH2O supplemented to 50μL. The PCR amplification program was as follows: 95℃5min; 94℃30s, 54℃30s, 72℃2min, 38cycles; 72℃2min, 4℃10min. The PCR product was recovered using the gel recovery kit of Jerri. The plasmid restriction system was as follows: 10×Q-cut buffer 5μL, endonuclease 2μL, plasmid 1μg, ddH2O supplemented to 50μL, and restriction digestion at 37℃ for 2 to 3 hours. The digestion product was recovered using Tiangen's gel recovery kit. The connection 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, ddH2O supplemented to 10 μL, and connected at 4°C overnight. Add 50 μL of TOP10 competent cells to the connection product, let it stand on ice for 30 minutes, heat shock at 37°C for 30 seconds, let it stand on ice for 2 minutes, add 1 mL of non-antibiotic LB medium, resuscitate at 37°C for 45 minutes, and then spread on the corresponding resistance LB plate and culture at 37°C overnight. Colony PCR identified positive clone colonies.

[0029] Table 1 Primers for MAPK3 gene

[0030]

[0031] 1.2 Agrobacterium transformation:

[0032] Take 0.5 μl of plasmid vector and add it to 50 μl of GV3101 Agrobacterium competent cells, mix well, transfer the mixture to the electroporation cup, and place it on ice for 1 min. The electroporation program is 2.2 kV, 5.0 ms, and immediately add 1 mL of antibiotic-free liquid LB after the electroporation. After shaking at 30°C for 1 hour, spread it on the solid LB selection medium containing Kan+Gen and culture it at 30°C for 48 hours.

[0033] 1.3 Arabidopsis transformation and transgenic plant screening:

[0034] Arabidopsis transformation adopts the inflorescence infection method, and Arabidopsis in the early flowering stage with good growth status is selected as the material. Agrobacterium GV3101 containing the target vector is shaken at 30℃ for 12h, shaken vigorously until the OD is 0.8-1.0, centrifuged for 10min, and the Agrobacterium cells are resuspended in the newly prepared transformation solution. During transformation, all the flowers of Arabidopsis are immersed in the transformation solution containing Agrobacterium for 45s, and the transformed Arabidopsis is wrapped in plastic wrap and kept away from light for 24 hours before being taken out.

[0035] The T0 generation Arabidopsis seeds received from transformation were dried in a 30°C incubator for 3-7 days, and after sterilization, were plated on MS plates containing screening antibiotics. The positive Arabidopsis were identified and the T1 generation Arabidopsis individual plants were harvested. The T1 generation seeds were screened on a medium containing screening antibiotics, and the strains with a 3:1 separation ratio, i.e., the strains with single copy insertion, were selected for passage and subsequent experiments.

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

[0037] Prepare 1 / 2MS medium, 30% sucrose, adjust the medium pH to 5.8 and 8.2 with 1M / L KOH solution, add 7g agar powder and sterilize by high pressure. After sterilization, cool to about 40°, add 0.8M / L NaHCO3 to make alkaline stress medium, pour into culture dish, wait for cooling and solidification, and then carry out subsequent experiments. The formula of stress medium is shown in Table 2.

[0038] Arabidopsis wild-type and T2 generation seeds overexpressing the RpMAPK3 gene were selected and spotted on alkaline stress medium plates, with 10 seeds spotted on each plate, 3 experimental replicates, 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 Real-time fluorescence quantitative PCR verification of RpMAPK3 gene expression in transgenic plants

[0042] SnapGene was used to design quantitative primers for the RpMAPK3 gene (Table 3). After the T2 transgenic Arabidopsis seeds were cultured on 1 / 2MS medium for 14 days, RNA from the whole plant was extracted and reverse transcribed into cDNA using the TransScript All-in-One First-Strand cDNASynthesis SuperMIX for qPCR Kit. The reagents used for real-time PCR were SYBY Green Master Mix (Takara). The reaction system was as follows: 2×SYBR 10μL, Forward Primer (10u M) 0.5μL, Reverse Primer (10u M) 0.5μL, cDNA 1μL, H2O 8μL. PCR program: 94℃30s; 94℃5s, 60℃30s, 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 and T2 seeds overexpressing the RpMAPK3 gene were planted on pH 5.8 (0mM NaHCO3) and pH 8.2 (5mM NaHCO3) culture media, and three overexpression plants were selected as three biological replicates. Ten seeds of each plant were planted on a plate, and three plates were repeated as three technical replicates. The germination rate was counted after 7 days.

[0049] The results are as follows Figure 1 As shown, the germination rate of Arabidopsis wild type on pH 5.8 (0mM NaHCO3) medium was 93.33%, and the germination rate on pH 8.2 (5mM NaHCO3) medium was 36.67%. Compared with the two, the germination rate under saline-alkali stress conditions was significantly reduced. There was no significant difference in the germination rate of seeds of plants transfected with RpMAPK3 gene at pH 5.8 (0mM NaHCO3) and pH 8.2 (5mM NaHCO3), which were 92.21% and 95.57%, respectively, indicating that overexpression of RpMAPK3 gene can improve the germination rate of plants under saline-alkali stress.

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

[0051] The T2 seeds of wild-type Arabidopsis and transgenic RpMAPK3 plants were planted on pH 5.8 (0mM NaHCO3) and pH 8.2 (3mM NaHCO3, 5mM NaHCO3) culture media, respectively. Three overexpression plants were selected as three biological replicates. Ten seeds of each plant were planted on plates, and three plates were repeated as three technical replicates. The root phenotype was observed after 14 days.

[0052] The results are as follows Figure 2 and Figure 3 As shown, in the medium without salt-alkali stress at pH 5.8 (0mM NaHCO3), there was no significant difference in the length and surface area of ​​the roots between the wild type and the overexpression plants, and the number of root tips in the wild type was 9.09% more than that in the overexpression plants. In the medium with salt-alkali stress at pH 8.2 (5mM NaHCO3), the root length, root surface area and number of root tips of the transgenic plants were higher than those of the wild type, which were 27.27%, 47.71% and 36.21% higher than those of the wild type, respectively. This indicates that under salt-alkali stress conditions, the root development of the wild type is significantly inhibited, while overexpression of the RpMAPK3 gene can improve the salt-alkali stress resistance of the plant.

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

[0054] In order to verify the phenotype of transgenic plants in improving 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, the whole plant was taken to extract RNA, which was reverse transcribed into cDNA and then used for quantitative PCR detection.

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

[0056] Therefore, 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 expanding the planting range of azalea, but also improving its growth conditions under unfavorable soil conditions, providing more choices 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.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. 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 solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. The salt-alkali tolerance RpMAPK3 gene of Rhododendron splendidum is characterized by: The nucleotide sequence of the salt-alkali tolerance RpMAPK3 gene of Rhododendron splendidum is shown in SEQ ID NO.

1.

2. The RpMAPK3 protein encoded by the salt-alkali tolerant RpMAPK3 gene of Rhododendron splendidum as claimed in claim 1, characterized in that: The amino acid sequence of RpMAPK3 protein is shown in SEQ ID NO.

2.

3. A recombinant vector containing the salt-alkali tolerant RpMAPK3 gene of Rhododendron splendidum, characterized in that: The recombinant vector contains the nucleotide sequence shown as SEQ ID NO.

1.

4. The recombinant vector according to claim 3, characterized in that: Recombinant vectors include cloning vectors or plant overexpression vectors.

5. The recombinant vector according to claim 4, characterized in that: The recombinant vector is a CaMV35S-RpMAPK3-GFP plant expression vector.

6. Use of the salt-alkali tolerance RpMAPK3 gene of Rhododendron splendidum as claimed in claim 1 in improving the salt-alkali tolerance of Rhododendron splendidum.

7. Use of the recombinant vector according to any one of claims 3 to 5 in improving the salt-alkali tolerance of Rhododendron splendidum.

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