RpHARDY gene and application of RpHARDY gene in improvement of saline-alkaline tolerance of rhododendron

Abstract

The invention relates to the technical field of gene engineering, and discloses an RpHARDY gene and application thereof in improvement of saline-alkaline tolerance of rhododendron simsii. The nucleotide sequence of the RpHARDY gene is as shown in SEQ ID NO. 1. 1, and the amino acid sequence of the RpHARDY protein coded by the RpHARDY gene is as shown in SEQ ID NO. 2. By adopting the RpHARDY gene and the application of the RpHARDY gene in improving the saline-alkaline tolerance of the rhododendron, the overexpression of the RpHARDY gene can improve the germination rate of seeds, so that the saline-alkaline tolerance is remarkably improved, the growth condition of the rhododendron pulchrum under adverse soil conditions is improved, and an effective gene resource is provided for the cultivation of a new variety of the saline-alkaline tolerant rhododendron pulchrum.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, in particular to a RpHARDY gene and an application thereof in improving the salt-alkali tolerance of azalea. Background Art

[0002] Salt-alkali stress seriously restricts the growth and yield of plants. The effects of salt-alkali stress on plant growth include both water stress and ion imbalance and toxicity. Under salt-alkali stress, the high salt concentration in the soil makes it difficult for plant roots to absorb water, which hinders the cell expansion of roots, stems and young leaves and causes stomata to close. + and Cl - The large amount of accumulation in cells affects the normal metabolism of cells and hinders the growth and development of plants. The response of plants to salt-alkali stress is a complex physiological and molecular regulatory process involving multiple mechanisms and signal pathways to maintain plant growth and survival.

[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] AP2 / ERF is a type of transcription factor unique to plants, which is mainly involved in the expression and regulation of genes related to plant growth and development as well as biotic and abiotic stresses. HARDY belongs to the AP2 / ERF-like subfamily in the AP2 / ERF (APETALA2 / ethylene responsive element binding factors) transcription factor family, encoding transcription factor proteins, and plays an important role in regulating biotic and abiotic stresses. When HARDY is overexpressed in Arabidopsis, the leaves become thicker and the lateral roots become more numerous. When HARDY was transferred into Egyptian clover (Trifolium alexandrinum L.), the dry weight and fresh weight of the transgenic plants were significantly increased compared with the wild type under the combined stress of drought and high salt. Summary of the invention

[0005] The purpose of the present invention is to provide a RpHARDY gene and its application in improving the salt-alkali tolerance of azalea. Overexpression of the RpHARDY gene of Rhododendron splendidum can increase the seed germination rate, thereby significantly improving the salt-alkali tolerance, improving the growth of Rhododendron splendidum under adverse soil conditions, and providing effective gene resources for the cultivation of new salt-alkali tolerant azalea varieties.

[0006] To achieve the above object, the present invention provides a RpHARDY gene, which is a Rhododendron splendidum RpHARDY gene, and its nucleotide sequence is shown in SEQ ID NO.1.

[0007] The present invention also provides the RpHARDY protein encoded by the RpHARDY gene. The amino acid sequence of the RpHARDY protein is shown in SEQ ID NO.2.

[0008] The present invention also provides application of the RpHARDY gene in improving the salt-alkali tolerance of Rhododendron splendidum.

[0009] The present invention also provides a recombinant vector comprising the RpHARDY gene, wherein the recombinant vector comprises a nucleotide sequence as shown in SEQ ID NO.1.

[0010] Furthermore, recombinant vectors include cloning vectors and plant overexpression vectors.

[0011] Furthermore, the recombinant vector is a CaMV35S-RpHARDY-GFP plant expression vector.

[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 RpHARDY gene of the present invention and its application in improving the salt-alkali tolerance of azalea are:

[0014] 1. The present invention cloned the RpHARDY gene of Rhododendron splendidum, constructed the CaMV35S-RpHARDY-GFP plant expression vector, and then obtained the transgenic material of Arabidopsis thaliana by Agrobacterium infection, and verified the salt-alkali tolerance function of RpHARDY in Arabidopsis thaliana.

[0015] 2. Overexpression of the RpHARDY gene of Rhododendron splendidum can increase the seed germination rate, thereby significantly improving the salt-alkali tolerance and the growth of Rhododendron splendidum under adverse soil conditions, providing effective genetic resources for the cultivation of new salt-alkali-tolerant Rhododendron 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 RpHARDY gene transgenic plants on seed germination rate in the embodiment of the present invention, wherein A is the phenotype of the germination rate of the RpHARDY gene transgenic plants under control and saline-alkali stress; B is the statistical result of the germination rate of the RpHARDY gene transgenic plants under control and saline-alkali stress, Col is the wild type control, and 35S:RpHARDY is the transgenic strain;

[0018] Figure 2 : The root phenotype of Arabidopsis thaliana overexpressing the RpHARDY gene under saline-alkali stress in the embodiment of 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:RpHARDY is a transgenic strain;

[0019] Figure 3 The effect of overexpressing the RpHARDY 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; 35S:RpHARDY is the transgenic strain;

[0020] Figure 4 : The quantitative PCR results of the RpHARDY gene in the examples of the present invention, wherein A is the expression of the RpHARDY gene in the wild type and overexpressing plants under pH 5.8 (0 mM NaHCO3), B is the expression of the RpHARDY 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] Example 1 Method Steps

[0024] 1.1 Cloning and vector construction of Rhododendron splendidum RpHARDY gene:

[0025] Primers for amplifying the HARDY gene were designed (Table 1). The PCR reaction system was as follows: 5×PCR mix 10μL, dNTP (2.5mM) 4μL, Prime F (10μM / L) 1.5μL, Prime R (10μM / L) 1.5μL, template (cDNA) 2μL, Prime star 0.2μL, ddH2O 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 a gel recovery kit. The plasmid digestion system was as follows: 10×Q-cut buffer 5μL, endonuclease 2μL, plasmid 1μg, ddH2O to 50μL, digestion at 37℃ for 2 to 3 hours. The digestion product was recovered using the Tiangen Company's gel recovery kit. The ligation system is as follows: 6 μL of PCR product, 2 μL of plasmid, 1 μL of 10×T4 ligasebuffer, 0.5 μL of T4 ligase, ddH2O to 10 μL, and ligation at 4°C overnight. Add 50 μL of TOP10 competent cells to the ligation product, let stand on ice for 30 minutes, heat shock at 37°C for 30 seconds, let stand on ice for 2 minutes, add 1 mL of LB medium without resistance, resuscitate at 37°C for 45 minutes, and then spread on the LB plate with the corresponding resistance, and culture at 37°C overnight. Colony PCR identified the positive clone colonies.

[0026] Table 1 RpHARDY gene primers

[0027]

[0028] 1.2 Agrobacterium transformation:

[0029] 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.

[0030] 1.3 Arabidopsis transformation and transgenic plant screening:

[0031] 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, and centrifuged for 10min when the OD is 0.8-1.0, and the Agrobacterium cells are resuspended in the newly prepared transformation solution. During the 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.

[0032] 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 strains with a 3:1 separation ratio, i.e., strains with single copy insertion, were selected for passage and subsequent experiments.

[0033] 1.4 Phenotypic experiments of overexpression of RpHARDY gene:

[0034] Prepare 1 / 2MS culture medium, 30% sucrose, adjust the pH of the culture medium to 5.8 and 8.2 respectively 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 culture medium, pour into the culture dish and wait for cooling and solidification before subsequent experiments. The formula of stress culture medium is shown in Table 2.

[0035] T2 generation Arabidopsis seeds overexpressing the RpHARDY gene and Arabidopsis wild-type seeds 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.

[0036] Table 2 Stress culture medium formula (1L)

[0037]

[0038] 1.5 Real-time fluorescence quantitative PCR verification of RpHARDY gene expression in transgenic plants:

[0039] SnapGene was used to design quantitative primers for the RpHARDY gene (Table 3). After the T2 transgenic Arabidopsis seeds were cultured on 1 / 2MS medium for 14 days, RNA was extracted from the whole plant 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 (10μM) 0.5μL, Reverse Primer (10μM) 0.5μL, cDNA 1μL, H2O 8μL. PCR program: 94℃30s; 94℃5s, 60℃30s, 45 cycles.

[0040] Table 3 RT-PCR primers for RpHARDY gene

[0041]

[0042] Example 2 Results

[0043] 2.1 Effect of overexpression of RpHARDY gene on germination rate of Arabidopsis thaliana under saline-alkali stress conditions:

[0044] Wild-type Arabidopsis and T2 seeds overexpressing the RpHARDY 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.

[0045] The results are as follows Figure 1 As shown, the germination rate of Arabidopsis wild type on pH5.8 (0mM NaHCO3) medium was 93.33%, and the germination rate on pH8.2 (5mM NaHCO3) medium was 36.67%, which was significantly lower than the germination rate under no salt-alkali stress conditions. There was no significant difference in the germination rate of RpHARDY-transformed plant seeds at pH5.8 (0mM NaHCO3) and pH8.2 (5mM NaHCO3), which were 93.33% and 92.2%, respectively, indicating that overexpression of the RpHARDY gene can increase the germination rate of plants under salt-alkali stress ( Figure 1 ).

[0046] 2.2 Effects of overexpression of RpHARDY gene on Arabidopsis root system under saline-alkali stress conditions:

[0047] The T2 seeds of wild-type Arabidopsis and transgenic RpHARDY plants were planted on pH 5.8 (0mM NaHCO3) and pH 8.2 (5mM NaHCO3) media, respectively, and 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.

[0048] The results are as follows Figure 2 and Figure 3 As shown in the figure, on the medium without salt-alkali stress, 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 overexpressing plants. On the medium with salt-alkali stress, pH 8.2 (5mM NaHCO3), the root length, root surface area and number of root tips of the transgenic plants were 3.5 times, 6.2 times and 2.7 times more than those of the wild type, respectively. Figure 2 , Figure 3). This indicates that under saline-alkali stress conditions, the root development of the wild type is significantly inhibited, while overexpression of the RpHARDY gene can improve the plant's resistance to saline-alkali stress.

[0049] 2.3 Quantitative PCR detection of RpHARDY gene expression:

[0050] 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.

[0051] The results are as follows Figure 4 As shown in the figure, under no salt-alkali stress, the expression level of RpHARDY gene in overexpressed plants was 205 times higher than that in wild type. Under high salt and high alkali treatment conditions, the expression level of RpHARDY gene in overexpressed plants was significantly higher than that in wild type, reaching 4600 times ( Figure 4 ). This indicates that overexpression of the RpHARDY gene can significantly improve the salt-alkali tolerance of plants.

[0052] Therefore, the present invention adopts the above-mentioned Rhododendron splendidum RpHARDY gene, encoded protein and application. Overexpression of the Rhododendron splendidum RpHARDY gene can increase the seed germination rate, thereby significantly improving the salt-alkali tolerance, improving the growth of Rhododendron splendidum under adverse soil conditions, and providing effective gene resources for the cultivation of new salt-alkali tolerant azalea varieties.

[0053] 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. RpHARDY gene, characterized in that: It is the RpHARDY gene of Rhododendron splendidum, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The RpHARDY protein encoded by the RpHARDY gene according to claim 1, characterized in that: The amino acid sequence of the RpHARDY protein is shown in SEQ ID NO.

2.

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

4. A recombinant vector comprising the RpHARDY gene, characterized in that: The recombinant vector contains the nucleotide sequence shown as SEQ ID NO.

1.

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

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

7. Use of the recombinant vector according to any one of claims 4 to 6 in improving the salt-alkali tolerance of Rhododendron splendidum.

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