The RpHARDY gene and its application in improving the salt and alkali tolerance of rhododendrons

Abstract

This invention relates to the field of genetic engineering technology, and discloses the RpHARDY gene and its application in improving the salt and alkali tolerance of rhododendrons. The nucleotide sequence of the RpHARDY gene is shown in SEQ ID NO.1. The amino acid sequence of the RpHARDY protein encoded by the RpHARDY gene is shown in SEQ ID NO.2. This invention utilizes the aforementioned RpHARDY gene and its application in improving the salt and alkali tolerance of rhododendrons. Overexpression of the RpHARDY gene can increase seed germination rate, thereby significantly enhancing salt and alkali tolerance, improving the growth of Rhododendron simsii under unfavorable soil conditions, and providing effective genetic resources for the breeding of new salt-tolerant rhododendron varieties.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the RpHARDY gene and its application in improving the salt and alkali tolerance of rhododendrons. Background Technology

[0002] Salt and alkali stress severely restricts plant growth and yield. The effects of salt and alkali stress on plant growth include water stress, ion imbalance, and toxicity. Under salt and alkali stress, excessively high salt concentrations in the soil make it difficult for plant roots to absorb water, hindering cell expansion in roots, stems, and young leaves and causing stomatal closure. + and Cl - The excessive accumulation of salt and alkali within cells affects normal cellular metabolism, thus hindering plant growth and development. The plant response to salt and alkali stress is a complex physiological and molecular regulatory process involving multiple mechanisms and signaling pathways to maintain plant growth and survival.

[0003] Rhododendrons are evergreen shrubs belonging to the genus *Rhododendron* in the family Ericaceae. They are characterized by large, brightly colored flowers and year-round green foliage, making them highly valuable for both ornamental and economic purposes. Plants in this genus have strict ecological requirements, preferring acidic environments with an optimal soil pH of 4.5 to 6. They exhibit poor tolerance to alkaline conditions, and salt-alkali stress is a major limiting factor for their growth, development, ex-situ conservation, and landscaping applications. *Rhododendron pulchrum*, belonging to the subgenus *Rhododendron* (*Tsutsusi*) in the family Ericaceae, is a common cultivated rhododendron group in my country. Due to its strong resilience, it is widely used in green space cultivation in the Yangtze River Delta region, as well as in Fujian, Hunan, and Guangdong provinces. However, *Rhododendron pulchrum* has few varieties, and those used in green spaces are primarily purple-flowered varieties, resulting in a significant lack of variety in ornamental appeal.

[0004] AP2 / ERF are a class of plant-specific transcription factors that primarily participate in plant growth and development, as well as the regulation of gene expression related to biotic and abiotic stresses. HARDY belongs to the AP2 / ERF-like subfamily within the AP2 / ERF (APETALA2 / ethyleneresponsive element-binding factors) family, encoding a transcription factor protein that plays a crucial role in regulating biotic and abiotic stresses. Overexpression of HARDY in Arabidopsis thaliana results in thicker leaves and more lateral roots. When HARDY is transfected into Egyptian clover (Trifolium alexandrinum L.), the transgenic plants, under combined drought and high-salt stress, exhibit significantly higher dry and fresh weights compared to the wild type. Summary of the Invention

[0005] The purpose of this invention is to provide the RpHARDY gene and its application in improving the salt and alkali tolerance of azaleas. Overexpression of the RpHARDY gene in Rhododendron simsii can improve seed germination rate, thereby significantly enhancing salt and alkali tolerance and improving the growth of Rhododendron simsii under unfavorable soil conditions, providing an effective gene resource for the breeding of new salt-tolerant azalea varieties.

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

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

[0008] This invention also provides the application of the above-mentioned RpHARDY gene in improving the salt and alkali tolerance of Rhododendron simsii.

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

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

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

[0012] This invention also provides the application of the above-mentioned recombinant carrier in improving the salt and alkali tolerance of Rhododendron simsii.

[0013] The advantages and positive effects of the RpHARDY gene described in this invention and its application in improving the salt and alkali tolerance of rhododendrons are as follows:

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

[0015] 2. Overexpression of the RpHARDY gene in Rhododendron simsii can improve seed germination rate, thereby significantly enhancing salt and alkali tolerance and improving the growth of Rhododendron simsii under unfavorable soil conditions, providing effective gene resources for the breeding of new salt-tolerant Rhododendron varieties.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This invention illustrates the effect of RpHARDY gene-transgenic plants on seed germination rate in various embodiments. A represents the phenotype of RpHARDY gene-transgenic plants under control and salt-alkali stress conditions; B represents the statistical results of RpHARDY gene-transgenic plants under control and salt-alkali stress conditions; Col represents the wild-type control; and 35S:RpHARDY represents the transgenic line.

[0018] Figure 2 The following are root phenotypes of Arabidopsis thaliana overexpressing the RpHARDY gene under salt-alkali stress in this embodiment of the invention. A represents the root phenotypes of wild-type and overexpressing plants under pH 5.8 (0 mM NaHCO3) conditions, B represents the root phenotypes of wild-type and overexpressing plants under pH 8.2 (5 mM NaHCO3) conditions, Col represents the wild-type control, and 35S:RpHARDY represents the transgenic line.

[0019] Figure 3 This invention illustrates the effect of RpHARDY gene overexpression on Arabidopsis roots under salt-alkali stress. A represents the root length, root surface area, and root tip number of wild-type and overexpressing plants under pH 5.8 (0 mM NaHCO3) conditions; B represents the root length, root surface area, and root tip number of wild-type and overexpressing plants under pH 8.2 (5 mM NaHCO3) conditions; Col represents the wild-type control; 35S:RpHARDY represents the transgenic line.

[0020] Figure 4 The following are quantitative PCR results of the RpHARDY gene in the embodiments of the present invention, wherein A represents the expression of the RpHARDY gene in wild-type and overexpressing plants under pH 5.8 (0mM NaHCO3) conditions, B represents the expression of the RpHARDY gene in wild-type and overexpressing plants under pH 8.2 (5mM NaHCO3) conditions, Col represents the wild-type control, and OE represents the transgenic line. Detailed Implementation

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

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

[0023] Example 1 Method Steps

[0024] 1.1 Cloning and vector construction of the RpHARDY gene in Rhododendron simsii:

[0025] Primers for amplifying the HARDY gene were designed (Table 1). The PCR reaction system was as follows: 10 μL 5×PCR mix, 4 μL dNTP (2.5 mM), 1.5 μL Prime F (10 μM / L), 1.5 μL Prime R (10 μM / L), 2 μL template (cDNA), 0.2 μL Prime star, and ddH2O to a final volume of 50 μL. The PCR amplification program was as follows: 95℃ for 5 min; 94℃ for 30 s, 54℃ for 30 s, 72℃ for 2 min, 38 cycles; 72℃ for 2 min, 4℃ for 10 min. PCR products were recovered using a gel extraction kit. The plasmid digestion system was as follows: 5 μL 10×Q-cut buffer, 2 μL restriction enzyme, 1 μg plasmid, and ddH2O to a final volume of 50 μL, digested at 37℃ for 2–3 hours. The digestion products were recovered using a gel extraction kit from Tiangen Pharmaceuticals. The ligation system was as follows: 6 μL PCR product, 2 μL plasmid, 1 μL 10×T4 ligase buffer, 0.5 μL T4 ligase, and ddH2O to a final volume of 10 μL. Ligation was performed overnight at 4°C. The ligation product was then added to 50 μL of TOP10 competent cells, incubated on ice for 30 min, heat-shocked at 37°C for 30 s, incubated on ice for 2 min, and then 1 mL of antibiotic-free LB medium was added. The cells were incubated at 37°C for 45 min, then plated onto LB plates with the appropriate antibiotic and incubated overnight at 37°C. Positive colonies were identified by colony PCR.

[0026] Table 1 Primers for the RpHARDY gene

[0027]

[0028] 1.2 Agrobacterium-mediated transformation:

[0029] Add 0.5 μl of plasmid vector to 50 μl of GV3101 Agrobacterium competent cells and mix well. Transfer the mixture to an electroporation cuvette and place on ice for 1 min. The electroporation program is 2.2 kV, 5.0 ms. Immediately after electroporation, add 1 mL of antibiotic-free LB medium and incubate at 30°C for 1 h on a shaker. Then, spread the mixture on solid LB selective medium containing Kan+Gen and incubate at 30°C for 48 h.

[0030] 1.3 Arabidopsis transformation and transgenic plant screening:

[0031] Arabidopsis transformation was performed using the inflorescence infection method, selecting Arabidopsis thaliana in its early flowering stage with good growth as material. Agrobacterium tumefaciens containing the target vector was gently shaken at 30℃ for 12 hours, then vigorously shaken until the OD reached 0.8–1.0, and centrifuged for 10 minutes. The Agrobacterium cells were resuspended in freshly prepared transformation buffer. During transformation, the entire Arabidopsis flower was immersed in the transformation buffer containing Agrobacterium tumefaciens for 45 seconds. The transformed Arabidopsis was then wrapped in plastic wrap and kept in the dark for 24 hours before being removed.

[0032] The T0 generation Arabidopsis seeds received after transformation were dried in a 30°C incubator for 3-7 days, sterilized, and then spread on MS plates containing screening antibiotics. Positive Arabidopsis plants were identified, and T1 generation Arabidopsis individual plants were harvested. The T1 generation seeds were screened on a medium containing screening antibiotics, and lines exhibiting a 3:1 segregation ratio (i.e., single-copy insertion lines) were selected for subculturing and subsequent experiments.

[0033] 1.4 Phenotypic experiments of RpHARDY gene overexpression:

[0034] Prepare 1 / 2 MS medium with 30% sucrose. Adjust the pH of the medium to 5.8 and 8.2 respectively using 1M / L KOH solution, then add 7g of agar powder and autoclave. After sterilization, cool to approximately 40°C, add 0.8M / L NaHCO3 to prepare alkali stress medium, pour into petri dishes, and allow to cool and solidify before proceeding with subsequent experiments. The formulation of the stress medium is shown in Table 2.

[0035] T2 generation Arabidopsis seeds overexpressing the RpHARDY gene and wild-type Arabidopsis seeds were selected. Ten seeds were spotted on alkaline stress medium plates, with three replicates. The plates were placed in a light incubator for 14 days to observe their phenotype.

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

[0037]

[0038] 1.5 Real-time quantitative PCR was used to verify the expression level of the RpHARDY gene in transgenic plants:

[0039] SnapGene was used to design quantitative primers for the RpHARDY gene (Table 3). After culturing T2 generation transgenic Arabidopsis seeds on 1 / 2 MS medium for 14 days, whole-plant RNA was extracted and reverse transcribed into cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMIX for qPCR kit. Real-time PCR was performed using SYBY GreenMaster Mix (Takara). The reaction mixture was as follows: 10 μL 2×SYBR, 0.5 μL Forward Primer (10 μM), 0.5 μL Reverse Primer (10 μM), 1 μL cDNA, and 8 μL H2O. The PCR program was: 94℃ for 30 s; 94℃ for 5 s, 60℃ for 30 s, for 45 cycles.

[0040] Table 3 Primers for RT-PCR of the RpHARDY gene

[0041]

[0042] Results of Example 2

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

[0044] Wild-type Arabidopsis thaliana and T2 generation seeds overexpressing the RpHARDY gene were seeded on medium at pH 5.8 (0 mM NaHCO3) and pH 8.2 (5 mM NaHCO3), respectively. Three overexpressing plants were selected as three biological replicates. Ten seeds from each plant were seeded on a plate, and three plates were used as three technical replicates. Germination rate was recorded after 7 days.

[0045] The results are as follows Figure 1 As shown, the germination rate of wild-type Arabidopsis thaliana was 93.33% on medium at pH 5.8 (0 mM NaHCO3) and 36.67% on medium at pH 8.2 (5 mM NaHCO3), which was significantly lower than the germination rate under no salt-alkali stress conditions. The germination rate of seeds from RpHARDY transgenic plants was not significantly different between pH 5.8 (0 mM NaHCO3) and pH 8.2 (5 mM NaHCO3), with germination rates of 93.33% and 92.2%, respectively. This indicates that overexpression of the RpHARDY gene can increase the germination rate of plants under salt-alkali stress. Figure 1 ).

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

[0047] Seeds from wild-type Arabidopsis thaliana and the T2 generation of RpHARDY transgenic plants were sown on medium at pH 5.8 (0 mM NaHCO3) and pH 8.2 (5 mM NaHCO3), respectively. Three overexpression plants were selected as three biological replicates. Ten seeds from each plant were sown on a plate, and three plates were used as three technical replicates. Root phenotypes were observed after 14 days.

[0048] The results are as follows Figure 2 and Figure 3 As shown, on a salt- and alkali-free medium at pH 5.8 (0 mM NaHCO3), there were no significant differences in root length and surface area between wild-type and overexpression plants. On a salt- and alkali-stressed medium at pH 8.2 (5 mM NaHCO3), the transgenic plants had 3.5 times more root length, 6.2 times more root surface area, and 2.7 times more root tips than the wild-type plants, respectively. Figure 2 , Figure 3This indicates that root development in wild-type plants is significantly inhibited under salt-alkali stress, while overexpression of the RpHARDY gene can improve the plant's resistance to salt-alkali stress.

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

[0050] To verify the phenotype of transgenic plants that enhance plant resistance under salt-alkali stress, the selected T2 generation seeds were sown on culture media with pH 5.8 (0 mM NaHCO3) and pH 8.2 (5 mM NaHCO3), respectively. After 14 days of culture, RNA was extracted from whole plants, reverse transcribed into cDNA, and then quantitative PCR was performed.

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

[0052] Therefore, this invention utilizes the RpHARDY gene, encoded protein, and application of Rhododendron simsii. Overexpression of the RpHARDY gene can improve seed germination rate, thereby significantly enhancing salt and alkali tolerance, improving the growth of Rhododendron simsii under unfavorable soil conditions, and providing effective gene resources for the breeding of new salt-tolerant Rhododendron varieties.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of the RpHARDY gene in improving the salt and alkali tolerance of Rhododendron simsii, characterized by: The nucleotide sequence of the RpHARDY gene of Rhododendron simsii is shown in SEQ ID NO.1, and the amino acid sequence of the RpHARDY protein encoded by the RpHARDY gene is shown in SEQ ID NO.

2. Overexpression of the RpHARDY gene of Rhododendron simsii can improve the salt and alkali tolerance of Rhododendron simsii.

2. The application of recombinant carriers in improving the salt and alkali tolerance of Rhododendron simsii, characterized by: The recombinant vector contains the RpHARDY gene of Rhododendron simsii as shown in SEQ ID NO.

1. The recombinant vector improves the salt and alkali tolerance of Rhododendron simsii by overexpressing the RpHARDY gene.

Images