Rhododendron salt-tolerant gene RpBOR2 and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and discloses a salt and alkali tolerant gene RpBOR2 of Rhododendron and application thereof.The nucleotide sequence of the salt and alkali tolerant gene RpBOR2 of Rhododendron is shown as SEQ ID NO.1.The amino acid sequence of the RpBOR2 protein encoded by the salt and alkali tolerant gene RpBOR2 of Rhododendron is shown as SEQ ID NO.2.The salt and alkali tolerant gene RpBOR2 of Rhododendron and application thereof are used for cultivating new Rhododendron varieties with salt and alkali tolerance, improving the adaptability and growth conditions of Rhododendron in alkaline soil, expanding the planting range of Rhododendron and improving the growth conditions of Rhododendron in adverse soil conditions, providing more choices for ornamental horticulture and ecological environment protection, and having important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a rhododendron salt-alkali tolerance gene RpBOR2 and an application thereof. Background Art

[0002] Salt-alkali stress is one of the important abiotic factors affecting global agricultural production. High salt and alkaline substances in the soil can disrupt the ion balance inside and outside plant cells, leading to an imbalance in cell osmotic pressure and difficulty in water absorption, which in turn affects the normal physiological metabolic processes of plants. The response of plants to salt-alkali stress includes a variety of regulatory mechanisms at the physiological and molecular levels. For example, plants will eliminate salt from cells by accumulating organic acids and ion pumps. Plants will activate a series of salt-alkali stress response genes. The expression of these genes can enhance the plant's antioxidant capacity, regulate the synthesis of osmotic substances, and promote the activity of certain detoxification enzymes. Salt-alkali stress is an important factor affecting plant growth and agricultural production. Understanding its impact mechanism on plants and the plant's response strategy is of great significance for improving the salt-alkali tolerance of crops.

[0003] Azaleas are evergreen shrubs of the genus Rhododendron in the Ericaceae family. Their large, colorful flowers remain green year-round, making them highly ornamental and economically valuable. Plants in this genus have strict ecological requirements and prefer acidic environments, ideally requiring a soil pH of 4.5 to 6. They have poor alkalinity tolerance, and saline-alkali stress is a major limiting factor in their growth, development, ex situ conservation, and landscape applications. Rhododendron pulchrum, a member of the subgenus Tsutsusi in the Ericaceae family, is a commonly cultivated azalea group in my country. 'Purple Crane' is a purple-flowered variety of Rhododendron pulchrum. Due to its strong stress resistance, it is most widely used in green space cultivation in the Yangtze River Delta region, as well as in Fujian, Hunan, and Guangdong.

[0004] The BOR2 gene is one of the genes closely associated with boron absorption and transport in plants. Boron is an essential trace element for plant growth and plays a crucial role in many physiological processes, including cell wall formation, cell membrane stability, and pollen germination and growth. The protein encoded by the BOR2 gene is a boron efflux transporter primarily responsible for transferring boron from the epidermis to the cortex. It participates in the radial transport of boron in plant roots and plays a crucial role in root cell elongation under low-boron conditions. Studies have found that the expression of the Arabidopsis BOR2 gene is induced by NaHCO3, and overexpression of the BOR2 gene enhances plant resistance. The role of BOR2 homologs in azalea under saline-alkali stress has not been reported. To investigate the expression changes of BOR2 homologs in different azalea species under saline-alkali stress, this experiment used the saline-alkali-tolerant azalea cultivar 'Zihe' and the saline-intolerant azalea cultivar 'Qilin'. Both cultivars were subjected to saline-alkali stress in hydroponics, and transcriptome sequencing and qPCR were used to measure changes in BOR2 gene expression. The RpBOR2 gene from the saline-alkali-tolerant cultivar 'Zihe' was cloned, and the phenotypes of Arabidopsis overexpressing the RpBOR2 gene under different saline-alkali treatments were examined, laying the foundation for further elucidating the mechanism of saline-alkali tolerance in azalea. Summary of the Invention

[0005] The purpose of the present invention is to provide a rhododendron salt-alkali tolerance gene RpBOR2 and its application, which are used to cultivate new salt-alkali tolerance rhododendron varieties, while improving the adaptability and growth conditions of rhododendrons in alkaline soils, expanding the planting range of rhododendrons and improving their growth conditions under adverse soil conditions, providing more options for ornamental gardening and ecological environment protection, and having important application value.

[0006] To achieve the above object, the present invention provides a rhododendron salt-alkali tolerance gene RpBOR2, the nucleotide sequence of the gene RpBOR2 is shown in SEQ ID NO.1.

[0007] The present invention also provides an RpBOR2 protein encoded by the salt-alkali tolerance gene RpBOR2 of rhododendron. The amino acid sequence of the RpBOR2 protein is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector comprising the azalea salt-alkali tolerance gene RpBOR2, wherein the recombinant vector comprises the nucleotide sequence shown as SEQ ID NO.1.

[0009] Furthermore, recombinant vectors include cloning vectors and plant overexpression vectors.

[0010] Furthermore, the recombinant vector is a CaMV35S-RpBOR2-GFP plant overexpression vector.

[0011] The present invention also provides an application of azalea salt-alkali tolerance gene RpBOR2 in improving the salt-alkali tolerance of azalea.

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

[0013] The advantages and positive effects of the azalea salt-alkali tolerance gene RpBOR2 and its application described in the present invention are:

[0014] 1. This study conducted transcriptome sequencing on saline- and alkali-tolerant azalea varieties under saline- and alkali-intolerant conditions. The authors found that the BOR2 gene is highly expressed in saline- and alkali-tolerant azalea varieties, while it is lowly expressed in saline- and alkali-intolerant varieties. The RpBOR2 gene from Rhododendron 'Zihe' was cloned, and the CaMV35S-RpBOR2-GFP plant expression vector was constructed. Transgenic Arabidopsis thaliana was obtained through Agrobacterium infection, and the function of RpBOR2 was verified in Arabidopsis thaliana.

[0015] 2. The azalea salt-alkali tolerance gene RpBOR2 can be used to breed new salt-alkali tolerant azalea varieties. At the same time, it can improve the adaptability and growth conditions of azalea in alkaline soil, expand the planting range of azalea and improve its growth under adverse soil conditions, providing more options for ornamental gardening and ecological environment protection, and has important application value.

[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 This is a heat map of the BOR2 gene in two azalea varieties under saline-alkali stress in the embodiment of the present invention, where ZH-0: 'Zihe' saline-alkali treatment for 0 h; ZH-6: 'Zihe' saline-alkali treatment for 6 h; ZH-24: 'Zihe' saline-alkali treatment for 24 h; QL-0: 'Qilin' saline-alkali treatment for 0 h; QL-6: 'Qilin' saline-alkali treatment for 6 h; QL-24: 'Qilin' saline-alkali treatment for 24 h, each group was repeated 3 times;

[0018] Figure 2 The quantitative results of the BOR2 gene in two azalea varieties in the embodiment of the present invention are shown, among which ZH-0: 'Zihe' treated with saline-alkali for 0 hours; ZH-6: 'Zihe' treated with saline-alkali for 6 hours; ZH-24: 'Zihe' treated with saline-alkali for 24 hours; QL-0: 'Qilin' treated with saline-alkali for 0 hours; QL-6: 'Qilin' treated with saline-alkali for 6 hours; QL-24: 'Qilin' treated with saline-alkali for 24 hours;

[0019] Figure 3Figure 1 shows the effect of the transgenic RpBOR2 gene on the germination rate of the plants in the embodiments of the present application, wherein A is a phenotype diagram of the transgenic RpBOR2 gene plants under control and salt-alkali stress; B is a statistical result diagram of the transgenic RpBOR2 gene plants under control and salt-alkali stress, Col: wild type control; 35S: RpBOR2: transgenic line;

[0020] Figure 4 Figure 2 shows the root system phenotype of the Arabidopsis thaliana overexpressing the RpBOR2 gene under salt-alkali stress in the embodiments of the present application, wherein A is the root system phenotype of the wild type and overexpression plants under pH 5.8 (0 mM NaHCO3) conditions; B is the root system phenotype of the wild type and overexpression plants under pH 8.2 (5 mM NaHCO3) conditions, Col: wild type control; 35S: RpBOR2: transgenic line;

[0021] Figure 5 Figure 3 shows the effect of the overexpression of the RpBOR2 gene on the root system of Arabidopsis thaliana under salt-alkali stress in the embodiments of the present application, wherein A is a statistical diagram of the root length, root surface area and root tip number of the wild type and overexpression plants under pH 5.8 (0 mM NaHCO3) conditions; B is a statistical diagram of the root length, root surface area and root tip number of the wild type and overexpression plants under pH 8.2 (5 mM NaHCO3) conditions, Col: wild type control; 35S: RpBOR2: transgenic line. DETAILED DESCRIPTION

[0022] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0023] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs.

[0024] Unless otherwise defined, the instruments, equipment and reagents used in the present application are all conventional commercially available.

[0025] In the following examples, the salt-tolerant gene RpBOR2 is referred to as BOR2 gene.

[0026] Example 1 Experimental method

[0027] 1. Culture and salt stress treatment of rhododendron cuttings:

[0028] Four-month-old cuttings of the salt- and alkali-tolerant azalea cultivar 'Zihe' and the salt- and alkali-sensitive azalea cultivar 'Qilin' were used as test materials and cultured in a hydroponic system until new roots developed. Before hydroponic culture, the cuttings were rinsed with clean water and incubated with deionized water for three days to acclimate to the water environment. The cuttings were then incubated with 1 / 4 Hoagland's nutrient solution (pH = 5.85 ± 0.05), with the solution replaced every seven days. The hydroponic culture conditions included a 20-minute nutrient solution cycle (approximately 5 minutes), continuous oxygenation with an aerator, 16 hours of light / 8 hours of darkness, a temperature of 20-25°C, and a humidity of 40-50%. The cuttings were cultured for approximately 20 days until new roots approximately 1-2 cm in length were formed. The alkaline stress test group was treated by adjusting the pH of the 1 / 4 Hoagland's nutrient solution to 8.3 using a buffer solution consisting of an equal mixture of 0.1M Na₂CO₃ and 0.1M NaHCO₃ (pH = 10.10 ± 0.02).

[0029] 2. Transcriptome sequencing and screening of differentially expressed genes:

[0030] After saline-alkali stress treatment of two rhododendron varieties, 5-6 cuttings were randomly selected at 0h, 6h, and 24h, and white tender root segments of about 1cm were collected to form a sample for transcriptome sequencing. Total RNA from the six groups of sample materials was extracted using TRIzol reagent according to the instructions. The transcriptome library was constructed using the VAHTS Universal V5 RNA-seq Library Prep Kit, and second-generation sequencing was performed using the Illumina platform. The differentially expressed genes obtained were analyzed according to the conditions of qValue < 0.05, 3 <FPKM<50、FC> 2. Screening is performed to obtain data that can be used for subsequent analysis.

[0031] 3. Real-time fluorescence quantitative PCR verification of BOR2 gene expression:

[0032] A heat map of differentially expressed genes was created using TBtools. Target genes were selected based on their expression trends under alkaline stress for subsequent functional verification. Five to six cuttings of two hydroponic azalea varieties were randomly selected at 0, 6, and 24 hours after saline-alkali stress treatment. Approximately 1 cm long white root segments were pooled together for RNA extraction. RNA was reverse transcribed into cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMIX for qPCR Kit. Quantitative primers for the BOR2 gene were designed using SnapGene (Table 1). 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 10 μM forward primer, 0.5 μL 10 μM reverse primer, 1 μL cDNA, and 8 μL HO. PCR program: 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 5 min.

[0033] Table 1 BOR2 gene RT-PCR primers

[0034]

[0035] 4. Cloning and vector construction of Rhododendron splendidum RpBOR2 gene:

[0036] Primers were designed to amplify the RpBOR2 gene (Table 2). 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 Jerex. 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. The digestion was performed at 37°C for 2–3 hours. The digestion product was recovered using a gel extraction kit from Tiangen. 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 cultured at 37°C overnight. Positive clones were identified by colony PCR.

[0037] Table 2 RpBOR2 gene primers

[0038]

[0039] 5. Agrobacterium transformation:

[0040] 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.

[0041] 6. Arabidopsis transformation and transgenic plant screening:

[0042] 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.

[0043] 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.

[0044] 7. Phenotypic experiments of overexpression of RpBOR2 gene:

[0045] 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 3.

[0046] T2 generation Arabidopsis seeds overexpressing the RpBOR2 gene and wild-type Arabidopsis seeds were selected and planted on alkaline stress culture medium plates, with 10 seeds planted on each plate. The experiment was repeated 3 times and the seeds were placed in a light incubator for 14 days to observe their phenotypes.

[0047] Table 3 Stress culture medium formula (1L)

[0048]

[0049] Experimental results of Example 2

[0050] 1. Expression of BOR2 gene under saline-alkali stress conditions:

[0051] like Figure 1 As shown in the study, two hydroponic seedlings of azalea were subjected to saline-alkali treatment at pH 8.3. Samples were collected at 0, 6, and 24 hours of treatment for transcriptome analysis. The results showed that the BOR2 gene was highly expressed in the salt-alkali-tolerant cultivar 'Zihe'. BOR2 expression was high in 'Zihe' 6 hours after treatment, but decreased after 24 hours, although still higher than at 0 hours. This suggests that in the early stages of saline-alkali stress, BOR2 expression in 'Zihe' is transiently high in response to stress. In the salt-alkali-sensitive cultivar 'Qilin', BOR2 expression was low under all stress conditions, with similar expression levels at 0 and 6 hours, followed by a significant decrease after 24 hours.

[0052] To verify the transcriptome results, quantitative PCR detection of BOR2 gene was carried out on two varieties and three treatment gradients (0h, 6h, 24h) ( Figure 2 Results and transcriptome heatmaps Figure 1The results showed that the BOR2 gene was highly expressed in the salt-alkali tolerant variety 'Zihe' within 24 h, but was lowly expressed in the salt-alkali sensitive variety 'Qilin'.

[0053] 2. Effect of overexpression of RpBOR2 gene on germination rate of Arabidopsis thaliana under saline-alkali stress conditions:

[0054] Wild-type Arabidopsis thaliana and T2 seeds overexpressing the RpBOR2 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.

[0055] The results are as follows Figure 3 As shown, the germination rate of wild-type Arabidopsis thaliana on a medium with pH 5.8 (0 mM NaHCO₃) was 93.33%, and on a medium with pH 8.2 (5 mM NaHCO₃) it was 36.67%, significantly lower than the germination rate under no saline-alkali stress conditions. The germination rates of transgenic RpBOR2 plants at pH 5.8 (0 mM NaHCO₃) and pH 8.2 (5 mM NaHCO₃) showed no significant difference, reaching 95.53% and 96.66%, respectively. This suggests that overexpressing RpBOR2 can increase the germination rate of plants under saline-alkali stress.

[0056] 3. Effects of overexpression of RpBOR2 gene on Arabidopsis roots under saline-alkali stress conditions:

[0057] T2 seeds of wild-type Arabidopsis and transgenic RpBOR2 plants were seeded on media 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 onto plates, with three replicates forming three technical replicates. Root phenotypes were observed 14 days later.

[0058] The results are as follows Figure 4 and Figure 5 As shown, on a medium without salinity or alkali stress (pH 5.8 (0 mM NaHCO₃), the root length and surface area of ​​the overexpressing plants were slightly greater than those of the wild type, while the number of root tips was less. On a salinity-stress medium (pH 8.2 (5 mM NaHCO₃), the root length of the transgenic plants was 59.15% greater than that of the wild type, while the root surface area and root tip number were 2.01 and 1.67 times greater, respectively. This suggests that under salinity-alkali stress conditions, root development in the wild type is significantly inhibited, while overexpressing the RpBOR2 gene can promote new root growth, thereby improving the plant's resistance to salinity or alkali stress.

[0059] Therefore, the present invention adopts the above-mentioned azalea salt-alkali tolerance gene RpBOR2 and its application to cultivate new salt-alkali tolerance azalea varieties, while improving the adaptability and growth conditions of azalea in alkaline soil, expanding the planting range of azalea and improving its growth under adverse soil conditions, providing more options for ornamental gardening and ecological environment protection, and has important application value.

[0060] 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. A salt-alkali tolerance gene for rhododendron RpBOR2 The application of the invention in improving the salt and alkali tolerance of Rhododendron splendidum is characterized by: Overexpression in Rhododendron splendidum RpBOR2 Genes to improve the salt and alkali tolerance of Rhododendron splendidum; RpBOR2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. RpBOR2 Genetically encoded RpBOR2 The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. The application of the recombinant vector in improving the salt-alkali tolerance of Rhododendron splendidum is characterized by: The recombinant vector contains the RpBOR2 Gene sequence, overexpressed in Rhododendron splendidum RpBOR2 Genes to improve the salt and alkali tolerance of Rhododendron splendidum.

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