Azalea saline-alkaline tolerant gene RpBOR2 and application thereof

Abstract

The invention relates to the technical field of gene engineering, and discloses an azalea saline-alkaline tolerant gene RpBOR2 and application thereof. The nucleotide sequence of the azalea saline-alkaline tolerant gene RpBOR2 is as shown in SEQ ID NO. 1. The amino acid sequence of the RpBOR2 protein coded by the azalea saline-alkaline tolerant gene RpBOR2 is as shown in SEQ ID NO. 2. The rhododendron saline-alkaline tolerant gene RpBOR2 and the application thereof are used for cultivating a new variety of saline-alkaline tolerant rhododendron, meanwhile, the adaptability and the growth condition of the rhododendron in alkaline soil are improved, the planting range of the rhododendron is expanded, the growth condition of the rhododendron under adverse soil conditions is improved, and the yield of the rhododendron is improved. More choices are provided for ornamental horticulture and ecological environment protection, and the method has important application value.

Description

Technical Field

[0001] The 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 destroy 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 process 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 antioxidant capacity of plants, 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 coping strategies of plants is of great significance for improving the salt-alkali tolerance of crops.

[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 and is a commonly cultivated rhododendron group in my country. 'Purple Crane' is a purple flower 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 and Fujian, Hunan, and Guangdong.

[0004] The BOR2 gene is one of the genes closely related to the absorption and transport of boron in plants. Boron is a trace element necessary for plant growth and plays an important role in many physiological processes of plants, including the formation of cell walls, the stability of cell membranes, the germination and growth of pollen, etc. The protein encoded by the BOR2 gene is a boron efflux transporter, which is mainly responsible for the transfer of boron from the epidermis to the cortex, and participates in the radial transport of boron in plant roots. It plays an important role in the elongation of root cells 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 will increase plant resistance. The role of BOR2 homologous genes in azalea under saline-alkali stress has not been reported. In order to study the changes in the expression of BOR2 homologous genes in different azaleas under saline-alkali stress, the materials used in this experiment were the azalea variety 'Zihe' that is resistant to saline-alkali stress and the azalea variety 'Qilin' that is not resistant to saline-alkali stress. 'Zihe' and 'Qilin' were treated with saline-alkali stress under hydroponic conditions, and the changes in the expression of BOR2 gene were determined by transcriptome sequencing and qPCR technology. The RpBOR2 gene of the salt-alkali tolerant variety 'Zihe' was cloned, and the phenotype of Arabidopsis overexpressing the RpBOR2 gene was detected under different saline-alkali treatment conditions, laying a foundation for further determining the salt-alkali tolerance mechanism of azalea. Summary of the invention

[0005] The purpose of the present invention is to provide a rhododendron salt-alkali tolerance gene RpBOR2 and an application thereof, which are used to cultivate new salt-alkali tolerant 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 unfavorable soil conditions, providing more options for ornamental horticulture 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 a RpBOR2 protein encoded by the salt-alkali tolerance gene RpBOR2 of azalea. 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 contains 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. The present invention carried out transcriptome sequencing on salt-alkali tolerant and salt-alkali intolerant azalea varieties under salt-alkali stress, and found that the BOR2 gene was highly expressed in salt-alkali tolerant azalea varieties, but was lowly expressed in salt-alkali intolerant azalea varieties. The RpBOR2 gene of Rhododendron splendidum 'Zihe' was cloned, and the CaMV35S-RpBOR2-GFP plant expression vector was constructed. The transgenic material of Arabidopsis thaliana was obtained by Agrobacterium infection, and the function of RpBOR2 was verified in Arabidopsis thaliana.

[0015] 2. The salt-alkali tolerance gene RpBOR2 of azalea 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, provide 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 It is a heat map of BOR2 gene in two azalea varieties under saline-alkali stress in the embodiment of the present invention, wherein ZH-0: 'Zihe' saline-alkali treatment 0h; ZH-6: 'Zihe' saline-alkali treatment 6h; ZH-24: 'Zihe' saline-alkali treatment 24h; QL-0: 'Qilin' saline-alkali treatment 0h; QL-6: 'Qilin' saline-alkali treatment 6h; QL-24: 'Qilin' saline-alkali treatment 24h, each group was repeated 3 times;

[0018] Figure 2 The quantitative results of BOR2 gene in two azalea varieties in the embodiment of the present invention, wherein ZH-0: 'Zihe' saline-alkali treatment 0h; ZH-6: 'Zihe' saline-alkali treatment 6h; ZH-24: 'Zihe' saline-alkali treatment 24h; QL-0: 'Qilin' saline-alkali treatment 0h; QL-6: 'Qilin' saline-alkali treatment 6h; QL-24: 'Qilin' saline-alkali treatment 24h;

[0019] Figure 3: The effect of the transgenic RpBOR2 plant on the germination rate in the embodiment of the present invention, wherein A is a phenotype diagram of the germination rate of the transgenic RpBOR2 plant under the control and saline-alkali stress; B is a statistical result diagram of the germination rate of the transgenic RpBOR2 plant under the control and saline-alkali stress, Col: wild type control; 35S: RpBOR2: transgenic strain;

[0020] Figure 4 : The root phenotype of Arabidopsis thaliana overexpressing the RpBOR2 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: wild type control; 35S: RpBOR2: transgenic strain;

[0021] Figure 5 : This is the effect of overexpressing the RpBOR2 gene on the root system of Arabidopsis thaliana under saline-alkali stress in the examples of the present invention, wherein A is a statistical graph of root length, root surface area and root tip number of wild-type and overexpressing plants under pH 5.8 (0 mM NaHCO3); B is a statistical graph of root length, root surface area and root tip number of wild-type and overexpressing plants under pH 8.2 (5 mM NaHCO3), Col: wild-type control; 35S: RpBOR2: transgenic strain. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0023] 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.

[0024] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are conventionally commercially available.

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

[0026] Example 1 Experimental method

[0027] 1. Cultivation and salt stress treatment of azalea cuttings:

[0028] Four-month-old cuttings of the salt-alkali tolerant variety 'Zihe' and the salt-alkali sensitive variety 'Qilin' of Rhododendron were used as test materials and cultured in a hydroponic system until new roots grew out. Before hydroponic culture, the culture substrate on the roots of the cuttings was rinsed with clean water, and then cultured with deionized water for 3 days to adapt to the water environment, and then cultured with 1 / 4 Hoagland nutrient solution pH = (5.85 ± 0.05), and the nutrient solution was replaced every 7 days. The hydroponic conditions were that the nutrient solution was circulated every 20 minutes (about 5 minutes), the oxygen pump continued to add oxygen, 16 hours of light / 8 hours of darkness, temperature 20-25℃, humidity 40-50%, and cultured for about 20 days until new roots of about 1-2 cm grew out. The pH of 1 / 4 Hoagland nutrient solution was adjusted to 8.3 with a buffer solution of 0.1M Na2CO3 and 0.1MNaHCO3 mixed in equal proportions (pH = 10.10 ± 0.02) as the alkaline stress experimental group.

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

[0030] After the two rhododendron varieties were treated with saline-alkali stress, 5-6 cuttings were randomly selected at 0h, 6h and 24h, and the white tender root segments of about 1cm were collected into one sample for transcriptome sequencing. The total RNA of the six groups of sample materials was extracted using TRIzol reagent according to the instructions. The transcriptome library was constructed using the VAHTS Universal V5RNA-seq Library Prep Kit, and the 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] The differentially expressed genes were plotted with TBtools to draw a heat map, and the target genes were selected according to the trend of gene expression under alkaline stress for subsequent gene function verification. After the hydroponic seedlings of two varieties of azalea were treated with saline-alkali stress, 5-6 cuttings were randomly selected at 0h, 6h and 24h, and the white tender root segments of about 1cm were collected into one sample to extract RNA. The RNA to be tested was reverse transcribed into cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMIX for qPCR kit. SnapGene was used to design the quantitative primers for the BOR2 gene (Table 1), and the reagents for Real-time PCR were SYBY GreenMaster 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: 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 RT-PCR primers for BOR2 gene

[0034]

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

[0036] Primers for amplifying the RpBOR2 gene were designed (Table 2). 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 the gel recovery kit of Jierui Company. The plasmid enzyme digestion system was as follows: 10×Q-cut buffer 5μL, endonuclease 2μL, plasmid 1μg, ddH2O to 50μL, 37℃ enzyme digestion for 2-3 hours. The digestion product was recovered using the gel recovery kit of Tiangen Company. The ligation system is as follows: 6 μL of PCR product, 2 μL of plasmid, 1 μL of 10×T4ligase buffer, 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 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 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.

[0037] Table 2 Primers for RpBOR2 gene

[0038]

[0039] 5. Agrobacterium transformation:

[0040] 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.

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

[0042] 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.

[0043] 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.

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

[0045] 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 3.

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

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

[0048]

[0049] Example 2 Experimental results

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

[0051] like Figure 1 As shown in the figure, two varieties of azalea hydroponic seedlings were treated with saline-alkali at pH 8.3, and samples at 0h, 6h and 24h were selected for transcriptome analysis. The results showed that the BOR2 gene was highly expressed in the salt-alkali tolerant variety 'Zihe'. The BOR2 gene in 'Zihe' showed high expression 6h after stress treatment, and the expression level decreased after 24h, but its expression level was also higher than 0h, indicating that in the early stage of salt-alkali stress, the BOR2 gene in 'Zihe' would show transient high expression in response to stress. In the salt-alkali sensitive variety 'Qilin', the BOR2 gene showed low expression under stress treatment, and its expression level was similar at 0h and 6h of stress treatment, but it decreased significantly after 24h.

[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 heatmap Figure 1The results showed that BOR2 gene was highly expressed in the salt-alkali tolerant variety 'Zihe' within 24 h, and 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 and T2 seeds overexpressing the RpBOR2 gene were planted on pH 5.8 (0mM NaHCO3) and pH 8.2 (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 germination rate was counted after 7 days.

[0055] The results are as follows Figure 3 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%, which was significantly lower than the germination rate under no salt-alkali stress conditions. There was no significant difference in the germination rate of seeds of plants transfected with RpBOR2 gene at pH 5.8 (0mM NaHCO3) and pH 8.2 (5mM NaHCO3), with the germination rates being 95.53% and 96.66%, respectively, indicating that overexpression of RpBOR2 gene can increase the germination rate of plants under salt-alkali stress.

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

[0057] The T2 seeds of wild-type Arabidopsis and transgenic RpBOR2 plants were planted on pH 5.8 (0mM NaHCO3) and pH 8.2 (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.

[0058] The results are as follows Figure 4 and Figure 5 As shown in the figure, on the medium without salt-alkali stress at pH 5.8 (0mM NaHCO3), the root length and surface area of ​​the overexpressing plants were slightly higher than those of the wild type, while the number of root tips was less than that of the wild type. On the medium with salt-alkali stress at pH 8.2 (5mM NaHCO3), the root length of the transgenic plants was 59.15% longer than that of the wild type, while the root surface area and the number of root tips were 2.01 and 1.67 times higher than those of the wild type. This indicates that under salt-alkali stress conditions, the root development of the wild type is significantly inhibited, while overexpressing the RpBOR2 gene can promote the growth of new roots, thereby improving the plant's resistance to salt-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 tolerant 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 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. A salt-alkali tolerance gene RpBOR2 of azalea, characterized by: The nucleotide sequence of gene RpBOR2 is shown as SEQ ID NO.

1.

2. The RpBOR2 protein encoded by the salt-alkali tolerance gene RpBOR2 of azalea according to claim 1, characterized in that: The amino acid sequence of the RpBOR2 protein is shown in SEQ ID NO.

2.

3. A recombinant vector comprising the azalea salt-alkali tolerance gene RpBOR2, 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 and plant overexpression vectors.

5. The recombinant vector according to claim 4, characterized in that: The recombinant vector is a CaMV35S-RpBOR2-GFP plant overexpression vector.

6. Use of the azalea salt-alkali tolerance gene RpBOR2 as claimed in claim 1 in improving the salt-alkali tolerance of azalea.

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

Images