A salt-tolerant gene BvRING-HC9 and its applications

Abstract

This invention relates to a salt-tolerance gene, BvRING-HC9, and its application. The invention focuses on the application of the BvRING-HC9 salt-tolerance gene in improving plant salt tolerance. The CDS sequence of the BvRING-HC9 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2. The BvRING-HC9 gene in heterologous overexpression plants prepared using this invention is stably inherited. The heterologously expressed BvRING-HC9 gene can alleviate the inhibitory effect of salt stress on plant germination, significantly reducing the hindered root and leaf growth of heterologously expressed BvRING-HC9 gene plants under salt stress, thus improving their adaptability to salt stress.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a salt tolerance gene BvRING-HC9 and its applications. Background Technology

[0003] Salt stress is primarily caused by neutral salts such as NaCl and Na₂SO₄. Excessive accumulation of Na⁺, Cl⁻, and SO₄²⁻ ions in the soil enters cells, inhibiting the absorption of essential cations such as K⁺, Ca²⁺, and Mg²⁺, interfering with enzymatic reactions, and damaging membrane structure. Simultaneously, high extracellular ion concentrations lead to a decrease in rhizosphere water potential, hindering water absorption and triggering osmotic stress. Under these stresses, reactive oxygen species (ROS) accumulate in large quantities, causing membrane lipid peroxidation, significantly increasing malondialdehyde (MDA) content and relative conductivity (REC), and impairing cell integrity. Furthermore, salt stress causes a sharp decline in chlorophyll content, reduces PSII photochemical efficiency, and significantly decreases net photosynthetic rate, stomatal conductance, and transpiration rate, ultimately leading to stunted plant growth, reduced biomass, and even wilting and death.

[0004] Faced with osmotic imbalance, ion toxicity, and oxidative damage caused by salt stress, plants have evolved multi-layered defense and adaptation mechanisms. At the osmotic regulation level, cells synthesize proline, betaine, soluble sugars, and alcohols to maintain osmotic potential and protect protein structures. Excess Na⁺ / H⁺ antitransporters (SOS1, NHX) and high-affinity K⁺ transporters (HKT1;1) on the plasma membrane and vacuolar membrane separate excess Na⁺ into the vacuoles or expel it from the extracellular space, maintaining the Na⁺ / K⁺ ratio. In terms of redox regulation, enzymes such as superoxide dismutase (SOD) and peroxidase (POD) scavenge reactive oxygen species (ROS). Furthermore, salt stress induces a significant increase in the levels of abscisic acid (ABA), jasmonic acid (JA), the ethylene precursor ACC, and ethylene release, indicating that salt stress comprehensively regulates plant physiological metabolism through a multi-level signaling network.

[0005] Salt stress is one of the factors limiting soil productivity. Compared to traditional salt-tolerant breeding methods, which are time-consuming and inefficient, genetic engineering can precisely improve salt tolerance traits in plants by introducing or regulating specific salt-tolerant genes. Therefore, discovering and applying key salt-tolerant genes through genetic engineering is an important technical means for breeding salt-tolerant crop varieties. Summary of the Invention

[0006] This invention provides a salt-tolerant gene BvRING-HC9 and its application. The application described in this invention can effectively improve the salt tolerance of plants.

[0007] This invention relates to the application of the salt-tolerant gene BvRING-HC9 in improving plant salt tolerance.

[0008] Furthermore, the application method is performed according to the following steps:

[0009] I. Construction of pCAMBIA1300S-3×FLAG-BvRING-HC9 expression vector;

[0010] II. Obtaining heterologous expression plants of the salt-tolerant gene BvRING-HC9: Wild-type plants were stably transformed using the Agrobacterium-mediated transformation method with the pCAMBIA1300S-3×FLAG-BvRING-HC9 recombinant vector, thus obtaining plants with salt tolerance.

[0011] Furthermore, the CDS sequence of the salt tolerance gene BvRING-HC9 is shown in SEQ ID NO.1.

[0012] Furthermore, the amino acid sequence of the protein encoded by the gene BvRING-HC9 is shown in SEQ ID No. 2.

[0013] The salt stress tolerance gene BvRING-HC9 used in this invention has a CDS of 561 bp and encodes 186 amino acids.

[0014] Furthermore, the wild-type plant is wild-type Arabidopsis thaliana.

[0015] Furthermore, the method for constructing the pCAMBIA1300S-3×FLAG-BvRING-HC9 expression vector is characterized by:

[0016] A. Obtain the gene BvRING-HC9;

[0017] B. Extract the plasmid of the pCAMBIA1300S-3×FLAG vector and digest the pCAMBIA1300S-3×FLAG vector with BamHI restriction endonuclease.

[0018] C. The single-enzyme-digested pCAMBIA1300S-3×FLAG vector was linearly ligated with the BvRING-HC9 gene CDS.

[0019] D. Transform the recombinant vector obtained above into Escherichia coli DH5α competent cells; then screen for positive recombinants and extract the recombinant vector; thus obtaining the pCAMBIA1300S-3×FLAG-BvRING-HC9 expression vector.

[0020] The protein molecular formula of the protein encoded by the salt stress tolerance gene BvRING-HC9 used in this invention (BvRING-HC9) is C 942 H 1510 N 270 O 252 S16 The relative molecular mass is 21.16 kDa, the isoelectric point is 8.33, and the amino acid sequence is shown in SEQ ID No. 2. The amino acid composition of BvRING-HC9 protein has the highest proportion of Leu (L), which is 15.6%, and the instability index is 48.46. Therefore, BvRING-HC9 protein is classified as unstable.

[0021] The application described in this invention utilizes the BvRING-HC9 gene to prepare heterologous overexpression plants in which the BvRING-HC9 gene can be stably inherited. The heterologous expression of the BvRING-HC9 gene can alleviate the inhibitory effect of salt stress on plant germination, and can significantly reduce the hindering effect on root and leaf growth of heterologous BvRING-HC9 gene expression plants under salt stress, thereby improving their adaptability under salt stress. Attached Figure Description

[0022] Figure 1 This is a 1% agarose gel electrophoresis image of the full-length CDS PCR amplification product of the BvRING-HC9 gene in Example 1;

[0023] Figure 2 This is a tertiary structure diagram of the BvRING-HC9 protein in Example 2;

[0024] Figure 3 This is the relative expression of the BvRING-HC9 gene in the leaves and roots of the sugar beet T710MU strain in Example 3 after treatment with 280 mM NaCl;

[0025] Figure 4 This is a 1% agarose gel electrophoresis image of the double enzyme digestion product of the pCAMBIA1300S-BvRING-HC9-3×FLAG recombinant plasmid in Example 4;

[0026] Figure 5 This is the screening of T3 generation positive Arabidopsis thaliana plants transfected with the BvRING-HC9 gene in Example 4;

[0027] Figure 6 This is a 1% agarose gel electrophoresis image of the PCR products of Arabidopsis thaliana plants transgenic with the BvRING-HC9 gene in Example 4. Lanes 1 to 10 are the PCR products of Arabidopsis thaliana plants transgenic with the BvRING-HC9 gene; lane 11 is the wild-type Arabidopsis thaliana genome control.

[0028] Figure 7This is a 1% agarose gel electrophoresis image of the RT-PCR verification product of the Arabidopsis thaliana plant transgenic with the BvRING-HC9 gene in Example 4. Lanes OE#1, OE#2, and OE#3 are the RT-PCR products of T3 generation Arabidopsis thaliana transgenic with the BvRING-HC9 gene, and WT is the wild-type Arabidopsis thaliana RT-PCR product.

[0029] Figure 8 The germination profiles of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana in Example 4 were obtained in the control group, culture medium under 100 mM (mol / L) and 150 mM (mol / L) NaCl stress.

[0030] Figure 9 This is a graph showing the statistical results of the germination rates of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana in Example 4 under control, 100 mM (mol / L), and 150 mM (mol / L) NaCl stress.

[0031] Figure 10 The root length phenotypes of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana in Example 4 were obtained by culturing in control, 100 mM (mol / L) and 150 mM (mol / L) NaCl stress media.

[0032] Figure 11 The results are statistical analysis of root length phenotypes in wild-type and BvRING-HC9 transgenic Arabidopsis thaliana in Example 4, under control, 100 mM (mol / L), and 150 mM (mol / L) NaCl stress.

[0033] Figure 12 This is the plant phenotype of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana in Example 4 in mixed soil under control and 150 mM (mol / L) NaCl stress.

[0034] Figure 13 The results are the statistical results of the fresh weight of leaves of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana plants in mixed soil under control and 150 mM (mol / L) NaCl stress in Example 4.

[0035] Figure 14 The results are the statistical results of leaf dry weight of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana plants in mixed soil under control and 150 mM (mol / L) NaCl stress in Example 4.

[0036] Figure 15 Na in Example 4 + Ion content standard curve;

[0037] Figure 16In the fourth specific implementation method, wild-type and BvRING-HC9 transgenic Arabidopsis thaliana plants under control and 150 mM (mol / L) NaCl stress showed Na + Graph of ion content determination results;

[0038] Figure 17 K in Example 4 + Ion content standard curve;

[0039] Figure 18 In Example 4, wild-type and BvRING-HC9 transgenic Arabidopsis thaliana plants were subjected to K under control and 150 mM (mol / L) NaCl stress. + Graph of ion content determination results;

[0040] Figure 19 In Example 4, wild-type and BvRING-HC9 transgenic Arabidopsis thaliana plants were subjected to NaCl stress under control and 150 mM (mol / L) NaCl stress. + / K + Graph showing the results of ion content determination. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] The beet T710MU strain described in the following examples was disclosed in the literature “Geng G, Lv C, Stevanato P, Li R, Liu H, Yu L, Wang Y. Transcriptome Analysis of Salt-Sensitive and Tolerant Genotypes Reveals Salt-Tolerance Metabolic Pathways in Sugar Beet. Int J MolSci. 2019 Nov 25;20(23):5910”, which is available to the public from Heilongjiang University.

[0046] The DH5α competent cells and Agrobacterium GV3101 competent cells used in the following examples are products of Shanghai Weidi Biotechnology Co., Ltd.

[0047] Example 1: Obtaining the salt tolerance gene BvRING-HC9

[0048] The CDS sequence of the salt stress tolerance gene BvRING-HC9 in this embodiment is shown in SEQ ID NO.1.

[0049] 1. Cloning of the salt tolerance gene BvRING-HC9:

[0050] In this embodiment, total RNA was extracted from the leaves of sugar beet plants. Using the total RNA as a template, full-length sugar beet cDNA was obtained through reverse transcription using the First Strand cDNA Synthesis Kit. Then, PCR amplification was performed using specific primers BvRING-HC9-s and BvRING-HC9-as with KOD high-fidelity enzyme. The PCR reaction system is shown in Table 1.

[0051] The primer sequences are as follows:

[0052] BvRING-HC9-s: 5'-ATGGAGGGAGATGGACCACC-3';

[0053] BvRING-HC9-as: 5'-CTAGCCTCCATGACGATTAACAAGA-3';

[0054] Table 1. PCR reaction system for BvRING-HC9 gene amplification

[0055]

[0056] The PCR reaction program was as follows: 94℃ for 2 min; 98℃ for 10 s, 54℃ for 30 s, 68℃ for 1 min, 30 cycles; stored at 4℃.

[0057] 2. Purify the PCR product (BvRING-HC9) using the following steps:

[0058] (1) Cut the target strip in the UV analysis rubber tapping instrument and put it into a pre-weighed 1.5 mL centrifuge tube;

[0059] (2) Weigh the gel block, add 3 times the volume of Buffer GM solution, place it in a 55℃ water bath for 10 min, tap the centrifuge tube every 5 min until the gel block is completely dissolved, add the gel solution to the adsorption column, and centrifuge at 8000 rpm for 30 s.

[0060] (3) Pour the filtrate in the collection tube back into the adsorption column and centrifuge at 8000 rpm for 30 s;

[0061] (4) Discard the filtrate, add 400 μL of Buffer B2 solution, and centrifuge at 8000 rpm for 30 s;

[0062] (5) Discard the filtrate, add 700 μL of Buffer WB solution, and centrifuge at 10000 rpm for 30 s;

[0063] (6) Repeat (5) once;

[0064] (7) Discard the filtrate in the collection tube and centrifuge at 11,000 rpm for 1 min;

[0065] (8) Place the adsorption column into a new 1.5 mL centrifuge tube, add 20 μL of ddH2O preheated at 50℃, incubate for 3 min, centrifuge at 12000 rpm for 1 min, collect the purified target fragment (BvRING-HC9 gene DNA fragment) and use the NanoDrop instrument to determine the DNA concentration.

[0066] 3. Ligation of DNA fragments with the pMD18-T vector:

[0067] The recovered purified PCR product (BvRING-HC9 gene DNA fragment) was tailed. The reaction system is shown in Table 2:

[0068] Table 2. Tailed PCR reaction system for BvRING-HC9 gene amplification

[0069]

[0070] The tailed BvRING-HC9 gene DNA fragment was mixed with the pMD18-T vector and ligated in a 16℃ metal bath for 16 h. The ligation system is shown in Table 3.

[0071] Table 3. T-vector ligation system for BvRING-HC9 gene amplification

[0072]

[0073] 4. The above ligation product was transformed into E. coli DH5α competent cells, as follows:

[0074] (1) In a sterile laminar flow hood, 10 μL of the ligation product was gently added to 50 μL of Escherichia coli DH5α competent cells, and then slowly aspirated to mix thoroughly. The mixture was then placed on ice and allowed to stand for 30 min.

[0075] (2) The conversion product was placed in a 42℃ water bath for 90 s for heat shock;

[0076] (3) Place the conversion product on ice for 30 min;

[0077] (4) Add 750 μL of LB liquid culture medium (antibiotic-free) to the conversion product in a sterile laminar flow hood and revive it in an air shaker at 37°C for 2 h;

[0078] (5) Spread the transformed bacterial culture onto LB plates (containing 50 mg·L⁻¹ Amp) and incubate upside down at 37°C for 16 h.

[0079] 5. Screening of positive recombinants:

[0080] Single colonies from the LB agar plates were transferred to 1 mL of LB liquid medium (containing 50 mg·L⁻¹ Amp) and incubated at 37°C on an air shaker for 4 h. PCR verification was performed using the bacterial culture as a template, followed by 1% agarose gel electrophoresis. The PCR system is shown in Table 4. Positive bacterial cultures were then transferred to 50 mL of LB liquid medium (containing 50 mg·L⁻¹ Amp) and incubated at 37°C on an air shaker for 16 h.

[0081] Table 4. Bacterial PCR system for BvRING-HC9 gene amplification

[0082]

[0083] The PCR reaction program was as follows: 94℃ for 2 min; 98℃ for 10 s, 54℃ for 30 s, 68℃ for 1 min, 30 cycles; stored at 4℃.

[0084] 6. Extraction and sequencing of recombinant plasmids, the specific steps are as follows:

[0085] (1) Take 5 mL of the bacterial culture from the previous step into a 10 mL centrifuge tube and centrifuge at 13000 rpm for 10 min at 4℃;

[0086] (2) Discard the supernatant, add 200 μL of pre-cooled Solution I solution to resuspend the bacterial cells, and mix by pipetting;

[0087] (3) Slowly add 300 μL of Solution II solution to lyse the cells, and gently invert to mix;

[0088] (4) Slowly add 200 μL of Solution Ⅲ solution to renature the DNA, gently invert to mix, let stand for 10 min, and centrifuge at 13000 rpm for 10 min;

[0089] (5) Transfer the supernatant to a new 1.5 mL centrifuge tube, add an equal volume of DNA extraction buffer (phenol: chloroform: isoamyl alcohol = 25: 24: 1), mix gently, and centrifuge at 13000 rpm for 5 min;

[0090] (6) Transfer the supernatant to a new 1.5 mL centrifuge tube, add an equal amount of pre-cooled isopropanol, gently invert to mix, let stand at -20℃ for 30 min, and you will see flocculent DNA. Centrifuge at 13000 rpm for 5 min.

[0091] (7) Discard the supernatant, add 1 mL of pre-cooled 75% ethanol to wash the precipitate, and centrifuge at 13000 rpm for 1 min;

[0092] (8) Repeat (7) once;

[0093] (9) Discard the supernatant and place the centrifuge tube containing the DNA precipitate in a laminar flow hood and blow until the ethanol completely evaporates;

[0094] (10) Add 50 μL of ddH2O preheated at 50℃ and add 1 μL of RNase enzyme to remove RNA;

[0095] (11) The concentration was determined using a NanoDrop instrument and detected by 1% agarose gel electrophoresis.

[0096] The full-length PCR amplification product of the BvRING-HC9 gene coding sequence (CDS) was obtained by 1% agarose gel electrophoresis. Figure 1 As shown ( Figure 1 This is a 1% agarose gel electrophoresis image of the full-length PCR amplification product of the BvRING-HC9 gene CDS (lane M is DL 2000 DNA Maker; lane 1 is the full-length PCR amplification product of the BvRING-HC9 gene CDS). The results show that the target band appears between 1000 bp and 750 bp, which is consistent with the expected full-length BvRING-HC9 gene CDS.

[0097] The recombinant plasmid that successfully passed electrophoresis was sent to Shanghai Bioengineering Co., Ltd. for sequencing. Sequencing revealed the CDS sequence of the BvRING-HC9 gene as shown in SEQ ID NO.1. The CDS of the BvRING-HC9 gene is 561 bp and encodes 186 amino acids.

[0098] Example 2 The protein encoded by the salt stress tolerance gene BvRING-HC9 in this embodiment has the amino acid sequence shown in SEQ ID No. 2.

[0099] Bioinformatics analysis of the salt tolerance gene BvRING-HC9:

[0100] In this embodiment, the protein encoded by the salt stress tolerance gene BvRING-HC9 contains 186 amino acids. The physicochemical properties of the protein encoded by gene BvRING-HC9 (BvRING-HC9) were predicted using the ProtParam online software. The results show that the molecular formula of BvRING-HC9 protein is C1. 942 H 1510 N 270 O 252 S 16 The relative molecular mass is 21.16 kDa, and the isoelectric point is 8.33. The amino acid composition of BvRING-HC9 protein has the highest proportion of Leu (L), which is 15.6%, and the instability index is 48.46. Therefore, BvRING-HC9 protein is classified as stable.

[0101] By inputting the amino acid sequence of the BvRING-HC9 protein into the SWISS-MODEL website, the tertiary structure of the BvRING-HC9 protein was predicted, and the potential Zn content in the tertiary structure of the BvRING-HC9 protein was identified. 2+ Binding sites such as Figure 2 As shown ( Figure 2 This is a tertiary structure diagram of the BvRING-HC9 protein.

[0102] Example 3: Expression analysis of the salt tolerance gene BvRING-HC9 in the leaves / roots of the sugar beet T710MU cultivar

[0103] 1. RNA extraction and reverse transcription, the specific methods are as follows:

[0104] Total RNA was extracted from leaves and roots of the sugar beet T710MU strain treated with 0 and 280 mM NaCl using the Trizol method. The RNA concentration was adjusted to 500 ng / μL using enzyme-free ddH2O. Reverse transcription was then performed, and the reverse transcription system is shown in Table 5.

[0105] Table 5 Reverse Transcription System

[0106]

[0107] 2. Real-time quantitative PCR of the salt stress tolerance gene BvRING-HC9, the specific method is as follows:

[0108] Primers BvRING-HC9-qs and BvRING-HC9-q-as for real-time quantitative PCR of the salt-stress-tolerant gene BvRING-HC9 were designed using the Primer 3 Plus website (https: / / www.primer3plus.com / ), and their specificity was verified using NCBI-Primerblast. Primers Bv18S-s and Bv18S-as were designed using sugar beet 18S rRNA as an internal reference gene. Salt stress response analysis of the BvRING-HC9 gene was performed using the first strand of cDNA from root and leaf tissues of the sugar beet T710MU line subjected to salt stress at 0 mM NaCl and 280 mM NaCl. Sampling time points for salt stress treatment were 0 h, 1 h, 3 h, 6 h, and 12 h. The reverse transcription reaction system is shown in Table 6.

[0109] The primer sequences are as follows:

[0110] BvRING-HC9-qs: 5'-AGAGACTGCGAGACCTTC-3';

[0111] BvRING-HC9-q-as: 5'-TTACTCCTACAATTCCTTCAGG-3';

[0112] Bv18S-s: 5'-CCCCAATGGATCCCGTTA-3';

[0113] Bv18S-as: 5'-TGACGGAGAATTAGGGTTCG-3'.

[0114] Table 6 Reverse transcription reaction system

[0115]

[0116] Set the fluorescence quantification program as shown in Table 7.

[0117] Table 7 Quantitative Fluorescence Procedure

[0118]

[0119] The data results were analyzed, and after calculation and integration using Excel, 2 -ΔΔCT The method is used to calculate the relative expression level of genes.

[0120] The results are as follows Figure 3 As shown ( Figure 3 The figure shows the relative expression of the BvRING-HC9 gene in the leaves and roots of the sugar beet T710MU variety under 280 mM NaCl treatment (red line represents expression in roots, green line represents expression in leaves). With 0 h as the control, the relative expression level of the BvRING-HC9 gene in leaves was highest at 6 h and highest in roots at 1 h under 280 mM salt treatment, indicating that the BvRING-HC9 gene can respond to salt stress.

[0121] Example 4: Application of the salt tolerance gene BvRING-HC9 in improving plant salt tolerance

[0122] 1. Construction of the pCAMBIA1300S-3×FLAG-BvRING-HC9 expression vector, the specific method is as follows:

[0123] The plasmid of the pCAMBIA1300S-3×FLAG vector was extracted using the alkaline lysis method. The pCAMBIA1300S-3×FLAG vector was then digested with BamHI restriction endonuclease. The digestion system is shown in Table 8.

[0124] Table 8. Enzyme digestion system of pCAMBIA1300S-3×FLAG vector

[0125]

[0126] The obtained pCAMBIA1300S-3×FLAG linearized vector was purified and ligated with the BvRING-HC9 gene CDS fragment in a 16℃ metal bath for 14 h. The ligation system is shown in Table 9.

[0127] Table 9 pCAMBIA1300S-3×FLAG-BvRING-HC9 Carrier Linkage System

[0128] The recombinant vector obtained by the above ligation is transformed into Escherichia coli DH5α competent cells (the transformation method of the recombinant vector in Example 1 can be used); then positive recombinants are screened and the recombinant vector is extracted (the positive recombinant screening method and recombinant plasmid extraction method in Example 1 can be used).

[0129] The recombinant vector pCAMBIA1300S-3×FLAG-BvRING-HC9 was obtained and transformed into Agrobacterium tumefaciens GV3101. The specific method is as follows:

[0130] (1) Take 1 μg of pCAMBIA1300S-3×FLAG-BvRING-HC9 recombinant plasmid, add 50 μL of Agrobacterium tumefaciens GV3101 competent cells, and freeze, thaw, and incubate in a 28℃ water bath, and freeze again for 5 min each time.

[0131] (2) After transformation, add 750 μL of LB liquid medium (antibiotic-free) and revive in an air shaker at 28°C for 4 h;

[0132] (3) Centrifuge the transformed bacterial culture at 6000 rpm for 1 min to collect the bacterial cells, take 100 μL of culture medium, gently aspirate and resuspend the bacterial cells, and spread them on LB solid medium (containing 50 mg·L⁻¹ Kan and 30 mg·L⁻¹ Rif), and incubate upside down at 28℃ for 48 h.

[0133] (4) Pick a single colony from the above LB solid medium and transfer it to 1 mL of sterile LB medium (containing 50 mg·L⁻¹ Kan and 30 mg·L⁻¹ Rif). Incubate at 28°C in an air shaker for 12 h. Use the bacterial culture as a template for PCR. Detect the PCR product by 1% agarose gel electrophoresis. The PCR system is shown in Table 10.

[0134] Table 10 PCR System

[0135]

[0136] The primer sequences are as follows:

[0137] FLAG-BvRING-HC9-s: 5'-CGCGGATCCATGGAGGGAGATGGACCACC-3';

[0138] FLAG-BvRING-HC9-as: 5'-CGGACTAGTGCCTCCATGACGATTAACAAGAGTA-3';

[0139] The PCR reaction program was as follows: 94℃ for 2 min; 98℃ for 10 s, 55℃ for 30 s, 68℃ for 1 min 50 s, 30 cycles; stored at 4℃.

[0140] After amplifying the positive bacterial culture identified by PCR and extracting the plasmid (using the extraction method for recombinant plasmids in Example 1), double enzyme digestion was performed for identification, such as... Figure 4 As shown ( Figure 4This is a 1% agarose gel electrophoresis image of the double digestion products of the pCAMBIA1300S-BvRING-HC9-3×FLAG recombinant plasmid. Lane M is the DL 15000 marker; lanes 1 and 2 are the double digestion products of the pCAMBIA1300S-BvRING-HC9-3×FLAG recombinant plasmid. Figure 4 Clear bands were observed between the 250 bp and 1000 bp markers, validating the BvRING-HC9 plasmid constructed on pCAMBIA1300S-3×FLAG. The successfully validated plasmid was then sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0141] 2. Obtaining heterologous expression plants of the salt-tolerant gene BvRING-HC9:

[0142] The pCAMBIA1300S-3×FLAG-BvRING-HC9 recombinant vector was stably transformed into wild-type Arabidopsis thaliana using Agrobacterium-mediated transformation. The specific method is as follows:

[0143] (1) A single positive colony of Agrobacterium tumefaciens GV3101 containing the recombinant vector pCAMBIA1300S-3×FLAG-BvRING-HC9 was amplified into 50 mL LB liquid medium (containing 50 mg·L⁻¹ Kan and 30 mg·L⁻¹ Rif) and cultured in an air shaker at 28°C until OD. 600 To reach a pH of 0.8-1.0; centrifuge 50 mL of bacterial culture at 6000 rpm for 10 min;

[0144] (2) Discard the supernatant, add 25 mL of osmotic conversion buffer to resuspend the bacterial cells, and centrifuge at 6000 rpm for 10 min;

[0145] (3) Discard the supernatant and resuspend the bacterial cells in 25 mL of osmotic conversion buffer until the OD value is reached. 600 Once the value reaches 1.0, it is ready to be used for immersion in Arabidopsis inflorescences;

[0146] (4) Water thoroughly the day before soaking and cut off any open flowers and pods of Arabidopsis thaliana;

[0147] (5) Immerse the terminal inflorescence of Arabidopsis thaliana completely in the transformation solution for 1 min, blot off the excess transformation solution with filter paper, and then culture the Arabidopsis thaliana in the dark for 24 h and under light for 24 h while keeping it moist. Repeat the immersion twice.

[0148] (6) Harvest T0 generation transgenic seeds, vernalize them at 4℃, and then culture them in 1 / 2 MS medium (containing 25 mg·L⁻¹ HYG) for 12 days. Transgenic seedlings with normal root growth and no whitening are then transplanted into the soil to continue growing. The above screening process is repeated to obtain T3 generation seeds.

[0149] (7) Collect T3 generation seeds, and after culturing for a certain period of time, select transgenic seedlings with normal root growth and no whitening, and transplant them into soil (e.g. Figure 5 As shown, Figure 5 (This involves screening T3 generation positive Arabidopsis thaliana plants transgenic with the BvRING-HC9 gene) and identifying them at the DNA and RNA levels.

[0150] The above method was used to screen heterologous expression plants up to the T3 generation. The DNA levels of 10 selected plants were then validated, and the results are as follows: Figure 6 As shown ( Figure 6 This is a 1% agarose gel electrophoresis image of the PCR products of the Arabidopsis thaliana plants transgenic with the BvRING-HC9 gene in Specific Implementation Method 4. Lanes 1 to 10 represent the PCR products of the Arabidopsis thaliana plants transgenic with the BvRING-HC9 gene; lane 11 represents the wild-type Arabidopsis thaliana genome control. From the 10 obtained T3 generation heterologous expression plants, three plants with high PCR brightness (lanes 1 and 6, and named OE#1, OE#2, and OE#3) were selected for RT-PCR experiments. RT-PCR validation results showed that bands were observed only in the overexpression lines, while no bands were observed in the WT (e.g., ...). Figure 7 As shown, Figure 7 This is a 1% agarose gel electrophoresis image of the RT-PCR verification product of Arabidopsis thaliana plants transgenic with the BvRING-HC9 gene. Lanes OE#1, OE#2, and OE#3 are the RT-PCR products of T3 generation Arabidopsis thaliana transgenic with the BvRING-HC9 gene, and WT is the wild-type Arabidopsis thaliana transgenic with RT-PCR. This proves that three transgenic lines of the BvRING-HC9 gene were successfully obtained.

[0151] 3. Determination of germination rate of plants heterologously expressing the salt-tolerant gene BvRING-HC9:

[0152] Seeds from BvRING-HC9 gene heterologous overexpression positive plants (OE#1, OE#2, OE#3) and wild-type plants (WT) were sown in 1 / 2 MS medium and subjected to stress treatments with 100 mM and 150 mM NaCl. Germination rate was measured with a control group (no stress). Three biological replicates were performed. Figure 8 The germination profiles of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana in control, 100 mM and 150 mM NaCl stress media; such as Figure 8 As shown, from Figure 8 It can be seen that under 100 mM and 150 mM NaCl stress, the germination rate and growth status of the overexpressing positive plants (OE#1, OE#2, OE#3) were better than those of the WT plants.

[0153] Figure 9 This is a statistical figure showing the germination rates of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana in control, 100 mM, and 150 mM NaCl stress media; the results are as follows. Figure 9 As shown, there was no significant difference in germination rate between wild-type and heterologous expression plants under no NaCl stress treatment; however, under 100 mM and 150 mM NaCl stress, the germination rate of heterologous expression plants was significantly higher than that of wild-type plants (P < 0.05). The results indicate that heterologous expression of the BvRING-HC9 gene can effectively improve the seed germination rate of plants under salt stress.

[0154] 4. Measurement of root length in plants heterologously expressing the salt-tolerance gene BvRING-HC9:

[0155] Seeds from heterologous BvRING-HC9 gene-expressing plants (OE#1, OE#2, OE#3) and wild-type plants (WT) were sown in 1 / 2 MS medium and cultured for 7 days. They were then transferred to fresh 1 / 2 MS medium and subjected to salt stress treatments of 100 mM and 150 mM NaCl, with no stress serving as a control. Root length under salt stress was measured after 5 days of culture, using three biological replicates. Figure 10 The root length phenotypes of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana cultured in control, 100 mM and 150 mM NaCl stress media are as follows: Figure 10 As shown, from Figure 10 It can be seen that under 150 mM NaCl stress, the roots of the overexpressing positive plants (OE#1, OE#2, OE#3) were longer than WT.

[0156] Figure 11 These are the statistical results of root length phenotypes in wild-type and BvRING-HC9 transgenic Arabidopsis thaliana in control, 100 mM and 150 mM NaCl stress media; the results are as follows: Figure 11 As shown, the root growth status of the heterologous expression lines was not significantly different from that of the wild type under both NaCl stress and 100 mM NaCl stress. However, under 150 mM NaCl stress, both wild-type and heterologous expression plants had shorter roots, indicating that salt stress affects plant root growth. However, compared to the wild type, the root length of the heterologous expression plants was significantly longer (P < 0.05). These results indicate that heterologous expression of the BvRING-HC9 gene can effectively increase root length in plants under high salt stress.

[0157] 5. Phenotypic observation and leaf dry and fresh weight determination of plants heterologously expressing the salt stress tolerance gene BvRING-HC9:

[0158] Seeds from heterologous BvRING-HC9 gene-expressing plants (OE#1, OE#2, OE#3) and wild-type plants (WT) were planted in a mixed soil (vermiculite:soil = 1:2). After four weeks of growth, the plants were subjected to 150 mM NaCl stress treatment (150 mM NaCl solution was applied every other day), with no stress serving as a control. Phenotypic results and leaf dry and fresh weights were observed on day 6. Three biological replicates were performed (e.g., Figure 12 (As shown).

[0159] Figure 12 The plant phenotypes of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana in mixed soil under control and 150 mM NaCl stress were compared. Figure 13 The results are statistical results of the fresh weight of leaves of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana plants in mixed soil under control and 150 mM NaCl stress. Figure 14 These are the statistical results of leaf dry weight of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana plants in mixed soil under control and 150mM NaCl stress conditions; the experimental results are as follows: Figures 12-14 As shown, the plants grew well under the absence of NaCl stress. However, under 150 mM NaCl stress, compared with the wild type, the heterologous expression plants exhibited better growth under salt stress, and the fresh and dry weight of leaves were significantly increased (P < 0.05). These results indicate that heterologous expression of the BvRING-HC9 gene can effectively increase plant biomass and improve its adaptability to salt stress.

[0160] 6. Determination of ion content in plants heterologously expressing the salt-tolerance gene BvRING-HC9:

[0161] Seeds from heterologous BvRING-HC9 gene-expressing plants (OE#1, OE#2, OE#3) and wild-type plants (WT) were sown in a mixed soil (vermiculite:soil = 1:2). After four weeks of growth, the plants were subjected to 150 mM NaCl stress treatment (150 mM NaCl solution was applied every other day), with no stress as a control. Ion content was determined by flame atomic absorption spectrometry. Three biological replicates were performed. The specific methods are as follows:

[0162] (1) Take 0.01 g of Arabidopsis leaf tissue, dry it at 70℃, grind it into powder and pass it through a 60-mesh sieve, and add 5 mL of concentrated sulfuric acid;

[0163] (2) Digest in a fume hood overnight at 180°C. When no brown gas is produced, add H2O2, 1.5 mL of cesium chloride / lanthanum chloride solution, and add ddH2O to make up to 10 mL. Filter with a filter membrane and then test.

[0164] (3) K was measured at wavelengths of 766.5 nm and 589 nm using atomic absorption spectrometry. + and Na + content.

[0165] (4) Prepare standard solutions, the specific formulas are shown in Tables 11 and 12. Determine the ion content using a flame atomic absorption spectrometer, and calculate the ion content of each strain using the standard curve. The ion content standard curve is shown in Tables 11 and 12. Figure 15 and Figure 17 As shown ( Figure 15 Yes + Ion content standard curve; Figure 17 It is K + (Standard curve of ion content).

[0166] Table 11 Determination of Na + Ion content standard solution

[0167] Table 12 Determination of K + Ion content standard solution

[0168] Figure 16 This refers to the Na+ levels in wild-type and BvRING-HC9 transgenic Arabidopsis thaliana plants under control and 150 mM NaCl stress. + Graph of ion content determination results; Figure 18 The K plants of wild-type and BvRING-HC9 transgenic Arabidopsis thaliana under control and 150 mM NaCl stress were... + The results of ion content determination are shown in the figure; the results are as follows. Figure 16 and Figure 18 As shown, the proportion of Na+ in the roots of BvRING-HC9 heterologous expression plants was significantly lower than that of wild type under salt stress, while the proportion of K+ in the roots of BvRING-HC9 heterologous expression plants was not significantly different between the wild type and the wild type under salt stress.

[0169] Figure 19 The wild-type and BvRING-HC9 transgenic Arabidopsis thaliana plants under control and 150 mM NaCl stress showed low Na2 content. + / K + The results of ion content determination are shown in the figure. Figure 19 As shown, under salt stress, BvRING-HC9 heterologous expression plants had a lower Na+ / K+ ratio, and these plants maintained a better physiological state under salt stress, showing less impact from it. This suggests that the BvRING-HC9 gene may enhance plant salt tolerance by reducing Na+ content.

Claims

1. Application of a salt-tolerant gene, BvRING-HC9, in improving plant salt tolerance.

2. The application according to claim 1, characterized in that, The application method is performed according to the following steps: I. Construction of pCAMBIA1300S-3×FLAG-BvRING-HC9 expression vector; II. Obtaining heterologous expression plants of the salt-tolerant gene BvRING-HC9: Wild-type plants were stably transformed using the Agrobacterium-mediated transformation method with the pCAMBIA1300S-3×FLAG-BvRING-HC9 recombinant vector, thus obtaining plants with salt tolerance.

3. The application according to claim 1 or 2, characterized in that, The CDS sequence of the salt stress tolerance gene BvRING-HC9 is shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, The amino acid sequence of the protein encoded by the gene BvRING-HC9 is shown in SEQ ID No.

2.

5. The application according to claim 2, characterized in that, The wild-type plant is wild-type Arabidopsis thaliana.

6. The application according to claim 2, characterized in that, Construction method of pCAMBIA1300S-3×FLAG-BvRING-HC9 expression vector: A. Obtain the gene BvRING-HC9; B. Extract the plasmid of the pCAMBIA1300S-3×FLAG vector and digest the pCAMBIA1300S-3×FLAG vector with BamHI restriction endonuclease. C. The single-enzyme-digested pCAMBIA1300S-3×FLAG vector was linearly ligated with the BvRING-HC9 gene CDS. D. Transform the recombinant vector obtained by the above ligation into Escherichia coli DH5α competent cells; Then, positive recombinants were screened and the recombinant vector was extracted; thus, the pCAMBIA1300S-3×FLAG-BvRING-HC9 expression vector was obtained.

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