Chrysanthemum morifolium cv. 'hangbaiju' nhx sodium-hydrogen antiporter protein cmnhx1, encoding gene and application

Abstract

The application discloses a chrysanthemum 'Huangbaijuyun' NHX sodium hydrogen reverse transport protein CmNHX1, a coding gene and application, wherein the sodium hydrogen reverse transport protein CmNHX1 comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO. 2. The application further provides a nucleotide sequence SEQ ID NO. 1 for coding the above protein. The application carries out chrysanthemum salt tolerance trait improvement research through genetic engineering technology, and the result is that the salt stress tolerance of the chrysanthemum is enhanced, and the practical value is outstanding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering application technology, and relates to an important structural protein in the salt stress resistance process of chrysanthemum growth and development, specifically involving the NHX sodium-hydrogen reverse transporter CmNHX1 of chrysanthemum 'Hangbaiju', its encoding gene and application. Background Technology

[0002] Hangzhou white chrysanthemum ( Chrysanthemum morifolium Ramat. 'Hangbaiju' is a traditional Chinese medicinal plant. Its dried capitulum is rich in various active ingredients and is widely used in traditional Chinese medicine and health products. Currently, the control over the non-agricultural and non-grain use of arable land is becoming increasingly strict, and usable arable land resources are becoming increasingly scarce. Against this backdrop, developing and utilizing reserve arable land resources such as saline-alkali land is of great significance for ensuring the production space of medicinal plants. However, salt stress caused by soil salinization affects the growth and development of Hangzhou white chrysanthemum through ion toxicity and oxidative damage, limiting its cultivation and promotion on marginal lands such as saline-alkali land, and also hindering the expansion of medicinal plant planting space. The NHX (Na⁺ / H⁺ reverse transporter) gene family is a core regulatory factor in plant salt stress response, regulating plant salt tolerance through mediating processes such as ion homeostasis maintenance and signal transduction, but its specific function in medicinal chrysanthemum remains unclear. Summary of the Invention

[0003] To fill the gap in the chrysanthemum 'Hangzhou White Chrysanthemum' CmNHX1 This invention discloses the cloning and expression patterns of the gene, as well as the blank of the sodium-hydrogen reverse transporter CmNHX1 in chrysanthemum 'Hangbaiju'; CmNHX1 Gene sequence and encoded amino acid sequence, protein subcellular localization, and growth phenotype of transgenic plants are used to guide future regulation using genetic engineering techniques. CmNHX1 Gene expression provides a theoretical basis for improving the salt tolerance of chrysanthemum 'Hangbaiju'.

[0004] On one hand, the present invention provides a sodium-hydrogen reverse transporter CmNHX1 from chrysanthemum 'Hangbaiju' that participates in the salt stress response. The sodium-hydrogen reverse transporter CmNHX1 comprises a polypeptide (protein) having the amino acid sequence shown in SEQ ID NO.2; or a protein having the characteristics of chrysanthemum 'Hangbaiju' sodium-hydrogen reverse transporter CmNHX1, obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2.

[0005] On the other hand, the present invention provides a coding gene encoding the NHX sodium-hydrogen reverse transporter CmNHX1 of the above-mentioned chrysanthemum 'Hangbaiju', wherein the nucleotide sequence of the coding gene is specifically: (a) the base sequence is as shown in positions 1 to 1575 of SEQ ID NO.1; or (b) a sequence having at least 80% homology with the nucleic acid shown in positions 1 to 1575 of SEQ ID NO.1.

[0006] In this invention, "isolated DNA" and "purified DNA" refer to DNA or fragments that have been isolated from sequences flanking them in their natural state, and also to DNA or fragments that have been separated from components that accompany nucleic acids in their natural state, and from proteins that accompany them in the cell.

[0007] In this invention, the gene encoding the NHX sodium-hydrogen reverse transporter CmNHX1 of chrysanthemum 'Hangbaiju' refers to the nucleotide sequence encoding the protein activity of chrysanthemum 'Hangbaiju', such as the nucleotide sequence from position 1 to 1575 shown in SEQ ID NO.1 and its degenerate sequence. This degenerate sequence refers to a sequence generated when one or more codons in nucleotides from position 1 to 1575 of SEQ ID NO.1 are replaced by degenerate codons encoding the same amino acid. Due to codon degeneracy, a degenerate sequence with less than 80% homology to the nucleotide sequence from position 1 to 1575 of SEQ ID NO.1 can also encode the sequence shown in SEQ ID NO.2. The aforementioned encoding gene can also refer to a nucleotide sequence with at least 80% homology to the nucleotide sequence shown in SEQ ID NO.1.

[0008] The aforementioned coding gene may also refer to variant forms that encode the same function as the NHX sodium-hydrogen reverse transporter CmNHX1 of chrysanthemum 'Hangbaiju', with the sequence shown in SEQ ID NO.1; these variant forms include (but are not limited to): deletions, insertions and / or substitutions of 1 to 120 nucleotides, and additions of up to 90 nucleotides at the 5′ and / or 3′ ends.

[0009] In this invention, real-time quantitative PCR can be used to analyze chrysanthemum 'Hangzhou white chrysanthemum'. CmNHX1 The expression pattern of gene products, i.e., analysis of chrysanthemum 'Hangzhou white chrysanthemum'. CmNHX1 The presence and quantity of gene mRNA transcripts in cells.

[0010] Furthermore, the chrysanthemum 'Hangzhou White Chrysanthemum' according to the present invention CmNHX1 Gene sequences and amino acid sequences can be used to screen for 'Hangzhou White Chrysanthemum' based on nucleic acid homology or expressed protein homology. CmNHX1 Gene-related homologous genes or homologous proteins.

[0011] The chrysanthemum 'Hangzhou White Chrysanthemum' of this invention CmNHX1 Full-length sequences or fragments of gene-related nucleotides can typically be obtained using PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0012] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods.

[0013] In addition, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0014] Besides being produced by recombinant methods, fragments of the protein of the present invention can also be produced by solid-phase technology through direct peptide synthesis. The individual fragments of the protein of the present invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

[0015] This invention also provides a recombinant expression vector containing the encoding gene of the NHX sodium-hydrogen reverse transporter CmNHX1 from the chrysanthemum 'Hangbaiju' variety. The recombinant expression vector is pBI121- CmNHX1 .

[0016] This invention also provides the application of the gene encoding the NHX sodium-hydrogen reverse transporter CmNHX1 of the above-mentioned chrysanthemum 'Hangbaiju' in enhancing the salt tolerance of chrysanthemum.

[0017] The above applications include: constructing a recombinant expression vector containing the coding gene of the sodium-hydrogen reverse transporter CmNHX1, transforming it into a plant host, and culturing and screening to obtain transgenic plants.

[0018] Beneficial Effects: Hangzhou white chrysanthemum, a traditional Chinese medicinal plant and one of the "Eight Treasures of Zhejiang," possesses both medicinal and health-promoting value. Market demand is strong and the planting scale is large. However, its cultivation and promotion are limited by salt stress, and the scarcity of arable land further restricts its production space. This invention is the first to clearly demonstrate the salt tolerance advantage of Hangzhou white chrysanthemum under salt stress, systematically analyzes the function of the CmNHXs gene family, and confirms... CmNHX1 This invention significantly enhances the host's salt tolerance. For the first time, it cloned the coding sequence of CmNHX1, a crucial sodium-hydrogen reverse transporter protein regulating the growth and development of chrysanthemum 'Hangbai Ju', and analyzed it using real-time quantitative PCR. CmNHX1Gene expression patterns, transient expression analysis of tobacco leaf epidermal cells, and subcellular localization of the sodium-hydrogen reverse transporter CmNHX1, provide insights for future regulation using genetic engineering techniques. CmNHX1 The expression of genes provides a theoretical basis for improving the salt tolerance of Hangzhou white chrysanthemum and has great application value. Attached Figure Description

[0019] Figure 1 The chrysanthemum 'Hangzhou White Chrysanthemum' of this invention CmNHX1 Homology comparison results (DNAMAN) of the gene and Arabidopsis thaliana NHX protein sequence and phylogenetic tree analysis of homologous genes; where A is the phylogenetic tree and B is the homologous sequence alignment diagram; Figure 2 The localization diagram of the sodium-hydrogen reverse transporter CmNHX1 in tobacco leaf epidermal cells of chrysanthemum 'Hangbaiju'. Figure 3 For chrysanthemum 'Hangzhou White Chrysanthemum' CmNHX1 A diagram illustrating the expression patterns of genes in different tissues of Hangzhou white chrysanthemum. Figure 4 for CmNHX1 Phenotypic diagrams of transgenic Arabidopsis thaliana; where A is the identification diagram of positive lines overexpressing tobacco; B is the diagram of wild-type (WT) and... CmNHX1 Graph showing root length measurements of seedlings on MS medium, MS medium supplemented with 100 mM NaCl, and MS medium supplemented with 100 mM Sorbitol; C represents WT and CmNHX1 Figures showing the growth of seedlings on MS medium with 100 mM NaCl, MS medium with 100 mM Orbitol, and MS medium. Detailed Implementation

[0020] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0021] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (4th Edition), or as recommended in the reagent instructions.

[0022] Example 1: Chrysanthemum 'Hangzhou White Chrysanthemum' CmNHX1 Cloning of genes 1. Obtaining plant materials Total RNA was extracted from leaf tissue of normally growing chrysanthemum 'Hangbaiju'.

[0023] 2. RNA extraction Using Hunan Aikerui Biotechnology Co., Ltd. SteadyPureThe "Plant RNA Extraction Kit" extracts total RNA, gel electrophoresis is used to identify the integrity of the RNA, and a spectrophotometer (Nanodrop 2000) is used to determine the purity and concentration of the RNA.

[0024] 3. Full-length cloning of genes Based on the nucleotide sequence and protein function annotation results provided by the laboratory's previous full-length transcriptome analysis, the chrysanthemum 'Hangzhou White Chrysanthemum' was obtained. CmNHX1 Full-length gene. The extracted RNA was reverse transcribed (using the HiScript III 1st Strand cDNA Synthesis Kit) to obtain cDNA. Using the first-strand cDNA as a template, PCR amplification was performed using primers CmNHX1-F (5'-ATGCTATGACGGAAAAATTAGACG- 3') and CmNHX1-R (5'-CGAGCTTCGGCATGGCCATCACCC- 3'), yielding a 1575 bp fragment. This fragment was recovered and ligated into the pMD18-T vector. Using M13-47 and RV-M as universal primers, the sequence was sent to Hangzhou Youkang Sequencing Center.

[0025] Combining the sequencing results with NCBI's ORF Finding (http: / / www.ncbi.nlm.nih.gov / gorf) predictions, chrysanthemums were discovered. CmNHX1 The ORF reading frame of the gene was obtained. The full-length coding sequence (SEQ ID NO.1) of 1575 bp was amplified. Sequencing results were aligned to the NCBI BLAST database (GenBank, http: / / blast.ncbi.nlm.nih.gov / ). The nucleotide sequence and encoded protein showed high homology with the known Arabidopsis thaliana AtNHX4 protein, suggesting it is preliminarily considered to be a... NHX Gene.

[0026] Example 2: Chrysanthemum 'Hangzhou White Chrysanthemum' CmNHX1 Gene sequence information and homology analysis The chrysanthemum 'Hangzhou White Chrysanthemum' of this invention CmNHX1 The full-length open reading frame (OPF) sequence of the gene is 1575 bp, and the detailed sequence is shown in SEQ ID NO.1. Based on the ORF sequence, the amino acid sequence of the sodium-hydrogen reverse transporter CmNHX1 from chrysanthemum 'Hangbaiju' was deduced, consisting of 524 amino acids, and the detailed sequence is shown in SEQ ID NO.2.

[0027] Chrysanthemum 'Hangzhou White Chrysanthemum' CmNHX1The open reading frame sequence of the gene and the amino acid sequence of its encoded protein were subjected to nucleotide and protein homology searches in NCBI using the BLAST program. The results showed that it had extremely high amino acid similarity to NHX from Arabidopsis thaliana. Figure 1 As shown in B. Phylogenetic analysis showed that the sodium-hydrogen reverse transporter CmNHX1 in chrysanthemum 'Hangbaiju' has high homology with the NHX4 protein in Arabidopsis thaliana, such as... Figure 1 As shown in Figure A.

[0028] Example 3: Subcellular localization analysis of the sodium-hydrogen reverse transporter CmNHX1 from 'Hangbaiju' chrysanthemum in tobacco leaves Specific primers CmNHX1-pHB-GFP-F (5'-TCTCTCTCTCAAGCTTGGATCCATGGATGCAGAGGCCGAGTTTG-3') and CmNHX1-pHB-GFP-R (5'-TGCTCACCATGTCGACAGTCAATGGATCAACGAATC-3') were designed at the start and stop codons, respectively, and introduced onto both sides of the full-length gene sequence. BamH I and SalI Enzyme restriction sites. The plasmid containing the target fragment with enzyme restriction sites is transformed into the pHB-GFP binary transformation vector. BamH I and SalI Double enzyme digestion, recovery of the digested pHB vector and CmNHX1 The fragment was subjected to homologous recombination with recombinase Exnase II at 37°C for 30 min to construct the recombinant expression vector pHB- CmNHX1- GFP (denoted as CmNHX1 -GFP), and the correctly identified recombinant expression vector was transformed into Agrobacterium GV3101.

[0029] The identified GV3101 strain was inoculated into 5 mL LB (containing 50 mg / L Kan and 50 mg / L Rif) and cultured at 28°C and 180 rpm until OD. 600 The value is approximately 0.6; 1 mL of bacterial culture is added to 25 mL of LB liquid medium and incubated at 28°C until the OD value reaches 0.6. 600 The OD value was approximately 0.6; 10 mL of bacterial culture was centrifuged at 4500 rpm for 15 min; the bacterial cells were resuspended in MS liquid medium until the OD value reached approximately 0.6. 600 Approximately 0.5 mg / L was added, along with AS and MES, and incubated at room temperature for at least 3 hours. The mixture was then injected into tobacco leaves and cultured in the dark for 48 hours. Observation was performed using a laser confocal microscope at an excitation wavelength of 600 nm. The results are as follows: Figure 2 As shown, in tobacco leaves infected with pHB-GFP empty vector bacterial solution, GFP fluorescence signals can be clearly observed to be evenly distributed in the cell membrane and nucleus, while those transformed into recombinant plasmids... CmNHX1 In the leaves of -GFP, the fluorescent signal was localized to the cell membrane, indicating that CmNHX1 is localized to the cell membrane.

[0030] Example 4: Chrysanthemum 'Hangzhou White Chrysanthemum' CmNHX1 Gene expression patterns in different tissues of Hangzhou white chrysanthemum Material acquisition and processing: for exploration CmNHX1 To investigate gene expression characteristics in different tissues of Hangzhou white chrysanthemum, this invention subjected 14-day-old tissue-cultured Hangzhou white chrysanthemum seedlings to salt stress treatment (hydroponics, salt concentration 7 mM). Root, stem, and leaf tissue samples (0.1 g each) were collected at 0 hours (0H), 3 hours, 6 hours, 12 hours, 24 hours, and 36 hours of stress. The samples were sealed in aluminum foil, flash-frozen in liquid nitrogen, and then stored at -80℃. Subsequent qRT-PCR was used for systemic detection. CmNHX1 Gene expression dynamics.

[0031] 2. RNA extraction, determination of RNA integrity, purity, and concentration, and cDNA acquisition are described in Example 1.

[0032] 3. Design specific primers for real-time quantitative PCR analysis of gene expression levels in various tissues, based on the obtained chrysanthemum 'Hangbaiju'. CmNHX1 Gene sequence designed for use in real-time quantitative PCR CmNHX1 Specific primers for gene quantification analysis, primer q CmNHX1 -F(5'-GCTATGACGGAAAAATTAGACGAAT-3'), primer q CmNHX1 -R(5'-GCTATGACGGAAAAATTAGACGAAT-3'), the primer for the internal reference gene PP2A is PP2A. - F (5'-GCTAGTGGCCGTACAACTGG-3'), PP2A-R (5'-GCCAGCAAGGTCCAATCGAA-3').

[0033] 4. Standard curves for the target gene and internal reference gene: The standard cDNA solution was serially diluted with ddH2O. Then, using the diluted cDNA as templates, real-time quantitative PCR amplification was performed with specific primers for the target gene and internal reference gene, respectively. Melting curves and standard curves were plotted. The melting curves were analyzed to determine whether a single peak was obtained for the target gene and internal reference gene, in order to determine whether a single PCR amplification product could be obtained using the primers. The appropriate dilution factor of the template cDNA was determined by the standard curves.

[0034] 5. Real-time quantitative analysis of the target gene in the test sample: Using the first strand of the synthesized cDNA as a template, the target gene and the internal reference gene were amplified with specific primers for real-time quantitative PCR. The reaction was performed using a Bio-Rad CFX real-time quantitative PCR instrument. The reaction volume was 20 µL, and the reaction program was as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing for 30 s, 40 cycles.

[0035] 6. Use 2 -△△Ct Methods for relative quantitative analysis, such as Figure 3 As shown, the results indicate that... CmNHX1 The expression levels of the gene in roots and leaves gradually increased with stress duration, with the most significant response at 24 hours (24H). CmNHX1 The expression characteristics of genes that are continuously activated specifically in the root system and the changes in the overall salt tolerance phenotype of the plant (see...) Figure 4 C) Highly consistent. The above results provide molecular-level evidence for the important role of CmNHX1 in the salt stress response, demonstrating that this gene plays a key and conserved regulatory role in the chrysanthemum salt stress response pathway.

[0036] Example 5, pBI121- CmNHX1 Construction of recombinant expression vectors and CmNHX1 Gene-transformed Arabidopsis thaliana 1. Construct pBI121- CmNHX1 Recombinant expression vector XbaI and SmaI restriction sites were introduced. The plasmid containing the target fragment with the restriction sites was double-digested with XbaI and SmaI into the pBI121 binary transformation vector. The digested vector was then recovered and ligated via homologous recombination to obtain the recombinant expression vector pBI121-, which expresses the target gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter. CmNHX1 (The expression framework of this vector is abbreviated as 35S::CmNHX1), and it was transformed into Agrobacterium GV3101. After picking and shaking, molecular identification confirmed that Agrobacterium transformation was positive.

[0037] 2. Transformation of Arabidopsis thaliana Transformation of Arabidopsis thaliana by inflorescence immersion: The specific steps for infecting Arabidopsis thaliana using the Agrobacterium-mediated inflorescence immersion method are as follows.

[0038] ① The best time for Arabidopsis thaliana infection is when wild-type Arabidopsis thaliana grows to the middle and early stages of flowering. The day before infection, cut off the flowers and pods that have already bloomed, leaving only the buds. Water and fertilize them thoroughly in preparation for infection.

[0039] ② Take 100 μL of Agrobacterium tumefaciens positive bacterial culture and add it to 25 mL of LB liquid medium containing Kana (50 mg / L) and Rif (25 mg / L) for expansion culture. Incubate on a constant temperature shaker (28°C, 220 rpm) until the bacterial culture reaches OD. 600 The value is 0.8-1.0.

[0040] ③ OD of Agrobacterium tumefaciens culture medium to be expanded 600 When the bacterial concentration is between 0.8 and 1.0, start centrifugation to collect bacterial cells at 6000 rpm for 10 min, and discard the supernatant.

[0041] ④ Prepare a resuspension using the formula MS + 1 mM MES + 2 mM MgCl2 + 200 μM AS. The resuspension should be prepared fresh for use. Resuspend the bacterial cells in the resuspension and adjust the OD. 600 Values ​​range from 0.8 to 1.0, OD 600 After adjusting the concentration, add Tween-20 to bring the final concentration to 0.02%, and shake well to obtain the infection solution.

[0042] ⑤ Immerse the entire inflorescence of each Arabidopsis thaliana plant in the infection solution for 10 minutes.

[0043] ⑥ After infection, place the Arabidopsis bag flat in a dark room for 1 day to maintain humidity. After dark culture, place it in a greenhouse at 22℃, 65% humidity, 16 hours of light and 8 hours of darkness for normal culture.

[0044] ⑦ One week later, repeat the process to infect the sample again to improve the conversion success rate.

[0045] ⑧ After the secondary infection is completed, cultivate it normally and manage it using conventional cultivation methods until harvest.

[0046] 3. Screening of transgenic positive lines Genomic DNA and RNA were extracted from Arabidopsis thaliana to identify positive plants.

[0047] Example 6: Phenotypic Analysis of Transgenic Arabidopsis Material transformation verification: Seeds obtained in Example 5 were cultured to obtain T3 generation transgenic lines, denoted as OX1-1, OX1-2, and OX1-3. Genomic DNA was extracted from the T3 generation of these transgenic Arabidopsis thaliana (Actin was selected as the endogenous internal reference gene in Arabidopsis thaliana), and PCR amplification was performed. Wild-type Arabidopsis thaliana was used as a control (denoted as WT). The results are as follows: Figure 4 As shown in Figure A, the wild-type (WT) strains showed no amplification bands. CmNHX1 Clear target bands were observed in all transgenic lines, confirming... CmNHX1 The gene has completed genome integration and is able to be stably inherited.

[0048] Stress treatment: Mix wild-type (WT) germination 3 days after germination with CmNHX1 Overexpression line seedlings were transferred to normal MS medium, MS medium containing 100 mM NaCl (salt stress), and MS medium containing 100 mM sorbitol (osmotic stress) and cultured for 14 days. The growth under different treatments was analyzed using taproot length as an indicator. The results are as follows: Figure 4 B. Figure 4 As shown in C.

[0049] Growth performance under normal conditions: According to Figure 4 B. Figure 4 C, in normal MS medium. CmNHX1 There was no significant difference in taproot length between the overexpression lines and the wild type, indicating that... CmNHX1 Overexpression of the gene did not have a significant effect on the basic growth and development of Arabidopsis roots.

[0050] Phenotypic and root length analysis under salt stress: Growth of all lines was inhibited under 100 mM NaCl treatment. CmNHX1 Overexpression lines showed reduced leaf area and weakened growth, but their overall condition was better than the wild type; meanwhile, CmNHX1 The taproot length of the overexpressing lines was significantly longer than that of the wild type (e.g., Figure 4 B. Figure 4 C), indicating CmNHX1 Overexpression of sorbitol effectively alleviated the inhibitory effect of salt stress on root elongation in Arabidopsis thaliana and significantly improved the plant's salt tolerance. Furthermore, it was found that root length in Arabidopsis thaliana under 100 mM sorbitol osmotic stress did not change significantly, indicating... CmNHX1 The gene overexpression lines did not respond significantly to sorbitol osmotic stress.

[0051] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A chrysanthemum 'Hangbaiju' NHX sodium-hydrogen reverse transporter CmNHX1, characterized in that, The amino acid sequence of the sodium-hydrogen reverse transporter CmNHX1 is shown in SEQ ID NO.

2.

2. A gene encoding the NHX sodium-hydrogen reverse transporter CmNHX1 of chrysanthemum 'Hangbaiju' as described in claim 1, characterized in that, The encoding gene is a nucleotide sequence as shown in SEQ ID NO.

1.

3. A recombinant expression vector, characterized in that, It contains the gene encoding the NHX sodium-hydrogen reverse transporter CmNHX1 of chrysanthemum 'Hangbaiju' as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is pBI121- CmNHX1 .

5. The application of the gene encoding the NHX sodium-hydrogen reverse transporter CmNHX1 of chrysanthemum 'Hangbaiju' as described in claim 2 in enhancing the salt tolerance of chrysanthemum.

6. The application of the gene encoding the NHX sodium-hydrogen reverse transporter CmNHX1 of chrysanthemum 'Hangbaiju' according to claim 5 in enhancing the salt tolerance of chrysanthemum, characterized in that, The application includes: constructing a recombinant expression vector containing the coding gene of the sodium-hydrogen reverse transporter CmNHX1, transforming it into a plant host, and culturing and screening to obtain transgenic plants.

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