Hg49737 gene and its salt tolerance application

Abstract

This invention provides the Hg49737 gene of Haloxylon ammodendron and its regulation of Na+. + In terms of efflux applications, the function of the *Haloxylon ammodendron* Hg49737 gene was verified by cloning the gene, constructing a subcellular localization vector, and performing genetic transformation on a yeast defective variant. It was found that the *Haloxylon ammodendron* Hg49737 gene can promote Na+ excretion. + External drainage significantly improves plant salt tolerance. This will provide relevant candidate genes and theoretical basis for the improvement of saline-alkali soils and the breeding of new salt-tolerant plant (crop) varieties.

Description

Technical Field

[0001] This invention belongs to the fields of plant bioengineering and transgenic technology, specifically relating to the salt tolerance gene Hg49737 of the halophyte Haloxylon ammodendron and its regulation of Na+. + Applications in external discharge. Background Technology

[0002] In recent years, industrial pollution and improper irrigation methods have led to increased soil salinity and increasingly severe soil salinization in arable land. Related studies indicate that by 2050, more than half of arable land will be affected by soil salinization. [1,2] Soil salinization is one of the most significant abiotic stresses affecting crop growth and yield. Excessive accumulation of soluble salts in the topsoil layer negatively impacts plant growth and development; this phenomenon is known as salt damage. [3] Salt damage severely restricts seed germination, plant growth, and overall productivity. [4] Almost all crops important to humans are sensitive to high concentrations of salt in the soil. The presence of high salt concentrations in the soil causes crops to accumulate large amounts of sodium (Na). + and Cl -[5] Soil salinization hinders crops' absorption of nutrients and water, and in severe cases, can lead to crop dehydration, ultimately resulting in reduced yields or crop failure. Decreased soil nutrients, physical and biological indicators, ecological degradation, and reduced crop productivity are all caused by soil salinization, which has become the primary abiotic factor limiting sustainable ecological development and agricultural production. [6,7] Therefore, improving crop salt tolerance and increasing the yield and quality of crops in saline-alkali land has become a top priority in agricultural research.

[0003] Halophytes have attracted widespread attention from breeders due to their abundance of salt-tolerant genes and unique salt-tolerant mechanisms. Halogeton glomeratus, belonging to the Chenopodiaceae family and the Halogeton genus, is an annual herbaceous desert halophyte widely distributed in arid and semi-arid regions of my country. Its leaves are highly succulent, with large central vacuoles in the mesophyll cells, serving as important sites for salt accumulation. The root system of Halogeton glomeratus is the first organ to sense salt stress, exhibiting restricted salt absorption characteristics and containing a large number of salt stress response genes. This study cloned the CDS sequence of the salt-induced expression gene Hg49737 from Halogeton glomeratus roots; constructed a subcellular localization vector for transient expression analysis in tobacco; and constructed a yeast expression vector, transformed into a defective yeast strain, and analyzed its Na+ expression in a yeast heterologous expression system. +The study aims to investigate the absorption characteristics of salt-tolerant plants (crops) to provide relevant candidate genes and theoretical basis for the breeding of new salt-tolerant plant (crop) varieties. Extensive practical experience has demonstrated that planting salt-tolerant plants (crops) in saline-alkali land can improve soil conditions, especially salt-tolerant crops, which require relatively little investment and yield quick results. Therefore, breeding new salt-tolerant varieties is of great significance for agriculture in saline-alkali land.

[0004] Problems with existing technologies: There are no reports in existing technologies regarding the function and application of the Haloxylon ammodendron gene Hg49737 in regulating the salt tolerance of Haloxylon ammodendron. Summary of the Invention

[0005] The key technical problem this invention aims to solve is to provide the Haworthia maughanii gene Hg49737 and its application in regulating salt tolerance. To solve the above technical problem, this invention adopts the following technical solution:

[0006] 1. The Hg49737 gene of Haloxylon ammodendron, wherein the transcript sequence of the Hg49737 gene is shown in SEQ No. 1 of the sequence listing, and the fragment of the Hg49737 gene that exerts its salt-resistance function is shown in SEQ No. 2 of the sequence listing.

[0007] 2. A vector comprising a nucleotide fragment as shown in sequence listing SEQ No. 1 or SEQ No. 2.

[0008] 3. Methods for cloning and constructing vectors of the Hg49737 halophyte gene, including: (1) selection of plant materials and reagents, (2) RNA extraction and reverse transcription, (3) primer design and gene cloning, and (4) construction of subcellular localization vectors, overexpression vectors and yeast expression vectors.

[0009] 4. Methods for transforming the Hg49737 gene of Haloxylon ammodendron into Agrobacterium and yeast, including: (1) transforming Agrobacterium, (2) transforming yeast defective strains with vectors.

[0010] 5. Methods for verifying the function of the Haloxylon ammodendron Hg49737 gene, including: (1) subcellular localization, Arabidopsis thaliana genetic transformation and yeast defective genetic transformation; (2) obtaining transgenic Arabidopsis thaliana and phenotypic analysis; (3) phenotypic analysis of transgenic yeast and determination of sodium ion content.

[0011] 6. The Hg49737 gene of Haloxylon ammodendron in Na + In terms of efflux applications, the functional fragment of the Haloxylon ammodendron Hg49737 gene is shown in the sequence listing SEQ No. 2.

[0012] 7. Application of the Halophytum Hg49737 gene in enhancing the salt tolerance of Arabidopsis thaliana, wherein the functional fragment of the Halophytum Hg49737 gene is shown in the sequence listing SEQ No. 2.

[0013] 8. Application of the expression of the Haloxylon ammodendron Hg49737 gene in the nucleus of tobacco cells, wherein the functional fragment of the Haloxylon ammodendron Hg49737 gene is shown in the sequence listing SEQ No. 2.

[0014] Beneficial Effects: This invention successfully cloned the Hg49737 gene using gene cloning. Subcellular localization vectors, overexpression vectors, and yeast expression vectors were constructed. Subcellular localization in tobacco revealed that Hg49737 is mainly expressed in the nucleus, but may also be expressed in the cell wall. After successful transformation into Arabidopsis thaliana, 30-day-old T1 generation seedlings were subjected to 200mM NaCl salt stress. The transgenic seedlings showed better growth than wild-type Arabidopsis thaliana, indicating that Hg49737 enhances its salt tolerance. The effect of increasing NaCl concentration in the culture medium was further observed. + The increase in Na, which transforms the empty vector pYES2 + In vivo Na of sensitive yeast strain AXT3 + The content of Na increased, while the conversion of Hg49737... + In vivo Na of sensitive yeast strain AXT3 + The content of Na was reduced in all samples, and significantly lower than that in yeast strains transformed with the empty vector pYES2. + The content of Hg49737 indicates that Hg49737 participates in Na+ expression in the yeast heterologous expression system. + The external discharge. Attached Figure Description

[0015] Figure 1 This is a structural diagram of the subcellular localization vector pBI121-eGFP.

[0016] In the figure, the Hg49737 gene is linked between the two restriction enzyme sites XbaI and SmaI.

[0017] Figure 2 This is a structural diagram of the overexpression vector pBI121-GUS.

[0018] In the figure, the Hg49737 gene is linked between the two restriction enzyme sites XbaI and SmaI.

[0019] Figure 3 This is a structural diagram of the yeast expression vector pYES2.

[0020] In the figure, the Hg49737 gene is linked between the EcoRI and XbaI restriction sites.

[0021] Figure 4 This is an image of RNA agarose gel electrophoresis.

[0022] In the diagram, M: D15000Marker.

[0023] Figure 5The target gene was amplified by PCR.

[0024] In the figure, M: D15000 Marker; 1-3 are PCR products of the Hg49737 gene.

[0025] Figure 6 This is a diagram illustrating the subcellular localization of genes.

[0026] In the image, the cell edges are brighter (actually green).

[0027] Figure 7 Figure 1 shows the resistance screening of transgenic T0 generation seeds and the salt stress phenotype analysis of T1 generation seedlings.

[0028] In the figure, A: Screening of transgenic T0 generation seed-resistant seedlings (positive plants are unaffected, non-positive plants turn yellow); B: Phenotypic comparison between wild-type Arabidopsis thaliana WT and transgenic Arabidopsis thaliana T1 generation under 200mM NaCl stress for 0d and 14d.

[0029] Figure 8 The growth and sodium ion content of yeast strain AXT3 transformed with Hg49737 and empty vector (pYES2) on AP medium were measured.

[0030] In the diagram, 1 and 10 –1 10 –2 10 –3 The figures represent yeast cultures diluted 1, 10, 100, and 1000 times, respectively. A: Growth of yeast strain AXT3 transformed with Hg49737 and the empty vector (pYES2) on AP medium containing 1 mM KCl and different concentrations of NaCl (0, 10, 30, 50 mM); B: NaCl concentration in the transgenic yeast. + content.

[0031] Figure 9 This is a schematic diagram of the Hg49737 gene and its effective fragment structure. The bold text in the diagram represents the effective fragment. Specific implementation methods

[0032] Unless otherwise specified, the methods and apparatus used in the following embodiments of this invention are conventional methods and apparatus; the equipment and reagents used are all conventional equipment and reagents purchased from reagent companies. To make the objectives, technical solutions, and advantages of this invention clearer, the specific implementation methods of this invention are described in detail below with reference to specific embodiments. Examples of these preferred embodiments are illustrated in the specific embodiments. It should also be noted that, in order to avoid obscuring the technical solution of this invention due to unnecessary details, only technical solutions and / or processing steps closely related to the solution according to this invention are shown in the embodiments, while other details that are not closely related are omitted.

[0033] Example 1

[0034] This embodiment provides the Hg49737 gene sequence of Haloxylon ammodendron, the transcript sequence of which is shown in SEQ No. 1 of the sequence listing, and the fragment of the Hg49737 gene that exerts its salt-tolerant function is shown in SEQ No. 2 of the sequence listing (e.g.). Figure 9 ).

[0035] Example 2

[0036] This embodiment provides a method for cloning the Hg49737 halophyte gene and constructing a vector, including:

[0037] 1. Selection of plant materials and reagents

[0038] The halophyte and tobacco used in this invention were both preserved in our laboratory and cultivated in the laboratory plant culture room and artificial climate incubator. The polysaccharide and polyphenol plant total RNA extraction kit, rapid plasmid mini-preparation kit, and universal DNA purification and recovery kit used in this invention were all purchased from Tiangen Biotech Co., Ltd. The reverse transcription kit (TranscriptorFirstStrand cDNA Synthesis Kit) and the fluorescence quantitative reagent kit (FastStart Essential DNA GreenMaster) were purchased from Roche. Antibiotics were purchased from Solarbio. Restriction enzymes were purchased from BioNTech Biotechnology Co., Ltd. Taq premixed solution was purchased from Acrel Biotech.

[0039] Na + Sensitive yeast strain AXT3 (due to the presence of Na+ in the plasma membrane of the strain) + -ATPases ScENA1-4, plasma membrane Na + K + / H + Antiporter ScENA1 and vacuolar membrane Na + K + / H + The absence of the antitransporter ScEHX1 leads to the sensitivity of yeast strain AXT3 to high salt concentrations, K + The absorption-deficient yeast strain CY162 (the absence of TRK1 and TRK2 in the strain results in a lack of potassium ion uptake capacity; yeast strain CY162 cannot grow under conditions where potassium ion concentration is below 7 mM), and the subcellular localization vector pBI121-eGFP ( Figure 1 ) and overexpression vector pBI121-GUS ( Figure 2 The cloning vector pMD19-T vector, E. coli Trans-T1 competent cells, and yeast expression vector pYES2 were provided by the Triticeae Research Group of the College of Agriculture, Gansu Agricultural University. Figure 3 Purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0040] 2. RNA extraction and reverse transcription

[0041] Roots were collected at the six-leaf stage of Haloxylon ammodendron. RNA was extracted using the Tiangen Polysaccharide and Polyphenol Plant Total RNA Extraction Kit, and then the RNA was reverse transcribed into cDNA using the Roche Transcriptor First Strand cDNA Synthesis Kit. For detailed instructions, please refer to the Roche manual.

[0042] The results showed that RNA extracted from the roots of *Salvia splendens* at the six-leaf stage was analyzed by ultraviolet spectrophotometry, and the 260 / 280 ratios of each RNA were all between 1.9 and 2.1, indicating good purity. Electrophoresis on a 1.2% agarose gel at 150V for 15 min also showed good RNA quality, suitable for subsequent experiments. Figure 4 ).

[0043] 3. Primer design and gene cloning

[0044] Cloning primers, subcellular localization primers, overexpression primers (same as subcellular localization primers), and yeast expression primers were designed using NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The results are shown in Table 1.

[0045] Table 1. Primers for Hg49737 cloning, subcellular localization (including restriction enzyme sites and protective bases), and yeast expression primers (including restriction enzyme sites and protective bases).

[0046]

[0047] The target gene was amplified by PCR using Aikerui Taq premixed buffer. The target fragment was recovered using the Tiangen Universal DNA Purification and Recovery Kit, ligated into the cloning vector, and transformed into E. coli competent cells Trans-T1 for blue-white screening. Finally, positive clone plasmids were extracted using the Tiangen Rapid Plasmid Mini-Prep Kit and sent to Sangon Biotech for sequencing. Specific steps are detailed in the instructions for each kit.

[0048] The results showed that using cDNA from *Salvia splendens* as a template, the target gene was amplified by cloning primer PCR. The amplified product was electrophoresed on a 1.5% agarose gel at 100V for 30 min. Figure 5After recovering the amplified products using the Tiangen universal agarose gel extraction kit, the target gene amplification product and the pMD19-T vector were ligated overnight at 16℃. On the second day, the overnight ligated product was transformed into Trans-T1 E. coli. After 12–16 hours, blue-white screening was performed. White spots were picked and placed in 5 ml of LB liquid medium containing kanamycin (Kan). Colony PCR was performed at 37℃, 150 rpm for 12 hours. Simultaneously, plasmids were extracted and sent for sequencing. Suitable colonies were sent to the company for sequencing. After successful sequencing, the colonies were stored at -80℃ for later use.

[0049] The PCR amplification system and reaction procedure are as follows.

[0050] PCR amplification system:

[0051] Premix Taq 12.5μL <![CDATA[ddH2O]]> 9.5μL Primer-F 1.0μL Primer-R 1.0μL cDNA 1μL

[0052] PCR reaction procedure:

[0053]

[0054] 4. Construction of subcellular localization vectors, overexpression vectors, and yeast expression vectors

[0055] Subcellular localization primers, overexpression primers, and yeast expression primers were designed, and protective bases and restriction enzyme sites were added to the 5' end of the primers (Table 1). The target gene was amplified by PCR using the positive plasmid from 1.2.3 as a template. The amplification products, subcellular localization vector pBI121-eGFP, overexpression vector pBI121-GUS, and yeast expression vector pYES2 were digested with the following systems: In sterilized PCR tubes, 10 μL of the target gene and pBI121-eGFP or pBI121-GUS vector plasmid, 2 μL of 10x T Buffer, 2 μL of BSA, 1 μL of XbaI, 1 μL of SmaI, and 4 μL of ddH2O were added sequentially; in sterilized PCR tubes, 10 μL of the target gene and pYES2 vector plasmid, 4 μL of 10x M Buffer, 1 μL of XbaI, 1 μL of EcoRI, and 4 μL of ddH2O were added respectively. The samples were briefly centrifuged, sealed with sealing film, and incubated overnight in a 37°C water bath for enzyme digestion. Six tubes of each plasmid were double-digested. The next day, the samples were detected by 1% agarose gel electrophoresis. The small target gene fragment and the large vector fragment were recovered from the gel. The enzyme digestion products were recovered using the Tiangen Universal DNA Purification and Recovery Kit. The target gene was ligated into the subcellular localization vector pBI121-eGFP, the overexpression vector pBI121-GUS, and the yeast expression vector pYES2, respectively, and then sequenced. The samples were then transformed into Trans-T1 cells, screened for blue-white spots, detected by colony PCR, and sequenced. Plasmids were extracted after successful sequencing.

[0056] The results showed that, using the positive clone vector plasmid as a template, the target gene was amplified using subcellular localization primers, overexpression primers, and yeast expression primers, respectively. The amplification products were recovered using the Tiangen universal DNA agarose gel extraction kit. The target gene, subcellular localization vector, overexpression vector, and yeast expression vector were double-digested at 37℃. Then, the target gene was ligated into the subcellular localization vector, overexpression vector, and yeast expression vector using T4 DNA ligase at 16℃, respectively.

[0057] Example 3

[0058] This embodiment provides the transformation of the Haloxylon ammodendron Hg49737 gene in Agrobacterium and yeast, including:

[0059] 1. Transformation of Agrobacterium

[0060] After successfully obtaining the subcellular localization vector and overexpression vector, the subcellular localization vector and overexpression vector were transformed into Agrobacterium GV3101 using the freeze-thaw method. The specific steps were as follows: Agrobacterium competent cells were aliquoted into sterile 1.5 mL centrifuge tubes, 50 μL per tube; 10 μL of plasmid was added, and the mixture was mixed by pipetting, incubated on ice for 5 min, immediately placed in liquid nitrogen for 5 min, incubated in a 37°C water bath for 5 min, and then placed on ice for 5 min. 700 μL of antibiotic-free LB liquid medium was added, and the mixture was incubated at 28°C and 200 rpm with shaking for 2-3 h. After centrifugation at 6000 rpm for 1 min, 500 μL of supernatant was discarded, and the remaining supernatant was mixed with the bacterial precipitate. 150 μL was spread onto LB solid medium containing Kans and Rif, sealed, and incubated upside down at 28°C for 48-72 h until plaques appeared. Select single clones and culture them in LB liquid medium containing the corresponding antibiotics at 28°C and 200 rpm for 2-3 days. Perform bacterial PCR detection and sequencing, and store the successfully sequenced positive strains at -80°C.

[0061] 2. Transformation of yeast auxotroph strains using vectors

[0062] Yeast expression vectors were transformed into defective strains using the lithium acetate conversion method. The specific steps were as follows: Ura solid selection medium was heated until completely melted, then dispensed into culture dishes, solidified, and cooled for later use; CY162 and AXT3 competent cells were dispensed into 1.5 mL sterile centrifuge tubes, 50 μL per tube, with four tubes containing each competent cell, and the tubes were labeled; 240 μL of 50% PEG and 36 μL of 1M were added sequentially to the centrifuge tubes. LiAc, 65 μL sterile water, 5 μL salmon sperm DNA, 2 μL each of empty vector plasmid and recombinant plasmid, were briefly centrifuged; each centrifuge tube was vigorously vortexed for about 1 min to ensure complete mixing of the added samples, sealed, and incubated at 28℃ for 30 min, followed by heat shock at 42℃ for 20 min. After removal, the tubes were centrifuged at 10000 rpm for 1 min, and the supernatant was discarded; 500 μL of sterile water was added to the centrifuge tubes, and the mixture was gently mixed by pipetting with a 1000 mL pipette, and centrifuged at 10000 rpm for 1 min; the supernatant was discarded, and the previous step was repeated, removing a portion of the supernatant to make the tube volume 50 μL; 30 μL of the transformation mixture was evenly spread on Ura solid medium, sealed, and incubated upside down at 28℃ for 3 days; after single plaques appeared, a single plaque was picked from 10 mL of Ura liquid selective medium, expanded, and verified by bacterial PCR to confirm the appropriate bacterial culture for subsequent experiments.

[0063] Example 3

[0064] This embodiment provides the Haematococcus pluvialis Hg49737 gene in Na... + Applications in external discharge include:

[0065] 1. The Agrobacterium tumefaciens bacterial solution was transiently converted into tobacco leaves using the injection permeation method. The specific steps were as follows: One day before injection, the Agrobacterium tumefaciens bacterial solution stored at -80℃ was removed and thawed on ice; 20 μL of Agrobacterium tumefaciens bacterial solution was added to 10 mL of LB liquid medium containing Kans and Rif, and cultured at 28℃ and 220 rpm with shaking for at least 16 hours; the corresponding antibiotic, 5 mL of 10 mM MES, and 400 μL of 20 μmol ACE were added to 50 mL of LB liquid medium, followed by 4 mL of activated bacterial solution, and cultured at 28℃ and 220 rpm in the dark with shaking, during which the OD value of the bacterial solution was measured; when the OD value of the bacterial solution reached approximately 0.5, it was aliquoted into sterilized 50 mL centrifuge tubes, centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the bacterial precipitate was collected; 5 mL of 10 mM MES, 3 mL of 150 μmol ACE, and 1 mL of [unspecified substance] were added to 50 mL of 1% MS liquid medium. The bacterial cells were resuspended in 10 mM MgCl₂ until the OD₆₀ = 0.5. After incubating on ice for 2 hours, the bacteria could be used to infect tobacco. Select healthy tobacco plants with suitable leaf size and inject the bacterial solution into the underside of the leaves using a 2.5 mL syringe (needle removed), avoiding the main veins as much as possible. An empty GFP vector was used as a control and clearly labeled. The injected tobacco plants were cultured in the dark for 2 days, and the distribution of fluorescence signals was observed under a laser scanning confocal microscope (LSM800).

[0066] 2. Transformation of Arabidopsis thaliana by flower infusion method: Take Agrobacterium tumefaciens culture from a -80°C freezer, add 20 μl to 10 mL of LB broth containing Rif and Kan, and incubate in the dark at 28°C and 200 rpm for at least 18 hours, until the culture becomes turbid and orange-yellow. Add 100 μl of the activated culture to 100 mL of LB broth containing Rif and Kan, and incubate in the dark at 28°C and 200 rpm with shaking until the culture becomes turbid and orange-yellow. Perform two replicates for each gene. Aliquot the above culture into 50 mL sterile centrifuge tubes, centrifuge at 4000 rpm for 15 min, discard the supernatant, collect the bacterial pellet, and then add 200 mL of... Prepare a 5% sucrose solution by pipetting and resuspending the precipitate. Centrifuge again at 4000 rpm for 15 minutes, discard the supernatant, and resuspend the bacterial precipitate in a 5% sucrose solution to achieve a final OD600 of 0.5-0.8. Protect from light and add 0.04% Silwet-77. For infection, immerse the inflorescence in the infusion solution for 1 minute, cover with a black plastic bag, and incubate in the dark for 24 hours. Remove the plastic bag and incubate normally. Depending on the plant growth, reinfection can be performed after one week. When the siliques turn yellow or brown, cover with bags and stop watering. Harvest the seeds when the plants die or most of the siliques split open. After removing leaves and other impurities, the transgenic T0 generation seeds were placed in a 4℃ refrigerator for about one week. After vernalization, they were sown on MS medium containing 50 mg·L⁻¹ Kan. The sterilized seeds were spread on MS medium containing Kan and placed under light / dark conditions of 16 / 8 h at 22-24℃ to allow germination. After the seedlings developed their first pair of true leaves and roots, resistance screening was performed: resistant seedlings showed root growth at the same time as true leaves, with better seedling growth and green leaves; non-resistant seedlings had yellow leaves, inhibited growth, and even died. Positive seedlings were transplanted into seedling soil mixed with vermiculite, nutrient soil, and coconut coir. Once it was basically confirmed that the resistant seedlings could flower and be harvested normally, leaves were randomly selected from the plants, marked, and DNA was extracted from transgenic Arabidopsis and wild-type Arabidopsis using a plant genomic DNA extraction kit, following the instructions. PCR amplification was performed using the primers in Table 1, following the same PCR amplification method as above, and detected by 1% agarose gel electrophoresis. After disinfection, the T1 generation seeds collected from positive plants were sown on MS medium. After three true leaves emerged, the seedlings were transplanted into seedling trays and cultured for 30 days under light / dark conditions of 16 / 8h and 22-24℃. Then, they were subjected to salt stress with 200mM NaCl. The phenotype was observed and photographed at 0, 7, and 14 days of stress.

[0067] 3. Take 50 μL of the successfully cultured bacterial solution into 10 mL of Ura liquid selective medium and incubate at 28°C and 200 rpm until the OD600 of the bacterial solution reaches 0.5. Then, dilute the bacterial solution 1, 10, 100, and 1000 times respectively. Take 5 μL of the diluted bacterial solution and spot it onto a container containing different concentrations of Na. +On AP medium, specifically: pYES2 empty vector, pYES2-Hg49737, and AXT3 were sequentially spot-transformed from top to bottom on AP medium containing 1 mM KCl and supplemented with 0, 10, 30, and 50 mM NaCl. After drying the bacterial colonies, the cultures were sealed and incubated upside down in a 28°C incubator for two days. The yeast colony growth was then observed and photographed. Simultaneously, 1 mL of bacterial suspension with an OD600 of 0.5 was added to 100 mL of liquid AP medium containing 10 and 50 mM NaCl. The cultures were incubated at 8°C and 220 rpm in the dark for 3 days. The bacterial suspension was centrifuged, the supernatant was removed, and the precipitate was resuspended and washed three times. The sodium ion content in the yeast was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0068] The results showed that Hg49737 is expressed on the cell membrane and may also be expressed on the cell wall, so it may be localized on the cell membrane and may also have a small amount localized on the cell wall. Figure 6 After salt stress, wild-type Arabidopsis thaliana exhibited yellowing and wilting symptoms, while transgenic Arabidopsis thaliana showed significantly better growth than wild-type Arabidopsis thaliana, indicating that Hg49737 improved the salt tolerance of the plants. Figure 7 ); in Na + At a concentration of 0 mM, all strains showed consistent and good growth. With increasing Na+ concentration in the culture medium, the AXT3 yeast strain transformed with pYES2-Hg49737 showed essentially the same growth pattern compared to the strain transformed with the empty vector pYES2, with no significant difference. Figure 8 A). Further analysis of Na+ in yeast strains transformed with empty vectors pYES2 and Hg49737. + The content was found to increase with the amount of Na in the culture medium. + The increase in Na+ in yeast strains transformed with the empty vector pYES2 + The content of Na increased, while the yeast strain that transformed Hg49737 had Na in its body. + The content of Na was reduced and significantly lower than that in yeast strains transformed with the empty vector pYES2. + content ( Figure 8 B) indicates that Hg49737 is involved in Na in the yeast heterologous expression system. + The external discharge.

[0069] The above description is only a specific embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

[0070] References:

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Claims

1. A Haloxylon ammodendron Hg49737 gene, characterized in that The Hg49737 gene sequence is shown as SEQ ID No.

2.

2. A vector, characterized by The vector comprises a nucleotide fragment shown as SEQ ID No.

2.

3. The use of overexpressing Puccinellia tenuiflora Hg49737 gene to enhance the salt tolerance of Arabidopsis thaliana, characterized in that The Hg49737 gene sequence of the halophyte is shown as SEQ ID No. 2.

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