Application of the Hg52609 gene of Halogeton glomeratus in improving the salt tolerance of Arabidopsis thaliana

Abstract

The present invention relates to the gene cloning and functional application of the salt-tolerant gene Hg52609 in the roots of Halogeton glomeratus, belonging to the field of plant bioengineering technology. The salt-tolerant gene Hg52609 was cloned from the roots of Halogeton glomeratus for the first time. By cloning the salt-tolerant related gene Hg52609 in the roots of Halogeton glomeratus, a subcellular localization expression vector was constructed, and gene localization was carried out through transient expression in tobacco; an overexpression vector was constructed, and Arabidopsis thaliana was stably genetically transformed for salt tolerance identification; a yeast heterologous expression vector was constructed to further verify the function of the Hg52609 gene. It was found that the Hg52609 gene is involved in the efflux of Na+ but does not mediate the absorption of K+ under high concentrations of Na+. The present invention provides the cloning and functional application of the salt-tolerant gene Hg52609 in the roots of Halogeton glomeratus, which will be a good material for exploring plant salt-tolerant genes and studying the salt-tolerant regulation mechanism, and has important application value for improving the stress resistance of crops and cultivating salt-tolerant and alkali-tolerant crops.

Description

Technical Field

[0001] The present invention belongs to the fields of plant bioengineering and transgenic technology, and particularly relates to the salt tolerance gene Hg52609 of the root system of the halophyte Halogeton glomeratus and its application. Background Art

[0002] Salt is a major environmental stress factor for plants, and the exacerbation of cultivated land salinization will have a serious impact on the globe. Soil salinization will affect plants such as reducing growth efficiency, weakening photosynthesis and decreasing yield (Yang Jinsong et al., 2022). The salt in the soil will reduce the ability of plants to absorb water, inhibit the growth of leaves and roots, resulting in slow plant growth and reduced growth rate. Salt may also enter the transpiration stream, further damaging the cells in the leaves, and ultimately reducing the growth rate. The possible reason is that the salt load exceeds the ability of the cells to compartmentalize salt in the vacuole. Salt will rapidly accumulate in the cells causing cell dehydration and inhibiting the activity of enzymes. The excessive salt concentration increases the osmotic potential of the soil and limits the ability of plants to absorb water (Munns R et al., 2005).

[0003] Most plants cannot tolerate high salt content in the soil, but halophytes are plants that can grow normally and complete their life cycles in soils with a salt content of more than 70 mmol / L. Halogeton glomeratus is an annual dicotyledonous plant of the genus Halogeton in the Chenopodiaceae family, which is widely distributed in the arid and semi-arid regions of northwestern China. Its stems and leaves are highly succulent and can grow normally in saline-alkali environments. Its main salt tolerance mechanism is to compartmentalize salt-toxic ions in the cell vacuoles. Therefore, its salt tolerance mechanism can be studied at the molecular level (Zhou Zhenguo et al., 2018).

[0004] In the prior art, there is no report on the function and application of the salt tolerance gene Hg52609 of the root system of Halogeton glomeratus in participating in the regulation of salt stress response. The present invention takes the salt tolerance gene Hg52609 of the root system of Halogeton glomeratus as the starting point and clones the gene Hg52609. Further, through methods such as vector construction, subcellular localization, Arabidopsis transformation, construction of yeast expression vectors and yeast growth phenotype analysis, the function of the salt tolerance gene Hg52609 of the root system of Halogeton glomeratus is studied, aiming to further analyze the salt tolerance regulation mechanism of the root system of Halogeton glomeratus and provide salt tolerance candidate genes for the cultivation of salt-tolerant varieties in the later stage, and provide a scientific basis for improving the salt tolerance of cultivated crops in the future. Summary of the Invention

[0005] The key technical problem to be solved by the present invention is to provide the cloning of the salt tolerance gene Hg52609 of the root system of Halogeton glomeratus and the functional verification of its response to salt stress. To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] 1. The salt tolerance gene sequence of the roots of Halogeton glomeratus, wherein the transcript sequence of the Hg52609 gene is shown in SEQ No.1 of the sequence listing, and the fragment of the Hg52609 gene that plays the salt resistance function is shown in SEQ No.2 of the sequence listing.

[0007] 2. The gene cloning and vector construction method of the salt tolerance gene Hg52609 of the roots of Halogeton glomeratus, including:

[0008] (1) Selection of plant materials and reagents; (2) RNA extraction and reverse transcription; (3) Primer design and gene cloning; (4) Construction of overexpression vector, subcellular localization expression vector and yeast heterologous expression vector.

[0009] 3. The function verification method of the salt tolerance gene Hg52609 of the roots of Halogeton glomeratus, including:

[0010] (1) Arabidopsis thaliana genetic transformation and transgenic Arabidopsis thaliana resistance screening; (2) Obtaining transgenic Arabidopsis thaliana and growth phenotype analysis; (3) Yeast growth phenotype analysis.

[0011] 4. The function verification method of the salt tolerance gene Hg52609 of the roots of Halogeton glomeratus, including: (1) Subcellular localization of pBI121-GFP-Hg52609 in Nicotiana benthamiana, (2) Arabidopsis thaliana genetic transformation and salt tolerance analysis, (3) Transformation of the vector into yeast and yeast growth phenotype analysis.

[0012] 5. Application of the Hg52609 gene of Halogeton glomeratus in + Na efflux, wherein the fragment of the Hg52609 gene of Halogeton glomeratus that plays the function is shown in SEQ No.2 of the sequence listing.

[0013] 6. Application of the Hg52609 gene of Halogeton glomeratus in enhancing the salt resistance of Arabidopsis thaliana, wherein the fragment of the Hg52609 gene of Halogeton glomeratus that plays the function is shown in SEQ No.2 of the sequence listing.

[0014] 7. Application of the Hg52609 gene of Halogeton glomeratus in expressing on the tobacco cell nucleus, wherein the fragment of the Hg52609 gene of Halogeton glomeratus that plays the function is shown in SEQ No.2 of the sequence listing.

[0015] Beneficial effects: Physiological analysis shows that Halogeton glomeratus shows strong salt tolerance under salt stress and is an excellent wild resource for obtaining salt tolerance genes. Based on the transcriptomics and proteomics analysis of the roots of Halogeton glomeratus, the transcript sequence of the salt-induced expression gene is selected, and the Hg52609 gene is successfully cloned by gene cloning, and an overexpression vector is constructed to infect Arabidopsis thaliana for resistance screening and phenotype analysis; a yeast heterologous expression vector is constructed and phenotype analysis is carried out.

[0016] It was found that under 200 mM NaCl treatment, the growth rate of Arabidopsis thaliana slowed down, and symptoms such as leaf atrophy and withering appeared in wild-type plants, but the transgenic plants with Hg52609 gene could grow normally. This indicates that the Hg52609 gene can significantly improve the salt tolerance of Arabidopsis thaliana, and this gene can be applied to the cultivation of new varieties of salt-tolerant crops.

[0017] AXT3 is a yeast strain sensitive to Na + and does not contain ATPase (ENA1-ENA4), so it is extremely sensitive to Na + When no Na + is added to the AP medium with 1 mM KCl, both transformants can grow. When 10, 30, or 50 mM KCl is added, compared with the yeast strain transformed with the Hg52609 gene, the yeast strain transformed with the empty vector pYES2 grows better, and as the concentration of Na + increases, this contrast phenomenon becomes more obvious, indicating that the introduction of the Hg52609 gene increases the sensitivity of the yeast strain AXT3 to Na + , suggesting that the Hg52609 gene is involved in the efflux of Na + .

[0018] The introduction of the Hg52609 gene into the Na + -sensitive yeast strain AXT3 can reduce the toxicity of Na + to yeast cells, indicating that the Hg52609 gene is involved in the efflux of Na; it shows that the salt tolerance gene Hg52609 in the roots of Halogeton glomeratus can participate in the response to salt stress by regulating the concentration of Na + , and then play a role in improving the salt tolerance of Halogeton glomeratus. + Description of the Drawings

[0019] Figure 1 is the RNA agarose gel electrophoresis map of the roots of Halogeton glomeratus

[0020] Figure 2 is the agarose gel electrophoresis map of PCR amplification of the target gene; among them, A: Gel extraction verification for the subcellular localization and overexpression vector parts; B: Gel extraction verification for the yeast heterologous expression part.

[0021] Figure 3 is the agarose gel electrophoresis map of PCR amplification of the target fragment from the bacterial liquid; among them, A: Bacterial liquid PCR amplification for the subcellular localization part and overexpression vector part; B: Bacterial liquid PCR amplification for the yeast heterologous expression part.

[0022] Figure 4Agarose gel electrophoresis diagrams for double digestion verification of subcellular localization expression vectors and overexpression vectors; among them, A: Double digestion verification result of gene Hg52609; B: Double digestion verification result of GFP; C: Double digestion verification result of GUS.

[0023] Figure 5 Agarose gel electrophoresis diagrams for gel extraction of subcellular localization expression vectors and overexpression vectors; among them, A: Gel extraction verification of PCR of the bacterial liquid of the expression vector constructed with the Hg52609 gene; B: Gel extraction verification of GFP; C: Gel extraction verification result of GUS.

[0024] Figure 6 Agarose gel electrophoresis diagrams for identification of positive recombinant subcellular localization expression vectors and overexpression vectors; among them, A: PCR verification of the bacterial liquid of pBI121-GFP constructed with the Hg52609 gene; B: Double digestion verification of the GFP recombinant plasmid; C: PCR verification of the bacterial liquid of pBI121-GUS constructed with the Hg52609 gene; D: Double digestion verification of the GUS recombinant plasmid.

[0025] Figure 7 Diagrams related to subcellular localization; among them, A: Schematic diagram of the subcellular vector; B: Subcellular localization diagram of the salt tolerance gene Hg52609 in the roots of Halogeton glomeratus.

[0026] Figure 8 Schematic diagram of the overexpression vector.

[0027] Figure 9 Flow chart of Arabidopsis thaliana infection.

[0028] Figure 10 Phenotypes of transgenic Arabidopsis thaliana overexpressing Hg52609 and wild-type Arabidopsis thaliana after 15 days of 200 mM NaCl stress.

[0029] Figure 11 Agarose gel electrophoresis verification diagrams for constructing yeast heterologous expression vectors; among them, A: Gel extraction verification diagram of the gene; B: Colony PCR verification diagram of the pYES2-Hg52609 recombinant expression vector; C: Double digestion verification of the recombinant plasmid

[0030] Figure 12 Schematic diagram of the yeast heterologous expression vector.

[0031] Figure 13 Agarose gel electrophoresis verification diagrams for the transformation of the vector into yeast; among them, A: Colony PCR verification diagram of the transformation of the Hg52609 gene into AXT3 yeast; B: Colony PCR verification diagram of the transformation of the Hg52609 gene into CY162 yeast.

[0032] Figure 14 For the overexpression of the Hg52609 gene on Na + 、K+ Absorption characteristic diagram; wherein, A: Expression of Hg52609 gene and pYES2 empty vector in mutant yeast AXT3; B: Expression of Hg52609 gene and pYES2 empty vector in mutant yeast CY162; C: Na + content accumulated in yeast strains; D: K + content accumulated in yeast strains.

[0033] Figure 15 It is a schematic diagram of the structure of Hg52609 gene and effective fragments. The bold part in the figure is the effective fragment. Specific implementation method

[0034] In the following embodiments of the present invention, the methods and devices used are conventional methods and devices unless otherwise specified; the equipment and reagents used are all conventional equipment and reagents purchased from reagent companies. To make the objectives, technical solutions and advantages of the present invention clearer, the following specific embodiments are used to elaborate on the specific implementation manners of the present invention in detail. Examples of these preferred implementation manners are illustrated in the specific embodiments. Here, it should also be noted that in order to avoid obscuring the technical solutions of the present invention due to unnecessary details, only the technical solutions and / or processing steps closely related to the solutions of the present invention are shown in the embodiments, while other details with little relevance are omitted.

[0035] Example 1

[0036] This example provides the Hg52609 gene of Halogeton glomeratus. The transcript sequence of the Hg52609 gene is shown in Sequence Listing SEQ No. 1, and the fragment of the Hg52609 gene that exerts the salt tolerance function is shown in Sequence Listing SEQ No. 2 (such as Figure 15 ).

[0037] Example 2

[0038] This example provides a method for cloning and vector construction of the salt tolerance gene Hg52609 of Halogeton glomeratus roots, including:

[0039] 1. Selection of plant materials and reagents

[0040] Both Halogeton glomeratus and wild-type Arabidopsis thaliana used in the present invention are preserved in this laboratory and planted in the laboratory plant cultivation room. They are cultured with Hoagland nutrient solution. When the plants grow to 8 weeks old, they are treated with 100 mmol / L NaCl stress for 7 days, and then total RNA is extracted. The polysaccharide polyphenol plant total RNA extraction kit, reverse transcription kit, rapid plasmid miniprep kit, and DNA gel recovery kit used in the present invention are all purchased from Tiangen Biochemical Technology Co., Ltd. The synthesis of primers and the sequencing of samples are completed by Sangon (Shanghai) Biotech Co., Ltd.

[0041] 2. RNA Extraction and Reverse Transcription

[0042] When planting Halogeton glomeratus, mix the seeds with clean fine sand and evenly sow them in plastic flower pots filled with culture substrate (fine sand: vermiculite = 1:1 evenly mixed). Spray distilled water with a spray bottle until saturated, and culture in a plant culture room. After 3 - 4 days, cover the exposed roots of the seedlings with fine sand, and then water an appropriate amount of Hoagland's nutrient solution every day. After the seedlings grow for 2 months, add 100 mmol / L NaCl to the Hoagland's nutrient solution and perform salt stress treatment for 7 days (Wang Juncheng, 2017). After extracting RNA using the Tiangen Total Plant RNA Extraction Kit, use the Tiangen reverse transcription kit PrimeScript TM RT reagent Kit with Gdna Eraser to reverse transcribe into cDNA, which is used as a template for cloning. The specific operation process refers to its instruction manual.

[0043] The results showed that: extracting the RNA of Halogeton glomeratus roots and detecting by a ultra - micro ultraviolet spectrophotometer, the results were good. Using 1% agarose gel, the electrophoresis detection results showed that the RNA quality was good and could be used for subsequent reverse transcription and functional verification experiments ( Figure 1 ).

[0044] 3. Primer Design and Gene Cloning

[0045] Use NCBI Primer - BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer - blast / ) to design cloning primers, subcellular localization expression primers, and overexpression vector primers based on the transcript sequence of the Hg.35760 gene. Add corresponding restriction enzyme sites and protection bases according to the expression vector used later, set the temperature according to the Tm value of the synthesized primers, and perform gene amplification. Subsequently, detect by 1% agarose gel electrophoresis and observe with the help of a gel electrophoresis imaging instrument. The primer design, amplification system, and reaction program are shown in the following table

[0046] Table 1 Cloning Primers, Subcellular Localization Expression Vector Primers, and Overexpression Vector Primers

[0047]

[0048] Table 2 Yeast Heterologous Expression Vector Primers

[0049]

[0050]

[0051] The target gene was amplified by PCR using Premix Taq, and the PCR amplification system was as follows:

[0052]

[0053] The PCR reaction program for the Hg52609 gene (for subsequent construction of transient expression vectors, overexpression vector part) was:

[0054]

[0055] The PCR reaction program for the Hg52609 gene (for subsequent construction of yeast heterologous expression vectors part) was:

[0056]

[0057] The target fragment was recovered using a DNA gel extraction kit (TIANgel Purification Kit), the target gene fragment was ligated to the pMD19-T vector, and Escherichia coli competent cells Trans1-T1 were transformed for blue-white screening and PCR detection. Finally, the positive clone plasmid was extracted using the Tiangen rapid plasmid miniprep kit and sent to Sangon Biotech Co., Ltd. for sequencing. See the kit instructions for specific steps.

[0058] The results showed that using the cDNA of the roots of Halogeton glomeratus as a template, the target gene was amplified by PCR using cloning primers. The amplification product was electrophoresed on a 1% agarose gel at 120 V for 20 min ( Figure 2 ). After recovering the amplification product using a DNA gel extraction kit (TIANgel Purification Kit), the gel-extracted product, Solution Ⅰ, and pMD19-T vector were successively added to the PCR tube, and it was mixed by centrifuging for a few seconds using a microcentrifuge and then taken out and placed in a metal bath (16 °C). After 12 - 16 h, Escherichia coli competent cells Trans1-T1 were transformed. LB medium supplemented with Amp was poured into the culture dish and dried. A mixed solution was prepared by mixing the competent cells (Trans1-T1) with X-gal (20 mg / mL) and IPTG (24 mg / mL) according to a volume ratio of X-gal:IPTG = 4:1. The mixed solution was pipetted onto the solid culture dish and spread evenly with a spreader and left to dry. The well-shaken bacterial solution was pipetted and quickly spread on the plate. After drying, it was sealed. First, it was placed face up in the dark at 37 °C for 1 h, and then incubated upside down until white single colonies grew for blue-white screening. The white colonies were picked into 5 ml of LB liquid medium containing ampicillin (Amp) and cultured at 37 °C and 200 rpm in the dark until the tube became turbid, and then the bacterial solution was subjected to PCR detection ( Figure 3 ).

[0059] 1. Construction of subcellular localization expression vectors, overexpression vectors and yeast heterologous expression vectors

[0060] Vector activation: Under aseptic conditions, first activate the bacterial solutions of pBI121-GFP vector and pBI121-GUS vector on LB solid medium (Jefferson R A et al., 1987), culture at 37°C, pick white monoclonal colonies into LB liquid medium (added with 50 mg / mL Kana), and shake the bacterial solution until it becomes uniformly turbid; add pYES2 vector to Escherichia coli competent cells, through ice bath, heat shock at 42°C, add LB liquid medium (without antibiotics) after ice bath, culture at 37°C and 180 rpm for 45 min. Centrifuge to retain about 100 μL of supernatant, mix with the cells and spread them on LB medium plates (added with Amp), seal the mouth. Culture at 37°C for about 15 h. Pick monoclonal colonies with better growth into LB liquid medium (added with Amp), and culture by shaking at 37°C for about 14 h.

[0061] Extraction of target gene and expression vector plasmid: Extract the plasmids from the appropriate bacterial solutions of clone sequencing and vector-activated bacteria respectively. The specific operation is carried out according to the instructions of TIANGEN plasmid extraction kit. Then measure the concentration of the extracted plasmids using a ultra-micro spectrophotometer.

[0062] Double digestion of target gene and vector plasmid: Select the target gene with the highest concentration and pBI121-GFP vector plasmid, pBI121-GUS vector plasmid and pYES2 vector plasmid, and perform double digestion with Xba I and Sma I respectively, then place them in a metal bath at 37°C overnight. The double digestion system is shown in Table 3-1.

[0063] Table 3-1 Double digestion system of vector plasmid and Hg52609

[0064]

[0065] Gel recovery of target gene and vector and gel recovery verification: Detect the enzyme digestion products by 1% agarose gel electrophoresis. Cut the gel containing the target gene band and vector band, use a gel recovery kit to recover the target fragment and vector, and perform electrophoresis running gel verification.

[0066] Ligation of target gene and vector: Use T4 ligase to ligate the target gene with pBI121-GFP vector, pBI121-GUS vector and pYES2 vector. Add them to the PCR tube in the order of ddH2O, target fragment, vector, 10×T4 DNA ligase Buffer, centrifuge transiently and then place it in a PCR instrument, incubate at 65°C for 3 min, then add T4 DNA ligase after ice bath for several seconds, seal the mouth, and perform overnight ligation in a 16°C metal bath. The ligation system is shown in Table 3-2.

[0067] Table 3-2 Ligation System of Target Gene Fragment and Vector

[0068]

[0069] Transformation of Escherichia coli competent cells: Through the transformation of Escherichia coli competent cells, blue-white screening, and PCR detection, the specific steps are the same as 1.2.3. After successful sequencing of the bacterial liquid, plasmids were extracted and double digestion verification was performed using Xba I and Sma I. The specific steps are the same as 1.2.4.

[0070] After successful verification, transform Agrobacterium tumefaciens GV3101: In a laminar flow hood, add melted LB solid medium to Rif and Kana in sequence, mix well, and pour it into a culture dish. Add GV3101 competent cells and gene plasmids to a centrifuge tube, mix gently, and then perform ice bath, addition of liquid nitrogen, 37 °C water bath, and ice bath again in sequence. Add LB liquid medium (without adding Rif and Kana), and culture on a shaker. Centrifuge to remove the excess supernatant, and pipette the precipitate evenly. Pipette the bacterial liquid and spread it on the plate, dry the bacterial liquid, and culture it inverted at 28 °C for about 48 h. After single colonies grow out, select multiple monoclonal colonies and verify them using PCR. Select the positive monoclonal bacterial liquid that amplifies the target gene band, preserve the bacterial strain, and send it to a sequencing company for sequencing. Compare the sequencing results with the transcript sequence of the gene (with the help of DNAMAIN software).

[0071] The results showed that the target gene, pBI121-GFP expression vector, and pBI121-GUS expression vector were respectively digested with two restriction endonucleases, Xba I and Sma I, and double digestion verification was performed using 1% agarose gel ( Figure 4 ); The target gene was digested with two restriction endonucleases, EcoR I and Xba I. Detected by 1% agarose gel electrophoresis. The target gene and vector were respectively recovered using a DNA gel recovery kit (TIANgel Purification Kit), and gel recovery detection was performed using 1% agarose gel, showing that both the target gene and vector were successfully recovered ( Figure 5 、 Figure 11 A). The target gene and clone were ligated overnight using T4 ligase, and then transformed into Escherichia coli and cultured overnight at 37 °C until single colonies grew out. Select multiple monoclonal colonies and verify them using PCR. Select the positive monoclonal bacterial liquid that amplifies the target gene band and send it to a biological company for sequencing (Luo Jin et al., 2022).

[0072] Identification of positive recombinants: Randomly pick single colonies and add them to LB liquid medium (with Kana antibiotic added) and culture until the bacterial liquid becomes uniformly turbid, and perform colony PCR verification; ligate the target gene with the pYES2 vector using T4 ligase and transform Escherichia coli, pick monoclonal colonies and perform bacterial liquid PCR amplification. The results show that the colony PCR of the Hg52609 gene shows an amplified fragment of the expected size as the target gene ( Figure 6 AC, Figure 11 B). Then, use a kit (TIANGEN) to extract the recombinant plasmid from the bacterial liquid with the amplified target fragment by PCR and perform double digestion verification ( Figure 6 BD). From the above results, it can be concluded that the subcellular localization expression vector pBI121-GFP-Hg52609, the plant expression vector pBI121-GFP-Hg52609, and the yeast heterologous expression vector were successfully constructed ( Figure 7 , Figure 8 , Figure 12 ).

[0073] Example 3

[0074] This example provides the application of the salt tolerance gene Hg52609 in the roots of Halogeton glomeratus in participating in plant salt tolerance, including:

[0075] 1. Subcellular localization of pBI121-GFP-Hg52609 in Nicotiana benthamiana

[0076] Transiently transform tobacco with pBI121-GFP and pBI121-GFP-Hg52609, and observe the subcellular localization of the gene in tobacco cells by using a laser confocal microscope. It can be seen from the figure that the pBI121-GFP empty vector is expressed at various positions in tobacco cells, while pBI121-GFP-Hg52609 mainly shows green fluorescence in the cytoplasm, determining that the Hg52609 gene is mainly expressed in the cytoplasm ( Figure 7 B).

[0077] 2. Genetic transformation of Arabidopsis thaliana

[0078] Sow Arabidopsis thaliana seeds in a substrate of nutrient soil: vermiculite = 1:1 and culture until the flowering stage for standby. When Arabidopsis thaliana grows more inflorescences, remove the pods of the plants to be infected in advance, and select plants with strong growth for infection. One day before infection, place the Arabidopsis thaliana seedlings in a weak light environment. During infection, infect the inflorescences with the infiltration buffer for 1 min. After the infection is completed, wrap the plants with plastic wrap, place them in a cardboard box in the dark for 24 h, then remove the plastic wrap and place them in the culture room for cultivation ( Figure 9 ). After 2 days of dark culture (Dong Wanchun et al., 2015), uncover the covering and culture normally without using too strong light.

[0079] When the siliques of the transgenic Arabidopsis thaliana turn yellow or brown, the T0 generation seeds are gently cut and collected in centrifuge tubes in a timely manner, and antibiotic Kan screening is carried out on the 1 / 2MS medium containing Kana. The obtained T1 generation transgenic Hg52609 gene seeds and wild-type Arabidopsis thaliana seeds (Col-1) are sown into the culture medium respectively. After one month, a 200 mmol / L salt stress treatment is carried out. By observing the growth status of the seedlings, it is found that under the NaCl stress treatment, the growth rate of Arabidopsis thaliana plants is slow, the growth of wild-type plants is weak, and symptoms such as withering and yellowing appear 2-3 days earlier than that of transgenic plants. However, the transgenic Hg52609 gene plants can grow normally ( Figure 10 ). It shows that the Hg52609 gene can significantly improve the salt tolerance of Arabidopsis thaliana, and this gene can be applied to the cultivation of new salt-tolerant crop varieties.

[0080] 3. Transformation of vectors into yeast and analysis of yeast growth phenotypes

[0081] Taking the pYES2 empty plasmid as a control, the pYES2 and the plasmid of the target gene are transformed into AXT3 and CY162 yeast by the lithium acetate transformation method, and monoclonal colonies are selected through auxotrophic media. Positive identification is carried out by bacterial liquid PCR amplification and electrophoresis detection. The results show that for the transformation of the Hg52609 gene into AXT3 yeast, a band consistent with the size of the target gene is amplified in the bacterial liquid; ( Figure 13 A). For the transformation of the Hg52609 gene into CY162 yeast, the target band is amplified in the bacterial liquid ( Figure 13 B).

[0082] AXT3 is a yeast strain sensitive to Na+, without ATPase (ENA1-ENA4), so it is extremely sensitive to Na+ (Amtmann et al., 2001). When no Na is added to the AP medium with 1 mM KCl + , both transformants can grow. When 10, 30, 50 mM KCl are added, compared with the yeast strain transformed with the Hg52609 gene, the yeast strain transformed with the empty vector pYES2 grows better, and with the increase of the Na+ concentration, this contrast phenomenon becomes more obvious, indicating that the introduction of the Hg52609 gene increases the sensitivity of the yeast strain AXT3 to Na+, suggesting that the Hg52609 gene is involved in the + efflux of Na. ( Figure 14 A).

[0083] CY162 is a K + -deficient yeast strain, without the K + transporter proteins TRK1 and TRK2, and cannot grow under low potassium conditions (K + <7 mM) ( et al., 2002). In K+ When the concentration is 100 mM, both the yeast strain transformed with the empty vector pYES2 and the yeast strain transformed with the Hg52609 gene can grow. However, when K + < 7 mM (0, 0.2 mM, 1 mM), neither the yeast strain transformed with the empty vector pYES2 nor the yeast strain transformed with the Hg52609 gene can grow, indicating that this gene does not have the function of compensating for the K + deficient yeast strain CY162, that is, it does not mediate K + uptake in yeast ( Figure 14 B).

[0084] When the Na + concentration in the growth environment of the yeast strain increases to 100 mM, the K + content and Na + content in the AXT3-Hg52609 strain are significantly reduced compared with those at a Na + concentration of 10 mM ( Figure 14 C); when the K+ < 7 mM (0, 0.2 mM, 1 mM) in the growth environment of the yeast strain, neither the CY162-Hg52609 strain nor the yeast strain transformed with the empty vector pYES2 can grow ( Figure 14 D).

[0085] In summary, the salt tolerance gene Hg52609 in the roots of Halogeton glomeratus participates in the efflux of Na + without mediating K + uptake, and thus participates in the response to plant salt stress, thereby improving plant salt tolerance.

[0086] The above are only the specific embodiments of the present application. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present application, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present application.

[0087] References:

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[0089] [2] Munns R. Genes and salt tolerance: bringing them together [J]. Newphytologist, 2005, 167(3): 645-663.

[0090] [3] Zhou Zhenguo, Meng Yaxiong, Song Zhanshu, et al. Cloning and Analysis of the Unigene7547 Fragment of the Halogeton glomeratus Gene [J]. Molecular Plant Breeding, 2018, 16(22): 7281-7288.

[0091] [4] Wang Juncheng. Study on the Salt Compartmentalization Tolerance Mechanism of Halogeton glomeratus [J]. Lanzhou: Gansu Agricultural University, 2017.

[0092] Mahajan S, Tuteja N. Cold, Salinity and Drought Stresses: An Overview [J]. Arch Biochem Biophys, 2005, 444(2): 139-158.

[0093] [5] Luo Jin, Zhou Min, Li Ailing, et al. Cloning, Bioinformatics Analysis, Subcellular Localization and Expression Analysis of AhBI-1 in Peanut under Aluminum Stress [J]. Molecular Plant Breeding. 2022, 4(02): 1-25.

[0094] [6] Dong Wanchun, Fan Tingting, Yang Libo, et al. Construction of the Overexpression Vector of the Arabidopsis CDR6 Gene and Screening and Identification of Overexpressing Plants [J]. Journal of Anhui Agricultural Sciences, 2015, 43(13): 63-64.

[0095] [7] Amtmann A, Fischer M, Marsh E L, et al. The Wheat cDNA LCT1 Generates Hypersensitivity to Sodium in a Salt-sensitive Yeast Strain [J]. Plant Physiology, 2001, 126(3): 1061-1071.

[0096] [8] p, Eckelman B, Vaidyanathan R, et al. Altered Shoot / Root Na+ Distribution and Bifurcating Salt Sensitivity in Arabidopsis by Genetic Disruption of the Na+ Transporter AtHKT1 [J].

[0097] FEBS Letters, 2002, 531(2): 157-161.

Claims

1. The Hg52609 gene of Halogeton glomeratus, characterized in that The Hg52609 gene transcript sequence is as shown in Sequence Listing SEQ No.1, and the fragment of the Hg52609 gene that exerts the salt resistance function is as shown in Sequence Listing SEQ No.

2.

2. Application in promoting yeast cell Na efflux by overexpressing the Hg52609 gene of Halogeton glomeratus, characterized in that + The fragment of the Hg52609 gene that exerts the Na + efflux function is shown in Sequence Listing SEQ No.

2. ​ 3. Application of overexpressing the Hg52609 gene of Halogeton glomeratus to enhance the salt tolerance of Arabidopsis thaliana, characterized in that The fragment of the Hg52609 gene that exerts the function of enhancing the salt resistance of Arabidopsis thaliana is as shown in Sequence Listing SEQ No.2.

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