Application of Halophyte Hg35760 Gene and Its Salt-Tolerance

Abstract

The present invention relates to the gene cloning and functional application of the salt-tolerance gene Hg35760 in the root system of halophyte grass, and belongs to the field of plant bioengineering technology. The present invention cloned the salt-tolerance gene Hg35760 from the root system of halophyte grass for the first time. By cloning the salt-tolerance-related gene Hg35760 in the root system of halophyte grass, a subcellular localization expression vector was constructed to perform gene subcellular localization; an overexpression vector was constructed and stably genetically transformed into Arabidopsis thaliana for salt tolerance identification; a yeast heterologous expression vector was constructed to further verify the function of the Hg35760 gene, and it was found that the Hg35760 gene is involved in the regulation of high concentrations of Na + efflux and mediate K + The present invention provides the cloning and functional application of the salt-tolerant gene Hg35760 in the root system of halophyte, which will provide a reference for studying the salt tolerance mechanism of plants and improving their salt tolerance, and provide a valuable resource for exploring genes for salt-tolerant breeding.

Description

Technical Field

[0001] The invention belongs to the field of plant bioengineering and transgenic technology, and particularly relates to a salt-tolerant gene Hg35760 in the root system of a halophyte halophyte and its functional application. Background Art

[0002] Salinization refers to the process in which soluble salts enter the soil surface from the bottom of the soil and accumulate under the influence of natural or human factors. Soil salinization has a significant hindering effect on agricultural production, causing serious deterioration of the ecological environment and insufficient food supply. With the continuous development of my country's economy and the continuous improvement of people's requirements for agricultural production, the contradiction between increasing crop yields through irrigation and fertilization and further aggravation of soil salinization has become increasingly prominent. Among them, salt stress is one of the most important abiotic stresses to plants. In essence, the high external salt content changes the water potential and increases the osmotic pressure of plant cells or tissues, thereby affecting plants (Yang Jinsong et al., 2022).

[0003] For most plants that are not salt-tolerant, their growth will be inhibited when subjected to salt stress. Salt will have a certain impact on plant roots, stems, leaves, and plant height. When plants take in a lot of Na + 、Cl - When plants are under salt stress, Na+ accumulates in large quantities and converts Ca+ located on the cell membrane into 2+ Replacement, destruction of cell membrane structure, affecting the normal development of plants, and limiting the plant's ability to Ca 2+ 、Fe 2+ , K + and absorption of nutrient ions (Liu Jia et al., 2021).

[0004] Plants cope with salt stress through environmental, hormonal, and genetic regulation. Improving plant salt tolerance using traditional breeding techniques is not only time-consuming and labor-intensive, but also ineffective. Numerous studies have shown that improved plant salt tolerance is due to the overexpression of related genes in plants. Since the 1950s, with the development of molecular biology, the use of modern molecular biotechnology to identify salt-tolerant genes, improve plant salt tolerance, and thereby increase plant yield has become a hot topic of research. Several genes related to plant salt tolerance have been cloned and identified. As researchers continue to deepen their genomic research, more salt-tolerant genes will be identified in the future.

[0005] Halophytes, through their long history of biological evolution, have accumulated a wealth of salt- and alkali-tolerant genes, enabling them to thrive in saline-alkali soils. This has attracted considerable attention from researchers. Studying the salt-tolerance mechanisms of halophytes is crucial for improving the salt tolerance of crops (Xue Qiongqiong et al., 2021).

[0006] Halophytes appeared in people's vision before the 18th century. They are plants that can survive in high-salt environments. At present, researchers have discovered a large number of salt-tolerant genes from halophytes and verified their salt-tolerant functions by transforming model plants Arabidopsis thaliana and tobacco. The halophyte halophyte (Halogeton glomeratus) is an annual herbaceous plant of the Chenopodiaceae family, widely distributed in the arid areas of western Gansu. Halophytes survive in arid saline-alkali environments for a long time. The aboveground stems and leaves are highly fleshy and have strong water retention, windbreak and sand fixation, drought resistance and salt tolerance. They are of great significance in the discovery of salt-resistant genes, the study of salt and drought tolerance, and the provision of candidate genes for crop breeding (Zhou Zhenguo et al., 2018).

[0007] Problems with the existing technology: The existing technology has not reported on the function and application of the salt-tolerance gene Hg35760 in the root system of halophytes in regulating responses to salt stress. The present invention uses the salt-tolerance gene Hg35760 in the root system of halophytes as a starting point and clones the gene Hg35760. The function of the salt-tolerance gene Hg35760 in the root system of halophytes is further studied through methods such as vector construction, subcellular localization, Arabidopsis transformation, construction of yeast expression vectors, and yeast growth phenotypic analysis. The purpose is to further analyze the salt-tolerance mechanism of the root system of halophytes and provide salt-tolerance candidate genes for the later breeding of salt-tolerant varieties, thereby providing a technical basis for improving the salt tolerance of cultivated crops in the future. Summary of the Invention

[0008] The key technical problem to be solved by the present invention is to provide cloning of the salt-tolerance gene Hg35760 in the root system of halophyte and to verify its function in response to salt stress. To solve the above technical problem, the present invention adopts the following technical solution:

[0009] 1. The salt-tolerance gene sequence of the root system of halophyte. The gene transcript sequence is shown in the sequence listing SEQ No. 1, and the fragment of the Hg35760 gene that exerts salt-tolerance function is shown in the sequence listing SEQ No. 2.

[0010] 2. The gene cloning and vector construction method of the salt-tolerance gene Hg35760 in the root system of halophyte includes:

[0011] (1) Selection of plant materials and reagents; (2) RNA extraction and reverse transcription; (3) Primer design and gene cloning; (4) Construction of overexpression vectors, subcellular localization expression vectors, and yeast heterologous expression vectors.

[0012] 3. Functional verification of the salt-tolerance gene Hg35760 in the root system of halophytes, including:

[0013] (1) Arabidopsis genetic transformation and resistance screening of transgenic Arabidopsis; (2) Acquisition of transgenic Arabidopsis and growth phenotype analysis; (3) Yeast growth phenotype analysis.

[0014] 4. Functional verification methods for the salt-tolerance gene Hg35760 in the roots of halophytes, including: (1) subcellular localization of pBI121-GFP-Hg35760 in Nicotiana benthamiana, (2) genetic transformation and salt tolerance analysis of Arabidopsis thaliana, and (3) vector transformation of yeast and analysis of yeast growth phenotypes.

[0015] 5. The Hg35760 gene of halophyte is expressed in Na + For the application in efflux, the functional fragment of the Hg35760 gene of Halophyte is shown in SEQ No. 2 in the sequence table.

[0016] 6. Application of the Halophyte Hg35760 gene to enhance salt resistance in Arabidopsis thaliana. The functional fragment of the Halophyte Hg35760 gene is shown in SEQ No. 2 in the sequence listing.

[0017] 7. Application of the Halophyte Hg35760 gene for expression in tobacco cell nuclei, wherein the functional fragment of the Halophyte Hg35760 gene is shown in SEQ No. 2 in the sequence listing.

[0018] Beneficial effects: Halophyte has the characteristics of adapting to harsh environments such as drought, salinity, and high temperature, and is an excellent wild resource for obtaining salt-tolerant genes. Based on the transcriptomics and proteomics analysis of the Halophyte root system, the present invention selects the transcript sequence of the salt-induced expression gene, and successfully clones the Hg35760 gene by gene cloning. An overexpression vector is constructed to infect Arabidopsis for resistance screening and phenotypic analysis; a yeast heterologous expression vector is constructed and phenotypic analysis is performed.

[0019] The researchers found that under 200 mM NaCl treatment, Arabidopsis thaliana grew slowly, with wild-type plants showing symptoms such as withering and yellowing, while transgenic plants expressing the Hg35760 gene grew normally. This suggests that the Hg35760 gene can significantly improve salt tolerance in Arabidopsis thaliana and could be used in the development of new salt-tolerant crop varieties.

[0020] Found in Na + When Na is 0, 10, or 30 mM, the growth of the yeast strain transformed with the empty vector pYES2 is basically the same as that of the yeast strain transformed with the Hg35760 gene. + The growth of yeast strain transformed with Hg35760 gene was slightly better than that of yeast strain transformed with empty vector pYES2 after 10-fold dilution at 50mM, indicating that + Under these conditions, the introduction of Hg35760 gene reduced Na +Toxicity to yeast cells suggests that the Hg35760 gene may be involved in the Na + 's external discharge.

[0021] In K + When the K + When the concentration of Hg35760 gene was less than 7mM (0, 0.2mM, 1mM), the yeast strain transformed with the empty vector pYES2 did not grow, while the yeast strain transformed with the Hg35760 gene grew, indicating that the Hg35760 gene has the function of compensating for K + The function of the defective yeast strain CY162, which mediates K + absorption.

[0022] Hg35760 gene was transferred into Na + The sensitive yeast strain AXT3 can reduce high concentrations of Na + Toxicity to yeast cells indicates that the Hg35760 gene is involved in the high concentration of Na + efflux; Hg35760 gene was transferred into K + Defective yeast strain CY162, overexpression in yeast has the ability to compensate for K + The function of the defective yeast strain CY162, which mediates K + This indicates that the salt-tolerance gene Hg35760 in the root system of halophyte can participate in the response to salt stress and thus play a role in improving the salt tolerance of halophyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the agarose gel electrophoresis diagram of RNA from the roots of halophyte.

[0024] Figure 2 Agarose electrophoresis diagram of PCR amplified target gene; A: gel recovery verification for subcellular localization and overexpression vector; B: gel recovery verification for yeast heterologous expression.

[0025] Figure 3 Agarose gel electrophoresis diagram of bacterial solution PCR amplification of target fragments; A: bacterial solution PCR amplification of subcellular localization part and overexpression vector part; B: bacterial solution PCR amplification of yeast heterologous expression part.

[0026] Figure 4 Agarose gel electrophoresis diagram for double enzyme digestion verification of the construction of subcellular localization expression vector and overexpression vector; A: Hg35760 gene double enzyme digestion verification result; B: GFP double enzyme digestion verification result; C: GUS gel recovery verification result.

[0027] Figure 5Agarose gel electrophoresis diagram for gel recovery of subcellular localization expression vector; A: Hg35760 gene gel recovery verification; B: GFP gel recovery verification; C: GUS gel recovery verification results.

[0028] Figure 6 Agarose gel electrophoresis diagram for the construction of subcellular localization expression vector and identification of positive recombinants of overexpression vector; A: PCR verification of Hg35760 gene construction in pBI121-GFP bacterial solution; B: Double enzyme digestion verification of GFP recombinant plasmid; C: PCR verification of Hg35760 gene construction in pBI121-GUS bacterial solution; D: Double enzyme digestion verification of GUS recombinant plasmid.

[0029] Figure 7 These are subcellular localization-related diagrams; A: schematic diagram of subcellular vectors; B: subcellular localization diagram of the salt-tolerance gene Hg35760 in the roots of halophyte.

[0030] Figure 8 Schematic diagram of the overexpression vector.

[0031] Figure 9 Flowchart of Arabidopsis infection.

[0032] Figure 10 The phenotypes of Hg35760-transfected Arabidopsis and wild-type Arabidopsis after 15 days of 200 mM NaCl stress.

[0033] Figure 11 Agarose gel electrophoresis verification diagram for constructing yeast heterologous expression vector; A: Gene gel recovery verification diagram; B: Colony PCR verification diagram of pYES2-Genes recombinant expression vector; C: Double enzyme digestion verification of recombinant plasmid

[0034] Figure 12 Schematic diagram of yeast heterologous expression vector.

[0035] Figure 13 Agarose gel electrophoresis verification diagram of yeast transformation by the vector; A: PCR verification diagram of AXT3 yeast colony transformation by Hg35760 gene; B: PCR verification diagram of CY162 yeast colony transformation by Hg35760 gene.

[0036] Figure 14 The overexpression of Hg35760 gene has an effect on Na + , K + Absorption characteristics of the yeast strain; A: expression of Hg35760 gene and pYES2 empty vector in mutant yeast AXT3; B: expression of Hg35760 gene and pYES2 empty vector in mutant yeast CY162; C: Na accumulated in yeast strain + Content; D: K accumulated in yeast strain+ content.

[0037] Figure 15 Schematic diagram of the structure of the Hg35760 gene and its effective fragments. The effective fragments are in bold. Specific implementation methods

[0038] The methods and apparatus used in the following embodiments of the present invention are conventional methods and apparatus unless otherwise specified; the equipment and reagents used are conventional equipment and reagents purchased from a reagent company. In order to make the purpose, technical solutions and advantages of the present invention clearer, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments. Examples of these preferred implementations are illustrated in the specific embodiments. It should also be noted that in order to avoid obscuring the technical solutions of the present invention due to unnecessary details, only technical solutions and / or processing steps closely related to the solutions of the present invention are shown in the embodiments, and other details that are not very relevant are omitted.

[0039] Example 1

[0040] This embodiment provides the halophyte Hg35760 gene, the transcript sequence of the Hg35760 gene is shown in the sequence table SEQ No. 1, and the fragment of the Hg35760 gene that exerts salt resistance is shown in the sequence table SEQ No. 2 ( Figure 15 ).

[0041] Example 2

[0042] This embodiment provides a method for cloning the salt-tolerance gene Hg35760 in the root system of halophyte and constructing a vector, comprising:

[0043] 1. Plant material and reagent selection

[0044] The halophyte and wild-type Arabidopsis thaliana used in this study were both maintained in our laboratory and cultivated in an artificial climate chamber. The total RNA extraction kit (RNA Plant Plus Reagent), reverse transcription kit, rapid plasmid miniprep kit, and DNA gel recovery kit used in this study were all purchased from Tiangen Biochemical Technology Co., Ltd. Primer synthesis and sample sequencing were performed by Sangon (Shanghai) Bioengineering Co., Ltd.

[0045] 2. RNA Extraction and Reverse Transcription

[0046] Halophyte seeds were cultured in sterilized medium (sand: vermiculite = 1:1) for two weeks and then the roots of the halophyte were collected. RNA was extracted using the Tiangen Plant Total RNA Extraction Kit and then reverse transcription was performed using the Tiangen PrimeScript Reverse Transcription Kit. TMUse RTreagentKit with GDNA Eraser to reverse transcribe into cDNA, which will be used as a cloning template. Wear gloves and operate on ice throughout the process. For detailed operation procedures, refer to the instruction manual.

[0047] The results showed that: RNA was extracted from the roots of halophyte and OD was detected by ultra-micro UV spectrophotometer. 260 and OD 280 The purity and content of total RNA were determined by calculating their ratio and developing OD 260 / OD 280 In the range of 1.8 to 2.0, it indicates that the extracted RNA has not been significantly degraded and has good purity. Using 1% agarose gel, 120 V electrophoresis for 20 min, the test results show that the RNA quality is good and can be used for subsequent reverse transcription and functional verification experiments ( Figure 1 ).

[0048] 3. Primer Design and Gene Cloning

[0049] Primers were designed using the NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) using the transcript sequence of the Hg35760 gene. Cloning primers, subcellular localization expression primers, and overexpression vector primers were designed. Restriction sites and protective bases were added based on the expression vector to be used. The temperature was set according to the Tm value of the synthesized primers for gene amplification. The results were then analyzed by 1% agarose gel electrophoresis and visualized using a gel electrophoresis imaging instrument. The primer design, amplification system, and reaction procedure are shown in the table below.

[0050] Table 1 Cloning primers, subcellular localization expression vector primers and overexpression vector primers

[0051]

[0052] Table 2 Yeast heterologous expression vector primers

[0053]

[0054] Use Premix Taq premix solution to PCR amplify the target gene. The PCR amplification system is:

[0055]

[0056] The PCR reaction procedure for the Hg35760 gene is as follows (for subsequent construction of transient expression vectors and overexpression vectors):

[0057]

[0058]

[0059] The PCR reaction procedure for the Hg35760 gene is as follows (for subsequent construction of yeast heterologous expression vector):

[0060]

[0061] On a UV transilluminator, the agarose gel containing the target fragment was cut out with a blade and recovered using a DNA gel recovery kit (TIANgel Purification Kit). The target gene fragment was connected to the pMD19-T vector and transformed into the competent E. coli Trans1-T1 for blue-white screening and PCR detection. The obtained PCR product was tested for the positive strain by agarose gel electrophoresis, and 3 bacterial solutions with positive PCR test results were selected and sent to the company for sequencing. The positive bacterial solution with correct sequencing results was added to 15mL LB+Amp liquid culture medium on a clean bench, and expanded and cultured on a constant temperature shaker at 37°C and 200-300r. Finally, a bacterial culture was made and the positive clone plasmid was extracted using the Tiangen Rapid Plasmid Mini Extraction Kit. For specific steps, please refer to the kit instructions.

[0062] The results showed that: using the cDNA of the root system of halophyte as a template, the target gene was amplified by PCR using cloning primers, and the amplified product was electrophoresed on 1% agarose gel at 120V for 20min ( Figure 2 After recovering the amplified product using the DNA gel purification kit (TIAN gel Purification Kit), add the gel recovery product, Solution I, and pMD19-T vector to the PCR tube in sequence. Use a flash centrifuge to mix them thoroughly, then remove them and place them in a metal bath (16°C) for ligation for 12-16 hours. After ligation, transform the competent E. coli cells and pour 20-25 mL of LB medium supplemented with Amp into the culture dish and blow dry. Prepare a mixture of competent cells (Trans1-T1) with X-gal (20 mg / mL) and IPTG (24 mg / mL) at a volume ratio of X-gal:IPTG = 4:1. Pipette the mixture onto a solid culture dish, spread it evenly with a spreader, and let it dry. Pipette the shaken bacterial solution and quickly spread it on the plate. After drying, seal it. Place it in the dark at 37°C with the front side facing up for 1 hour, then invert and culture until single white colonies grow for blue-white screening. The white spots were picked up and placed in 5 ml of LB liquid culture medium containing ampicillin (Amp), and cultured at 37°C and 200 rpm under light-shielding conditions until the tube became turbid. Then the bacterial solution was tested by PCR ( Figure 3 ).

[0063] 1. Construction of subcellular localization expression vectors, overexpression vectors and yeast heterologous expression vectors

[0064] Vector Activation: Under sterile conditions, activate the pBI121-GFP and pBI121-GUS vectors on LB solid medium. Culture at 37°C, select a single white colony, and transfer it to LB liquid medium (supplemented with 50 mg / mL Kana). Shake the culture until uniformly turbid. Add the pYES2 vector to the competent E. coli cells, incubate them on ice, heat shock them at 42°C, and then incubate them on ice again. Then, transfer them to LB liquid medium (without antibiotics) and incubate them at 37°C at 180 rpm for 45 minutes. Centrifuge and retain approximately 100 μL of the supernatant to mix the cells. Plate the cells onto LB medium plates (supplemented with Amino Acids), seal the plates, and incubate at 37°C for approximately 15 hours. Select a single colony with good growth and transfer it to LB liquid medium (supplemented with Amino Acids) and incubate them at 37°C with shaking for approximately 14 hours.

[0065] Extraction of target gene and expression vector plasmid: Extract plasmids from bacterial cultures suitable for sequencing and vector activation, respectively. Follow the instructions for the TIANGEN Plasmid Extraction Kit. Measure the concentration of the extracted plasmid using an ultramicrospectrophotometer.

[0066] Double enzyme 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 use Xba I and Sma I to perform double enzyme digestion respectively, then place in a metal bath at 37°C overnight. The double enzyme digestion system is shown in Table 3-1.

[0067] Table 3-1 Vector plasmid and Hg35760 double enzyme digestion system

[0068]

[0069] Gel recovery and verification of target gene and vector: Analyze the digestion products by electrophoresis on a 1% agarose gel. Cut out the gel containing the target gene band and vector band, and use a gel recovery kit to recover the target fragment and vector. Verify the gel run by electrophoresis.

[0070] Ligation of the target gene and vector: Use T4 ligase to ligate the target gene with the pBI121-GFP vector, pBI121-GUS vector, and pYES2 vector. Add ddH2O, target fragment, vector, and 10× T4 DNA ligase buffer to a PCR tube in that order. Centrifuge briefly, place in a PCR instrument, and incubate at 65°C for 3 minutes. Place on ice for a few seconds, then add T4 DNA ligase. Seal the tube and incubate overnight in a 16°C metal bath. The ligation system is shown in Table 3-2.

[0071] Table 3-2 Target gene fragment and vector connection system

[0072]

[0073] Transformation into competent E. coli cells: Follow the same steps as in 1.2.3 for transformation into competent E. coli cells, screening with blue-white plate plasmids, and PCR detection. After successful sequencing of the bacterial solution, extract the plasmid and verify it by double digestion with Xba I and Sma I, following the same steps as in 1.2.4.

[0074] After successful verification, transform Agrobacterium GV3101: In an ultra-clean workbench, add Rif and Kana to the melted LB solid culture medium, mix well, and pour into a culture dish. Add GV3101 competent cells and gene plasmids to the centrifuge tube, mix slowly, and then place in an ice bath, liquid nitrogen, 37°C water bath, and ice bath again. Add LB liquid culture medium (without adding Rif and Kana), and culture on a shaker at 28°C and 220rpm for 2 to 3 hours. Centrifuge to remove excess supernatant and pipette the precipitate evenly. Pipette the bacterial solution onto the plate, blow dry the bacterial solution, and culture it upside down at 28°C for about 48 hours. After single colonies grow, select multiple single clones and verify them using PCR. Select the positive monoclonal bacterial solution that amplifies the target gene band and save the strain, and send it to a sequencing company for sequencing. Compare the sequencing results with the gene transcript sequence (with the help of DNAMAIN software).

[0075] The results showed that the target gene, pBI121-GFP expression vector and pBI121-GUS expression vector were cut by two restriction endonucleases, Xba I and Sma I, respectively, and double enzyme digestion was verified by 1% agarose gel. Figure 4 ); EcoR I and Xba I restriction endonucleases were used to cut the target gene. The target gene and vector were recovered separately using a DNA gel purification kit (TIANgel Purification Kit), and gel recovery detection was performed on a 1% agarose gel, showing that both the target gene and the vector were successfully recovered ( Figure 5 、 Figure 11 A) Use T4 ligase to ligate the target gene and clone overnight. Then, transform E. coli and culture at 37°C overnight until single colonies form. Select multiple single colonies and verify them using PCR. Select positive single colonies that amplify the target gene band and send them to a biotechnology company for sequencing.

[0076] Identification of positive recombinants: Randomly pick a single colony and add it to LB liquid medium (with Kana antibiotics) and culture until the bacterial solution is uniformly turbid, and perform colony PCR verification; connect the target gene to the pYES2 vector using T4 ligase and transform E. coli, pick a single colony and perform bacterial PCR amplification. The results show that the Hg35760 gene colony PCR shows that a fragment with the expected size of the target gene is amplified ( Figure 6 AC, Figure 11 B). Then, the bacterial solution from which the target fragment was amplified by PCR was used to extract the recombinant plasmid using a kit (TIANGEN), and the recombinant plasmid was double-enzyme digested for verification ( Figure 6 BD, Figure 11 C), from the above results, it can be concluded that the subcellular localization expression vector pBI121-GFP-Hg35760, the plant expression vector pBI121-GFP-Hg35760 and the yeast heterologous expression vector were successfully constructed ( Figure 7 A. Figure 8 、 Figure 12 ).

[0077] Example 3

[0078] This embodiment provides the application of the salt-tolerance gene Hg35760 in the root system of halophytes in participating in plant salt tolerance, including:

[0079] 1. Subcellular Localization of pBI121-GFP-Gene in Nicotiana benthamiana

[0080] Tobacco was transiently transformed with pBI121-GFP and pBI121-GFP-Hg35760, and the subcellular localization of the genes in tobacco cells was observed using a laser confocal microscope (Guo J et al., 2022). It was found that the empty pBI121-GFP vector was expressed in all locations of tobacco cells, while pBI121-GFP-Hg35760 showed green fluorescence mainly in the cytoplasm, confirming that the Hg35760 gene was mainly expressed in the cytoplasm ( Figure 7 B).

[0081] 2. Arabidopsis genetic transformation and phenotypic analysis of transgenic Arabidopsis

[0082] Vermiculite, nutrient soil, and perlite are mixed evenly in a volume ratio of 9:3:1 and divided into flower pots. After the soil has absorbed moisture, use tweezers to evenly spot the Arabidopsis seeds on the surface of the soil layer, then seal the pot with plastic wrap, and then place it in a light culture room for cultivation. When the Arabidopsis germinates and grows to 4 leaves, the film can be removed. The conditions of the light culture room are a culture temperature of 25°C, 16 hours of light, and 8 hours of darkness (Koh LD et al., 2015). When Arabidopsis grows more inflorescences, cut off the grown siliques one day in advance, select strong plants for infection, and place the Arabidopsis seedlings in a low-light environment the day before infection. During infection, the Arabidopsis plants are infected in the infection solution in the plastic cup for 1 minute, and the excess infection solution is absorbed by absorbent paper. After the infection, the plants are wrapped with sealing film. The infection process is as follows: Figure 9 After culturing in the dark for 2 days, uncover the cover and culture normally without using too strong light.

[0083] When the siliques of the transgenic Arabidopsis thaliana turned yellow or brown, the T0 generation seeds were gently cut and collected in a centrifuge tube for antibiotic Kan screening. The obtained T1 transgenic Hg35760 seeds and wild-type Arabidopsis seeds (Col-1) were sown in the culture medium respectively. After one month, they were subjected to 200mmol / L salt stress. The growth of the seedlings was observed and it was found that under 200mmol / L NaCl treatment, the plants grew slowly. The wild-type plants grew weakly and turned yellow and died 4 to 5 days earlier than the transgenic Arabidopsis thaliana. The transgenic Hg35760 plants could tolerate salt stress ( Figure 10 ). This indicates that the Hg35760 gene can significantly improve the salt tolerance of Arabidopsis thaliana and can be used in the breeding of new salt-tolerant crop varieties.

[0084] 3. Transformation of Yeast with Vectors and Analysis of Yeast Growth Phenotype

[0085] Using the pYES2 empty plasmid as a control, pYES2 and the target gene plasmid were transformed into AXT3 and CY162 yeast using the lithium acetate transformation method. Single clones were selected using nutrient-deficient culture medium, and positive identification was performed using bacterial liquid PCR amplification and electrophoresis detection. The results showed that when the Hg35760 gene was transformed into AXT3 yeast, a band of the target gene size was amplified from the bacterial liquid; ( Figure 13 A). The target band was amplified from the bacterial solution of CY162 yeast transformed with Hg35760 gene ( Figure 13 B).

[0086] The AXT3K strain lacks the endogenous tonoplast NHX1 protein and Na + The efflux proteins NHA1 and ENA1-4 lose the ability to efflux Na+, so the Na + Particularly sensitive (Amtmann et al., 2001). + When Na is 0, 10, or 30 mM, the growth of the yeast strain transformed with the empty vector pYES2 is basically the same as that of the yeast strain transformed with the Hg35760 gene. + The growth of yeast strain transformed with Hg35760 gene was slightly better than that of yeast strain transformed with empty vector pYES2 after 10-fold dilution at 50mM, indicating that + Under these conditions, the introduction of Hg35760 gene reduced Na + Toxicity to yeast cells indicates that Hg35760 gene is involved in the regulation of Na + The efflux ( Figure 14 A).

[0087] CY162 is a K+ deficient yeast strain that lacks K + Efflux proteins TRK1, TRK2 ( et al., 2002), in K + When the K + When the concentration of Hg35760 gene was less than 7mM (0, 0.2mM, 1mM), the yeast strain transformed with the empty vector pYES2 did not grow, while the yeast strain transformed with the Hg35760 gene grew, indicating that the gene has the function of compensating for K + The function of the defective yeast strain CY162, which mediates K + Absorption ( Figure 14 B).

[0088] When yeast strains are placed in a growth environment with Na + When the concentration increased to 100 mM, the K + Content, Na + Content and 10mM concentration of Na + Compared with the Figure 14 C); When the yeast strain is placed in the growth environment K + When the concentration increased, K + The content of Na + Content reduction ( Figure 14 D).

[0089] In summary, the salt tolerance gene Hg35760 in the root system of halophytes participates in the + efflux and mediate K + absorption, and then participate in the response to plant salt stress, thereby improving plant salt tolerance.

[0090] The above is only a specific implementation method of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

[0091] References:

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Claims

1. Halophyte Hg35760 gene, characterized in that The gene transcript sequence is shown in SEQ No. 1 in the sequence listing, and the fragment of the Hg35760 gene that exerts salt resistance function is shown in SEQ No. 2 in the sequence listing.

2. Overexpression of the halophyte Hg35760 gene in yeast cells promotes Na + The application of efflux is characterized by The transcript sequence of the halophyte Hg35760 gene is shown in the sequence table SEQ No.

1.

3. Application of overexpressing the halophyte Hg35760 gene in Arabidopsis thaliana to enhance salt resistance, characterized in that The transcript sequence of the halophyte Hg35760 gene is shown in the sequence table SEQ No. 1.

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