Use of potri.018g082500 gene in improving salt tolerance of plants

Abstract

The application discloses an application of a Potri.018G082500 gene in improving salt tolerance of plants, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the Potri.018G082500 gene is shown in SEQ ID NO. 1. An expression vector of the Potri.018G082500 gene is constructed, and the expression vector is used to transform Arabidopsis thaliana and 84K poplar. The results of examples show that under salt stress, the cell membrane of the Arabidopsis thaliana overexpressing the Potri.018G082500 gene has low damage degree, high stability, an effective antioxidant defense system, and enhanced synthesis capacity of osmotic adjustment substances; the leaf of the 84K poplar overexpressing the Potri.018G082500 gene is slightly damaged, the plant height, leaf number and biomass are higher, the photosynthetic system has high salt tolerance, the cell ion distribution can be balanced, water loss is reduced, the antioxidant enzyme activity is high, the stress resistance is significantly enhanced, and the salt tolerance of the plant is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to the application of the Potri.018G082500 gene in improving the salt tolerance of plants. Background Technology

[0002] Soil salinization is a global environmental stressor that poses a severe challenge to agricultural and forestry production worldwide. In high-salt environments, plants' ability to acquire water and nutrients is severely impaired, and their growth, development, and maintenance of ion homeostasis are also severely damaged, leading to stunted growth and development.

[0003] Poplar, as a typical fast-growing pioneer tree species, plays an irreplaceable and crucial role in ecological fields such as windbreak and sand fixation, soil remediation, and soil and water conservation due to its efficient biomass accumulation capacity and wide ecological adaptability. However, the increasingly serious problem of secondary soil salinization not only significantly weakens the productivity and timber quality of poplar plantations but also poses a serious threat to the sustainable functioning of their ecosystem services. To address this issue, exploring the molecular mechanisms of poplar salt tolerance and cultivating new poplar varieties with high salt resistance has become one of the key issues in current poplar breeding work.

[0004] AspAT (Aspartate aminotransferase) is located at the initiation point of the Asp metabolic pathway in organisms, playing a crucial role in gluconeogenesis and the tricarboxylic acid cycle. It is an important intermediate in amino acid biosynthesis and degradation, as well as a source of the carbon skeleton, and participates in regulating carbon and nitrogen balance and energy synthesis. It plays a vital role in plant growth, development, and stress response. Recent studies have revealed that AspAT possesses multiple biological functions in abiotic stress adaptation, promoting adaptive growth in plants by enhancing antioxidant defense systems and inducing metabolic remodeling. However, methods for improving plant tolerance to high salt stress using AspAT have not been reported in current technologies. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide the Potri.018G082500 gene (encoding the AspAT enzyme protein). Another technical problem this invention aims to solve is to provide the expressed protein of the Potri.018G082500 gene. A further technical problem this invention aims to solve is to provide the application of the Potri.018G082500 gene in regulating plant growth under salt stress, for the purpose of regulating plant growth under salt stress. Yet another technical problem this invention aims to solve is to provide the application of the Potri.018G082500 gene in improving plant salt tolerance, for the purpose of regulating plant stress resistance.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A Potri.018G082500 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The expressed protein of the Potri.018G082500 gene has the amino acid sequence shown in SEQ ID NO.2.

[0009] Vectors and recombinant bacteria containing the Potri.018G082500 gene.

[0010] Application of the Potri.018G082500 gene in promoting plant growth under salt stress, wherein the plant is 84K poplar or Arabidopsis thaliana, and the salt stress is 75-300mM NaCl stress.

[0011] Applications of the Potri.018G082500 gene in promoting improved salt tolerance in plants include:

[0012] 1) Construct an expression vector for the Potri.018G082500 gene;

[0013] 2) The expression vector of the constructed Potri.018G082500 gene was transformed into 84K poplar or Arabidopsis thaliana;

[0014] 3) Cultivate, screen and obtain transgenic 84K poplar or Arabidopsis plants with improved salt tolerance.

[0015] The Potri.018G082500 gene regulates relative water content and relative electrical conductivity, and / or chlorophyll content, and / or net photosynthetic rate, and / or stomatal conductance, and / or intercellular CO2 concentration, and / or transpiration rate, and / or chlorophyll fluorescence parameters, and / or MDA content and antioxidant enzyme activity, and / or H2O2 and O2 in leaves of 84K poplar under salt stress. 2- Application in the content of, and / or the stomatal morphology of leaves, and / or the content of inorganic ions in leaves, wherein the salt stress is 75-300 mM NaCl stress.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1) This invention constructs an expression vector for the Potri.018G082500 gene and transforms it into Arabidopsis thaliana. The results show that under 150 mM NaCl stress, the relative conductivity of Arabidopsis thaliana overexpressing Potri.018G082500 is significantly lower than that of the wild type, indicating that its cell membrane system is less damaged and its stability is stronger; the overexpressing lines also show lower levels of MDA (malondialdehyde), H2O2 (hydrogen peroxide), and O2.2- The superoxide anion content was significantly lower than that of the wild type, indicating that its antioxidant defense system was more effective and could reduce salt stress-induced oxidative stress damage. Under salt stress, the content of soluble sugar, soluble protein and proline (Pro) in the overexpressing plants was significantly higher than that in the wild type, indicating that its ability to synthesize osmotic regulators was enhanced, which helped maintain cell osmotic pressure balance.

[0018] 2) This invention constructs an expression vector for the Potri.018G082500 gene and transforms it into 84K poplar. The results show that under short-term stress (3 days) of 300mM NaCl, almost all the lower and middle leaves of the wild type withered, while the overexpressing plants only had slight edge wrinkling, and the number of remaining leaves was more than twice that of the wild type. Under long-term stress (24 days) of 75mM NaCl, the plant height, number of leaves, and biomass (fresh weight and dry weight) of the overexpressing plants were significantly higher than those of the wild type, with a particularly significant increase in root biomass. Under salt stress, the chlorophyll content (total chlorophyll, chlorophyll a / b) of the overexpressing plants was significantly higher than that of the wild type, and the decrease in photosynthetic parameters such as net photosynthetic rate, stomatal conductance, and transpiration rate was smaller. The increase in intercellular CO2 concentration was significantly lower than that of the wild type, indicating that its photosynthetic system is more tolerant to salt stress. The overexpressing plants can maintain higher K under salt stress. + Ca 2+ Mg 2+ Content and K + / Na + The ratio helps balance the distribution of ions within cells and reduce Na+. + Toxicity; Under salt stress, the stomatal aperture of overexpressing plants was significantly reduced, decreasing water loss; simultaneously, H2O2 (hydrogen peroxide) and O 2- The content of superoxide anion was significantly lower than that of wild type, and the activity of antioxidant enzymes (SOD, POD, CAT) was significantly increased, further enhancing the stress resistance. Attached Figure Description

[0019] Figure 1 Figures showing the screening and identification of overexpression lines (A shows the growth of WT and Arabidopsis thaliana T3 generation seeds overexpressing Potri.018G082500 on a medium containing kanamycin; B shows the results of RT-PCR screening of T3 generation overexpression homozygotes; C shows the relative expression level of the Potri.018G082500 gene in the overexpression homozygotes).

[0020] Figure 2 The growth status of transgenic Arabidopsis thaliana lines under stress with 150 mM NaCl solution (2 weeks) is shown in the figure (A is phenotype; B is relative water content; C is relative electrical conductivity).

[0021] Figure 3The graph shows the chlorophyll content and chlorophyll fluorescence parameters of transgenic Arabidopsis lines under 150mM NaCl solution stress (2 weeks). (A: total chlorophyll content; B: chlorophyll a content; C: chlorophyll b content; D: Fv / Fm; E: chlorophyll b content) F stands for ETR; G for qP; H for qN; I for NPQ.

[0022] Figure 4 MDA, H2O2, and O2 levels in transgenic Arabidopsis lines under 150 mM NaCl solution stress (2 weeks) 2- Content diagram (A represents MDA content; B represents H2O2 content; C represents O content) 2- content);

[0023] Figure 5 The graph shows the soluble sugar, soluble protein, and proline content of transgenic Arabidopsis thaliana lines under 150 mM NaCl solution stress (2 weeks) (A is soluble sugar content; B is soluble protein content; C is proline content).

[0024] Figure 6 The growth status of the transgenic 84K poplar line subjected to continuous stress with 300mM NaCl solution for 3 days is shown in the figure (A is the phenotypic change of the control (water treatment); B is the phenotypic change of the salt stress (300mM NaCl solution) treatment).

[0025] Figure 7 The growth status of transgenic 84K poplar lines subjected to continuous stress with 75mM NaCl solution for 24 days is shown in the figure (A is the phenotypic change of the control (water treatment); B is the phenotypic change of the salt stress (75mM NaCl solution) treatment; C is the phenotype of detached leaves; D is the phenotype of roots).

[0026] Figure 8 Growth indicators of the transgenic 84K poplar line subjected to continuous stress with 75mM NaCl solution for 24 days (A: plant height; B: total fresh weight; C: root fresh weight; D: number of leaves; E: total dry weight; F: root dry weight);

[0027] Figure 9 The relative moisture content and relative conductivity of Populus tomentosa seedlings overexpressing Potri.018G082500 at 84K were plotted in soil (A represents relative moisture content; B represents relative conductivity).

[0028] Figure 10 The chlorophyll content of Populus tomentosa seedlings overexpressing Potri.018G082500 at 84K is shown in the figure (A is the total chlorophyll content; B is the chlorophyll a content; C is the chlorophyll b content).

[0029] Figure 11The net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate of Populus tomentosa seedlings overexpressing Potri.018G082500 in soil were plotted (A: net photosynthetic rate; B: stomatal conductance; C: intercellular CO2 concentration; D: transpiration rate).

[0030] Figure 12 The chlorophyll fluorescence parameters of 84K poplar seedlings overexpressing Potri.018G082500 were plotted in soil (A represents Fv / Fm; B represents Fv / Fm). C stands for ETR; D stands for qP; E stands for qN; F stands for NPQ.

[0031] Figure 13 The graph shows the MDA content and antioxidant enzyme activity of Populus tomentosa seedlings overexpressing Potri.018G082500 84K in soil culture (A is MDA content; B is SOD enzyme activity; C is POD enzyme activity; D is CAT enzyme activity).

[0032] Figure 14 To overexpress H2O2 and O2 in the leaves of Populus tomentosa seedlings grown in soil at 84K. 2- Content diagram (A represents DAB and NBT staining; B represents H2O2 and O2 staining) 2- content);

[0033] Figure 15 The images show the stomatal morphology of leaves from Populus tomentosa overexpressing Potri.018G082500 84K (AB represents the stomatal aperture of tissue-cultured seedlings after treatment in stomatal opening buffer for 1 h; A is magnified 1000×, B is magnified 400×); CE represents the stomatal morphology of soil-cultured seedlings after treatment in stomatal opening buffer for 1 h; C is magnified 1000×, D is magnified 400×, E is magnified 100×; the scale bar in the images represents 10 μm).

[0034] Figure 16 The effect of overexpression of Potri.018G082500 on water loss rate and stomatal aperture of 84K poplar leaves under salt stress is shown in the figure (A is the water loss rate of detached leaves exposed to air for 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, and 12 h; B is the stomatal length; C is the stomatal width; and D is the stomatal area).

[0035] Figure 17 The graph shows the content of inorganic ions in the leaves of Populus tomentosa seedlings overexpressing Potri.018G082500 at 84K (A represents Na). + Content; B is K + Content; C is K + / Na + The ratio; D is Ca 2+ Content; E represents Mg 2+content). Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0037] Example 1

[0038] 1. Total RNA extraction and cDNA acquisition

[0039] Total RNA was extracted from leaves of *Populus tomentosa* using a plant RNA extraction kit (Tiangen). Specific experimental procedures were performed according to the kit's instructions. RNA concentration and purity were determined using a micro spectrophotometer. Reverse transcription of RNA and synthesis of cDNA were performed using an All-in-One First-Strand Synthesis MasterMix (with dsDNase) reverse transcription kit (Jiangsu Yugong) according to its instructions. The reaction mixture was prepared on ice, mixed, and incubated at 37°C for 2 min, 55°C for 15 min, and 85°C for 5 min.

[0040] 2. Cloning the target gene

[0041] Based on the Potri.018G082500 gene sequence (Gene ID: Potri.018G082500) from the Populus tomentosa genome database, gene-specific primers were designed, and recombinant arm gene sequences CTTCACTGTTGATACAT and CCTTGCTCACCATGGATCC were added to the 5' end of the primers, respectively. PrimeScan was used to analyze the sequence. Max DNAPolymerase's high-fidelity PCR amplification kit was used to amplify full-length cDNA. Primer sequences are shown below:

[0042] Potri.018G082500-F:

[0043] 5'-ATGGAGTCTTCTTCTGTGTTTG-3';

[0044] Potri.018G082500-R:

[0045] 5'-GCCAACACGGGTAACAG-3'.

[0046] The final sequencing yielded the nucleotide sequence of the Potri.018G082500 gene, as shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein, as shown in SEQ ID NO.2.

[0047] Example 2

[0048] 1. Constructing an expression carrier

[0049] The obtained cDNA pairs were double-digested with NdeI and BamHI endonucleases, and then ligated into a linearized pRI101(GFP) vector (preserved in our laboratory) using a seamless cloning kit (Beyotime). After transformation of E. coli by heat shock, single clones were picked, plasmids were extracted, and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification.

[0050] 2. Identification of transformed Arabidopsis thaliana and overexpression lines

[0051] 1) Activation culture of Agrobacterium GV3101

[0052] Glycerol-containing Agrobacterium tumefaciens GV3101 was streaked onto LB agar containing 50 mg / L kanamycin and 50 mg / L rifampin, and incubated at 28°C for 24-48 h. Single colonies were picked for PCR verification (primers P35 and EGFPN3). PCR-positive colonies were inoculated into LB liquid medium containing kanamycin and rifampin, and cultured overnight at 28°C and 200 rpm with shaking. 1 mL of the bacterial culture was added to 100 mL of LB liquid medium and incubated overnight until the culture turned orange-yellow and the OD value... 600 The OD value was between 0.8 and 1.6. The bacterial culture was centrifuged at 4000 rpm for 10 min, and the bacterial cells were collected. 50 mL of a 5% sucrose solution (containing 0.05% Silwet L-77) was prepared, and the collected bacterial cells were resuspended in the sucrose solution and mixed by pipetting. 600 The value is between 0.8 and 1.0.

[0053] 2) Transformation of Arabidopsis thaliana by inflorescence infection method

[0054] First, remove the fully opened inflorescences and siliques. Place the unopened inflorescences in the bacterial solution for 30 seconds to 2 minutes. After infection, wrap the plants in a black plastic bag and maintain humidity, then incubate in the dark for 24 hours. Remove the plastic bag and allow the plants to grow normally. Infect once a week until the siliques form and mature, then dry them and harvest the T0 generation seeds.

[0055] 3) Screening and identification of overexpression lines

[0056] First, sterilize the seeds by adding 1 mL of 70% ethanol to a centrifuge tube, vortexing for 30 seconds, discarding the supernatant, and repeating the sterilization process 3-4 times. Then, wash with 1 mL of sterile distilled water, repeating 3-5 times. Finally, sow the sterilized T0 generation seeds on 1 / 2 MS solid medium containing 50 mg / L Kan, treat at 4°C for 2-3 days, and then transfer to a light incubator for resistance screening (culture conditions: light intensity 100 mol / m²). 2 Under a photoperiod of 16 hours of light / 8 hours of darkness, at a temperature of 22℃ and a relative humidity of 70%, healthy positive plants were obtained and designated as the T1 generation. These were then transferred to soil for cultivation. After seed harvesting, Kan resistance selection was repeated until the T3 generation, during which all selected plants were green (homozygous). When the T3 generation plants reached 7-8 leaves in the soil, RT-PCR was performed to detect the expression of the Potri.018G082500 gene overexpressing Arabidopsis thaliana. Quantitative real-time PCR was used to determine the relative expression level of the Potri.018G082500 gene in some T3 generation overexpressing lines. The primer sequences used for obtaining and identifying transgenic Arabidopsis thaliana are shown below:

[0057] EGFPN3:

[0058] 5'-CTGGTCGAGCTGGACGGCGACG-3',

[0059] P35:

[0060] 5'-GTGCGTCATCCCTTACGTCAGT-3',

[0061] Potri.018G082500_OE_FW:

[0062] 5'-CTTCACTGTTGATACATATGGAGTCTTCTTCTGTGTTTG-3',

[0063] Potri.018G082500_OE_REV:

[0064] 5'-CCTTGCTCACCATGGATCCGCCAACACGGGTAACAG-3',

[0065] AtUbi6_qFW:

[0066] 5'-GGTCTCACCTACGTTTTACCAGA-3',

[0067] AtUbi6_qREV:

[0068] ATCCACAACATCCAAAAACAAC-3'.

[0069] The results are as follows Figure 1 As shown, homozygous T3 lines were obtained through Kan screening. The plants grew well and no phenotypic segregation occurred. Figure 1 (A); Preliminary verification was performed by RT-PCR on 17 T3 generation homozygous plants cultured in soil. Electrophoresis results showed that all 17 lines were overexpressing the gene, with 8 lines showing relatively bright bands (A). Figure 1 (B) ; Quantitative real-time PCR was performed on these 8 lines. Compared with the wild type, the expression levels of the Potri.018G082500 gene in all 8 overexpression lines were significantly higher than those in the wild-type plants, and their log... 10 (Relative expression level) is between 4.5 and 5.1 times. Figure 1 (C).

[0070] 3. Functional identification of transgenic Arabidopsis thaliana

[0071] 1) Phenotypic analysis

[0072] Seeds were evenly sown on the surface of soil culture medium (vermiculite: perlite: nutrient soil = 1:1:3) using a 1 mL pipette tip, and cultured for four weeks while maintaining humidity. Plants with uniform growth were randomly divided into a control group and a treatment group. The soil of the plants was irrigated with water and 150 mM NaCl solution, respectively. After two weeks of treatment, photographs were taken and the relative conductivity, relative water content, chlorophyll content, and chlorophyll fluorescence parameters were measured. Different lowercase letters in the figure indicate significant differences (P < 0.05).

[0073] The results are as follows Figure 2 As shown, after treatment with 150 mM NaCl solution, the transgenic plants exhibited significantly better growth than the wild type, with higher biomass, less leaf chlorosis, significantly higher relative water content (29.9%–37.9%), and significantly lower relative conductivity (17.5%–34.1%). These results indicate that the membrane system damage in plants overexpressing Potri.018G082500 was significantly less than that in wild-type plants.

[0074] The results are as follows Figure 3 As shown, under NaCl treatment, the total chlorophyll content, chlorophyll a content, and chlorophyll b content of plants overexpressing Potri.018G082500 were 26.8%–32.3%, 20.2%–22.7%, and 36.7%–49.4% higher than those of the wild type, respectively, and the differences were significant, indicating that overexpression of Potri.018G082500 alleviated the effect of salt stress on chlorophyll content; the chlorophyll fluorescence parameters Fv / Fm of plants overexpressing Potri.018G082500 were also significantly higher. ETR and qP were 16.8%–35.4%, 20.3%–36.5%, 26.8%–32.3%, and 58.7%–77.8% higher than those of the wild type, respectively, while qN and NPQ were lower. This indicates that the photosystem II of the plants overexpressing Potri.018G082500 is less affected by salt stress, and their need for chlorophyll fluorescence quenching mechanisms is less than that of the wild type, making them more adaptable to salt stress.

[0075] 2) Analysis of physiological and biochemical indicators

[0076] The levels of MDA, H2O2, and O2 in wild-type and overexpressing Potri.018G082500 Arabidopsis thaliana plants under salt stress were measured. 2- The content was tested, and the differences between different lowercase letters in the figure are significant (P<0.05).

[0077] The results are as follows Figure 4 As shown, under normal growth conditions, there was no significant difference between wild-type and *Arabidopsis thaliana* plants overexpressing *Potri.018G082500*. After salt stress treatment, the accumulation of MDA and H2O2, and O2 in *Potri.018G082500*-overexpressing plants were significantly increased. 2- The production of oxidative stress was significantly lower than that of wild type by 8.8%–12.1%, 5.2%–13.6%, and 55.1%–58.6%, respectively, indicating that the oxidative damage of the Potri.018G082500 overexpression line under salt stress was lower than that of wild type.

[0078] Soluble sugars, soluble proteins, and proline were measured in WT and Potri.018G082500 Arabidopsis thaliana plants under salt stress, respectively. Different lowercase letters in the figure indicate significant differences (P<0.05).

[0079] The results are as follows Figure 5 As shown, under no stress, the contents of soluble sugar, soluble protein, and Pro in all Arabidopsis strains were at low levels with no significant differences. Under salt stress, the overexpressing strains accumulated soluble sugar, soluble protein, and Pro at levels 16.6%–29.2%, 11.2%–12.8%, and 82.4%–102.5% higher than the wild type, respectively, indicating that overexpression of Potri.018G082500 can improve the synthesis of osmotic regulatory substances and better resist salt stress.

[0080] Example 3

[0081] 1. Obtaining genetically modified 84K poplar

[0082] Agrobacterium tumefaciens GV3101 glycerol bacillus was removed from a -80℃ freezer and spread onto agar plates. Single colonies were picked and inoculated into LB broth (containing Kan and Rif), and incubated overnight at 28℃ and 220 rpm in the dark. The bacterial cells were collected by centrifugation, resuspended in resuspended solution, and OD was adjusted. 600 The bacterial culture medium was 0.6-0.8, and the sample was placed at room temperature for 1 hour. A wound was gently made on the underside of the leaf with a scalpel, and the leaf was immersed in the resuspension with shaking for 10-15 minutes. Excess bacterial culture was wiped dry with filter paper, and the leaf and stem segments were spread evenly on a co-culture medium and incubated in the dark for 2 days. The leaf was then transferred to a selection medium and incubated in the dark for approximately 14 days. After callus growth, the callus was cut along the leaf edge and transferred to a selection medium, subcultured every 4-5 days. The callus tissue reached a length of 0.5 cm. 2 Afterward, the cells were transferred to differentiation medium and subcultured once every 14 days; when the adventitious shoots grew to about 1.5 cm, they were transferred to rooting medium for further culture.

[0083] 2. Functional identification of transgenic 84K poplar

[0084] 1) Phenotyping of soil-grown seedlings under short-term stress

[0085] Two-month-old wild-type and overexpression plants were selected for short-term (3-day) salt stress treatment with 300mM NaCl solution.

[0086] The results are as follows Figure 6 As shown, after 3 days of NaCl stress, almost all the middle and lower leaves of the wild-type plants withered, leaving only 5 to 6 leaves at the top, with severe drying and almost complete growth stagnation; while the edges of the middle and lower leaves of the overexpressing plants were only slightly wrinkled, and the top leaves remained normally extended, with more than twice the number of remaining leaves as the wild type, and the overall growth status was significantly better than that of the wild type.

[0087] 2) Phenotypic characteristics of soil-grown seedlings under long-term stress

[0088] One-month-old wild-type and transgenic plants grown in soil were subjected to long-term (24-day) salt stress treatment with 75 mM NaCl solution. Key morphological characteristics such as plant height, number of leaves, and fresh weight were measured. Different lowercase letters in the figure indicate significant differences (P < 0.05).

[0089] The results are as follows Figure 7 As shown, after 12 days of NaCl treatment, the growth rate of all plants was significantly inhibited, and the number of leaves decreased. This was more pronounced in the wild-type plants, where stem elongation was slowed and leaf drop was more severe. At 24 days of stress, the growth inhibition in the wild-type plants intensified, leaf damage became more severe, and necrotic spots appeared. The transgenic plants maintained a higher plant height and biomass, with only slight wrinkling of leaf edges, and their root biomass was higher than that of the wild-type.

[0090] The results are as follows Figure 8 As shown, after 12 and 24 days of salt stress, compared with the wild type, the plant height of the overexpressing plants increased by 6.4%–10.6% and 12.9%–19.2%, and the number of leaves increased by 29.8%–41.5% and 27.8%–44.4%, respectively, all at a significant level. After salt stress, the fresh weight of all lines of the overexpressing plants increased by 43.2%–56.9% compared with the wild type, and the dry weight was approximately 1.50 times that of the wild type; the fresh and dry weight of the roots increased by 69.9%–84.0% and 88.9%–118.1% respectively compared with the wild type; the fresh and dry weight of the aboveground parts were 41.4%–55.7% and 41.6%–56.4% higher than that of the wild type, respectively.

[0091] 3) Relative water content and relative electrical conductivity of Populus tomentosa seedlings overexpressing Potri.018G082500 at 84K K soil.

[0092] The relative water content and relative electrical conductivity of wild-type and overexpressing Potri.018G082500 84K poplar seedlings were measured after 3 days of salt stress. Different lowercase letters in the figure indicate significant differences (P<0.05).

[0093] The results are as follows Figure 9 As shown, under salt stress, the relative water content of wild-type plants decreased to 34.4%, while that of overexpressing plants increased by 57.1%–70.7% compared to wild-type plants; the relative electrical conductivity of wild-type plants increased to 76.4%, while that of overexpressing plants was 35.2%–49.2% lower than that of wild-type plants.

[0094] 4) Chlorophyll content of 84K poplar seedlings overexpressing Potri.018G082500

[0095] The chlorophyll content of wild-type and overexpressing Potri.018G082500 84K poplar seedlings in soil culture under salt stress was measured in different treatment groups after 3 days of salt stress. Different lowercase letters in the figure indicate significant differences (P<0.05).

[0096] The results are as follows Figure 10 As shown, compared to normal growth conditions, the total chlorophyll content of wild-type plants decreased by 36.7% under salt stress, while the overexpression plants showed chlorophyll content increases of 24.5%–34.2% in each line compared to the wild type. The chlorophyll a and chlorophyll b contents of the wild type decreased by 39.0% and 31.2%, respectively, while those of the overexpression plants decreased by only 4.5%–13.0% and 11.7%–22.4%, respectively.

[0097] 5) Net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate of 84K poplar seedlings overexpressing Potri.018G082500 in soil.

[0098] Net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate of wild-type and overexpressing Potri.018G082500 soil-cultured seedlings in different treatment groups were measured after 3 days of salt stress. Different lowercase letters in the figure indicate significant differences (P<0.05).

[0099] The results are as follows Figure 11 As shown, compared with normal growth conditions, the net photosynthetic rate, stomatal conductance, and transpiration rate of wild-type plants decreased by 82.1%, 95.7%, and 92.4%, respectively, under salt stress. In contrast, the photosynthetic parameters of the overexpressing plants were 85.5%–98.2%, 116.9%–323.3%, and 75.6%–173.9% higher, respectively, than those of the wild-type plants. The intercellular CO2 concentration in the leaves of wild-type plants increased significantly by 40.9%, while the intercellular CO2 concentration in the overexpressing lines increased only by 4.8%–9.9%.

[0100] 6) Chlorophyll fluorescence parameters of 84K poplar seedlings overexpressing Potri.018G082500

[0101] Chlorophyll fluorescence parameters of wild-type and overexpressing Potri.018G082500 soil-grown seedlings in different treatment groups were detected after 3 days of salt stress. Different lowercase letters in the figure indicate significant differences (P<0.05).

[0102] The results are as follows Figure 12 As shown, after 3 days of salt stress, the Fv / Fm ratio of each line of overexpressing plants was significantly reduced. ETR and qP were 43.5%–78.5%, 90.7%–110.8%, and 76.7%–105.6% higher than wild type, respectively, while qN and NPQ were lower than wild type.

[0103] 7) MDA content and antioxidant enzyme activity of 84K poplar seedlings overexpressing Potri.018G082500

[0104] The MDA content and the activities of antioxidant enzymes such as SOD, POD, and CAT in wild-type and overexpressing Potri.018G082500 soil-cultured seedlings were measured in different treatment groups after 3 days of salt stress. Different lowercase letters in the figure indicate significant differences (P<0.05).

[0105] The results are as follows Figure 13 As shown, under salt stress, the MDA content of transgenic plants was significantly lower than that of wild type by 38.7%–47.2%; the activities of SOD, POD, and CAT were higher than those of wild type by 10.3%–19.7%, 7.11%–15.4%, and 60.6%–149.9%, respectively.

[0106] 8) Overexpression of H2O2 and O2 in leaves of Populus tomentosa seedlings grown in soil at 84K 2- content

[0107] Leaves were stained with 3,3'-diaminobenzidine (DAB) and nitrocyanin tetrazolium (NBT), and H2O2 and O2 were measured. 2- The content is shown in the figure, with different lowercase letters indicating significant differences (P<0.05).

[0108] The results are as follows Figure 14 As shown, wild-type plants exhibited larger and darker DAB and NBT staining areas, indicating a higher concentration of reactive oxygen species (ROS) in their cells. In contrast, the overexpressing plants showed significantly reduced staining intensity, with H2O2 and O2 staining being more concentrated. 2- The content was reduced by 5.8%–11.4% and 18.6%–27.6% respectively compared to the wild type.

[0109] 9) Stomatal morphology of leaves of poplar seedlings overexpressing Potri.018G082500 84K soil culture

[0110] The stomatal morphology of leaves of wild-type and overexpressing Potri.018G082500 84K poplar tissue culture seedlings and soil-cultured seedlings was observed using an optical microscope after treatment with stomatal opening buffer of 0 mM and 150 mM NaCl for 1 h. The stomatal diameter, length, width and open area were measured. Different lowercase letters in the figure indicate significant differences (P<0.05).

[0111] The results are as follows Figure 15 and 16 As shown, under salt stress, the stomatal aperture of overexpression tissue culture seedlings and soil culture seedlings was significantly lower than that of wild type, and the stomatal length was shortened by 21.1% to 24.3%, the stomatal width was reduced by 39.3% to 41.2%, and the stomatal area was reduced by 45.2% to 51.0% compared with wild type.

[0112] 10) Inorganic ion content in leaves of poplar seedlings overexpressing Potri.018G082500 84K

[0113] Na+ levels in wild-type and 84K-overexpressing poplar leaves treated with 0mM, 50mM, and 100mM NaCl were measured. + K + Ca 2+ Mg 2+ The content of inorganic ions is shown in the figure, and the differences between different lowercase letters indicate significant differences (P<0.05).

[0114] The results are as follows Figure 17 As shown, under treatment with 50 mM and 100 mM NaCl, the K content of each overexpressing plant line decreased.+ Ca 2+ Mg 2+ Content and K + / Na + The levels were significantly higher than those of the wild type, indicating that overexpression of Potri.018G082500 plays a role in regulating ion homeostasis.

[0115] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Application of the Potri.018G082500 gene with the nucleotide sequence shown in SEQ ID NO.1 in promoting plant growth under salt stress, wherein the plant is 84K poplar or Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The salt stress is 75-300 mM NaCl stress.

3. Application of the Potri.018G082500 gene, whose nucleotide sequence is shown in SEQ ID NO.1, in promoting salt tolerance in 84K poplar or Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, include: 1) Construct an expression vector for the Potri.018G082500 gene; 2) The constructed expression vector of the Potri.018G082500 gene was transformed into 84K poplar or Arabidopsis thaliana; 3) Cultivate, screen and obtain transgenic 84K poplar or Arabidopsis plants with improved salt tolerance.

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