Application of PdbHLH57 gene in improving plant salt tolerance

Abstract

The application provides a method for improving salt tolerance of plants PdbHLH57 The application relates to the field of plant genetic engineering technology. PdbHLH57 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2. PdbHLH57 The application clones a gene from Populus nigra var. 'Zhongshancaiyun' PdbHLH57 The transgenic P. simonii is obtained through a Populus genetic transformation system mediated by Agrobacterium. PdbHLH57 The transgenic P. simonii has significantly improved salt tolerance, and provides a gene resource for cultivating salt-tolerant plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and particularly relates to... PdbHLH57 Application of genes in improving plant salt tolerance. Background Technology

[0002] With climate change and the expansion of irrigated agriculture, the global area of ​​salinized soil has exceeded 1 billion hectares, resulting in limited forest growth and hindered ecological restoration. (Poplar trees) Populus spp. As a fast-growing tree species used for afforestation and economic purposes, its root system is highly sensitive to salt ions. When soil Na+... + When the concentration is >100 mM, the biomass decreases by an average of 40%, which seriously restricts its promotion and planting in saline-alkali areas.

[0003] The bHLH (basic Helix-Loop-Helix) family is one of the largest classes of transcription factors in plants. The poplar genome identified 185 bHLH genes, divided into 18 subgroups, among which… PdbHLH57 Belonging to subgroup III(f), it may be involved in anthocyanin biosynthesis. To date, no studies have revealed the involvement of genes in the poplar salt stress response, and its downstream target gene network and physiological regulatory mechanisms remain completely unknown. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide PdbHLH57 Application of genes in improving plant salt tolerance.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides overexpression PdbHLH57 The application of genes in improving plant salt tolerance, the PdbHLH57 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Preferably, the PdbHLH57 The amino acid sequence of the gene is shown in SEQ ID NO.2.

[0008] Preferably, the improvement in plant salt tolerance can increase the plant's tolerance to NaCl concentration up to 300 mM.

[0009] Preferably, the plant includes poplar.

[0010] Overexpression PdbHLH57 Application of genes in enhancing the expression of salt tolerance genes in plants, wherein the salt tolerance genes include PdSOD1 , PdPOD1 , PdABA2 , PdGA1 , PdHRG1 , PdP5CS1 , PdWRKY77 and / or PdSOS1 .

[0011] Overexpression PdbHLH57 Application of genes in improving POD activity, SOD activity and / or proline content in plants in saline-alkali land.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This invention discloses PdbHLH57 The application of genes in improving plant salt tolerance involves the field of plant genetic engineering technology. PdbHLH57 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO. 2. This invention cloned from the black poplar variety "Zhongshan Caiyun". PdbHLH57 Overexpression was obtained through an Agrobacterium-mediated poplar genetic transformation system. PdbHLH57 Transgenic poplar, through salt tolerance evaluation, was found to have overexpression... PdbHLH57 The transgenic poplar showed significantly improved salt tolerance, providing genetic resources for cultivating salt-tolerant plants. Attached Figure Description

[0014] Figure 1 For the identification of genetically modified leaves;

[0015] Figure 2 Transformation in the case of separation PdbHLH57 Salt stress tolerance analysis of Populus tomentosa plants;

[0016] Figure 3 For the transfer PdbHLH57 Salt stress tolerance analysis of *Populus tomentosa* plants; WT: wild type; OE-1, OE-3 and OE-8: transgenic lines; where a is the front view and top view; b is the plant height and fresh weight;

[0017] Figure 4 Physiological indicators of transgenic poplar under salt stress; POD: peroxidase; SOD: superoxide dismutase; MDA: malondialdehyde; REC: relative conductivity; H2O2: hydrogen peroxide; Proline: proline;

[0018] Figure 5 Transformation under salt stress PdbHLH57 DAB staining of gene-modified Populus tomentosa and wild-type; WT: wild-type; OE-1, OE-3 and OE-8: transgenic lines;

[0019] Figure 6 Transformation under salt stress PdbHLH57NBT staining of gene-modified Populus tomentosa and wild-type; WT: wild-type; OE-1, OE-3 and OE-8: transgenic lines;

[0020] Figure 7 Genes related to stress resistance in wild-type and transgenic PdbHLH57 Relative expression levels in *Populus alba* gene; Detailed Implementation

[0021] This invention provides overexpression PdbHLH57 The application of genes in improving plant salt tolerance, the PdbHLH57 The nucleotide sequence of the gene is shown in SEQ ID NO.1:

[0022] SEQ ID NO.1:

[0023]

[0024] The PdbHLH57 The amino acid sequence of the gene is shown in SEQ ID NO.2:

[0025] SEQ ID NO.2:

[0026] .

[0027] In this invention, the improvement of plant salt tolerance can increase the plant's tolerance to NaCl concentration up to 300 mM.

[0028] In this invention, the plant preferably includes poplar.

[0029] Overexpression PdbHLH57 Application of genes in enhancing the expression of salt tolerance genes in plants, wherein the salt tolerance genes include PdSOD1 , PdPOD1 , PdABA2 , PdGA1 , PdHRG1, PdP5CS1 , PdWRKY77 and / or PdSOS1 .

[0030] Overexpression PdbHLH57 Application of genes in improving POD activity, SOD activity and / or proline content in plants in saline-alkali land.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Material Specifications

[0033] The poplar is a new variety of colorful-leaved poplar, "Zhongshan Caiyun", which is planted in the Jiangsu Provincial Institute of Botany, Chinese Academy of Sciences.

[0034] RNApreppure plant total RNA extraction kit was purchased from TIANGEN Biotech.

[0035] The PCR kit was purchased from Novizan Biotechnology (Beijing) Co., Ltd.

[0036] Example 1: Preparation and Identification of Transgenic Plants

[0037] (1.1) Total RNA extraction and cDNA acquisition

[0038] Total RNA was extracted from the leaves of *Cymbidium goeringii* using the RNApreppure plant total RNA extraction kit. Using the extracted total RNA as a template, reverse transcription was performed using the FastKing one-step method for first-strand genomic cDNA synthesis premixed reagent to obtain cDNA.

[0039] (1.2) Target gene amplification

[0040] Using cDNA as a template, PCR amplification of the target gene was performed. The forward primer was PdbHLH57-F, the sequence of which is shown in SEQ ID NO.3:

[0041] SEQ ID NO.3: 5'-ATGGCTACTAAGCTCCACAA-3';

[0042] The reverse primer PdbHLH57-R, the sequence of which is shown in SEQ ID NO.4:

[0043] SEQ ID NO. 4: 5'-TCAACATTTCCCAGCAACTC-3'.

[0044] The PCR reaction system consisted of: 1 µL cDNA template, 0.8 µL each of forward and reverse primers (10 µM), 10 µL buffer, 0.4 µL dNTPs, 0.4 µL novizan high-fidelity enzyme, and 6.6 µL ddH2O.

[0045] The PCR reaction conditions were: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 35 cycles; final extension at 72℃ for 10 min.

[0046] The obtained amplification products were subjected to agarose gel electrophoresis, and then the gel was extracted using a gel extraction kit. After the concentration of the recovered products was determined, they were stored at -20℃, or directly subjected to enzyme digestion, ligation and other reactions.

[0047] The final sequencing yielded the nucleotide sequence of the PdbHLH57 gene as shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein as shown in SEQ ID NO.2.

[0048] (1.3) Constructing an overexpression vector for the PdbHLH57 gene

[0049] The pCAMBIA2300 empty vector plasmid and the PCR product of the enzyme digestion primers were double-digested using Xba I and Sal I restriction enzymes.

[0050] The double enzyme digestion reaction system consisted of: Xba I 2 µL, Sal I 2 µL, 10 µL of 1000 ng / µL pCAMBIA2300 empty vector plasmid, 6 µL of 1×T+BSA, and ddH2O up to 40 µL; and Xba I 2 µL, Sal I 2 µL, 20 µL of 150 ng / µL cDNA, 6 µL of 1×T+BSA, and ddH2O up to 40 µL. The concentrations of Xba I and Sal I were 20,000 units / ml.

[0051] The double enzyme digestion reaction conditions were: 37℃ for 4 hours and 30℃ for 4 hours.

[0052] The target gene fragment was recombined and ligated with the linearized vector fragment to obtain the plant expression vector pCAMBIA2300-PdbHLH57.

[0053] The ligation reaction system consisted of: 1.0 µL T4 DNA Ligase, 3.0 µL of 150 ng / µL target fragment digestion product, 3.0 µL of 1000 ng / µL pCAMBIA2300 plasmid digestion product, 1.0 µL 10×T4 Ligase Buffer, and up to 10 µL ddH2O. The concentration of T4 DNA Ligase was 10 mg / mL. The reaction conditions were: mix thoroughly, centrifuge, place the sample in a metal bath, and incubate overnight at 16°C.

[0054] (1.4) Transformation of Agrobacterium tumefaciens EHA105

[0055] Take Agrobacterium competent cells EHA105 stored at -80℃ and hold them in your palm for a moment until partially thawed. While in an ice-water mixture, insert them into ice. Take 100 µL of competent cells and add 0.01-1.0 µg of plasmid. Gently tap the bottom of the tube to mix. Incubate sequentially on ice, in liquid nitrogen, in a 37℃ water bath, and in an ice bath for 5 min each. Add sterile LB broth and incubate at 28℃ with shaking for 2-3 hours. Centrifuge at 6000 rpm for 1 min to collect the bacteria. Take 50 µL of the supernatant, gently pipette to resuspend the bacterial block, and spread it onto an LB agar plate containing 50 mg / L kanamycin. Invert the plate and incubate on a shaker at 28℃ for 2-3 days. Pick white single colonies and inoculate them into LB broth containing 50 mg / L kanamycin. Incubate at 250 rpm and 28℃ for 4 hours with shaking. Perform PCR detection using primers to identify positive clones. After successful identification, incubate the cells for transformation of poplar leaves.

[0056] (1.5) Staining poplar leaves

[0057] The identified Agrobacterium transformant pCAMBIA2300-PdbHLH57 was inoculated into 30 mL LB liquid medium (containing 50 mg / mL kanamycin) and cultured in a shaker at 28°C until the OD600 reached 0.5-0.8. The cells were collected by centrifugation, and Agrobacterium was resuspended in 30 mL of transformation medium MSO. Poplar leaves were transformed using the soaking method. Fresh leaves were taken, cut into small pieces, and soaked in Agrobacterium medium for about ten minutes. The leaves were then removed and placed on MS solid medium without antibiotics and cultured in the dark at 28°C for 2-3 days. After that, they were transferred to MS differentiation medium containing kanamycin and allowed to differentiate. The medium was changed once a week. After the leaves differentiated and grew resistant buds, they were tested.

[0058] (1.6) Identification of transgenic positive plants

[0059] (1.6.1) Staining identification

[0060] Leaves from wild-type and transgenic plants were placed in GUS staining solution and stained at 37°C for at least 12 hours. Destaining was then performed using anhydrous ethanol.

[0061] The results are as follows Figure 1 As shown in Figure A, the leaves of wild-type poplar plants were not stained blue, while the leaves of transgenic lines OE-1 and OE-3 turned blue; the results indicate that the reporter gene was successfully integrated into the plant genome, and the plant is a transgenic plant.

[0062] (1.6.2) PCR identification

[0063] PCR kits were used to verify the transgenic plants by PCR on plants stained blue. The forward primer was Q-PdbHLH57-F, and the primer sequence is shown in SEQ ID NO.5.

[0064] SEQ ID NO.5: 5'-TTACGACTCAATGACAAGAAG-3'

[0065] The reverse primer is Q-PdbHLH57-R, and the primer sequence is shown in SEQ ID NO.6:

[0066] SEQ ID NO. 6: 5'-GCAGTTAGGGGAAGTTATG-3'.

[0067] The amplification conditions were: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, for 35 cycles; and a final extension at 72℃ for 10 min.

[0068] The reaction system consisted of: 1 μL cDNA template, 0.8 μL each of upstream and downstream primers (10 μM), 10 μL buffer, 0.4 μL dNTPs, 0.4 μL novizan high-fidelity enzyme, and 6.6 μL ddH2O.

[0069] The results are as follows Figure 1 As shown in Figure B, the target band could not be amplified using wild-type poplar leaves and water as templates, but the target band could be amplified using leaves of transgenic lines OE-1 and OE-3 as templates, indicating that the gene has been transferred into transgenic lines OE-1 and OE-3.

[0070] Example 2: Determination of Salt Stress Resistance of Transgenic Populus tomentosa Leaves

[0071] Wild-type Populus alba and transgenic Populus alba (prepared and verified using the same method as in Example 1 to increase accuracy, resulting in transgenic plants OE-5, OE-8, and OE-10) of similar growth were perforated into 1 cm diameter circles. Each treatment of each line was cultured for 3 days in aqueous solutions of water, 100 mM NaCl, 200 mM NaCl, and 300 mM NaCl. The leaf condition was observed and the chlorophyll content was measured. Leaves from poplar seedlings under different treatments were weighed, veins removed, chopped, and washed. Leaf fragments were collected and stored in 80% acetone at 28°C in the dark for 24 hours until the leaves turned white to ensure complete chlorophyll extraction. The chlorophyll extract was collected and quantified. 3 ml of the chlorophyll extract was measured colorimetrically at wavelengths of 663 nm, 645 nm, and 470 nm to determine the absorbance and calculate the total chlorophyll content. 80% acetone was used as a blank control.

[0072] Wild-type and transgenic *Populus alba* plants with uniform growth after two months of rooting culture were transplanted to a substrate after one week of hydroponic acclimatization and cultured in a greenhouse for two months. Irrigation was performed using either water (control) or 300 mM NaCl solution (once daily for 7 days), with three biological replicates for each line. Plant fresh weight, plant height, and other growth parameters were measured, and phenotypic differences under salt stress were systematically analyzed. Results are shown below. Figure 2 As shown.

[0073] Figure 2 The 'a' in the text indicates that there was no significant difference in the leaves of wild-type Populus tomentosa and transgenic Populus tomentosa in normal aqueous solution. After culturing in 100 mM NaCl, 200 mM NaCl and 300 mM NaCl aqueous solutions for 3 days, the leaves of the transgenic line were greener than those of the wild-type line. Figure 2 The 'b' indicates that in normal aqueous solutions, there was no significant difference in chlorophyll concentration between wild-type Populus tomentosa and transgenic Populus tomentosa lines. However, after culturing in 100 mM NaCl, 200 mM NaCl, and 300 mM NaCl aqueous solutions for 3 days, the chlorophyll concentration of the transgenic lines was significantly higher than that of the wild type, indicating that PdbHLH57 can significantly enhance the salt tolerance of Populus tomentosa in vitro.

[0074] To investigate the salt stress resistance of transgenic lines, leaves from wild-type Populus tomentosa and transgenic lines (PdbHLH57 gene) were cultured in NaCl solutions of different concentrations. The total chlorophyll content was then measured to determine the plants' salt stress resistance. The results showed that under various NaCl treatments, the chlorophyll concentration of the transgenic lines was significantly higher than that of the wild-type lines (WT), indicating that PdbHLH57 can significantly enhance the salt tolerance of poplar trees in vitro.

[0075] Example 3: Observation of Salt Tolerance Morphology in Transgenic Populus tomentosa

[0076] Wild-type and transgenic plants (OE1, OE3, OE8) with roughly the same growth rate were selected and treated with water and 300 mM NaCl solution for 7 days respectively. Plant growth differences were observed, and fresh weight and plant height were recorded. Under normal water treatment, the growth of wild-type and transgenic *Populus tomentosa* was basically the same. After 7 days of stress with 300 mM NaCl solution, all poplar lines showed symptoms of damage, but the transgenic lines showed less damage and fewer wilted leaves than the wild-type. Under normal conditions, there was no significant difference in plant height and fresh weight among the poplar lines. Under salt stress, the transgenic lines were 10.5%-14.4% and 27.7%-38.2% higher in plant height and fresh weight than the wild-type, respectively. Figure 3 As shown.

[0077] Example 4: Analysis of physiological and biochemical indicators of transgenic Populus alba under salt stress

[0078] Leaves from the middle part of the plant after salt stress were taken, and the leaf tissue was thoroughly ground with liquid nitrogen for the determination of various physiological and biochemical indicators.

[0079] (4.1) Determination of malondialdehyde (MDA) content

[0080] Take one leaf from the same part of each plant, mix and take 0.1 g of the sample, cut it into small pieces, add 2 mL of 10% trichloroacetic acid and a small amount of quartz sand, grind into a homogenate, add another 8 mL of 10% trichloroacetic acid and grind further, transfer the homogenate to a 10 mL centrifuge tube, centrifuge at 4000 rpm / min for 10 min, and the supernatant is the malondialdehyde extract.

[0081] Colorimetric reaction and determination

[0082] Take four clean test tubes, label them, and use three as sample tubes (three replicates). Add 2 mL of malondialdehyde extraction solution to each tube. Use one as a control tube and add 2 mL of ultrapure water. Then add 2 mL of 0.6% thiobarbituric acid solution to each tube and shake well. Incubate the mixture in a boiling water bath for 10 min (starting the timer when small bubbles appear in the solution). Remove the test tubes and cool them rapidly. Centrifuge at 4000 rpm / min for 10 min. Take the supernatant and measure the absorbance (A) at wavelengths of 532 nm, 450 nm, and 600 nm.

[0083] Calculation results: MDA concentration calculation formula: CMDA = 6.45(A532 - A600) - A450 (μmol / L), where A450, A532, and A600 are the absorbance values ​​measured at wavelengths of 450 nm, 532 nm, and 600 nm, respectively. MDA content (μmol / LFW) = (CMDA) (extraction volume) / (fresh weight of leaves) 1000).

[0084] (4.2) Determination of proline (Pro) content

[0085] Extraction of proline: Select leaves from the same position on each plant, take 0.1 g of the mixture, cut them into small pieces, and place them in stoppered test tubes. Then add 5 mL of 3% sulfosalicylic acid solution to each tube and extract in a boiling water bath for 10 min (shaking frequently during the extraction process). After cooling, filter into a clean test tube. The filtrate is the proline extract.

[0086] Determination of proline: Pipette 2 mL of proline extract into a stoppered test tube, add 2 mL of glacial acetic acid and 2 mL of 2.5% acidic ninhydrin reagent, and heat in a boiling water bath for 30 min until the solution turns red. After cooling, add 4 mL of toluene, shake for 30 s, let stand for a moment, and transfer the supernatant to a 10 mL centrifuge tube. Centrifuge at 3000 rpm / min for 5 min. Gently pipette the red proline toluene solution from the supernatant into a cuvette, using the toluene solution as a blank control, and measure the absorbance at a wavelength of 520 nm.

[0087] Result calculation: Find the proline content X in the sample solution from the standard curve, and calculate the proline content according to the following formula.

[0088] Proline content (μg / g FW) = (X (Total extract) / (Fresh weight of sample) (Amount of extract during measurement).

[0089] (4.3) Superoxide dismutase (SOD) activity assay:

[0090] Crude enzyme extraction: Cut 0.1 g of treated poplar leaves with scissors and place them in a mortar. Under ice bath conditions, add 1 mL of 50 mmol / L pH 7.8 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (4°C pre-cooled) to the mortar. Grind rapidly at low temperature until homogenized, then transfer to a centrifuge tube. Rinse the mortar with 3 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer and transfer to a centrifuge tube. Centrifuge at 10000 rpm / min for 20 min at 4°C. The supernatant is the crude enzyme solution, which should be stored at 4°C.

[0091] The SOD reaction system and sample addition order are as follows: 1.5 mL of 50 mmol / L pH 7.8 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, 0.3 mL of 130 mmol / L methionine solution, 0.3 mL of 750 μmol / L nitroblue tetrazolium solution, 0.3 mL of 100 μmol / L disodium ethylenediaminetetraacetate solution, 0.3 mL of 0.2 μmol / L riboflavin solution, 0.1 mL of crude enzyme solution, and 0.2 mL of ultrapure water are thoroughly mixed in a 20 mL glass test tube. The test tube is then reacted under 4000 lx light for 10 min. Two test tubes are used as controls (using ultrapure water instead of enzyme solution). After mixing, one control test tube is placed in the dark, while the other control test tube, along with the other test tubes containing enzyme solution, is placed under a fluorescent lamp for 10 min. After the reaction, the tubes are immediately protected from light. The absorbance of the unlit control test tube is measured at a wavelength of 560 nm as a blank reference.

[0092] SOD enzyme activity calculation: One unit of superoxide dismutase activity is defined as the inhibition of nitroblue tetrazolium photoreduction by 50% per minute per gram fresh weight (FW) of leaves.

[0093] SOD enzyme activity calculation formula: SOD activity = [(Ac - As)] V] / (0.5 Ac Vs t W), where Ac: absorbance value of the reaction solution in the light-illuminated control tube; As: absorbance value of the reaction solution in the sample tube; V: total volume of the sample extract; Vs: volume of the sample extract taken during the measurement; t: light-illuminated reaction time; W: sample mass.

[0094] (4.4) Peroxidase (POD) activity assay

[0095] Crude enzyme solution extraction: Use the crude enzyme solution described above.

[0096] POD enzyme reaction system and sample addition sequence: 3 mL of 25 mmol / L guaiacol solution, 0.2 mL of 250 mmol / L hydrogen peroxide solution, and 0.1 mL of crude enzyme solution. Starting from 30 s after adding the crude enzyme solution, the absorbance value of the reaction system at a wavelength of 470 nm was recorded every 30 s, and the measurement was continued for 10 min.

[0097] POD enzyme activity calculation: One unit of peroxidase activity is defined as an increase of 0.001 in absorbance per minute per gram of fresh weight (FW) of leaf sample.

[0098] POD enzyme activity calculation formula: POD activity = (ΔA470) / (ΔA470) V) / (W) Vs 0.001 t), where ΔA470: change in absorbance of the reaction mixture; V: total volume of sample extract; W: sample mass; Vs: volume of sample extract taken during measurement; t: enzyme reaction time.

[0099] (4.5) Determination of H2O2 content

[0100] The determination was performed according to the method of Christense et al. (1997): Leaves from poplar seedlings under different treatments were taken, and approximately 0.5 g were weighed after removing the veins. The exact weight was recorded. The leaves were ground into a homogenate in a pre-cooled 0.1% trichloroacetic acid (TCA) ice bath. The homogenate was pipetted into centrifuge tubes, and the mortar was rinsed repeatedly with TCA and the rinse water was transferred to the centrifuge tubes. The mixture was centrifuged at 1200 g for 10 min at 4°C, and the precipitate was discarded, retaining the supernatant. 1 ml of the supernatant was taken, and 0.1 ml of a 95% hydrochloric acid solution containing titanium tetrachloride (TiCl4) (containing 20% ​​w / v TiCl4) was added sequentially. While mixing, 0.2 ml of ammonia water (NH4OH) was added, and the mixture was allowed to stand for 2 min. The mixture was then centrifuged at 1000 g for 10 min at 4°C, and the supernatant was discarded, retaining the precipitate. The precipitate was washed at least three times with pre-cooled acetone to ensure it was colorless. Finally, 1 mol·L⁻¹ of TCA was added... -1 After dissolving the precipitate with 3 ml of sulfuric acid, the volume was adjusted to 5 ml. The resulting solution was then analyzed colorimetrically at a wavelength of 415 nm, and the H2O2 content was calculated.

[0101] Relative conductivity determination: Take a 50 mL conical flask, wash it with ddH2O, sterilize it under high temperature and high pressure, dry it, and set it aside; wash the dust off the surface of the leaf with tap water, then rinse it 2-3 times with ddH2O, wipe it dry with sterile paper, and use a clean punch to punch out a 1 cm diameter disc from the leaf; put the discs into 50 mL conical flasks, add 50 mL ddH2O, evacuate for 15 min, and measure its conductivity S1 using a conductivity meter; put the evacuated conical flasks into a boiling water bath and heat (20 min), cool to room temperature, and measure its conductivity S2; the formula for calculating relative conductivity is: relative conductivity = S1 / S2.

[0102] High-salt environments induce excessive accumulation of reactive oxygen species (ROS) in plants, and the resulting oxidative damage significantly inhibits normal physiological activities. The activity levels of peroxidase (POD) and superoxide dismutase (SOD) serve as key biochemical indicators for assessing plant ROS scavenging efficiency. Proline, as an important marker of stress resistance, shows a positive correlation between its accumulation and plant stress resistance. Malondialdehyde (MDA), as an end product of membrane lipid peroxidation, directly reflects the degree of damage to the cell membrane system and is a core parameter for determining plant resistance. Comparison of physiological indicators between transgenic and wild-type Populus alba plants revealed no statistically significant differences in SOD activity, POD activity, H2O2 content, proline content, MDA concentration, and relative conductivity under normal growth conditions. After salt stress treatment, the POD activity, proline content, and H2O2 content of both transgenic and wild-type lines showed an increasing trend. However, the POD activity, SOD activity, and proline content of transgenic *Populus tomentosa* were 25.9%-37.6%, 25.7%-32%, and 52%-92.8% higher than those of the wild type, respectively, while the H2O2, MDA content, and relative conductivity were 38.2%-41.1%, 18.1%-22.9%, and 21.1%-27.6% lower, respectively. These data confirm that overexpression of the PdbHLH57 gene significantly enhances the salt stress tolerance of *Populus tomentosa* (see details). Figure 4 ).

[0103] Example 5: Histochemical staining analysis

[0104] Wild-type and transgenic Populus alba tissue culture seedlings with roughly the same growth rate and two months of growth were collected and subjected to stress treatment with water and 200 mM NaCl solution for 24 hours, respectively. The leaves were then cut off and immersed in DAB and NBT staining solutions, treated at room temperature in the dark, and stained overnight. After staining, the stained leaves were rinsed with water and then destained with 75% anhydrous ethanol in a boiling water bath.

[0105] NBT and DAB staining showed that, under water treatment conditions, the leaf staining of PdbHLH57-overexpressing transgenic plants and wild-type plants was basically the same; under 300 mM NaCl stress, the leaves of PdbHLH57-overexpressing transgenic plants were lighter in color than those of wild-type plants. Figure 5 and Figure 6 The results showed that the content of hydrogen peroxide and superoxide anion in the leaves of transgenic plants was lower than that of wild-type plants, and the degree of damage was less. The ROS accumulation level in transgenic plants was lower than that in wild-type plants, indicating that overexpression of PdbHLH57 can enhance the tolerance of Populus tomentosa to salt stress.

[0106] Example 6: Determination of expression levels of salt tolerance-related genes

[0107] Total RNA was extracted from wild-type and transgenic Populus alba after one week of salt stress and reverse transcribed into cDNA. Homologous genes of salt stress-related genes such as superoxide dismutase (SOD) and peroxidase (POD) were screened based on the Populus genome database, and specific quantitative primers (Table 1) were designed for qRT-PCR detection (with Actin2 as an internal control).

[0108] Table 1 Primer sequences for quantitative fluorescence detection

[0109]

[0110] In the process of plant resistance to salt stress, genes do not function alone; multiple genes need to work together. To understand the response mechanism of transgenic lines under salt stress, the expression of eight salt stress-related genes was analyzed. The results showed that the expression levels of salt-tolerance-related genes in transgenic plants were significantly higher than those in wild-type plants under salt stress, and their salt tolerance was also significantly higher. Figure 7 This suggests that PdbHLH57 may further enhance the salt tolerance of transgenic plants by regulating salt-tolerance genes. Actin2 The gene is a housekeeping gene, used to indicate the relative abundance of the other 8 genes.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpression PdbHLH57 The application of genes in improving the salt tolerance of poplar trees is characterized by, The PdbHLH57 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The PdbHLH57 The amino acid sequence of the gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The method of improving the salt tolerance of poplar trees can increase the poplar trees' tolerance to NaCl concentrations up to 300 mM.

4. The application of overexpression of the PdbHLH57 gene as described in claim 1 in improving the POD activity, SOD activity and / or proline content of poplar trees in saline-alkali land.

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