Use of expression promoter of pubhlh47 gene in improving salt stress tolerance of populus nigra

Abstract

This invention belongs to the field of genetic engineering technology, specifically involving PubHLH47 The application of gene expression promoters in improving the salt stress tolerance of Populus tomentosa. PubHLH47 The nucleotide sequence of the gene is shown in SEQ ID NO.1. Using the method provided by this invention... PubHLH47 Gene-constructed plant expression vectors enable PubHLH47 Genes are overexpressed in Populus tomentosa. PubHLH47 Gene overexpression in plants effectively enhances their tolerance to high salt stress. This invention is the first to discover a gene that can improve salt stress tolerance in forest trees. PubHLH47 Genes provide excellent genetic resources for promoting genetic engineering breeding of forest trees and provide a scientific basis for the future improvement of Populus tomentosa.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving PubHLH47 Application of gene expression promoters in improving the salt stress tolerance of Populus tomentosa. Background Technology

[0002] Salt stress is one of the most severe environmental stresses faced by plants in nature. With the exacerbation of climate change, rising sea levels, and inadequate irrigation practices, increased soil salinity directly inhibits tree and crop growth, weakening agricultural and forestry productivity. The anchoring nature of trees inevitably subjects them to complex environmental stresses. To adapt to environmental changes, they have evolved a series of physiological defense mechanisms to resist various abiotic damages. Transcription factors play a crucial regulatory role in this process. Therefore, screening and identifying key genes related to salt tolerance, studying plant responses to salt stress, and cultivating plants with strong salt tolerance are urgent problems to be solved.

[0003] bHLH (basic helix-loop-helix) is one of the largest transcription factor families in plants. Its members are widely involved in important biological processes such as plant metabolism, bioactive ingredient synthesis, metal ion homeostasis, and signaling hormone regulation. They also play crucial roles in plant growth, development, and environmental stress. The basic domain, located at the N-terminus of a 60-amino acid region and approximately 15 amino acids long, allows this transcription factor to bind to E-box elements (5'-CANNTG-3'), G-box (5'-CACGTG-3'), and T / G-box (5'-CACGTT-3'). Studies have shown that bHLH family genes play key roles in plant growth and development, such as root development, flowering regulation, leaf senescence, secondary growth, secondary metabolism, and seed germination. Furthermore, some bHLH proteins, such as those involved in hormone metabolism, play important roles in plant defense against pests and pathogens. In addition, bHLH proteins are also involved in responses to abiotic stresses. Drought, salt stress, cold, or abscisic acid (ABA) can induce changes in wheat. TabHLH1 Cucumber bHLH041 grapes VvbHLH1 Rice OsbHLH148 Corn bHLH55 And in Apple MdbHLH3 The expression of these genes suggests that these bHLH transcription factors may play a role in resisting drought, salt, and cold stress. Although these studies provide a foundation for understanding the function of bHLH in plants, the bHLH family contains numerous genes, and bHLH genes in different species may exhibit functional differentiation. Therefore, whether bHLH has a corresponding function in *Populus alba* is unknown. HLH47Whether the gene has salt tolerance is unknown. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the present invention provides PubHLH47 Application of gene expression promoters in improving the salt stress tolerance of Populus tomentosa.

[0005] PubHLH47 The application of gene expression promoters in improving the salt stress tolerance of Populus tomentosa. PubHLH47 The nucleotide sequence of the gene is shown in SEQ ID NO.1, which is:

[0006] Using the invention provided PubHLH47 Gene-constructed plant expression vectors enable PubHLH47 Genes are overexpressed in Populus tomentosa. PubHLH47Gene overexpression in plants effectively enhances their tolerance to high salt stress, thus proposing... PubHLH47 Application of gene expression promoters in improving the salt stress tolerance of Populus tomentosa.

[0007] PubHLH47 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2, which is: MSMGSELNETVEDVTENRCFPAGENKGKVPKRIHKSEREKLKREQLNELFLELASALELSQPNNGKASMLCETTRLLKDLHTQIESLKKENVALLSESHYVTVEKNELREESSALEHQIGKLHSELEMRAASQSKPDLNVPPPEFLQPPHFPVDSFRLPAVDAVALQQTSTVFVVPISPDHVQGFPMATSNVSKPHPRYPTAADSWPSQLLREQQIGRKEV.

[0008] Preferably, the improvement of Populus tomentosa's salt stress tolerance includes: Reduce the relative electrical conductivity of Populus tomentosa leaves under salt stress; Reduce malondialdehyde content in Populus tomentosa leaves under salt stress; Reduce the hydrogen peroxide content in the leaves of Populus tomentosa under salt stress; To enhance the superoxide dismutase activity of Populus tomentosa under salt stress; To enhance the peroxidase activity of Populus tomentosa under salt stress.

[0009] Preferably, the expression promoter comprises: containing the PubHLH47 Recombinant plasmids containing the gene or containing the above PubHLH47 Recombinant bacteria.

[0010] Preferably, the recombinant plasmid is the product of the... PubHLH47 The gene is obtained by linking it to a plant expression vector.

[0011] Preferably, the plant expression vector is pBI121-GFP.

[0012] Preferably, the recombinant bacteria are obtained by introducing the recombinant plasmid into Agrobacterium.

[0013] Preferably, a method for improving the salt stress tolerance of Populus tomentosa includes introducing the recombinant bacteria into Populus tomentosa cells or plants.

[0014] Preferably, the import method includes transient transfection.

[0015] Preferably, during transient transfection, the transfection solution consists of the following final concentrations of substances: 10 mM CaCl2, 2% sucrose, 120 μM AS, 20 μM 5-Azac, 0.02% Tween 20, 200 mg / L DTT, 2 mM MES-KOH, and 270 mM mannitol.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides PubHLH47 The protein product of gene expression is the *Populus alba* bHLH transcription factor, which is utilized using the protein product provided by this invention. PubHLH47 Gene-constructed plant expression vectors enable PubHLH47 Genes are overexpressed in Populus tomentosa. PubHLH47 The gene overexpression effectively improved the plant's tolerance to high salt stress, indicating that this gene is an important key factor in regulating salt tolerance. Attached Figure Description

[0017] Figure 1 Populus tomentosa PubHLH47 Electrophoretic detection image of wild-type Populus tomentosa RNA extracted using the CTAB method.

[0018] Figure 2 Populus tomentosa PubHLH47 Gene clone electrophoresis diagram, where M is DL2000 DNA Marker, 1 is... PubHLH47 Gene products.

[0019] Figure 3 Populus tomentosa PubHLH47 PCR electrophoresis detection image of gene constructed into pCloneEZ-TOPO vector, where M is DL2000 DNA Marker and 1-6 are PCR products.

[0020] Figure 4 Populus tomentosa PubHLH47 PCR electrophoresis results of gene construction into the pBI121-GFP overexpression vector, where M represents the DL2000 DNA Marker, and 1-10 represent... PubHLH47 Gene.

[0021] Figure 5 for PubHLH47 Subcellular localization map of the gene and pBI121-GFP.

[0022] Figure 6 Populus tomentosa PubHLH47 Analysis of relative electrical conductivity (A), malondialdehyde content (B), hydrogen peroxide content (C), superoxide dismutase activity (D), and peroxidase activity (E) in plants with transient gene transformation.

[0023] Figure 7 for PubHLH47 NBT (nitrotetrazole blue) and DAB (diaminobenzidine method) staining of leaves from transiently transformed plants.

[0024] Figure 8 Populus tomentosa PubHLH47 The graph shows the expression levels of antioxidant-related genes, where A represents... PuSOD1 B is PuSOD2 C is PuPOD1 D is PuPOD2 E is PuP5CS1 F is PuP5CS2 . Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0026] Populus tomentosa, mainly distributed in Northeast China, is characterized by its ease of asexual reproduction and gene transformation, making it a valuable genetic resource for selective breeding. Studies in Populus tomentosa have explored transcription factors involved in drought, zinc, phosphorus, or nitrate responses, but their role under salt stress remains largely unexplored. In particular, functional studies on bHLH transcription factors have primarily focused on model plants and economic crops such as Arabidopsis thaliana, tobacco, rice, and maize. However, research on members of this gene family in woody plants is relatively limited, with reports only in a few plants. This invention is the first to discover a gene in forest trees that can enhance salt stress response. PubHLH47 Genes, for PubHLH47 The functional study of the gene in Populus tomentosa provides a research basis, and its overexpression can improve the salt tolerance of Populus tomentosa, providing a scientific basis for future improvement of Populus tomentosa.

[0027] Example 1 Populus alba PubHLH47 Acquisition of genes 1. Obtaining the target gene sequence Extraction of whole wild-type Populus tomentosa (P. davidii) using the CTAB method Populus ussurensisTotal RNA from *kof.* was used as a template, and cDNA was synthesized using the cDNA Synthesis SuperMix Kit (Novizan) according to the instructions. This cDNA was diluted tenfold and used as a template for cloning the target gene using KOD enzyme (Toyobo) (primers for target gene cloning are detailed in Table 1). The reaction conditions were: 94℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 54℃ annealing for 30 s, 68℃ extension for 50 s, 35 cycles, and a final reverse extension at 68℃ for 5 min, stored at 4℃. The product was detected by 1.0% agarose gel electrophoresis. The PCR cloning system is shown in Table 2.

[0028] Table 1: Primer sequences for target gene cloning in this invention Table 2: PCR reaction system for target gene cloning in this invention 2. Target gene ligated into pCloneEZ-TOPO vector Will PubHLH47 The recovered target fragment was ligated with the pCloneEZ-TOPO vector, and the ligation system is shown in Table 3.

[0029] Table 3: pCloneEZ-TOPO Linkage System Reaction conditions: 25℃, 5 min.

[0030] Following the heat shock transformation method, *E. coli* DH5α competent cells were thawed on ice. 5 μL of ligation product solution was added and gently mixed. The mixture was then incubated on ice for 30 min, with two gentle taps during this period. A 42°C water bath for 90 s followed by a 2 min ice bath was then performed. 600 μL of LB liquid medium was added to the mixture, and the mixture was incubated at 37°C with shaking at 220 rpm for 1 h. After incubation, the culture was centrifuged at 4000 rpm for 5 min, the supernatant was removed, and 200 μL of the bacterial suspension was evenly spread onto a solid culture medium and incubated upside down at 37°C for 16 h. Single colony selection was performed the following day. The colonies were placed in 1 mL of LB liquid medium containing 50 mg / L Kan and the PCR amplification products were analyzed by agarose gel electrophoresis.

[0031] result Total RNA was extracted from leaves using the CTAB method. RNA was detected by agarose gel electrophoresis, and the results showed... Figure 1The bands were clear, with no diffusion observed. Furthermore, the A260 / A280 ratio remained stable at 1.9, indicating a good RNA template suitable for subsequent experiments.

[0032] like Figure 2 As shown in the gel electrophoresis image, the quality-tested cDNA was used as the PCR template, and according to... PubHLH47 Gene-specific primers were designed, successfully amplifying the 666 bp target fragment. Electrophoresis analysis using a DL 2000 DNA Marker as a reference showed the target band was located in the 500-750 bp range, exhibiting good specificity and clear color development, meeting the requirements for subsequent experiments. After ligation of the pCloneEZ-TOPO vector with the gel-recovered product, the recombinant plasmid was transformed into *E. coli* DH5α competent cells and cultured under suitable conditions. Several single colonies were randomly selected for colony PCR screening; PCR detection revealed a single and clear band. Electrophoresis results are shown below. Figure 3 As shown, samples confirmed as positive for bacterial colonies by preliminary testing were randomly selected and sent to a biotechnology company for sequencing and sequence alignment analysis. The sequencing results, as shown in SEQ ID NO.1, confirmed correct alignment, proving... PubHLH47 The gene has been successfully constructed into the pCloneEZ-TOPO vector, resulting in pCloneEZ-TOPO- PubHLH47 The aforementioned PubHLH47 The gene was isolated and cloned from Populus tomentosa, as shown in SEQ ID NO.1, with a full-length nucleotide sequence of 666 bp.

[0033] Example 2 PubHLH47 Construction of plant gene expression vectors PubHLH47 Gene overexpression vectors ( PubHLH47 - OE Construction of ) Based on the full-length sequence characteristics of the successfully sequenced *Populus alba* bHLH and the pBI121-GFP multiple cloning site, a pair of primers was designed: pBI121- PubHLH47 -GFP-F: 5'-gctctagaatgagcatgggttcagagctga-3', recorded as SEQ ID NO.5.

[0034] pBI121- PubHLH47 -GFP-R: 5'-gagtcgacccacttccttcctgccaatttg-3', recorded as SEQ ID NO.6.

[0035] The above steps successfully constructed the cloning vector recombinant plasmid pCloneEZ-TOPO- PubHLH47 The plasmid was used as a template to introduce restriction sites. The PCR reaction system is shown in Table 4. The reaction system consisted of 35 cycles: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 50 s, and a final extension at 72℃ for 5 min.

[0036] Table 4. PCR reaction system for introducing homologous arms of the target gene in this invention. Subsequently, the plant expression vector pBI121-GFP was ligated using a double enzyme digestion method, and then transformed into *E. coli* via heat shock. Plasmids were extracted from the successfully constructed bacterial culture and transformed into *Agrobacterium* EHA105 using liquid nitrogen. The transformed bacteria were plated on LB agar containing kanamycin (Kan) and rifampin (Rif) for screening. Successful transformation was verified by PCR and sequencing comparison, and the transformed bacterial cultures were used for subsequent transgenic applications. The specific method for liquid nitrogen transformation of *Agrobacterium* is as follows:

[0037] (1) Using 2 μL of recombinant plasmid pBI121- PubHLH47 -GFP was mixed with thawed EHA105 Agrobacterium competent cells and thoroughly mixed by gentle pipetting. The mixture was then placed in an ice bath for 10 min, followed by liquid nitrogen flash freezing for 5 min. It was then transferred to a 37°C constant temperature water bath for 10 min incubation. Finally, the mixture was placed on ice for 5 min to complete the transformation process.

[0038] (2) After mixing 600 μL of LB liquid medium with the bacterial culture, the mixture was placed in a constant temperature environment at 28℃ and shaken at 220 rpm for 2 h. Then, the culture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 300 μL of fresh LB medium was added to resuspend the bacterial pellet. Finally, the resuspended bacterial culture was evenly spread on the surface of LB solid medium containing Kan and Rif, and placed in a constant temperature incubator at 28℃ for 48 h.

[0039] (3) After picking a single colony and amplifying it in liquid LB medium with added kanamycin, the culture product was detected by PCR technology, and then the amplified product was analyzed by electrophoresis, so as to achieve effective screening of positive clones. Finally, the positive bacterial solution containing the target band was expanded and stored at -80℃ for a long time.

[0040] result PubHLH47 - OE Obtaining overexpression vectors Using pCloneEZ-TOPO-PubHLH47 The plasmid was used as a template for PCR amplification. After purification by gel electrophoresis, the target DNA fragment was double-digested with the pBI121-GFP vector. Recombination ligation was then performed using T4 DNA ligase to obtain pBI121-GFP. PubHLH47 The constructed recombinant plasmid (-GFP) was introduced into *E. coli* DH5α competent cells. After 12 hours of culture, positive monoclonal strains were screened, and the results were verified by shaking culture and culture PCR. Figure 4 The position of the target band is consistent with that of the target band, which proves that the band has been ligated into a positive recombinant. After sequencing, the sequence alignment is correct, and the bacteria can be transformed into Agrobacterium.

[0041] Example 3 PubHLH47 Subcellular Localization Fresh onion inner epidermal tissue was peeled off with a sterile blade and spread evenly on differentiation medium for 24 h. The pBI121-GFP (control) plasmid and pBI121- PubHLH47 -GFP recombinant plasmids were embedded in tungsten powder to construct two different DNA microbullets. Onion epidermis was bombarded with nitrogen using a gene gun, and then the treated onion epidermis was incubated in a dark incubator for 24 h. Subcellular localization characteristics were then observed using a fluorescence inverted microscope.

[0042] result like Figure 5 As shown in the figure, microscopic observation revealed that pBI121-GFP exhibited a green fluorescent signal throughout the cell. PubHLH47 -GFP vectors only show green fluorescence in the cell nucleus, which coincides with staining under the DAPI (nuclear localization signal) pattern, indicating that the PubHLH47 protein is localized in the cell nucleus.

[0043] Example 4 Measurement of transient transformation of Populus tomentosa and physiological indicators under salt stress 1. The instantaneous transformation of Populus tomentosa (1) PubHLH47 - OE After obtaining single clones of Agrobacterium and pBI121-GFP through streak screening, they were inoculated into 5 mL LB liquid medium containing Kan and Rif, respectively, and then transferred to a shaker at 28°C for shaking culture.

[0044] (2) Transfer 1 mL of the above bacterial culture to 50 mL of LB liquid medium containing the same antibiotic combination and continue to expand the culture. Incubate overnight at 28°C and 220 rpm until the bacterial culture becomes obviously turbid. During this process, the pBI121-GFP empty vector is used as a control group and is cultured simultaneously.

[0045] (3) Transfer 2 mL of the turbid bacterial culture to 50 mL of fresh LB liquid medium containing Kan (50 mg / L), and continue to culture at 28℃ and 220 rpm until OD. 600 The value is between 0.6 and 0.7.

[0046] (4) After the culture is completed, the culture medium is placed in a centrifuge and run continuously at 3000 rpm for 20 min to obtain and collect the microbial cell precipitate.

[0047] (5) The bacterial cells were resuspended and adjusted using conversion solution (components: 10 mM CaCl2, 2% sucrose, 120 μM AS, 20 μM 5-Azac, 0.02% Tween 20, 200 mg / L DTT, 2 mM MES-KOH and 270 mM mannitol) to achieve the final OD. 600 The value stabilized at 0.7. Wild *Populus alba* tissue culture plants with a growth cycle of three weeks and excellent phenotype were selected as recipient materials. After thoroughly removing residual culture medium from the roots, the intact plants were immersed in a 50 mL mixture containing transformation solution and bacterial culture. After treatment at 25℃ and 90 rpm for 1 h, 1 / 3 volume of fresh transformation solution was added, and the culture was continued under the same conditions for another 1.5 h.

[0048] (6) After treatment, the plants were transferred to rehydration solution (120 μM AS, 10 mM CaCl2, 200 mg / L DTT and 2% sucrose) and shaken for 30 s to complete surface cleaning. After drying, they were placed in co-culture medium for 2 days.

[0049] 2. Measurement of physiological indicators under salt stress Selected transiently transformed empty vector control plants (pBI121-GFP) and PubHLH47 Overexpression lines ( PubHLH47 - OE Using these materials as experimental materials, the plants were subjected to stress treatment with 150 mmol / L NaCl solution for 12 h at a constant temperature of 25℃. Two transgenic lines were obtained, one under normal conditions and the other under stress conditions. RNA was extracted from these two lines, reverse transcribed into cDNA, and then detected by real-time quantitative PCR to confirm that the gene was successfully transferred into Populus tomentosa.

[0050] (1) Determination of relative conductivity Equal masses of leaf and root samples were weighed and placed in 50 mL centrifuge tubes. After adding 40 mL of deionized water, the tubes were first vacuum-treated for 15 min, then shaken on a shaker at 140 rpm for 2 h. When using a conductivity meter, the initial conductivity value C1 was recorded first. The samples were then placed in a boiling water bath for 15 minutes and cooled to room temperature before being remeasured to obtain the final value C2. Deionized water was used as a control to determine the blank value C0. The relative conductivity value of the samples was finally calculated using a formula.

[0051] Calculation formula: Relative conductivity % = (C1 - C) 01 ) / (C2-C 02 ) × 100%; Among them, C 01 C is the value measured for water before boiling. 02 The values ​​are measured after the water has been boiled.

[0052] (2) Determination of malondialdehyde (MDA) content Take pBI121-GFP (control) after normal culture and salt stress and PubHLH47 -OE strains were rapidly ground into powder in a liquid nitrogen environment. Approximately 0.05 g of each sample was placed in 1 mL of 0.1% TCA (w / v). The samples were incubated at 4°C for 30 minutes, then centrifuged at 10000 rpm for 15 minutes. 0.2 mL of the supernatant was added to 0.8 mL of a mixture of 20% (w / v) TCA and 0.5% (w / v) TBA. The mixture was then placed in a 95°C constant temperature water bath for 15 minutes, followed by cooling on ice. Finally, the mixture was centrifuged at 12000 rpm for 1.5 mL of 20% (w / v) TCA and 0.5% (w / v) TBA, and the absorbance (OD) value was measured. 450 OD 532 and OD 600 The formula for calculating MDA content is: MDA (mmol / g FW) = 1 / 4 [6.543 × (OD)]. 532 -OD 600 -0.559×OD 450 ].

[0053] (3) Determination of hydrogen peroxide (H2O2) content (kit method) The H2O2 content was determined using an H2O2 content determination kit (Nanjing Jiancheng Bioengineering Institute). For specific experimental procedures, please refer to the kit's instruction manual.

[0054] (4) Superoxide dismutase (SOD) activity assay (kit method) Superoxide dismutase (SOD) activity assay kit (Nanjing Jiancheng Bioengineering Institute). For specific experimental procedures, please refer to the kit instructions.

[0055] (5) Peroxidase (POD) activity assay (kit method) The POD content was determined using a peroxidase (POD) activity assay kit (Nanjing Jiancheng Bioengineering Institute). For specific experimental procedures, please refer to the kit's instruction manual.

[0056] (6) Nitroblue tetrazolium (NBT) tissue staining Superoxide anion (O2) .- This method can reduce nitroblue tetrazolium (NBT) to produce a water-insoluble blue formaldehyde dye. It is highly sensitive and is often used with O2. .- Histochemical localization experiments.

[0057] The specific operating steps are as follows: a. Select leaves of Populus tomentosa that have been treated with salt stress. Care should be taken when handling the leaves to avoid secondary damage. b. Immerse the leaves completely in the pre-prepared NBT staining solution for 2 hours, then place them under light for 1 hour until the staining effect is clearly visible. c. Finally, the stained leaves were decolorized using Carno fixative (glacial acetic acid to anhydrous ethanol in a volume ratio of 1:3).

[0058] d. Treat with a clarifying agent (glycerol to anhydrous ethanol, volume ratio 1:4).

[0059] (7) Diaminobenzidine (DAB) tissue staining DAB infiltration staining of plant leaves is a widely used tissue staining technique, and its main operational steps are as follows: a. Select leaves of Populus tomentosa that have been treated with salt stress. Handle the leaves gently during the collection process to avoid causing secondary damage. b. Place the leaves in the pre-prepared DAB staining solution, stain them at room temperature for 12-16 h, and then place them under light for 1 h. c. Once reddish-brown spots appear on the leaves, decolorize them using Carno fixative (glacial acetic acid and anhydrous ethanol in a volume ratio of 1:3).

[0060] d. Treat with a clarifying agent (glycerol to anhydrous ethanol, volume ratio 1:4).

[0061] result (1) Analysis of physiological indicators under salt stress Relative electrical conductivity (EL) is a key physiological indicator for assessing the degree of cell membrane damage. When plants encounter stress, disruption of cell membrane integrity leads to increased electrolyte leakage and a rise in EL values; the structural integrity of the cell membrane directly relates to the plant's stress resistance. Figure 6 As shown in Figure A, after salt stress treatment... PubHLH47 - OE The increase in EL value of the strain was significantly smaller than that of the pBI121-GFP strain, indicating that plants overexpressing this gene can reduce cell membrane damage and have a certain resistance to salt stress.

[0062] Salt stress also promotes the production of large amounts of reactive oxygen species (ROS) in plants, including H2O2 and O2. .- These are two common types of ROS; malondialdehyde (MDA) is an indicator reflecting the degree of membrane lipid peroxidation. The lower its content, the less oxidative damage the plant suffers and the less affected it is by stress. In addition, superoxide dismutase (SOD) and peroxidase (POD) can scavenge ROS and other peroxide free radicals, reduce damage to the cell membrane system, and thus enhance plant stress resistance.

[0063] To further verify PubHLH47 The gene's effect on enhancing the salt tolerance of Populus tomentosa, and its effect on the pBI121-GFP line under salt stress conditions. PubHLH47-OE The transgenic lines were analyzed for MDA and H2O2 content, and SOD and POD activities were determined. The results are as follows: Figure 6 As shown in BE. Data shows that... PubHLH47 The leaves of the -OE strain showed significantly reduced MDA and H2O2 content and significantly enhanced SOD and POD activity, effectively reducing ROS accumulation in vivo.

[0064] In conclusion, PubHLH47 Genes can enhance the salt tolerance of Populus tomentosa.

[0065] (2) Analysis of leaf staining under salt stress In the detection of reactive oxygen species (ROS), superoxide anions (O2) are... .- It can bind with nitroblue tetrazolium (NBT), reducing it to a water-insoluble blue polymer. The intensity of the blue color can visually reflect the O2 content within the plant. .- The lighter the color, the less severe the damage to the plant. For example... Figure 7 As shown, under non-stress conditions, the pBI121-GFP strain and PubHLH47-OE There was no difference in the degree of NBT staining in the leaves of the two strains, indicating that O2 levels were similar. .- The content was basically the same; after salt stress treatment, the staining of both strains deepened, but PubHLH47-OE The staining intensity of the pBI121-GFP line was significantly lower than that of the pBI121-GFP line. This indicates that salt stress leads to increased O2 in the plant. .-The content increased, while PubHLH47 Overexpression of plant O2 .- With less accumulation and less damage, its salt tolerance is improved.

[0066] Hydrogen peroxide (H₂O₂) can oxidize 3,3'-diaminobenzidine (DAB) to form a brown precipitate; therefore, DAB staining results can be used to assess the degree of plant damage and stress resistance. pBI121-GFP and pBI121-GFP were cut from plants treated with non-stress and salt stress, respectively. PubHLH47-OE Transformed plant leaves were immersed in an appropriate amount of DAB staining solution, and the staining was observed. The results are as follows: Figure 7 As shown. Under normal culture conditions, there was no significant difference in leaf staining intensity between the two strains, indicating similar H2O2 content; after salt stress, the staining intensity of both strains increased to varying degrees, but... PubHLH47-OE The leaf color deepening of the pBI121-GFP strain was significantly less than that of the pBI121-GFP strain. This indicates that under salt stress, PubHLH47 Overexpressing the gene results in less H2O2 production, less damage, and stronger stress resistance in the plants.

[0067] Example 5 Populus alba PubHLH47 Effects of antioxidant gene expression 1. RNA was extracted using the CTAB method and reverse transcribed into cDNA. Primers for quantitative real-time PCR were designed using NCBI online software. The total PCR reaction volume was 20 µL, containing 1 µL of cDNA template, 1 µL each of forward and reverse primers, 10 µL of 2×ChamQ Universal SYBR qPCR Master Mix enzyme, and 7 µL of ddH2O. Table 5 below shows the designed qRT-PCR primer sequences.

[0068] Table 5: qRT-PCR primer sequences 2. Populus tomentosa PubHLH47 Analysis of the expression of antioxidant-related genes Quantitative results show that under non-stress conditions, PubHLH47-OE and empty carrier plants PuSOD , PuPOD The expression levels of the gene and the PuP5CS gene are basically the same; under salt stress, PubHLH47-OE plant PuSOD Gene, PuPOD Genes and PuP5CS Gene expression levels were significantly higher in pBI121-GFP plants than in pBI121-GFP plants. Figure 8 (AD). The above results indicate that Populus tomentosa PubHLH47 can affect the expression of antioxidant-related genes and improve the salt tolerance of Populus tomentosa.

[0069] After salt stress PubHLH47-OE The expression levels of PuSOD, PuPOD, and PuP5CS genes in the plant compared to the pBI121-GFP plant further validated that PubHLH47 can enhance the salt tolerance of Populus tomentosa.

[0070] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0071] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0072] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. PubHLH47 The application of genes in improving the salt stress tolerance of Populus tomentosa is characterized by, Through overexpression PubHLH47 Genes enhance the salt tolerance of Populus tomentosa. PubHLH47 The nucleotide sequence of the gene is shown in SEQ ID NO.1; Improving the salt tolerance of Populus tomentosa includes alleviating the degree of damage to Populus tomentosa plants caused by salt stress.

2. The application according to claim 1, characterized in that, Use containing the above PubHLH47 Recombinant plasmids containing the gene or containing the above PubHLH47 Recombinant bacterial overexpression of genes PubHLH47 Gene.

3. The application according to claim 2, characterized in that, The recombinant plasmid is made by... PubHLH47 The gene is obtained by linking it to a plant expression vector.

4. The application according to claim 3, characterized in that, The plant expression vector is pBI121-GFP.

5. The application according to claim 2, characterized in that, The recombinant bacteria are obtained by introducing the recombinant plasmid into Agrobacterium.

6. The application according to claim 5, characterized in that, Methods to improve the salt stress tolerance of Populus tomentosa include introducing the recombinant bacteria into Populus tomentosa cells or plants.

7. The application according to claim 6, characterized in that, The import method includes transient transfection.

8. The application according to claim 7, characterized in that, For transient transfection, the transfection solution consists of the following final concentrations: 10 mM CaCl2, 2% sucrose, 120 μM AS, 20 μM 5-Azac, 0.02% Tween 20, 200 mg / L DTT, 2 mM MES-KOH and 270 mM mannitol.

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