Populus davidiana pbd rap2 gene and application thereof

Abstract

This invention relates to the field of genetic breeding technology, and specifically discloses a type of Populus tomentosa. PdbRAP2 Genes and their applications, the aforementioned PdbRAP2 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2. This invention utilizes genetic engineering to overexpress *Populus sanguisorbus*. PdbRAP2 Genes have improved the drought resistance of Populus tomentosa.

Description

Technical Field

[0001] This invention relates to the field of genetic breeding technology, specifically to a type of Populus tomentosa. PdbRAP2 Genes and their applications. Background Technology

[0002] Global warming is leading to a sustained increase in the frequency and intensity of droughts. Among numerous abiotic stresses, drought has the most direct impact on plants, inhibiting the growth and development of roots, stems, and leaves. Water deficiency not only significantly weakens photosynthesis in leaves but also causes stomatal closure, reducing carbon dioxide absorption and consequently lowering the rate of photosynthetic product synthesis. Secondly, drought disrupts the plant's water balance, leading to protoplast dehydration and damage to cell membranes and organelle membranes, affecting normal physiological functions. Drought also directly impacts the plant's nutrient transport processes. Due to insufficient water, nutrients absorbed by roots are difficult to transport upwards, and organic matter synthesized by leaves is difficult to transport downwards, thus affecting normal plant growth and metabolism and inhibiting plant development. Therefore, cultivating new drought-resistant tree varieties to mitigate plant damage under drought conditions is of great significance for forestry production.

[0003] To adapt to various environments, plants generate a series of physiological responses involving multiple genes. These responses form a complex gene regulatory network to mitigate or eliminate the damage caused by adverse environments, with transcription factors playing a crucial regulatory role. Transcription factors, by binding to specific cis-acting elements, regulate the transcription of downstream genes, directly activating or inhibiting their expression and controlling their timing and abundance, thereby producing various physiological and biochemical changes that enable plant responses to stress. Therefore, in the gene regulatory network for plant stress resistance, transcription factors act as molecular switches regulating the expression of stress-responsive genes. Compared to functional genes, transcription factors can regulate the expression of multiple related genes in stress and are considered excellent candidate genes for improving plant stress resistance through genetic engineering.

[0004] Shan Xinyang ( Populus davidiana × P. bolleana Populus tomentosa is an artificially hybridized superior variety from Northeast China, possessing excellent characteristics such as rapid growth, strong adaptability, and good stress resistance. It also features the unique characteristic of only flowering and not producing fluff, giving it significant economic and ecological value. However, arid environments severely limit the growth and development of Populus tomentosa. Therefore, developing new varieties of Populus tomentosa with enhanced drought resistance has become an urgent task in forestry production. Discovering a functional gene related to the drought resistance of Populus tomentosa is of great significance. Summary of the Invention

[0005] To discover a functional gene related to drought resistance in Populus tomentosa, this invention provides a Populus tomentosa gene. PdbRAP2Genes and their applications. This invention utilizes genetic engineering to overexpress *Populus spp.* PdbRAP2 Genes have improved the drought resistance of Populus tomentosa.

[0006] This invention provides a new type of poplar. PdbRAP2 Genes, the ones mentioned PdbRAP2 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2.

[0007] The present invention also provides an overexpression vector comprising the aforementioned Populus alba. PdbRAP2 Gene.

[0008] Furthermore, by Shanxin Yang PdbRAP2 The gene was obtained by ligating it into the pROKII vector.

[0009] The present invention also provides a genetically engineered bacterium, obtained by transforming Agrobacterium competent cells with the overexpression vector.

[0010] The present invention also provides a method for improving the drought resistance of Populus tomentosa, comprising the following steps:

[0011] Using the cDNA of *Populus spp.* as a template, the target gene fragment was amplified using the sequences shown in SEQ ID NO.3–SEQ ID NO.4 as primers;

[0012] The target gene fragment was ligated to a linearized pROKII plasmid to obtain... PdbRAP2 Gene overexpression vectors;

[0013] PdbRAP2 After the gene overexpression vector was transformed into Agrobacterium competent cells, it was then transformed into Populus alpinus using the Agrobacterium-mediated transformation method to obtain a new drought-resistant alpine transgenic line.

[0014] The present invention also provides the aforementioned *Populus alba*. PdbRAP2 The application of the gene, the overexpression vector, or the genetically engineered bacteria in improving the drought resistance of Populus tomentosa.

[0015] Furthermore, by overexpressing *Populus spp.* through genetic engineering... PdbRAP2 Genes, thereby improving the drought resistance of Populus tomentosa.

[0016] Furthermore, through the expression of Shanxin Yang's PdbRAP2 Genes enhance the SOD and POD activities of Populus tomentosa.

[0017] Furthermore, through the expression of Shanxin Yang's PdbRAP2 The gene reduces H2O2 concentration and conductivity.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] We found one in Shanxin Yangzhong PdbRAP2 The gene was cloned and constructed into a plant overexpression vector. After being transferred into *Populus tomentosa* using stable genetic transformation technology, it was found that overexpression of this gene improved the drought resistance of the poplar. The implementation of this invention will deepen the research on the molecular basis of target traits in forest tree breeding and is of great significance for creating new stress-resistant varieties using key genes and improving the local ecological environment of our province. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 For the purpose of this invention, overexpression transfer PdbRAP2 Screening of Populus tomentosa resistant seedlings; in the figure, a represents the callus induction process; b and c represent the bud induction process; d represents the rooting stage of the resistant seedlings.

[0022] Figure 2 Under PEG stress PdbRAP2 Chemical histochemical staining analysis of transgenic lines; from left to right: Evans blue staining, DAB staining, and NBT staining.

[0023] Figure 3 for PdbRAP2 Determination of superoxide dismutase (SOD) activity of transgenic poplar under drought stress.

[0024] Figure 4 for PdbRAP2 Determination of peroxidase (POD) activity of transgenic poplar under drought stress.

[0025] Figure 5 for PdbRAP2 Determination of hydrogen peroxide (H2O2) content in transgenic poplar under drought stress.

[0026] Figure 6 for PdbRAP2 Determination of the relative electrical conductivity of transgenic poplar under drought stress.

[0027] Figure 7 The effect of transgenic Populus alba on stomatal aperture;

[0028] In the diagram, A is... PdbRAP2Stomatal aperture detection of transgenic Populus tomentosa and wild-type Populus tomentosa;

[0029] B is PdbRAP2 Stomatal width / length values ​​of transgenic Populus tomentosa and wild-type Populus tomentosa.

[0030] Figure 8 for PdbRAP2 Phenotypic changes in transgenic poplar plants and leaves under drought stress. Detailed Implementation

[0031] 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. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative 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, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0032] Example 1: A type of poplar PdbRAP2 Genes and their applications.

[0033] I. Shanxin Yang PdbRAP2 Cloning of genes

[0034] (1) Shanxin Yang PdbRAP2 Gene amplification

[0035] Total RNA was extracted from wild-type *Populus tomentosa* tissue culture seedlings (25℃, 16h light / 8h dark) using an RNA extraction kit (Beijing Biotech Biotechnology Co., Ltd.). Primers were designed for PCR amplification. The primer sequences required for PCR amplification are shown in Table 1, and the reaction system is shown in Table 2. The target gene product was recovered using a purification and recovery kit (Guangzhou Feiyang Biotechnology Co., Ltd.), and sequenced to obtain the complete coding region (CDS) shown in SEQ ID NO.1. PdbRAP2 Gene sequence. The amino acid sequence of the encoded Populus alba PdbRAP2 is shown in SEQ ID NO.2.

[0036] SEQ ID NO.1:

[0037] ATGTGTGGAGGTGCCATCATTTCCGATTTCGTACCCGTTAAACGTGGCCGGAAACCGACCACTGAGGATCTCTGGTCTGAACTTGACTCCTTATCTGATTTTCTTGGACTTGATCATCGTTCTATGAACAATATTAATAATGGTAGCAAAAAAGAAAACCTATCAAATCTCAAGCTCGCTCAAAAGCCACGCCAGCCCAACCAAGTGATAACAGAGAGAGTTGAGAAGCCAAGCCAAGCAACAGAGCAAGAAGCCGGTAAAAAGAAGGTTCAGAGAACCAGAAAGAATGTGTACAGAGGAATAAGGCAAAGGCCATGGGGTAAATGGGCAGCTGAAATAAGAGACCCACACAAAGGTGTTAGAGTTTGGCTGGGCACTTACAACACAGCTGATGAAGCCGCTAAAGCTTATGATGAAGCCGCCAAGCGCATCCGTGGTGATAAAGCCAAGCTCAACTTCCCCCCTCAGCCACCACCAACGTCGGAGGCGGCGCCACCACCTGCCAAGAAGCGTTGCATCTTGGGTCCTGAAACGGCTGTTATGGCTAGTTTTGAGCAAATCTTGAACTCGGAGTCGTTTCATGGCCTGGAACCGCATCAGGCGGCGGCCCAGCTGAGTTGTGACGGAGGTGGAAGTGGTGATTATAATTGTGACCCTGTGGACCCTTGGATGCTTGATGATCTTATCAACGTCAGCTTAATTATTAGTAATATAATTAATTAA。

[0038] SEQ ID NO.2:

[0039] MCGGAIISDFVPVKRGRKPTTEDLWSELDSLSDFLGLDHRSMNNINNGSKKENLSNLKLAQKQRQPNQVITERVEKPSQATEQEAGKKKVQRTRKNVYRGIRQRPWGKWAAEIRDPHKGV RVWLGTYNTADEAAKAYDEAAKRIRGDKAKLNFPPQPPPTSEAAPPPAKKRCILGPETAVMASFEQILNSESFHGLEPHQAAAQLSCDGGGSGDYNCDPVDPWMLDDLINVSLIISNIIN.

[0040] Table 1 Gene-specific primers

[0041]

[0042] Table 2 Amplification reaction system

[0043]

[0044] PCR reaction program: 95℃ / 3min; 94℃ / 30s, 55℃ / 30s, 72℃ / 1min, 35 cycles; 72℃ / 7min.

[0045] II. Shanxin Yang PdbRAP2 Analysis of drought resistance genes

[0046] 1. Shanxin Yang PdbRAP2 Construction of gene overexpression vectors

[0047] (1) Shanxin Yang PdbRAP2 Gene amplification

[0048] according to PdbRAP2 Based on the CDS sequence of the gene, gene-specific primers were designed to deliver the S... ma I. Restriction sites were introduced at both ends of the RAP2 gene. The primers required for PCR amplification were pROKⅡ-RAP2-F (SEQ ID NO.5) and pROKⅡ-RAP2-R (SEQ ID NO.6). Using the recovered target gene product as a template, PCR amplification was performed using the primers in Table 3. The PCR reaction system is shown in Table 4, and the PCR reaction procedure is as follows.

[0049] Table 3 Gene-specific primers

[0050]

[0051] Table 4 Amplification reaction system

[0052]

[0053] PCR reaction program: 95℃ / 3min; 94℃ / 30s, 55℃ / 30s, 72℃ / 1min, 35 cycles; 72℃ / 7min.

[0054] (2) Shanxin Yang PdbRAP2 Gene gel recycling

[0055] After the PCR reaction, the position of the target band was detected by agarose gel electrophoresis, and then the target gene product was recovered using a purification and recovery kit (Guangzhou Feiyang Biotechnology Co., Ltd.) (see the instruction manual for specific experimental procedures). The recovered target gene product was then detected by agarose gel electrophoresis and its concentration was measured, and stored at -20℃ for later use.

[0056] (3) Extraction of pROKII plasmid

[0057] pROKII plasmid was extracted using a plasmid extraction kit (Guangzhou Feiyang Biotechnology Co., Ltd.) (see the instruction manual for specific experimental procedures). The extracted plasmid was detected and its concentration was measured by agarose gel electrophoresis and stored at -20℃.

[0058] (4) Enzyme digestion of pROKII plasmid

[0059] Using S ma I. The pROKII plasmid was digested with restriction endonucleases, and the reaction system is shown in Table 5.

[0060] Table 5 Enzyme digestion reaction system

[0061]

[0062] The reaction procedure was: 37℃, 2h.

[0063] After enzyme digestion, agarose gel electrophoresis was performed (the volume ratio of pre-digestion to post-digestion was 1:2 at the time of sample loading). After successful enzyme digestion, the digested vector was purified and recovered using a purification and recovery kit and stored at -20℃ for later use.

[0064] (5) Ligation of the target gene product with the post-digestion pROKII vector

[0065] Using homology fusion, the target gene product was ligated into the enzyme-digested pROKII vector using a one-step PCR cloning kit (Suzhou Nearshore Protein Technology Co., Ltd.) to obtain the ligation product. The reaction system is shown in Table 6, and the reaction program is: 37℃, 15 min; 50℃, 15 min.

[0066] Table 6. Ligation system of target gene product and post-digestion pROKII vector

[0067]

[0068] (6) Heat shock transformation of Escherichia coli

[0069] The ligation product was transferred into E. coli competent cells Top10 using a heat shock conversion method, as follows:

[0070] 0. Take 5 μL of ligation solution into 50 μL of E. coli competent cells, incubate on ice for 30 min, and gently tap to mix 3 times every 10 min.

[0071] ②In a 42℃ water bath for 90 seconds, then immediately place in an ice bath for 2 minutes;

[0072] ③ Add 400 μL LB liquid medium (antibiotic-free), incubate at 37℃ and 220 rpm for 1 h with shaking;

[0073] ④ Spread 200 μL of bacterial culture onto LB solid medium plates (containing 50 mg / L kanamycin sulfate (Kan)) and incubate overnight at 37°C.

[0074] (7) PCR detection of bacterial culture

[0075] Single colonies from the plates were picked and incubated in LB broth (containing 50 mg / L Kan) at 37°C with shaking at 220 rpm for 4 h. Then, using the bacterial culture as a template, PCR detection was performed using the vector primers pROKⅡ-F (SEQ ID NO.7) and pROKⅡ-R (SEQ ID NO.8). The vector primers are located at S... ma I. The enzyme cleavage site contains a total of 447 bp before and after the cleavage site; the reaction system and reaction procedure are shown below.

[0076] SEQ ID NO.7: 5'-AGACGTTCCAACCACGTCTT-3';

[0077] SEQ ID NO. 8: 5'-CCAGTGAATTCCCGATCTAG-3'.

[0078] Table 7 Bacterial PCR Reaction System

[0079]

[0080] PCR reaction program: 95℃ / 3 min; 94℃ / 30 s, 55℃ / 30 s, 72℃ / 1 min, 30 cycles; 72℃ / 7 min.

[0081] The recovered target gene was homologously fused with the enzyme-digested pROKII vector and then heat-shock transformed. Single colonies were picked from the plates, and bacterial culture PCR was performed using vector primers. Bacterial cultures with correctly positioned bands were sent to a biotechnology company for sequencing. The sequencing results were correct, indicating... PdbRAP2 The gene overexpression vector was successfully constructed and is named PdbRAP2. - pROKⅡ plasmid.

[0082] 2. Shanxin Yang PdbRAP2 Preparation of genetically engineered bacteria

[0083] Using a plasmid extraction kit, extract the constructed plasmid. PdbRAP2 Gene overexpression vector. PdbRAP2 was overexpressed via electroporation. - pROKⅡ plasmid was transformed into EHA105 Agrobacterium competent cells, and the experimental steps are as follows:

[0084] 1) Clean the electric shock cup with anhydrous ethanol and disinfect it under ultraviolet light in a clean bench for 30 minutes;

[0085] 2) Add 2 μL of plasmid to 50 μL of Agrobacterium competent cells, mix well and transfer to an electroporation cuvette;

[0086] 3) Administer an electric shock at 1700 V;

[0087] 4) Add 400 μL of LB liquid medium (antibiotic-free) to the electroporation vessel, mix well, transfer to a new 1.5 mL centrifuge tube, and incubate at 28°C and 220 rpm for 1 h with shaking.

[0088] 5) Spread 200 μL of bacterial culture onto LB solid medium (containing 50 mg / L Kan) and incubate at 28℃ for 2 days.

[0089] Pick a single colony from the plate onto LB liquid medium. After confirming the correct position of the band by electrophoresis, store the bacterial culture at -80°C.

[0090] 3. Stable genetic transformation of Populus tomentosa

[0091] (1) Agrobacterium-mediated transformation of Populus tomentosa

[0092] ① Activation of engineered bacteria: The bacteria were activated using a three-zone streak method. PdbRAP2 The engineered bacterial cultures of the gene overexpression vector and the editing vector were activated in LB solid medium (containing 50 mg / L Kan) and cultured at 28°C for 2 days;

[0093] ② Pick a single colony and place it in 50 mL of LB liquid medium (containing 50 mg / L Kan), incubate at 28°C with shaking at 220 rpm until OD reaches 50%. 600 =0.7;

[0094] ③ Transfer the bacterial culture to a 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 min; discard the supernatant and add an equal volume of 1 / 2 MS liquid medium (containing 150 μM acetylsuccinone (AS)) to resuspend the bacterial cells, and incubate at 28 ℃ with shaking at 180 rpm for 40 min.

[0095] ④ Pour the infection solution into a sterile petri dish, cut the leaves of the poplar in the infection solution to make wounds, and then soak them in the infection solution for 5 minutes.

[0096] ⑤ Place the cut leaves on sterile filter paper to absorb the bacterial solution, then place them in differentiation medium (containing 150 μM AS) and incubate in the dark at 25 ℃ for 3 days.

[0097] (2) Screening of resistant Populus tomentosa seedlings

[0098] ① Sterilization: After dark culture, the leaves of Populus tomentosa were transferred to differentiation medium (containing 30 mg / L Kan and 300 mg / L cephalosporin (Cef)) and cultured in an artificial climate chamber;

[0099] ② Subculture: Change the culture medium (differentiation medium + 30 mg / L Kan, 300 mg / L Cef) every 7 days until adventitious shoots appear; transfer to stem-growing medium (containing 40 mg / L Kan, 500 mg / L Cef) and culture for 14 days; then transfer individual seedlings to rooting medium (containing 50 mg / L Kan) and culture for 30 days.

[0100] Experimental results are as follows Figure 1 As shown, resistant seedlings of Populus tomentosa were obtained.

[0101] (3) PdbRAP2 Identification of transgenic strains

[0102] ① DNA extraction: DNA was extracted from the overexpressing transgenic plants using a plant DNA extraction kit (see the instruction manual for specific experimental steps). After DNA extraction, agarose gel electrophoresis was performed to detect the DNA and measure its concentration. The DNA was then stored at -20°C.

[0103] ②PCR identification: Using extracted transgenic plant DNA as a template, pROKⅡ-F and pROKⅡ-R were used as primers. After PCR, PCR identification was performed. Correct bands indicated the acquisition of overexpressing transgenic lines. The screened and identified overexpressing transgenic lines were recorded as follows: PdbRAP2 -OE.

[0104] 4. Shanxin Yang PdbRAP2 Analysis of the drought tolerance of genes

[0105] First, the 20-day-old poplar seedlings were... PdbRAP2 -OE and wild-type tissue culture seedlings (WT) were subjected to 20% PEG 6000 stress for 12 h, with water irrigation treatment as a control. Then, plant leaves were taken for histochemical staining with Evans blue, DAB, and NBT. The remaining materials were quick-frozen and ground in liquid nitrogen for physiological index testing.

[0106] (1) Histochemical staining

[0107] ①Evans blue staining:

[0108] Leaves of *Populus alba* var. *assamica* (plants irrigated) under both stress and non-stress treatments were taken and placed in centrifuge tubes. 0.5 mg / mL of Evans blue staining solution was added, and the tubes were stained under vacuum for 30 min, maintaining this state overnight. After staining, the leaves were decolorized by boiling in a mixture of 75% ethanol and 5% glycerol.

[0109] Figure 2 Evans blue staining is used. Evans blue staining solution can penetrate dead cells and stain them blue. The intensity of the staining indicates the number of dead cells; the more severe the cell damage, the more dead cells are present. As shown in the figure, under PEG stress conditions, compared to the wild type, PdbRAP2 The leaves of plants overexpressing the transgene are lighter in color, indicating that there are fewer dead cells in the leaves of the transgenic plants. PdbRAP2 The overexpressing transgenic lines showed low levels of damage after being subjected to stress.

[0110] ②DAB staining:

[0111] Leaves of *Populus alba* var. *assamica* (plants irrigated) under both stress and non-stress treatments were taken and placed in centrifuge tubes. 1 mg / mL DAB staining solution was added, and the tubes were stained overnight at room temperature. After staining, the leaves were destained by boiling in 75% ethanol and 5% glycerol. Figure 2 For DAB staining, oxygen ions released from H2O2 in cells oxidize DAB, forming a brown precipitate. The intensity of the staining indicates the amount of H2O2 released from the cells; the more severe the cell damage, the more H2O2 is released. As shown in the figure, under PEG stress conditions, compared to the wild type, PdbRAP2 Plants overexpressing the transgene have lighter-colored leaves, indicating... PdbRAP2 The overexpressing transgenic lines showed low levels of damage after being subjected to stress.

[0112] ③NBT staining:

[0113] Leaves of *Populus alba* var. *assamica* (plants irrigated) were taken from both the stress-treated and unstressed control groups and placed in centrifuge tubes. 0.25 mg / mL of NBT staining solution was added, and the tubes were stained overnight at room temperature. After staining, the leaves were destained by boiling in 75% ethanol and 5% glycerol.

[0114] Figure 2 NBT staining is used to detect superoxide anions (O2) in plants. - The content of superoxide anions (O2) in cells can be determined by the depth of staining. - The more severe the cell damage, the higher the concentration of superoxide anions (O2). - The more (the more). For example Figure 2 As shown, under PEG stress conditions, compared with the wild type, PdbRAP2 Plants overexpressing the transgene have lighter-colored leaves, indicating overexpression. RAP2 Superoxide anions (O2) in the leaves of genetically modified plants - The content of superoxide anions (O2) in the leaves of *Populus tomentosa* plants was lower than that in the control group. - Low content indicates conversion PdbRAP2 The genetic lines showed low levels of damage after being subjected to stress.

[0115] The above staining results indicate that PdbRAP2 Genes can positively regulate the drought resistance of Populus tomentosa.

[0116] (2) Measurement of physiological indicators

[0117] ① Superoxide dismutase (SOD) activity assay (kit method):

[0118] Accurately weigh 0.3 g of plant tissue and add four times the volume of Reagent VIII-Homogenizing Medium at a weight (g):volume (mL) ratio of 1:4. Cut the tissue into small pieces and homogenize it under ice-water bath conditions to prepare a 20% homogenate. Centrifuge at 3500 rpm for 10 minutes and collect the supernatant for analysis. Refer to the SOD assay kit (Nanjing Jiancheng Bioengineering Research Institute) for specific operating procedures and result calculations. Reagent VIII-Homogenizing Medium contains plant copper-zinc superoxide dismutase Cu... 2+ Zn 2+ -SOD assay kit, catalog number: A001-4-1.

[0119] Superoxide dismutase (SOD) can catalyze the dismutation reaction of superoxide anion free radicals, resisting damage to the cell membrane system caused by reactive oxygen species or other peroxide free radicals, thereby improving the stress resistance of plants. The measurement results are as follows: Figure 3 As shown. Under non-stress conditions (control), overexpression PdbRAP2The SOD activities of the genetically modified *Populus spp.* strain and the wild-type *Populus spp.* strain were roughly the same; under abiotic stress conditions of PEG, overexpression... PdbRAP2 The SOD activity of the gene-overexpressing plants was higher than that of the wild-type plants, indicating that the overexpressing plants after stable transformation had stronger resistance to abiotic stress than the wild-type plants. The experimental results show... PdbRAP2 The gene can positively regulate SOD activity, and thus positively regulate the drought resistance of Populus tomentosa.

[0120] ② Peroxidase (POD) activity assay (kit method):

[0121] Pretreatment: Preparation of homogenate from young plant tissue with high water content: Wipe the plant tissue clean of water and impurities, accurately weigh the plant tissue, and add 9 times the volume of homogenizing medium (physiological saline or phosphate buffer: 0.1 mol / L pH 7.2 is recommended) at a weight (g): volume (mL) ratio of 1:9. Prepare a 10% tissue homogenate under ice-water bath conditions, centrifuge at 3500 rpm for 10 minutes, and take the supernatant for analysis. Preparation of homogenate from dried plant tissue with low water content: Wipe the plant tissue clean of water and impurities, cut it into small pieces, put it in a mortar, add liquid nitrogen, grind it into powder, transfer it out, and accurately weigh it. Add 9 times the volume of homogenizing medium (physiological saline or phosphate buffer: 0.1 mol / L pH=7 is recommended) at a weight (g): volume (mL) ratio of 1:9, vortex to extract for 3-5 minutes, centrifuge at 3500 rpm for 10 minutes, and take the supernatant for analysis. For detailed operating procedures and result calculations, please refer to the POD assay kit (Nanjing Jiancheng Bioengineering Research Institute). Under the catalysis of peroxidase (POD), H2O2 oxidizes guaiacol to a brownish-yellow product. Peroxidase is an important protective enzyme in plants to reduce damage from oxygen free radicals and is closely related to the plant's ability to resist abiotic stress. The experimental results are as follows: Figure 4 As shown in the figure, under non-stress conditions (control), the POD activity of *Populus alba* lines overexpressing the RAP2 gene and wild-type *Populus alba* lines was roughly the same; under PEG abiotic stress conditions, the POD activity of the RAP2 gene overexpressing lines was higher than that of the wild type, indicating that the plants overexpressing the RAP2 gene have a stronger ability to resist abiotic stress than the wild type. The results suggest... PdbRAP2 The gene expression level is positively correlated with the enzyme activity of POD, indicating that it can improve the stress resistance of plants by regulating the activity of antioxidant enzymes in plants.

[0122] ③ Determination of H2O2 content (kit method)

[0123] Accurately weigh the tissue and add 9 volumes of 0.9% physiological saline at a weight (g):volume (mL) ratio of 1:9. Homogenize mechanically under ice-water bath conditions, centrifuge at 1000 rpm for 10 minutes, and collect 10% of the supernatant for analysis. Refer to the SOD assay kit (Nanjing Jiancheng Bioengineering Research Institute) for detailed operating procedures and result calculations.

[0124] H2O2, as a reactive oxygen species, is ubiquitous in living organisms and plays a crucial role in the conversion between different reactive oxygen species. Among numerous oxidative metabolic products, H2O2 accelerates cell aging and breakdown. This is because it damages cell membranes and directly or indirectly oxidizes biomolecules. Under abiotic stress, the stronger a plant's resistance to stress, the lower the accumulated H2O2 content within it. Experimental results show... Figure 5 As shown.

[0125] like Figure 5 As shown, under non-stress conditions (control), overexpression PdbRAP2 The hydrogen peroxide content of the gene-modified *Populus spp.* strain and the wild-type *Populus spp.* strain were approximately the same; under abiotic stress conditions of PEG, overexpression... PdbRAP2 The H2O2 concentration in the experimental group was lower than that in the wild type, indicating that the experimental group plants had a stronger ability to resist abiotic stress than the wild type. The results suggest that overexpression... PdbRAP2 Genes can enhance a plant's resistance to adverse conditions.

[0126] ④ Determination of relative conductivity:

[0127] Fresh leaves of uniform size, after stress treatment, were rinsed three times successively with double-distilled water and ultrapure water. The surface moisture was blotted dry with filter paper, and the leaves were placed in 50 mL centrifuge tubes. 30 mL of ultrapure water was added, and the tubes were vacuum-pumped for 15 min. The conductivity value was measured using a conductivity meter and recorded as S1. The centrifuge tubes were then placed in a 90℃ constant temperature water bath for 20 min, cooled to room temperature, and the conductivity value was measured and recorded as S2. The relative conductivity of plant mesophyll is a fundamental indicator of plant cell membrane permeability. When plants are subjected to stress, the cell membrane is damaged, increasing membrane permeability and causing electrolytes to leak out of the cell. A lower relative conductivity reflects a higher stress resistance. Calculation: Relative conductivity = S1 / S2 * 100%.

[0128] Experimental results are as follows Figure 6 As shown, under non-stress conditions (control), overexpression PdbRAP2 The relative electrical conductivity of the gene-modified *Populus spp.* strain and the wild-type *Populus spp.* strain were approximately the same; under abiotic stress conditions of PEG, overexpression... PdbRAP2 The relative conductivity of the gene line was lower than that of the wild type, indicating overexpression. PdbRAP2The genetically modified plant is more resistant to abiotic stress than the wild type, and overexpression... PdbRAP2 Genes can enhance a plant's resistance to adverse conditions.

[0129] (3) Determination of stomatal aperture

[0130] Leaves were collected from transgenic and wild-type Populus tomentosa before and after stress treatment. The lower epidermis of the leaves was torn off and placed in a stomatal opening solution (30 mM KCl, 10 mM Mes-KOH, pH adjusted to 6.15) for 2 h. The results were observed under a microscope. Figure 7 As shown in the figure, under drought stress, compared with the wild type... PdbRAP2 The overexpressing lines showed a higher degree of stomatal closure, indicating that the overexpressing lines of this gene can improve the plant's water retention capacity and thus its drought resistance by regulating stomatal opening and closing.

[0131] (4) Phenotypic analysis of drought stress

[0132] Those with uniform growth PdbRAP2 Transgenic plants with overexpressed genes and wild-type *Populus alba* tissue culture rooted seedlings were transplanted into sterilized soil (substrate soil / perlite / vermiculite = 3 / 1 / 1) and cultured in an artificial climate chamber for 20 days. Wild-type and transgenic lines were then treated with 20% PEG solution for 5 days, and phenotypic changes in the plants under PEG stress were observed.

[0133] Experimental results are as follows Figure 8 Under non-stress conditions, transgenic Populus tomentosa plants showed similar growth to wild-type (WT) Populus tomentosa; after PEG stress treatment, compared with the wild type, overexpression of [a specific gene] was significantly increased. PdbRAP2 The genetically modified plants grew better than the wild type, with less wilting of the leaves, indicating that... PdbRAP2 Genes can enhance a plant's resistance to drought stress.

[0134] Chemical tissue staining, physiological index detection, and stomatal aperture experiments showed that the RAP2 gene can significantly improve the drought resistance of transgenic Populus tomentosa, laying a foundation for the early research of drought-resistant new varieties of forest trees.

[0135] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0136] 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. A type of mountain poplar PdbRAP2 The application of genes, overexpression vectors, or genetically engineered bacteria in improving the drought resistance of Populus tomentosa is characterized by, Overexpressing Populus shanxiensis PdbRAP2 gene by genetic engineering to improve drought resistance of Populus shanxiensis; Improving the drought resistance of Populus tomentosa is manifested in: increasing the SOD and POD activities of Populus tomentosa plants, and reducing H2O2 concentration and electrical conductivity; The PdbRAP2 The CDS sequence of the gene is shown in SEQ ID NO.1; the overexpression vector contains the aforementioned *Populus alba*. PdbRAP2 Genes; the genetically engineered bacteria were obtained by transforming Agrobacterium competent cells with an overexpression vector.

2. The application according to claim 1, characterized in that, The overexpression vector was developed by Shanxi Yang PdbRAP2 The gene was obtained by ligating it into the pROKII vector.

3. A method for improving the drought resistance of Populus tomentosa, characterized in that, Includes the following steps: Using the cDNA of *Populus spp.* as a template, the target gene fragment shown in SEQ ID NO.1 was amplified using the sequences shown in SEQ ID NO.3 to SEQ ID NO.4 as primers; The target gene fragment was ligated to a linearized pROKII plasmid to obtain... PdbRAP2 Gene overexpression vectors; PdbRAP2 After the gene overexpression vector was transformed into Agrobacterium competent cells, it was transformed into Populus alba using Agrobacterium-mediated transformation to obtain drought-resistant transgenic Populus alba strains. The drought resistance is manifested in the following way: under drought stress, the transgenic Populus tomentosa variety improves the plant's water retention capacity by regulating the opening and closing of stomata, thereby enhancing the plant's drought resistance.

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