Populus sutchuenensis PdbRAP2 gene and application thereof

Abstract

The invention relates to the technical field of genetic breeding, and particularly discloses a PdbRAP2 gene of Populus sutchuenensis and application of the PdbRAP2 gene, the CDS sequence of the PdbRAP2 gene is as shown in SEQ ID NO.1, and the coded amino acid sequence of the PdbRAP2 gene is as shown in SEQ ID NO.2. The PdbRAP2 gene of the Populus sutchuenensis is over-expressed in a genetic engineering manner, so that the drought resistance of the Populus sutchuenensis is improved.

Description

Technical Field

[0001] The present invention relates to the field of genetic breeding technology, and in particular to a new poplar PdbRAP2 Genes and their applications. Background Art

[0002] Global warming is causing the frequency and intensity of droughts to increase continuously. Among the many abiotic stresses, drought has the most direct impact on plants. Under drought conditions, the growth and development of plant roots, stems, and leaves are all inhibited. Water loss not only significantly reduces leaf photosynthesis but also causes stomata to close, reducing carbon dioxide absorption and, in turn, the rate of photosynthetic product synthesis. Furthermore, drought disrupts plant water balance, leading to dehydration of cell protoplasts and, in turn, damage to cell and organelle membrane structures, impacting normal physiological function. Drought also directly impacts plant transport processes. Due to insufficient water, nutrients absorbed by the roots are difficult to transport upward, and organic matter synthesized by the leaves is also difficult to transport downward. This, in turn, impacts normal plant growth and metabolism, inhibiting growth and development. Therefore, cultivating new drought-tolerant tree varieties to mitigate damage to plants under drought conditions is of great significance to forestry production.

[0003] To adapt to various environments, plants produce a series of physiological responses involving multiple genes. These responses form a complex gene regulatory network to reduce or eliminate the damage caused by adverse environments. Transcription factors play an important regulatory role in this. Transcription factors achieve transcriptional regulation of downstream genes by binding to specific cis-acting elements. They can directly activate or inhibit the expression of downstream genes and regulate the timing and abundance of their expression, thereby producing various physiological and biochemical changes, thereby realizing the plant's response to adversity. Therefore, in the gene regulatory network of plants to resist adversity, transcription factors are molecular switches that regulate the expression of stress-responsive genes. Compared with functional genes, transcription factors can regulate the expression of multiple related genes in adversity and are considered to be excellent candidate genes for improving plant stress resistance through genetic engineering.

[0004] Populus serrata ( Populus davidiana × P. bolleana Populus shanxiensis is a hybrid variety grown in Northeast China. It boasts excellent traits such as rapid growth, strong adaptability, and excellent stress resistance. It also produces flowers without dropping catkins, making it of great economic and ecological value. However, drought significantly restricts its growth and development. Therefore, developing new varieties of Populus shanxiensis with enhanced drought resistance has become a pressing task in forestry production. Identifying a functional gene associated with drought tolerance in Populus shanxiensis is of great significance. Summary of the Invention

[0005] In order to explore a functional gene related to drought tolerance of Populus davidianus, the present invention provides a Populus davidianus PdbRAP2Gene and its application. The present invention overexpresses the gene of Populus davidianus by genetic engineering. PdbRAP2 Genes have improved the drought resistance of Populus altissima.

[0006] The present invention provides a new poplar PdbRAP2 gene, the PdbRAP2 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

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

[0008] Furthermore, by Shan Xinyang PdbRAP2 The gene was ligated with 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 drought resistance of Populus davidiana, comprising the following steps: Using the cDNA of Populus davidiana as a template and the sequences shown in SEQ ID NO.3 to SEQ ID NO.4 as primers, the target gene fragment was amplified; The target gene fragment was connected to the linearized pROKII plasmid to obtain PdbRAP2 Gene overexpression vector; PdbRAP2 After the gene overexpression vector was transformed into Agrobacterium competent cells, it was transformed into Populus davidianus using the Agrobacterium-mediated method to obtain a new drought-resistant high-altitude transgenic variety.

[0011] The present invention also provides a kind of Populus serrata PdbRAP2 Application of the gene, the overexpression vector or the genetically engineered bacteria in improving the drought resistance of Populus davidianus.

[0012] Furthermore, by genetic engineering, the PdbRAP2 genes, thereby improving the drought resistance of Populus davidiana.

[0013] Furthermore, by overexpressing PdbRAP2 Genes enhance SOD and POD activities in Populus davidianus plants.

[0014] Furthermore, by overexpressing PdbRAP2 Genes reduce H2O2 concentration and electrical conductivity.

[0015] Compared with the prior art, the present invention has the following beneficial effects: We found a PdbRAP2The researchers cloned the gene, constructed it into a plant overexpression vector, and transferred it into Populus serrata using stable genetic transformation technology. Overexpression of the gene was found to improve the tree's drought resistance. This invention will deepen research on the molecular basis of target traits in forestry breeding and has important implications for developing new stress-resistant varieties using key genes and improving the local ecological environment in our province. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The present invention overexpresses PdbRAP2 Screening of resistant Populus shanxin seedlings with the gene; in the figure, a is the callus induction process; b and c are the bud induction processes; d is the rooting stage of the resistant seedlings.

[0018] Figure 2 Under PEG stress PdbRAP2 Chemical tissue staining analysis of transgenic lines; from left to right, Evans blue staining, DAB staining, and NBT staining are shown.

[0019] Figure 3 for PdbRAP2 Determination of superoxide dismutase (SOD) activity in transgenic Populus davidiana under drought stress.

[0020] Figure 4 for PdbRAP2 Determination of peroxidase (POD) activity in transgenic Populus davidiana under drought stress.

[0021] Figure 5 for PdbRAP2 Determination of hydrogen peroxide (H2O2) content in transgenic Populus davidiana under drought stress.

[0022] Figure 6 for PdbRAP2 Determination of relative electrical conductivity of transgenic Populus davidiana under drought stress.

[0023] Figure 7 The effect of transgenic Populus davidianus on stomatal aperture; In the figure, A is PdbRAP2 Stomatal aperture detection of transgenic and wild-type Populus davidiana; B is PdbRAP2 Stomatal width / length values ​​of transgenic and wild-type Populus davidiana.

[0024] Figure 8 for PdbRAP2 Phenotypic changes of transgenic Populus davidiana plants and leaves under drought stress. DETAILED DESCRIPTION

[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 by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0026] Example 1: A new poplar PdbRAP2 Genes and their applications.

[0027] 1. Populus serrata PdbRAP2 Gene cloning (1) Populus dasyphylla PdbRAP2 Gene amplification Total RNA was extracted from wild-type Populus davidiana tissue culture seedlings (constant temperature 25°C, 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 Bioengineering Co., Ltd.) and sequenced to obtain the complete coding region (CDS) shown in SEQ ID NO.1. PdbRAP2 The amino acid sequence of the gene encoded by the Populus davidianus PdbRAP2 is shown in SEQ ID NO.2.

[0028] SEQ ID NO.1: ATGTGTGGAGGTGCCATCATTTCCGATTTCGTACCCGTTAAACGTGGCCGGAAACCGACCACTGAGGATCTCTGGTCTGAACTTGACTCCTTATCTGATTTTCTTGGACTTGATCATCGTTCTATGAACAATATTAATAATGGTAGCAAAAAAGAAAACCTATCAAATCTCAAGCTCGCTCAAAAGCCACGCCAGCCCAACCAAGTGATAACAGAGAGAGTTGAGAAGCCAAGCCAAGCAACAGAGCAAGAAGCCGGTAAAAAGAAGGTTCAGAGAACCAGAAAGAATGTGTACAGAGGAATAAGGCAAAGGCCATGGGGTAAATGGGCAGCTGAAATAAGAGACCCACACAAAGGTGTTAGAGTTTGGCTGGGCACTTACAACACAGCTGATGAAGCCGCTAAAGCTTATGATGAAGCCGCCAAGCGCATCCGTGGTGATAAAGCCAAGCTCAACTTCCCCCCTCAGCCACCACCAACGTCGGAGGCGGCGCCACCACCTGCCAAGAAGCGTTGCATCTTGGGTCCTGAAACGGCTGTTATGGCTAGTTTTGAGCAAATCTTGAACTCGGAGTCGTTTCATGGCCTGGAACCGCATCAGGCGGCGGCCCAGCTGAGTTGTGACGGAGGTGGAAGTGGTGATTATAATTGTGACCCTGTGGACCCTTGGATGCTTGATGATCTTATCAACGTCAGCTTAATTATTAGTAATATAATTAATTAA。

[0029] SEQ ID NO.2: MCGGAIISDFVPVKRGRKPTTEDLWSELDSLSDFLGLDHRSMNNINNGSKKENLSNLKLAQKQRQPNQVITERVEKPSQATEQEAGKKKVQRTRKNVYRGIRQRPWGKWAAEIRDPHKGV RVWLGTYNTADEAAKAYDEAAKRIRGDKAKLNFPPQPPPTSEAAPPPAKKRCILGPETAVMASFEQILNSESFHGLEPHQAAAQLSCDGGGSGDYNCDPVDPWMLDDLINVSLIISNIIN.

[0030] Table 1 Gene-specific primers Table 2 Amplification reaction system PCR reaction program: 95°C / 3 min; 94°C / 30 s, 55°C / 30 s, 72°C / 1 min, 35 cycles; 72°C / 7 min.

[0031] 2. Populus serrata PdbRAP2 Analysis of genes for drought tolerance 1. Populus dasyphylla PdbRAP2 Construction of gene overexpression vector (1) Populus dasyphylla PdbRAP2 Gene amplification according to PdbRAP2 The CDS sequence of the gene was determined and gene-specific primers were designed to ma I restriction sites were introduced at both ends of the RAP2 gene. The primers required for PCR amplification are pROKⅡ-RAP2-F (SEQ ID NO. 5) and pROKⅡ-RAP2-R (SEQ ID NO. 6). PCR amplification was performed using the primers listed in Table 3, using the recovered target gene product as a template. The PCR reaction system is shown in Table 4, and the PCR procedure is as follows.

[0032] Table 3 Gene-specific primers Table 4 Amplification reaction system PCR reaction program: 95°C / 3 min; 94°C / 30 s, 55°C / 30 s, 72°C / 1 min, 35 cycles; 72°C / 7 min.

[0033] (2) Populus dasyphylla PdbRAP2 Gene gel excision recovery After the PCR reaction, the target band was detected by agarose gel electrophoresis. The target gene product was then recovered using a purification and recovery kit (Guangzhou Feiyang Bioengineering Co., Ltd.) (see the instructions for specific experimental procedures). The recovered target gene product was detected by agarose gel electrophoresis and its concentration was measured. The product was then stored at -20°C until needed.

[0034] (3) Extraction of pROKII plasmid The pROKII plasmid was extracted using a plasmid extraction kit (Guangzhou Feiyang Bioengineering Co., Ltd.) (see the instruction manual for specific experimental steps). The extracted plasmid was subjected to agarose gel electrophoresis and concentration measurement and stored in a -20°C refrigerator.

[0035] (4) Enzyme digestion of pROKII plasmid Using S ma The pROKII plasmid was digested with restriction endonucleases I and the reaction system was shown in Table 5.

[0036] Table 5 Enzyme digestion reaction system The reaction procedure was: 37°C, 2h.

[0037] After the enzyme digestion is completed, agarose gel electrophoresis is performed (the volume ratio before and after the digestion is 1:2 when spotting). After successful enzyme digestion, the digested vector is purified and recovered using a purification and recovery kit and stored in a -20°C refrigerator for future use.

[0038] (5) Ligation of the target gene product with the excised pROKII vector Using the homologous fusion method, a one-step PCR cloning kit (Suzhou Nearshore Protein Technology Co., Ltd.) was used to ligate the target gene product with the digested pROKII vector to obtain a ligation product. The reaction system is shown in Table 6, and the reaction procedure was: 37°C for 15 min; 50°C for 15 min.

[0039] Table 6 Ligation system of target gene product and excised pROKII vector (6) Heat shock transformation of Escherichia coli The ligation product was transformed into E. coli Top10 competent cells using the heat shock transformation method as follows: 0. Take 5 μL of ligation solution and add it to 50 μL of E. coli competent cells, incubate on ice for 30 min, and gently tap to mix 3 times every 10 min; ② Place in a 42°C water bath for 90 seconds, then immediately place in an ice bath for 2 minutes; ③ Add 400 μL LB liquid medium (without antibiotics) and culture at 37°C with shaking at 220 rpm for 1 h; ④ Spread 200 μL of bacterial solution on LB solid medium plate (containing 50 mg / L kanamycin sulfate (Kan)) and culture at 37°C overnight.

[0040] (7) PCR detection of bacterial liquid Single colonies on the plate were picked and placed in LB liquid medium (containing 50 mg / L Kan) and cultured at 37°C with shaking at 220 rpm for 4 h. PCR detection was then performed using the bacterial solution as a template using vector primers pROKⅡ-F (SEQ ID NO. 7) and pROKⅡ-R (SEQ ID NO. 8). The vector primers were located at S ma The total length of the enzyme cutting site is 447 bp. The reaction system and procedure are as follows.

[0041] SEQ ID NO.7: 5'-AGACGTTCCAACCACGTCTT-3'; SEQ ID NO. 8: 5'-CCAGTGAATTCCCGATCTAG-3'.

[0042] Table 7 Bacterial liquid PCR reaction system PCR reaction program: 95°C / 3 min; 94°C / 30 s, 55°C / 30 s, 72°C / 1 min, 30 cycles; 72°C / 7 min. The recovered target gene was homologously fused with the pROKⅡ vector after enzyme digestion and heat-shocked. Single colonies on the plate were picked and the bacterial solution was PCR tested using vector primers. The bacterial solution with the correct band position was sent to a biological company for sequencing. The sequencing results were correct, indicating that PdbRAP2 The gene overexpression vector was successfully constructed and named PdbRAP2 - pROKⅡ plasmid.

[0043] 2. Populus dasyphylla PdbRAP2 Preparation of genetically engineered bacteria Use a plasmid extraction kit to extract the constructed PdbRAP2 Gene overexpression vector. PdbRAP2 was transformed into - The pROKⅡ plasmid was transformed into EHA105 Agrobacterium competent cells. The experimental steps are as follows:

[0044] 1) Clean the electric shock cup with anhydrous ethanol and sterilize it under ultraviolet light in a clean bench for 30 minutes. 2) Add 2 μL of plasmid to 50 μL of Agrobacterium competent cells, mix well, and transfer to an electroporation cuvette; 3) Electric shock with 1700 V voltage; 4) Add 400 μL of LB liquid medium (without antibiotics) to the cuvette, mix thoroughly, transfer to a new 1.5 mL centrifuge tube, and incubate at 28°C, 220 rpm, and shake for 1 h. 5) Spread 200 μL of bacterial solution on LB solid medium (containing 50 mg / L Kan) and culture at 28°C for 2 days.

[0045] Pick a single colony from the plate and place it in LB liquid culture medium. After the electrophoresis detection band position is correct, the strain is stored in a -80℃ refrigerator.

[0046] 3. Stable genetic transformation of Populus shanxinensis (1) Agrobacterium-mediated transformation of Populus shanxinensis ① Activation of engineered bacteria: Through the three-zone demarcation method, PdbRAP2 The engineered bacterial cultures of the gene overexpression vector and editing vector were activated in LB solid medium (containing 50 mg / L Kan) and cultured at 28°C for 2 days. ②Pick a single colony and place it in 50 mL LB liquid medium (containing 50 mg / L Kan) and culture at 28°C with shaking at 220 rpm until the OD 600 =0.7; ③ Transfer the bacterial suspension 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 acetosyringone (AS)) to resuspend the bacteria. Incubate at 28°C, shaking at 180 rpm for 40 min. ④ Pour the infection solution into a sterile culture dish, cut wounds on the leaves of Populus davidiana in the infection solution, and then soak them in the infection solution for 5 minutes; ⑤ Place the cut leaves on sterile filter paper to absorb the bacterial solution, place them in differentiation medium (containing 150 μM AS), and culture in the dark at 25°C for 3 days.

[0047] (2) Screening of resistant Populus shanxin seedlings ① Sterilization: After dark culture, transfer the leaves of Populus davidianus to differentiation medium (containing 30 mg / L Kan and 300 mg / L cephalosporin (Cef)) and culture in an artificial climate chamber; ② Subculture: Change the culture medium (differentiation medium + 30 mg / L Kan, 300 mg / L Cef) every 7 days until adventitious buds grow; transfer to stemming 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.

[0048] The experimental results are as follows Figure 1 As shown, resistant seedlings of Populus shanxin were obtained.

[0049] (3) PdbRAP2 Identification of transgenic lines ① DNA extraction: DNA of overexpressing transgenic plants was extracted using a plant DNA extraction kit (see the instructions for specific experimental steps). After DNA extraction, agarose gel electrophoresis was performed and the concentration was measured, and the DNA was stored in a -20°C refrigerator.

[0050] ②PCR identification: Using the extracted transgenic plant DNA as a template, using pROKⅡ-F and pROKⅡ-R as primers. After the PCR is completed, PCR identification is performed. The correct band proves that the overexpression transgenic line has been obtained. The overexpression transgenic line screened and identified is recorded as PdbRAP2 -OE.

[0051] 4. Populus dasyphylla PdbRAP2 Analysis of genes for drought tolerance First, the 20-day-old Populus davidianus PdbRAP2 -OE and wild-type tissue culture seedlings WT were subjected to 20% PEG 6000 stress for 12 h, and water irrigation was used as a control. Then, the leaves of the plants were used for histochemical staining with Evans blue, DAB, and NBT. The remaining materials were quick-frozen and ground in liquid nitrogen for detection of physiological indicators.

[0052] (1) Histochemical staining ①Evans blue staining: Leaves from stress-treated and unstressed plants (water-irrigated plants) were placed in centrifuge tubes. 0.5 mg / mL Evans blue staining solution was added and vacuumed for 30 minutes. The vacuum was maintained and the staining was continued overnight. After staining, the leaves were decolorized by boiling with 75% ethanol and 5% glycerol.

[0053] Figure 2 Evans blue staining solution can enter dead cells and dye them blue. The depth of the staining can be used to determine the number of dead cells in the cells. The more severely damaged the cells, the more dead cells there are. As shown in the figure, under PEG stress conditions, compared with the wild type, PdbRAP2 The leaves of the transgenic plants were lighter in color, indicating that the number of dead cells in the leaves of the transgenic plants was small. PdbRAP2 The overexpression transgenic lines showed low damage after stress.

[0054] ②DAB staining: Leaves from stress-treated and unstressed plants (water-irrigated plants) were placed in centrifuge tubes and stained overnight at room temperature with 1 mg / mL DAB solution. After staining, the leaves were decolorized by boiling with 75% ethanol and 5% glycerol. Figure 2 DAB staining, the oxygen ions released by H2O2 in the cells can oxidize DAB to form a brown precipitate. The depth of the staining can be used to determine the amount of H2O2 released in the cells. The more severely damaged the cells are, the more H2O2 is released. As shown in the figure, under PEG stress conditions, compared with the wild type, PdbRAP2 The leaves of plants overexpressing the transgenic gene are lighter in color, indicating that PdbRAP2 The overexpressing transgenic lines showed low damage after stress.

[0055] ③NBT staining: Leaves from stress-treated and unstressed plants (water-irrigated plants) were placed in centrifuge tubes and stained overnight at room temperature with 0.25 mg / mL NBT staining solution. After staining, the leaves were decolorized by boiling with 75% ethanol and 5% glycerol.

[0056] Figure 2 NBT staining is used to detect superoxide anions (O2 - ) content, and according to the depth of staining, the superoxide anion (O2 - ) is more serious, the more serious the cell damage is, the more superoxide anion (content O2 - ) more. Figure 2 As shown, under PEG stress conditions, compared with the wild type, PdbRAP2 The leaves of plants overexpressing the transgene were lighter in color, indicating that the overexpression RAP2 Superoxide anions (O2 - ) content was higher than that of superoxide anions (O2 - ) content is small, indicating that PdbRAP2 The damage degree of the gene strain after stress was low.

[0057] The above staining results show that PdbRAP2 The gene can positively regulate the drought tolerance of Populus davidiana.

[0058] (2) Measurement of physiological indicators ①Superoxide dismutase (SOD) activity assay (kit method): Accurately weigh plant tissue (0.3 g), add four times the volume of Reagent VIII - Homogenization Medium at a weight (g): volume (mL) ratio of 1:4, mince, homogenize under ice-water bath conditions to prepare a 20% homogenate, centrifuge at 3500 rpm for 10 minutes, and take the supernatant for testing. For specific operation steps and result calculation, refer to the SOD assay kit (Nanjing Jiancheng Bioengineering Research Institute). Reagent VIII - Homogenization Medium contains plant copper zinc - superoxide dismutase Cu 2+ Zn 2+ -SOD assay kit, catalog number: A001-4-1.

[0059] SOD can catalyze the dismutation reaction of superoxide anion free radicals, resist the damage of reactive oxygen or other peroxide free radicals to the cell membrane system, thereby improving the plant's resistance to stress. The test results are as follows: Figure 3 Under non-stress conditions (control), overexpression PdbRAP2 The SOD activities of the transgenic and wild-type Populus shanxin strains were roughly the same; under the abiotic stress conditions of PEG, the overexpression PdbRAP2 The SOD activity of the gene-transformed plants was higher than that of the wild type, indicating that the overexpressed plants after stable transformation have stronger resistance to adverse stress than the wild type plants. PdbRAP2 The gene can positively regulate SOD activity and drought tolerance in Populus davidiana.

[0060] ② Peroxidase (POD) activity assay (kit method): Pretreatment: Preparation of homogenates of young leaf plant tissues with high water content: Wipe clean the plant tissue of all moisture and impurities, accurately weigh the tissue, and add 9 volumes of homogenization medium (normal saline or phosphate buffer, 0.1 mol / L pH 7.2 is recommended) at a ratio of 1:9 (weight (g): volume (mL). Prepare a 10% homogenate in an ice-water bath. Centrifuge at 3500 rpm for 10 minutes, and remove the supernatant for analysis. Preparation of homogenates of dried plant tissues with low water content: Wipe clean the plant tissue of all moisture and impurities, mince, place in a mortar, add liquid nitrogen, grind into a powder, and transfer. Accurately weigh the tissue, add 9 volumes of homogenization medium (normal saline or phosphate buffer, 0.1 mol / L pH 7 is recommended) at a ratio of 1:9 (weight (g): volume (mL). Vortex mix and extract for 3-5 minutes, then centrifuge at 3500 rpm for 10 minutes, and remove the supernatant for analysis. The specific operation steps and result calculation refer to the POD determination kit (Nanjing Jiancheng Bioengineering Research Institute). Under the catalysis of peroxidase (POD), H2O2 oxidizes guaiacol into a dark brown product. Peroxidase is an important protective enzyme in plants that reduces the damage of oxygen free radicals and is closely related to the ability of plants to resist adverse stress. The experimental results are as follows: Figure 4 As shown. Under non-stress conditions (control), the POD activity of the RAP2 gene-overexpressing Populus shanxin and wild-type Populus shanxin was roughly the same; under the abiotic stress conditions of PEG, the POD activity of the RAP2 gene-overexpressing Populus shanxin was higher than that of the wild-type, indicating that the RAP2 gene-overexpressing plants have a stronger ability to resist adverse stress than the wild-type. PdbRAP2 The gene expression level is positively correlated with the enzyme activity of POD, indicating that it can improve the plant's stress resistance by regulating the activity of antioxidant enzymes in the plant body.

[0061] ③ Determination of H2O2 content (kit method) Accurately weigh the tissue and add 9 volumes of 0.9% saline at a weight (g): volume (mL) ratio of 1:9. Mechanically homogenize the tissue in an ice-water bath. Centrifuge at 1000 rpm for 10 minutes, and collect 10% of the supernatant for analysis. For detailed steps and calculation of results, refer to the SOD assay kit (Nanjing Jiancheng Bioengineering Research Institute).

[0062] H2O2, as a reactive oxygen species, is ubiquitous in organisms and is an important hub for the conversion of reactive oxygen species. Among the numerous oxidative metabolites, H2O2 accelerates the process of cell aging and disintegration. The principle is that it can damage cell membranes and directly or indirectly oxidize biological macromolecules. Under adverse stress, the stronger the plant's ability to resist adversity, the lower the H2O2 content accumulated in the body. The experimental results are as follows: Figure 5 shown.

[0063] like Figure 5 As shown, under non-stress conditions (control), overexpression PdbRAP2 The hydrogen peroxide content of the gene-expressing Populus shanxin and wild-type Populus shanxin was roughly the same; under the abiotic stress condition of PEG, the overexpression PdbRAP2 The H2O2 concentration of the gene strain was lower than that of the wild type, indicating that the experimental group plants had a stronger ability to resist adverse stress than the wild type. PdbRAP2 Genes can improve plants' ability to withstand stress.

[0064] ④ Determination of relative conductivity: Fresh leaves of uniform size, after stress treatment, were rinsed three times with double-distilled water and then ultrapure water. Surface moisture was removed with filter paper and placed in a 50 mL centrifuge tube. 30 mL of ultrapure water was added and vacuum pumped for 15 minutes. The conductivity was measured using a conductivity meter, which was recorded as S1. The centrifuge tube was then placed in a 90°C water bath for 20 minutes. After cooling to room temperature, the conductivity was measured and recorded as S2. The relative conductivity of plant mesophyll is a basic indicator of plant cell membrane permeability. When plants are exposed to stress, cell membranes are damaged, membrane permeability increases, and electrolytes leak out of the cells. Lower relative conductivity reflects higher stress tolerance. Calculation: Relative conductivity = S1 / S2 x 100%.

[0065] The experimental results are as follows Figure 6 As shown, under non-stress conditions (control), overexpression PdbRAP2 The relative conductivity of the genetically modified Populus shanxin strain and the wild-type Populus shanxin strain was roughly the same; under the abiotic stress condition of PEG, the overexpression PdbRAP2 The relative conductivity of the gene strain is lower than that of the wild type, indicating that overexpression PdbRAP2 The ability of gene plants to resist adverse stress is stronger than that of wild type. PdbRAP2 Genes can improve a plant's ability to withstand stress.

[0066] (3) Determination of stomatal aperture Take the leaves of transgenic and wild-type Populus dahliae before and after stress treatment, tear off the lower epidermis of the leaves, put them in stomatal opening solution (30 mM KCl, 10 mM Mes-KOH, pH adjusted to 6.15) for 2 hours, and observe them under a microscope. The experimental results are as follows: Figure 7 As shown in the figure, under drought stress, compared with the wild type PdbRAP2 The overexpression strain had a higher degree of stomatal closure. The results showed that the overexpression strain of this gene can improve the water retention capacity of the plant by regulating the opening and closing of stomatal pores, thereby improving the drought resistance of the plant.

[0067] (4) Phenotypic analysis of drought stress The growth will be consistent PdbRAP2 Gene-overexpressing transgenic and wild-type Populus davidiana rooted tissue culture seedlings were transplanted into sterilized soil (substrate soil / perlite / vermiculite = 3 / 1 / 1) and cultured in an artificial climate chamber for 20 days. The wild-type and transgenic lines were then treated with 20% PEG solution for 5 days, and the phenotypic changes of the plants under PEG stress were observed.

[0068] The experimental results are as follows Figure 8 Under non-stress conditions, the growth of transgenic Populus shanxinensis plants was similar to that of wild type (WT) Populus shanxinensis; after PEG stress treatment, the overexpression PdbRAP2 The growth of the genetically modified plants was better than that of the wild type, and the leaves wilted less severely, indicating that PdbRAP2 Genes can improve the plant's resistance to drought stress.

[0069] The results of chemical tissue staining, physiological index detection and stomatal aperture experiments showed that the RAP2 gene can significantly improve the drought resistance of transgenic Populus davidiana, laying the preliminary research foundation for the cultivation of new drought-resistant forest varieties.

[0070] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0071] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A new mountain poplar PdbRAP2 A gene characterized by described PdbRAP2 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

2. An overexpression vector, characterized in that: Containing the poplar of claim 1 PdbRAP2 Gene.

3. The overexpression vector according to claim 2, characterized in that You Shan Xinyang PdbRAP2 The gene was ligated with the pROKII vector.

4. A genetically engineered bacterium, characterized in that: Obtained by transforming Agrobacterium competent cells with the overexpression vector according to claim 3.

5. A method for improving drought tolerance of Populus davidiana, characterized in that: The steps include: Using the cDNA of Populus davidiana as a template and the sequences shown in SEQ ID NO.3 to SEQ ID NO.4 as primers, the target gene fragment was amplified; The target gene fragment was connected to the linearized pROKII plasmid to obtain PdbRAP2 Gene overexpression vector; PdbRAP2 After the gene overexpression vector was transformed into Agrobacterium competent cells, it was transformed into Populus davidianus using the Agrobacterium-mediated method to obtain a new drought-resistant high-altitude transgenic variety.

6. The new poplar of claim 1 PdbRAP2 Use of the gene, the overexpression vector according to any one of claims 2 to 3, or the genetically engineered bacteria according to claim 4 in improving the drought resistance of Populus davidianus.

7. The use according to claim 6, characterized in that By genetic engineering to overexpress the PdbRAP2 genes, thereby improving the drought resistance of Populus davidiana.

8. The use according to claim 7, characterized in that By overexpressing PdbRAP2 Genes enhance SOD and POD activities in Populus davidianus plants.

9. The use according to claim 7, characterized in that By overexpressing PdbRAP2 Genes reduce H2O2 concentration and electrical conductivity.

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