Application of PdbUNE12 gene in improving salt tolerance of populus sutchuenensis and method for improving salt tolerance of populus sutchuenensis

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a PdbUNE12 gene in improving the salt tolerance of populus sutchuenensis and a method for improving the salt tolerance of populus sutchuenensis. The nucleotide sequence of the PdbUNE12 gene is as shown in SEQ ID NO. 1 (Sequence Identifier Number 1). The invention finds that the overexpressed PdbUNE12 gene can reduce the electrolyte permeability of a plant under salt stress, can reduce the malondialdehyde content of the plant under salt stress, and can improve the SOD enzyme activity of the plant under salt stress, so that the invention provides the application of the PdbUNE12 gene in improving the salt tolerance of populus sutchuenensis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to PdbUNE12 the application of a gene in improving the salt tolerance of Populus davidiana × Populus bolleana and a method for improving the salt tolerance of Populus davidiana × Populus bolleana. Background Art

[0002] In the natural environment, the growth and development of plants may be restricted by various abiotic stresses. Among them, salt stress is one of the most common abiotic stresses, which can induce secondary stresses such as ion, osmotic, and oxidative stresses, and have a series of negative effects on plants, affecting the survival, growth, and development of plants. As the main regulators of stress-responsive genes, transcription factors have gradually become the preferred choice for plant improvement. Basic helix-loop-helix (bHLH) proteins are one of the most common types of transcription factors in eukaryotes and play a crucial role in the stress response of plants by regulating gene expression. They achieve this by recognizing specific motifs in the promoters of target genes; they are not only generally involved in the growth and metabolism of plants, but also play an important role in the response of plants to adversity. However, most studies on bHLH transcription factors have focused on a small number of model plants and crops. Although a variety of molecular biology methods have been used to explore the role of bHLH transcription factors in various stages of plant growth and development, as well as in metabolic pathways and stress responses, there is still much to be learned about the functions and regulatory roles of these proteins.

[0003] Populus davidiana × Populus bolleana ( Populus davidiana × Populus bolleana ), belonging to the Salicaceae family and the Populus genus, is an excellent clone selected by artificial hybridization of Populus davidiana and Populus bolleana. This hybrid grows rapidly, has strong adaptability and good stress resistance, and is a valuable ornamental tree species for urban and courtyard greening in cold northern regions, with significant economic and ecological value. Although bHLH proteins have been found to play a key role in the response of various plants to abiotic stresses, the research on the salt tolerance-related functions of bHLH in Populus davidiana × Populus bolleana is still limited. Exploring genes related to the stress resistance of forest plants, studying their physiological function regulation characteristics and morphological regulation response mechanisms, is of great significance for promoting the research of forest plant molecular biology technology and cultivating new forest plant varieties. Summary of the Invention

[0004] To solve the above problems, the present invention provides PdbUNE12 the application of a gene in improving the salt tolerance of Populus davidiana × Populus bolleana and a method for improving the salt tolerance of Populus davidiana × Populus bolleana.

[0005] PdbUNE12 The application of a gene in improving the salt tolerance of Populus davidiana × Populus bolleana, wherein the PdbUNE12

[0006] Populus davidiana × P. bolleana PdbUNE12 The amino acid sequence of

[0006] is shown in SEQ ID NO.2. Specifically, SEQ ID NO.2 is: MAGNPPPEGLGDDFFEQILAGQPPGYGGEAVGSTSLPMMGLQLGSSAANVGLMRSGANNNMGMMPLGLNLEHHGFLRQQQDDGSSSLDTNNSNNINNASPFSITSAGFLGRDSVHMTSLFPTFGQLQIHSSIRSTPPPLGPPQIHQFNSQPTSGAVSAVPQPPGIRPRVRARRGQATDPHSIAERLRRVRITERVKALQELVPTCNKTDRAAMLDEIVDYVKFLRLQVKVLSMSRLGAAGAVAQLVADVPLSSVQGEGIEGGGDQQAWENWSNDGTEQEVAKLMEEDVGAAMQLLQSKALCIMPVSLASAIFRARPPNAPTLVKPESNPPS.

[0007] Preferably, overexpressing PdbUNE12 the gene improves the salt tolerance of Populus davidiana × P. bolleana.

[0008] Preferably, the method for overexpressing PdbUNE12 the gene is as follows: Performing PCR amplification using Populus davidiana × P. bolleana cDNA as a template to obtain an amplification product; Connecting the amplification product to the vector pROKⅡ to obtain an overexpression vector; Transforming the overexpression vector into Agrobacterium to up-regulate the expression level of the MdTyDC gene.

[0009] Preferably, the primers used in the PCR amplification are shown in SEQ ID NO.9~SEQ ID NO.10.

[0010] Preferably, the amplification system is as follows: 10×Ex Taq PCR buffer, dNTP Mix, pROKⅡ-PdbUNE12-F, pROKⅡ-PdbUNE12-R, Populus davidiana × P. bolleana cDNA template, Ex Taq, ddH2O.

[0011] Preferably, when connecting the amplification product to the vector pROKⅡ, the pROKⅡ vector is linearized at Sma Ⅰ.

[0012] Preferably, the ligation reaction system is as follows: 10×T4 ligase buffer, pROKⅡ vector, PdbUNE12Gene, T4 DNA ligase.

[0013] A method for improving the salt tolerance of Populus davidiana×P. bolleana, which comprises introducing the PdbUNE12 gene into Populus davidiana×P. bolleana to obtain a transgenic plant, and overexpressing the PdbUNE12 gene, thereby improving the salt tolerance of Populus davidiana×P. bolleana.

[0014] Compared with the prior art, the beneficial effect of the present invention lies in: The present invention discovers a bHLH gene with salt tolerance function in Populus davidiana×P. bolleana PdbUNE12 , and discovers that overexpressing the PdbUNE12 gene can reduce the electrolyte leakage rate of the plant under salt stress, can reduce the malondialdehyde content of the plant under salt stress, and can increase the SOD enzyme activity of the plant under salt stress. Therefore, the present invention provides the PdbUNE12 application of the gene in improving the salt tolerance of Populus davidiana×P. bolleana. Brief Description of the Drawings

[0015] Figure 1 is the multiple sequence alignment of the PdbUNE12 gene of Populus davidiana×P. bolleana and the PdbUNE12 gene of Arabidopsis thaliana.

[0016] Figure 2 is the expression analysis of the PdbUNE12 gene in Populus davidiana×P. bolleana under salt stress.

[0017] Figure 3 is the acquisition of the PdbUNE12 gene overexpressing plants of Populus davidiana×P. bolleana.

[0018] Figure 4 is the sequencing of the PdbUNE12 gene knockout lines of Populus davidiana×P. bolleana.

[0019] Figure 5 is the stress phenotype of the PdbUNE12 transgenic plants of Populus davidiana×P. bolleana.

[0020] Figure 6 is the detection of the electrolyte leakage rate of the PdbUNE12 transgenic plants of Populus davidiana×P. bolleana.

[0021] Figure 7 is the detection of the malondialdehyde content of the PdbUNE12 transgenic plants of Populus davidiana×P. bolleana.

[0022] Figure 8 is the detection of the peroxidase activity of the PdbUNE12 transgenic plants of Populus davidiana×P. bolleana.

[0023] Figure 9 is the detection of the superoxide dismutase activity of the PdbUNE12 transgenic plants of Populus davidiana×P. bolleana. Detailed Embodiments

[0024] The following is a detailed description of the specific embodiments of the present invention. However, it should be understood that the protection scope 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 creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0025] The steps of stable transformation and resistance screening of correctly sequenced positive clones in the present invention are as follows: 1. Populus davidiana × P. bolleana PdbUNE1 2. Agrobacterium tumefaciens transformation of overexpression and knockout vector plasmids a. Preparation of Agrobacterium tumefaciens EHA105 competent cells (1) Dilute the activated strain 50-fold and shake it at 28°C and 220 rpm until OD 600 = 0.5 - 0.7.

[0026] (2) Transfer the bacterial solution into a 50 mL centrifuge tube, centrifuge at 4°C and 4000 rpm for 10 min, discard the supernatant, resuspend the cells with 20 mL of 10% sterile glycerol, centrifuge at 4°C and 6000 rpm for 10 min, and repeat this step 2 times.

[0027] (3) Resuspend the cells with 2 mL of 10% sterile glycerol, dispense 50 μL into each tube, quickly freeze in liquid nitrogen and store at -80°C for later use.

[0028] b. PdbUNE1 2. Transformation of overexpression and knockout vectors into Agrobacterium tumefaciens EHA105 competent cells by electroporation (1) Add 2 μL of the recombinant plasmid to the Agrobacterium tumefaciens competent cells, mix well and transfer to a clean electroporation cuvette.

[0029] (2) After electroporation with a voltage of 1700 V, add 600 μL of LB medium to the electroporation cuvette and mix well.

[0030] (3) Transfer the mixed medium to a 1.5 mL sterile centrifuge tube and culture at 28°C and 220 rpm for 1 h.

[0031] (4) Pipette 200 μL of the bacterial solution and spread it on the selection medium, and culture it upside down at 28°C for 2 days.

[0032] (5) Randomly pick colonies for enlarged culture and perform colony PCR detection with vector primers. Detect by 1% agarose gel electrophoresis, select positive clones and use them as engineering strains for later use.

[0033] 2. Stable genetic transformation and resistance screening of Populus davidiana × P. bolleana (1) PdbUNE1 2. Agrobacterium strains with gene overexpression and knockout vectors were activated to OD 600= 0.4-0.6, centrifuge at 3000 rpm for 10 min, resuspend the cells in 1 / 2MS liquid medium (containing AS at a final concentration of 150 μM), and adjust the OD 600 =0.2-0.4.

[0034] (2) Soak the Populus davidiana explants in the infection solution and gently shake them for 10 min.

[0035] (3) After soaking, blot the explants dry with sterilized filter paper, spread them on co-culture medium, and culture them in the dark for 3 days.

[0036] (4) After dark culture, transfer the explants to screening medium.

[0037] (5) After the resistant adventitious buds are produced, they are transferred to a rooting medium for seedling growth to obtain transgenic plants.

[0038] Example 1 Under salt stress PdbUNE12 Analysis of gene expression in Populus davidianus 1. Stress treatment of Populus shanxinensis potted seedlings The aseptic tissue culture seedlings of Populus shanxinensis that had been rooted for 2 weeks were transplanted into pots and placed at a temperature of 25°C, a photoperiod of 14 h light and 10 h dark, and a light intensity of 600 μmol / m 2 ·s soil culture room for 6 weeks. The 2-month-old P. shanxinensis potted seedlings with the same growth potential were divided into 6 groups, and the potted seedlings were treated with root irrigation stress using 500 mL of 250 mM NaCl solution. The treatment time was 0 h, 3 h, 6 h, 12 h, 24 h, and 48 h, respectively. At the same time, the P. shanxinensis potted seedlings with normal watering were used as controls, and 3 potted seedlings were treated at each time point.

[0039] 2. Extraction and reverse transcription of total RNA from stress-treated materials of Populus dahliae 2.1. Extraction of total RNA from stress-treated materials of Populus dahliae The total RNA of the stress-treated samples was extracted using a total RNA rapid extraction kit. The specific steps are as follows: (1) Add 600 μL of Buffer Rlysis-P solution to a 1.5 mL centrifuge tube.

[0040] (2) Take an appropriate amount of plant tissue material and grind it into powder using liquid nitrogen. Add it to a 1.5 mL centrifuge tube containing Buffer Rlysis-P solution and mix well.

[0041] (3) Add 60 μL of Buffer PCA solution to the well-mixed sample, mix well, and place it at -20 °C for 3 min.

[0042] (4) Centrifuge at 12,000 rpm at 4 °C for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0043] (5) Add 1 / 2 volume of phenol and chloroform to the supernatant respectively, and mix well. Centrifuge at 12,000 rpm at 4 °C for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0044] (6) Add an equal volume of chloroform to the supernatant, and mix well. Centrifuge at 12,000 rpm at 4 °C for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0045] (7) Add 1 / 3 volume of absolute ethanol to the supernatant, mix well, and let it stand at room temperature for 3 min. Centrifuge at 12,000 rpm at 4 °C for 5 min, and discard the supernatant.

[0046] (8) Aspirate 700 μL of 75% ethanol to wash the precipitate, centrifuge at 12,000 rpm at 4 °C for 3 min, and discard the supernatant.

[0047] (9) Repeat step (8) once.

[0048] (10) Invert at room temperature for 10 min, add 30 μL of DEPC-treated ddH2O to dissolve the precipitate, check the sample quality by 1% agarose gel electrophoresis, and simultaneously detect the sample concentration and purity using a nucleic acid concentration meter.

[0049] 2.2 Reverse transcription of total RNA from Populus davidiana × P. bolleana under stress treatment Since the RNA samples extracted using the phenol-chloroform plant total RNA rapid extraction kit may be contaminated with a certain proportion of genomic DNA molecules, it is necessary to remove the DNA molecules in the samples before reverse transcription of the sample RNA. The specific steps are as follows: (1) Genomic DNA removal reaction Reaction system: 2.0 μL of 5×gDNA Eraser Buffer, 1.0 μL of gDNA Eraser, 1.0 μg of Total RNA, supplemented with RNase Free ddH2O to 10.0 μL Reaction conditions: 42 °C for 2 min, 4 °C ∞.

[0050] (2) Reverse transcription reaction After the reaction of removing genomic DNA from the sample is completed, add the drugs required for the reverse transcription reaction, mix well and immediately carry out the reverse transcription reaction. The specific steps are as follows: Reaction system: 10.0 μL of the reaction solution in step 1, 1.0 μL of Prime Script RT Enzyme Mix I, 1.0 μL of RT Primer Mix, 4.0 μL of 5× Prime Script Buffer 2 (for Real Time), RNase Free ddH2O to 20.0 μL Reaction conditions: 37℃ for 15 min, 85℃ for 5 sec, 4℃ infinity.

[0051] The quality of the reverse transcription product was checked by 1% agarose gel electrophoresis, diluted 5 times and used as a template for RT-qPCR, and the remaining sample was stored at -20°C.

[0052] 3. Under salt stress PdbUNE12 Analysis of gene expression in different tissues of Populus davidianus Populus shanxiensis PdbUNE12 The full-length gene sequence was obtained, RT-qPCR primers were designed, and RT-qPCR technology was used to analyze the stress-treated Populus shanxinensis materials. PdbUNE12 The expression pattern of the gene was analyzed. The actin gene (Actin, Genbank accession number: KR180380) and the elongation factor gene (Ubiquitin, Genbank accession number: XM_035064935) of Populus shanxin were selected as internal reference genes for RT-qPCR. Three biological replicates were set for each sample, and the relative expression of the target gene was analyzed using the Livak method. The primers required for the reaction are shown in Table 2.

[0053] The RT-qPCR reaction system was as follows: cDNA template 2.0 μL, PdbUNE12-F (10 μM) 1.5 μL, PdbUNE12-R (10 μM) 1.5 μL, 2×SYBR Green Real time PCR Master Mix 10.0 μL, ddH2O to 20.0 μL The RT-qPCR reaction procedure is shown in Table 1.

[0054] Table 1 RT-qPCR reaction procedure Table 2 Primer sequences used for RT-qPCR The results are as follows Figure 2 As shown, PdbUNE12The gene showed a significant up - regulation under salt stress treatment. The research results indicate that Populus davidiana×P. bolleana PdbUNE12 gene responds significantly to salt stress.

[0055] Example 2 Populus davidiana×P. bolleana PdbUNE12 Obtaining of over - expression plants 1. Cloning of Populus davidiana×P. bolleana PdbUNE12 gene Based on the full - length sequence of Populus davidiana×P. bolleana PdbUNE12 gene, specific gene primers were designed. A homologous arm upstream of the Sma Ⅰ restriction site was introduced at the 5' end, and a homologous arm downstream of the Sma Ⅰ restriction site was introduced at the 3' end. At the same time, vector primers pROKII - F and pROKII - R were designed, as shown in Table 3.

[0056] Table 3 Primer sequences used for constructing over - expression vector Using Populus davidiana×P. bolleana cDNA as a template, the Populus davidiana×P. bolleana PdbUNE1 gene was amplified using the designed gene - specific primers. The gene cloning reaction system is as follows: 10×Ex Taq PCR buffer (20 mM) 2.0 μL, dNTP Mix (2.5 mM) 1.6 μL, pROKⅡ - PdbUNE12 - F (10 μM) 0.5 μL, pROKⅡ - PdbUNE12 - R (10 μM) 0.5 μL, Populus davidiana×P. bolleana cDNA template (200 ng / μL) 1.0 μL, Ex Taq (5 U / μL) 0.3 μL, ddH2O was added to make up to 20.0 μL.

[0057] The RT - qPCR reaction program is shown in Table 4.

[0058] Table 4 Reaction program 2. Ligation of Populus davidiana×P. bolleana PdbUNE12 gene with pROKⅡ vector The PdbUNE12 gene digested with enzymes and purified and recovered was ligated with Sma Ⅰ - linearized pROKⅡ vector. The ligation reaction system is as follows: 10×T4 ligase buffer 1.0 μL, pROKⅡ vector 5.0 μL, PdbUNE12 gene 3.0 μL, T4 DNA ligase 1.0 μL.

[0059] Reaction conditions: 16℃ for 12 h, 4℃ for 12 h.

[0060] 3. Transformation of the ligation product into Escherichia coli Prepare competent Escherichia coli Top10 strain. The specific steps are as follows: (0) Pipette 1 mL of the activated bacterial solution and dilute it 50-fold. Incubate at 37 °C with shaking at 220 rpm until OD 600 = 0.4 - 0.5.

[0061] (2) Transfer the bacterial solution to a pre-cooled 50 mL centrifuge tube and place it on ice for 10 min.

[0062] (3) Centrifuge at 3000×g for 10 min at 4 °C and discard the supernatant.

[0063] (4) Add 10 mL of pre-cooled 0.1 M CaCl2 to the centrifuge tube and place it on ice for 30 min.

[0064] (5) Centrifuge at 3000×g for 5 min at 4 °C and discard the supernatant.

[0065] (6) Add 1400 μL of pre-cooled CaCl2 and 600 μL of 50% glycerol to the pellet and mix well.

[0066] (7) Aliquot 100 μL per tube, and store at -80 °C under liquid nitrogen quick-freezing conditions.

[0067] 4. Heat shock transformation of the ligation product into competent Escherichia coli Top10. The specific steps are as follows: (1) Pipette 5 μL of the ligation solution into the competent cells, mix well, and place on ice for 30 min.

[0068] (2) Incubate in a 42 °C water bath for 1.5 min, then place on ice for 3 min. Add 500 μL of fresh LB, incubate at 37 °C with shaking at 220 rpm for 40 min.

[0069] (3) Centrifuge at 3500 rpm for 3 min. Discard 400 μL of the supernatant. Resuspend the cells and spread them on the resistance screening medium. Incubate at 37 °C upside down for 12 h.

[0070] 4. Identification of positive clones Pick a single colony and expand it in the liquid screening medium. Pipette the bacterial solution as a template for colony PCR detection. The reaction system is as follows: 2×Taq Master Mix 10.0 μL, pROKⅡ-F (10 μM) 0.8 μL, pROKⅡ-R (10 μM) 0.8 μL, Populus davidiana × P. bolleana cDNA template 1.0 μL, ddH2O to make up to 20.0 μL The reaction conditions are the same as in Table 4.

[0071] The PCR products were detected by 1% agarose gel electrophoresis. The positive clones were sent for sequencing. The strains with correct sequencing results were propagated, and the plasmids were extracted for standby.

[0072] After stable transformation, multiple overexpression PdbUNE12 plants, namely OE1 - OE5, were gradually obtained through resistance screening, as Figure 3 shown.

[0073] Example 3 Populus davidiana × P. bolleana PdbUNE12 Obtaining of knockout plants Construction of knockout expression vector pEG237 - PdbUNE1 2 1. Design of knockout expression targets The full - length sequence of the Populus davidiana × P. bolleana PdbUNE12 gene was input into the online processing website target Design (http: / / skl.scau.edu.cn / targetdesign / result / ). After calculation, the gene knockout targets were output. By comparing with the genomic sequence, the PdbUNE12 exon composition of the gene was obtained. The target position needed to be completely located within a whole exon and avoid the conserved domain of the gene. The selected Populus davidiana × P. bolleana PdbUNE12 gene knockout targets were analyzed for secondary structure using the online processing website RNA Folding Form (http: / / mfold.rna.albany.edu / ?q=mfold / RNAFolding - Form2.3) to avoid continuous pairing between the target sequence and the sgRNA sequence. The sequences of the 3 PdbUNE12 gene knockout targets designed were shown in Table 5.

[0074] Table 5 Target sequences used for constructing knockout expression vectors Note: The underlined part is the target sequence.

[0075] 2. Renaturation of knockout targets The designed targets were sent to a biological company for synthesis. The synthesized targets were single - stranded DNA and needed to be renatured into double - stranded DNA for the construction of knockout expression vectors. The renaturation reaction system was as follows: Primer F (100 μM) 9.0 μL, Primer R (100 μM) 9.0 μL, Ex Taq Buffer (20 mM) 2.0 μL Renaturation reaction program: 95℃ 5 min, 85℃ 5 min, 75℃ 5 min, 65℃ 5 min, 55℃ 5 min, 45℃ 5 min, 35℃ 5 min, 25℃ ∞.

[0076] The product after renaturation was diluted 100 times for standby.

[0077] 3. Populus davidiana × P. bolleana PdbUNE12 Ligation of the gene knockout target with the pEG237 vector Populus davidiana × P. bolleana after renaturation PdbUNE12 The gene knockout target was ligated with the linearized pEG237 vector. The specific system and conditions were the same as those in Example 2.

[0078] 4. Transformation of the ligation product into Escherichia coli and identification of positive clones were the same as those in Example 2.

[0079] Extraction PdbUNE12 DNA of the gene knockout plants was extracted. Molecular detection primers were used to amplify the fragment at the target site. The amplified fragment was sent for sequencing, and the gene knockout lines une1, une2, and une3 were obtained through sequencing detection. The sequencing results were as PdbUNE12 shown. The targets all Figure 4 knocked out the PdbUNE12 gene, resulting in mutations of stop codons, insertions of single bases, and deletions.

[0080] Example 4 Molecular detection of transgenic Populus davidiana × P. bolleana Through stable transformation, overexpression and knockout plants of the PdbUNE12 gene in Populus davidiana × P. bolleana were obtained. Molecular detection primers were designed according to the PdbUNE12 gene in Populus davidiana × P. bolleana. Overexpression plants were detected by RT-qPCR, and knockout plants were detected by sequencing of PCR products. The primer sequences are shown in Table 6.

[0081] Table 6 Primer sequences for detecting transgenic plants The results were as Figure 3 and Figure 4 shown.

[0082] Example 5 Detection of physiological and biochemical indexes of transgenic Populus davidiana × P. bolleana plants Transgenic Populus davidiana × P. bolleana PdbUNE12 gene overexpression, knockout, and wild-type potted seedlings were treated with 250 mM NaCl for 12 h. Plants without stress treatment were used as controls. After treatment, the electrolyte leakage rate, malondialdehyde content, POD enzyme activity, SOD enzyme activity, and chlorophyll content of the whole plants were measured.

[0083] 1. Determination of electrolyte leakage rate The measurement steps of the electrolyte leakage rate were as follows: (1) Clean the dust on the surface of the test material and dry it with filter paper. Take leaves of equal size and put them into a clean 50 mL centrifuge tube, add 30 mL of ultrapure water, pump for 2 minutes and maintain the vacuum state for 13 minutes. Measure the conductivity value with a conductivity meter and record it as S1.

[0084] (2) Place the centrifuge tube after S1 measurement in a 90℃ water bath and heat for 20 min. Cool to room temperature and measure its conductivity value, which is recorded as S2.

[0085] (3) Electrolyte permeability = S1 / S2.

[0086] 2. Determination of malondialdehyde content The measurement steps of MDA are as follows: (1) Take about 0.05-0.1 g of plant material, freeze it quickly with liquid nitrogen, grind it, and place it in a 2 mL centrifuge tube.

[0087] (2) Add 1.5 mL of 10% trichloroacetic acid (TCA), incubate at 4°C for 30 min, and centrifuge at 11,000 rpm for 10 min.

[0088] (3) Take 1 mL of the extract, add 1 mL of 0.6% TBA, mix well, boil in a boiling water bath for 15 min, cool rapidly, and centrifuge at 12,000 rpm for 20 min.

[0089] (4) Take the supernatant and measure the OD values at 600 nm, 532 nm and 450 nm respectively. The control tube contains 1 mL of water + 1 mL of 0.6% TBA.

[0090] (5) MDA (mmol / g) = [6.452 (OD 532 -OD 600 )-0.559OD 450 ] / W 3. Peroxidase activity assay The steps for measuring peroxidase activity are as follows: (1) Take 0.05-0.1 g of plant material, freeze it quickly in liquid nitrogen, and then grind it.

[0091] (2) Add 1.5 mL of 0.01 M phosphate buffer, react for 30 min, centrifuge at 11,000 rpm for 20 min, and collect the supernatant.

[0092] (3) Enzyme solution 0.5 mL + 0.8% H2O2 0.5 mL + 0.1 M phosphate buffer 0.5 mL + 0.1 M guaiacol 0.5 mL.

[0093] (4) Incubate in a 30°C water bath for 10 min.

[0094] (5) Measure the absorbance value (OD) of the sample at 470 nm, using double-distilled water instead of H2O2 as a control.

[0095] (6) POD activity = (N × △A) / W N: Dilution factor; △A: Absorbance; W: Weight of the material 4. Determination of superoxide dismutase (SOD) enzyme activity The measurement steps of superoxide dismutase (SOD) enzyme activity are as follows: (1) Take 0.05 g - 0.1 g of plant material, grind it in liquid nitrogen, add 1.5 mL of 1 / 15 M phosphate buffer, incubate at 4°C for 30 min, and centrifuge at 11000 rpm for 20 min.

[0096] (2) Enzyme solution + 1.5 mL of reaction solution. (3) After reacting for 10 min in a 6-level light incubator at 30°C, immediately measure the absorbance value. Use the reaction solution as a control and zero with the diluted enzyme solution.

[0097] (4) OD 560 0.5 mL H2O + 1.5 mL SOD reaction solution ------- Measured value (1 per batch) = A1 0.5 mL of enzyme solution (already diluted 3 - 5 times) + 1.5 mL H2O ------- Zero adjustment (1 per sample) 0.5 mL of enzyme solution (already diluted 3 - 5 times) + 1.5 mL of reaction solution ------- Measured value (1 per sample) = A2 (5) A 560 %: Percentage of absorbance value inhibited at 560 nm; V : Total volume of the extracted enzyme solution (mL); v : Volume of the enzyme solution in the reaction (mL); T : Reaction time (min); W : Net weight of the plant material powder.

[0098] Results 1. Determination of electrolyte leakage rate Relative conductivity is a physiological index indicating damage to the cell membrane. Damage to the cell membrane leads to an increase in electrolyte leakage, and the integrity of the cell membrane structure affects the stress resistance of plants.

[0099] Take Populus davidiana × P. bolleana seedlings with a growth amount meeting the detection requirements PdbUNE12 Measure the electrolyte leakage rate of the overexpressed and knockout potted seedlings of the gene, using wild-type Populus davidiana × P. bolleana plants as a control. As Figure 6It can be seen that for Populus davidiana × P. bolleana plants without stress, the values of electrolyte leakage rate are basically the same. For Populus davidiana × P. bolleana plants treated with stress, compared with the control, the electrolyte leakage of the overexpression plants of gene PdbUNE12 is significantly reduced, while the electrolyte leakage of the knockout lines is significantly increased. The above experimental results show that the overexpression of Populus davidiana × P. bolleana PdbUNE12 gene can reduce the electrolyte leakage rate of plants under salt stress.

[0100] 2. Determination of malondialdehyde content Malondialdehyde is the final decomposition product of membrane lipid peroxidation, and its content change can reflect the degree of damage of plants under stress.

[0101] The measurement results of malondialdehyde content are as Figure 7 It can be seen that for Populus davidiana × P. bolleana plants without stress, the values of malondialdehyde content are basically the same. For Populus davidiana × P. bolleana plants treated with stress, compared with the control, the malondialdehyde content of the overexpression plants of gene PdbUNE12 is significantly reduced, while the malondialdehyde content of the knockout lines is significantly increased. The above experimental results show that the overexpression of Populus davidiana × P. bolleana PdbUNE12 gene can reduce the malondialdehyde content of plants under salt stress.

[0102] 3. Determination of peroxidase activity Under the catalysis of peroxidase (POD), H2O2 oxidizes guaiacol into a tea-brown product. Peroxidase is an important protective enzyme in plants to reduce the damage of oxygen free radicals and is closely related to the ability of plants to resist stress.

[0103] The measurement of POD enzyme activity is as Figure 8 It can be seen that for Populus davidiana × P. bolleana plants without stress, the POD enzyme activities are basically the same. For Populus davidiana × P. bolleana plants treated with stress, compared with the control, the POD enzyme activities of the overexpression plants of gene PdbUNE12 are significantly increased, while the POD enzyme activities of the knockout lines are significantly reduced. The above experimental results show that the overexpression of Populus davidiana × P. bolleana PdbUNE12 gene can increase the POD enzyme activity of plants under salt stress.

[0104] 4. Determination of superoxide dismutase activity SOD can catalyze the dismutation reaction of superoxide anion radicals, resist the damage of reactive oxygen species or other peroxide radicals to the cell membrane system, and thus improve the stress resistance of plants.

[0105] The measurement of SOD enzyme activity is as Figure 9 It can be seen that for Populus davidiana × P. bolleana plants without stress, the SOD enzyme activities are basically the same. For Populus davidiana × P. bolleana plants treated with stress, compared with the control, the SOD enzyme activities of the overexpression plants of gene PdbUNE12The SOD enzyme activity of the overexpression plants was significantly increased, while that of the knockout lines was significantly decreased. The above experimental results indicate that Populus davidiana × P. bolleana PdbUNE12 Overexpression of the gene can increase the SOD enzyme activity of plants under salt stress.

[0106] Populus davidiana × P. bolleana PdbUNE12 The stress phenotypes of Populus davidiana × P. bolleana transgenic plants are as Figure 5 shown. Under salt stress, the knockout lines showed obvious wilting, while the wild-type plants showed less wilting. In contrast, there was no obvious change in the plant condition of the overexpression lines.

[0107] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value at both ends of each numerical range and any value between the two ends can be selected. To prevent redundancy, the present invention describes preferred embodiments.

[0108] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0109] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. PdbUNE12 Application of gene in improving salt tolerance of Populus davidiana×P. bolleana, wherein PdbUNE12 the nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. The application according to claim 1, wherein Improve the salt tolerance of Populus davidiana×P. bolleana by overexpressing PdbUNE12 genes.

3. The application according to claim 2, wherein The method of overexpression PdbUNE12 is as follows: Performing PCR amplification using cDNA of Populus davidiana×Populus bolleana as a template to obtain an amplification product; Linking the amplification product to the vector pROKⅡ to obtain an overexpression vector; Transforming the overexpression vector into Agrobacterium to up-regulate the expression level of the MdTyDC gene.

4. The application according to claim 3, wherein The primers used in the PCR amplification are shown as SEQ ID NO.9~SEQ ID NO.

10.

5. The application according to claim 3, wherein The amplification system is as follows: 10×Ex Taq PCR buffer, dNTP Mix, pROKⅡ-PdbUNE12-F, pROKⅡ-PdbUNE12-R, Populus davidiana×Populus bolleana cDNA template, Ex Taq, ddH2O.

6. The application according to claim 3, wherein When ligating the amplification product to the vector pROKⅡ, the pROKⅡ vector is linearized at Sma site Ⅰ.

7. The application according to claim 3, characterized in that, The ligation reaction system is as follows: 10×T4 ligase buffer, pROKⅡ vector, PdbUNE12 gene, T4 DNA ligase.

8. A method for improving the salt tolerance of Populus davidiana × P. bolleana, characterized in that, As described in claim 1 PdbUNE12 The gene is introduced into Populus davidiana×P. bolleana to obtain transgenic plants, and the PdbUNE12 gene is overexpressed, thereby improving the salt tolerance of Populus davidiana×P. bolleana.

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