SlBOR2 gene for improving basic salt resistance of plant, application of SlBOR2 gene, protein, plant overexpression vector and method

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to an SlBOR2 gene for improving basic salt resistance of plants, application of the SlBOR2 gene, protein, a plant overexpression vector and a method. The nucleotide sequence of the SlBOR2 gene is as shown in SEQ ID No.1 (Sequence Identifier Number 1). The invention finds that the gene SlBOR2 in salix mongolica can respond to the stress of alkaline salt for the first time, in the poplar with the SlBOR2 gene transferred, compared with the wild poplar, under the stress of sodium bicarbonate with different concentrations, the growth states of a transgenic line and the wild type are inhibited to different degrees, but the growth condition of the transgenic poplar is slightly influenced. In a word, compared with wild poplar, the SlBOR2 transgenic poplar strain is larger in biomass. Therefore, overexpression of the SlBOR2 improves the resistance of the poplar under the stress of the alkaline salt.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to an SlBOR2 gene for improving plant alkaline salt resistance, and its application, protein, plant overexpression vector and method. Background Art

[0002] With rapid urbanization, ecological and environmental challenges are becoming increasingly severe. Against this backdrop, demand for landscape plants in ecological restoration, landscape creation, and environmental greening continues to grow. Among the various adverse stresses that impact the growth of landscape plants, salinity is one of the most severe. Salinized soils, characterized by nutrient imbalance, low biodiversity, and limited water availability, significantly restrict the normal growth of plants in salinized areas.

[0003] The "soda" saline-alkali soil in the Songnen Plain in Northeast China is named because its main components are Na2CO3 and NaHCO3. The pH value of some soils is even higher than 9.2. The overall soil structure in this area is poor, and only a small number of salt-alkali tolerant grass species and shrubs can survive under such conditions, such as sheep fescue and Mongolian willow.

[0004] In recent years, more and more studies have confirmed that the negative impact of alkaline salt stress caused by Na2CO3 and NaHCO3 on crops is more complex and harmful than that of neutral salt stress. It was previously generally believed that alkaline salt stress was caused by HCO3 - and CO3 2- The presence of HCO3 brings additional high pH stress to plants, and the latest research shows that in addition to the high pH environment, - It is the main factor that makes alkaline salt stress more harmful.

[0005] Genetic improvement is crucial for creating new, more alkaline-tolerant and adaptable landscaping varieties for soda saline-alkali lands, enriching the landscaping plant resources there, improving the regional ecological environment, and promoting local ecological restoration and sustainable development. Therefore, studying the resistance of Mongolian willow to alkaline salt stress is a crucial task for improving the greening effect and diversity of ornamental plants in the region. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides an SlBOR2 gene for improving plant alkaline salt resistance and its application, protein, plant overexpression vector and method. Overexpression of the SlBOR2 gene can improve plant alkaline salt resistance.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a SlBOR2 gene for improving plant alkaline salt resistance. The nucleotide sequence of the SlBOR2 gene is shown in SEQ ID No. 1.

[0009] The present invention also provides the use of overexpressing the S1BOR2 gene described in the above technical solution in improving the alkaline salt resistance of plants.

[0010] The present invention also provides the use of overexpressing the S1BOR2 gene described in the above technical solution in increasing plant height, root length, fresh weight and dry weight of plants under alkaline salt conditions.

[0011] The present invention also provides the use of overexpressing the S1BOR2 gene described in the above technical solution in reducing the content of superoxide anions and / or hydrogen peroxide in plants under alkaline conditions.

[0012] The present invention also provides the S1BOR2 gene described in the above technical solution or the use described in the above technical solution, wherein the plant includes poplar.

[0013] The present invention also provides the S1BOR2 gene described in the above technical solution or the use described in the above technical solution, wherein the alkaline salt includes bicarbonate and / or carbonate.

[0014] The present invention also provides a protein encoded by the S1BOR2 gene described in the above technical solution, and the amino acid sequence of the protein is shown in SEQ ID No. 2.

[0015] The present invention also provides a plant overexpression vector, which is obtained by connecting the S1BOR2 gene described in the above technical solution to the plant expression vector pCAMBIA1300.

[0016] The present invention also provides a method for obtaining alkaline salt-resistant plants, comprising the following steps:

[0017] 1) Transforming the plant overexpression vector described in the above technical solution into Escherichia coli, extracting the plasmid, and obtaining a recombinant vector;

[0018] 2) transforming the recombinant vector obtained in step 1) into Agrobacterium to obtain transformed bacteria;

[0019] 3) Infecting plant leaves with the transformed bacteria obtained in step 2) to obtain alkaline salt-resistant plants.

[0020] Preferably, in step 3), the transformed bacteria infect plant leaves in the form of bacterial liquid, and the OD of the bacterial liquid is 600 The value is 0.6~0.8.

[0021] Beneficial effects:

[0022] This study, published in the journal Nature Communications, discovered for the first time that the SlBOR2 gene in Mongolian willow can respond to alkaline salt stress. In poplars transgenic with the SlBOR2 gene, growth was inhibited to varying degrees under different concentrations of sodium bicarbonate stress compared to wild-type poplars, although the transgenic poplars showed less of an effect. Overall, the SlBOR2 transgenic poplars had greater biomass than the wild-types. This suggests that overexpressing SlBOR2 improves poplar resistance to alkaline salt stress.

[0023] Experiments presented in this paper demonstrate that overexpressing the SlBOR2 gene in poplars resulted in transgenic poplars with greater biomass compared to wild-type plants. This suggests that SlBOR2 is differentially expressed under alkaline salt treatment. Subsequently, it was found that transgenic poplars overexpressing SlBOR2 exhibited enhanced alkaline tolerance compared to wild-type plants.

[0024] In this study, transgenic poplar plants grew better under sodium bicarbonate treatment than wild-type poplars. NBT, DAB, and Evans blue root staining also revealed that the transgenic plants suffered less damage. Therefore, the SlBOR2 gene has important application value in genetically improving salt and alkali tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0026] Figure 1 This is the electrophoresis diagram of SlBOR2 gene cloning;

[0027] Figure 2 for the subcellular localization of SlBOR2 protein;

[0028] Figure 3 This is the molecular identification result of transgenic poplar;

[0029] Figure 4 The phenotypes of wild-type poplar and transgenic poplar under treatment with different concentrations of sodium bicarbonate;

[0030] Figure 5 NBT, DAB and Evans blue staining of the roots of wild-type poplar and transgenic poplar plants under alkaline salt stress. DETAILED DESCRIPTION

[0031] The present invention provides a SlBOR2 gene for improving plant alkaline salt resistance. The nucleotide sequence of the SlBOR2 gene is shown in SEQ ID No. 1, and is specifically as follows:

[0032] SEQ ID No. 1:

[0033]

[0034] The SlBOR2 gene provided by the present invention can be used in the following applications: A. Confirming that the SlBOR2 gene has the ability to regulate plant resistance to alkaline salts; B. Breeding poplars with enhanced resistance to alkaline salts; C. Applying transgenic plants carrying the SlBOR2 gene to saline-alkali soils for soil remediation. The present invention utilizes the SlBOR2 gene to enhance plant resistance to alkaline salt stress. Transgenic plants exhibit less restricted plant height, root length, fresh weight, and dry weight after sodium bicarbonate stress. Poplar plants overexpressing the SlBOR2 gene may indirectly affect H through related ion transport. + The systemic absorption rate can resist the alkaline environment, maintain ion balance and improve the alkali resistance of the plant.

[0035] The present invention also provides the use of overexpressing the S1BOR2 gene described in the above technical solution in improving the alkaline salt resistance of plants.

[0036] The present invention also provides the use of overexpressing the S1BOR2 gene described in the above technical solution in increasing plant height, root length, fresh weight and dry weight of plants under alkaline salt conditions.

[0037] The present invention also provides the use of overexpressing the S1BOR2 gene described in the above technical solution in reducing the content of superoxide anions and / or hydrogen peroxide in plants under alkaline conditions.

[0038] In the present invention, the plant preferably includes poplar. In the present invention, the alkaline salt preferably includes bicarbonate and / or carbonate.

[0039] The present invention also provides a protein encoded by the S1BOR2 gene described in the above technical solution, the amino acid sequence of the protein is shown in SEQ ID No. 2, and is as follows:

[0040] SEQ ID No. 2:

[0041] .

[0042] The present invention also provides a plant overexpression vector, which is obtained by ligating the SlBOR2 gene described in the above technical solution to the plant expression vector pCAMBIA1300. The present invention does not specifically limit the method for ligating the SlBOR2 gene to the plant expression vector pCAMBIA1300, and those skilled in the art can use conventional methods.

[0043] The present invention also provides a method for obtaining alkaline salt-resistant plants, comprising the following steps:

[0044] 1) Transforming the plant overexpression vector described in the above technical solution into Escherichia coli, extracting the plasmid, and obtaining a recombinant vector;

[0045] 2) transforming the recombinant vector obtained in step 1) into Agrobacterium to obtain transformed bacteria;

[0046] 3) Infecting plant leaves with the transformed bacteria obtained in step 2) to obtain alkaline salt-resistant plants.

[0047] The present invention does not specifically limit the methods for transforming the plant overexpression vector into Escherichia coli and extracting the plasmid, and those skilled in the art may adopt conventional methods.

[0048] The present invention does not particularly limit the method for transforming the recombinant vector into Agrobacterium, and those skilled in the art can use conventional methods.

[0049] In the present invention, the transformant is preferably inoculated into plant leaves in the form of bacterial solution, and the OD of the bacterial solution is 600 The present invention has no particular limitation on the method for the transformed bacteria to infect plant leaves, and those skilled in the art can use conventional methods.

[0050] The invention inserts the SlBOR2 gene into the T-DNA region on the Ti plasmid of Agrobacterium, uses the Agrobacterium carrying the SlBOR2 gene to infect cut poplar leaves, transfers the SlBOR2 gene into poplars through the leaf disc method, identifies the transgenic poplars, and finally obtains poplars with the SlBOR2 gene transferred therein.

[0051] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] Genetic transformation methods of poplars and molecular identification of transgenic poplars

[0054] Polymerase chain reaction (PCR amplification) of SlBOR2 gene and construction of plant expression vector:

[0055] Based on the transcriptome sequencing results of Salix mongolica, upstream and downstream primers were designed using the full-length SlBOR2 gene as a template. Polymerase chain reaction (PCR) of the SlBOR2 gene was performed using Salix mongolica cDNA as a template. The amplification system consisted of 50 μl of 10× Ex Taq Buffer, 0.25 μl of Ex Taq enzyme, 1.25 μl of dNTPs, 1 μl of a 10 μM upstream primer, 1 μl of a 10 μM downstream primer, 1 μl of the cDNA template, and 40.5 μl of sterile water. The PCR amplification procedure was 95°C pre-denaturation for 3 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 60.5°C for 30 s, and extension at 72°C for 45 s, followed by full-length extension at 72°C for 10 min, and storage at 4°C. The PCR amplification product (SEQ ID No. 1) was obtained through these steps.

[0056] The upstream primer for PCR amplification of the SlBOR2 gene (SEQ ID No. 3) is: 5′-ACTTGGATGTTGGAGGTG-3′.

[0057] The downstream primer for PCR amplification of the SlBOR2 gene (SEQ ID No. 4) is 5′-GTAAGAATCGGCTGTGAA-3′.

[0058] The gel recovery kit was purchased from Kangwei Century Biotechnology Co., Ltd. with the product number CW2302. For specific experimental methods, please refer to the instructions.

[0059] Using TaKaRa's pMD19-T Vector Cloning Kit, ligate the recovered PCR product to the pMD19-T vector. Combine 4 µl of recovered PCR product, 1 µl of pMD19-T vector, and 5 µl of Solution I for a total of 20 µl. Ligation was performed overnight in a 16°C water bath.

[0060] To transform E. coli, thaw 100 μl of competent E. coli (DH5α) on ice. Competent cells are manufactured by Weidi Biotechnology, catalog number DL1001. Add 10 μl of the ligation product to the thawed competent cells, mix gently, and place on ice for 30 minutes. Heat shock the cells in a 42°C water bath for 1 minute, then quickly cool them on ice for 2 minutes. Be gentle and avoid shaking to prevent the competent cells from rupturing. Add 400 μl of LB liquid medium (preheated at 37°C for 2 minutes) to a centrifuge tube and incubate in a shaker at 37°C, 200 rpm, for 1 hour. Evenly spread the transformed E. coli culture on LB selection solid medium (50 mg / ml Amp). Once the culture is completely absorbed by the LB selection solid medium, incubate the cells in an inverted position overnight at 37°C.

[0061] A single strain of appropriate size was selected and placed in 5 ml of LB liquid medium (50 mg / ml Amp) and cultured in a shaker at 37°C, 200 rpm, for 12 hours. A 1 μl aliquot of the bacterial suspension was used for PCR identification. The PCR reaction system consisted of 10 μl of 2× Hieff PCR Master Mix, 1 μl of a 10 μM upstream primer, 1 μl of a 10 μM downstream primer, 1 μl of the monoclonal bacterial suspension as template, and 7 μl of sterile water. The PCR amplification procedure was 30 cycles of pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 30 seconds, annealing at 60.5°C for 30 seconds, and extension at 72°C for 45 seconds, followed by full-length extension at 72°C for 10 minutes. The target gene band was then confirmed by 1.0% agarose gel electrophoresis.

[0062] The positive bacterial solution was sent to a biological company for sequencing. The sequencing results were compared with the template, and the correct positive bacterial solution was retained for subsequent work. The plasmid extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd., with the catalog number DP103-03. The experimental method was referred to the instruction manual.

[0063] The PCR amplification product was ligated to the plant expression vector pCAMBIA1300 using a double enzyme digestion method. The manufacturer of T4 DNA ligase was Takara, with the product number 2011A.

[0064] The double enzyme digestion and ligation steps are as follows: A total of 50 μl of double enzyme digestion reaction system was used: 5 μl of 10×FD Green Buffer for the SlBOR2 gene, 1.2 μl of each of the upstream and downstream restriction enzymes, 20 μl of PCR amplification product, and 23 μl of sterile water. The double enzyme digestion reaction system for the pCAMBIA1300 vector was 5 μl of 10×FD Green Buffer, 1.2 μl of each of the upstream and downstream restriction enzymes, 20 μl of the pCAMBIA1300 vector plasmid, and 23 μl of sterile water. The double enzyme digestion reaction was carried out at 37°C for 1 hour. After gel electrophoresis, the target gene and vector digestion products were recovered using a gel recovery kit to recover the correct target band.

[0065] The ligation system consisted of 1 μl of T4 Buffer, 1 μl of T4 DNA ligase, 3 μl of the gene gel recovery product, and 5 μl of the vector gel recovery product after enzyme digestion. The ligation conditions were 16°C overnight.

[0066] The vector carrying the target gene was transformed into competent Escherichia coli DH5α using the principle of thermal expansion and contraction. The cells were evenly plated onto Kana-resistant LB solid medium and grown in an inverted position overnight at 37°C. Single colonies were then picked and identified by PCR. The PCR reaction system consisted of 10 μl of 2× Hieff PCR Master Mix, 1 μl of a 10 μM upstream primer, 1 μl of a 10 μM downstream primer, 1 μl of the template (a single clone of the bacterial culture), and 7 μl of sterile water. The PCR amplification protocol consisted of 30 cycles of initial denaturation at 95°C for 3 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60.5°C for 30 seconds, and extension at 72°C for 45 seconds. The final full-length extension at 72°C was performed for 10 minutes, and the cells were stored at 4°C. Positive colonies harboring the target gene were screened by gel electrophoresis using a 2000 DNA marker as a control. Positive colonies were cultured overnight in 5 ml of LB liquid medium supplemented with 5 μl of Kana antibiotic at 37°C and 200 rpm with shaking. The plasmid is extracted from the positive bacterial solution after amplification and sequenced for detection.

[0067] The positive bacterial solution is sent to a biological company for sequencing, the sequencing results are compared with the template, and the correct positive bacterial solution is retained for subsequent work.

[0068] Agrobacterium-mediated genetic transformation of poplar

[0069] Transformation of Agrobacterium competent cells: The correctly sequenced recombinant vector was transformed into Agrobacterium competent cell EHA105. Agrobacterium competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. with the product number pCAMBIA1300. Thaw the Agrobacterium competent cells stored at -80°C at room temperature. Once partially thawed, place them on ice to continue thawing. Generally, add 5μl of plasmid DNA to every 100μl of Agrobacterium competent cells, stir and mix thoroughly with a pipette tip, place them on ice and let them stand for 5 minutes, then place them in liquid nitrogen for 5 minutes, then place them in a 37°C water bath for 5 minutes, and finally place them on ice again for 5 minutes. Add 400μl of YEP liquid medium and culture at 28°C with shaking for 3 hours. Take about 150μl and spread it on a plate with Rif and Kana antibiotics. Culture them upside down in a 28°C incubator for 2-3 days. After a single colony grows, it is picked and inoculated in 1 μl of liquid culture medium containing Rif, 1 μl of Kana, and 1 ml of YEP. Culture is shaken at 28°C for one day. The resulting bacterial suspension is then subjected to PCR analysis. The PCR reaction system consists of 10 μl of 2× Hieff PCR Master Mix, 1 μl of a 10 μM upstream primer, 1 μl of a 10 μM downstream primer, 1 μl of the single-clone bacterial suspension as template, and 7 μl of sterile water. The PCR amplification procedure is 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 60.5°C for 30 s, and extension at 72°C for 45 s. The final full-length extension is performed at 72°C for 10 min, and storage is performed at 4°C. Using a 2000 DNA marker as a control, positive colonies harboring the target gene are screened by agarose gel electrophoresis. 200 μl of the positive bacterial suspension is then inoculated in 5 ml of YEP liquid culture medium, 5 μl of Kana, and 5 μl of Rif antibiotics, shaking for one day at 28°C and 200 rpm. 200 μl of the obtained bacterial solution was taken out and added into 200 μl of glycerol in a new centrifuge tube and stored in a -80°C refrigerator.

[0070] Agrobacterium-mediated leaf disc infection of poplars: First, prepare 50 ml OD 600 After activating the Agrobacterium containing the expression vector stored in a -80℃ refrigerator, pick a single colony and amplify it in YEP medium containing antibiotics. 600=0.6-0.8, transfer the bacterial solution into a centrifuge tube in a clean bench to infect the leaves. In the clean bench, use tissue culture scissors to cut 2-3 leaves from the top of a young, well-grown, 3-4-week-old 84K poplar tree of appropriate size and size, soak them in pre-prepared liquid differentiation medium (to prevent the leaves from air-drying). Use a tissue culture knife to carefully cut the leaves, removing the tip, surrounding leaves, base, and edge of the leaves, and make 3-4 cuts perpendicular to the veins. This operation is performed by pouring an appropriate amount of liquid differentiation medium into a flat plate covered with handkerchief paper to prevent the leaves from air-drying and the tissue culture knife from hitting the wall. At the same time, the Agrobacterium bacterial suspension transferred to the centrifuge tube was centrifuged at 5000 rpm for 10 minutes at room temperature. The supernatant was removed and the bacterial suspension was resuspended in liquid differentiation medium. The suspension was added to the plate with the cut leaves (the original medium was removed or the leaves were transferred to a new plate). The leaves were infected for 25 minutes (which can be adjusted appropriately according to the bacterial suspension concentration). The infected leaves were slightly blotted with filter paper and placed face down (to facilitate breathing) in differentiation medium (Agar) and co-cultured (inverted culture). The sealed plates were placed in a cardboard box and then placed in a plant incubator. Incubated at 25°C in the dark for three days. And the following points should be noted during the experiment: when cutting leaves, try to use large leaves, the incision must be neat, and make another cut at the petiole, but do not completely remove the petiole (the wound caused by scissors is not neat, and buds are likely to grow here); burned tissue culture equipment must be cooled before use (scissors, tweezers, and bottle mouth); do not use tweezers to clamp the part that is prone to callus growth, and discard the part that is clamped at the leaf corner; avoid contamination of Agrobacterium with other bacteria, and do not use Agrobacterium with poor vitality; mark the co-culture plus gene name and date on the sealing film.

[0071] Washing the leaves after co-cultivation: After three days of co-cultivation, wash away the Agrobacterium from the leaves and transfer them to resistance selection medium. First, prepare the resistance selection differentiation medium. In a laminar flow hood, add the pre-prepared hormone and antibiotic stock solutions to warm WPM medium to a final concentration of 0.03 mg / L 6-BA, 0.01 mg / L 1BA, 0.0008 mg / LTDZ, 200 mg / L Cef, 200 mg / L LTim, and 3 mg / L Hyg. Transfer the co-cultivated leaves to liquid differentiation medium and wash off any surface bacteria for 3-5 minutes. Prepare six bottles of 200 mg / L cefuroxime solution, 200 ml each. Wash the co-cultivated leaves approximately six times (depending on the severity of the infection; excessive washing will cause the leaves to turn black), each time for 5-8 minutes. Blot the leaves dry with filter paper and place them face down in the resistance selection differentiation medium, spacing the leaves widely to prevent Agrobacterium from spreading between leaves. Inverting the plates is not necessary. Incubate in a dark incubator to encourage the growth of white callus on the leaves. Once small buds emerge, place the plate under light conditions. If Agrobacterium appears on any leaves or if fluid flow is observed, change the plate immediately. During the experiment, be sure to disinfect the tweezers after each cephalosporin wash and allow them to cool before use. Gently shake the bottle when washing with cephalosporin to minimize damage to the leaves.

[0072] Screening and culture of transgenic poplars: Thereafter, the culture medium was changed weekly, and the culture medium formula was adjusted according to the state of the buds: Screening and culture for 7 days (second plate change): WPM + 0.03mg / L 6-BA + 0.01mg / L IBA + 0.001mg / L TDZ; Screening and culture for 14 days (third plate change): WPM + 0.03mg / L 6-BA + 0.01mg / L IBA + 0.0005mg / L TDZ; Screening and culture for about 21 days (fourth plate change): WPM + 0.03mg / L 6-BA + 0.01mg / L IBA + 0.0004mg / L TDZ; Screening and culture for 28 days (fifth plate change): WPM + 0.03mg / L 6-BA + 0.01mg / L IBA + 0.0004mg / L TDZ; Screening and culture for 35 days (sixth plate change): WPM + 0.03mg / L 6-BA+0.02mg / L IBA+0.0002mg / LTDZ, and gradually discard the leaf discs that have been screened out, turned yellow and black, and separate the small buds from the leaf discs. After the leaves differentiate and grow strong adventitious buds, the buds are separated and placed in rooting medium to induce their rooting; that is, screening and culture for 42 days (the seventh bottle change): MS+0.02mg / LNAA+0.05mg / L IBA; screening and culture for 49 days (the eighth bottle change): MS+0.02mg / LNAA+0.05mg / L IBA. The rooting process can also screen out some false positive plants that cannot take root. It should be noted that agar should be used to prepare the screening medium instead of plant gel during screening. The rooted plants are subcultured in a plant incubator.

[0073] Identification of transgenic poplars

[0074] DNA-Level Identification of Transgenic Poplars

[0075] The upstream primer for PCR identification of SlBOR2 gene was 5′-ACTTGGATGTTGGAGGTG-3′.

[0076] The downstream primer for PCR identification of SlBOR2 gene was 5′-GTAAGAATCGGCTGTGAA-3′.

[0077] The amplified target band size was 2145 bp.

[0078] CTAB DNA extraction from poplars: Pre-set a water bath to 65°C. First, select healthy transgenic poplar leaves and sample them in a laminar flow hood. Place the leaves in a 2.0ml centrifuge tube and freeze in liquid nitrogen. Grind the leaves into a white powder using an oscillating grinder. Add 500μl of CTAB, preheated to 65°C in a water bath, to the ground sample and mix thoroughly by inverting. Incubate the sample in a 65°C water bath for 20 minutes, carefully inverting the tube every 5 minutes to mix thoroughly. Centrifuge at 12,000 rpm for 10 minutes at room temperature, and transfer the supernatant to a fresh 1.5ml centrifuge tube. Add 250μl of chloroform and 250μl of Tris-saturated phenol, mix thoroughly, and centrifuge at 12,000 rpm for 5 minutes at room temperature. Add another 250μl of chloroform and 250μl of Tris-saturated phenol, mix thoroughly, and centrifuge at 12,000 rpm for 5 minutes at room temperature. Slowly aspirate the supernatant (being careful not to touch the lower layer) and place it into a new 1.5ml centrifuge tube. Add 800μl of anhydrous ethanol and 100μl of 3M NaAc and precipitate the DNA for 20 minutes. Centrifuge at 12,000 rpm for 20 minutes at room temperature. Discard the supernatant and wash the DNA with 500μl of 75% ethanol. Remove the ethanol with a pipette. Dissolve the DNA in 30-50μl of sterile water.

[0079] DNA from transgenic and wild-type poplars was extracted using the CTAB method. PCR identification was performed using these DNA templates. The PCR reaction system consisted of 10 μl of 2× Hieff PCR Master Mix, 1 μl of a 10 μM upstream primer, 1 μl of a 10 μM downstream primer, 1 μl of template DNA, and 7 μl of sterile water. The PCR amplification procedure included 30 cycles of initial denaturation at 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 60.5°C for 30 s, and extension at 72°C for 45 s. The DNA was then extended to the full length at 72°C for 10 min, and then stored at 4°C. PCR products were subjected to gel electrophoresis, and a 2000 bp DNA marker was used as a control to identify transgenic poplars harboring the target gene.

[0080] RNA level identification of transgenic poplars

[0081] First, select healthy transgenic poplar leaves. Samples are collected in a laminar flow hood. Leaves are placed in 2ml RNase-free centrifuge tubes and quickly frozen in liquid nitrogen. Grind the leaves into a white powder using a vibrating grinder. Add 1ml of Trizol reagent to the ground sample to lyse the cells, and let it stand at room temperature for 5 minutes. Add 200μl of chloroform to the sample, shake and mix thoroughly, and let it stand at room temperature for 2-3 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes. Transfer the supernatant to a new centrifuge tube, and record the approximate volume. Add an equal volume of isopropanol to the supernatant, let it stand at room temperature for 10 minutes, and then centrifuge at 12,000 rpm at 4°C for 10 minutes. Discard the supernatant, add 500μl of 75% DEPC ethanol solution, flick the pellet, mix with the solution, and centrifuge at 12,000 rpm at 4°C for 2 minutes. Discard the supernatant and add 500 μl of 75% DEPC ethanol solution. Flip the pellet to mix with the solution. Centrifuge at 12,000 rpm for 2 minutes at 4°C. Discard the supernatant. Centrifuge at 12,000 rpm for 2 minutes at 4°C. Remove the ethanol with a pipette. Dissolve the DNA in 30-50 μl of sterile water.

[0082] PCR identification was performed using cDNA from transgenic and wild-type poplars as templates. The PCR reaction system consisted of 10 μl of 2× Hieff PCR Master Mix, 1 μl of a 10 μM upstream primer, 1 μl of a 10 μM downstream primer, 1 μl of the template cDNA, and 7 μl of sterile water. The PCR amplification procedure included 30 cycles of pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 30 seconds, annealing at 60.5°C for 30 seconds, and extension at 72°C for 45 seconds, followed by a full-length extension at 72°C for 10 minutes, and storage at 4°C. PCR products were analyzed by gel electrophoresis using a 2000 DNA Marker as a control to identify transgenic poplars harboring the target gene.

[0083] To determine the subcellular localization of the SlBOR2 gene, the pCAMBIA1300-GFP expression vector contained the GFP sequence. The recombinant vector in Agrobacterium was used to infect tobacco. The expression position was observed using a laser confocal microscope. The protein encoded by the SlBOR2 gene was expressed in the cytoplasmic membrane.

[0084] Example 2

[0085] Phenotype of SlBOR2 gene overexpression under treatment with different concentrations of sodium bicarbonate

[0086] Wild-type poplars and the transgenic poplars in Example 1 were subcultured in 1 / 2 MS medium, and the poplars took root after 7 days.

[0087] Wild-type poplars and the transgenic poplars described in Example 1 were selected for their similar growth (plant height, leaf mass, number of fibrous roots, and root length comparable to 1-2 cm) and transplanted into 1 / 2 MS medium supplemented with 5, 10, and 15 mM NaHCO for stress treatment. After 10 days, the plants were removed from the medium, and the roots were washed with distilled water to remove any residual medium. The wild-type and transgenic poplars were photographed, and their taproot and aerial lengths were measured. Fresh and dry weights were also determined.

[0088] Example 3

[0089] Effects of SlBOR2 gene overexpression on bicarbonate resistance in plants

[0090] Wild-type poplars and the transgenic poplars in Example 1 were subcultured in 1 / 2 MS medium, and the poplars took root after 7 days.

[0091] Wild-type poplars and transgenic poplars in Example 1 with similar growth characteristics (plant height, leaf mass, number of fibrous roots, and root length of 1-2 cm) were selected. Plants stressed with 15 mM NaHCO3 were selected. Leaves were removed after 10 days of treatment. Leaves from plants cultured normally in 1 / 2 MS medium were used as controls. O2 was detected using a kit. - Determination of H2O2 content. After quantification by a kit, the results showed that under 15mM NaHCO3 stress, SlBOR2 transgenic plants contained less superoxide anions and hydrogen peroxide.

[0092] Place the plant in a 50ml large tube, add NBT dye solution, immerse the leaves, observe that the root tip is blue and the leaves are brown. Generally, the roots are dyed for about 1 hour and the leaves are dyed for about 0.5 hours. Pour out the dye solution and add Carnoy's fixative. Try not to move the seedlings during this process. After 2 hours, pour out the fixative solution and add a transparent agent. Observe and take pictures 1 day after the transparent agent is added.

[0093] Carnoy's fixative: 1 ml of glacial acetic acid is added to 3 ml of anhydrous ethanol and mixed.

[0094] Clarifying agent: add 1ml glycerol to 4ml anhydrous ethanol and mix

[0095] The results of NBT staining showed that the wild-type poplar had a stronger staining degree than the transgenic poplar after bicarbonate stress, indicating that the roots of the wild-type poplar suffered greater damage under stress. The transgenic poplar was more resistant to alkaline salt stress.

[0096] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A SlBOR2 gene for improving plant alkaline salt resistance, characterized in that: The nucleotide sequence of the S1BOR2 gene is shown in SEQ ID No.

1.

2. Use of the S1BOR2 gene according to claim 1 in improving plant alkaline salt resistance by overexpression.

3. Use of overexpressing the S1BOR2 gene according to claim 1 in increasing plant height, root length, fresh weight and dry weight under alkaline saline conditions.

4. Use of overexpressing the S1BOR2 gene according to claim 1 in reducing the content of superoxide anions and / or hydrogen peroxide in plants under alkaline conditions.

5. The S1BOR2 gene according to claim 1 or the use according to any one of claims 2 to 4, characterized in that: The plants include poplars.

6. The S1BOR2 gene according to claim 1 or the use according to any one of claims 2 to 4, characterized in that: The basic salts include bicarbonates and / or carbonates.

7. A protein encoded by the S1BOR2 gene according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No.

2.

8. A plant overexpression vector, characterized in that: The S1BOR2 gene according to claim 1 is connected to the plant expression vector pmCAMBIA1300 to obtain a plant overexpression vector.

9. A method for obtaining alkaline salt-resistant plants, characterized in that: The following steps are involved: 1) transforming the plant overexpression vector according to claim 8 into Escherichia coli, extracting the plasmid, and obtaining a recombinant vector; 2) transforming the recombinant vector obtained in step 1) into Agrobacterium to obtain transformed bacteria; 3) Infecting plant leaves with the transformed bacteria obtained in step 2) to obtain alkaline salt-resistant plants.

10. The method according to claim 9, characterized in that In step 3, the transformed bacteria infect plant leaves in the form of bacterial liquid, and the OD 600 The value is 0.6~0.8.

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