Application and method of betula luminifera RIPK1 gene in improving plant low-phosphorus stress tolerance

Abstract

The invention discloses a betula luminifera RIPK1 gene which has a nucleotide sequence as shown in SEQ ID No: 1, or a sequence which is obtained by deleting, adding and / or substituting one or more nucleotides of the sequence and has unchanged functions. The invention further discloses application of the betula luminifera RIPK1 gene, and the betula luminifera RIPK1 gene is used for improving the tolerance of plants to low-phosphorus stress. The invention also discloses a method for improving the tolerance of plants under low phosphorus stress. Technicists in the field negatively regulate and control expression of the betula luminifera RIPK1 gene or knock out the betula luminifera RIPK1 through a gene editing technology, the low-phosphorus tolerance of the betula luminifera can be improved, and the phosphorus utilization efficiency of plants can be improved.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology and relates to the use and method of a Betula luminifera RIPK1 gene in improving plant low-phosphorus stress tolerance. Background Art

[0002] With the increase in global agricultural production demand, crops are facing a variety of environmental pressures, among which low phosphorus stress has become one of the important factors restricting plant growth and yield. Phosphorus is an essential nutrient element in the growth and development of plants, especially in energy metabolism, nucleic acid synthesis and cell membrane structure. However, the distribution of phosphorus resources in nature is extremely limited, and the available phosphorus in the soil is usually insufficient, resulting in plants often facing the dilemma of low phosphorus stress in the natural environment. In order to improve the growth ability of plants in low phosphorus environments, scholars have carried out a lot of research. At present, the response mechanism of plants to low phosphorus stress mainly includes phosphorus absorption, transport and regulation of phosphorus utilization efficiency. Among these mechanisms, the morphological changes and physiological responses of plant roots in low phosphorus environments are particularly important. Many plants respond to low phosphorus stress by increasing the organic acids secreted by the roots, promoting the dissolution and absorption of phosphorus in the soil, or by changing the root structure to increase the absorption area of ​​the roots. However, the regulatory ability of these natural adaptabilities is controlled by genes, and there are significant differences in the response mechanisms of different plant species and different environmental conditions.

[0003] In recent years, scientists have discovered through genomics and molecular biology research that certain plant genes play an important role in the response to low-phosphorus stress. As an important signal transduction gene, the RIPK1 gene (Receptor-interacting Protein Kinase 1) plays a vital role in the immune response and stress response of plants. As a barren-tolerant plant, the function of the RIPK1 gene of Betula luminifera in a low-phosphorus environment has not been deeply studied. Existing studies have shown that the RIPK1 gene plays a key role in the pathological response and physiological regulation of plants, but its function under low-phosphorus stress in plants has not yet been clearly revealed.

[0004] At present, although some crop varieties such as rice, corn and soybeans have improved their tolerance to low-phosphorus stress through transgenic technology or conventional breeding, the research on low-phosphorus tolerance genes of specific plant species such as Betula luminifera is still in its preliminary stage. Therefore, in-depth research on the function of the Betula luminifera RIPK1 gene and its mechanism of action under low-phosphorus stress has important theoretical value and application prospects for improving the low-phosphorus tolerance of plants, especially in improving the growth and yield of important economic plants. Although the existing technology has proposed some gene improvement methods, how to effectively improve the phosphorus absorption and utilization efficiency of plants through genetic engineering means is still an urgent problem to be solved in the current field of agricultural biotechnology. Therefore, the development of new low-phosphorus stress tolerance regulatory genes and their applications have become an important direction in plant biotechnology research. Summary of the invention

[0005] The purpose of the present invention is to provide the use and method of the Betula luminifera RIPK1 gene in improving the tolerance of plants to low-phosphorus stress, so as to reveal the function of the Betula luminifera RIPK1 gene under low-phosphorus stress.

[0006] The present invention provides a RIPK1 gene of Betula luminifera, which has a nucleotide sequence shown in SEQ ID No: 1, or a sequence in which one or more nucleotides are deleted, added and / or substituted, but the function remains unchanged. Preferably, the nucleotide sequence of the RIPK1 gene of Betula luminifera is SEQ ID No: 1. The present invention uses RT-PCR technology to extract total RNA from Betula luminifera leaves through specific primer design, and after reverse transcription into cDNA, amplifies the complete coding sequence of the RIPK1 gene. After genome alignment and sequence analysis, it is confirmed that the gene is the RIPK1 gene.

[0007] The present invention also provides a use of the Betula luminifera RIPK1 gene, which is used to improve the tolerance of plants to low-phosphorus stress, and to negatively regulate the expression of the Betula luminifera RIPK1 gene or knock out the Betula luminifera RIPK1 gene through gene editing technology, wherein the nucleotide sequence of the Betula luminifera RIPK1 gene is SEQ ID No: 1.

[0008] In one embodiment, the plant is Betula luminifera.

[0009] The present invention also provides a method for improving the tolerance of plants under low phosphorus stress, the method comprising:

[0010] a) obtaining a gene knockout plant by knocking out the Betula luminifera RIPK1 gene;

[0011] b) cultivating the plant under low phosphorus stress conditions, thereby improving its tolerance to low phosphorus stress.

[0012] In one embodiment, the plant is Betula luminifera.

[0013] In one embodiment, the method further comprises screening plants in which the Betula luminifera RIPK1 gene is knocked out to determine gene knockout plants with stronger tolerance.

[0014] In order to reveal the function of the RIPK1 gene of Betula luminifera under low-phosphorus stress, the present application overexpressed the RIPK1 gene of Betula luminifera in Betula luminifera through transgenic technology, and compared the phenotypes of transgenic plants and wild types under low-phosphorus stress and found that the root system (root length, surface area, volume, number of forks) of the wild-type control grew better than that of the transgenic plants, indicating that the growth and development of the transgenic plant strains overexpressing the RIPK1 gene of Betula luminifera were more affected under low-phosphorus stress, indicating that the RIPK1 gene of Betula luminifera has a negative regulatory effect in plant tolerance to low-phosphorus stress. Therefore, those skilled in the art can enhance the low-phosphorus tolerance of Betula luminifera by negatively regulating the expression of the RIPK1 gene of Betula luminifera or knocking out RIPK1 of Betula luminifera through gene editing technology, which helps to improve the phosphorus utilization efficiency of the plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings described below are used to provide further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0016] Figure 1 This is a total RNA electrophoresis detection diagram of the Betula luminifera leaves of the present invention.

[0017] Figure 2 This is an electrophoresis diagram of the construction of the overexpression vector of the Betula luminifera RIPK1 gene of the present invention; Figure 2 A The first lane is a negative control, and the second lane is the amplified product of the Betula luminifera RIPK1 gene; Figure 2 B The first lane is a negative control, and the second to fourth lanes (labeled as 1, 2, 3) are the amplification products of the monoclonal bacterial solution templates 1-3.

[0018] Figure 3 These are fluorescence images of zygotic embryo-induced transformation of Betula luminifera of the present invention. a and b are bright field observation and GFP detection during induction of cluster seedlings, respectively; c and d are bright field observation and GFP detection during rooting culture; e and f are bright field observation and GFP detection during matrix culture. GFP detection uses a handheld fluorescent lamp (LUYOR-3415RG Hand-held Lamp).

[0019] Figure 4 This is the identification of the Betula luminifera RIPK1 gene overexpressing plant of the present invention, EV is the wild type, and OE is the Betula luminifera RIPK1 gene overexpressing plant.

[0020] Figure 5 These are phenotypic observation diagrams of the Betula luminifera RIPK1 gene overexpressing plants under low-phosphorus stress of the present invention, A is the phenotype diagram after 35 days of low-phosphorus stress, B is the plant height, and C is the root length.

[0021] Figure 6 It is the inorganic phosphorus (Pi) content of the Betula luminifera RIPK1 gene overexpressing plants under low phosphorus stress of the present invention.

[0022] Figure 7 It is the hydrogen peroxide content of the Betula luminifera RIPK1 gene overexpressing plants under low phosphorus stress of the present invention. DETAILED DESCRIPTION

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] 1. Materials

[0027] 1.1 Experimental Materials

[0028] Betula luminifera seeds were collected from the Pingshan Experimental Base of Zhejiang A&F University. On a sunny day, branches with mature seeds were cut with high-branch shears. The seeds were placed in 50 ml centrifuge tubes, dried at 37 degrees, and stored in a -20 refrigerator. The zygotic embryos required for subsequent experiments were obtained through tissue culture.

[0029] Five-year-old mature leaves of Betula luminifera were collected from the Pingshan Experimental Base of Zhejiang Agricultural and Forestry University and immediately placed in liquid nitrogen for transportation until RNA extraction. The remaining leaf samples can be stored at -80℃ for later use.

[0030] 1.2 Experimental reagents and instruments

[0031] DNA polymerase, various restriction endonucleases, T4 ligase, Marker and TRIzol reagents were purchased from Bao Biotechnology (Dalian) Co., Ltd.; plasmid extraction kit and DNA gel recovery kit were purchased from Shanghai Shenggong Biotechnology Co., Ltd. The PCR instrument was a US PE9700 PCR instrument, and the clean bench was purchased from Suzhou Chengjing Purification Technology Co., Ltd.

[0032] 1.3 Primer synthesis and sequencing

[0033] Primer synthesis and sequencing were completed by Sangon Biotechnology (Shanghai) Co., Ltd.

[0034] 2. Methods

[0035] 2.1 Extraction of total RNA from Betula luminifera

[0036] Total RNA from Betula luminifera leaves was extracted using a modified CTAB+TRIzol method, as follows:

[0037] (1) Add 3 mL of CTAB extraction buffer (10% CTAB, 10% PVP40, 1.0 M Tris-HCl (pH 8.0), 5 M NaCl, 0.6 M EDTA (pH 8.0)) preheated at 65°C to a 10 mL centrifuge tube and add 200 μL of β-mercaptoethanol;

[0038] (2) Take 2 g of leaves stored at -70°C, put them into a mortar fully cooled with liquid nitrogen, and grind them thoroughly in liquid nitrogen until they become a white powder;

[0039] (3) Transfer the white powder into the preheated extract and immediately shake thoroughly to mix. Place in a 65°C water bath for 30 min, shaking 3-4 times during this period.

[0040] (4) Add an equal volume of 25:24:1 (acidic saturated phenol: chloroform: isoamyl alcohol), about 3 ml, to the mixture. Mix well and centrifuge at 14,000 rpm for 10 min (room temperature);

[0041] (5) Transfer the supernatant to a new 10 ml centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (24:1), mix by inversion, centrifuge at 12000 rpm and 4°C for 10 min, and take the supernatant;

[0042] (6) Repeat step 4 once until the middle layer disappears;

[0043] (7) Transfer the supernatant to a 1.5 mL centrifuge tube, add an equal volume of isopropanol, and place in a -20°C refrigerator for 30 min. Centrifuge at 12,000 rpm and 4°C for 10 min, discard the supernatant, and add 400 μL of SSTE solution preheated at 65°C to the precipitate until it is completely dissolved;

[0044] (8) Add 1 mL of TRIzol reagent to the solution and let it stand at room temperature for 5 min. Then add 200 μL of chloroform, shake well, and let it stand at room temperature for 2-3 min.

[0045] (9) Centrifuge at 12000 rpm for 15 min at 4°C;

[0046] (10) Aspirate the supernatant, add an equal volume of isopropanol, and let stand at room temperature for 10 min;

[0047] (11) Centrifuge at 12000 rpm at 4°C for 10 min, discard the supernatant, and RNA will be visible at the bottom of the tube;

[0048] (12) Add 1 mL of pre-cooled 75% ethanol to wash the precipitate, shake gently, centrifuge at 8000 rpm at 4°C for 5 min, and discard the supernatant;

[0049] (13) Repeat step 11 and vacuum dry the precipitate for 7-10 min; add appropriate amount of DEPC water to dissolve the precipitate and store at -80°C for later use.

[0050] 2.2 Gene cloning

[0051] The RNA of Betula luminifera (Total RNA) was diluted with an appropriate amount of DEPC ddH2O, and the first-strand cDNA synthesis was performed by reverse transcription according to the instructions of PrimeScriptTM RTReagent Kit (Perfect Real Time) (TaKaRa). The reverse transcription was completed in two steps, and the system was as follows:

[0052] (1) The reaction system for removing genomic DNA is as follows:

[0053]

[0054]

[0055] (2) The reverse transcription reaction system is as follows:

[0056]

[0057] The overexpression primers of the Betula luminifera RIPK1 gene were designed (Table 1), and the gene cloning PCR system was as follows (50 μL):

[0058]

[0059] The reaction program was: 98°C for 10 s, 52°C for 5 s, 72°C for 1 min 40 s, 35 cycles, and 72°C for 5 min.

[0060] Table 1 Primers used for gene cloning

[0061]

[0062] 2.3 Target fragment recovery

[0063] Utilizing AxyPrep TM The target fragments were recovered using DNA gel recovery kit (AXYGEN).

[0064] (1) Cut the agarose gel containing the target gene fragment under ultraviolet light and weigh the gel. This weight is regarded as the gel volume (e.g. 100 mg = 100 μL).

[0065] (2) Add Buffer DE-A solution, about 3 gel volumes, and heat in a 75°C water bath for 6-8 min, shaking every 2-3 min until the gel is completely melted.

[0066] (3) Add Buffer DE-B solution, about 1 / 2 the volume of Buffer DE-A, and mix well. When the target fragment length is less than 400 bp, add an appropriate amount of isopropanol, about 1 gel volume.

[0067] (4) Pipette the mixed solution from the previous step into a DNA preparation tube (placed in a 2 ml centrifuge tube), centrifuge at 12000 rpm for 1 min, and remove the filtrate.

[0068] (5) Add 500 μL of Buffer W1 to the prepared tube and incubate at 12,000 rpm for 1 min. Discard the filtrate.

[0069] (6) Add 700 μL of Buffer W2 to the prepared tube and incubate at 12,000 rpm for 1 min. Discard the filtrate and repeat once.

[0070] (7) Place the prepared tube in a 2 mL centrifuge tube and centrifuge at 12,000 rpm for 1 min. Discard the filtrate.

[0071] (8) Place the prepared tube into a clean 1.5 ml centrifuge tube, add 25-30 μL of ddH2O preheated to 65°C to the center of the prepared tube, let stand at room temperature for 1-3 minutes, and centrifuge at 12,000 rpm for 1 minute to elute the DNA.

[0072] (9) Take 2-3 μL of DNA solution for agarose gel electrophoresis and store the remaining solution at -20°C.

[0073] 2.4 Target fragment connection

[0074] Mix the following solutions, mix gently, centrifuge briefly, and PCR program: 25℃20min.

[0075]

[0076] 2.5 Target fragment transformation

[0077] (1) Add 5 μL of ligation product to 50 μL of competent TransT1 and incubate on ice for 25-30 min.

[0078] (2) Heat shock in a 42°C water bath for 40 seconds. Place on ice for 2 minutes.

[0079] (3) Add 300-500 μL of hormone-free LB liquid culture medium and culture at 200 rpm and 37°C for 30-60 min.

[0080] (4) Incubate at 4000 rpm for 2 min, remove part of the supernatant, retain 50-100 μL of the bacterial culture, add 10 μL of isopropylthiogalactoside (IPTG, 100 mg / mL) and 8 μL of 5-bromo-4-chloro-3-indole-α-D-galactoside (X-gal, 100 mg / ml), and mix well.

[0081] (5) Apply the remaining solution to a plate containing kanamycin (Kan, 50 mg L -1 ) solid medium and cultured upside down at 37°C overnight.

[0082] 2.6 PCR verification of target fragment

[0083] (1) Pick the above white single colony, place it in 500 μL of liquid LB medium containing the same concentration of antibiotics, culture at 37°C for 3-6 hours, and take 1 μL for bacterial liquid PCR.

[0084] (2) Use gene cloning primers to perform bacterial liquid PCR detection. The reaction system (10 μL) is as follows:

[0085]

[0086] (3) PCR program: 94°C for 5 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 5 min, 35 cycles; 72°C for 10 min.

[0087] (4) PCR products were detected by agarose gel electrophoresis, and positive clones were selected and sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing.

[0088] (5) Extract the plasmid using the EasyPure Plasmid MiniPrep Kit (Transgene, Beijing). The resulting positive plasmid was stored in a -20°C refrigerator for use in the next step.

[0089] 2.7 Construction of overexpression vector

[0090] (1) Double digestion was performed using restriction endonucleases Hind III and Nco I. The digestion reaction system was as follows (50 μL):

[0091]

[0092] The enzyme digestion was carried out at 37℃ for 3-4h. After the reaction, the fragments were detected by electrophoresis and the concentrations were determined (see 2.3 for details).

[0093] The recovered target fragment and the vector fragment are connected, and the system is as follows:

[0094]

[0095] (3) Perform transformation and bacterial testing according to 2.5-2.6 to obtain the correct positive clone.

[0096] (4) Extract the plasmid of the strain with the correct sequencing result and set aside for use.

[0097] 2.8 Chemical transformation and bacterial testing

[0098] (1) Take out the Agrobacterium stored at -80°C and thaw it on ice;

[0099] (2) Take 1 μL of the recombinant plasmid and add it to the competent Agrobacterium.

[0100] Gently blow and stir with the tip of the gun, mix slightly, and place on ice, in liquid nitrogen, in a 37°C water bath, or in ice water for 5 minutes;

[0101] (3) Add 700 μL-1 mL of fresh YEP liquid culture medium without any resistance to the centrifuge tube, and place the bacterial liquid in a shaker (28°C, 200 rpm) for 2-3 h;

[0102] (4) Centrifuge (6000 rpm, 2 min), carefully pour off part of the supernatant, leaving the remaining liquid volume in the tube at about 70-100 μL, and pipette evenly;

[0103] (5) Spread the bacterial solution evenly on YEP solid medium containing antibiotics (50 mg / L Kana and 50 mg / L Rif) and incubate inverted in a biochemical incubator at 28°C for 48-72 h;

[0104] (6) Pick the monoclonal bacteria on the plate for testing, and store the monoclonal bacterial liquid with positive test results at -80°C.

[0105] 2.9 Agrobacterium-mediated transformation of Betula luminifera by zygotic embryo induction

[0106] (1) Add the Agrobacterium culture medium containing the RIPK1 overexpression vector and the control empty vector (pK2GW7-eYGFP) to 30 mL of LB liquid medium (50 mg / L Spec + 30 mg / L Rif) at a ratio of 1:30 and culture overnight at 28°C in a shaking incubator (200 rpm);

[0107] (2) On the second day, take the bacterial solution obtained above and add it to the antibiotic-free LB liquid medium at a ratio of 1:30 and continue to culture until the OD600 reaches 0.5-0.6. The obtained result is the infection solution;

[0108] (3) Place the seeds of Betula glabra, which have been swollen for 2 days after being soaked in running water, into a sterile clean bench and sterilize them by washing with 75% alcohol for 30 seconds, 30% hydrogen peroxide for 13 minutes, and sterile water three times in sequence. Use a sterile scalpel to cut the embryos of the sterilized seeds longitudinally, remove the seed coat, and soak the embryos in the bacterial solution for 10-15 minutes.

[0109] (4) The infected zygotic embryos were placed in the dark for co-culture (WPM + 2.0 mg / L 6-BA + 0.2 mg / L NAA) for 2 to 3 days, during which the plates were changed in time according to the status of Agrobacterium Pantoea;

[0110] (5) Then, selection culture (WPM+2.0mg / L 6-BA+0.2mg / L NAA+50mg / L Kana+200mg / L cephalosporin) was carried out. After 15 days, resistant and fluorescent callus tissues appeared one after another.

[0111] (6) After 20 days, the resistant calli were transferred to differentiation medium for further culture (WPM+0.8mg / L 6-BA+0.02mg / LNAA+0.5mg / L GA3+50mg / L Kana+200mg / L cephalosporin); after adventitious buds grew out, they were subcultured in tissue culture bottles (WPM+0.8mg / L 6-BA+50mg / L Kana); after 20 to 30 days, when the clustered seedlings grown from the adventitious buds grew to about 3 cm, they were transferred to rooting medium (WPM+0.2mg / L NAA) and asexual propagation was continued to obtain sufficient transgenic seedlings.

[0112] The bottle caps of the transgenic lines were loosened, and the plants were placed in a tissue culture room and an incubator for 7 days of hardening. They were then moved to a substrate (peat soil: perlite: vermiculite = 2:1:1) for cultivation and growth in preparation for subsequent low-phosphorus treatment.

[0113] 2.10 Low-phosphorus treatment method for Betula luminifera

[0114] After one month of substrate culture, the empty load and two overexpression lines were randomly divided into two groups and moved to a hydroponic box for culture. During the whole process, oxygen was supplied and the hydroponic nutrient solution was changed regularly (5 days / time). After being cultured in a normal phosphorus concentration solution for 3 weeks and then moved into pure water for starvation treatment for one week, the seedlings were randomly moved to normal phosphorus concentration and low phosphorus nutrient solution (Table 2). Phosphorus starvation test: Physiological indicators were measured and samples were taken one day before treatment and 1 day, 3 days, 7 days, 14 days, 21 days, 28 days, and 35 days after treatment. At least 3 biological replicates were performed in each period. After sampling, RNA extraction was performed immediately, and the remaining samples were stored in a -80℃ refrigerator.

[0115] The transgenic birch treated above was photographed and observed, the plant height and root length were measured with a ruler, and the biomass was weighed with a 1 / 10,000 balance. Each sample had at least 3 replicates, and the data were processed using EXCEL 2019 and analyzed for significance using T-test or Turkey's multiple comparisons.

[0116] The inorganic phosphorus content, ACP activity and H2O2 content of the treated transgenic birch were measured.

[0117] During the treatment period, photosynthetic parameters were measured at 1 day, 3 days, 7 days, 14 days, 21 days, 28 days, and 35 days. Each strain had at least 3 biological replicates, and at least 3 locations were selected for measurement in each replicate. The photosynthetic parameters were measured using a portable photosynthesis system Li-6800XTR (Li-Cor, USA). The measurements were taken at room temperature of 25 ± 2 °C from 8:00 to 11:00 in the morning, and the light intensity was set to 1000 μmol m -2 s -1 , CO2 concentration was set to 400 μmol / mol, and flow rate was set to 500 μmol s -1 , and then measured four indicators including net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and calculated water use efficiency (WUE = Pn / E).

[0118] Table 2 Hydroponic nutrient solution formula

[0119]

[0120] 2.11 Data Analysis

[0121] All experimental data were repeated at least three times and statistically analyzed (such as t-test or ANOVA analysis). Data such as photosynthesis and inorganic phosphorus content were processed using Excel 2019 software, and significance analysis was performed based on the statistical results.

[0122] 3. Experimental results

[0123] 3.1 Total RNA extraction from Betula luminifera

[0124] Betula luminifera leaves were collected from the Pingshan Experimental Base of Zhejiang Agricultural and Forestry University, transported in liquid nitrogen, and immediately used for RNA extraction. The remaining samples were stored in a -80℃ refrigerator. The extracted RNA was observed by electrophoresis and the bands were clear and without dragging ( Figure 1 ), and OD260 / OD280 was 1.8-2.1, indicating that the RNA quality was good and could be used for the next step of reverse transcription and gene cloning.

[0125] 3.2 Cloning and vector construction of Betula luminifera RIPK1 gene

[0126] Using Betula luminifera cDNA as template for amplification, a band of about 1500 bp was obtained ( Figure 2 A), gel cutting and recovery, ligation, transformation and bacterial detection. After the sequence is correct, the Betula luminifera RIPK1 gene overexpression vector is constructed, and the bacterial detection is correct after transformation into Escherichia coli ( Figure 2 B), extract the positive plasmid and transform Agrobacterium, and keep the bacteria at -80℃.

[0127] 3.3 Obtaining and identifying Betula luminifera RIPK1 gene overexpressing plants

[0128] Through Agrobacterium-mediated Betula luminifera zygotic embryo induction transformation, a total of one empty wild-type strain (named EV) and two Betula luminifera overexpressing strains (named OE1 and OE3) were obtained. Fluorescence observation of these plants revealed that all of them fluoresced ( Figure 3 ). At the same time, RNA extraction, reverse transcription and semi-quantitative analysis were performed on the transgenic lines. It was found that the expression level of RIPK1 gene in OE1 and OE3 was significantly higher than that in EV ( Figure 4 ), which were 6.56 and 5.54 times of EV, respectively, and were identified as positive plants.

[0129] 3.4 Phenotypes of Betula luminifera RIPK1 overexpressing plants under low phosphorus stress

[0130] Phenotypic observations revealed that plant growth was inhibited by low phosphorus stress, but wild-type EV was less affected by low phosphorus than transgenic OE1 and OE3. Under conditions of sufficient Pi, OE1, OE3, and EV all grew well; but under conditions of Pi deficiency, the plant height of OE1 and OE3 was significantly lower than that of EV, and the total root length, root surface area, number of nodes, and number of forks of OE1 and OE3 were significantly lower than those of EV ( Figure 5 ), indicating that plants overexpressing the RIPK1 gene in Betula luminifera have reduced adaptability to low phosphorus.

[0131] 3.5 Analysis of inorganic phosphorus content in Betula luminifera RIPK1 overexpressing plants

[0132] The Pi content of EV, OE1 and OE3 decreased under low phosphorus stress, but the decrease in inorganic phosphorus in OE1 and OE3 was significantly lower than that in EV, and OE was more affected by low phosphorus ( Figure 6 ).

[0133] 3.6 Detection of reactive oxygen species (ROS) accumulation in Betula luminifera RIPK1 gene overexpression plants

[0134] When the hydrogen peroxide content in the plants was measured, it was found that the H2O2 content in OE1 and OE3 under low phosphorus stress was significantly higher than that in EV ( Figure 7). This indicates that the oxidative damage of OE plants under low-P stress was significantly higher than that of EV, suggesting that it has a lower tolerance to low-P.

[0135] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A Betula luminifera RIPK1 gene, characterized in that: The gene has the nucleotide sequence shown in SEQ ID No: 1, or a sequence in which one or several nucleotides are deleted, added and / or substituted but the function remains unchanged.

2. The gene according to claim 1, characterized in that The nucleotide sequence of the Betula luminifera RIPK1 gene is SEQ ID No:

1.

3. The use of the Betula luminifera RIPK1 gene according to claim 1, for improving the tolerance of plants to low phosphorus stress, characterized in that: The expression of the Betula luminifera RIPK1 gene is negatively regulated or the Betula luminifera RIPK1 gene is knocked out by gene editing technology, wherein the nucleotide sequence of the Betula luminifera RIPK1 gene is SEQ ID No:

1.

4. The use according to claim 3, characterized in that The plant is Betula luminifera.

5. A method for improving the tolerance of plants under low phosphorus stress, characterized in that The method comprises: a) obtaining a gene knockout plant by knocking out the Betula luminifera RIPK1 gene; b) cultivating the plant under low phosphorus stress conditions, thereby improving its tolerance to low phosphorus stress.

6. The method according to claim 5, characterized in that The plant is Betula luminifera.

7. The method according to claim 5 or 6, characterized in that: The method further includes screening plants in which the RIPK1 gene of Betula luminifera has been knocked out to identify gene-knockout plants with stronger tolerance.

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