Application of betula luminifera NRT2.1 gene in promoting plant nitrogen absorption and preparation method of transgenic plant of betula luminifera NRT2.1 gene

Abstract

The invention discloses a betula luminifera NRT2.1 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 also discloses an application of the betula luminifera NRT2.1 gene in promoting nitrogen absorption of plants. The invention also discloses a transgenic plant and a preparation method thereof. The invention provides the application of the betula luminifera NRT2.1 gene in promoting the nitrogen absorption of the plant, and the nitrogen absorption and utilization rate of the root system of the plant can be obviously promoted by increasing the betula luminifera NRT2.1 gene in the plant.

Description

Technical Field

[0001] The invention relates to application of Betula luminifera NRT2.1 gene in promoting plant nitrogen absorption and a preparation method of a transgenic plant thereof. Background Art

[0002] Nitrogen absorption and transport by plants is a key process for their normal growth and development. Nitrate (NO3 - ) is one of the main forms of nitrogen sources required for plant growth, and the absorption of nitrate by plants mainly depends on the nitrate transporter (NRT) family. The NRT family mainly includes two types of transporters, NRT1 and NRT2, which play different functions under different nitrogen concentrations and environmental conditions. Among them, NRT1 proteins are generally involved in the absorption of low-affinity nitrates, while the NRT2 family is a high-affinity nitrate transporter that can effectively absorb nitrates at low nitrate concentrations.

[0003] In model plants such as Arabidopsis, members of the NRT2 family, such as AtNRT2.1, AtNRT2.2 and AtNRT2.5, all have high nitrate affinity and can play an important role in low nitrate environments. In particular, the AtNRT2.1 gene is mainly expressed in the mature zone of the root and plays a key transport role under low nitrate conditions. Studies have shown that mutations in AtNRT2.1 can lead to a significant decrease in the high-affinity influx of nitrate, thereby affecting the plant's nitrogen absorption capacity.

[0004] Although the functions of the NRT2 family have been widely studied in many plants, related research in forest trees is still weak. In particular, the nitrogen absorption mechanism and the function of related transport proteins of Betula luminifera, an important forest tree species, are still unclear. Therefore, exploring the function of the NRT2.1 gene of Betula luminifera and its role under low nitrogen stress will not only help reveal its important role in nitrogen absorption, but also provide new ideas for improving the nitrogen utilization efficiency of forest trees through genetic engineering.

[0005] In the natural environment, soil nitrogen resources are often scarce or uneven, especially in poor soils or under low nitrogen stress, it becomes more difficult for plants to effectively absorb and utilize nitrogen. By improving the nitrogen absorption efficiency of plants, the growth performance of plants in low nitrogen environments can be significantly improved, thereby increasing the productivity of agriculture and forestry.

[0006] Therefore, the present invention aims to provide a new method to improve the nitrogen absorption capacity of plants under low nitrogen stress by studying and applying the Betula luminifera NRT2.1 gene, thereby enhancing its growth advantage and yield under low nitrogen conditions. Summary of the invention

[0007] The purpose of the present invention is to provide an application of the Betula luminifera NRT2.1 gene in promoting plant nitrogen absorption and a method for preparing a transgenic plant thereof, so as to reveal the function of the Betula luminifera NRT2.1 gene and to create a transgenic plant using the gene.

[0008] The present invention provides a Betula luminifera NRT2.1 gene, which has a nucleotide sequence as 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 Betula luminifera NRT2.1 gene is SEQ ID No: 1.

[0009] In a specific embodiment, the amino acid sequence encoded by the Betula luminifera NRT2.1 gene is SEQ ID No: 2.

[0010] The present invention also provides the use of the Betula luminifera NRT2.1 gene in promoting nitrogen absorption by plants, and prepares Betula luminifera NRT2.1 gene modified plants, wherein the expression of the Betula luminifera NRT2.1 gene in the modified plants is increased relative to the wild-type plants, thereby promoting nitrogen absorption by plants; wherein the nucleotide sequence of the Betula luminifera NRT2.1 gene is shown in SEQ ID No: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, specifically, an increase in the product obtained by transcription and the product obtained by translation. Conversely, in the concept of "reduced expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; a decrease in the intensity of the above process is a decrease in expression, specifically, a decrease in the product obtained by transcription and the product obtained by translation.

[0011] In a specific embodiment, the plant is Arabidopsis thaliana or Betula luminifera. Preferably, the plant is Betula luminifera.

[0012] In a specific embodiment, the application includes connecting the Betula luminifera NRT2.1 gene to a vector, transforming it into Betula luminifera through Agrobacterium-mediated transformation, screening, culturing and obtaining transgenic strains.

[0013] In a specific embodiment, the nitrogen absorption activity of the roots of the above-mentioned plant is significantly promoted by overexpressing the Betula luminifera NRT2.1 gene under low nitrogen stress.

[0014] The present invention also provides a transgenic plant, in which the Betula luminifera NRT2.1 gene is transformed by Agrobacterium-mediated transformation, and the expression of the Betula luminifera NRT2.1 gene in the transgenic plant is significantly increased compared with that in the wild-type plant.

[0015] The present invention also provides a method for preparing the above transgenic plant, comprising the following steps:

[0016] (a) cloning the Betula luminifera NRT2.1 gene into an appropriate vector;

[0017] (b) using Agrobacterium-mediated method to introduce the vector into plant cells to obtain transgenic plants;

[0018] (c) Screening and culturing transgenic plants to obtain transgenic plants overexpressing the Betula luminifera NRT2.1 gene.

[0019] In a specific embodiment, the method further comprises low nitrogen treatment to evaluate the performance of the transgenic plant under low nitrogen stress.

[0020] In a specific embodiment, the low nitrogen treatment comprises planting the transgenic plant in a low nitrogen nutrient solution and observing the root growth, leaf phenotype and nitrogen absorption activity thereof.

[0021] In order to reveal the function of the Betula luminifera NRT2.1 gene under low nitrogen stress, the present application overexpressed the Betula luminifera NRT2.1 gene in Betula luminifera through transgenic technology, and compared the performance of transgenic plants and empty control under low nitrogen stress. It was found that the root system (root length, surface area, volume, number of forks) of the transgenic plants grew better than that of the wild-type control, indicating that the transgenic plant strains overexpressing the Betula luminifera NRT2.1 gene have a better coping mechanism under low nitrogen stress and can obtain more nitrogen by elongating the root system.

[0022] The present application provides the use of the Betula luminifera NRT2.1 gene in promoting nitrogen absorption by plants. By increasing the Betula luminifera NRT2.1 gene in plants, the nitrogen absorption and utilization rate of the plant's root system can be significantly promoted. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings described below are used to provide a 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 an improper limitation on the present application. In the following drawings, LN in the drawings means growth under low nitrogen stress, and CK means growth under normal nitrogen supply of the plant.

[0024] Figure 1 This is the electrophoresis diagram of the Betula luminifera NRT2.1 gene cloning of the present invention. Figure 1 A: The first lane is a negative control, and the second to third lanes are cDNA template amplification products (from left to right); Figure 1 B The first lane is a negative control, and lanes 2 to 5 are the amplification products of monoclonal bacterial solution templates 1-4 (from left to right).

[0025] Figure 2 The electrophoresis diagram of the Betula luminifera NRT2.1 gene vector constructed by the present invention is shown in FIG. Figure 2 A: The first lane is a negative control, and the second and third lanes are all plasmid template amplification products; Figure 2 B The first lane is a negative control, and lanes 2 to 6 are the amplification products of monoclonal bacterial solution templates 1-5 (from left to right).

[0026] Figure 3 This is a fluorescence image of the zygotic embryo-induced transformation of Betula luminescens according to the present invention.

[0027] Figure 4 This is a diagram for detecting the root RNA quality of the Betula luminifera NRT2.1 gene-overexpressing strain of the present invention.

[0028] Figure 5 This is a phenotype diagram of the Betula luminifera NRT2.1 gene overexpressing strain of the present invention.

[0029] Figure 6 This is a leaf phenotype diagram of the Betula luminifera NRT2.1 gene overexpressing strain of the present invention.

[0030] Figure 7 It is the biomass increment of the Betula luminifera NRT2.1 gene overexpressing strain of the present invention.

[0031] Figure 8 It is the chlorophyll content of the Betula luminifera NRT2.1 gene overexpressing strain of the present invention.

[0032] Fig. 9 This is an analysis of the net photosynthetic rate of the Betula luminifera NRT2.1 gene overexpressing strain of the present invention.

[0033] Fig.10 The present invention is an analysis of chlorophyll fluorescence parameters of the Betula luminifera NRT2.1 gene overexpressing strain.

[0034] Fig.11 It is the C, N and H content of the Betula luminifera NRT2.1 gene overexpressing strain of the present invention.

[0035] Fig.12 The nitrate reductase (NR) and nitrite reductase (NIR) activities in the roots of the Betula luminifera NRT2.1 gene-overexpressing strain under low nitrogen stress of the present invention.

[0036] Fig.13 The expression level of NRT2.1 gene in the roots of the Betula luminifera NRT2.1 gene-overexpressing strain of the present invention is shown.

[0037] Fig.14The present invention provides an analysis of the nitrate nitrogen content and related enzyme gene expression in the roots of the Betula luminifera NRT2.1 gene-overexpressing strain.

[0038] Fig.15 The present invention provides an observation of the root morphology of the Betula luminifera NRT2.1 gene transgenic strain under low nitrogen stress.

[0039] Fig.16 This is the observation of root morphology of the Betula luminifera NRT2.1 gene-transformed strain under normal nitrogen supply of the present invention. DETAILED DESCRIPTION

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] 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.

[0043] 1. Materials

[0044] 1.1 Experimental Materials

[0045] Betula luminifera seeds were collected from the Pingshan Experimental Base of Zhejiang Agricultural and Forestry University. On a sunny day, branches with mature seeds were cut with high-branch shears. The seeds were placed in 50ml centrifuge tubes, dried at 37 degrees, and stored in a -20 refrigerator. The zygotic embryos required for subsequent experiments were obtained through tissue culture. After 7 days, 14 days, and 21 days of low nitrogen stress treatment, the roots, stems, and leaves of the 5-month-old Betula luminifera tissue culture seedlings G49-3 were taken for RNA extraction.

[0046] 1.2 Experimental reagents and instruments

[0047] 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.

[0048] 1.3 Primer synthesis and sequencing

[0049] Primer synthesis and sequencing were completed by Sangon Biotechnology (Shanghai) Co., Ltd.

[0050] 2. Methods

[0051] 2.1 Extraction of total RNA from Betula luminifera

[0052] The RNA (Total RNA) of Betula luminifera leaves was extracted using the modified CTAB+Trizol method, and the steps are as follows:

[0053] (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;

[0054] (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 white powder;

[0055] (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.

[0056] (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);

[0057] (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;

[0058] (6) Repeat step 4 once until the middle layer disappears;

[0059] (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;

[0060] (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.

[0061] (9) Centrifuge at 12000 rpm for 15 min at 4°C;

[0062] (10) Aspirate the supernatant, add an equal volume of isopropanol, and let stand at room temperature for 10 min;

[0063] (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;

[0064] (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;

[0065] (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.

[0066] 2.2 Gene cloning

[0067] After the total RNA of Betula luminifera was diluted with an appropriate amount of DEPC ddH2O, the first strand of reverse transcription cDNA synthesis was performed according to the instructions of PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa). Reverse transcription was completed in two steps, and the system was as follows:

[0068] (1) The reaction system for removing genomic DNA is as follows:

[0069]

[0070] (2) The reverse transcription reaction system is as follows:

[0071]

[0072] The overexpression primers of Betula luminifera NRT2.1 gene were designed (Table 1), and the gene cloning PCR system was as follows (50 μL):

[0073]

[0074]

[0075] 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.

[0076] Table 1. Primers used for gene cloning

[0077]

[0078] 2.3 Target fragment recovery

[0079] Utilizing AxyPrep TM DNA gel recovery kit (AXYGEN) was used to recover the target fragment;

[0080] (1) Cut the agarose gel containing the target gene fragment under ultraviolet light and weigh the gel. This weight is regarded as a gel volume (e.g., 100 mg = 100 μL);

[0081] (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;

[0082] (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;

[0083] (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;

[0084] (5) Add 500 μL of Buffer W1 to the prepared tube, incubate at 12,000 rpm for 1 min, and discard the filtrate;

[0085] (6) Add 700 μL of Buffer W2 to the prepared tube, incubate at 12,000 rpm for 1 min, discard the filtrate, and repeat once;

[0086] (7) Place the prepared tube in a 2 mL centrifuge tube, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0087] (8) Place the preparation tube into a clean 1.5 ml centrifuge tube, add 25-30 μL of ddH2O preheated to 65°C to the center of the preparation tube, let stand at room temperature for 1-3 min, and centrifuge at 12,000 rpm for 1 min to elute the DNA;

[0088] (9) Take 2-3 μL of DNA solution for agarose gel electrophoresis and store the remaining solution at -20°C.

[0089] 2.4 Target fragment connection

[0090] Mix the following solutions, mix gently, centrifuge briefly, and PCR program: 25℃20min.

[0091]

[0092] 2.5 Target fragment transformation

[0093] (1) Add 5 μL of ligation product to 50 μL of competent TransT1 and place on ice for 25-30 min;

[0094] (2) Heat shock at 42°C in a water bath for 40 seconds and place on ice for 2 minutes;

[0095] (3) Add 300-500 μL of hormone-free LB liquid medium and culture at 200 rpm and 37°C for 30-60 min;

[0096] (4) Incubate at 4000 rpm for 2 min, remove part of the supernatant, retain 50-100 μL of the bacterial solution, 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;

[0097] (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.

[0098] 2.6 PCR verification of target fragment

[0099] (1) Pick the above white single colony, place it in 500 μL of liquid LB medium with the same concentration of antibiotics, culture at 37°C for 3-6 hours, and take 1 μL for bacterial liquid PCR;

[0100] (2) Use gene cloning primers to perform bacterial liquid PCR detection. The reaction system (10 μL) is as follows:

[0101]

[0102] (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;

[0103] (4) PCR products were detected by agarose gel electrophoresis, and positive clones were selected and sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing;

[0104] (5) Extract the plasmid and name the obtained recombinant plasmid containing the target gene: OE-NRT2.1.

[0105] 2.7 Construction of overexpression vector

[0106] (1) Double digestion was performed using restriction endonucleases Hind III and Nco I. The digestion reaction system was as follows (50 μL):

[0107]

[0108] Digest at 37℃ for 3-4h. After the reaction, detect and recover the fragments by electrophoresis and determine the concentration (see 2.3 for details).

[0109] (2) Connect the recovered target fragment and the vector fragment. The system is as follows:

[0110]

[0111] (3) Perform transformation and bacterial testing according to 2.5-2.6 to obtain the correct positive clone;

[0112] (4) Extract the plasmid of the strain with the correct sequencing result and set aside for use.

[0113] 2.8 The steps of chemical transformation and bacterial testing are as follows:

[0114] (1) Take out the Agrobacterium stored at -80°C and thaw it on ice;

[0115] (2) Take 1 μL of the recombinant plasmid and add it to the competent Agrobacterium. Stir it gently with the pipette tip. After mixing it slightly, place it on ice, in liquid nitrogen, in a 37°C water bath, or in ice water for 5 min.

[0116] (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;

[0117] (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;

[0118] (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;

[0119] (6) Pick the monoclonal bacteria on the plate for testing, and store the monoclonal bacterial liquid with positive test results at -80°C.

[0120] 2.9 Agrobacterium-mediated transformation of Betula luminifera by zygotic embryo induction

[0121] (1) Take the Agrobacterium culture medium containing the NRT2.1 overexpression vector and the control empty vector (pK2GW7-eYGFP) at a ratio of 1:30 and add it to 30 mL of LB liquid medium (50 mg / L Spec + 30 mg / L Rif), and culture it overnight in a shaking incubator (200 rpm) at 28°C;

[0122] (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;

[0123] (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.

[0124] (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;

[0125] (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.

[0126] (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.

[0127] 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, and then moved to a substrate (peat soil: perlite: vermiculite = 2:1:1) for cultivation and growth in preparation for subsequent low-nitrogen treatment.

[0128] 2.10 Low nitrogen treatment method for Betula luminifera

[0129] 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 nitrogen concentration solution for 3 weeks, the seedlings were moved to pure water for starvation treatment for one week, and then the seedlings were randomly moved to normal nitrogen concentration and low nitrogen nutrient solution (Table 2). Nitrogen starvation test: Physiological indicators were measured and samples were taken one day before treatment and 1 day, 7 days, and 21 days after treatment. At least 3 biological replicates were performed in each period. N supplementation test: Nitrogen was supplemented to the seedlings in the low nitrogen culture solution on the 21st day, and photosynthetic fluorescence indicators were measured and samples were taken after 1h and 24h of supplementation. After sampling, RNA extraction was performed immediately, and the remaining samples were stored in a -80℃ refrigerator.

[0130] Table 2 Hydroponic nutrient solution formula

[0131]

[0132] Note: All components are added in mol / L.

[0133] 3. Experimental results

[0134] 3.1 Cloning of the Betula luminifera NRT2.1 gene

[0135] Total RNA was extracted from roots of Betula luminifera under low nitrogen stress. Electrophoresis showed that the bands were clear and without dragging, and the OD260 / OD280 was 1.8-2.1, indicating that the RNA quality was good. Amplification was performed using cDNA as a template, and a 1600 bp band was obtained ( Figure 1 A), gel extraction, ligation, transformation and bacterial testing, a 1600 bp band was obtained ( Figure 1 B), extract the plasmid.

[0136] The final plasmid pK2GW7 was connected and transformed. After bacterial testing, the plasmid was successfully connected ( Figure 2 ), the plasmid was extracted and transformed into Agrobacterium tumefaciens GV3103, and used to infect zygotic embryos of Betula luminifera.

[0137] 3.2 Identification and performance observation of transgenic plants

[0138] Through Agrobacterium-mediated Betula luminifera zygotic embryo induction transformation, a total of one empty control strain (named EV) and two Betula luminifera overexpression strains (named OE5 and OE6) were obtained. Fluorescence observation of these plants showed 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, and it was found that the expression level of NRT2.1 gene in OE5 and OE6 of Betula luminifera was significantly increased, and they were identified as positive plants.

[0139] RNA was extracted and reverse transcribed from the root samples of the overexpression strain and the empty vector strain before treatment (0d), 1d, 7d, 21d after treatment, and 1h and 24h after nitrogen supplementation, according to the methods in 2.1 and 2.2. 2 μL RNA was diluted 3 times for electrophoresis observation, and the bands were clear and without dragging ( Figure 4 ), 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 qRT-PCR analysis.

[0140] Through low nitrogen treatment of Betula luminifera NRT2.1 gene plants, it was found that the overexpression strain OE grew better and was more tolerant to low nitrogen than the empty EV control strain. Specifically, in a low nitrogen environment (LN), the leaves of plants overexpressing the Betula luminifera NRT2.1 gene were larger and greener, and the biomass growth was significantly higher than that of the empty control strain. The chlorophyll a, chlorophyll b and total chlorophyll contents were significantly higher than those of the empty control strain ( Figure 5-8 ). The net photosynthetic rate and Fv / Fm data showed that the overexpression of the NRT2.1 gene of Betula luminifera was significantly higher than that of the empty control under low nitrogen conditions, and the decrease was not obvious compared with the normal nitrogen supply (CK) ( Fig. 9, 10 ). The results of elemental analysis of C, N, and H content showed that the nitrogen content in the overexpression plants was always significantly higher than that in the empty control strains ( Fig.11 ). Low nitrogen induced the expression of the Betula luminifera NRT2.1 gene in plants, and the expression level of plants overexpressing the Betula luminifera NRT2.1 gene was significantly higher than that of the empty control strain. At the same time, the expression levels of nitrate reductase (NR) and nitrite reductase (NIR) in the roots of Betula luminifera overexpressed in a low nitrogen environment decreased significantly less than those under normal nitrogen supply (CK), indicating that the nitrate content in the overexpressed strains was higher under low nitrogen conditions, which is consistent with the results of nitrate content determination ( Figure 12-14 ). All plants were scanned for roots, and the results showed that low nitrogen stress inhibited root growth but promoted root elongation. Under low nitrogen stress, the root growth of the transgenic Betula NRT2.1 gene strain was better, that is, the root length, surface area, volume, number of forks, etc. were significantly higher than those of the empty EV root system, indicating that the strain overexpressing the Betula NRT2.1 gene has a better coping mechanism in a low nitrogen environment, that is, to obtain more nitrogen by elongating the root system ( Fig.15 , 16 , Table 3).

[0141] Table 3 Analysis of root morphological characteristics of transgenic Betula luminifera

[0142]

[0143] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Betula luminifera NRT2.1 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 NRT2.1 gene is SEQ ID No:

1.

3. The gene according to claim 1, characterized in that The amino acid sequence encoded by the Betula luminifera NRT2.1 gene is SEQ ID No:

2.

4. The application of the Betula luminifera NRT2.1 gene in promoting nitrogen absorption by plants is characterized in that: Prepare a Betula luminifera NRT2.1 gene-modified plant, wherein the expression of the Betula luminifera NRT2.1 gene in the modified plant is increased compared to the wild-type plant, thereby promoting the plant's absorption of nitrogen; wherein the nucleotide sequence of the Betula luminifera NRT2.1 gene is shown in SEQ ID No:

1.

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

6. The use according to claim 4, characterized in that: The root system of the plant significantly promotes nitrogen absorption activity of the root system of the plant under low nitrogen stress by overexpressing the Betula luminifera NRT2.1 gene.

7. A transgenic plant, characterized in that: The transgenic plant is transformed with the Betula luminifera NRT2.1 gene of claim 1 or 2 through Agrobacterium-mediated transformation, and the expression of the gene in the transgenic plant is significantly increased compared with that in the wild-type plant.

8. A method for preparing the transgenic plant of claim 7, comprising the following steps: (a) cloning the Betula luminifera NRT2.1 gene according to claim 1 or 2 into an appropriate vector; (b) introducing the vector into plant cells using Agrobacterium-mediated method to obtain transgenic plants; (c) screening and culturing the transgenic plants to obtain transgenic plants overexpressing the Betula luminifera NRT2.1 gene.

9. The method according to claim 8, characterized in that The method also includes a low nitrogen treatment to evaluate the performance of the transgenic plants in a low nitrogen environment.

10. The method according to claim 9, characterized in that The low nitrogen treatment includes planting the transgenic plants in a low nitrogen nutrient solution and observing the root growth, leaf phenotype and nitrogen absorption activity of the transgenic plants.

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