Application of TGA7 gene and / or encoded protein thereof in regulation and control of plant growth and low nitrogen stress adaptability

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a TGA7 gene and / or an encoded protein thereof in regulation and control of plant growth and low nitrogen stress adaptability. Amplification primers of the TGA7 gene comprise an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2, and the nucleotide sequence of the regulatory gene TGA7 is as shown in SEQ ID NO.3. The amplification primer provided by the invention can improve plant growth and low nitrogen stress adaptability, promote growth of plant root systems, further promote nutrient absorption of plants and improve the yield of crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to the application of TGA7 gene and / or its encoded protein in regulating plant growth and low nitrogen stress adaptability. Background Art

[0002] Nitrogen is the mineral element with the largest demand for plants and one of the most critical factors in determining crop yield. Comprehensive and systematic analysis of plant responses and strategies for utilizing nitrogen nutrition is an important basis for promoting sustainable agricultural development through molecular breeding in the future. The nitrogen level in the soil is extremely heterogeneous in both time and space. In order to better adapt to this environment, plants have evolved a nitrogen nutrition regulatory network with transcription factors as the core to optimize their growth. For example, NO3 - The core signaling transcription factor NLP7 directly acts as NO3 - Receptors sense NO3 - level, and transport NO3 via nuclear-cytoplasmic shuttling - signal, activating downstream NO3 - In addition, transcription factors such as STOP1, HY5, BES1, NIGT1 / HRS1, and TCP20 have also been reported to directly regulate nitrogen absorption. Therefore, exploring new transcription factors is an effective strategy to analyze the mechanism of efficient nitrogen utilization in plants.

[0003] TGACG-Binding (TGA) transcription factors belong to the bZIP transcription factor family. The TGA family of Arabidopsis has a total of 10 members, which play an important role in plant defense and plant development. Among the 10 TGAs in Arabidopsis, NPR1 can interact with 7 TGAs, and these 7 TGAs can be further divided into 3 subbranches. TGA1 and TGA4 belong to the first class; TGA2, TGA5, and TGA6 constitute the second class; the third class includes TGA3 and TGA7. At present, the prior art discloses the application of the TGA7 gene in regulating the flowering period of plants, such as Chinese patent CN111996200A, which was published on November 27, 2020, but there have been no research reports on the TGA7 gene in regulating nitrogen absorption and adaptability to low nitrogen stress. Summary of the invention

[0004] The object of the present invention is to provide an application of the TGA7 gene and / or its encoded protein in regulating plant growth and low nitrogen stress adaptability. The amplification primers of the TGA7 gene can improve plant growth and low nitrogen stress adaptability, promote the growth of plant roots, and then promote the plant's absorption of nutrients and increase the yield of crops.

[0005] The present invention provides amplification primers for a regulatory gene TGA7 for plant growth and low nitrogen stress adaptability, the primers comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2, and the nucleotide sequence of the regulatory gene TGA7 is shown in SEQ ID NO.3.

[0006] As a preferred embodiment, the plant includes a cruciferous plant.

[0007] The present invention provides a vector for overexpressing the regulatory gene TGA7 prepared based on the amplification primers.

[0008] As a preferred embodiment, the backbone vector of the overexpression vector includes 35s-pCAMBIA1300.

[0009] The present invention provides the use of the amplification primers of the regulatory gene TGA7 or the vector in improving the tolerance of plants to low nitrogen stress.

[0010] The present invention provides the use of the amplification primers of the regulatory gene TGA7 or the vector in regulating the root elongation of plants under low nitrogen stress.

[0011] As a preferred embodiment, the regulation includes increasing.

[0012] As a preferred embodiment, the root system includes a taproot.

[0013] The present invention provides the use of the amplification primers of the regulatory gene TGA7 or the vector in regulating the root nitrate absorption rate of plants under low nitrogen stress.

[0014] As a preferred embodiment, the regulation includes promotion.

[0015] Beneficial effects: The present invention provides the application of TGA7 gene and / or its encoded protein in regulating plant growth and low nitrogen stress adaptability. The transcription factor TGA7 is cloned from a model plant using the amplification primer of the TGA7 gene, and two mutant strains are constructed by knocking out and overexpressing the TGA7 gene. The functional mutagenesis mutant strain and the knockout strain of the gene have significantly reduced growth and low nitrogen stress adaptability, while the overexpression strain shows significantly improved adaptability to low nitrogen stress. The test results show that under low nitrogen stress, the transgenic overexpression Arabidopsis has significantly increased taproot elongation, significantly improved aboveground growth, and root NO3 - The absorption rate was significantly improved, while the taproot elongation and aboveground growth of the mutant strain of the TGA7 gene were significantly lower than those of the wild type. -The absorption rate was significantly lower than that of the wild type. Increasing the expression of TGA7 was beneficial to plant growth and the improvement of adaptability to low nitrogen stress.

[0016] The present invention provides the use of the above-mentioned amplification primers of the regulatory gene TGA7 or the above-mentioned vectors in improving plant tolerance to low nitrogen stress. The application of the present invention is of great significance for improving plant tolerance to low nitrogen stress, promoting plant growth, and then promoting plant absorption of nutrients, and increasing the yield of crops. The use of plant seeds obtained by using the amplification primers provided by the present invention and the amplified genes or proteins as nitrogen efficient utilization plants can greatly simplify industrial production operations, open up channels for genetic breeding of nitrogen efficient utilization plants, provide new production ideas for genetic breeding of nitrogen efficient utilization plants, reduce the screening work in the traditional breeding process, and at the same time, the use of nitrogen efficient utilization plants reduces the input of nitrogen fertilizers, greatly reducing production costs and environmental costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A schematic diagram of the binary vector 35s-pCAMBIA1300 provided by the present invention;

[0018] Figure 2 A schematic diagram of the transgenic vector pOETGA7 provided by the present invention;

[0019] Figure 3 The comparison result of the constructed sequence table of the TGA7 gene knockout vector provided by the present invention;

[0020] Figure 4 A comparison chart of TGA7 gene expression levels between the wild type and TGA7 overexpressing transgenic strains provided by the present invention;

[0021] Figure 5 A comparison chart of the fresh weight of the aboveground parts of the wild type, TGA7 low expression mutant and knockout transgenic strain provided by the present invention;

[0022] Figure 6 A comparison chart of the fresh weight of roots of the wild type, TGA7 low expression mutant and knockout transgenic strain provided by the present invention;

[0023] Figure 7 A comparison chart of the growth of the wild type, TGA7 low-expression mutant and over-expression transgenic strain provided by the present invention; wherein the scale length is 15 cm;

[0024] Figure 8 A comparison chart of the relative biomass of the aboveground parts and roots of the wild type, TGA7 low-expression mutant and over-expression transgenic strain provided by the present invention;

[0025] Fig. 9A comparison chart of root nitrate absorption rates of the wild type, TGA7 low-expression mutant and over-expression transgenic strain provided by the present invention. DETAILED DESCRIPTION

[0026]

[0027] In the present invention, the plant may include all types of plants, such as cruciferous plants, commonly but not limited to Arabidopsis thaliana. In order to illustrate the regulation of the TGA7 gene, the present invention uses the model plant Arabidopsis thaliana as material for experiments, and the amplified gene is recorded as AtTGA7 in the examples.

[0028] The present invention provides a vector for overexpressing the regulatory gene TGA7 prepared based on the above-mentioned amplification primers. As a preferred embodiment, the vector uses 35s-pCAMBIA1300 as a backbone vector. The vector for overexpressing the Arabidopsis transcription factor TGA7 gene of the present invention can achieve overexpression of the Arabidopsis growth and low nitrogen stress adaptability regulatory gene TGA7. The present invention has no special restrictions on the construction method of the vector for overexpressing the regulatory gene TGA7, and conventional vector construction methods well known to those skilled in the art can be used, such as enzyme ligation.

[0029] In a specific embodiment of the present invention, the present invention can first use the upstream primer of the nucleotide sequence as shown in SEQ ID NO.1 and the downstream primer of the nucleotide sequence as shown in SEQ ID NO.2 to amplify to obtain the TGA7 coding region sequence containing restriction sites and protective bases at both ends, and then insert this sequence into the overexpression vector pCAMBIA1300, that is, insert it between the two identical restriction endonucleases on the vector used, to obtain the following. Figure 2 The plant expression vector pOETGA7 containing the AtTGA7 gene is shown. Figure 2 The fragment (marked in brownish red) inserted at the rear side of the two tandem 35S promoters is labeled as TGA7. The vector for overexpression regulation gene TGA7 of the present invention can be introduced into plants by Agrobacterium-mediated method to achieve gene overexpression. Figure 1 The 35s-pCAMBIA1300 shown is a plant constitutive overexpression vector, and the constitutive promoter can be composed of two CaMV35S promoters, for example, two cauliflower mosaic virus CaMV35S promoters connected in series.

[0030] The present invention provides the use of the amplification primers of the regulatory gene TGA7 or the vector in improving plant tolerance to low nitrogen stress. As a preferred embodiment, the nucleotide sequence of the TGA7 gene is shown in SEQ ID NO.3, and the amino acid sequence of the TGA7 protein is shown in SEQ ID NO.4 (MMSSSSPTQLASLRDMGIYEPFQQIVGWGNVFKSDINDHSPNTATSSIIQVD PRIDDHNNNIKINYDSSHNQIEAEQPSSNDNQDDDGRIHDKMKRRLAQNREAARKSRLRKKAYVQQLEESRLKLSQLEQELEKVKQQGHLGPSGSINTGIASFEMEYSHWLQEQSRRVSELRTALQSHISDIELKMLVESCLNHYANLFQMKSDAAKADVFYLISGMWRTSTERFFQWIGGFRPSELLNVVMPYLQPLTDQQILEVRNLQQSSQQAEDALSQGIDKLQQSLAESIVIDAVIESTHYPTHMAAAIENLQALEGFVNQADHLRQQTLQQMAKILTTRQSARGLLALGEYLHRLRALSSLWAARPQEPT). In the present invention, the TGA7 protein is a sequence composed of 368 amino acids. In the present invention, the evaluation method for plant growth and low nitrogen stress tolerance can be used for low nitrogen treatment to detect plant biomass and nitrate absorption rate for evaluation. As a preferred embodiment, the overexpression method may include the following steps: cloning the gene TGA7 into a plant constitutive overexpression vector to obtain a recombinant expression vector; and transforming the recombinant expression vector into a plant by Agrobacterium-mediated. In the present invention, the plant constitutive overexpression vector may be 35s-pCAMBIA1300. The present invention does not specifically limit the method of transformation, and conventional operation methods well known to those skilled in the art may be used. After transformation, the present invention may perform operations of culturing, screening and harvesting transgenic seeds. The present invention does not specifically limit the methods of culturing, screening and harvesting, and conventional operation methods well known to those skilled in the art may be used. In the present invention, the plant may include all types of plants, such as Arabidopsis. In order to illustrate the overexpression method of the TGA7 gene, the present invention uses the model plant Arabidopsis as a material for experiment.

[0031] The present invention provides the use of the amplification primers of the above-mentioned regulatory gene TGA7 or the above-mentioned vector in regulating the root elongation of low nitrogen stress plants, the regulation may include improvement, and the root system may include a taproot. As a preferred embodiment, the nucleotide sequence of the TGA7 gene is shown in SEQ ID NO.3, and the amino acid sequence of the TGA7 protein is shown in SEQ ID NO.4. In the present invention, the regulation may include promoting the biomass of the aboveground part and the root system of the plant by overexpression of the TGA7 gene or inhibiting the biomass of the aboveground part and the root system of the plant by knocking out or silencing the TGA7 gene. As a specific embodiment, the forward and complementary single-stranded DNA of the sgRNA containing the complementary sticky ends of the BsaI restriction enzyme digestion product are synthesized, and the nucleotide sequence is specifically the forward primer shown in SEQ ID NO.5 (5'-TGCAGTTTAGCGCAGAACCGA GAAG-3') and the nucleotide sequence is as shown in SEQ ID NO.6 (5'-AAACCTTCTCG GTTCTGCGCTAAAC-3').

[0032] In the present invention, the plant may include all types of plants, such as Arabidopsis. In order to illustrate the regulation mode of the TGA7 gene, the present invention uses the model plant Arabidopsis as a material for experiments. As a specific embodiment, overexpression of the TGA7 gene increases the biomass of the aboveground part and root system of Arabidopsis under low nitrogen stress conditions, and the relative biomass of the aboveground part and root system of Arabidopsis is significantly higher than the relative biomass of the wild type and Arabidopsis mutant materials with reduced TGA7 gene expression. In the present invention, the aboveground part is all aboveground parts above the stem, and the root system is the main root. In an embodiment of the present invention, overexpression of the TGA7 gene increases the biomass of the aboveground part and root system of Arabidopsis under low nitrogen stress conditions, and the relative biomass of the aboveground part and main root of Arabidopsis is significantly higher than the relative biomass of the aboveground part and main root of the wild type and Arabidopsis mutant materials with reduced TGA7 gene expression. As a specific embodiment, the biomass detection method can include disinfecting the surface of the plant seeds with 75% alcohol by mass concentration, then washing with sterile water for 3 to 5 times, sowing the seeds on a 1 / 2MS solid culture medium plate, and placing the plate in a 4°C refrigerator for 3 days, and then placing the plate in a light incubator (light 16h / dark 8h) for 5 days; then fix the stem of the seedling with a sponge and transfer it to a hydroponic pot containing 1 / 2MS liquid culture medium to continue growing for 14 days; when the plant grows to a certain size, transfer it to a low nitrogen treatment condition (0.01mM KNO3+0.01mM NH4NO3) or a control treatment condition (2mM KNO3+2mM NH4NO3) to continue growing for 14 days. Use a balance to measure the biomass of the aboveground part and root system of each material seedling, and calculate the relative biomass on the control plate of the corresponding material, that is, the biomass under the low nitrogen treatment condition / the biomass under the control treatment condition.

[0033] The present invention provides the use of the amplification primers of the above-mentioned regulatory gene TGA7 or the above-mentioned vector in regulating the root nitrate absorption rate of low nitrogen stress plants, and the regulation may include promotion. As a preferred embodiment, the nucleotide sequence of the TGA7 gene is shown in SEQ ID NO.3, and the amino acid sequence of the TGA7 protein is shown in SEQ ID NO.4. In the present invention, the regulation preferably includes increasing the root nitrate absorption rate by overexpressing the TGA7 gene or increasing the root nitrate absorption rate of the plant by knocking out or silencing the TGA7 gene. In the present invention, the plant preferably includes all types of plants, such as Arabidopsis. In order to illustrate the regulation mode of the TGA7 gene, the present invention uses the model plant Arabidopsis as a material for experiment. In the embodiment of the present invention, overexpression of the TGA7 gene increases the nitrate absorption rate of the root system of Arabidopsis under low nitrogen stress conditions. As a specific embodiment, the method for detecting the root nitrate absorption rate preferably includes transferring 5-day-old seedlings to a low nitrogen treatment plate or a control treatment plate and continuing to grow for 5 days. After washing in 0.05 mM CaSO4 solution for 1 min, the seedlings were transferred to a 0.2 mM 15KNO3 ( 15 The roots were then washed with 0.05 mM CaSO4 for 1 minute and the aboveground and roots were harvested separately. The samples were fully dried and the dry weight was measured. The aboveground and underground samples of the same plant were mixed and ground, and the mass spectrometer (Isorime100; Hanau, Germany) was used to determine the mass spectrometry in the plant. 15 N abundance.

[0034] To further illustrate the present invention, the application of the TGA7 gene and / or its encoded protein provided by the present invention in regulating plant growth and low nitrogen stress adaptability is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 Cloning of TGA7 gene

[0036] The surface-sterilized Arabidopsis seeds were planted in 1 / 2MS solid culture medium, purified for 2 days in the dark at 4°C, and then cultured under light conditions for 8 days. The roots of the seedlings were collected for RNA extraction, and a kit was used to perform reverse transcription to synthesize cDNA sequences as templates for subsequent gene cloning.

[0037] According to the sequencing results of the currently published Arabidopsis thaliana genome (gene number AT1G77920), upstream primers and downstream primers were designed respectively:

[0038] Upstream primer: 5'-ATGATGAGTTCTTCTTCTCCAACACA-3' (as shown in SEQ ID NO.7);

[0039] Downstream primer: 5'-AGTTGGTTCTTGTGGACGAGCT-3' (as shown in SEQ ID NO. 8).

[0040] The PCR amplification was performed using KOD FX enzyme from TOYOBO. The reaction procedure for PCR amplification was as follows: pre-denaturation: 94°C, 2 minutes; denaturation: 98°C, 10 seconds; annealing at 57°C, 30 seconds; extension at 68°C, 2 minutes (35 cycles); final extension: 68°C, 5 minutes. The reaction system for PCR amplification was as follows:

[0041]

[0042] The PCR amplification product was sent for sequencing to obtain the nucleotide sequence of AtTGA7 (SEQ ID NO. 3).

[0043] Example 2 Construction of constitutive overexpression transgenic vector

[0044] The constitutive overexpression vector pCAMBIA1300 (see Figure 1 ) was linearized. Primers TGA7-F (as shown in SEQ ID NO.1) and TGA7-R (as shown in SEQ ID NO.2) were used, and the nucleotide sequence of AtTGA7 obtained in Example 1 was used as a template. The PCR amplification reaction procedure in Example 1 was referred to to amplify the coding region sequence of Arabidopsis thaliana AtTGA7 containing restriction sites at both ends. The coding region sequence of AtTGA7 was inserted into the pCAMBIA1300 vector using a ligase-independent rapid cloning kit (ClonExpress II One Step Cloning Kit, C115; VazymeBiotech Co., Ltd.), and the transgenic vector pOETGA7 (see FIG. 1 ) in which the Arabidopsis thaliana gene AtTGA7 is driven by the promoter CaMV35S was obtained. Figure 2 ), wherein the promoter CaMV35S is composed of two cauliflower mosaic virus CaMV35S promoters in series.

[0045] Example 3 Transformation of Arabidopsis thaliana

[0046] 0.5 μg of the vector pOETGA7 plasmid prepared in Example 2 was transferred into the competent cells of Agrobacterium tumefaciens strain GV3101, and the cells were successively placed in an ice bath for 5 minutes, liquid nitrogen for 5 minutes, a 37°C water bath for 5 minutes, and an ice bath for 5 minutes. Antibiotic-free LB culture medium was added and activated in a shaker at 28°C for 1 hour to obtain an Agrobacterium strain containing the vector pOETGA7.

[0047] The steps of Arabidopsis flower dipping are as follows: Agrobacterium carrying the pOETGA7 vector is inoculated into fresh LB liquid medium containing 50 mg / L Kana and 25 mg / L R Rif antibiotics at a ratio of 1:100, and shaken overnight at 28°C. The cells are collected by centrifugation at 5000 rpm for 5 minutes, and resuspended in transformation solution (water, 10% w / v sucrose, 0.02% v / v Silwet-77) to a bacterial solution OD of 0. 600 The wild type (Col-0) Arabidopsis thaliana that had bolted and partially completed flowering was selected as the transgenic material, the mature pods were subtracted, and the flowers and buds were retained. The floral dipping transformation method was used, and the aerial part of Arabidopsis thaliana was dipped into the above-prepared bacterial solution for 2 minutes, and then cultured in the dark at 23°C for 24 hours and then transferred to normal light culture to harvest T1 generation seeds.

[0048] The steps for screening positive homozygous lines are as follows: sow T1 generation seeds on a plate containing hygromycin for screening, select positive plants with normal growth to harvest T2 generation seeds; continue to sow T2 generation seeds on a plate containing hygromycin for screening, calculate the segregation ratio, and select single copy insertion lines with a segregation ratio that basically meets 3:1 to harvest T3 generation seeds; select a certain number of T3 generation seeds, if the T3 generation seeds of a certain line can grow normally on a plate containing hygromycin, then the line is a homozygous line with a single copy insertion (TGA7OX).

[0049] Example 4 Construction of gene knockout transgenic material

[0050] The forward primer and its complementary single-stranded DNA containing sgRNA complementary to the sticky end of the BsaI restriction enzyme digestion product were synthesized, and the nucleotide sequence was specifically the forward primer shown in SEQ ID NO.5 (5'-TGCAGTTTAGCGCAGAACCGA GAAG-3') and the nucleotide sequence was the reverse primer shown in SEQ ID NO.6 (5'-AAACCTTCTCG GTTCTGCGCTAAAC-3'). The two single-stranded DNA fragments were phosphorylated and annealed to form a double-stranded sgRNA with sticky ends; at the same time, pMgPaef-237-2A-gfp was linearized with BsaI restriction endonuclease [Hashimoto, R., Ueta, R., Abe, C., Osakabe, Y. & Osakabe, K. Efficient multiplex genome editing induces precise, and self-ligated type mutations in tomato plants. Front Plant Sci. 9, 916 (2018)]; the double-stranded sgRNA and the linearized vector were connected using TOYOBO's ligase Ligation high (Code No. LGK-100) to obtain the cloning vector pMgPaef-237-2A-gfp-sgRNA for TGA7 gene knockout. The cloning vector pMg Paef-237-2A-gfp-sgRNA was transferred into Agrobacterium GV3101, and then the constructed vector was transferred into Arabidopsis by the floral transformation method, and positive seedlings were screened on a 1 / 2MS plate containing 50 mg / L Kana. The leaves of the seedlings of the positive seedlings were cut to extract the plant genomic DNA, and the TGA 7genomic DNA of all genetically transformed seedlings was amplified. The PCR product was recovered by electrophoresis and gel cutting to recover the target band, and the upstream primer (SEQ ID NO.7) and the downstream primer (SEQ ID NO.8) were used as sequencing primers to send to a sequencing company for sequencing; at the same time, the possible off-target sites were analyzed by conventional operation methods familiar to those skilled in the art, and the homozygous mutants were selected for single plant harvesting to obtain T2 generation homozygous positive plants. In this process, a homozygous AtTGA7 mutant strain with AtTGA7 gene mutation and no off-target was obtained. The sequence table comparison results are shown as follows: Figure 3 shown.

[0051] Example 5 Molecular Detection of Target Gene Expression

[0052] RNA was extracted from young seedlings of wild-type and overexpressing transgenic plants, and reverse transcribed. Real-time quantitative PCR was performed using SYBR Green Realtime PCR Master Mix from TOYOBO, with Actin2 gene as internal reference. The detection system and primers used are as follows:

[0053] The fluorescent real-time quantitative PCR reaction primers used were:

[0054] qTGA7-F: 5'-ATCATAGTCCCAATACTGCTA-3' (as shown in SEQ ID NO. 9);

[0055] qTGA7-R: 5'-ATCCTGCCATCATCATCTT-3' (as shown in SEQ ID NO. 10);

[0056] qUBQ10-F: 5'-ACCCTAACGGGAAAGACGA-3' (as shown in SEQ ID NO. 11);

[0057] qUBQ10-R: 5′-GGAGCCTGAGAACAAGATGAA-3′ (as shown in SEQ ID NO. 12).

[0058] The reaction procedure of fluorescent real-time quantitative PCR is as follows:

[0059] Pre-denaturation: 95°C, 1 min; PCR cycle: 95°C, 15 sec; 60°C, 15 sec; 72°C, 45 sec (40 cycles).

[0060] The reaction system of fluorescent real-time quantitative PCR is:

[0061]

[0062]

[0063] The detection results of the molecules expressed by the target gene are shown in Tables 1 and Figure 4 , where WT refers to wild type and OX refers to overexpression, i.e., TGA7 OX1 and TGA7 OX2 represent two independent lines of TGA7 overexpressing transgenic lines, respectively.

[0064] Table 1 Relative expression levels of TGA7 gene in wild-type and TGA7 overexpressing transgenic lines

[0065] genotype Relative expression WT 1.00±0.11 TGA7OX1 11.57±0.70 TGA7OX2 14.24±0.27

[0066] As shown in Table 1 and Figure 4As shown in the figure, the expression level of TGA7 gene in the overexpression transgenic plants was 11.57±0.70 and 14.24±0.27, relative to the expression level of TGA7 gene in the non-transgenic lines. Compared with the non-transgenic lines, the TGA7 gene was 10-15 times more expressed in the overexpression transgenic plants.

[0067] Example 6 Detection of plant growth and low nitrogen stress tolerance

[0068] The seeds of the wild type, the Arabidopsis TGA7 gene mutant material (tga7) purchased from the Arabidopsis Biological Resource Center (ABRC), the overexpressed transgenic material (TGA7OX) obtained in Example 3, and the gene knockout material (tga7-KO) obtained in Example 4 were disinfected with 75% alcohol surface, then washed 3 to 5 times with sterile water, and the seeds were sown on a 1 / 2MS solid culture medium plate, and then the plate was placed in a 4°C refrigerator for 3 days. The plate was placed in a light incubator (light 16h / dark 8h) for 5 days; then it was transferred to a low nitrogen treatment (0.01mM KNO3+0.01mM NH4NO3) plate or a control treatment (2mM KNO3+2mM NH4NO3) plate and the biomass was measured after continued growth for 5 days to complete the plate phenotype observation. Alternatively, the stem of the seedling was fixed with a sponge and transferred to a hydroponic pot containing 1 / 2MS liquid culture medium for further growth for 14 days; when the plant grew to a certain size, it was transferred to a low nitrogen treatment condition or a control treatment condition for further growth for 14 days. The biomass of the aboveground part and the root system of the seedlings of each material was measured with a balance, and the relative biomass on the control plate of the corresponding material was calculated, that is, the biomass under the low nitrogen treatment condition / the biomass under the control treatment condition. The test results are shown in Tables 2 to 4 and Figures 5 to 8 .

[0069] Table 2 Aboveground biomass of wild-type, TGA7 low expression and TGA7 gene knockout transgenic lines

[0070]

[0071]

[0072] Table 3 Root biomass of wild-type, TGA7 low expression and TGA7 gene knockout transgenic lines

[0073] Root biomass (mg) WT tga7 tga7-KO Normal nitrogen 2.33+0.34 0.94+0.23 1.37+0.17 Low nitrogen 1.98+0.24 0.47+0.22 0.90+0.26

[0074] Table 4 Relative biomass of wild-type, TGA7 low-expression and TGA7 over-expression transgenic lines

[0075] genotype Fresh weight of aboveground parts (mg) Relative aboveground biomass Root fresh weight (mg) Relative root biomass WT 160.6±15.9 0.318±0.066 31.7±4.0 0.776±0.098 tga7 46.2±12.7 0.221±0.062 4.3±1.6 0.359±0.089 TGA7OX1 209.6±19.2 0.491±0.062 34.7±5.0 0.916±0.102 TGA7OX2 206.2±13.6 0.441±0.055 36.7±6.2 0.925±0.106

[0076] As shown in Tables 2 to 4 and Figures 5 to 8 As shown in the figure, under normal conditions and low nitrogen stress conditions, compared with the wild type, the growth of the TGA7 low-expression mutant was significantly slow, the taproot was shorter, the aboveground part was smaller and yellowed, while the growth of the overexpression transgenic strain was significantly enhanced, and the taproot growth and aboveground growth were significantly stronger than the wild type. In addition, overexpression of the TGA7 gene increased the biomass of the aboveground and root system of Arabidopsis under low nitrogen stress conditions, and the relative biomass of the aboveground and root system of Arabidopsis was significantly higher than that of the wild type and the Arabidopsis mutant material with reduced TGA7 gene expression.

[0077] Example 7 Detection of nitrate absorption rate of roots

[0078] The wild type, the Arabidopsis TGA7 gene mutant material (tga7) purchased from the Arabidopsis Biological Resource Center (ABRC), and the overexpression transgenic material (TGA7 OX) obtained in Example 3 of the present invention that had been grown on the plate for 5 days were transferred to the low nitrogen treatment plate or the control treatment plate and continued to grow for 5 days. After washing in 0.05 mM CaSO4 solution for 1 minute, the seedlings were transferred to a plate containing 0.2 mM 15 KNO3( 15 The roots were then washed with 0.05 mM CaSO4 for 1 minute and the aboveground and roots were harvested separately. The samples were fully dried and the dry weight was measured. The aboveground and underground samples of the same plant were mixed and ground, and the mass spectrometer (Isorime100; Hanau, Germany) was used to determine the mass spectrometry in the plant. 15 N abundance, the test results are shown in Table 5 and Fig. 9 .

[0079] Table 5 Root nitrate absorption rate

[0080]

[0081]

[0082] As shown in Table 5 and Fig. 9 As shown, under low nitrogen stress, the root nitrate uptake rate of transgenic overexpressing (TGA7 OX2) Arabidopsis was significantly increased compared with the wild-type control, while the root nitrate uptake rate of the TGA7 gene low-expression mutant was significantly lower than that of the wild-type.

[0083] It can be seen that the tolerance of transgenic overexpression (TGA7 OX2) Arabidopsis to low nitrogen stress is significantly improved compared with the wild-type control, while the tolerance of the TGA7 gene low-expression mutant strain to low nitrogen stress is significantly lower than that of the wild-type, indicating that the gene TGA7 is indeed involved in the regulation of plant tolerance to low nitrogen stress.

[0084] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A primer for amplifying the regulatory gene TGA7 for plant growth and low nitrogen stress adaptability, characterized in that: The primers include: an upstream primer whose nucleotide sequence is shown as SEQ ID NO.1 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO.

2. The nucleotide sequence of the regulatory gene TGA7 is shown as SEQ ID NO.

3.

2. The amplification primer according to claim 2, characterized in that The plants include plants of the family Cruciferae.

3. A vector for overexpressing the regulatory gene TGA7 prepared based on the amplification primers according to claim 1 or 2.

4. The carrier according to claim 3, characterized in that The backbone vector of the overexpression vector includes 35s-pCAMBIA1300.

5. Use of the amplification primers of the regulatory gene TGA7 according to claim 1 or 2 or the vector according to claim 3 or 4 in improving plant tolerance to low nitrogen stress.

6. Use of the amplification primers for the regulatory gene TGA7 according to claim 1 or 2 or the vector according to claim 3 or 4 in regulating the root elongation of plants under low nitrogen stress.

7. The use according to claim 6, characterized in that: Said regulation includes increasing.

8. The use according to claim 6, characterized in that: The root system includes a taproot.

9. Use of the amplification primers for the regulatory gene TGA7 according to claim 1 or 2 or the vector according to claim 3 or 4 in regulating the root nitrate absorption rate of plants under low nitrogen stress.

10. The use according to claim 9, characterized in that: Said regulation includes promotion.

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