Application of GmASR2 gene in regulation and control of plant phosphorus nutrition

Abstract

The invention discloses an application of a GmASR2 gene in regulation and control of plant phosphorus nutrition. The relation between the soybean ASR transcription factor GmASR2 gene and the important nutrient element phosphorus is defined, it is proved that the gene participates in the low-phosphorus adaptation mechanism of plants, and the fact that the GmASR2 gene participates in soybean phosphorus nutrition regulation and control is disclosed for the first time. Researches show that the GmASR2 gene is obviously subjected to up-regulation expression under low phosphorus stress in roots and leaves, plant growth and phosphorus nutrition absorption are promoted by overexpression of the GmASR2 gene, and plant growth and phosphorus nutrition are positively regulated; under low phosphorus stress, the overexpression of the GmASR2 gene can improve the phosphorus deficiency symptom of the plant, improve the absorption of phosphorus nutrients and the accumulation of biomass of the plant, significantly promote the growth of the root system, and increase the phosphorus content and chlorophyll content of the plant. The GmASR2 gene plays an important role in improving plant phosphorus efficiency, promoting plant growth under low-phosphorus stress and increasing phosphorus content, and provides an important gene resource for plants to adapt to low-phosphorus environmental stress.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and plant breeding. More specifically, it relates to the application of the GmASR2 gene in regulating plant phosphorus nutrition. Background Art

[0002] Phosphorus is an essential element required by plants. Its effects on soybeans are mainly reflected in promoting the growth and development of soybeans, improving yield and quality, and enhancing stress resistance. Phosphorus can promote the development of soybean roots, making the roots more developed, which is beneficial for soybeans to absorb more water and nutrients from the soil, providing a solid foundation for the growth and development of soybeans. Phosphorus participates in various metabolic processes in soybeans, such as energy transfer, material transformation and transportation, etc., and can promote the reproductive growth of soybeans, including processes such as flower bud differentiation, flowering and fruiting. Adequate supply of phosphorus can shorten the formation process of reproductive organs, increase the pod-setting rate and grain weight of soybeans, thereby increasing the yield. Phosphorus can also improve the quality of soybeans. It helps in the synthesis and accumulation of proteins and fats in soybeans, improving the nutritional value of soybeans. Phosphorus can improve the drought resistance and cold resistance of soybeans and promote the development of soybean root nodules. Therefore, regulating soybean phosphorus nutrition is very important for soybean production.

[0003] Soybean genes play a crucial role in regulating plant phosphorus nutrition. Their advantages are mainly reflected in improving phosphorus absorption, participating in regulating the transportation, distribution and utilization of phosphorus in plants, and enhancing phosphorus tolerance. Soybeans adapt to phosphorus-deficient soils through a series of complex genetic mechanisms, including the activation, absorption, transportation, storage and reuse of phosphates. For example, the GmGDPD2 gene in soybeans has been identified as a major gene for low-phosphorus tolerance. Overexpression of this gene can significantly promote root growth and development and organic acid secretion, thereby improving phosphorus absorption efficiency. Under phosphorus-deficient conditions, soybeans can adapt to this adversity by regulating their own gene expression. For example, the GmGDPD2 gene not only promotes phosphorus absorption, but also forms a regulatory module together with genes such as GmMyb73 and GmGA2ox1, affecting root cell division by regulating root hormone levels, thereby enhancing the tolerance of soybeans to low-phosphorus stress. Therefore, by genetically improving soybean genes, the absorption and utilization efficiency of phosphorus by soybeans can be significantly increased, thereby increasing the yield of soybeans. Improving the absorption and utilization efficiency of phosphorus by soybeans helps to reduce the application amount of phosphate fertilizers, thereby saving phosphorus resources, which is of great significance for alleviating the global phosphorus resource crisis. Reducing the application amount of phosphate fertilizers can not only save resources, but also reduce environmental pollution caused by phosphorus loss. Therefore, the genetic improvement of soybean genes provides the possibility for achieving environmentally friendly agricultural production.

[0004] ASR genes are plant-specific transcription factors that play important roles in regulating plant senescence, fruit ripening, and responses to abiotic stresses. These genes are expressed in plant organs and growth stages and play important roles in plant responses to developmental and environmental conditions, especially involving ABA signaling. ASR proteins have strong hydrophilicity, indicating that they may play a role in drought stress responses. Since the first ASR gene was discovered in tomato, ASR genes have been identified in various monocotyledonous and dicotyledonous plants. However, homologous genes have not been found in the model plant Arabidopsis thaliana. Studies have shown that ASR proteins usually act as molecular chaperones, osmoregulatory proteins, metal-binding proteins, and antioxidant or detoxifying proteins. Some ASRs can also act as unique transcription factors. However, the functions of the vast majority of soybean ASR transcription factors are unclear, the relationship with the lack of important nutrient elements has not been clarified, and it is unknown whether they are also involved in the mechanism of plant adaptation to low phosphorus. Therefore, it is necessary to provide more gene resources that can regulate plant phosphorus nutrition and enable plants to adapt to low phosphorus environmental stress, which is of great significance for improving the efficiency of plant phosphorus nutrition absorption in phosphorus-deficient soils. Summary of the Invention

[0005] The present invention clarifies the relationship between the function of soybean ASR transcription factors and the mechanism of plant adaptation to low phosphorus, and provides the application of the GmASR2 gene in regulating plant phosphorus nutrition.

[0006] The first object of the present invention is to provide the application of the GmASR2 gene in positively regulating plant growth and phosphorus nutrition.

[0007] The second object of the present invention is to provide the application of a preparation that promotes the expression of the GmASR2 gene.

[0008] The third object of the present invention is to provide a method for promoting plant growth and nutrient absorption, or increasing the phosphorus content and / or chlorophyll content of plants.

[0009] The fourth object of the present invention is to provide a method for cultivating plants resistant to low phosphorus or plants with high phosphorus content.

[0010] The above objects of the present invention are achieved by the following technical solutions.

[0011] The present invention clarifies the relationship between the soybean ASR transcription factor GmASR2 gene (gene number Glyma.20g167500) and the important nutrient element phosphorus, and proves that it is involved in the mechanism of plant adaptation to low phosphorus. For the first time, it is publicly disclosed that the GmASR2 gene is involved in the regulation of phosphorus nutrition in soybeans. The nucleotide sequence of the GmASR2 gene is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.2. Research shows that the expression of the GmASR2 gene is significantly up-regulated by low phosphorus stress in both roots and leaves. Overexpression of the GmASR2 gene promotes plant growth and phosphorus nutrition absorption, promotes the absorption and accumulation of phosphorus in plants by positively regulating plant growth and phosphorus nutrition, and alleviates the inhibitory effect of plants under low phosphorus stress; under low phosphorus conditions, overexpression of the GmASR2 gene can improve the phosphorus deficiency symptoms of soybeans and Arabidopsis thaliana, increase the absorption of phosphorus nutrients and the accumulation of biomass in plants, significantly promote the growth of roots, increase the phosphorus content and chlorophyll content of plants, and regulate plant growth. The GmASR2 gene plays an important role in improving plant phosphorus efficiency, promoting plant growth and development under low phosphorus stress, and increasing phosphorus content, providing an important gene resource for plants to adapt to low phosphorus environmental stress, and is of great significance for improving the growth status of crops in phosphorus-deficient areas and increasing food production.

[0012] Therefore, the present invention provides the application of the GmASR2 gene in positively regulating plant growth and phosphorus nutrition.

[0013] Furthermore, regulating plant phosphorus nutrition is to increase the phosphorus content of plants and promote the absorption of phosphorus by plants.

[0014] Preferably, the plant is soybean or Arabidopsis thaliana.

[0015] The present invention provides the application of the GmASR2 gene in promoting plant growth under low phosphorus stress.

[0016] The present invention provides the application of the GmASR2 gene in cultivating plants tolerant to low phosphorus stress.

[0017] Preferably, the GmASR2 gene is overexpressed in plants.

[0018] The present invention provides the application of the GmASR2 gene in increasing the phosphorus content and / or chlorophyll content of plants under low phosphorus stress, or in the preparation of preparations for increasing the phosphorus content and / or chlorophyll content of plants.

[0019] The present invention provides the application of the preparation for promoting the expression of the GmASR2 gene in promoting plant growth under low phosphorus stress.

[0020] The present invention provides the application of the preparation for promoting the expression of the GmASR2 gene in the preparation of products for promoting the absorption of plant phosphorus nutrition or products tolerant to low phosphorus stress.

[0021] The present invention provides the use of an agent for promoting the expression of the GmASR2 gene in increasing the phosphorus content and / or chlorophyll content of plants, or in the preparation of a product for increasing the phosphorus content and / or chlorophyll content of plants.

[0022] The present invention provides the use of an agent for promoting the expression of the GmASR2 gene in cultivating low-phosphorus tolerant plants.

[0023] Preferably, the agent is a plasmid, vector or recombinant bacterium overexpressing the GmASR2 gene.

[0024] The present invention provides a method for increasing the phosphorus content and / or chlorophyll content of plants, which is to promote the expression of the GmASR2 gene in plants, or to treat plants with an agent for promoting the expression of the GmASR2 gene.

[0025] The present invention also provides a method for cultivating low-phosphorus tolerant plants or high-phosphorus content plants, which is to promote the expression of the GmASR2 gene in plants, or to introduce a recombinant vector or recombinant bacterium containing the overexpressed GmASR2 gene into plants.

[0026] Furthermore, by transferring the overexpression vector containing the GmASR2 gene into a plant expression vector and transforming plant materials exogenously, transgenic materials tolerant to low-phosphorus stress or with high phosphorus content can be obtained.

[0027] The present invention has the following beneficial effects:

[0028] The present invention clarifies the relationship between the soybean ASR transcription factor GmASR2 gene and the important nutrient element phosphorus, proves that it is involved in the mechanism of plant adaptation to low phosphorus, and first discloses that the GmASR2 gene is involved in the regulation of soybean phosphorus nutrition. Research shows that overexpressing the GmASR2 gene can promote plant growth under low-phosphorus stress, relieve the inhibitory effect of low-phosphorus stress on plant growth and development, and alleviate the phosphorus deficiency symptoms of plants. Under low-phosphorus stress, overexpressing the GmASR2 gene can improve the phosphorus deficiency symptoms of soybean and Arabidopsis, promote root growth, increase the phosphorus content, and improve the absorption of phosphorus nutrients and the accumulation of biomass by plants; it shows that the GmASR2 gene plays an important role in improving plant phosphorus efficiency and promoting plant growth and development and increasing phosphorus content under low-phosphorus stress, and provides an important gene resource for plants to adapt to low-phosphorus environmental stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a result graph of quantitative PCR of the GmASR2 gene in different tissues (HP represents normal phosphorus nutrition, LP represents low-phosphorus treatment; the left figure is the quantitative result in the middle root, and the right figure is the quantitative result in the leaf).

[0030] Figure 2Results of transient expression of 35S:GFP and 35S:GmASR2-GFP in tobacco leaf cells, respectively (excitation wavelength of GFP channel: 488 nm; DAPI is the ultraviolet channel, excitation wavelength 360 nm, which can bind to DNA and emit blue fluorescence; Merge represents the fusion of GFP and DAPI; Bright field is the bright field).

[0031] Figure 3 Phenotype diagrams of whole plants of different soybean chimeras after 25 days of treatment under different phosphorus conditions (OE is the plant overexpressing the GmASR2 gene; EV is the empty vector plant; HP represents normal phosphorus nutrition; LP represents low phosphorus treatment, the same below, the scale bar in the figure is 10 cm).

[0032] Figure 4 Phenotype diagrams of leaves of different ages of different soybean chimeras after 25 days of treatment under different phosphorus conditions (the first leaf represents the oldest trifoliate leaf, the second leaf represents the second trifoliate leaf, and so on, the scale bar in the figure is 10 cm).

[0033] Figure 5 Results of differences in biomass indexes of different soybean chimeras after 25 days of different phosphorus treatments (ASR2_OE is overexpression of the GmASR2 gene; EV is the empty vector).

[0034] Figure 6 Results of differences in biomass indexes of different soybean chimeras after 25 days of different phosphorus treatments (ASR2_OE is overexpression of the GmASR2 gene; EV is the empty vector).

[0035] Figure 7 Phenotypes of different plant materials after 10 days of treatment under high and low phosphorus conditions (OX-5 and OX-9 are GmASR2 gene materials; Col-0 is the Columbia wild type material; the scale bar in the figure is 1 cm).

[0036] Figure 8 Results of differences in main root length, number of lateral roots, lateral root density, fresh weight, and chlorophyll content of different plant materials after 10 days of different phosphorus treatments (OX-5 and OX-9 are materials overexpressing the GmASR2 gene; Col-0 is the Columbia wild type material; the scale bar in the figure is 1 cm).

[0037] The data in the above figures are the average values and standard errors of 4 biological replicates; "*", "**", and "***" respectively indicate significant differences (Student’s t-test, 0.01 < P ≤ 0.05), extremely significant differences (Student’s t-test, 0.001 < P ≤ 0.01), and extremely extremely significant differences (Student’s t-test, P ≤ 0.001) between HP and LP treatments). DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] Example 1 Expression pattern of GmASR2 gene under phosphorus stress

[0041] 1. Soybean material processing

[0042] The full soybean variety YC03-3 of the same period was selected as the plant material. After being sterilized by fumigation with chlorine (50 mL sodium hypochlorite + 2.1 mL concentrated hydrochloric acid) for 4 h, the soybean was placed in a fume hood to remove the residual chlorine. Then the soybean was placed in an artificial culture room for sand culture for 4 to 5 days. After the cotyledons were slightly opened, it was transferred to normal nutrient solution hydroponics (Hoagland nutrient solution). After acclimatization for 7 days, the soybean was given normal phosphorus nutrition HP (500 μM PO4 2- ) and low phosphorus treatment LP (5 μM PO4 2- ) treatment, and samples were collected after short-term (2h) and long-term (240h) treatment, and the collected samples were stored at -80℃. RNA was extracted from soybean root and leaf samples obtained after 2h and 240h of different phosphorus treatments, and reverse transcribed into cDNA, which was used as a template for real-time fluorescence quantitative PCR.

[0043] 2. RNA extraction and reverse transcription

[0044] Cut about 100 mg of plant material into a 2 mL centrifuge tube and immediately place it in liquid nitrogen; add 1 mL of Trizol reagent to the centrifuge tube and put a steel ball in it, shake it on a shaker for 2 minutes, mix it thoroughly and let it stand for 5 minutes; add 200 μL of chloroform to the centrifuge tube, shake it vigorously for 10 seconds (to thoroughly mix the two phases), let it stand for 3 minutes, and then centrifuge it at 4°C, 12000 r / min for 15 minutes; take about 400 μL of supernatant to another new 1.5 mL centrifuge tube, add an equal volume of isopropanol, turn it upside down evenly, and let it stand for 10 minutes; centrifuge it at 4°C, 12000 r / min for 10 minutes, at this time the RNA sinks to the bottom; discard the supernatant, add 500 μL Wash the precipitate twice with 75% ethanol (upside down), centrifuge at 4°C, 12000r / min, for 5min each time; air-dry the precipitate in a fume hood, add 20μL sterile water to dissolve the precipitate, and store at -80°C for later use; determine the RNA concentration.

[0045] Refer to the reverse transcription kit instruction manual, calculate the RNA dosage (10 pg - 1 μg) based on the OD value of the extracted RNA. First, remove genomic DNA at 42 °C for 2 min, and the reaction system is shown in Table 1 below; then perform reverse transcription, and the reaction program is: 42 °C for 2 min; 85 °C for 5 s reaction system, and the reaction system for reverse transcription is shown in Table 2 below; all operations must be completed on ice. After the reaction, store the cDNA at -20 °C.

[0046] Table 1 Reaction System for Removing Genomic DNA

[0047]

[0048] Table 2 Reaction System for RNA Reverse Transcription

[0049]

[0050] 3. Quantitative PCR Primer Design

[0051] Download the genomic CDS sequence of GmASR2 as shown in SEQ ID NO.1 and the GmASR2 protein sequence as shown in SEQ ID NO.2 from the website; and design and synthesize its specific quantitative amplification primers based on the genomic sequence: GmASR2_qF (SEQ ID NO.3): 5’-GATGATGAGGTTGACTATAAGAAGG-3’; GmASR2_qR (SEQ ID NO.4): 3’-GGAAGGCAAACCCACCA-5’.

[0052] 4. Real-time Fluorescent Quantitative PCR

[0053] Use GmEF1a (gene ID Glyma.17G186600) and its specific quantitative PCR primers as the reference gene; take equal volumes of the cDNA of all the above samples and mix them evenly as the standard curve sample 1, and then dilute it 3 times with ddH2O successively until a total of 7 samples are obtained; dilute the cDNA of all samples 10 times with ddH2O as the template for the quantitative PCR reaction; use the Applied Biosystems StepOnePlus real-time PCR system to perform fluorescent quantitative PCR according to the reaction system shown in Table 3 below, and the reaction program is: pre-denaturation at 95 °C for 30 s; 40 cycles (denaturation at 95 °C for 30 s; annealing / extension at 60 °C for 30 s); after the reaction, try to adjust the standard curve R 2 to be infinitely close to 0.999, and after the efficiency value is between 80% and 120%, export the data; process the data, calculate the relative expression level of the gene, and perform differential analysis.

[0054] Table 3 Reaction System for Real-time Fluorescent Quantitative PCR

[0055]

[0056]

[0057] 5. Result Analysis

[0058] The expression results of the GmASR2 gene in different tissues are as Figure 1 shown, indicating that the GmASR2 gene is induced to express under low phosphorus stress in the roots and leaves of soybeans under long-term treatment. Under normal phosphorus conditions, the expression level of the GmASR2 gene is down-regulated in leaves and up-regulated in roots. Under low phosphorus stress, the expression level of the GmASR2 gene is significantly up-regulated.

[0059] Example 2 Construction of the GmASR2 Gene Overexpression Vector

[0060] 1. Overexpression Vector of Soybean GmASR2 Fused with GFP Tag

[0061] ① Amplify the target fragment: Using the cDNA of soybean YC03-3 as a template, design and synthesize primers: pTF101S_GmASR2_OX_GFP_F (SEQ ID NO.5): 5’-AGGACAGGGTACCCGGGGATCCATGGCCGAAGAGAAACACCACAAG-3’, pTF101S_GmASR2_OX_GFP_R (SEQ ID NO.6): 3’-CACCATGGTACTAGTGTCGACGCCAAAGAGATGGTGGTGCTTC-5’. Amplify the full-length CDs sequence of GmASR2 according to the reaction system in Table 4 below. The reaction program is: pre-denaturation at 94°C for 2 min; 32 cycles (denaturation at 94°C for 15 s; annealing at 57°C for 30 s; extension at 68°C for 1 min 10 s); final extension at 68°C for 5 min; store at 16°C.

[0062] Table 4 Fragment Amplification Reaction System

[0063]

[0064] Perform agarose gel electrophoresis on the PCR product, cut the target fragment from the gel, and purify the target fragment using a DNA recovery kit.

[0065] ② Linearize the vector: Use the restriction endonuclease SpeI to digest the pTF101S plasmid DNA alone.

[0066] ③ Ligation: Ligate the target fragment with the intermediate vector pTF101S according to the reaction system in Table 5 below. The reaction program is: 37°C for 30 min.

[0067] Table 5 Ligation Reaction System

[0068]

[0069] ④Transformation of Escherichia coli: Pipette 10 μL of the ligation product into 100 μL of DH5α Escherichia coli competent cells, gently pipette and mix well, incubate on ice for 30 min, heat shock in a 42 °C water bath for 1 min, and immediately transfer to ice for cooling for 2 min. Add 500 - 700 μL of LB liquid medium in a laminar flow hood, culture in a shaker at 37 °C and 220 rpm for 1 h, spread the bacterial solution on a plate containing the corresponding antibiotic, culture overnight at 37 °C, pick monoclonal colonies for shaking culture, and perform detection and sequencing.

[0070] ⑤Transformation of Agrobacterium tumefaciens: After successful comparison by monoclonal sequencing, extract the plasmid using a plasmid extraction kit. Pipette 2 μL of the plasmid into 100 μL of GV3101 Agrobacterium tumefaciens competent cells, incubate on ice for 30 min, then freeze in liquid nitrogen for 1 min, heat shock at 37 °C for 5 min, and then transfer to ice for cooling for 3 min. Add 500 - 700 μL of YEP liquid medium in a laminar flow hood, culture in a shaker at 28 °C and 220 rpm for 2 - 3 h, spread the bacterial solution on a plate containing the corresponding antibiotic, culture at 28 °C for 1 - 2 d, pick monoclonal colonies for shaking culture and detection, and add an equal volume of 50% glycerol to the successfully detected bacterial solution and store at -80 °C.

[0071] 2. Overexpression transformation vector of soybean GmASR2

[0072] Transfer the plasmid extracted after transforming Escherichia coli above into K599 Agrobacterium tumefaciens competent cells by the same method, and add an equal volume of 50% glycerol to the successfully transferred bacterial solution after detection and store at -80 °C.

[0073] Example 3 Subcellular localization of GmASR2

[0074] Perform transient expression in tobacco leaf cells. Cultivate Nicotiana benthamiana in advance. When it grows to 4 - 5 weeks, it can be used for experiments; Inoculate 50 μL of GV3101 Agrobacterium tumefaciens carrying P19 (enhanced protein expression) and the target vector (GmASR2 gene overexpression vector) into 10 mL of YEP liquid medium respectively, add the corresponding antibiotics, and culture in a shaker at 28 °C and 180 - 220 rpm for 16 h; The next day, centrifuge the bacterial solution at 5000 rpm for 10 min and discard the supernatant; Resuspend the bacterial cells with 2 mL of infiltration solution, centrifuge at 5000 rpm for 10 min, and repeat this step once; Resuspend the bacterial cells again with 1 mL of infiltration solution, and adjust the OD 600Adjust to 0.45 - 0.55, incubate in the dark at 28°C for 4 h; invert the bacteria containing P19 and the target expression vector up and down in equal volumes and mix well, aspirate the mixed bacterial liquid with a 1 mL syringe, and inject it from the back of the tobacco leaves; after injection, incubate in the dark for 12 h, continue normal culture for 2 - 3 d, and then observe the green fluorescence signal at a wavelength of 488 nm in tobacco epidermal cells under a laser confocal microscope.

[0075] The results are as Figure 2 shown, indicating that the GmASR2 protein is localized in the nucleus after transient expression in tobacco leaf cells.

[0076] Example 4 GmASR2 Soybean Chimeric Transformation Experiment

[0077] 1. Plant Sample Treatment

[0078] Select the same period, plump soybean variety YC03 - 3 as the plant material, sterilize it by fumigating with chlorine gas (50 mL sodium hypochlorite + 2.1 mL concentrated hydrochloric acid) for 4 h, and then place it in a fume hood to remove the residual chlorine gas. Place the soybeans at a spacing of 10 seeds per piece of paper on the top of the moist germination paper (39×18 cm), roll up the germination paper with the seeds, place it vertically in a 1 L sterile plastic beaker (10 rolls / each), pour about 600 mL of sterile water, cover it with plastic wrap to prevent excessive evaporation of water, make several holes in the plastic wrap, and grow it in an artificial culture room for 4 d. During this period, inoculate the Agrobacterium rhizogenes K599 containing the target gene (overexpression GmASR2 gene vector) and the empty vector on the YEP plate containing the screening antibiotic, and incubate at 28°C for 2 d.

[0079] Use a knife to cut off the roots below the green part of the 4 - day-old soybean seedlings (retain the stem 5 - 7 cm), collect a layer of bacterial paste on the plate with the cut surface of the stem, place it on the new moist germination paper at a spacing of 8 seedlings per piece of paper, keep the cotyledons unfolded outside the paper, roll it up and put it into a sterile plastic beaker, finally pour about 600 mL of sterile water, cover it with plastic wrap and make holes, and put it back into the artificial culture room. Remove the plastic wrap as the plants grow.

[0080] After callus grows on the cut surface and roots emerge, conduct detection. Transfer the successfully transformed soybean chimeras (after overexpression and its empty vector materials are treated with low phosphorus for 10 d, sample to extract RNA and perform semi - quantitative analysis with relevant primers, the method is the same as in Example 1) to normal nutrient solution hydroponics (Hoagland nutrient solution). After acclimating for 7 d, conduct normal phosphorus nutrition HP (500 μM PO4 2- ) and low phosphorus treatment LP (5 μM PO4 2-)Treatment, samples were collected after 25 days. The plant height, number of leaves, SPAD value, fresh weight of aboveground and underground parts of soybean plants were measured according to the following methods. The total root length, root surface area and average root diameter data were obtained by scanning the soybean roots. After the plant samples were completely dried, the dry weight was weighed. Fresh and dry samples were taken to determine the soluble phosphorus and total phosphorus contents. Each data was taken at least three biological replicates, and the collected samples were stored at -80 °C.

[0081] 2. Measurement methods for plant parameter indicators

[0082] (1) Measurement of SPAD value: First, use the SPAD502 chlorophyll meter with an empty clip for calibration and zeroing. Select three different sites on the same leaf and clamp with the instrument for 2 - 3 s. The value displayed at this time is the relative chlorophyll content at this site. Take the average of the three data as the SPAD value of one leaf, and at least three leaves of each seedling were measured.

[0083] (2) Measurement of soluble phosphorus content: ① Weigh an appropriate amount of plant material (0.1 g of leaf sample, 0.2 g of root sample), place it in a clean mortar, and grind it after adding 600 μL of Tris-HCl (0.1 M); ② Transfer the ground sample to a 2 mL centrifuge tube, add another 600 μL of Tris-HCl (0.1 M) to the mortar to wash the remaining sample, and transfer it to the same centrifuge tube, making three replicates; ③ Centrifuge at 4 °C and 13,000 rpm for 40 min; ④ Take the supernatant (20 μL of leaf sample, 100 μL of root sample) and the standard curve solution (1.8 mL, without adding ddH2O) into another 2 mL centrifuge tube, add ddH2O (make up to 1.8 mL) and 200 μL of color reagent in sequence, mix well, and react in the dark for 30 min; ⑤ Pipette 200 μL of the reaction solution into the microplate to measure the 700 absorbance value; ⑥ After subtracting the blank absorbance value from all absorbance values, calculate the standard curve, and substitute the sample absorbance value to obtain the soluble phosphorus content of the sample.

[0084] (3) Measurement of total phosphorus content: ① Place the dry plant sample in an oven at 60 °C and dry it for 1 - 2 days, then crush it with a grinding machine; ② Weigh about 0.1 g of the sample and put it into a crucible, mark the serial number at the bottom with a pencil, and carbonize the sample with an electric furnace; ③ Put the sample with the lid on into the muffle furnace in sequence, ash it at 600 °C for 8 h. The sample becomes grayish-white to be considered completely ashed, otherwise the time needs to be extended; ④ After cooling, take out the sample, add 8 ml of 100 mM HCl, fully dissolve it, and measure the total phosphorus content by the molybdenum antimony anti-colorimetric method. The specific measurement method is the same as that of soluble phosphorus content.

[0085] 3. Result analysis

[0086] The phenotypes of the overexpressed GmASR2 gene (OX) materials are as Figure 3As shown in the figure, under normal phosphorus conditions, there was no significant difference between the plants overexpressing the GmASR2 gene and the empty vector-transformed plants; under low phosphorus stress, overexpression of the GmASR2 gene promoted plant growth, indicating that overexpression of GmASR2 could enhance the tolerance of soybeans to low phosphorus stress.

[0087] The phenotypes of leaves with different leaf ages of the material overexpressing the GmASR2 gene (OX) are as Figure 4 shown. Under low phosphorus stress, the leaves of soybean plants overexpressing GmASR2 grew better than those of the empty vector-transformed plants.

[0088] The specific statistical results of plant biomass indexes are as Figure 5 and Figure 6 shown. Under low phosphorus treatment, compared with the empty vector-transformed plants, the plant height of the material overexpressing GmASR2 increased by 22.88%, the fresh weight increased by 23.48%, the aboveground fresh weight increased by 25.64%, the dry weight increased by 80.78%, the aboveground dry weight increased by 82.95%, the underground dry weight increased by 71.15%, and the total phosphorus in the aboveground part increased by 12.57%. It shows that compared with the empty vector-transformed plants, overexpression of GmASR2 can increase the plant height, fresh weight (aboveground), and dry weight (underground and aboveground) of plants to promote the growth of soybean plants; and it can increase the total phosphorus content in the aboveground part to relieve the inhibitory effect of low phosphorus stress on soybean plants and maintain the growth and development of soybeans under low phosphorus conditions.

[0089] Example 5 Transformation and Screening of Arabidopsis thaliana Materials Overexpressing GmASR2

[0090] 1. Transformation of Arabidopsis thaliana

[0091] In this study, the floral dip method was used to transform Arabidopsis thaliana: GV3101 carrying the target gene (the vector overexpressing the GmASR2 gene) was inoculated into 350 mL of YEP liquid medium, and the corresponding antibiotics were added. The culture was carried out at 28 °C and 180 - 220 rpm on a shaker for 16 - 24 h; the bacterial solution was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, 2 mL of infiltration solution was added to resuspend the cells, and after pipetting and mixing evenly, it was all poured back into 200 mL of infiltration solution and stirred until the liquid surface foamed; the Arabidopsis thaliana pods that had grown to 6 - 7 cm were cut clean, and their inflorescences were all immersed in the infiltration solution, gently shaken for about 30 s and taken out, and then dark-treated with a black bag for about 12 h and then returned to normal culture.

[0092] 2. Screening of Transgenic Materials

[0093] After harvesting and drying the Arabidopsis thaliana T0 generation seeds, they were sown in the soil substrate, placed at 4°C for stratification for 2 days, and then put into the culture room. After almost all of them germinated, herbicide (Basta, 10% glufosinate-ammonium solution diluted 2000 times before use) was sprayed 2 - 3 times. After waiting for 4 - 6 days, the non-yellowing Arabidopsis thaliana lines were selected and cultured in a new soil substrate. After the pods matured, the T1 generation seeds were harvested. The T1 generation seeds were sown in the substrate and screened with herbicide. The single-copy inserted T2 lines that met the 3:1 segregation ratio were selected. After harvesting the seeds, this step was repeated to screen out the homozygous T3 lines that were fully resistant.

[0094] 3. Phenotypic analysis of overexpressing GmASR2 in Arabidopsis thaliana

[0095] For different phosphorus treatments (HP: 0.625 mM PO4 2- ; LP: 5 μM PO4 2- ): The dried and disinfected Arabidopsis thaliana seeds of the wild type and two overexpressing lines were sown in normal 1 / 2 MS medium. After stratification at 4°C for 2 days, they were put into an artificial climate incubator for about 3 days. Arabidopsis thaliana seedlings with consistent growth were selected and transferred to different phosphorus culture dishes. Four replicates were set for different treatments, with 4 seedlings in one replicate. After being put back into the artificial climate incubator for 10 days, samples were collected and relevant data such as the main root length, lateral root number, lateral root density, fresh weight, and chlorophyll content were measured.

[0096] 4. Result analysis

[0097] The phenotypes of the materials after being treated for 10 days under different phosphorus conditions are as Figure 7 shown. Under normal phosphorus conditions, there was no obvious difference between Arabidopsis thaliana overexpressing the GmASR2 gene and the wild type. Under low phosphorus stress, heterologous overexpression of the GmASR2 gene in Arabidopsis thaliana could promote the growth of Arabidopsis thaliana, relieve the inhibitory effect of low phosphorus stress on the growth of Arabidopsis thaliana, and alleviate the impact of low phosphorus stress on Arabidopsis thaliana. The statistical results of the biomass indexes of Arabidopsis thaliana are as Figure 8 shown. Compared with the wild type, heterologous overexpression of the GmASR2 gene in Arabidopsis thaliana could increase the main root length, lateral root number, fresh weight, and chlorophyll content of the plants, and enhance the tolerance of the plants to low phosphorus.

[0098] In summary, the present invention clarifies the relationship between the soybean ASR transcription factor GmASR2 gene and the important nutrient element phosphorus, and proves that it is involved in the mechanism of plant adaptation to low phosphorus. For the first time, it is publicly disclosed that the GmASR2 gene is involved in the regulation of phosphorus nutrition in soybeans. The research shows that overexpressing the GmASR2 gene can promote plant growth under low phosphorus stress, relieve the inhibition of low phosphorus stress on plant growth and development, and alleviate the phosphorus deficiency symptoms of plants. Under low phosphorus stress, overexpressing the GmASR2 gene can improve the phosphorus deficiency symptoms of soybeans and Arabidopsis, promote root growth, increase phosphorus content, and improve the absorption of phosphorus nutrients and the accumulation of biomass by plants; it shows that the GmASR2 gene plays an important role in improving plant phosphorus efficiency and promoting plant growth and development and increasing phosphorus content under low phosphorus stress, providing an important gene resource for plants to adapt to low phosphorus environmental stress.

[0099] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of the GmASR2 gene in positively regulating plant growth and phosphorus nutrition, characterized in that, The sequence of the GmASR2 gene is shown as SEQ ID NO.

1.

2. Use of the GmASR2 gene in promoting plant growth under low phosphorus stress, characterized in that, Overexpress the GmASR2 gene in plants, and the sequence of the GmASR2 gene is shown as SEQ ID NO.

1.

3. Use of the GmASR2 gene in cultivating plants tolerant to low phosphorus stress, characterized in that, Overexpress the GmASR2 gene in plants, and the sequence of the GmASR2 gene is shown as SEQ ID NO.

1.

4. Use of the GmASR2 gene to increase the phosphorus content in plants under low phosphorus stress or in the preparation of a preparation for increasing the phosphorus content in plants, characterized in that, The sequence of the GmASR2 gene is shown as SEQ ID NO.

1.

5. Application of a preparation for promoting the expression of the GmASR2 gene shown as SEQ ID NO.1 in promoting plant growth under low phosphorus stress.

6. Application of a preparation for promoting the expression of the GmASR2 gene shown as SEQ ID NO.1 in preparing a product for promoting plant phosphorus nutrition absorption or in preparing a product for tolerating low phosphorus stress.

7. Application of a preparation for promoting the expression of the GmASR2 gene shown as SEQ ID NO.1 in increasing the phosphorus content and / or chlorophyll content of plants or in preparing a product for increasing the phosphorus content and / or chlorophyll content of plants.

8. Application of a preparation for promoting the expression of the GmASR2 gene shown as SEQ ID NO.1 in cultivating plants tolerant to low phosphorus stress.

9. A method for promoting plant growth and nutrient absorption, or increasing the phosphorus content and / or chlorophyll content of plants, characterized in that, Promote the expression of the GmASR2 gene in plants, or treat the plants with a preparation for promoting the expression of the GmASR2 gene; the sequence of the GmASR2 gene is shown as SEQ ID NO.

1.

10. A method for cultivating low-phosphorus tolerant plants or high-phosphorus content plants, characterized in that, Promote the expression of the GmASR2 gene in plants, or introduce a recombinant vector or recombinant bacterium containing the overexpressed GmASR2 gene into the plants; the sequence of the GmASR2 gene is shown as SEQ ID NO.1.

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