Application of soybean PSK oligopeptide gene GmPSK alpha14 in regulation and control of soybean growth and phosphorus nutrition thereof

Abstract

The invention discloses an application of a soybean PSK oligopeptide gene GmPSK alpha 14 in regulation and control of soybean growth and phosphorus nutrition thereof. The invention provides new application of the GmPSK alpha14 gene, and research shows that the GmPSK alpha14 gene can respond to low-phosphorus stress, is obviously subjected to up-regulation expression of low-phosphorus stress in soybean roots and leaves, and participates in regulation and control of soybean phosphorus nutrition and growth under low-phosphorus stress; the GmPSK alpha14 gene is cloned to prepare a recombinant vector, the recombinant vector is transformed into soybean for overexpression, it is shown that the GmPSK alpha14 gene is capable of regulating plant phosphorus nutrition, under the condition of phosphorus nutrient deficiency, overexpression of the GmPSK alpha14 gene can improve the phosphorus deficiency symptom of soybean, growth of soybean and soybean root systems can be remarkably promoted, absorption of soybean to phosphorus nutrient and accumulation of biomass can be improved, and the yield of the soybean can be increased. The plant phosphorus content is increased, the soybean growth is regulated, and more gene resources are provided for the plants to adapt to environmental low-phosphorus 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 soybean PSK short peptide gene GmPSKα14 in regulating soybean growth and its phosphorus nutrition. Background Art

[0002] Soybean is one of the main food and oil crops in China. It has a long growth cycle and high growth intensity, and thus has a relatively large demand for phosphate fertilizer. Phosphorus plays a crucial role in the growth process of soybeans. It can not only promote the development and growth of soybean roots, increase the growth rate of soybeans, but also enhance the immunity of soybeans, helping soybeans better absorb other nutrients, thereby increasing the yield and quality of soybeans. Therefore, regulating the phosphorus nutrition of soybeans is of great significance for the growth and development of soybeans and agricultural production.

[0003] In the initial stage of soybean growth, phosphorus mainly promotes root growth; before flowering, phosphorus mainly acts on the vegetative growth of soybean plants, shortens the formation process of reproductive organs, and prevents flower and pod drop. However, excessive fertilization will also have negative effects, resulting in a decrease in soybean yield. Therefore, reasonably controlling the input amount of phosphate fertilizer and improving the absorption and utilization efficiency of soil phosphorus by soybeans through genetic improvement are effective ways to solve the contradiction between phosphorus supply and phosphorus loss.

[0004] In recent years, scientists have made remarkable progress in the research on soybean phosphorus nutrition regulatory genes. Researchers obtained a major QTL gene GmGDPD2 for low phosphorus tolerance by integrating GWAS and linkage analysis. Overexpression of this gene can significantly promote root growth and development and organic acid secretion of the optimal haplotype, improve phosphorus absorption and soybean yield of soybeans; knocking out this gene inhibits soybean root growth and reduces phosphorus utilization efficiency, and the root development blockage can be restored by exogenous spraying of GA synthesis inhibitors. These studies aim to deeply understand the mechanisms of phosphorus absorption, transport and utilization in soybeans, providing theoretical support for the cultivation of new high-yield and high-efficiency soybean varieties. Therefore, regulating soybean phosphorus nutrition is of great significance for increasing the yield and quality of soybeans. Improving the absorption and utilization efficiency of soil phosphorus by soybeans through genetic improvement is an effective way to solve the contradiction between phosphorus supply and phosphorus loss. In order to more precisely regulate soybean phosphorus nutrition, achieve a breakthrough in soybean yield potential and reduce phosphate fertilizer application, it is necessary to develop more gene resources that can be used for soybean phosphorus nutrition regulation.

[0005] PSKs (Phytosulfokines) are the first peptide growth factors discovered in plants and play crucial roles in various physiological processes such as cell proliferation and differentiation, as well as in plant growth and development, response to biotic and abiotic stresses, and the balance between growth and immunity. PSK is synthesized from a preproprotein with an N-terminal hydrophobic signal sequence for targeting the secretory pathway. The preprotein has an acidic region that overlaps with the typical PSK signal. Conserved amino acids are predicted to play roles in proteolytic cleavage or sulfation. The PSK peptide backbone is located near the C-terminus, followed by 4 to 6 mainly basic amino acids. Two tyrosyl side chains in the PSK pentapeptide backbone of the preprotein are sulfated by the Golgi-localized tyrosine protein sulfotransferase TPST, which is encoded by a single-copy gene in Arabidopsis thaliana. After secretion, the preprotein is proteolytically cleaved to produce bioactive PSK. PSK regulates various physiological and biochemical reactions in plants, including in vitro differentiation of plant cells, cell growth, regulation of root apical meristem cells and quiescent center cells, development of pollen tube cells, regulation of plant immunity, etc.; it is involved in abiotic stress responses, as well as in co-regulation with plant hormones such as auxin, cytokinin, jasmonic acid, brassinosteroid, etc. However, currently, the functions of the vast majority of soybean PSK short peptide genes are unclear, and there are few studies on the functional role of GmPSKα. Whether it is also involved in the mechanism of plant adaptation to low phosphorus is unknown. Therefore, in order to explore more gene resources and more precisely regulate the phosphorus nutrition of soybeans, it is necessary to provide more gene resources that can regulate plant phosphorus nutrition and enable plants to adapt to low phosphorus environments, which is of great significance for improving the phosphorus nutrition absorption efficiency of plants, alleviating the inhibition of plants under low phosphorus stress, and application in phosphorus-deficient soils. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of existing soybean phosphorus nutrition regulatory genes and provide the application of the soybean PSK short peptide gene GmPSKα14 in regulating soybean growth and its phosphorus nutrition.

[0007] The object of the present invention is to provide a new application of the GmPSKα14 gene.

[0008] Another object of the present invention is to provide the application of a preparation for promoting the expression of the GmPSKα14 gene.

[0009] Still another object of the present invention is to provide a method for promoting soybean growth, or increasing the phosphorus content of soybeans, or cultivating soybean plants resistant to low phosphorus stress.

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

[0011] The present invention has discovered more gene resources involved in regulating soybean phosphorus nutrition, and provided a new application of the GmPSKα14 gene (gene ID: Glyma.20G135700). The nucleotide sequence of the GmPSKα14 gene is shown as SEQ ID NO:1, and its protein sequence is shown as SEQ ID NO:2. Research shows that the GmPSKα14 gene can respond to low phosphorus stress and is involved in regulating soybean phosphorus nutrition and growth under low phosphorus stress. The expression of the GmPSKα14 gene is significantly up-regulated by low phosphorus stress in both soybean roots and leaves. By cloning the GmPSKα14 gene, inserting it into the pC1300 plasmid with a 35S promoter to prepare a recombinant vector, and transforming it into soybeans for overexpression, it is shown that overexpressing the GmPSKα14 gene positively regulates plant phosphorus nutrition, can promote the absorption and accumulation of phosphorus in plants, and alleviate the impact of low phosphorus stress on plant growth and development. Under the condition of phosphorus nutrient deficiency, overexpressing the GmPSKα14 gene can improve the phosphorus deficiency symptoms of soybeans, significantly promote the growth of soybeans and their roots, increase the absorption of phosphorus nutrients and the accumulation of biomass in soybeans, increase the phosphorus content in plants, and regulate the growth of soybeans. The GmPSKα14 gene plays an important role in improving plant phosphorus efficiency and promoting plant growth and root development under low phosphorus stress, providing more gene resources for plants to adapt to environmental low phosphorus stress.

[0012] Therefore, the present invention provides the application of the GmPSKα14 gene in positively regulating soybean phosphorus nutrition.

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

[0014] Preferably, it is to improve the absorption and utilization of soluble phosphorus by soybeans.

[0015] The present invention provides the application of the GmPSKα14 gene in increasing the phosphorus content of soybeans.

[0016] The present invention provides the application of the GmPSKα14 gene in promoting the growth of soybeans, or in promoting the growth of soybeans under low phosphorus stress.

[0017] The present invention provides the application of the GmPSKα14 gene in relieving the inhibition of soybean growth by low phosphorus stress.

[0018] Furthermore, overexpress the GmPSKα14 gene in soybeans.

[0019] The present invention provides the application of the GmPSKα14 gene in cultivating soybean plants resistant to low phosphorus stress.

[0020] The present invention provides the application of a preparation for promoting the expression of the GmPSKα14 gene in increasing the phosphorus content of soybeans, or in the preparation of a product for increasing the phosphorus content of soybeans.

[0021] The present invention provides the application of a preparation for promoting the expression of the GmPSKα14 gene in promoting the growth of soybeans under low phosphorus stress or in promoting the growth of soybean roots.

[0022] The present invention provides the application of a preparation for promoting the expression of the GmPSKα14 gene in the preparation of a product for promoting the growth of soybeans.

[0023] The present invention provides the application of a preparation for promoting the expression of the GmPSKα14 gene in cultivating soybean plants resistant to low phosphorus stress.

[0024] Preferably, the preparation is a plasmid, vector or recombinant bacterium overexpressing the GmPSKα14 gene.

[0025] The present invention also provides a method for promoting the growth of soybeans, or increasing the phosphorus content of soybeans, or cultivating soybean plants resistant to low phosphorus stress, by promoting the expression of the GmPSKα14 gene in soybeans, or transferring a recombinant vector or recombinant bacterium containing the overexpressed GmPSKα14 gene into soybeans.

[0026] Preferably, the method can also directly treat the plants with a preparation for promoting the expression of the GmPSKα14 gene.

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

[0028] The present invention has discovered more gene resources involved in regulating phosphorus nutrition in soybeans and provided new applications for the GmPSKα14 gene. Research shows that the GmPSKα14 gene can respond to low phosphorus stress and participate in regulating phosphorus nutrition and growth in soybeans under low phosphorus stress. The GmPSKα14 gene is significantly up-regulated by low phosphorus stress in both soybean roots and leaves, positively regulates plant phosphorus nutrition, can promote the absorption and accumulation of phosphorus by plants, and relieve the impact of low phosphorus stress on plant growth and development; under conditions of phosphorus nutrient deficiency, overexpressing the GmPSKα14 gene can improve the phosphorus deficiency symptoms of soybeans, significantly promote the growth of soybeans and their roots, increase the absorption of phosphorus nutrients and the accumulation of biomass by soybeans, increase the phosphorus content of plants, and regulate the growth of soybeans. The GmPSKα14 gene plays an important role in improving plant phosphorus efficiency and promoting plant growth and root development under low phosphorus stress, providing more gene resources for plants to adapt to environmental low phosphorus stress. Description of the Drawings

[0029] Figure 1Quantitative PCR results of GmPSKα14 gene under different phosphorus conditions (HP represents normal phosphorus nutrition, LP represents low phosphorus treatment; the left figure shows the quantitative results in leaves, and the right figure shows the quantitative results in roots; among them, 24h represents the first trifoliate leaf after 24h of high and low phosphorus treatment, and 14d represents the first trifoliate leaf closest to the root after 14d of high and low phosphorus treatment; the data are the average and standard error of 4 biological replicates, and "*", "**" 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).

[0030] Figure 2 Phenotypes of overexpressing GmPSKα14 gene (OX) and empty vector (EV) soybean chimeras under high and low phosphorus conditions after 25 days of treatment (HP represents normal phosphorus nutrition, LP represents low phosphorus treatment, the same below; the scale bar in the figure is 10 cm).

[0031] Figure 3 Phenotypes of leaves with different leaf ages of overexpressing GmPSKα14 gene (OX) and empty vector (EV) soybean chimeras under high and low phosphorus conditions after 25 days of treatment (the first leaf represents the oldest first trifoliate leaf, the second leaf represents the second trifoliate leaf, and so on; the scale bar in the figure is 10 cm).

[0032] Figure 4 Statistical results of the differences in fresh weight, aboveground fresh weight, underground fresh weight, dry weight, aboveground dry weight, and underground dry weight of overexpressing GmPSKα14 gene (PSKα14_OX) and empty vector (EV) soybean chimeras under different phosphorus treatments for 25 days (the data in the figure are the average and standard error of 4 biological replicates, and "*", "**" 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).

[0033] Figure 5Statistical results of differences in plant height, number of compound leaves, SPAD value, total root length, root surface area, and average root diameter of soybean chimeras overexpressing the GmPSKα14 gene (PSKα14_OX) and empty vector (EV) under different phosphorus treatments for 25 days (data in the figure are the mean and standard error of 4 biological replicates, and "*", "**", and "***" 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, respectively).

[0034] Figure 6 Statistical results of differences in soluble phosphorus content in the third leaf, soluble phosphorus content in roots, total phosphorus content in shoots, and total phosphorus content in roots of soybean chimeras overexpressing the GmPSKα14 gene (PSKα14_OX) and empty vector (EV) under different phosphorus treatments for 25 days (data in the figure are the mean and standard error of 4 biological replicates, and "*", "**", and "***" 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, respectively). Detailed implementation manners

[0035] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments 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 technical field of the present invention.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0037] Example 1 Expression pattern of GmPSKα14 gene under different phosphorus levels

[0038] 1. Treatment of soybean materials

[0039] Select the same period, plump soybean variety YC03-3 as the plant material, sterilize it by fumigating with chlorine gas (50 mL of sodium hypochlorite + 2.1 mL of concentrated hydrochloric acid) for 4 h, and then place it in a fume hood to remove the residual chlorine gas. Subsequently, place the soybean in a sand culture in an artificial culture room for 4-5 days. After the cotyledons slightly open, transfer it to normal nutrient solution hydroponics (Hoagland nutrient solution). After acclimating for 7 days, apply normal phosphorus nutrition HP (625 μM PO4 2- ) and low phosphorus treatment LP (5 μM PO4 2-)Samples were collected after short-term (24 h) and long-term (14 d) treatments, and the collected samples were stored at -80°C. RNA was extracted from soybean root and leaf samples obtained after hydroponics for 24 h in the short term and 14 d in the long term under different phosphorus treatments, and reverse-transcribed into cDNA, which was used as a template for real-time fluorescence quantitative PCR.

[0040] 2. RNA Extraction and Reverse Transcription

[0041] (1) RNA extraction: Cut about 100 mg of plant material and put it 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 in a steel bead, shake on a shaker for 2 min, let it stand for 5 min after thorough mixing; add 200 μL of chloroform to the centrifuge tube, shake vigorously for 10 s (thoroughly mix the two phases), let it stand for 3 min, then centrifuge at 4°C, 12,000 r / min for 15 min; take about 400 μL of the supernatant to another new 1.5 mL centrifuge tube, add an equal volume of isopropanol, invert it up and down evenly, and let it stand for 10 min; centrifuge at 4°C, 12,000 r / min for 10 min, at this time the RNA sinks to the bottom; discard the supernatant, add 500 μL of 75% ethanol to wash the precipitate twice (invert up and down), centrifuge at 4°C, 12,000 r / min each time for 5 min; air-dry the precipitate in a fume hood, add 20 μL of sterile water to dissolve the precipitate, store at -80°C for later use; measure the concentration value of RNA.

[0042] (2) Reverse transcription: Use the one-step method to remove genomic DNA and perform RNA reverse transcription. The reaction system for reverse transcription is: 2.5 μL of 4X ABScriptⅢ RT Mix, 0.5 μL of 20X gDNA Remover Mix, 1000 μg of RNA, RNase-Free Water up to 10 μL; the reaction conditions are: 37°C for 2 min, 55°C for 15 min, 85°C for 5 min, and hold at 4°C.

[0043] 3. Primer Design

[0044] Download the genomic CDS sequence of GmPSKα14 from the website, as shown in SEQ ID NO.1, and its protein sequence is shown in SEQ ID NO.2; design and synthesize its specific quantitative amplification primers according to the genomic sequence:

[0045] GmPSKα14_qF (SEQ ID NO.3): 5’-AAACTCAACATGGGGTTTTGG-3’;

[0046] GmPSKα14_qR (SEQ ID NO.4): 5’-GAGAACCAGCTATATATGAATAT-3’.

[0047] 4. Real-time fluorescence quantitative PCR

[0048] The specific quantitative PCR primers used were SEQ ID NO.3 - 4, and the reference gene was GmEF1a (gene ID Glyma.17G186600). Equal volumes of the cDNA of all the above samples were mixed evenly as the standard sample 1 for the standard curve. Subsequently, it was diluted 3-fold with ddH2O successively until a total of 7 standard samples were obtained; the cDNA of all samples was diluted 10-fold with ddH2O as the template for the quantitative PCR reaction.

[0049] The quantitative PCR reaction system was (20 μL reaction system): 10 μL of BrightCycle Universal SYBR Green qPCR Mi4 with UDG, 2 μL of cDNA, 0.4 μL of qF, 0.4 μL of qR, and RNase-Free Water up to 20 μL; the quantitative PCR reaction conditions were: 95°C for 1 min (15 min for the hot start enzyme); 95°C for 15 sec, 60°C for 15 sec, 40 cycles; 72°C for 30 sec. After the real-time quantitative PCR reaction was completed, try to adjust the standard curve so that R2 was infinitely close to 0.999 and the efficiency value was between 80% - 120%, and then export the data; process the data, calculate the relative expression level of the gene, and perform differential analysis.

[0050] 5. Result analysis

[0051] The expression result of the GmPSKα14 gene was as Figure 1 shown. This result showed that the GmPSKα14 gene responded to low phosphorus stress and was significantly up-regulated in soybean leaves under long-term and short-term low phosphorus treatment conditions; in soybean roots, under normal phosphorus conditions, the expression level of the GmPSKα14 gene decreased after long-term treatment, while under low phosphorus stress, the expression level of the GmPSKα14 gene was significantly up-regulated.

[0052] Example 2 Construction of an overexpression vector of soybean GmPSKα14 fused with GFP tag

[0053] 1. Amplify the target fragment: Using the cDNA of soybean YC03-3 as the template, primers were designed and synthesized as shown in Table 1, and the full-length CDs sequence of GmPSKα14 was amplified by PCR. The amplification reaction system was as shown in Table 2, and the reaction program was as shown in Table 3. Subsequently, the PCR product was subjected to agarose gel electrophoresis, the target fragment was cut out of the gel, and the target fragment was purified using a DNA recovery kit.

[0054] Table 1 Amplification Primers

[0055]

[0056] Table 2 Fragment Amplification Reaction System

[0057]

[0058] Table 3 Fragment Amplification Reaction Conditions

[0059]

[0060] 2. Linearized Vector: Digest the plasmid DNA of pC1300 with the restriction endonuclease XbaI alone.

[0061] 3. Ligation: Ligate the target fragment with the intermediate vector pC1300. The reaction system is: 3 ng of linearized vector plasmid, 2 ng of purified target fragment, 5 μL of 2×MultiF Seamless Assembly Mix. Reaction program: 50°C, 15 min.

[0062] 4. Escherichia coli Transformation: Pipette 10 μL of the ligation product into 100 μL of DH5α Escherichia coli competent cells, gently pipette and mix well, ice-bath 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 at 37°C in a shaker at 220 rpm for 1 h, spread the bacterial solution on a plate containing the corresponding antibiotic, culture overnight at 37°C, pick single colonies for shaking culture, and perform detection and sequencing.

[0063] 5. Agrobacterium tumefaciens Transformation: After successful comparison of monoclonal sequencing, extract the plasmid using a plasmid extraction kit. Pipette 2 μL of the plasmid into 100 μL of K599 Agrobacterium tumefaciens competent cells, ice-bath 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 at 28°C in a shaker at 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 single 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 for future use.

[0064] Example 3 Soybean Chimeric Transformation

[0065] 1. Plant Sample Treatment

[0066] Select the plump soybean variety YC03-3 in the same period as the plant material. After sterilizing with chlorine gas (50 mL of sodium hypochlorite + 2.1 mL of concentrated hydrochloric acid) for 4 h, 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 and place it vertically in a 1 L sterile plastic beaker (10 rolls per beaker). Pour about 600 mL of sterile water and cover it with plastic wrap to prevent excessive evaporation of water. Poke 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 GmPSKα14 gene vector) and the empty vector on a YEP plate containing screening antibiotics and incubate it at 28 °C for 2 d.

[0067] Use a knife to cut off the roots below the green part of the 4-day-old soybean seedlings (retain a 5-7 cm stem). Collect the bacterial paste on a single-layer plate with the cut surface of the stem. Place it on a new moist germination paper at a spacing of 8 seedlings per piece of paper, keeping 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 poke holes, and put it back into the artificial culture room. Remove the plastic wrap as the plant grows. After callus grows from the cut surface and roots emerge, conduct detection. For the successfully transformed soybean chimeras (the overexpression and its empty vector materials are treated with low phosphorus for 10 d, then sampled to extract DNA for semi-quantification, and the method is the same as in Example 1).

[0068] Transfer the successfully transformed soybean chimeras to normal nutrient solution hydroponics (Hoagland nutrient solution). After acclimating for 7 d, conduct normal phosphorus nutrition HP (625 μM PO4 2- ) and low phosphorus treatment LP (5 μM PO4 2- ) treatments. After 25 d, collect samples, measure the plant height, number of leaves, SPAD value, and fresh weights of the aboveground and underground parts of the soybean plants. Scan the soybean roots to obtain data on total root length, root surface area, and average root diameter. After completely drying the plant samples, weigh the dry weight. Take fresh and dry samples to measure the soluble phosphorus and total phosphorus contents. Each data should have at least three biological replicates, and the collected samples are stored at -80 °C.

[0069] 2. Determination of plant phosphorus content

[0070] Determination of soluble phosphorus content: Weigh an appropriate amount of plant materials (0.1 g of leaf samples and 0.2 g of root samples), place them in a clean mortar, add 600 μL of Tris-HCl (0.1 M), and grind; 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; centrifuge at 4 °C and 13,000 rpm for 40 min; take the supernatant (20 μL of leaf samples and 100 μL of root samples) and the standard curve solution (1.8 mL, without adding ddH2O) into another 2 mL centrifuge tube, successively add ddH2O (make up to 1.8 mL) and 200 μL of chromogenic reagent, mix well, react in the dark for 30 min; pipette 200 μL of the reaction solution into the microplate, OD 700 Measure the absorbance value; 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.

[0071] Determination of total phosphorus content: Dry the plant dry samples in an oven at 60 °C for 1 - 2 days, then crush them with a grinding machine; weigh about 0.1 g of the sample and put it into a crucible, mark the serial number at the bottom with a pencil, carbonize the sample with an electric furnace; cover the sample with a lid and put it into the muffle furnace in sequence, ash at 600 °C for 8 h. The sample becomes grayish-white to be considered completely ashed, otherwise, the time needs to be extended; take out the sample after cooling, 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.

[0072] 3. Result analysis

[0073] Under different phosphorus conditions, the phenotypes of the overexpressed GmPSKα14 gene (OX) materials are as Figure 2 shown. Compared with the normal phosphorus condition, the plants are shorter and the old leaves show phosphorus deficiency symptoms such as chlorosis under low phosphorus conditions. Under normal phosphorus conditions, overexpression of GmPSKα14 promoted plant growth, while under low phosphorus stress, overexpression of GmPSKα14 alleviated the low phosphorus stress symptoms, and the lateral root formation of its overexpressed plants was more sensitive to low phosphorus; it showed that overexpression of GmPSKα14 could enhance the tolerance of soybean to low phosphorus stress.

[0074] Under different phosphorus conditions, the phenotypes of the leaves of different leaf ages of the overexpressed GmPSKα14 gene (OX) materials are as Figure 3 shown. After low phosphorus treatment, the old leaves of the plants are lighter in green color and even show phosphorus deficiency symptoms such as chlorosis compared with high phosphorus treatment. Compared with the empty vector transformed plants, overexpression of GmPSKα14 can promote the growth of soybean leaves, and under low phosphorus conditions, it relieves the inhibitory effect of low phosphorus stress on the leaves.

[0075] Under different phosphorus conditions, the statistical results of the biomass indexes of the materials overexpressing the GmPSKα14 gene (OX) are as follows Figure 4 shown. Compared with the empty vector-transformed plants, overexpression of GmPSKα14 can promote the growth of plants under low phosphorus stress, increase the fresh weight of the whole soybean plant by 21.97%, the fresh weight of the above-ground part by 20.86%, and the fresh weight of the underground part by 25.22%; increase the dry weight of the whole soybean plant by 13.38%, the dry weight of the above-ground part by 20.48%, and the dry weight of the underground part by 27.12%; increase the plant height of soybean under low phosphorus stress by 15.27%, the number of compound leaves by 11.43%, and the SPAD value by 5.22%. At the same time, whether under normal phosphorus or low phosphorus conditions, overexpression of GmPSKα14 can promote the root development of plants, increase the total root length, root surface area, and average root diameter. Compared with the empty vector-transformed plants, overexpression of GmPSKα14 can promote the increase of the total root length of soybean by 36.50%, the root surface area by 33.23%, and the average root diameter by 4.56% under normal phosphorus conditions; compared with the empty vector-transformed plants, overexpression of GmPSKα14 can promote the growth of plants under low phosphorus stress, increase the total root length of soybean by 40.37%, the root surface area by 66.45%, and the average root diameter by 6.67% ( Figure 5 ).

[0076] Furthermore, by measuring the phosphorus content of the plants, the results are as follows Figure 6 shown, indicating that compared with the empty vector-transformed plants, overexpression of GmPSKα14 can increase the total phosphorus content and soluble phosphorus content in the above-ground and underground parts of soybean plants under normal phosphorus conditions. The total phosphorus content in the above-ground part increases by 1.44%, the total phosphorus content in the underground part increases by 5.53%, the soluble phosphorus content in the above-ground part increases by 18.44%, and the soluble phosphorus content in the underground part increases by 11.14%. Compared with the empty vector-transformed plants, overexpression of GmPSKα14 can increase the total phosphorus content in the underground part of plants by 7.58%, the soluble phosphorus content in the above-ground part by 110.80%, and the soluble phosphorus content in the underground part by 19.84% under low phosphorus conditions. Overexpression of GmPSKα14 can significantly increase the phosphorus content of plants under low phosphorus stress to maintain the growth and development of soybean under low phosphorus conditions.

[0077] In summary, the present invention has discovered more gene resources involved in regulating soybean phosphorus nutrition and provided a new application of the GmPSKα14 gene. Research shows that the GmPSKα14 gene can respond to low phosphorus stress, participate in regulating the absorption of soybean phosphorus nutrition under low phosphorus stress, and alleviate the impact of low phosphorus stress on plant growth and development; under the condition of phosphorus nutrient deficiency, overexpressing the GmPSKα14 gene can improve the phosphorus deficiency symptoms of soybeans, significantly promote the growth of soybeans and their roots, increase the absorption of phosphorus nutrients and the accumulation of biomass by soybeans, increase the phosphorus content in plants, and regulate the growth of soybeans. The GmPSKα14 gene plays an important role in improving plant phosphorus efficiency and promoting plant growth and root development under low phosphorus stress, providing more gene resources for plants to adapt to environmental low phosphorus stress.

[0078] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to 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. Use of the GmPSKα14 gene shown in SEQ ID NO:1 in positively regulating soybean phosphorus nutrition.

2. Use of the GmPSKα14 gene shown in SEQ ID NO:1 in increasing the phosphorus content of soybeans, characterized in that, Overexpress the GmPSKα14 gene in soybean.

3. Use of the GmPSKα14 gene shown in SEQ ID NO:1 in promoting the growth of soybeans or promoting the growth of soybeans under low phosphorus stress, characterized in that, Overexpress the GmPSKα14 gene in soybean.

4. Use of the GmPSKα14 gene shown in SEQ ID NO:1 in relieving the inhibition of soybean growth by low phosphorus stress, characterized in that, Overexpress the GmPSKα14 gene in soybean.

5. Use of the GmPSKα14 gene shown in SEQ ID NO:1 in cultivating soybean plants resistant to low phosphorus stress.

6. Use of a preparation for promoting the expression of the GmPSKα14 gene shown in SEQ ID NO:1 in increasing the phosphorus content of soybean or in preparing a product for increasing the phosphorus content of soybean.

7. Use of a preparation for promoting the expression of the GmPSKα14 gene shown in SEQ ID NO:1 in promoting the growth of soybean under low phosphorus stress.

8. Use of a preparation for promoting the expression of the GmPSKα14 gene shown in SEQ ID NO:1 in preparing a product for promoting the growth of soybean.

9. Use of a preparation for promoting the expression of the GmPSKα14 gene shown in SEQ ID NO:1 in cultivating soybean plants resistant to low phosphorus stress.

10. A method for promoting the growth of soybeans, or increasing the phosphorus content of soybeans, or cultivating soybean plants resistant to low-phosphorus stress, characterized in that, Promote the expression of the GmPSKα14 gene in soybean, or transfer a recombinant vector or recombinant bacterium containing the overexpressed GmPSKα14 gene into soybean; the sequence of the GmPSKα14 gene is as shown in SEQ ID NO:1.

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