Application of plant low-phosphorus-stress-resistant gene GmGLK66

Abstract

The invention discloses an application of a low phosphorus stress resistant gene GmGLK66 of a plant. Researches find that the GmGLK66 gene participates in phosphorus balance regulation and control of soybean root systems and responds to low phosphorus stress, and the transcription level of the gene is obviously up-regulated under the low phosphorus stress. Meanwhile, the GmGLK66 gene has positive regulation and control of soybean root growth and phosphorus efficiency, and can significantly improve the total phosphorus content of soybeans. Under normal phosphorus conditions and low-phosphorus stress, growth of soybean in-vitro hair roots can be remarkably promoted by over-expression of the GmGLK66 gene, the fresh weight of the hair roots is increased, and the total phosphorus content is increased; and after the GmGLK66 gene is knocked out, the effects are just opposite, and the fresh weight of hair roots and the total phosphorus content can be reduced. Therefore, the GmGLK66 gene plays an important role in promoting the growth of soybean roots and improving the phosphorus efficiency of the soybeans, and has important significance in illuminating the biological function of the GLK gene in leguminous crops adapting to low-phosphorus stress regulation.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology. More specifically, it relates to the application of the plant low phosphorus stress tolerance gene GmGLK66. Background Technology

[0002] Phosphorus is essential for all life on Earth. In plants, it is a crucial structural component of DNA, RNA, ATP, and phospholipids in biological membranes, playing a vital role in the regulation of photosynthesis, energy metabolism, and various other biochemical reactions, as well as cell signaling. Plants transmit phosphorus via Pi transporters located in the plasma membrane of root cells, using orthophosphate (H₂PO₄). - Phosphorus is obtained from the soil in the form of phosphorus. However, in many natural ecosystems and arable lands, phosphorus levels are below those required for optimal plant growth, which greatly limits agricultural production worldwide.

[0003] Soybeans are an important dual-purpose crop for grain and oil in my country, rich in nutrients. Soybean protein content is 36-46%, similar to animal protein, and 3-5 times that of cereal crops. Phosphorus is a crucial nutrient for soybean growth; phosphorus deficiency inhibits growth and reduces yield. In the long-term adaptation to low phosphorus stress, plants have evolved a series of physiological and molecular mechanisms to enhance soil phosphorus absorption and utilization. These mechanisms mainly include changes in root morphology, activation of soil phosphorus by root exudates (organic acids, acid phosphatase, etc.), regulation of plant adaptation to low phosphorus stress through local and systemic phosphorus signaling pathways, and the formation of plant-microbe symbiotic systems through interactions with microorganisms (such as mycorrhizal fungi). Among these, changes in root morphology are key to maximizing nutrient acquisition by plants. Past research has shown that low phosphorus can induce adaptive changes in plant root morphology to acquire more soluble phosphorus from the soil. For example, in Arabidopsis thaliana, low phosphorus leads to shorter taproots, more lateral roots, and increased root hairs. Similarly, under low phosphorus conditions, the root-to-shoot ratio, root length, root hair density, and number of lateral roots in rice were significantly increased. These changes in root morphology increased the contact area between rice roots and soil, promoting the absorption and utilization of phosphorus by the plant. However, there are currently few gene resources available for regulating soybean root growth and adapting to low phosphorus stress.

[0004] The Golden2-like (GLK) gene is a member of the transcription factor GARP (Golden2, ARR-B, Psr1) family. Current research indicates that the GLK gene plays a role in disease defense and is functionally conserved in regulating chloroplast development, controlling chloroplast development in both green and non-green tissues. In Arabidopsis, it has been shown to play an important role in chloroplast development and photosynthesis. However, whether the GLK gene affects soybean's response to phosphorus regulation has not been reported, and its biological function in the phosphorus signaling network of leguminous crops is unclear. Currently, there is a lack of gene resources to improve soybean phosphorus efficiency. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the lack of existing gene resources that are tolerant to low phosphorus stress and can improve the phosphorus efficiency of soybeans, and to provide the application of the plant low phosphorus stress tolerance gene GmGLK66.

[0006] The purpose of this invention is to provide the application of the plant low phosphorus stress tolerance gene GmGLK66.

[0007] Another objective of this invention is to provide an application of a formulation that promotes the expression of the GmGLK66 gene in the breeding of soybean plants to withstand low phosphorus stress.

[0008] Another object of the present invention is to provide a method for promoting soybean growth or increasing the phosphorus content of soybean plants.

[0009] Another object of the present invention is to provide a method for cultivating soybean plants resistant to low phosphorus stress.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention provides a systematic functional analysis of the soybean GmGLK66 gene, demonstrating its role in regulating soybean root growth and phosphorus efficiency. The study found that the GmGLK66 gene participates in the regulation of phosphorus homeostasis in soybean roots, and its transcriptional expression is significantly upregulated under low phosphorus stress. The cDNA nucleotide sequence of the GmGLK66 gene is shown in SEQ ID NO. 1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO. 2. This indicates that the GmGLK66 gene positively regulates soybean root growth and phosphorus efficiency, significantly increasing the total phosphorus content of soybeans. Furthermore, under normal phosphorus conditions and low phosphorus stress, overexpression of the GmGLK66 gene significantly promotes the growth of detached soybean hairy roots, increases fresh root weight, and improves total phosphorus content; conversely, knocking out the GmGLK66 gene has the opposite effect, inhibiting the growth of detached soybean hairy roots and reducing fresh weight and total phosphorus content. Therefore, the GmGLK66 gene plays an important role in promoting soybean root growth and improving soybean phosphorus efficiency, which is of great significance for elucidating the biological function of the GLK gene in regulating the adaptation of leguminous crops to low phosphorus stress.

[0012] Therefore, this invention provides the application of the GmGLK66 gene in positively regulating soybean growth and improving soybean phosphorus efficiency.

[0013] This invention provides the application of the GmGLK66 gene in promoting soybean growth under low phosphorus stress, or in the preparation of growth promoters.

[0014] This invention provides the application of the GmGLK66 gene in increasing the phosphorus content of soybeans, or in the preparation of products with increased phosphorus content in soybeans.

[0015] This invention provides the application of the GmGLK66 gene in the cultivation of soybean plants resistant to low phosphorus stress and / or high phosphorus content.

[0016] Meanwhile, this invention provides formulations that promote the expression of the GmGLK66 gene for promoting soybean growth, or promoting soybean growth under low phosphorus stress, for preparing products that promote soybean growth, for increasing the phosphorus content of soybeans, for preparing products that increase the phosphorus content of soybeans, and for use in breeding soybean plants to tolerate low phosphorus stress.

[0017] Preferably, the preparation is a plasmid, vector, or recombinant bacteria that promotes the expression of the GmGLK66 gene. An overexpression vector containing the GmGLK66 gene can be used to construct a recombinant expression vector containing the GmGLK66 gene from existing plant expression vectors. The plant expression vector includes, for example, an overexpression vector or other derived plant expression vectors.

[0018] This invention provides a method for promoting soybean growth or increasing phosphorus content in soybean plants by overexpressing the GmGLK66 gene in soybean plants.

[0019] This invention provides a method for cultivating soybean plants resistant to low phosphorus stress, which involves transferring a recombinant vector containing GmGLK66 gene overexpression or its recombinant bacteria into the plant, or treating the plant with an agent that promotes GmGLK66 gene expression.

[0020] Preferably, the plant expression vector carrying the GmGLK66 gene can be transformed into soybeans by, for example, Agrobacterium-mediated transformation.

[0021] The present invention has the following beneficial effects:

[0022] This invention conducted a systematic functional analysis of the soybean GmGLK66 gene, demonstrating that GmGLK66 regulates soybean root growth and phosphorus efficiency. The study found that the GmGLK66 gene participates in the regulation of phosphorus homeostasis in soybean roots, and its transcriptional expression is significantly upregulated under low phosphorus stress. This indicates that the GmGLK66 gene positively regulates soybean root growth and phosphorus efficiency, significantly increasing the total phosphorus content of soybeans. Furthermore, under normal phosphorus conditions and low phosphorus stress, overexpression of the GmGLK66 gene significantly promotes the growth of detached soybean hairy roots, increases fresh root weight, and improves total phosphorus content; conversely, knocking out the GmGLK66 gene has the opposite effect, inhibiting the growth of detached soybean hairy roots and reducing fresh weight and total phosphorus content. Therefore, the GmGLK66 gene plays an important role in promoting soybean root growth and improving soybean phosphorus efficiency, which is significant for elucidating the biological function of the GLK gene in the regulation of low phosphorus stress in leguminous crops. Attached Figure Description

[0023] Figure 1 Figure 1 shows the results of the analysis of the expression pattern of GmGLK66 in different tissues of soybean under different phosphorus concentration treatments (+P in the figure represents normal phosphorus treatment, -P represents low phosphorus treatment, the data are the mean and standard error of 3 replicates, asterisks indicate that the difference between control (+P) and treatment (-P) is significant (Student's t-test), *: P<0.05, **: P<0.01, ***: P<0.001).

[0024] Figure 2 Subcellular localization analysis of GmGLK66 fusion GFP protein in rice protoplasts (the figures show the green fluorescent channel (GFP), red fluorescent channel (RFP), bright field, and merged image, with a scale bar of 10 μm).

[0025] Figure 3 The self-activation activity analysis of GmGLK66 protein (SD in the figure: yeast defective screening medium; -Trp: tryptophan defective; -His: histidine defective; X-α-Gal: 5-bromo-4-chloro-3-indolyl-β-D-galactoside, histochemical substrate of β-galactosidase, which produces a blue precipitate upon hydrolysis).

[0026] Figure 4The effect of GmGLK66 overexpression on phosphorus availability in isolated hairy roots of transgenic soybean was investigated. (A represents the phenotypes of the empty vector control (CK) and GmGLK66 overexpression (OX) transgenic hairy roots under normal and low phosphorus conditions; B represents the relative expression level of the GmGLK66 gene; C represents the fresh weight of hairy roots; D represents the total phosphorus content; (+P) indicates normal phosphorus, (-P) indicates low phosphorus; OX represents the overexpression line; CK represents the transgenic line transformed with the empty vector; scale bar is 1.2 cm; all data are the mean and standard error of 10 replicates; asterisk (*) indicates a significant difference between the overexpression line and the control line (Student's t-test; *: P < 0.05, **: 0.001) <P<0.01,***:P<0.001)。

[0027] Figure 5 To investigate the effect of GmGLK66 knockout on the phosphorus availability of detached soybean root hairs (A: phenotype of empty vector control (CK) and GmGLK66 knockout transgenic root hairs under normal and low phosphorus conditions; B: fresh weight of root hairs; C: total root length of root hairs; D: total phosphorus content; glk66 represents the knockout line; CK represents the transgenic line transformed with the empty vector; (+P) represents normal phosphorus, (-P) represents low phosphorus; scale bar is 1.2 cm; data are the mean and standard error of 10 replicates; asterisk (*) indicates significant difference between overexpression lines and control lines (Student's t-test; *: P < 0.05, **: 0.001)). <P<0.01,***:P<0.001)。 Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] The soybean (Glycine max (L.) Merr.) used in the examples was Yuechun 03-3 (YC03-3), sourced from the Root Biology Research Center of South China Agricultural University. The rice used was Nipponbare, also sourced from the Root Biology Research Center of South China Agricultural University.

[0031] Example 1: Expression pattern of GmGLK66 gene under low phosphorus stress

[0032] 1. Plant samples

[0033] Soybean YC03-3 seeds with intact seed coats and uniform size were selected using the paper-roll seedling method. The seeds were sterilized for 12 hours with chlorine gas generated from the reaction of 100 mL sodium hypochlorite and 4.2 mL hydrochloric acid, and then bleached in a clean bench for 1 hour before use. Square filter paper (20×20 cm) was cut, and a 1 / 4 soybean total nutrient solution with a pH of 5.8 and sterile water were prepared and sterilized for later use.

[0034] For paper roll culture, lay plastic wrap on the experimental table, soak filter paper in a 1 / 4 soybean nutrient solution, and place 7 sterilized soybeans about 1 cm from one side of the filter paper, with the hilum facing down. Roll the filter paper from the first soybean to the end. Place the rolled filter paper, with the end without soybeans facing down, into a 500 mL beaker containing 1 / 4 soybean nutrient solution, and wrap the top of the rolled filter paper with plastic wrap. Place the beaker in an incubator at 24–26°C, first in the dark for 1 day, then in a light / dark (12h / 12h) cycle for 3–4 days, until the radicle reaches 5–6 cm.

[0035] Seedlings with uniform growth were selected and transferred to soybean nutrient solutions with different phosphorus concentrations: normal phosphorus treatment (+P: 1250 μM KH2PO4) and low phosphorus treatment (-P: 12.5 μM KH2PO4). Eight replicates were performed for each treatment, with eight seedlings per replicate. The pH of the nutrient solution was adjusted to approximately 5.8 every two days, and the nutrient solution was changed weekly. Different tissue samples were harvested at 6, 12, 18, 24, and 36 days of growth. After being frozen in liquid nitrogen, the samples were stored at -80°C for later use.

[0036] 2. Real-time quantitative PCR analysis

[0037] Total RNA was extracted from plant samples treated with different phosphorus concentrations using the TRizol kit (Invitrogen, USA). The DNase I-treated RNA was reverse transcribed into cDNA using the MMLV-Reverse Transcription Kit (Promega, USA). qRT-PCR analysis was then performed using the SYBR (Promega, USA) kit. After reverse transcription, the samples were diluted 10-fold and analyzed using the Applied Biosystems StepOnePlus Real-Time PCR system.

[0038] GmGLK66 was selected as the candidate gene, its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2. Quantitative PCR primers for the GmGLK66 gene were designed and synthesized. The internal reference gene was the soybean housekeeping gene EF1-α (Glyma17g23900). The specific primers are shown below:

[0039] GmGLK66-RT1-F (SEQ ID NO:3): 5'-TGGTGCTTTCCAGCCATTCC-3';

[0040] GmGLK66-RT1-R (SEQ ID NO:4): 5'-CTAGAACTCGCGGCGGCT-3';

[0041] EF1-α-F (SEQ ID NO:5): 5'-TGCAAAGGAGGCTGCTAACT-3';

[0042] EF1-α-R (SEQ ID NO:6): 5'-CAGCATCACCGTTCTTCAAA-3'.

[0043] (1) Preparation of the reaction system: First, calculate the required amount of reaction mixture. Mix all reagents except cDNA, dispense 18 μL into each tube, and then add 2 μL of cDNA template to make the final reaction volume 20 μL. Each 20 μL reaction system contains: 0.5 μL forward / reverse primers, 10 μL SYBR Premix Ex Taq (2×), 2 μL template, and 7 μL ddH2O.

[0044] (2) Preparation of standard curve: First, take 1-2 μL of cDNA stock solution from each sample and put it into a new PCR centrifuge tube. Then, perform serial dilution of the reaction system. Each 10-fold dilution is a standard sample. Continue this process to obtain 5 concentration gradient standard samples.

[0045] (3) Quantitative PCR reaction program: 95℃ pre-denaturation for 30s, PCR reaction (95℃ denaturation for 30s, 60℃ annealing for 15s, 72℃ extension for 30s) for 40 cycles.

[0046] The expression levels of each sample were calculated using Rotor-Gene's Real-Time Analysis Software based on the quantitative PCR results.

[0047] Test results as follows Figure 1 As shown, the GmGLK66 gene is expressed in soybean roots, leaves, flowers, pods, and root nodules. Compared with normal phosphorus treatment, low phosphorus treatment significantly enhanced the expression level of the GmGLK66 gene in soybean roots, with low phosphorus stress leading to an approximately 4-fold upregulation of GmGLK66 gene expression in roots. This indicates that GmGLK66 expression is significantly upregulated at the transcriptional level under low phosphorus stress.

[0048] Example 2: Construction of expression vector

[0049] 1. Construction of the overexpression vector pTF101s-GmGLK66-OX

[0050] Based on the full-length CDS sequence of the GmGLK66 gene, specific primers were designed: GmGLK66-OX-F (SEQ ID NO:7): 5'-GTACCCGGGGATCCTCTAGAATGTATTTTTTCACTGAGAA A-3', and GmGLK66-OX-R (SEQ ID NO:8): 5'-GCCTGCAGGTCGACTCTAGAT CAATAGCCAAGGGAAGTAGA-3'. Using soybean YC03-3 cDNA as a template, the gene fragment was amplified using the Phanta Max Super Fidelity DNA Polymerase kit (Novizan, China). The reaction system (50 μL) consisted of: 2×phata max buffer 25 μL, forward and reverse primers (10 mM) 2 μL each, 2 mM dNTPs 1 μL, Phanta Max Super Fidelity DNA Polymerase 1 μL, cDNA template 2 μL, and ddH2O 18 μL. PCR program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 sec, 58℃ annealing for 30 s, 72℃ annealing for 1 min, 72℃ final extension for 5 min, with the denaturation-annealing process repeated 30 times.

[0051] After PCR products were subjected to gel electrophoresis, the target band was recovered and purified using an agarose gel electrophoresis kit (Meiji Biotechnology, China) following the manufacturer's instructions. Subsequently, using the Clone Express II One Step Cloning Kit 29 recombination exchange kit (Novizan, China), the fragment was inserted into the Sam I restriction site of the linearized pTF101s plasmid, then transformed into E. coli DH5α. After incubation at 37°C for 12 h, positive clones were cultured and sent to the company for sequencing. After successful sequencing, the plasmid was extracted, and the target gene fragment was ligated into the pTF101s vector using a one-step cloning method, successfully preparing the overexpression vector pTF101s-GmGLK66-OX.

[0052] 2. Construction of the knockout expression vector pGES401-GmGLK66-KO

[0053] Using the CRISPR / Cas9 system, a knockout vector was constructed following the method of Bai et al. (Bai M, Yuan J, Kuang H, et al. Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean[J]. Plant Biotechnology Journal, 2020, 18(3): 721-731). First, the sgRNA target of GmGLK66 was designed using the website TargetDesign (http: / / skl.scau.edu.cn / ta-rgetdesign / ). GmGLK66-sgRNA-F / R oligonucleotide chains were synthesized by Qingke Biotechnology Co., Ltd., namely GmGLK66-sgRNA-F (SEQ ID NO:9): 5'-TGGTCTCGTGCAA TTCACATCCTCCTCAGCTGGTTTTAGAGCTAGAAATAGC-3' and GmGLK66-sgRNA-R (SEQ ID NO:10): 5'-TGGTCTCGAAACCACTAGAAGGCCAATTCCCATGCACCAGCCGGGAATCGAA-3', and then annealed to double strands. The annealing system consisted of (50 μL): 5 μL each of the forward and reverse primers (GmGLK66-sgRNA-F / R, 10 Mm) (final concentration 100 mM), 5 μL of Tris-HCl solution (pH 7.4, final concentration 50 Mm), and ddH2O was added to bring the total to 50 μL. The annealing program was: 95℃ for 4 min, followed by RAMP gradient cooling from 0.1℃ / s to 95℃ to 16℃, and storage at 16℃. The annealed double-linked gene was inserted into the BsaⅠ restriction site of the linearized pGES401 vector, transformed into competent E. coli cells, and after successful sequencing, the GmGLK66-KO plasmid was extracted and transformed into Agrobacterium tumefaciens EHA105 to obtain the knockout expression vector pGES401-GmGLK66-KO.

[0054] 3. Construction of the BD-GmGLK66 vector for verifying the self-activation activity of GmGLK66 protein

[0055] The construction method and conditions were the same as for the overexpression vector pTF101s-GmGLK66-OX. The primers used were BD-Gm GLK66-F (SEQ ID NO:11): 5'-CATGGAGGCCGAATTCATGTATTTTTTCACT GAGAA-3', and BD-GmGLK66-R (SEQ ID NO:12): 5'-GCAGGTCGACGGATCC TCAATAGCCAAGGGAAGTAG-3'. The recovered PCR fragment was ligated into the linearized vector pGBKT 7 restriction site, transformed into competent intestinal cells, and after sequencing confirmed to be correct, the plasmid was extracted for later use.

[0056] 4. Construction of the subcellular localization expression vector 35S::GmGLK66-GFP

[0057] Using soybean YC03-3 cDNA as a template, the full-length CDS sequence of the GmGLK66-GFP gene was amplified using the forward-specific primer (SEQ ID NO:13): 5'-CACGGGGACTCTAGCGCTACCGGTATGTATTTTTTC ACTGAGAA-3' and the reverse-specific primer (SEQ ID NO:14): 5'-CATGGTGGCGACC GGTAGATAGCCAAGGGAAGTAGAGGTAT-3'. The PCR amplification system consisted of: 25 μL 2×Vazyme phata buffer, 1 μL Vazyme phataase, 1 μL dNTP, 1 μL each of the forward and reverse primers, 3 μL cDNA template, and finally 16 μL ddH2O to a final volume of 50 μL. The PCR amplification program was as follows: 94℃ pre-denaturation for 2 minutes, 94℃ denaturation for 30 seconds, 58℃ annealing for 40 seconds, 72℃ extension for 30 seconds, 30 cycles from denaturation to extension, and PCR products were stored at 16℃.

[0058] The amplified products were then detected by gel electrophoresis. If the band size was correct, the target fragment was further purified using an agarose gel DNA extraction kit (Meiji Biotechnology, China). AgeI was selected as the single restriction enzyme site for the pEGAD vector, and the target gene fragment was ligated into the pEGAD vector using a one-step cloning method. The ligation reaction system was the same as above, and the ligation product was stored at -20°C for later use. Finally, the ligation product was transformed into E. coli Trans1-T1 competent cells and cultured at 37°C for 12 hours. After the positive clones were cultured and sent to the company for sequencing, the subcellular localization expression vector 35s::GmGLK66-GFP was obtained. The positive strain was stored at -80°C.

[0059] Example 3: Subcellular localization and self-activation activity verification of GmGLK66 protein

[0060] 1. Subcellular localization analysis of GmGLK66 protein transient expression in rice protoplasts

[0061] The subcellular localization expression vector 35s::GmGLK66-GFP was transformed into rice protoplasts. The preparation and transformation of rice protoplasts followed existing techniques (Mai H, Qin T, Wei H, et al. Overexpression of OsACL5triggers environmentally-dependent leaf rolling and reduces grain size in rice[J]. Plant Biotechnology Journal, 2024, 22(4):833-847.). Fluorescence of tobacco leaves was observed and fluorescence images were captured using a laser confocal scanning microscope (Zeisee, Germany), with excitation wavelengths of 488 nm (GFP green fluorescence) and 568 nm (mcherry red fluorescence). Images were analyzed using Zen2011 software.

[0062] The results are as follows Figure 2 As shown, the 35s::GmGLK66-GFP fluorescent protein is mainly expressed in the nucleus of rice protoplasts.

[0063] 2. Verification of the self-activation activity of GmGLK66 protein

[0064] Using the BD-GmGLK66 vector constructed in Example 2, the self-activation activity of GmGLK66 protein in yeast cells was analyzed by a colorimetric reaction method with X-α-Gal as the substrate. The BD-GmGLK66 plasmid was transformed into yeast Y2HGold and plated on yeast plates with single absence (SD / His) and containing X-α-Gal. Yeast transformed with pGBKT7(BD) plasmid was used as a negative control group, and yeast transformed with pGBKT7(BD-53) plasmid was used as a positive control group.

[0065] The results are as follows Figure 3 As shown, BD-GLK66 is identical to the negative control pGBKT7 and cannot grow normally on SD / -His+X-α-gal medium, indicating that GmGLK66 does not have self-activation activity.

[0066] Example 4: Obtaining Transgenic Material

[0067] 1. Obtaining in vitro root hair material from genetically modified soybeans

[0068] (1) Seed germination: Select uniform, clean, and plump soybean seeds (WT) and sterilize them with chlorine in a desiccator. The chlorine generation reaction is as follows: add 4.2 mL of concentrated hydrochloric acid to 100 mL of sodium hypochlorite solution. After sterilization for 12 hours, remove the seeds from the desiccator and place them in a laminar flow hood to remove residual chlorine. Then, plant the seeds in sterile MS germination medium and incubate them in a constant temperature and light incubator at 24°C.

[0069] (2) Culturing: During germination, Agrobacterium K599 carrying the vector and plasmid, stored at -80℃ in glycerol, was streaked onto YEP solid medium containing the corresponding antibiotic based on the resistance of the vector and plasmid. The empty vector Agrobacterium K599 was activated by streaking onto YEP solid medium containing streptomycin and spectinomycin resistance. After streaking, the cultures were incubated in the dark at 28℃. After 2 days, single colonies were picked for testing. Positive strains were reserved for use. The night before infection, positive strains were inoculated into 50mL centrifuge tubes containing 5mL of YEP liquid medium and incubated in the dark at 28℃ for 12–16 hours.

[0070] (3) Infection and co-culture: In a clean bench, use a scalpel to cut the germinated seeds about 0.5 cm from the cotyledon node along the hypocotyl region, peel off the seed coat, cut the seeds in half along the hypocotyl, remove the buds at the cotyledon node, and use a scalpel to cut the cotyledons in half along the hypocotyl. Then, use a scalpel to dip in bacterial solution and make incisions at the cotyledon node and hypocotyl. Place the explants with wounds in a glass petri dish containing moistened filter paper, seal with sterile plastic wrap, and incubate at 24℃ under constant temperature and light for 5-7 days. After that, transfer to a culture dish containing termethin (final concentration 50 mg / L). -1 ) and glufosinate (final concentration 20 μg / L) -1 Incubate on MS solid medium at 24°C in the dark.

[0071] (4) Treatment: Hairy roots were observed to be produced after culturing on MS solid medium for 7-10 days. Hairy roots of uniform size were selected and transplanted into rooting medium with different phosphorus contents (normal phosphorus (+P: 1250 μM KH2PO4) and low phosphorus (-P: 12.5 μM KH2PO4)). After culturing in the dark at 24℃ for 12 days, the hairy roots were harvested.

[0072] 2. Detection of isolated hairy roots from genetically modified soybeans

[0073] (1) Total RNA was extracted from the hairy roots of the obtained transgenic lines and reverse transcribed into cDNA for later use. Real-time quantitative PCR analysis was performed using the AppliedBiosystems StepOnePlus Real-Time PCR system, with quantitative PCR using a Promega kit. The primers used for quantitative PCR were the same as in Example 1: GmGLK66 quantitative primers (SEQ ID NO: 2-3), soybean housekeeping gene GmEF1-α (SEQ ID NO: 3-4). The reaction system (20 μL) consisted of: 10 μL 2×Go Tap qPCR Master Mix, 0.4 μL each of forward and reverse primers, 2 μL cDNA template, and 7.2 μL Nuclease-free water. The reaction conditions were: 95℃ pre-denaturation for 1 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, and 72℃ extension for 30 s, repeated 40 times from denaturation to extension. The relative expression level was the ratio of the expression level of the target gene GmGLK66 to the expression level of the soybean housekeeping gene EF1-α.

[0074] The results showed that the expression level of GmGLK66 in the detached hairy roots of transgenic soybean was significantly higher than that in the control group, indicating that the detached hairy roots of transgenic soybean were successfully obtained.

[0075] Example 5: Functional Analysis of GmGLK66

[0076] 1. Effects of GmGLK66 overexpression on phosphorus availability in isolated hairy roots of transgenic soybean

[0077] To investigate the effect of GmGLK66 on the phosphorus responsiveness of transgenic soybean rootlets in vitro, approximately 0.2 g of transgenic rootlets were inoculated into solid MS medium with normal phosphorus (+P: 1250 μM KH2PO4) and low phosphorus (-P: 12.5 μM KH2PO4) concentrations and cultured for 14 days. Ten independent biological replicates were set up for each treatment. After treatment, the expression level of GmGLK66 was detected (by real-time quantitative PCR, the method was the same as in Example 1), and the fresh weight and total phosphorus content of the rootlets were measured.

[0078] The results are as follows Figure 4 As shown, after 14 days of culture, the phenotypes of different root hairs revealed that under normal phosphorus (+P) conditions, overexpression of GmGLK66 significantly promoted the growth of isolated soybean root hairs, and under low phosphorus (-P) conditions, overexpression of GmGLK66 also significantly increased the growth of isolated soybean root hairs. Figure 4 A). Real-time quantitative PCR results showed that under normal phosphorus supply and low phosphorus conditions, the expression level of GmGLK66 in detached soybean hairy roots overexpressing GmGLK66 was significantly higher than that in the empty vector control (CK), with expression levels increasing by approximately 1.5-fold and 4-fold, respectively. Figure 4 B). Under normal phosphorus (+P) conditions, compared with the empty control (CK), the fresh weight of GmGLK66 transgenic hairy roots overexpressed increased by 60.4%; under low phosphorus (-P) conditions, compared with the empty control (CK), the fresh weight of GmGLK66 transgenic detached hairy roots overexpressed increased by 18.4%. Figure 4 C).

[0079] Under normal phosphorus (+P) conditions, overexpression of GmGLK66 significantly increased the total phosphorus content of transgenic detached hair roots, with a 231.6% increase compared to the empty control (CK). Under low phosphorus (-P) conditions, overexpression of GmGLK66 also significantly increased the total phosphorus content of transgenic detached hair roots, with a 143.7% increase compared to the empty control (CK). Figure 4 D). These results indicate that the soybean GmGLK66 gene positively regulates soybean root growth and phosphorus efficiency.

[0080] 2. The effect of knocking out GmGLK66 on the phosphorus availability of isolated hairy roots of transgenic soybean.

[0081] To investigate the effect of GmGLK66 on the phosphorus availability of transgenic soybean rootlets in vitro, approximately 0.2 g of transgenic rootlets were inoculated into solid MS medium with normal phosphorus (+P: 1250 μM KH2PO4) and low phosphorus (-P: 12.5 μM KH2PO4) concentrations and cultured for 14 days. Ten independent biological replicates were set up for each treatment. After harvest, the fresh weight, total root length, and total phosphorus content of the rootlets were measured.

[0082] The results are as follows Figure 5 As shown, under normal phosphorus (+P) conditions, knocking out the expression of GmGLK66 did not significantly inhibit the growth of isolated soybean root hairs. Figure 5 A), compared with the empty vector control (CK), the fresh weight of GmGLK66 transgenic hairy roots knocked out was reduced by 26.3% ( Figure 5 B), the total root length of the GmGLK66 transgenic hairy roots knocked out decreased by 22.2% ( Figure 5 C). Under low phosphorus (-P) conditions, knockout of GmGLK66 also significantly inhibited the growth of detached soybean hairy roots. Compared with the empty control (CK), the fresh weight of GmGLK66 knockout transgenic hairy roots was reduced by 15.6% ( Figure 5 B), the total root length of the GmGLK66 transgenic hairy roots knocked out decreased by 9.2% ( Figure 5 C).

[0083] Under normal phosphorus (+P) conditions, knocking out GmGLK66 significantly reduced the total phosphorus content in transgenic hairy roots. Figure 5 D), compared with the empty control (CK), the total phosphorus content of the GmGLK66 transgenic hairy roots knocked out was reduced by 9.01% ( Figure 5 D). Under low phosphorus (-P) conditions, knockout of GmGLK66 also significantly reduced the total phosphorus content of transgenic hairy roots. Compared with the empty control (CK), the total phosphorus content of GmGLK66 knockout transgenic hairy roots decreased by 21.55%. Figure 5 D). These results indicate that the soybean GmGLK66 gene is involved in the regulation of phosphorus balance in soybean roots.

[0084] In summary, this study found that the GmGLK66 gene participates in the regulation of phosphorus balance in soybean roots. In response to low phosphorus stress, its transcriptional expression is significantly upregulated under low phosphorus stress. This indicates that the GmGLK66 gene positively regulates soybean root growth and phosphorus efficiency, significantly increasing the total phosphorus content of soybeans. Furthermore, under normal phosphorus conditions and low phosphorus stress, overexpression of the GmGLK66 gene significantly promotes the growth of detached soybean hairy roots, increases fresh weight, and improves total phosphorus content; conversely, knocking out the GmGLK66 gene has the opposite effect, inhibiting the growth of detached soybean hairy roots and reducing fresh weight and total phosphorus content. Therefore, the GmGLK66 gene plays an important role in promoting soybean root growth and improving soybean phosphorus efficiency.

[0085] 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 changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of the GmGLK66 gene, as shown in SEQ ID NO.1, in positively regulating soybean growth and improving soybean phosphorus efficiency.

2. The GmGLK66 gene, as shown in SEQ ID NO.1, promotes soybean growth under low phosphorus stress, or its application in the preparation of growth promoters.

3. The application of the GmGLK66 gene, as shown in SEQ ID NO.1, in increasing the phosphorus content of soybeans, or in the preparation of products with increased phosphorus content of soybeans.

4. Application of the GmGLK66 gene, as shown in SEQ ID NO.1, in the cultivation of soybean plants resistant to low phosphorus stress and / or high phosphorus content.

5. Application of formulations that promote the expression of the GmGLK66 gene as shown in SEQ ID NO.1 in promoting soybean growth or in promoting soybean growth under low phosphorus stress.

6. Application of a formulation that promotes the expression of the GmGLK66 gene as shown in SEQ ID NO.1 in the preparation of products that promote soybean growth.

7. The use of a formulation that promotes the expression of the GmGLK66 gene as shown in SEQ ID NO.1 in increasing the phosphorus content of soybeans, or in the preparation of products that increase the phosphorus content of soybeans.

8. Application of a formulation that promotes the expression of the GmGLK66 gene as shown in SEQ ID NO.1 in the breeding of soybean plants to tolerate low phosphorus stress.

9. A method for promoting soybean growth or increasing the phosphorus content of soybean plants, characterized in that, The GmGLK66 gene was overexpressed in soybean plants.

10. A method for cultivating soybean plants tolerant to low phosphorus stress, characterized in that, Transform plants into recombinant vectors containing GmGLK66 gene overexpression or their recombinant bacteria, or treat plants with agents that promote GmGLK66 gene expression.

Images