Application of GmCXIP4 gene in regulation and control of plant phosphorus nutrition and growth promotion

Abstract

The invention discloses application of a GmCXIP4 gene in regulation and control of plant phosphorus nutrition and growth promotion. Research shows that the GmCXIP4 gene participates in regulation of soybean root growth and phosphorus nutrition absorption, and through construction and identification of overexpression and knockout carrier materials, the overexpression GmCXIP4 gene can inhibit plant growth and reduce absorption and accumulation of phosphorus nutrition by plants; after the GmCXIP4 gene is knocked out, plant growth can be remarkably promoted, absorption and accumulation of phosphorus nutrition by the plant are promoted, and the phosphorus content of the plant is increased; under low phosphorus stress, plant growth and phosphorus nutrition absorption can be promoted. As a new gene resource for regulating plant growth and phosphorus nutrition, the GmCXIP4 gene plays an important role in improving plant phosphorus nutrition and promoting plant growth, provides an important gene resource for cultivating low-phosphorus-resistant plants, and plays an important role in adapting to low-phosphorus-condition growth of plants and improving phosphorus utilization efficiency.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and plant breeding technology. More specifically, it relates to the application of the GmCXIP4 gene in regulating phosphorus nutrition and promoting plant growth. Background Technology

[0002] Phosphorus is an essential nutrient for plant growth and development. It is not only a major component of cell structure but also directly or indirectly participates in various metabolic processes, such as membrane and nucleotide synthesis, photosynthesis, energy transfer, and signal transduction. In soil, soluble inorganic phosphorus is easily adsorbed and fixed into insoluble forms such as calcium phosphate, aluminum phosphate, and iron phosphate, leading to reduced phosphorus availability and making it difficult for plants to absorb and utilize. Low available phosphorus content is one of the main factors limiting crop yields in most soils, especially acidic soils. Statistics show that low phosphorus stress causes at least 30-40% of crop yields to be reduced worldwide. In current agricultural production, the main approach is to alleviate the problem of insufficient phosphorus supply in the soil by applying phosphate fertilizers. However, because crops utilize less than 25% of phosphate fertilizers in the current season, and excessive application of phosphate fertilizers can cause eutrophication of water bodies and environmental pollution, improving crop utilization of phosphate fertilizers based on optimized nutrient management models is an effective way to maintain sustainable agricultural development.

[0003] In recent years, significant progress has been made both domestically and internationally in the study of mechanisms by which root traits regulate the efficient absorption and utilization of phosphorus in plants. This includes regulating root morphology to increase the plant's ability to acquire soil phosphorus, the physiological and molecular mechanisms by which root exudates, such as organic acids, activate and utilize insoluble phosphorus, and the phosphorus signaling regulatory network. Calcium ions (Ca...) 2+ Calcium (CAS) are important second messengers in cell signaling, and the calcium signaling network is involved in the growth and adaptation of all organisms. CAS are commonly referred to as Ca2+. 2+ / H+ antitransporter proteins, plant CAXs are located on the vacuolar membrane and are mainly involved in Ca2+ transport. 2+ Transport to the vacuole also involves the transport of other cations. CAX interacting protein 4 (CXIP4) is a protein that can activate H+ / Ca2+ transport. 2+ A novel Arabidopsis protein, the reverse transporter CAX, can regulate the transport of CAX signaling molecules in plants and also plays an important role in ion transport systems.

[0004] The CXIP4 gene is specifically expressed in plants, and although it has been cloned and reported in Arabidopsis and grape, CXIP4 plays a role in regulating calcium. 2+The function of the CXIP4 gene in signal transduction is still unknown. Whether it participates in the molecular mechanisms regulating soybean root growth and phosphorus uptake remains unclear. Therefore, there is an urgent need to provide more gene resources to develop new low-phosphorus-tolerant genes, cultivate low-phosphorus-tolerant plants, and improve plant growth and phosphorus uptake under low-phosphorus stress. This is of great significance for the regulation of plant growth and metabolism. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the GmCXIP4 gene in regulating phosphorus nutrition and promoting plant growth.

[0006] The first objective of this invention is to provide applications of the GmCXIP4 gene.

[0007] A second objective of this invention is to provide the application of formulations that delete or knock out the GmCXIP4 gene.

[0008] A third objective of this invention is to provide a method for promoting plant growth and / or increasing the phosphorus content of plants.

[0009] A fourth objective of this invention is to provide a method for cultivating plants resistant to low phosphorus stress and / or high phosphorus content.

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

[0011] This invention, through the construction of transgenic soybean whole-plant materials, systematically analyzed the function of the GmCXIP4 gene, demonstrating its function in regulating soybean root growth and phosphorus content. The study showed that the GmCXIP4 gene participates in regulating soybean root growth and phosphorus nutrient absorption, negatively regulating plant phosphorus nutrition and plant growth. Through the construction and identification of overexpression and knockout vector materials, it was shown that overexpression of the GmCXIP4 gene inhibits plant growth and reduces the accumulation of phosphorus nutrients; while knockout of the GmCXIP4 gene significantly promotes plant growth, increases phosphorus nutrient absorption and accumulation, and increases plant phosphorus content; even under low phosphorus stress, it also promotes plant growth and phosphorus nutrient absorption. The GmCXIP4 gene has a significant impact on the growth and phosphorus content of transgenic soybeans, which is of great significance for elucidating the biological function of the CXIP gene in the synergistic regulation of acid and phosphorus in leguminous crops.

[0012] Therefore, this invention provides the application of the GmCXIP4 gene, as shown in SEQ ID NO:1, in the negative regulation of plant growth and / or phosphorus nutrient uptake.

[0013] This invention provides the application of the GmCXIP4 gene as a target in the cultivation of plants tolerant to low phosphorus stress or plants with high phosphorus content.

[0014] Furthermore, the amino acid sequence of the protein encoded by the GmCXIP4 gene is shown in SEQ ID NO.2.

[0015] This invention provides preparations containing the GmCXIP4 gene, as shown in SEQ ID NO:1, for use in promoting plant growth, or promoting plant growth under low phosphorus conditions, increasing the phosphorus content of plants, or preparing products that increase the phosphorus content of plants, preparing products that promote plant growth or improve plant tolerance to low phosphorus stress, cultivating plants tolerant to low phosphorus stress and / or plants with high phosphorus content, and promoting the absorption of phosphorus nutrients by plants.

[0016] Preferably, the preparation is a plasmid, vector, or recombinant bacteria that has the GmCXIP4 gene deleted or knocked out.

[0017] More preferably, expression vectors for the GmCXIP4 gene can be used to construct recombinant expression vectors containing the GmCXIP4 gene from existing plant expression vectors; plant expression vectors include binary Agrobacterium vectors, such as pTF101s, pEGAD, pGES201 or other derived plant expression vectors.

[0018] This invention also relates to cells containing the GmCXIP4 gene, which is missing or knocked out, comprising the GmCXIP4 gene or a recombinant vector; the cells can be plant cells, such as legume cells, or microbial cells, such as bacterial or fungal cells, such as yeast cells. The cells can be isolated, ex vivo, cultured, or part of a plant.

[0019] The plant involved in this invention can be a plant part, plant material, or plant seed. The plant is a legume, such as a common bean or a soybean; it can also be other plants, such as monocotyledons, such as rice, wheat, barley, corn, sorghum, sugarcane, oats, or rye; or other dicotyledons, such as tobacco, sunflower, beet, chili pepper, potato, or tomato.

[0020] This invention provides a method for promoting plant growth and / or increasing plant phosphorus content by deleting or knocking out the GmCXIP4 gene in the plant, or by treating the plant with an agent that deletes or knocks out the GmCXIP4 gene as shown in SEQ ID NO:1.

[0021] This invention provides a method for cultivating plants tolerant to low phosphorus stress and / or high phosphorus content by deleting or knocking out the GmCXIP4 gene in the plant, or by transferring a recombinant expression vector that knocks out the GmCXIP4 gene into the plant, thereby obtaining plants tolerant to low phosphorus stress and / or high phosphorus content.

[0022] Preferably, the recombinant expression vector can be transformed into plants by, for example, Agrobacterium-mediated transformation.

[0023] The present invention has the following beneficial effects:

[0024] This invention provides the application of the GmCXIP4 gene in regulating phosphorus nutrition and promoting plant growth. Studies show that the GmCXIP4 gene participates in regulating soybean root growth and phosphorus uptake, negatively regulating phosphorus nutrition and plant growth. Through the construction and identification of overexpression and knockout vector materials, it was shown that overexpression of the GmCXIP4 gene inhibits plant growth and reduces phosphorus uptake and accumulation; while knockout of the GmCXIP4 gene significantly promotes plant growth, phosphorus uptake and accumulation, and increases phosphorus content. Under low phosphorus stress, it also promotes plant growth and phosphorus uptake. The GmCXIP4 gene has a significant impact on the growth and phosphorus content of transgenic soybeans, which is of great significance for elucidating the biological function of the CXIP gene in the synergistic regulation of acid and phosphorus in leguminous crops. As a novel gene resource regulating plant growth and phosphorus nutrition, the GmCXIP4 gene plays an important role in improving plant phosphorus nutrition and promoting plant growth, providing an important gene resource for breeding low-phosphorus tolerant plants. Attached Figure Description

[0025] Figure 1 To construct overexpression and knockout GmCXIP4 expression vectors and identify transgenic lines (A is a schematic diagram of the overexpression GmCXIP4 vector structure; B is the herbicide resistance test of GmCXIP4 transgenic soybean leaves, scale bar = 1.2 cm; C is the PCR detection of the gene; D is the relative expression level of GmCXIP4 in the roots of wild-type (WT) and overexpression lines (OX1 and OX2), the data in the figure are the mean and standard error of 4 replicates; asterisk * indicates significant difference compared with WT (Student's t-test; "***", P<0.001); E is a schematic diagram of the GmCXIP4 knockout vector and target sequence, sgRNA represents guide RNA, UTR represents untranslated region). Regions), yellow boxes indicate exons, Up / downstream: upstream / downstream regions; F shows the nucleotide sequences and peak diagrams of the CRISPR / Cas9 target regions for wild-type (YC03-3) and two homozygous knockout lines (KO1 and KO2), with red horizontal lines indicating missing bases; G shows the amino acid sequences for WT and KO lines, with red horizontal lines indicating omitted amino acids, and asterisks * indicating stop codons).

[0026] Figure 2 Subcellular localization analysis results of GmCXIP4 in rice protoplasts (GFP: green fluorescent signal channel, Bright: bright field channel, DAPI: blue nuclear fluorescent signal channel, mCherry: red nuclear fluorescent signal channel. Scale bar is 50 μm).

[0027] Figure 3 The effects of different phosphorus concentrations on GmCXIP4 overexpression on soybean growth were analyzed (A: Phenotypic analysis of soybean growth under different phosphorus concentrations of GmCXIP4 overexpression, scale bar 10cm; B: Aboveground dry weight; C: Underground dry weight; D: Total root length; E: Lateral root length; F: Aboveground phosphorus content; G: Root phosphorus content. Asterisk WT indicates significant difference between the WT and OX lines ("*", P<0.05; "**", P<0.01)).

[0028] Figure 4 The effects of GmCXIP4 knockout at different phosphorus concentrations on soybean growth were analyzed (A: Phenotypic analysis of GmCXIP4 knockout at different phosphorus concentrations on soybean growth, scale bar 10 cm; B: Aboveground dry weight; C: Root dry weight; D: Total root length; E: Lateral root length; F: Aboveground phosphorus content; G: Root phosphorus content. Asterisk WT indicates significant difference from KO lines ("*", P<0.05; "**", P<0.01)). Detailed Implementation

[0029] 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.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] Example 1: Cloning of the GmCXIP4 gene and construction of the vector

[0032] 1. Construction of pTF101s-GmCXIP4-OX with overdose vector expression

[0033] Based on the full-length CDS sequence of GmCXIP4 (gene number LOC100794760, the nucleotide sequence of the GmCXIP4 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2), specific primers were designed using Primer software: a forward primer for the GmCXIP4-OX gene (SEQ ID NO:3:5'-ATGCCAGCTACAGCAGGAAGATGCCAGCTACAGCAGGAAG-3') and a reverse primer for the GmCXIP4-OX gene (SEQ ID NO:4:5'-TCATTCATCATCCCCATGGTGACTCATTCATCATCCCCATGGTGAC-3'), to amplify the gene CDS sequence.

[0034] Using soybean YC03-3 cDNA as a template, the gene fragment was amplified using Phanta Max Super-Fidelity DNA Polymerase (Novizan, China). The PCR 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. The PCR system consisted of: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 sec, 58℃ annealing for 30 s, 72℃ annealing for 1 min, and 72℃ final extension for 5 min, with the denaturation-annealing process repeated 30 times.

[0035] Next, the PCR products were subjected to gel electrophoresis, and the target band was recovered and purified according to the instructions of the agarose gel electrophoresis kit (Meiji Biotechnology, China). Then, using the Clone Express II One Step Cloning Kit (Novizan, China), the fragment was inserted into the Sam I restriction site of the linearized pTF101s plasmid, transformed into competent E. coli cells, and after sequencing confirmation, the GmCXIP4-OX plasmid was extracted and transformed into Agrobacterium tumefaciens EHA101 to obtain the overexpression vector pTF101s-GmCXIP4-OX. A schematic diagram of the GmCXIP4 overexpression vector structure is shown below. Figure 1 As shown in Figure A.

[0036] 2. Construction of the knockout vector pGES201-GmCXIP4-KO:

[0037] First, the target site of GmCXIP4, namely sgRNA, was designed using the website TargetDesign (http: / / skl.scau.edu.cn / ta-rgetdesign / ). Then, Qingke Biotechnology Co., Ltd. was commissioned to synthesize the forward primer GmCXIP4-sgRNA-F (SEQ ID NO:5:5'-GCTGAGGTGGATGTATCCCG-3') and the reverse primer GmCXIP4-sgRNA-R (SEQ ID NO:6:5'-CGGGATACATCCACCTCAGC-3') oligonucleotide chains, which were then annealed to double strands. The annealing system (50 μL) consisted of: 5 μL each of the forward and reverse primers (GmCXIP4-sgRNA-F / R, 10 μM), 5 μL of NaCl solution (final concentration 100 mM), 5 μL of Tris-HCl solution (pH 7.4, final concentration 50 mM), and ddH2O was added to make up to 50 μL. The annealing procedure is as follows: 95℃ for 4 min, RAMP 0.1℃ / s -95℃ gradient cooling to 16℃, and store at 16℃.

[0038] Then, the annealed double-linked vector was inserted into the Bsa I restriction site of the linearized pGES201z vector, transformed into competent E. coli cells, and after sequencing confirmed to be correct, the GmCXIP4-KO plasmid was extracted and transformed into Agrobacterium tumefaciens EHA105 to obtain the knockout vector pGES201-GmCXIP4-KO. The structural diagrams of the GmCXIP4 knockout vector and target sequence are shown below. Figure 1 As shown in E.

[0039] 3. Construction of the GmCXIP4-pEGAD-GFP expression vector fused with GmCXIP4

[0040] Based on the GmCXIP4 gene, specific primers (SEQ ID NO:7:5'-CTCTAGCGCTACCGGTATGCCAGCTACAGCAGGAAG-3', SEQ ID NO:8:5'-CATGGTGGCGACCGGTGCTTCATCATCCCCATGGTGAC-3') were designed and synthesized to amplify the gene promoter sequence. Using soybean YC03-3 genomic DNA as a template, the OFR fragment of the GmCXIP4 gene was amplified using the GmCXIP4-GFP-F / R primers. The OFR fragment of the GmCXIP4 gene was amplified using Phanta Max Super-Fidelity DNA Polymerase (Novizan, China). The amplification reaction system (50 μL) consisted of: 25 μL 2×phata max buffer, 2 μL each of forward and reverse primers (10 mM), 1 μL 2 mM dNTPs, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 2 μL DNA template, 2 μL 50 mM MgCl2, and 16 μL ddH2O. The PCR system was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 sec, 58℃ annealing for 30 s, 72℃ annealing for 2 min, and 72℃ final extension for 5 min, with the denaturation-annealing process repeated 30 times.

[0041] After PCR products were subjected to gel electrophoresis, the target bands were recovered and purified according to the instructions of the agarose gel electrophoresis kit (Meiji Biotechnology, China). Next, using the Clone Express II One Step Cloning Kit (Novizan, China), the fragment was inserted into the Age I restriction site of the linearized pEGAD plasmid, transformed into competent E. coli cells, and after sequencing confirmed to be correct, the GmCXIP4-GFP plasmid was extracted and transformed into Agrobacterium GV3101 to obtain the GmCXIP4-pEGAD-GFP fusion expression vector.

[0042] Example 2: Research on Transgenic Materials

[0043] 1. Obtaining transgenic soybean whole plant material

[0044] (1) Seed germination: Select soybean YC03-3 seeds and sterilize them in chlorine (100mL sodium hypochlorite + 4.2mL hydrochloric acid) for 12-14h. After sterilization, place the seeds in a clean bench and blow them for 30-60min. After that, sow the seeds in MS germination medium and place them in a 28℃ constant temperature and light incubator for 4-5d.

[0045] (2) Preparation of bacterial culture: Agrobacterium EHA101 containing the GmCXIP4-OX plasmid or Agrobacterium EHA105 containing the GmCXIP4-KO plasmid were inoculated into 50 mL of YEP liquid medium and cultured in a shaker at 28 °C for 12–14 h. The colonies were collected by centrifugation at 6000 rpm and resuspended in CM liquid to OD. 650 It is around 1.0.

[0046] (3) Cotyledon node infection and co-culture: Use a sterile scalpel to cut off the cotyledon 5 cm away from the hypocotyl, cut the cotyledon vertically and remove the young shoots; make 7-8 wounds at the cotyledon node with a scalpel, and soak in CM liquid suspension for 30 min; finally, use forceps to transfer the explant to the co-culture solid medium (CM solid medium), with the cut side down, seal the culture dish with airtight plastic wrap, transfer to a 24℃ constant temperature incubator, and culture in the dark for 3 days.

[0047] (4) Shoot induction: Explants cultured in the dark for 3 days were transferred to liquid shoot induction SI medium and washed for 2-3 minutes. Then, the explants were transferred to SI solid medium containing herbicides and antibiotics. The hypocotyl and cotyledons of the explants were inserted into the medium, and the culture dish was sealed with medical breathable tape. The explants were then transferred to a 24℃ constant temperature culture room for two weeks of light culture (18h light / 6h darkness). After two weeks, the elongated hypocotyl was removed and the explants were transferred to fresh SI solid medium and cultured for another two weeks under the same conditions.

[0048] (5) Sprout elongation: Remove the cotyledons of the differentiated explants and transfer them to SE solid medium for sprout induction, and culture for 2 to 8 weeks. Change the SE solid medium every two weeks during this period.

[0049] (6) Inducing rooting: When the regenerated buds grow to about 3-5cm, cut them off from the explant and transfer them to the rooting RM medium for further culture. About two weeks later, when the roots growing on the stem are larger than 3cm, remove the regenerated plant from the RM culture and transplant it into a hydroponic bottle for further culture. Four weeks later, transfer it to a greenhouse and cultivate it in soil until pods form.

[0050] 2. Identification of transgenic soybean whole plant materials

[0051] (1) Identification of transgenic lines overexpressing GmCXIP4-OX:

[0052] Transgenic plants were obtained by infecting soybean cotyledonary nodes with Agrobacterium tumefaciens. Transgenic soybeans were then identified using three methods: herbicide resistance testing, transgenic soybean PCR detection, and quantitative PCR detection. Young trifoliate leaves were selected, and half of each leaf was evenly coated with herbicide and marked. The leaf reaction was observed after 2–3 days. If there was no significant change between the herbicide-coated and uncoated leaves, the plant was likely a positive result. Plant DNA was then extracted and used for further PCR verification.

[0053] Herbicide resistance screening results as follows Figure 1 As shown in Figure B, the leaves of wild-type (WT) plants turned yellow and withered after being treated with herbicides, while the leaves of transgenic lines showed no obvious changes.

[0054] Using extracted wild-type and transgenic plant DNA as templates, gene fragments were amplified by pairwise combinations of the forward and reverse primers pTF101s-JC-F / R (SEQ ID NO:11:5'-CATTTGGAGAGGACACGC-3', SEQ ID NO:12:5'-CAACACATGAGCGAAACC-3') for the pTF101s vector and the gene primers GmCXIP4-OX-F / R (SEQ ID NO:3-4) for GmCXIP4. If a gene fragment of approximately 950 bp can be amplified, the result is as follows: Figure 1 As shown in Figure C, the DNA of the transgenic line can be amplified to a fragment of approximately 950 bp, while the wild-type DNA does not show any amplification bands. Further verification will be conducted.

[0055] Finally, total RNA was extracted from wild-type and transgenic plants to be tested, reversed into cDNA, and the expression level of GmCXIP4 was detected by real-time PCR. If the expression level of GmCXIP4 was significantly upregulated compared with wild-type, it was a transgenic line with overdose effect.

[0056] qRT-PCR results are as follows Figure 1 As shown in Figure D, the expression levels of GmCXIP4 in the two overexpressed transgenic lines OX1 and OX2 were significantly increased compared with those in the WT line; these results indicate that the overexpression of GmCXIP4 in transgenic lines is significant.

[0057] (2) Identification of GmCXIP4 knockout transgenic lines:

[0058] Transgenic soybeans were identified sequentially using three methods: herbicide resistance testing, PCR testing, and Sanger sequencing. The herbicide resistance testing method was the same as above, and positive plants were then used for further PCR verification.

[0059] Using extracted DNA from wild-type and transgenic plants as templates, fragments were amplified using the pGES201 vector's Crispr-F and the GmCXIP4-sgRNA-R target primers. If a gene fragment of approximately 500 bp was amplified, it indicated that the vector had not off-target, and further verification was performed. DNA from the transgenic plants was extracted as a template, and fragments were amplified using the GmCXIP4-Cas9-F / R primers (SEQ ID NO:9:5'-TCAAGATGCCAGCTACAGCAGG-3', SEQ ID NO:10:5'-TCTCTCCATCTCTGAATCAATC-3'). The amplified products were sequenced using the Sanger sequencing method to verify the editing effect. If a base sequence insertion or deletion resulted in a sense mutation in an amino acid, the gene knockout transgenic line was considered successfully edited.

[0060] Sequencing results as follows Figure 1 As shown in F, the sequencing peak diagram clearly shows a single base peak, and the KO1 and KO2 lines have deletions of 1bp and 5bp, respectively, both of which lead to frameshifts and premature termination of the GmCXIP4 protein amino acid sequence. Figure 1 G). These results confirm the successful acquisition of homozygous transgenic lines with the GmCXIP4 gene knockout.

[0061] Example 3: Functional Analysis of GmCXIP4

[0062] 1. Subcellular localization of GmCXIP4 in rice protoplasts

[0063] The 35S::GmCXIP4-GFP vector, fused with GFP, was constructed using the same method as in Example 1 and transformed into rice protoplasts for subcellular localization analysis of GmCXIP4. The results are as follows: Figure 2 As shown, GFP green fluorescence signal and mcherry are mainly present in the cell nucleus in rice protoplasts, which also indicates that GmCXIP4 is mainly located in the cell nucleus.

[0064] 2. Effects of different phosphorus concentrations on GmCXIP4 overexpression on soybean growth

[0065] The effects of GmCXIP4 overexpression on soybean growth under different phosphorus levels were investigated. Sand-based seedling cultivation was employed: seeds of uniformly sized wild-type soybean (WT) and two overexpressing (GmCXIP4-OX1, GmCXIP4-OX2) T3 generation transgenic lines were selected and sown with the hilum facing down in quartz sand moistened with half a soybean hydroponic nutrient solution at a depth of approximately 2 cm. Four days after germination, seedlings were transplanted into a nutrient solution for further cultivation. Two phosphorus levels were established: a high-phosphorus treatment with a KH2PO4 concentration of 250 μM KH2PO4 and a low-phosphorus treatment with a KH2PO4 concentration of 5 μM KH2PO4. After 14 days of treatment, the plants were harvested, and plant biomass and root length were measured.

[0066] The measurement results are as follows Figure 3 As shown, under phosphorus-sufficient (+P) conditions, overexpression of GmCXIP4 inhibited plant growth. Figure 3 AG). Under +P conditions, compared with the wild type (WT), the aboveground dry weight of the two overexpression lines OX1 and OX2 decreased by 13.7% and 17.6%, respectively; root dry weight decreased by 17.9% and 18.9%, respectively; total root length decreased by 20.4% and 23.5%, respectively; lateral root length decreased by 21.4% and 24.8%, respectively; aboveground phosphorus content decreased by 29.1% and 31.2%, respectively; and root phosphorus content decreased by 24.2% and 22.5%, respectively. Figure 3 B). Under -P conditions, the aboveground dry weight, root dry weight, total root length, lateral root length, aboveground phosphorus content, and root phosphorus content of the transgenic lines were not different from those of the WT line.

[0067] 3. Effects of different phosphorus concentrations on GmCXIP4 knockout on soybean growth

[0068] The effects of GmCXIP4 knockout on soybean growth under different phosphorus levels were investigated. Sand-based seedling cultivation was employed: seeds of uniformly sized wild-type soybeans (WT) and two knockout expression (GmCXIP4-KO1, GmCXIP4-KO2) T3 generation transgenic lines were selected and sown with the hilum facing down in quartz sand moistened with half a soybean hydroponic nutrient solution at a depth of approximately 2 cm. Four days after germination, seedlings were transplanted into a nutrient solution for further cultivation. Two phosphorus levels were established: a high-phosphorus treatment with a KH2PO4 concentration of 250 μM KH2PO4 and a low-phosphorus treatment with a KH2PO4 concentration of 5 μM KH2PO4. After 14 days of treatment, the plants were harvested, and plant biomass and root length were measured.

[0069] The measurement results are as follows Figure 4 As shown, under phosphorus-sufficient (+P) and phosphorus-deficient (-P) conditions, knockout of GmCXIP4 promoted plant growth. Figure 4A). Under +P conditions, compared with the wild type (WT), the aboveground dry weight of the two knockout lines KO1 and KO2 increased by 20.6% and 18.8%, respectively; root dry weight increased by 27.2% and 19.8%, respectively; total root length increased by 30.0% and 27.6%, respectively; lateral root length increased by 29.2% and 26.4%, respectively; aboveground phosphorus content increased by 28.5% and 24.9%, respectively; and root phosphorus content increased by 34.6% and 31.9%, respectively. Figure 4 Under -P conditions, compared with the wild type (WT), the aboveground dry weight of the two knockout lines KO1 and KO2 increased by 16.4% and 27.7%, respectively; root dry weight increased by 25.0% and 30.7%, respectively; total root length increased by 25.3% and 20.6%, respectively; lateral root length increased by 26.3% and 21.2%, respectively; aboveground phosphorus content increased by 65.4% and 75.3%, respectively; and root phosphorus content increased by 24.6% and 27.8%, respectively.

[0070] In summary, the GmCXIP4 gene negatively regulates phosphorus nutrition and plant growth, belonging to a key regulatory gene in the phosphorus signaling network. Through the construction and identification of overexpression and knockout vector materials, it was shown that overexpression of the GmCXIP4 gene inhibits plant growth and reduces phosphorus uptake and accumulation; conversely, knockout of the GmCXIP4 gene significantly promotes plant growth, increases phosphorus uptake and accumulation, and increases phosphorus content. Even under low phosphorus stress, it also promotes plant growth and phosphorus uptake. Therefore, the GmCXIP4 gene, as a novel gene resource regulating plant growth and phosphorus nutrition, plays an important role in improving plant phosphorus nutrition and promoting plant growth, providing a crucial gene resource for breeding low-phosphorus-tolerant plants.

[0071] 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 GmCXIP4 gene, as shown in SEQ ID NO:1, in the negative regulation of plant growth and / or phosphorus nutrient uptake.

2. Application of the GmCXIP4 gene, as shown in SEQ ID NO:1, as a target in the cultivation of plants tolerant to low phosphorus stress or plants with high phosphorus content.

3. Application of preparations containing the GmCXIP4 gene (as shown in SEQ ID NO:1) that are missing or knocked out in promoting plant growth or in promoting plant growth under low phosphorus conditions.

4. Application of preparations containing the GmCXIP4 gene (as shown in SEQ ID NO:1) that lack or knock out the phosphorus content of plants or in the preparation of products that increase the phosphorus content of plants.

5. Application of formulations containing the GmCXIP4 gene (as shown in SEQ ID NO:1) that are missing or knocked out in the preparation of products that promote plant growth or improve plant tolerance to low phosphorus stress.

6. Application of formulations containing the GmCXIP4 gene (as shown in SEQ ID NO:1) that are missing or knocked out in the cultivation of plants tolerant to low phosphorus stress and / or plants with high phosphorus content.

7. Application of preparations containing the GmCXIP4 gene (as shown in SEQ ID NO:1) that are missing or knocked out in promoting phosphorus uptake in plants.

8. The application according to any one of claims 3 to 7, characterized in that, The preparation is a plasmid, vector, or recombinant bacteria that has the GmCXIP4 gene deleted or knocked out as shown in SEQ ID NO.

1.

9. A method for promoting plant growth and / or increasing plant phosphorus content, characterized in that, The GmCXIP4 gene in a plant is deleted or knocked out, or the plant is treated with an agent that deletes or knocks out the GmCXIP4 gene as shown in SEQ ID NO:1; the sequence of the GmCXIP4 gene is shown in SEQ ID NO:

1.

10. A method for cultivating plants tolerant to low phosphorus stress and / or high phosphorus content, characterized in that, The GmCXIP4 gene in a plant is deleted or knocked out, or a recombinant expression vector with the GmCXIP4 gene knocked out is transferred into a plant to obtain plants tolerant to low phosphorus stress and / or high phosphorus content; the sequence of the GmCXIP4 gene is shown in SEQ ID NO:1.

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