Plant low-phosphorus tolerance important gene gmcxip1-1 and application thereof

Abstract

The application discloses a plant low-phosphorus tolerance important gene GmCXIP1-1 and application thereof. The application first clones a GmCXIP1-1 gene of a CXIP family participating in cation transport in a plant body in soybean, and the nucleotide sequence of the GmCXIP1-1 gene is shown as SEQ ID NO:1. Research shows that the GmCXIP1-1 gene is induced to express up-regulation under low-phosphorus stress, under different phosphorus concentration treatment conditions, overexpression of the GmCXIP1-1 can obviously increase the biomass of a transgenic plant, and promote the growth of the plant under low-phosphorus conditions; meanwhile, overexpression of the GmCXIP1-1 can improve the tolerance of the plant to the low-phosphorus stress, and reduce the inhibition of low phosphorus on the growth of the root system of the plant. It is shown that the GmCXIP1-1 has an important role in the adaptation of the plant to low-phosphorus stress, and can improve the adaptation of the plant to low-phosphorus stress in acid soil through a transgenic technology.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. More specifically, it relates to an important plant gene for low phosphorus tolerance, GmCXIP1-1, and its applications. Background Technology

[0002] Soybean (Glycine max) is an important legume crop, a major source of protein and oil in animal feed, and a high-protein dual-purpose crop for both grain and feed. It has a long history of cultivation in my country and is widely planted. As one of the world's most widely cultivated crops, soybean is a crucial source of plant protein and oil for humans and a primary raw material for biodiesel production. With the continuous reduction of arable land nationwide, the soybean planting area has decreased significantly, while demand has increased substantially, leading to a supply shortage in China and a large influx of high-quality, inexpensive imported soybeans into the domestic market. Therefore, vigorously developing soybean cultivation is a crucial measure to address my country's soybean supply shortage.

[0003] Southern my country has abundant water and heat resources, suitable for soybean production. However, most soils in the south are acidic, and in acidic soils, low phosphorus stress severely limits soybean yield (Zhang and Xu, 2005; Wang et al., 2010; Liu et al., 2020). Applied phosphate fertilizers are easily fixed by iron and aluminum ions in the soil, forming insoluble phosphorus. Although the soil stores a large amount of phosphorus, the amount of available phosphorus that can be directly absorbed and utilized by plants is far from meeting the needs of normal soybean growth and development. Low phosphorus availability in acidic soils has become one of the important reasons limiting soybean production (Yan et al., 2000; Zhang et al., 2005; Wang et al., 2010; Ham et al., 2018). Therefore, improving the adaptability of soybeans to low phosphorus stress in acidic soils and promoting soybean production is of great significance to the sustainable development of my country's agriculture and economy.

[0004] Calcium (Ca 2+ Ca is a key cation and major nutrient element essential for plant growth and development, and also an important second messenger in cell signal transduction. 2+ The CAX gene family maintains homeostasis in the cytoplasm and vacuoles through various ion import and export mechanisms. 2+ Members of the cation antitransporter (CaCA) superfamily can transport metal cations such as Ca, Mn, Zn, and Cd, enhancing plant drought resistance through vacuolar membranes. et al., 2001; Cheng et al., 2002; Koren'kov et al., 2007; Socha and Guerinot, 2014; Yamada et al., 2014). The CXIP gene family is a group of genes that interact with the n-terminus of CAX to regulate the conversion of CAX to Ca2+. 2+ The CXIP gene is a unique transport protein, and research on it is currently limited, with only CXIP1 and CXIP4 studied in the model plant Arabidopsis thaliana (Cheng and Hirschi, 2003; Cheng et al., 2004). Currently, the CXIP gene has not been cloned and reported in any other plant species, and its specific functions and roles are unknown. Furthermore, it is unclear whether the CXIP gene is involved in the mechanisms of phosphorus stress in plants, and there are few reports on its analysis in soybean. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing CXIP genes and provide an important gene for plant low phosphorus tolerance, GmCXIP1-1, and its application.

[0006] The first objective of this invention is to provide a GmCXIP1-1 gene for plant tolerance to low phosphorus stress.

[0007] A second objective of this invention is to provide the protein encoded by the GmCXIP1-1 gene.

[0008] A third object of the present invention is to provide the application of the GmCXIP1-1 gene or an expression promoter thereof, or the protein encoded by claim 2.

[0009] The fourth objective of this invention is to provide a recombinant expression vector.

[0010] The fifth objective of this invention is to provide a genetically engineered bacterium.

[0011] The sixth objective of this invention is to provide a formulation that promotes plant growth under low phosphorus stress or enhances the ability of plant roots to tolerate low phosphorus stress.

[0012] A seventh objective of this invention is to provide a method for promoting plant root growth and / or improving plant tolerance to low phosphorus stress.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution:

[0014] This invention, using real-time quantitative PCR and homologous cloning, for the first time cloned the GmCXIP1-1 gene, a member of the CXIP family involved in cation transport (related to the CAX gene family), from soybean. Its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the GmCXIP1-1 gene is shown in SEQ ID NO:2. This study found that GmCXIP1-1 expression was significantly upregulated at the transcriptional level under low phosphorus stress, and its expression level increased significantly with prolonged phosphorus treatment time. Furthermore, transgenic Arabidopsis plants expressing excessive amounts of GmCXIP1-1 were obtained using Arabidopsis transgenic technology, demonstrating that the GmCXIP1-1 gene has the function of regulating soybean root adaptation to low phosphorus stress; simultaneously, it can reduce the inhibitory effect of low phosphorus stress on plant root growth.

[0015] Therefore, the present invention provides the following applications of the GmCXIP1-1 gene shown in SEQ ID NO:1 or its expression promoter, or the GmCXIP1-1 protein shown in SEQ ID NO:2:

[0016] Applications in promoting plant growth under low phosphorus stress or in the preparation of formulations that promote plant growth under low phosphorus stress.

[0017] Applications in reducing the inhibitory effect of low phosphorus stress on plant root growth.

[0018] Application in improving the ability of plant roots to tolerate low phosphorus stress.

[0019] Application in cultivating low-phosphorus tolerant plants.

[0020] This invention provides a recombinant expression vector containing the above-mentioned GmCXIP1-1 gene.

[0021] This invention provides a genetically engineered bacterium containing the above-mentioned recombinant expression vector.

[0022] The present invention also provides a preparation for promoting plant growth under low phosphorus stress or a preparation for improving the ability of plant roots to tolerate low phosphorus stress, containing the GmCXIP1-1 gene or its expression promoter.

[0023] The present invention also provides a method for promoting plant root growth and / or improving plant tolerance to low phosphorus stress by overexpressing the GmCXIP1-1 gene in plants.

[0024] Preferably, gene editing technology is used to positively regulate the expression level or protein activity of the GmCXIP1-1 gene in plants to promote root growth and / or improve plant tolerance to low phosphorus stress.

[0025] Preferably, an expression vector for overexpressing the GmCXIP1-1 gene is constructed, and plants are transformed to obtain transgenic plants that promote root growth and / or improve plant tolerance to low phosphorus stress.

[0026] The present invention has the following beneficial effects:

[0027] This invention discloses an important low-phosphorus tolerance gene, GmCXIP1-1, and its applications. This invention is the first to clone a CXIP family gene, GmCXIP1-1, involved in cation transport within plants, from soybean. Studies show that the expression of the GmCXIP1-1 gene is upregulated under low-phosphorus stress, and its expression level increases significantly with prolonged phosphorus treatment time. Under different phosphorus concentrations, overexpression of GmCXIP1-1 significantly increases the biomass of transgenic plants and promotes plant growth under low-phosphorus conditions. Simultaneously, overexpression of GmCXIP1-1 enhances the plant's tolerance to low-phosphorus stress and reduces the inhibitory effect of low phosphorus on root growth.

[0028] This invention discloses the application of the soybean GmCXIP1-1 gene in regulating plant adaptation to low phosphorus stress and promoting growth. It shows that the soybean CXIP gene is involved in the relevant mechanism of plant regulation of soil phosphorus stress. GmCXIP1-1 positively regulates the ability of plant roots to adapt to low phosphorus, indicating that GmCXIP1-1 plays an important role in plant adaptation to low phosphorus stress and can improve the plant's ability to adapt to low phosphorus stress in acidic soil through transgenic technology. Attached Figure Description

[0029] Figure 1 Analysis of GmCXIP1-1 expression pattern in soybean (Effect of low phosphorus treatment time on GmCXIP1-1 expression pattern in soybean roots; data are the mean and standard error of three replicates, asterisk indicates significant difference between control (+P) and treatment (-P) (Student's t-test), *: P<0.05, **: P<0.01, ***: P<0.001);

[0030] Figure 2Subcellular localization analysis of GmCXIP1-1 (A: Subcellular localization analysis results of GmCXIP1-1 fusion GFP protein in tobacco leaves; B: Subcellular localization analysis results of GmCXIP1-1 fusion GFP protein in onion epidermal cells; The first row of Figures A and B shows the subcellular localization map (35S::GFP) in tobacco or onion epidermal cells transformed with empty vector; the second row shows the subcellular localization map (35S::GmCXIP1-1-GFP) of GmCXIP1-1 fusion GFP protein in tobacco leaves or onion epidermal cells; Images A and B are observed under a laser confocal microscope using the green fluorescence channel (GFP), light microscopy channel (bright field), and the superimposed image (fusion), respectively, with a scale bar of 20 μm).

[0031] Figure 3 Identification of transgenic Arabidopsis plants overexpressing GmCXIP1-1 (A: GmCXIP1-1 PCR detection; B: GmCXIP1-1 expression level detection in leaves of different lines; WT: wild-type line; OX1 and OX2: two different transgenic soybean lines overexpressing GmCXIP1-1; data in the figure are the mean and standard error of four replicates; "*" indicates significant difference compared with WT (Student's t-test, P<0.05)).

[0032] Figure 4 The effects of high and low phosphorus treatments on the growth of Arabidopsis thaliana seedlings overexpressing GmCXIP1-1 were shown in the figure. (A: Phenotypic characteristics of wild-type (WT) and transgenic Arabidopsis thaliana lines (OX1, OX2) under high and low phosphorus treatments; scale bar in the figure is 1 cm; B: Number of lateral roots of wild-type (WT) and transgenic Arabidopsis thaliana lines (OX1, OX2) under high and low phosphorus treatments; C: Root growth of wild-type (WT) and transgenic Arabidopsis thaliana lines (OX1, OX2) under high and low phosphorus treatments; D: Fresh weight of aboveground parts of wild-type (WT) and transgenic Arabidopsis thaliana lines (OX1, OX2) under high and low phosphorus treatments; E: Fresh weight of roots of wild-type (WT) and transgenic Arabidopsis thaliana lines (OX1, OX2) under high and low phosphorus treatments; asterisks indicate significant differences between wild-type (WT) and transgenic Arabidopsis thaliana lines (OX1, OX2). (Student's...) t-test), *: P<0.05, **: P<0.01, ***: P<0.001). Detailed Implementation

[0033] 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.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] The soybean (Glycine max(L.)Merr.) material used in the following experiments was Yuechun 03-3 (YC03-3), which was obtained from the Root Biology Research Center of South China Agricultural University.

[0036] Example 1: Analysis of GmCXIP1-1 gene expression pattern

[0037] This invention marks the first cloning of a GmCXIP1-1 gene from soybean, belonging to the CXIP family and involved in cation transport within plants (associated with the CAX gene family). Its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the GmCXIP1-1 gene is shown in SEQ ID NO:2. This invention reveals that GmCXIP1-1 expression is regulated by low phosphorus stress at the transcriptional level. Further analysis is conducted to investigate the specific function of the GmCXIP1-1 gene in the synergistic response of soybean to low phosphorus stress in acidic soil.

[0038] 1. Plant samples

[0039] Soybean seeds of uniform size and without damaged seed coats were selected using the paper roll seedling method. The seeds were disinfected for 12 hours with chlorine gas generated from the reaction of 100 mL sodium hypochlorite and 4.2 mL hydrochloric acid, and then blown through a clean bench for 1 hour before use. Square filter paper of 20×20 cm was cut, and a 1 / 4 soybean complete nutrient solution with a pH of 5.8 was prepared (for specific formula, see: Liu Guoxuan, Chen Kang, Lu Xing, Tian Jiang & Liang Cuiyue. (2021). Functional study of soybean GmPIN2b regulating root response to low phosphorus stress. Journal of South China Agricultural University (04), 33-41.) and sterile water were also prepared and sterilized for later use.

[0040] 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.

[0041] Select seedlings with uniform growth and transfer them to nutrient solutions with high and low phosphorus treatments: +P (250 μM KH2PO4) and -P (5 μM KH2PO4). Each treatment was replicated in four batches, 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. Root samples were harvested at 6, 12, and 18 days, frozen in liquid nitrogen, and stored at -80°C for later use.

[0042] 2. Real-time quantitative PCR (qRT-PCR) analysis

[0043] Total RNA was extracted from the treated plant samples using a TRIzol kit (Invitrogen, USA). The DNase I-treated RNA was reverse transcribed into cDNA using an MMLV-Reverse Transcription Kit (Promega, USA). qRT-PCR analysis was then performed using a SYBR (Promega, USA) kit. After reverse transcription, the samples were diluted 15-fold and analyzed using an Applied Biosystems StepOnePlus Real-Time PCR system.

[0044] 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, dilute the mixture 10 times to get the first standard S1. Then, dilute the first standard 10 times to get the second standard S2. And so on, to get 5 concentration gradient standards, namely S1, S2, S3, S4 and S5.

[0045] The quantitative PCR primers used were the GmCXIP1-1 gene quantitative primers and the soybean housekeeping gene primers for the internal reference gene EF1-α, as shown below:

[0046] GmCXIP1-1-RT1-F: 5'-CAAACTCAAAACCGCTTCCA-3';

[0047] GmCXIP1-1-RT1-R: 5'-TACTCTTTCAGCCCTTGGCG-3';

[0048] EF1-α-F: 5'-TGCAAAGGAGGCTGCTAACT-3';

[0049] EF1-α-R: 5'-CAGCATCACCGTTCTTCAAA-3'.

[0050] Preparation of the reaction mixture: In every 20 μL reaction system, add 10 μL of SYBR Premix Ex Taq (2×), 0.5 μL of forward / reverse primers, 7 μL of ddH2O, and 2 μL of template. First calculate the required reaction volume, mix all reagents except cDNA, dispense 18 μL into each tube, and then add 2 μL of cDNA template to make a final volume of 20 μL.

[0051] The quantitative PCR reaction program was as follows: 95℃ pre-denaturation for 30 seconds, PCR reaction (95℃ denaturation for 30 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 30 seconds) for 40 cycles. The expression level of each sample was calculated using Rotor-Gene's Real-Time Analysis Software.

[0052] Test results as follows Figure 1 As shown, under high and low phosphorus treatment conditions, compared with high phosphorus treatment, the expression level of GmCXIP1-1 gene under low phosphorus treatment for 6d was significantly upregulated by low phosphorus stress, and its expression level increased significantly with the extension of phosphorus treatment time, and decreased by day 18.

[0053] Example 2: Subcellular localization analysis of GmCXIP1-1

[0054] Based on the GmCXIP1-1 gene sequence, specific primers were designed: GmCXIP1-1-GFP-F: 5'-CAGACCTTAATTAAGAGCT CATGTCGTGGTGTTACTGCGCTA-3', and GmCXIP1-1-GF PR: 5'-CATGGTGGCGACCGGTCGTCAGGACAACATTGCCTTCTCC-3'. The full-length ORF of the soybean GmCXIP1-1 gene was amplified using root cDNA of soybean genotype YC03-3 as a template. The reaction conditions were: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ annealing for 1–3 min, repeated 30 times, followed by 72℃ extension for 10 min.

[0055] The PCR amplification products were purified by gel electrophoresis using a kit. After obtaining the purified PCR products, a homologous recombination kit was used. II. The PCR product was recombinantly ligated with the linearized vector pEGAD, digested with Age I. The reaction mixture was 20 μL, containing 6 μL of PCR product, 8 μL of pEGAD linearized vector plasmid, 2 μL of recombinant ligase Exnase II, and 4 μL of reaction buffer. The reagent mixture was incubated at 37°C for 30 min. The recombinant plasmid was transformed into *E. coli* and sequenced. After confirming the sequence was correct, the plasmid was extracted. The 35S::GmCXIP1-1-GFP plasmid was transformed into *Agrobacterium tumefaciens* GV3101 and *Agrobacterium rhizogenes* K599. After confirming the sequence was correct, the plasmid was stored for later use.

[0056] Subcellular localization analysis of transient expression in tobacco epidermal cells was performed using Agrobacterium-mediated transformation. The vector 35S::GmCXIP1-1-GFP or the pEGAD empty vector (35S:GFP) was introduced into Agrobacterium strain GV3101. After successful transformation, GV3101 cells containing the vector were inoculated into YEP medium and cultured at 28°C with shaking for 16 h. The cells were then centrifuged (5000 rpm, 10 min) and resuspended in infiltration buffer (containing 10 mM MgCl2, 10 mM MES, and 10 mM acetylsyleugenol, pH 5.6) to OD200. 600 The spectrophotometric value was 0.4–0.5. After the bacterial suspension was incubated at 43°C in the dark for 3 hours, it was used to transform the lower epidermis of tobacco leaves from 5–6 weeks old by injecting the bacterial solution with a syringe. After the transformed tobacco was cultured normally for 3 days, the distribution of fluorescence signals in the tobacco epidermal cells was observed using a laser copolymer scanning microscope (Zeiss LSM780, Germany).

[0057] For subcellular localization of onion epidermal cells within onion scales, the OD value of the bacterial culture was adjusted to 0.6. The onion epidermal cells were pre-cultured on MS solid medium for 24 hours, and then immersed in the bacterial culture for 4–6 hours during transformation. After transformation, the onions were cultured for 48 hours and 24 hours, respectively, and the subcellular localization was observed under a laser confocal microscope (Zeiss LSM780, Germany). Plasmolysis was performed using 30% sucrose solution and 7% sodium chloride solution, respectively. The excitation wavelength of the laser confocal microscope was 488 nm.

[0058] The results are as follows Figure 2 As shown, after transient expression of the 35S::GmCXIP1-1-GFP vector in tobacco leaf epidermal cells, the empty vector control (35S::GFP) showed green fluorescence of GFP in the nucleus, cytoplasm, and plasma membrane, while the 35S::GmCXIP1-1-GFP showed strong GFP fluorescence at the cell edge, coinciding with the cell outline, indicating that GmCXIP1-1 may be localized on the cell wall. Figure 2 A). Similar results were observed in onion inner epidermal cells, where a strong GFP fluorescence signal was observed on their cell walls. Figure 2B). These results demonstrate that the GmCXIP1-1 protein is located in the cell wall and is a cell wall-binding protein. Example 3: Effects of high and low phosphorus treatments on the growth of Arabidopsis seedlings overexpressing GmCXIP1-1.

[0059] 1. Construction of the GmCXIP1-1 overexpression vector (OX-GmCXIP1-1-pTF101s)

[0060] Using soybean genotype YC03-3 root cDNA as a template, GmCXIP1-1 specific primers were designed. The coding region of GmCXIP1-1 was amplified using the upstream specific primer: 5'-CCGGGGATCCTCTAGAATGTCGTGGTGTTACTGCGCT A-3' and the downstream specific primer: 5'-GCAGGTCGACTCTAGATCAGGACAACATTGCCT TCTCC-3'. The PCR reaction system consisted of a total of 50 μL, including 43 μL Master mix (2×), 0.5 μL each of the forward and reverse primers, 2 μL cDNA template, 1 μL dNTPs (10 μM), and 1 μL high-fidelity enzyme, with the remainder made up with water. The PCR program was as follows: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ annealing for 1–3 min, repeated 30 times, followed by a 72℃ extension for 10 min. After confirming the PCR fragment recovery and sequencing were correct, the amplified G mCXIP1-1 CDS fragment was processed using a homologous recombination kit. II kits were ligated into the linearized vector pTF101s digested with XbaI enzyme. The resulting recombinant vector was transformed into E. coli and sequenced for verification. After confirming that the sequencing results were correct, the recombinant plasmid 35S::GmCXIP1-1 was extracted and transformed into Agrobacterium rhizogenes K599 and Agrobacterium GV3101 for later use.

[0061] 2. Obtaining transgenic Arabidopsis thaliana

[0062] Using the inflorescence infection method, the constructed OX-GmCXIP1-1-pTF101s vector plasmid was transformed into Agrobacterium GV3101. Positive clones were picked and cultured in 5 mL of YEP medium (containing spectinomycin and rifampin) overnight at 28°C; then, they were transferred to 100 mL of YEP medium for expansion culture to OD. 600The concentration was 1.6–2.0; then centrifuged at 6000 rpm for 10 min, the supernatant was discarded and the bacterial cells were collected. The bacterial cells were resuspended in an equal volume of 5% sucrose water or 1 / 2 MS culture medium, and 0.005–0.02% Silwet L-77 was added to prepare the transformation solution. During transformation, all Arabidopsis thaliana inflorescences (watered the day before transformation to keep the plants moist) were immersed in the transformation solution for 1 min. After removal, excess transformation solution was wiped off with filter paper, and then the plants were covered with plastic wrap to keep them moist. They were first covered with a black bag and cultured in the dark for 18 h, and then transferred to normal culture conditions until seed harvest (transformed once every week during the culture period, for a total of 3 transformations).

[0063] After harvesting T0 generation seeds from the transformed Arabidopsis thaliana, approximately 100 μL of seeds were taken for propagation identification. The specific steps are as follows: The entire operation is carried out in a clean bench. First, rinse with 70% ethanol for 1 minute, centrifuge to remove the ethanol, and then rinse once with sterile secondary water. Next, pre-wash with 10% sodium hypochlorite, centrifuge to remove the sodium hypochlorite, then rinse with 1 mL of 10% sodium hypochlorite for 5 minutes with shaking, centrifuge to remove the sodium hypochlorite, and rinse 5-6 times with sterile water. Finally, resuspend the seeds in sterile water and sow them evenly on MS medium containing herbicide. After low temperature treatment at 4℃ for 1 day, dormancy is broken, and the seeds are transferred to a plant light incubator with a photoperiod of 16h / 8h (light / dark) and a temperature of 22℃ / 20℃ (day / night). About 2 weeks later, the T1 generation seedlings that survive normally are transferred to the substrate to continue growing. After the plants grow, a few leaves are picked for DNA extraction, and PCR is performed to identify positive plants.

[0064] 3. Positive identification of transgenic Arabidopsis thaliana

[0065] The constructed overexpression vector (OX-GmCXIP1-1-pTF101s) plasmid was transformed into Agrobacterium GV3101. T3 generation transgenic Arabidopsis seeds were obtained using the inflorescence staining method and herbicide screening. Finally, quantitative PCR was used to confirm different transgenic Arabidopsis lines with high GmCXIP1-1 expression levels for subsequent gene function studies. The quantitative primers for Arabidopsis GmCXIP1-1 were: GmCXIP1-1-RT-F and GmCXIP1-1-RT-R. The Arabidopsis housekeeping gene EF1-α was used as an internal reference gene. The AtEF quantitative primers were used for Arabidopsis housekeeping gene analysis; the specific primer sequences are shown below.

[0066] GmCXIP1-1-RT-F: 5'-TTGCCGTTGCAATCAAGAGC-3';

[0067] GmCXIP1-1-RT-R: 5'-CCTTGGCGCAACAAGTCATT-3';

[0068] AtEF1-α-F: 5'-GTCGATTCTGGAAAGTCGACC-3';

[0069] AtEF1-α-R: 5'-AATGTCAATGGTGATACCACGC-3'.

[0070] The results are as follows Figure 3 As shown, compared with wild-type (WT) Arabidopsis thaliana, PCR results indicate that the transgenic lines express the GmCXIP1-1 gene ( Figure 3 A) Real-time quantitative PCR results showed that, compared with WT, the overexpression lines OX1, OX2 and OX3 all had GmCXIP1-1 expression levels (A). Figure 3 B), the difference is significant.

[0071] 4. High and low phosphorus treatment of transgenic Arabidopsis thaliana

[0072] Select a suitable amount of plump seeds from wild-type plants and two GmCXIP1-1 overexpressing positive lines, OX1 and OX2. After sterilization, sow the seeds on normal MS medium. After about 3-4 days, when the Arabidopsis roots reached about 1 cm, select uniform seedlings and transfer them to half MS medium square dishes for the corresponding treatments. Two phosphorus levels were set up: normal phosphorus supply treatment (1250 μM KH2PO4) and low phosphorus treatment (6.25 μM KH2PO4). Each square dish was a replicate, and each treatment had 5 replicates. On the 9th day after treatment, collect the samples and measure the aboveground fresh weight, underground fresh weight, number of lateral roots, and taproot length.

[0073] The results are as follows Figure 4 As shown, under low phosphorus treatment conditions, overexpression of GmCXIP1-1 significantly promoted root growth in transgenic Arabidopsis thaliana. Figure 4 A). Under low phosphorus conditions, the number of lateral roots in transgenic lines OX1 and OX2 increased by 50% and 20% respectively compared to WT. Figure 4 B). Under normal phosphorus treatment conditions, compared with WT, the primary root length of the transgenic lines OX1 and OX2, which overexpressed GmCXIP1-1, increased by about 9%. Under low phosphorus treatment conditions, the primary root length of the transgenic lines OX1 and OX2 increased by 17% and 23% respectively compared with WT. Figure 4 C). Meanwhile, under normal phosphorus treatment conditions, compared to WT, the aboveground and root fresh weights of the GmCXIP1-1 overexpressing transgenic lines OX1 and OX2 were reduced by approximately 20%, while under low phosphorus treatment conditions, the aboveground and root fresh weights of transgenic lines OX1 and OX2 increased by 20% and 40%, respectively, compared to WT. Figure 4 D and Figure 4 E).

[0074] 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. A plant tolerant to low phosphorus toxicity stress GmCXIP1-1 The use of a gene or its expression promoter, or the protein encoding it, in promoting plant growth under low phosphorus stress or in the preparation of formulations that promote plant growth under low phosphorus stress, is characterized by: GmCXIP1-1 The nucleotide sequence of the gene is shown in SEQ ID NO:1; GmCXIP1-1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2; the expression promoter is an overexpression agent. GmCXIP1-1 Gene expression vector; the plant is soybean or Arabidopsis thaliana.

2. A plant tolerant to low phosphorus toxicity stress GmCXIP1-1 The application of genes or their expression promoters, or encoded proteins, in reducing the inhibitory effect of phosphorus stress on plant root growth, characterized in that... GmCXIP1-1 The nucleotide sequence of the gene is shown in SEQ ID NO:1; GmCXIP1-1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2; the expression promoter is an overexpression agent. GmCXIP1-1 Gene expression vector; the plant is soybean or Arabidopsis thaliana.

3. A plant tolerant to low phosphorus toxicity stress GmCXIP1-1 The application of a gene or its expression promoter, or the protein encoding it, in improving the tolerance of plant roots to low phosphorus stress or in the preparation of formulations that improve the tolerance of plant roots to low phosphorus stress, is characterized by: GmCXIP1-1 The nucleotide sequence of the gene is shown in SEQ ID NO:1; GmCXIP1-1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2; the expression promoter is an overexpression agent. GmCXIP1-1 Gene expression vector; the plant is soybean or Arabidopsis thaliana.

4. A plant tolerant to low phosphorus toxicity stress GmCXIP1-1 The application of genes or their expression promoters, or encoded proteins, in the cultivation of low-phosphorus-tolerant plants, characterized in that... GmCXIP1-1 The nucleotide sequence of the gene is shown in SEQ ID NO:1; GmCXIP1-1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2; the expression promoter is an overexpression agent. GmCXIP1-1 Gene expression vector; the plant is soybean or Arabidopsis thaliana.

5. A method for promoting root growth and / or improving plant tolerance to low phosphorus stress under low phosphorus toxicity stress, characterized in that, Overexpression in plants GmCXIP1-1 Genes, characterized by, GmCXIP1-1 The nucleotide sequence of the gene is shown in SEQ ID NO:1; the plant is soybean or Arabidopsis thaliana.

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