Application of GmCHR1, a key regulatory gene in flavonoid metabolic pathway network, in Arabidopsis

Abstract

The application discloses application of a flavonoid metabolic pathway network key regulation gene GmCHR1 in Arabidopsis thaliana. Research shows that the expression amount of a soybean chalcone reductase (CHR) gene GmCHR1 in soybean roots is significantly up-regulated under low-phosphorus stress, and the low-phosphorus stress enhances the expression of GmCHR1 in the root system of Arabidopsis thaliana, and GmCHR1 influences the response of the root system of Arabidopsis thaliana to low-phosphorus stress. Through construction of a transgenic plant, overexpression of the GmCHR1 gene in Arabidopsis thaliana can promote the growth of Arabidopsis thaliana, significantly improve the biomass, total root length and total phosphorus content of the transgenic Arabidopsis thaliana under normal phosphorus conditions and low-phosphorus conditions; meanwhile, overexpression of GmCHR1 reduces the accumulation of flavonol in the root tip of the transgenic Arabidopsis thaliana, thereby enhancing the accumulation of auxin in the root tip of the transgenic Arabidopsis thaliana, positively regulating the synthesis of auxin in the root system of Arabidopsis thaliana and improving the content of auxin, and playing an important role in improving the growth and phosphorus efficiency of Arabidopsis thaliana.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding and genetic engineering technology. More specifically, it relates to the application of GmCHR1, a key regulatory gene in the flavonoid metabolic pathway network, in Arabidopsis thaliana. Background Technology

[0002] Phosphorus is an essential nutrient for plant growth and development, and a crucial component of biological macromolecules such as nucleic acids, lipids, and proteins. Low phosphorus levels are a significant factor limiting plant growth and production worldwide. In southern my country, where soils are predominantly acidic, applied phosphate fertilizers are easily fixed by soil particles, forming organic phosphorus or insoluble inorganic phosphorus that crops cannot utilize. Developing phosphorus-efficient crop varieties is essential for sustainable agricultural development in my country, which in turn requires a deeper understanding of the morphological, physiological, and molecular mechanisms by which plants adapt to low phosphorus stress.

[0003] To adapt to low phosphorus stress in soil, plants have evolved a series of precise and practical strategies to improve phosphorus absorption and utilization efficiency. These strategies include altering root morphology, such as increasing the number of lateral roots and root hairs while reducing taproot growth, thus increasing root distribution in the topsoil and effectively expanding the contact area between roots and the topsoil. Plant phosphorus acquisition typically relies on the root system's ability to efficiently dig into the soil; the root system plays a crucial role in improving crop phosphorus absorption efficiency. Under low phosphorus stress, plant root morphology generally changes, such as promoting the formation of adventitious and lateral roots, increasing root hair length and density, increasing the root-to-shoot ratio, and forming shallow root structures. Phosphorus distribution in the soil is uneven, with phosphorus content decreasing with increasing soil depth; shallow-rooted plants are more conducive to phosphorus absorption.

[0004] Auxins are key regulators of lateral root formation, playing a crucial role in lateral root initiation and development. In root research, flavonoids are considered important components of root exudates, interacting with hormones to participate in root phototropism and growth. Chalcone reductase (CHR) is one of the key enzymes in flavonoid biosynthesis. In soybean, CHR is an essential enzyme in the daidzein synthesis pathway, regulating the synthesis of the daidzein precursor isoglycyrrhizin. Existing studies have shown that overexpression or inhibition of CHRs can alter the resistance of transgenic plants to *Pseudomonas soybeanii*, *Phytophthora infestans*, and cyst nematodes, but the function of soybean CHR genes under low phosphorus stress remains unknown. Since the CHR gene family is rarely reported in *Arabidopsis thaliana*, the biological functions of CHR genes in the metabolic pathway network of *Arabidopsis thaliana* are currently unclear. Therefore, there is an urgent need to develop new applications for CHR genes to understand the morphological, physiological, and molecular mechanisms by which *Arabidopsis thaliana* adapts to low phosphorus stress. Summary of the Invention

[0005] This invention provides the application of GmCHR1, a key regulatory gene in the flavonoid metabolic pathway network, in Arabidopsis thaliana.

[0006] The first objective of this invention is to propose the application of the GmCHR1 gene in Arabidopsis thaliana.

[0007] A second objective of this invention is to provide the application of a formulation that promotes the expression of the GmCHR1 gene.

[0008] A third objective of this invention is to provide a method for increasing the auxin and / or phosphorus content in Arabidopsis roots.

[0009] The fourth objective of this invention is to provide a method for promoting the growth of Arabidopsis thaliana.

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

[0011] This invention discloses a novel application of the key regulatory gene GmCHR1 in the soybean flavonoid metabolic pathway network in Arabidopsis thaliana. The nucleotide sequence of the GmCHR1 gene is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2. Studies show that the expression level of the GmCHR1 gene in soybean roots is significantly upregulated by low phosphorus stress, while low phosphorus stress enhances the expression of the GmCHR1 gene in Arabidopsis roots. Therefore, the GmCHR1 gene influences the response of Arabidopsis roots to low phosphorus stress. By constructing transgenic plants, overexpression of the GmCHR1 gene in Arabidopsis thaliana promotes its growth and significantly increases biomass, total root length, and total phosphorus content under both normal and low phosphorus conditions. Simultaneously, overexpression of the GmCHR1 gene positively regulates auxin synthesis in Arabidopsis roots and increases auxin content. GmCHR1 enhances auxin accumulation in the root tips of transgenic Arabidopsis by reducing the accumulation of flavonols, thus playing a crucial role in improving the growth and phosphorus efficiency of Arabidopsis thaliana.

[0012] Therefore, this invention provides the following applications of the GmCHR1 gene:

[0013] Application of the GmCHR1 gene in positively regulating the synthesis of auxin in Arabidopsis roots and / or increasing the phosphorus content in Arabidopsis roots.

[0014] Application of the GmCHR1 gene in promoting Arabidopsis growth, or in promoting Arabidopsis growth under low phosphorus stress.

[0015] Application of the GmCHR1 gene in constructing Arabidopsis transgenic materials with high phosphorus and / or high auxin content.

[0016] Application of the GmCHR1 gene in the breeding of Arabidopsis thaliana to tolerate low phosphorus stress.

[0017] Applications of the GmCHR1 gene in increasing auxin content in Arabidopsis roots, or in the preparation of products that increase auxin content in Arabidopsis roots.

[0018] This invention provides the application of a formulation that promotes GmCHR1 gene expression in promoting Arabidopsis thaliana growth, or in promoting Arabidopsis thaliana growth under low phosphorus stress.

[0019] This invention provides the application of a formulation that promotes GmCHR1 gene expression in the preparation of products that promote Arabidopsis thaliana growth.

[0020] This invention provides the use of formulations that promote GmCHR1 gene expression in increasing the auxin and / or phosphorus content of Arabidopsis roots, or in the preparation of products that increase the auxin and / or phosphorus content of Arabidopsis roots.

[0021] Preferably, the agents for promoting GmCHR1 gene expression described herein include, but are not limited to, enzyme activators, compound promoters, plasmids, expression vectors, etc.

[0022] More preferably, the expression vector containing the GmCHR1 gene can be used to construct recombinant expression vectors containing the GmCHR1 gene from existing plant expression vectors. The plant expression vectors include, for example, overexpression vectors or other derived plant expression vectors.

[0023] This invention provides a method for increasing the auxin and / or phosphorus content in Arabidopsis roots by overexpressing the GmCHR1 gene in Arabidopsis or by transferring a preparation that promotes GmCHR1 gene expression into the plant, thereby increasing the auxin and / or phosphorus content in Arabidopsis roots.

[0024] In addition, this invention also provides a method for promoting Arabidopsis growth by overexpressing the GmCHR1 gene in Arabidopsis.

[0025] The present invention has the following beneficial effects:

[0026] This invention discloses a novel application of the key regulatory gene GmCHR1 in the soybean flavonoid metabolic pathway network in Arabidopsis thaliana. The nucleotide sequence of the GmCHR1 gene is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2. Studies show that the expression level of the GmCHR1 gene in soybean roots is significantly upregulated by low phosphorus stress, while low phosphorus stress enhances the expression of the GmCHR1 gene in Arabidopsis thaliana roots, indicating that the GmCHR1 gene affects the response of Arabidopsis thaliana roots to low phosphorus stress. By constructing transgenic plants, overexpression of the GmCHR1 gene in Arabidopsis thaliana promotes its growth and significantly increases biomass, total root length, and total phosphorus content under normal and low phosphorus conditions. Simultaneously, overexpression of the GmCHR1 gene positively regulates auxin synthesis in Arabidopsis roots and increases auxin content. GmCHR1 enhances auxin accumulation in the root tips of transgenic Arabidopsis by reducing the accumulation of flavonols, thus playing a crucial role in improving the growth and phosphorus efficiency of Arabidopsis thaliana.

[0027] This study shows that the GmCHR1 gene has a significant effect on the growth and total phosphorus content of Arabidopsis thaliana. This is of great significance for elucidating the biological function of the CHR gene in regulating the adaptation of Arabidopsis thaliana to low phosphorus stress. The ability of plants to adapt to low phosphorus stress in soil can be regulated through transgenic technology. Attached Figure Description

[0028] Figure 1 Figure 1 shows the results of the analysis of the expression pattern of GmCHR1 in different tissues of soybean under different phosphorus concentration treatments (+P indicates normal phosphorus treatment (+P: 1250 μM KH2PO4); -P indicates low phosphorus treatment (-P: 12.5 μM KH2PO4). Data are the mean and standard error of three replicates. Asterisks indicate that the difference between the control (+P) and the treatment (-P) is significant (Student's t-test), *: P<0.05, **: P<0.01, ***: P<0.001).

[0029] Figure 2 The staining results of GmCHR1 fusion GUS protein in isolated soybean hairy roots are shown in the figure (+P indicates normal phosphorus treatment (+P: 1250 μM KH2PO4), -P indicates low phosphorus treatment (-P: 12.5 μM KH2PO4); the scale bar is 1 cm, and the scale bar of the magnified root area is 0.2 mm).

[0030] Figure 3The staining results of GmCHR1 fusion GUS protein in transgenic Arabidopsis thaliana (+P indicates normal phosphorus treatment (+P: 1250 μM KH2PO4), -P indicates low phosphorus treatment (-P: 12.5 μM KH2PO4); the scale bar is 1 cm, and the scale bar of the magnified root area is 0.2 mm).

[0031] Figure 4 To investigate the effect of overexpression of GmCHR1 on phosphorus availability in transgenic Arabidopsis thaliana (A: Phenotypic characteristics of wild-type (WT) and transgenic Arabidopsis thaliana lines (OX1, OX2, OX3) under normal phosphorus (+P) and low phosphorus (-P) treatments, scale bar in the figure is 1 cm; B: Fresh weight of aboveground parts; C: Fresh weight of roots; D: Total root length; E: Taproot length; F: Number of lateral roots; G: Total phosphorus content of aboveground parts; H: Total phosphorus content of underground parts; Asterisks indicate significant differences between wild-type (WT) and transgenic Arabidopsis thaliana lines (OX1, OX2, OX3) (Student's-test), *: P<0.05, **: P<0.01, ***: P<0.001)).

[0032] Figure 5 The effect of GmCHR1 overexpression on the content of auxin and flavonols in the roots of transgenic Arabidopsis thaliana (A: Fluorescence intensity diagrams of flavonol and auxin distribution in the primary and lateral roots of wild-type (DR5-GFP) and transgenic Arabidopsis thaliana lines (OX1, OX2, OX3) under normal phosphorus treatment, with a scale bar of 20 μm; B: Relative fluorescence intensity of flavonols in the primary root; C: Relative fluorescence intensity of GFP in the primary root; D: Relative fluorescence intensity of flavonols in the lateral roots; E: Relative fluorescence intensity of GFP in the lateral roots). 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 materials used in this embodiment are sourced from:

[0036] The soybean (Glycine max(L.)Merr.) material was Yuechun 03-3 (YC03-3), which was obtained from the Root Biology Research Center of South China Agricultural University.

[0037] The Arabidopsis thaliana material, Colombian wild type, was obtained from the Root Biology Research Center of South China Agricultural University.

[0038] The Arabidopsis thaliana material DR5-GFP (containing an auxin reporter gene) was kindly provided by Professor Mao Chuanzao's research group at the College of Life Sciences, Zhejiang University.

[0039] Example 1: Analysis of the expression pattern of the GmCHR1 gene

[0040] Using the soybean chalcone reductase (CHR) gene GmCHR1 as a candidate gene (its nucleotide sequence is shown in SEQ ID NO:1), preliminary qRT-PCR analysis under normal phosphorus treatment (+P: 1250 μM KH2PO4) and low phosphorus treatment (-P: 12.5 μM KH2PO4) revealed that GmCHR1 expression was significantly upregulated at the transcriptional level under low phosphorus stress. Subsequently, the specific function of the GmCHR1 gene in response to soil low phosphorus stress was analyzed.

[0041] 1. Plant samples

[0042] Soybean seeds of uniform size and without damaged seed coats were selected using the paper roll seedling method. They 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 nutrient solution with a pH of 5.8 and sterile water were prepared and sterilized for later use.

[0043] 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.

[0044] Seedlings with uniform growth were selected and transferred to soybean nutrient solutions with different phosphorus concentrations: normal phosphorus treatment (+P: 250 μM KH2PO4) and low phosphorus treatment (-P: 5 μM KH2PO4). Eight replicates were made 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.

[0045] 2. Real-time quantitative PCR analysis

[0046] Total RNA was extracted from plant samples treated with different phosphorus concentrations 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 10-fold and analyzed using an Applied Biosystems StepOnePlus Real-Time PCR system.

[0047] The quantitative PCR primers used were those for the GmCHR1 gene, with the soybean housekeeping gene as the internal reference gene. The specific primers are shown below:

[0048] GmCHR1-RT1-F: 5'-AGCCCAGGTGAGTCTGAGAT-3';

[0049] GmCHR1-RT1-R: 5'-CAGCGAGTTGTGGTTTGGTG-3';

[0050] EF1-α-F: 5'-TGCAAAGGAGGCTGCTAACT-3';

[0051] EF1-α-R: 5'-CAGCATCACCGTTCTTCAAA-3'.

[0052] Preparation of the reaction mixture: For every 20 μL of 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 bring the final reaction volume to 20 μL.

[0053] 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.

[0054] 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.

[0055] The results are as follows Figure 1 As shown, the expression pattern of GmCHR1 in different tissues of soybean was first analyzed. The results showed that GmCHR1 was expressed in roots, leaves, flowers, pods, and root nodules. Moreover, compared with normal phosphorus treatment, low phosphorus treatment significantly enhanced the expression level of GmCHR1 in soybean roots. Low phosphorus stress led to a 208.89% upregulation of GmCHR1 expression in roots, but did not affect the expression level of GmCHR1 in other parts.

[0056] Example 2: Histochemical Localization Analysis of GmCHR1 Gene

[0057] 1. Histochemical localization in soybean

[0058] Using transgenic soybean detached hairy roots with the GmCHR1 promoter driving the GUS reporter gene (the method for obtaining transgenic soybean detached hairy roots is referenced from Mo Xiaohui (2019)), the roots were treated under different phosphorus concentrations (normal phosphorus treatment (+P: 1250 μM KH2PO4); low phosphorus treatment (-P: 12.5 μM KH2PO4)). The histochemical localization of GmCHR1 in detached soybean hairy roots was analyzed by GUS staining (gene histochemical localization analysis and GUS staining are referenced from Zhou Ming (2024)).

[0059] The results are as follows Figure 2 As shown, under both normal and low phosphorus conditions, obvious GUS staining was observed in the main rhizome, main root tip, lateral rhizome, lateral root tip, lateral root primordia, and pericycle. Low phosphorus stress enhanced the intensity of GUS staining, indicating that low phosphorus enhances the expression of GmCHR1 in soybean roots.

[0060] 2. Histochemical localization in Arabidopsis thaliana

[0061] Using the same method as described above, transgenic Arabidopsis thaliana with the constructed GmCHR1 promoter driving the GUS reporter gene was used as material, and the histochemical localization of GmCHR1 in transgenic Arabidopsis thaliana was analyzed by GUS staining.

[0062] The results are as follows Figure 3 As shown, under both normal and low phosphorus conditions, obvious GUS staining was observed in the main rhizome, main root tip, lateral rhizome, lateral root tip, lateral root primordia, and pericycle. Low phosphorus stress enhanced the intensity of GUS staining, indicating that low phosphorus enhances the expression of GmCHR1 in Arabidopsis roots.

[0063] Example 3: Construction of the carrier

[0064] 1. Construction of the overexpression vector pTF101s-GmCHR1-OX

[0065] Using soybean YC03-3 cDNA as a template, specific primers GmCHR1-OX-F / R were designed based on the full-length CDS sequence of GmCHR1. The gene fragment was amplified using Phanta Max Super Fidelity DNA Polymerase (Novizan, China). The 50 μL reaction system 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 cDNA template, and 18 μL ddH2O. The PCR amplification conditions were: 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.

[0066] The specific primers for GmCHR1-OX are shown below:

[0067] GmCHR1-OX-F: 5'-GTACCCGGGGATCCTCTAGAATGGCTGCTGCTATTG AAATC-3';

[0068] GmCHR1-OX-R: 5'-GCCTGCAGGTCGACTCTAGATTATATTTGATCATCCC AGAGA-3'.

[0069] 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. 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, transformed into E. coli DH5α, and cultured at 37°C for 12 h. Positive clones were then cultured in a shaking medium and sent to a 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 constructing the overexpression vector pTF101s-GmCHR1-OX.

[0070] 2. Construction of the GmCHR1-GUS fusion expression vector pTF102-GmCHR1-GUS

[0071] Using DNA from soybean variety YC03-3 as a template, the promoter sequence of the GmCHR1-GUS gene was amplified using forward and reverse primers. The PCR amplification system consisted of: 25 μL 2×Vazyme phata buffer, 1 μL Vazyme phataase, 1 μL dNTP, 1 μL each of 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 min, 94℃ denaturation for 30 s, 58℃ annealing for 3 min, and 72℃ extension for 3 min, repeated 30 times from denaturation to extension. The PCR products were stored at 16℃.

[0072] The specific primers for the GmCHR1-GUS gene are shown below:

[0073] Forward primer: 5'-CTATGACATGATTACGAATTCTACGCCATCGGACTTGACT T-3';

[0074] Reverse primer: 5'-GACTGACCTACCCGGGGATCCTCTAACTCTCAAAGGGTT GAC-3'.

[0075] The amplification products were detected by gel electrophoresis. If the band size was correct, the target fragment was further recovered and purified using an agarose gel DNA recovery kit (Megvii Biotechnology, China).

[0076] EcoRI and BamHI were selected as the double restriction sites for the pTF102 vector. The target gene fragment was ligated into the pTF102 vector using a one-step cloning method. The ligation product was transformed into E. coli DH5α competent cells and cultured at 37°C for 12 hours. The positive clones were then cultured in a shaker and sent to the company for sequencing. After successful sequencing, the positive strains were stored at -80°C. The GmCHR1 fusion GUS expression vector pTF102-GmCHR1-GUS was successfully constructed.

[0077] Example 4: Obtaining Transgenic Material

[0078] Transgenic Arabidopsis thaliana materials were prepared using the inflorescence infection method. The main steps were as follows: the constructed pTF102-GmCHR1-GUS vector plasmid was transformed into Agrobacterium GV3101; positive clones were picked and cultured overnight in 5 mL YEP medium (containing spectinomycin and rifampin) at 28°C; then, the culture was expanded to OD in 100 mL YEP medium. 600The concentration of the culture medium was 1.6–2.0. Then, the culture was 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. 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 (transformation was performed once a week during the culture period, for a total of 3 transformations).

[0079] After harvesting T0 generation seeds from the transformed Arabidopsis, approximately 100 μL of seeds were taken for seed propagation identification. The specific steps were as follows: The entire operation was performed in a clean bench. First, the seeds were rinsed with 75% ethanol for 1 min, centrifuged to remove the ethanol, and then rinsed twice with sterile secondary water. Next, they were pre-washed with 10% sodium hypochlorite, centrifuged to remove the sodium hypochlorite, and then rinsed with 1 mL of 10% sodium hypochlorite for 5 min with shaking, centrifuged to remove the sodium hypochlorite, and rinsed 5–6 times with sterile water. Finally, the seeds were resuspended in sterile water and evenly sown on 1 / 2 MS medium containing herbicide. After being treated at 4°C for 1 day to break dormancy, the seeds were transferred to… A plant growth chamber with a photoperiod of 16h / 8h (light / dark) and a temperature of 22℃ / 20℃ (day / night) was used. After about 2 weeks, the surviving T1 generation seedlings were transferred to the substrate to continue growing. After the plants grew, a few old leaves were picked for DNA extraction, and positive plants were identified by PCR. The seeds of the plants that tested positive were retained and harvested as the T1 generation. The same method was used to continuously screen and obtain T2 and T3 generation positive transgenic Arabidopsis seeds to ensure the authenticity of the transgenic plants.

[0080] Example 5: Effect of GmCHR1 overexpression on phosphorus responsiveness in transgenic Arabidopsis thaliana

[0081] Select appropriate amounts of plump seeds from wild-type Arabidopsis thaliana and three identified T3 generation GmCHR1-overexpressing positive lines (OX1, OX2, OX3). After sterilization, sow the seeds on normal MS solid germination medium. After approximately 3–4 days, when the Arabidopsis roots reached about 1 cm in length, 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 (12.5 μM KH2PO4). Each square dish was a replicate, and each treatment had 5 replicates. Samples were collected on day 14 after treatment, and the aboveground fresh weight, underground fresh weight, total root length, number of lateral roots, taproot length, and total phosphorus content were measured.

[0082] The results are as follows Figure 4As shown, overexpression of GmCHR1 significantly increased the total root length of transgenic Arabidopsis thaliana under both normal and low phosphorus treatments. Under normal phosphorus treatment, the total root lengths of OX1, OX2, and OX3 were increased by 62.8%, 93.3%, and 65.7% compared to WT, respectively. Furthermore, overexpression of GmCHR1 significantly increased the aboveground fresh weight and root fresh weight of transgenic Arabidopsis thaliana, while the taproot length and lateral root number were not significantly different from WT. This indicates that overexpression of GmCHR1 increased the total root length of transgenic Arabidopsis thaliana, and under low phosphorus conditions, it significantly promoted root growth.

[0083] Meanwhile, it was found that overexpression of GmCHR1 significantly increased the total phosphorus content in the roots of transgenic Arabidopsis thaliana. The total phosphorus content of OX1, OX2, and OX3 was increased by 48.0%, 98.5%, and 111.0% respectively compared with WT under normal phosphorus treatment; and by 89.1%, 87.5%, and 28.8% respectively compared with WT under low phosphorus treatment.

[0084] In summary, the results show that GmCHR1 affects the root response of Arabidopsis thaliana to low phosphorus stress, and overexpression of GmCHR1 significantly increases the biomass, total root length and total phosphorus content of transgenic Arabidopsis thaliana.

[0085] Example 6: Effects of GmCHR1 overexpression on auxin and flavonol content in transgenic Arabidopsis roots

[0086] 1. Germination and cultivation of Arabidopsis thaliana seeds

[0087] Select appropriate amounts of uniformly sized CK (Arabidopsis thaliana seeds containing the auxin reporter gene DR5-GFP) and GmCHR1-overexpressing positive lines (OX1, OX2, OX3), and aliquot them into 2mL centrifuge tubes, filling each tube to the 0.5mL mark. In a clean bench, add 1mL of deionized water to each centrifuge tube and soak for 15 minutes to break dormancy. Aspirate the deionized water, add 1mL of 75% ethanol, and shake the tubes up and down for 30 seconds. Aspirate the ethanol and rinse 3-4 times with sterile water. Add 1mL of 10% (v / v) sodium hypochlorite solution, shake for a few seconds, and let stand for 5 minutes to sterilize. Rinse 3-4 times with sterile water. Spread the seeds onto MS solid medium using a blue pipette tip for germination. After sowing, seal the tubes with breathable medical tape and place them at 23℃ under a light intensity of 120μmol·m⁻¹. -2 ·s -1 The Arabidopsis thaliana plants were germinated for 48 hours in a constant temperature incubator with 16 hours of light, 8 hours of darkness, and 60% relative humidity. After about 3 to 4 days, when the roots of Arabidopsis thaliana grew to about 1 cm, uniform seedlings were selected and transferred to 1 / 2 MS medium cubes with normal phosphorus supply (1250 μM KH2PO4) for 14 days of culture.

[0088] 2. DPBA staining of roots of GmCHR1-overexpressing transgenic Arabidopsis thaliana

[0089] Roots of GmCHR1-overexpressing transgenic Arabidopsis thaliana lines (OX1, OX2, OX3) and CK (DR5-GFP) under normal phosphorus supply treatment were immersed in a solution containing 0.01% (v / v) Triton X-100 and 2.52 mg / mL DPBA (flavonol-specific fluorescent dye diphenylboronic acid-2-aminoethyl ester) for 2.5 h. The samples were then washed in deionized water for 1 minute and fixed onto glass slides. The transgenic Arabidopsis roots were observed and photographed using a laser confocal scanning microscope (LSM780, Zeiss, Germany) with excitation wavelengths of 475 and 619 nm.

[0090] 3. Observation of auxin content in roots of GmCHR1-overexpressing transgenic Arabidopsis thaliana

[0091] The roots of GmCHR1-overexpressing transgenic Arabidopsis thaliana (OX1, OX2, OX3) and CK (DR5-GFP) were immersed in deionized water for 1 minute and then fixed on glass slides. They were observed and photographed using a laser confocal scanning microscope (LSM780, Zeiss, Germany) with an excitation wavelength of 488 nm.

[0092] The results are as follows Figure 5 As shown, under normal phosphorus treatment conditions, compared with the control (CK), the content of flavonols in the root tips of GmCHR1-overexpressing transgenic Arabidopsis thaliana (OX1, OX2, OX3) was significantly lower in the primary root than in the control (CK). Figure 5 The flavonol content in the lateral root tips of A and B was significantly lower than that in CK. Figure 5 A, D); while the auxin content in the root tip of the primary root of GmCHR1-overexpressing transgenic Arabidopsis (OX1, OX2, OX3) was significantly higher than that of the control (CK). Figure 5 In A and C), the auxin content in the lateral root tips was significantly higher than that in CK. These results indicate that GmCHR1 positively regulates the synthesis of auxin in Arabidopsis roots and increases auxin content. Overexpression of GmCHR1 enhances the accumulation of auxin in the root tips of transgenic Arabidopsis by reducing the accumulation of flavonols in the root tips.

[0093] 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. As shown in SEQ ID NO.1 GmCHR1 The application of genes in increasing phosphorus content in Arabidopsis roots is characterized by, Overexpression of the above in Arabidopsis thaliana GmCHR1 Gene.

2. As shown in SEQ ID NO.1 GmCHR1 The application of genes in promoting Arabidopsis growth is characterized by, Overexpression of the above in Arabidopsis thaliana GmCHR1 Gene.

3. As shown in SEQ ID NO.1 GmCHR1 The application of the gene in promoting Arabidopsis growth under low phosphorus stress is characterized by, Overexpression of the above in Arabidopsis thaliana GmCHR1 Gene.

4. As shown in SEQ ID NO.1 GmCHR1 The application of genes in constructing transgenic Arabidopsis thaliana materials with high phosphorus content is characterized by, Overexpression of the above in Arabidopsis thaliana GmCHR1 Gene.

5. As shown in SEQ ID NO.1 GmCHR1 The application of genes in the breeding of Arabidopsis thaliana to tolerate low phosphorus stress is characterized by, Overexpression of the above in Arabidopsis thaliana GmCHR1 Gene.

6. Promotes as shown in SEQ ID NO.1 GmCHR1 The application of gene expression agents in promoting Arabidopsis thaliana growth, characterized in that, The formulation is an overexpression of the [specific formulation]. GmCHR1 The carrier of genes.

7. Promotes as shown in SEQ ID NO.1 GmCHR1 The application of gene expression formulations in promoting Arabidopsis growth under low phosphorus stress, characterized in that, The formulation is an overexpression of the [specific formulation]. GmCHR1 The carrier of genes.

8. Promotes as shown in SEQ ID NO.1 GmCHR1 The application of gene expression formulations in the preparation of products that promote Arabidopsis thaliana growth, characterized in that, The formulation is an overexpression of the [specific formulation]. GmCHR1 The carrier of genes.

9. Promotes the effect shown in SEQ ID NO.1 GmCHR1 The application of gene-expressing agents in increasing phosphorus content in Arabidopsis roots or in the preparation of products that increase phosphorus content in Arabidopsis roots, characterized in that... The formulation is an overexpression of the [specific formulation]. GmCHR1 The carrier of genes.

10. A method for increasing the phosphorus content in Arabidopsis thaliana roots, characterized in that, Overexpression in Arabidopsis GmCHR1 Genes may promote GmCHR1 A gene-expressing agent is transferred into plants to increase the phosphorus content in Arabidopsis roots; the agent is an overexpression of the gene. GmCHR1 The vector of genes; GmCHR1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

11. A method for promoting the growth of Arabidopsis thaliana, characterized in that, By overexpressing in Arabidopsis GmCHR1 Genes, thereby promoting Arabidopsis growth; the aforementioned GmCHR1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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