Application of phaseolus vulgaris aurk and its interacting protein gene in plant salt tolerance regulation

Abstract

The application discloses a Phaseolus vulgaris AURK and application of the interacting protein genes in plant salt tolerance regulation. AURK The nucleotide sequence of the Phaseolus vulgaris AURK gene is shown as SEQ ID NO:1; the Phaseolus vulgaris GRF2 interacting protein gene is GRF5 The nucleotide sequences of the GRF2 gene and the GRF5 gene are shown as SEQ ID NO:2 and SEQ ID NO:3 respectively. The application clarifies the molecular mechanism of AURK-GRF2 and AURK-GRF5 modules in salt stress response through protein interaction verification and salt tolerance identification, provides theoretical support and gene resources for breeding of Phaseolus vulgaris salt-tolerant varieties, and provides a theoretical basis for crop salt tolerance improvement.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to common beans. AURK The application of their interacting protein genes in the regulation of plant salt tolerance. Background Technology

[0002] In recent years, soil salinization has become increasingly serious, posing a severe challenge to the sustainable development of global agriculture. High-salinity environments not only damage soil structure but also inhibit crop growth through multiple mechanisms, including osmotic stress, ion toxicity, and oxidative stress, leading to decreased yield and quality. Therefore, identifying crop salt-tolerant genes, elucidating salt-tolerant mechanisms, and improving crop salt tolerance through molecular breeding are of great significance for the effective utilization of saline-alkali land and ensuring food security.

[0003] Common beans (Phaseolus vulgaris L.) are one of the most commonly consumed legumes, originating in the Americas. After a long process of dissemination and domestication, they are now widely cultivated around the world. They are highly nutritious, rich in protein (approximately 20%-30%), dietary fiber, various vitamins, and minerals such as iron, zinc, and calcium, with a relatively balanced amino acid composition. When eaten with grains, they provide complementary proteins, significantly enhancing the nutritional value of the diet. Common beans are not only an important source of protein and carbohydrates for developing countries in Asia, Africa, and Latin America, but also a major export agricultural product for temperate regions such as China, the United States, and Canada. As a common food ingredient, common beans can be eaten fresh or processed into canned goods or various bean products, and are widely used in the food and feed industries, demonstrating significant economic and nutritional value. With the continuous improvement of living standards, global demand for common beans is steadily increasing.

[0004] Common beans are a crop sensitive to environmental stress, easily affected by saline-alkali stress, leading to yield decline. In recent years, with the impact of climate change and human activities, soil salinization has intensified, and the area affected by salinization is constantly increasing. Major common bean producing areas are gradually becoming saline. Given the scarcity of arable land resources and the need to reduce competition for land with staple crops, the efficient utilization of marginal soils such as saline-alkali land has become an important direction. Therefore, improving the salt tolerance of common beans has become an important research goal for agricultural researchers in recent years.

[0005] Over the past few decades, researchers have made limited progress in the study of salt tolerance in common bean, particularly in areas such as the discovery of stress-resistant germplasm resources, identification of functional genes, and analysis of salt tolerance mechanisms. Regarding the identification and screening of salt-tolerant germplasm resources, Bayuelo-Jiménez et al. systematically evaluated 132 wild germplasm accessions from 14 common bean species based on seedling phenotypes. Using indicators such as dry matter loss rate, salt sensitivity index, and root-to-shoot ratio, they found that highly salt-tolerant germplasm is mostly distributed in arid, semi-arid, coastal intertidal zones, and saline soil areas. Li Mengdi et al. evaluated the salt tolerance of 40 common bean varieties and screened out five highly salt-tolerant germplasm accessions: P16152, P19023, P18009, P18012, and P18051. Regarding the physiological mechanisms of common bean response to salt-alkali stress, studies have primarily employed physiological and biochemical methods, focusing on the accumulation of osmotic regulators (such as proline and betaine) and the dynamic changes in antioxidant enzyme systems (including superoxide dismutase and peroxidase), thus providing preliminary insights into the physiological adaptation strategies of common bean under salt stress. In terms of salt tolerance gene discovery, with the continuous development of molecular biology techniques, several genes related to common bean salt tolerance have been preliminarily identified, such as AOX, IQD, Trihelix, and P5CS1. However, whether these genes can be practically applied to molecular design breeding requires further verification. Therefore, in-depth exploration and functional research on the salt tolerance genes of common beans will not only help to elucidate their salt tolerance molecular mechanisms, but also provide theoretical basis and gene resources for the breeding of new salt-tolerant varieties, thereby improving the yield and quality of common beans on saline-alkali land, which is of great significance for ensuring food security and promoting sustainable agricultural development. Summary of the Invention

[0006] The purpose of this invention is to provide green beans AURK The application of their interacting protein genes in the regulation of plant salt tolerance.

[0007] kidney bean AURK The application of their interacting protein genes in the regulation of plant salt tolerance.

[0008] The green beans AURK The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0009] The green beans AURK Interacting protein genes are GRF2 Genes and GRF5 The genes, whose nucleotide sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0010] Includes the green beans AURK Gene recombination vectors.

[0011] Includes the green beans AURK Recombinant cells of genes.

[0012] One method to improve plant salt tolerance is to upregulate the expression levels of AURK protein, or the expression levels of GRF2 and GRF5 proteins, in plants.

[0013] The sequence of the AURK protein is shown in SEQ ID NO: 4, and the amino acid sequences of the GRF2 and GRF5 proteins are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0014] A method for cultivating salt-tolerant plants, using green beans AURK Genes are transferred into plants, or... GRF2 Genes and GRF5 Genes are transferred into plants.

[0015] The plant in question is a common bean, tobacco, corn, rice, or soybean.

[0016] The beneficial effects of this invention are as follows: This invention subjects 495 common bean germplasm accessions to salt stress, identifies salt tolerance at the seedling stage, and calculates the salt damage index; based on the genome resequencing data and salt damage index phenotypic data of the 495 common bean materials, a genome-wide association analysis is performed to identify candidate genes for salt tolerance in common bean; and salt tolerance is further identified and physiological and biochemical indicators are measured by constructing a common bean overexpression hairy root complex material to screen for candidate genes for salt tolerance in common bean. AURK We screened the interacting proteins GRF2 and GRF5 of salt-tolerant candidate genes using techniques such as yeast two-hybrid assays. Through protein interaction verification and salt tolerance identification, we elucidated the molecular mechanisms of the AURK-GRF2 and AURK-GRF5 modules in salt stress response, providing theoretical support and gene resources for the breeding of salt-tolerant bean varieties and a theoretical basis for improving crop salt tolerance. Attached Figure Description

[0017] Figure 1 Correlation analysis of phenotypes under salt stress treatment; A: Salt stress treatment of 495 materials, the upper figure shows the phenotype before salt stress treatment, and the lower figure shows the phenotype on day 9 of salt stress treatment; B, C: Distribution and dispersion of salt damage index of 495 materials from day 9 to day 16; D: Frequency distribution of salt damage index from day 9 to day 16.

[0018] Figure 2 Screening salt-tolerant candidate genes for GWAS and analyzing candidate gene expression patterns; A, B: Genome-wide association analysis of genome resequencing data to draw Manhattan plots and QQ plots; C: Distribution of SNP sites in 1 Mb chromosomal segments before and after significant loci; D: Linkage disequilibrium analysis of SNP sites in 107 kb chromosomal segments before and after significant loci; E: Analysis of candidate gene expression patterns.

[0019] Figure 3To identify salt tolerance and determine physiological and biochemical indicators in bean plants overexpressing the target gene hairy root complex; A: Phenotype of bean plants overexpressing the target gene hairy root complex after 24 h of salt stress treatment; B: Relative gene expression level in root tissue compared with control; C: Determination of physiological and biochemical indicators.

[0020] Figure 4 A: Interactions between AURK and growth regulatory factor proteins; B: Validation of AURK-GRF2 and GRF5 interactions in yeast; C: Subcellular localization of GFP, AURK, GRF2, and GRF5 in tobacco; D: Analysis of bimolecular fluorescence complementary interactions between AURK and GRF2, and AURK and GRF5; E: GST pull-down assay.

[0021] Figure 5 To analyze the salt tolerance of GRF2-overexpressing transgenic bean complexes: A: GRF2 expression level in bean root tissues at different times of salt stress; B: Phenotypic comparison of control and GRF2-overexpressing hairy root complex plants after 24 h of 1% NaCl salt stress treatment; C: Relative expression level of GRF2 gene in root tissues of bean GRF2-overexpressing hairy root complexes compared to the control after 24 h of salt stress; D: Relative expression level of AURK gene in root tissues of bean GRF2-overexpressing hairy root complexes compared to the control after 24 h of salt stress; E: Determination of physiological and biochemical indicators of bean GRF2-overexpressing hairy root complexes.

[0022] Figure 6 To analyze the salt tolerance of GRF5-overexpressing transgenic bean complexes: A: GRF5 expression level in bean root tissues at different times of salt stress; B: Phenotypic comparison of control and GRF5-overexpressing hairy root complex plants after 24 h of 1% NaCl salt stress treatment; C: Relative expression level of GRF5 gene in root tissues of bean GRF5-overexpressing hairy root complexes compared to the control after 24 h of salt stress; D: Relative expression level of AURK gene in root tissues of bean GRF5-overexpressing hairy root complexes compared to the control after 24 h of salt stress; E: Determination of physiological and biochemical indicators of bean GRF5-overexpressing hairy root complexes. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention. Example 1

[0024] I. Experimental Methods

[0025] 1. Experimental materials and genotype data

[0026] The 495 common bean materials used in this embodiment were obtained from the National Crop Germplasm Bank of the Institute of Crop Science, Chinese Academy of Agricultural Sciences. The original genome resequencing data were obtained from the NCBI SRA database (PRJNA515107).

[0027] 2. Salt tolerance assessment

[0028] Ten uniform and plump seeds were sown in small pots containing vermiculite of equal weight (each pot measuring 7 cm × 7 cm × 7 cm) at a depth of 2 cm. After 4 days of normal growth, the germinated seedlings were transferred to Hoagland culture medium for 3 days to allow them to recover. Then, they were subjected to salt stress treatment using a hydroponic system of Hoagland culture medium + 1% NaCl. They were grown in an artificial climate chamber at 25°C with a photoperiod of 16 h light / 8 h dark. Ten seedlings were planted per sample, and the experiment was repeated three times. On the 12th day of salt stress treatment, the salt tolerance of the seedlings was assessed according to the salt damage seedling condition grading survey standard (the salt tolerance grade classification is shown in Table 1). The salt damage index (SDI) was calculated according to the following formula, and the salt tolerance of the tested materials was determined using the SDI as the evaluation index (Table 2).

[0029] SDI=Σ (Ni×SRi) / (Nt×SRh)×100;

[0030] SDI: Salt damage index, in percentage (%); Ni: Number of plants at salt damage level i, in plants; SRi: Corresponding salt damage level value; Nt: Total number of plants surveyed, in plants; SRh: Highest salt damage level value.

[0031] Table 1 Classification of Salt Tolerance Levels of Seedling Conditions

[0032]

[0033] Table 2 Salt Tolerance Judgment Criteria

[0034]

[0035] 3. Genome-wide association analysis

[0036] The raw genome resequencing data from 495 common bean germplasms were first screened and filtered using vcftools. Principal component analysis was then performed using PLINK 1.9 software, phylogenetic relationships among germplasms were analyzed using TASSEL software, and the genetic structure of the population was analyzed using admixture software. Genome-wide association analysis was performed using the EMMAX model and GAPIT software, and Manhattan and QQ plots were generated. The significance threshold P was estimated using the Bonferroni correction method based on the number of analyzed single nucleotide polymorphisms (SNPs), and SNPs with a P value lower than the threshold were defined as significantly associated trait loci. Given the strong linkage disequilibrium (LD) between some significant SNPs, SNPs with a physical distance less than the LD decay distance were considered to originate from the same associated region, and the SNP with the lowest P value was selected as a candidate SNP for that region. Finally, the chromosomal intervals formed by the SNP ± LD distance were defined as candidate segments.

[0037] 4. Real-time quantitative PCR

[0038] To investigate the expression of salt-tolerant candidate genes, common bean seedlings that had grown for two weeks and had two fully expanded true leaves were subjected to salt stress treatment. Total RNA was extracted from the roots of common bean plants using the Trizol method and further purified using an RNA purification kit (Tiangen Biotech (Beijing) Co., Ltd.). Genomic DNA was then removed using the EasyScript one-step method, and cDNA was synthesized by reverse transcription using a cDNA synthesis kit (TransGen Biotech Co., Ltd., Beijing).

[0039] Primers for real-time quantitative PCR (RT-qPCR) were designed using Primer Premier 5 software (https: / / www.downza.cn / soft / 347277.html) (Table 3), with the bean Pv-action gene used as an internal control. Amplification and detection were performed using the Universal SYBR Green qPCR Supermix kit (Beijing Bairui Biotechnology Co., Ltd.) and the ROCGENE real-time quantitative PCR system. The total reaction volume was 10 µL, including: 5 µL 2×SYBR Green Mix, 0.2 µL each of forward and reverse primers (10 µmol / L), 1 µg cDNA, and finally, enzyme-free sterile water to make up to 10 µL. The reaction program was set as follows: 95℃ pre-denaturation for 30 s; followed by 45 cycles, including 95℃ denaturation for 5 s, 60℃ annealing for 34 s, 72℃ extension for 15 s, and 60℃ annealing for 60 s. The relative expression levels of candidate genes were calculated based on the expression level of the Pv-action gene in common bean under 0 h of salt stress.

[0040] 5. Creation of bean plants with hairy root complex overexpressing target gene and salt stress treatment

[0041] Primers were designed based on the CDS sequence of the target gene to amplify its complete coding region. This region was then cloned into the expression vector pJIT16318 containing a GFP tag via homologous recombination, constructing an overexpression vector for the target gene. This recombinant vector was transformed into competent Agrobacterium tumefaciens strain K599 for subsequent experiments; the empty vector control group did not contain the target gene. After the Agrobacterium culture reached the logarithmic growth phase with an OD600 of 0.8, the inoculum was infected approximately 1 cm below the cotyledons of a bean plant that had grown for 7 days and had two fully expanded true leaves. Once the bean hairy roots had emerged, bean hairy root complex plants overexpressing the candidate gene were obtained.

[0042] Infected bean hairy root complex plants were transferred to a 25°C incubator and cultured under a 16-h light / 8-h dark light cycle. After one week of closed, moist culture, the plants were transferred to 500 mL of 1.5 g / L Hoagland medium and cultured for 45 days at 25°C under a 16-h light / 8-h dark light cycle. The plants were then subjected to 24 h of salt stress treatment with 1% NaCl solution, and samples were taken at 0 h and 24 h of salt stress.

[0043] 7. Electrolyte leakage rate analysis

[0044] Fresh leaf samples of 0.5 g were collected from both the control group and the bean hairy root complex plants overexpressing the candidate gene, and placed in 50 mL centrifuge tubes. 20 mL of distilled water was then added to each tube, and the samples were allowed to stand at room temperature for 45 min. The initial electrolyte leakage rate was measured using a conductivity meter and recorded as A1. The samples were then boiled for 10 min, cooled to room temperature, and the electrolyte leakage rate was measured again and recorded as A2. The electrolyte leakage rate was calculated using the following formula: Electrolyte leakage rate (%) = (A1 × 100) / A2.

[0045] 8. Determination of malondialdehyde, glutathione, catalase, proline, and betaine content

[0046] Fresh root samples (0.1 g each) were collected from both the control group and the bean hairy root complex plants overexpressing the candidate gene. The samples were thoroughly ground in a mortar. Malondialdehyde (MDA), glutathione, catalase, proline, and betaine were extracted from the fresh roots of the bean hairy root complex plants according to the experimental methods provided by the following kits: Malondialdehyde (MDA) Content Assay Kit (Beijing Box Biotechnology Co., Ltd.), Reduced Glutathione (GSH) Content Assay Kit (Beijing Box Biotechnology Co., Ltd.), Catalase (CAT) Activity Assay Kit (Beijing Box Biotechnology Co., Ltd.), Proline (PRO) Content Assay Kit (Beijing Box Biotechnology Co., Ltd.), and Betaine Content Assay Kit (Beijing Box Biotechnology Co., Ltd.). The absorbance of the samples was measured using a Thermo Varioskan™ LUX multi-functional microplate reader (Thermo Fisher), and the contents of MDA, glutathione, catalase, proline, and betaine were calculated. All experiments were performed in triplicate.

[0047] 9. Yeast Two-Hybrid (Y2H) Experiment

[0048] The CDS sequence of AURK was cloned into the Y2H "bait" vector pGBKT7 to construct the BD-AURK expression vector, which was then transformed into yeast Y2HGold cells. The self-activation activity was verified and its toxicity was tested using the Y2HGold-GAL4 system yeast double hybrid screening library kit (plasmid transformation method) (Kulabo). The pGBKT7-AURK competent cells were constructed according to the kit method to screen cDNA libraries.

[0049] The CDS sequences of GRF2 and GRF5 were cloned into the "prey" vector pGADT7 to construct AD-GRF2 and AD-GRF5 expression vectors. Using BD-AURK as "bait" and AD-GRF2 and AD-GRF5 as "prey", positive clones were screened according to the Y2HGold-GAL4 system yeast dual-hybrid screening kit (plasmid transformation method) (Kulaibo). The growth of yeast cells was monitored using SD / -Leu-Trp and SD / -Leu-Trp-his-ade selective media supplemented with 250 ng / mL AbA and 100 μg / mL Xa-gal to identify interacting clones.

[0050] 10. Subcellular localization of tobacco

[0051] The CDS sequences of AURK, GRF2, and GRF5 were cloned into the subcellular localization vector pJIT16318 containing the cauliflower mosaic virus (CaMV) 35S promoter and GFP tag, and transformed into Agrobacterium tumefaciens competent cells (GV3101). Tobacco infection solution was prepared: pH adjusted to 5.6 with 100 ml of 10 mM MES, 10 mM MgCl2, and 100 mM AS. The Agrobacterium tumefaciens in the logarithmic growth phase (OD600 = 0.8) was resuspended and cultured in the dark for 2 h before infecting 4-week-old Tobacco Benedict's leaves. After infection, the tobacco plants were first cultured in the dark at 25°C for 24 h, then transferred to a photoperiod incubator at 25°C with 16 h light / 8 h dark for 42 h. Leaf epidermal cells were imaged using a confocal laser scanning microscope (LSM700; Zeiss).

[0052] 11. Bimolecular Fluorescence Complementary (BiFC) Experiment

[0053] The CDS sequence of AURK was cloned into the pXY106 vector to construct the AURK-nYFP expression vector; the CDS sequences of GRF2 and GRF5 were cloned into the pXY104 vector to construct the GRF2-cYFP and GRF5-cYFP expression vectors, respectively. These vectors were transformed into *Agrobacterium tumefaciens* GV3101 competent cells and cultured. Tobacco infection solution was prepared by resuspending *Agrobacterium tumefaciens* culture at OD600 = 0.8 during the logarithmic growth phase and culturing in the dark for 2 h, which was then used to infect leaves of 4-week-old *Nicotiana benthamiana*. Infected tobacco plants were first cultured in the dark at 25℃ for 24 h, then transferred to a 25℃, 16 h light / 8 h dark incubator for 36 h of further culture. Finally, the YFP fluorescence signal in the leaf epidermal cells was observed using a confocal laser scanning microscope (LSM700; Zeiss).

[0054] 12. Luciferase Complementation Imaging Experiment

[0055] Using the empty vectors pCAMBIA1300-nLUC and pCAMBIA1300-cLUC as negative controls, the CDS sequences of AURK, GRF2, and GRF5 were cloned into the nLUC and cLUC vectors, respectively, to construct recombinant expression vectors 35S:AURK-nLUC, 35S:GRF2-cLUC, and 35S:GRF5-cLUC. The recombinant expression vectors were then transformed into Agrobacterium tumefaciens competent cells (GV3101) for culture. Tobacco infection solutions were prepared by resuspending logarithmic-phase Agrobacterium suspension (OD600 = 0.8). Infection solutions were prepared by mixing 35S:AURK-nLUC with 35S:GRF2-cLUC, 35S:AURK-nLUC with 35S:GRF2-cLUC, nLUC with 35S:GRF2-cLUC, nLUC with 35S:GRF5-cLUC, and cLUC with 35S:AURK-nLUC at a 1:1 (volume / volume) ratio. After dark incubation for 2 hours, the infection solutions were used to infect leaves of 4-week-old *Nicotiana benthamiana* plants. Following infection, the tobacco plants were first incubated in the dark at 25°C for 24 hours, and then transferred to a 25°C incubator with a 16-hour light / 8-hour dark cycle for 36 hours. Finally, the epidermal cells of the tobacco leaves were observed using a confocal laser scanning microscope.

[0056] 13. External pull-down test

[0057] To verify the interaction between AURK and GRF2 and GRF5, AURK, GRF2, and GRF5 were constructed into expression vectors with GST or MBP tags, respectively, to create recombinant expression vectors AURK-GST, GRF2-MBP, and GRF5-MBP. The recombinant expression vectors AURK-GST, GRF2-MBP, GRF5-MBP, and the empty MBP plasmid were transformed into the protein expression strain *Escherichia coli* BL21(DE3), cultured at 37°C until OD600 = 0.6, and then induced for 20 h at 16°C and 110 rpm with 0.5 mM IPTG.

[0058] Centrifuge at 12000 rpm for 10 min at 4℃ to collect the supernatant. Add 100 µL of lysozyme and 20 µL of DNase I per mL of supernatant. Let stand at room temperature for 30 min to lyse the protein. Then, aliquot the sample into 2 mL centrifuge tubes at 250 µL each, freeze in liquid nitrogen for 25 s, and then thaw in a 28℃ water bath until completely thawed. Repeat this freeze-thaw cycle 25 times until the supernatant becomes clear to ensure complete protein lysis. Purify AUKR-GST protein with 100 mL ProteinIso GST Resin (Beijing Tiangen Biotech Co., Ltd.) and purify GRF2-MBP and GRF5-MBP proteins with 100 mL MBP tag affinity packing material (maltose-binding protein) (Lambolid Biotechnology). Incubate at 110 rpm for 10 h at 4℃ to allow the adsorption column to fully bind to the protein. Take 20µL of purified AURK-GST, GRF2-MBP, GRF5-MBP and MBP protein solutions and add 5µL of 5x SDS-PAGE protein loading buffer. Boil at 98℃ for 10 min. Prepare the protein gel using the TGX Stain-Free FastCast Acrylamide Kit, 12% kit (Beijing Box Biotechnology Co., Ltd.). Perform SDS-PAGE protein electrophoresis at 220V to detect the target protein.

[0059] The GST resin was eluted overnight at 4°C and 110 rpm using 70 µL of GST resin elution buffer. The supernatant was then collected after centrifugation at 4°C and 1200 rpm for 2 min to obtain the AURK-GST fusion protein. 20 µL of purified MBP, GRF2-MBP, and GRF5-MBP proteins were added to 20 µL of the AURK-GST fusion protein, along with 100 µL of pull-down buffer, and the mixture was co-incubated overnight at 4°C and 110 rpm. Finally, the supernatant was collected after centrifugation at 4°C and 1200 rpm for 10 min to obtain the fusion protein complexes of AURK-GST and GRF2-MBP, AURK-GST and GRF5-MBP, and AURK-GST and MBP.

[0060] Take 20 µL of the fusion protein complexes of AURK-GST and GRF2-MBP, AURK-GST and GRF5-MBP, and AURK-GST and MBP, add 5 µL of 5 x SDS-PAGE protein loading buffer, boil at 98 °C for 10 min, prepare the protein gel using the TGX Stain-Free FastCast Acrylamide Kit, 12% kit (Beijing Box Biotechnology Co., Ltd.), and perform SDS-PAGE protein electrophoresis at 220 V. After electrophoresis, protein bands were transferred to a nitrocellulose membrane at 20 V for 40 min using a transfer apparatus. The following steps were then performed: blocking with 15 mL Blocking Buffer at 40 rpm for 30 min at room temperature; elution with 15 mL 1x Wash Buffer at 40 rpm for 2 min at room temperature; incubation with 15 mL Dilution Buffer containing 5 µL of anti-GST antibody at 40 rpm for 3 h at room temperature; elution with 15 mL 1x Wash Buffer at 40 rpm for 2 min at room temperature; incubation with 15 mL Dilution Buffer containing 3 µL of anti-GST anti-mouse antibody at 40 rpm for 1 h at room temperature; and elution with 15 mL 1x Wash Buffer at 40 rpm for 2 min at room temperature, repeated 3 times. Finally, the protein-protein interaction results were viewed using an InvitrogeniBright imaging system.

[0061] 14. Data Analysis

[0062] One-way ANOVA was used to compare multiple datasets. The least significant difference (LSD) test was used after ANOVA, and the t-test was used to compare the datasets. SPSS statistical software (v25.0) was used to assess statistical significance, and Origin Pro (v2021) was used to generate bar charts.

[0063] II. Results and Analysis

[0064] 1. Phenotypic Analysis of Salt Stress in Kidney Beans

[0065] In this study, 495 genetically diverse common bean seedlings were subjected to salt stress treatment with 1% NaCl solution. On the 9th day of treatment, sensitive common bean materials began to show salt damage phenotypes. Figure 1 A). Based on the salt damage phenotype, the salt damage condition of seedlings was investigated daily from day 9 to day 16 of treatment, and the salt damage index was calculated. As the duration of salt stress increased, the average salt damage index of all materials continuously increased ( Figure 1 B), and the salt toxicity index varies widely among different test materials ( Figure 1(C) effectively reflects the differences in salt tolerance among different bean genotypes. The frequency distribution analysis of the salt damage index shows that this trait is normally or approximately normally distributed (Figure 1 D), which is consistent with the typical characteristics of quantitative traits.

[0066] 2. Genome-wide association analysis to identify salt tolerance-related candidate genes.

[0067] Using the salt damage index under salt stress in common bean resources as phenotypic data, a genome-wide association analysis (GWAS) was performed on its genome resequencing data, and Manhattan plots and QQ plots were constructed. Under the conditions of a maximum deletion rate of 0.99 and a minor allele frequency (MAF) of no less than 0.05, a total of 1,154,038 high-quality SNPs were selected for subsequent association analysis. Based on the total number of SNPs used (1,154,038), the significance threshold was estimated to be 6.06 (i.e., P = -log) using the Bonferroni correction method. 10 (1 / 1154038) ≈ 6.06). SNPs below this threshold were defined as significant trait-associated sites. During salt stress treatments on days 9, 10, and 16, significant signals exceeding the aforementioned significance threshold were detected at 16,839,389 bp on chromosome 9. Figure 2 A, B). Considering the strong linkage disequilibrium (LD) among some significant SNPs and their small decay distance, they cannot be considered independent sites. Therefore, the SNP with the lowest P-value was selected as the candidate SNP for this region. Using this candidate SNP as the center, an LD decay distance (107 kb) was extended to both sides. Linkage disequilibrium analysis was performed on this 214 kb chromosomal segment, and the results showed that this chromosomal region had significant correlation (…). Figure 2 (C, D) This interval was defined as a candidate interval. Gene annotation was performed on all protein-coding genes within the candidate interval Chr9:16,732,389-16,946,389. It was found that there were 14 genes in this interval (Table 3) that were the most promising candidate genes that were significantly associated with the salt damage index.

[0068] Table 3. Candidate gene information obtained from GWAS analysis

[0069]

[0070] 3. Screening and identification of candidate genes for salt tolerance in common beans

[0071] To analyze the function of 14 candidate genes in salt tolerance of common bean, their expression patterns were first investigated. RT-qPCR results showed that, among these 12 candidate genes—NADP-ME2, CRBN, TrpRS, HPT, RP-L17, AAP, AAT, LRRK2, PAP, PFK, AURK, and CABIN1—the expression levels generally showed a trend of initial upregulation followed by downregulation or sustained upregulation with increasing salt stress duration. Figure 2 E), while the other two genes showed low basal expression levels, with no significant change in expression levels under salt stress. Subsequently, the function of these genes in the salt stress response of common bean was further investigated using Agrobacterium tumefaciens K599-mediated hairy root transformation technology. The results showed that after 24 h of treatment with 1% NaCl, among the 12 candidate genes, transgenic hairy root composite plants overexpressing NADP-ME2, TrpRS, RP-L17, AAP, and RPPT exhibited severe leaf wilting (E). Figure 3 A), the transgenic complex exhibited a salt-sensitive phenotype; while plants overexpressing AURK showed stronger salt tolerance ( Figure 4 Other gene overexpression lines showed little phenotypic difference compared to the control. Corresponding physiological and biochemical analyses showed that, compared to the control, plants overexpressing NADP-ME2, TrpRS, RP-L17, AAP, and RPPT had decreased glutathione and catalase activity in their roots, and increased malondialdehyde content and relative electrolyte exudation rate. Figure 3 (C) Conversely, plants overexpressing AURK showed significantly increased levels of proline, glutathione, catalase, and betaine, while malondialdehyde (MDA) content and electrolyte leakage rate were significantly decreased. These results indicate that NADP-ME2, TrpRS, RP-L17, AAP, and RPPT may negatively regulate salt tolerance in common bean, while AURK plays a positive regulatory role, and the phenotype of plants overexpressing AURK was most pronounced compared to the control. Therefore, we selected AURK for further in-depth research.

[0072] 4. Interaction between AURK and growth regulatory factor proteins

[0073] To further elucidate the molecular mechanism by which AURK regulates salt tolerance in common beans, we analyzed the interacting proteins. Through yeast two-hybrid library screening, bacterial PCR sequencing, and protein sequence alignment, GRF2 and GRF5 were initially identified as potential interacting proteins of AURK. Further validation of the yeast two-hybrid interaction showed that yeast cells co-expressing BD-AURK with either GRF2-AD or GRF5-AD could grow on a synthesis-deficient medium lacking leucine, tryptophan, histidine, and adenine, while yeast cells expressing GRF2-AD or GRF5-AD alone could not grow under the same conditions. This result confirms a direct physical interaction between AURK and GRF2 and GRF5. Figure 4 A).

[0074] In addition, to investigate the subcellular localization of AURK, GRF2, and GRF5, we expressed GFP, AURK-GFP, GRF2-GFP, and GRF5-GFP fusion proteins, respectively, in the epidermal cells of transgenic tobacco leaves, and observed them using a confocal laser scanning microscope (LSM700, Zeiss). The results showed that these fusion proteins were mainly localized in the nucleus and cytoplasm. Figure 4 B). We performed bimolecular fluorescence complementation (BiFC) experiments in tobacco leaves. Co-transformation of AURK-nYFP with either GRF2-cYFP or GRF5-cYFP produced significant YFP fluorescence signals in both the nucleus and cytoplasm, while no fluorescence was detected in the negative control group. Figure 4 C). To further confirm the interaction between AURK and GRF2 and GRF5, we used luciferase complementation imaging (LCI) and GST in vitro pull-down assay. LCI results showed that samples co-expressing AURK-nLUC and either GRF2-cLUC or GRF5-cLUC produced strong luciferase signals, consistent with BiFC results, while no signal was detected in the control group. Figure 4 D). In the GST pull-down experiment, purified AURK-GST fusion protein was incubated with GRF2-MBP, GRF5-MBP, and MBP-tagged proteins, respectively. The results showed that GRF2-MBP and GRF5-MBP could effectively pull down AURK-GST, while MBP protein had no such effect, indicating that GRF2 and GRF5 can specifically bind to AURK in vitro. Figure 4 E). The above results collectively confirm that AURK interacts with GRF2 and GRF5 both in vivo and in vitro.

[0075] 5. Growth regulators positively regulate salt tolerance in green beans.

[0076] To investigate the functions of GRF2 and GRF5 in the response of common bean to salt stress, we systematically analyzed their expression dynamics under salt stress. RT-qPCR results showed that the expression of GRF2 and GRF5 was significantly upregulated in the early stages of salt stress, and then gradually downregulated with increasing treatment time, indicating that GRF2 and GRF5 respond to salt stress (…). Figure 5 (A, 6A). Through Agrobacterium tumefaciens K599-mediated transformation of bean hairy roots, we further evaluated the roles of GRF2 and GRF5 in the bean salt stress response. Phenotypic observation revealed that after 24 h of 1% NaCl treatment, control plants exhibited severe leaf wilting, while transgenic plants overexpressing GRF2 or GRF5 showed a significant growth advantage (Figs. 5B, 6B). Furthermore, in hairy root complex plants overexpressing GRF2 or GRF5, AURK gene expression was also induced to be upregulated (…). Figure 5 D, 6D). Physiological and biochemical analyses showed that, compared with the control, the transgenic plants had significantly increased levels of proline, glutathione, catalase, and betaine, while malondialdehyde content and relative electrolyte leaching rate were significantly decreased. Figure 5 E, 6 E). The above results indicate that GRF2 and GRF5 play a positive regulatory role in enhancing the salt tolerance of common beans.

[0077] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Phaseolus vulgaris AURK application of the gene in salt tolerance regulation of Phaseolus vulgaris, characterized in that, The bean AURK The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. A method of improving salt tolerance in Phaseolus vulgaris, characterized by, up-regulating the expression of AURK protein in the bean; the sequence of the AURK protein is shown as SEQ ID NO:

4.

3. A method of breeding salt tolerant beans, characterized in that, The bean of claim 1 AURK into the bean.

Images