Application of BRM gene and encoded protein thereof in regulating and controlling acid resistance of plants

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an application of a BRM gene and an encoding protein thereof in regulating and controlling the acid resistance of plants. And the login number of the BRM gene in a GenBank / EMBL database is AT2G46020. The invention further discloses a method for identifying the BRM gene. The BRM gene can physically interact with a core acid-resistant transcription factor STOP1 to inhibit the transcriptional activation activity of the STOP1 so as to influence the acid resistance of plants. Therefore, after the BRM gene is inhibited, the acid resistance of the plant can be improved, the growth of a plant root system and the absorption of nitrogen nutrition under an acidic condition are promoted, and meanwhile, the alkalization of a rhizosphere environment of the root system is promoted. By interpreting the functions of the BRM gene, an effective gene resource is provided for cultivating acid-resistant plants and improving acid soil while utilizing the acid soil.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to application of BRM gene and its coded protein in regulating plant acid resistance. BACKGROUND

[0002] High concentration of H + in acid soil can significantly inhibit the growth of plant roots and inhibit the absorption of plant to nitrogen, phosphorus, potassium and other mineral nutrients, reduce nutrient use efficiency, and increase the risk of environmental pollution. In addition, soil acidification can also lead to the release of a variety of metal ions to toxic concentrations, especially Al 3+ , Fe 3+ and Mn 2+ , which further inhibit plant growth. It is found by calculation of soil acidification model that if the nitrogen fertilizer application amount is increased at a rate of 1% per year, the average pH value of soil will be further reduced, which will affect the relative yield of major crops such as wheat, corn and rice. Therefore, the problem of soil acidification will bring great challenges to the sustainable development of agriculture and food security. Therefore, it is an urgent need to improve the tolerance of plants to H + / Al 3+ stress and cultivate new varieties of nitrogen-efficient crops suitable for acid soil.

[0003] As early as the beginning of the last century, researchers have begun to pay attention to the harm of soil acidification to crop production, and through water culture experiments and plate experiments, single low pH treatment proves that plant tolerance to H + may be an important basis for its tolerance to Al 3+ . For example, when the pH value of the solution culture system is reduced from 6.5 to 4.5, the root elongation rate of the plant is inhibited by nearly 78%; in the solid agar medium, the root elongation of Arabidopsis grown at pH 5.0 is less than half of that grown at pH 6.5. STOP1 is the first Cys2-His2 zinc finger transcription factor obtained by map-based cloning in the screening of acid-sensitive mutants of Arabidopsis, and the loss of function of STOP1 inhibits more than 70% of the root elongation of Arabidopsis under H + stress. It can be seen that STOP1 is a major gene for regulating plant tolerance to H + , but the upstream molecular mechanism of STOP1 for enhancing plant tolerance to H + stress is still not fully understood, which greatly hinders the cultivation process of nitrogen-efficient plants suitable for acid soil.

[0004] Therefore, it is of great significance to further explore the key genes for regulating plant tolerance to H + stress, to deeply analyze the molecular mechanism of plant acid stress tolerance and to cultivate new plant varieties with acid stress tolerance. SUMMARY

[0005] The application provides application of a BRM gene and a protein coded by the BRM gene in regulating plant acid resistance.

[0006] The application provides application of a BRM gene in regulating plant acid resistance, wherein the BRM gene has an accession number of AT2G46020 in a GenBank / EMBL database.

[0007] As a preferred solution, the acid resistance comprises at least one of the following effects in an acid environment: promoting plant root elongation, promoting nitrogen utilization rate of the plant, and promoting alkalinization of a rhizosphere environment of the plant.

[0008] As a preferred solution, the regulation comprises inhibiting expression of the BRM gene or knocking out the BRM gene to improve the acid resistance of the plant, or overexpressing the BRM gene to reduce the acid resistance of the plant.

[0009] The application further provides a protein coded by the BRM gene, wherein an amino acid sequence of the protein is shown as SEQ ID NO:1.

[0010] The application further provides a primer pair for amplifying the BRM gene, wherein the primer pair comprises an upstream primer and a downstream primer; a nucleotide sequence of the upstream primer is shown as SEQ ID NO:2, and a nucleotide sequence of the downstream primer is shown as SEQ ID NO:3.

[0011] The BRM gene has an accession number of AT2G46020 in the GenBank / EMBL database.

[0012] The application further provides a biological material capable of adjusting an expression amount of a BRM gene, wherein the BRM gene has an accession number of AT2G46020 in the GenBank / EMBL database.

[0013] As a preferred solution, the biological material comprises a biological material for overexpressing the BRM gene and a biological material for inhibiting expression of the BRM gene or knocking out the BRM gene.

[0014] The biological material for overexpressing the BRM gene comprises one or more of a recombinant expression vector containing the BRM gene, a recombinant microorganism containing the BRM gene, and a recombinant microorganism containing the recombinant expression vector.

[0015] The inhibition method comprises silencing.

[0016] The application also provides the use of the protein, the primer pair or the biomaterial in regulating the acid resistance of plants and / or creating a plant variety with target acid resistance.

[0017] The application also provides a method for improving the acid resistance of plants, comprising the following steps: knocking down the expression amount of a BRM gene in a target plant genome or knocking out the BRM gene in a target plant, to obtain a plant with improved acid resistance.

[0018] The BRM gene has an accession number of AT2G46020 in the GenBank / EMBL database.

[0019] The application also provides a method for improving acid soil, comprising planting the plant obtained by the method in an acid soil environment.

[0020] Beneficial effects: the application provides the use of the BRM gene in regulating the acid resistance of plants, and the BRM gene has an accession number of AT2G46020 in the GenBank / EMBL database. The BRM gene can physically interact with the core acid-tolerant transcription factor STOP1, inhibit the transcription activation activity of STOP1, and further affect the acid resistance of plants. Therefore, the BRM gene can improve the acid resistance of plants, promote the growth of plant roots and the absorption of nitrogen nutrients under acid conditions, and promote the alkalization of the rhizosphere environment of the roots. The application provides an effective gene resource for cultivating acid-tolerant plants and improving acid soil while utilizing acid soil by interpreting the function of the BRM gene.

[0021] The application clones the negative regulator BRM from model plants by using the amplification primer of the BRM gene, and constructs two mutant strains by knocking out and functionally complementing the BRM gene. The acid resistance of the functional mutant strain of the BRM gene is obviously improved, and the functional complement strain shows a similar acid-sensitive phenotype as the wild type plant. The test results show that under acid stress, the taproot elongation of the mutant Arabidopsis is obviously improved compared with the wild type control, and the nitrate absorption rate of the root system is obviously improved. Inhibiting the expression of BRM is beneficial to the improvement of the adaptability of plants to acid stress. The application has important significance for improving the acid resistance of plants, further promoting the absorption and growth of plants, and improving the yield of crops. The amplification primer and the gene or protein amplified by the amplification primer provided by the application are used as important negative regulatory genes for cultivating new acid-tolerant and nitrogen-efficient plants, which opens up the channel for genetic breeding of nitrogen-efficient plants adapted to acid soil, provides a new production idea for genetic breeding of nitrogen-efficient plants, reduces the screening work in the traditional breeding process, and the use of nitrogen-efficient plants reduces the input of nitrogen fertilizer, promotes the alkalization of the rhizosphere environment, slows down the acidification process of the soil, and provides an effective and sustainable biological strategy for the improvement of acid farmland soil. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1. BRM physically interacts with STOP1; A, schematic of full-length BRM and its various fragments used; numbers above the boxes indicate the amino acid positions of the corresponding fragments; B-C, yeast two-hybrid assay demonstrating the interaction between BRM and STOP1; D, co-immunoprecipitation (Co-IP) experiment showing the interaction of BRM with STOP1 in Arabidopsis;

[0023] Figure 2 Figure 2. Down-regulation of BRM expression in Example 2 can improve the tolerance of plants to low pH and the promotion of nitrate uptake by low pH; A and B, growth of Col-0, brm (brm-3 and brm-20) and pBRM:BRM-FLAG / brm-3 transgenic plants (COM) in agar medium; C and D, growth of Col-0, brm (brm-3 and brm-20) and stop1 mutant in medium supplemented with different doses of nitrate; E, RT-qPCR analysis of NRT1.1 expression in the roots of Col-0, brm (brm-3 and brm-20) and stop1 mutant, data represented as mean ± standard deviation of 4 biological replicates; F and G, root growth comparison of brm-20 and nrt1.1 single mutant and double mutant brm nrt1.1; scale bar: 1 cm;

[0024] Figure 3 Figure 3. Genotyping of brm nrt1.1 double mutant by PCR in Example 2; the left of the dashed line is the detection of BRM; the right of the dashed line is the detection of NRT1.1; brm-20 was used for the construction of double mutant; the gel map below shows the primers used for genomic PCR, LBb1.3, T-DNA left border primer of SALK line; LP, left primer of genomic DNA; RP, right primer of genomic DNA;

[0025] Figure 4 Figure 4. Effect of BRM on the accumulation of STOP1 under low pH conditions in Example 3; A, images of GFP-STOP1 in pSTOP1:GFP-STOP1 / stop1 and pSTOP1:GFP-STOP1 / brm-20 seedlings, scale bar: 0.1 mm; B, quantitative analysis of GFP-STOP1 fluorescence intensity in A, three-line graph presents the mean ± standard deviation of 12 seedlings for each line and each condition, different letters indicate significant differences between the means, determined by one-way ANOVA combined with Tukey's multiple comparison test (p < 0.05);

[0026] Figure 5 BRM antagonizes the regulation of NRT1.1 expression by STOP1; where A is ChIP-qPCR assay using Col-0 and brm-3 mutants; B is a genome browser screenshot showing the binding site of BRM in the NRT1.1 gene; C is ChIP-qPCR assay using 10-day-old Col-0 and pBRM:BRM-FLAG / brm-3 plants and anti-FLAG antibody, the enrichment fold represents the normalized value of immunoprecipitation efficiency in transgenic plants relative to Col-0 plants; D is open chromatin FAIRE-qPCR assay of P1-1 and P2-1 regions of NRT1.1 relative to UBQ10 coding sequence, the relative amplicon abundance represents the normalized value of results in mutants relative to Col-0 plants;

[0027] Figure 6 Genotyping of brm stop1 double mutants by PCR in Example 4; where A is detection of BRM; B is detection of STOP1, brm-20 was used for double mutant construction, the gel picture below shows primers used for genomic PCR, LBb1.3, T-DNA left border primer for SALK lines; LP, left primer for genomic DNA; RP, right primer for genomic DNA;

[0028] Figure 7 STOP1 acts downstream of BRM in the low-pH response in Example 4; where A and B are root growth comparison of stop1 and brm-20 single mutants and double mutants, whiskers on the upper and lower sides of the box plot represent the minimum and maximum values; the box represents the mean ± standard deviation of 10 seedlings, scale bar: 1 cm; C is nitrate uptake rate comparison of stop1 and brm-20 single mutants and double mutants, data is represented as the mean ± standard deviation of 4 biological replicates for each line under different conditions;

[0029] Figure 8 Root growth of Col-0 and brm-3 seedlings in agar medium with single species and mixed species planting in Example 5; where A is a schematic diagram showing the growth response of single species and mixed species planting seedlings; B-C are pictures of single species and mixed species planting seedlings in neutral pH and low pH medium; D is root elongation, whiskers on the upper and lower sides of the box plot represent the minimum and maximum values; the box is presented as the mean ± standard deviation of 12 seedlings for each line and each condition; scale bar: 1 cm, different letters indicate significant differences between the means (p < 0.05; multi-factorial ANOVA combined with Tukey's multiple comparison test);

[0030] Figure 9 BRM deletion can enhance the response of H +Stress rhizosphere alkalization and H + Tolerance; wherein A-B is the root growth of seedlings in single and mixed planting in acid agar medium, the whiskers on the upper and lower sides of the box plot represent the minimum and maximum values; the box is presented with the average value ± standard deviation of 7 seedlings per line and per condition; C and D are the growth of Col-0, brm-3 and stop1 seedlings in agar medium with or without pH buffer; E is the visualization of rhizosphere alkalization by staining with bromocresol purple for 12h in basal agar medium with initial pH of 4.7; F is the pH value in rooting agar medium; scale: 1cm;

[0031] Figure 10 Inhibition of BRM expression at low pH in Example 6; wherein A is the RT-qPCR analysis of BRM in wild type plant roots at 2h, 4h and 24h after low pH treatment (n=4 biological replicates); B-C is the effect of BRM-GFP accumulation in pBRM:BRM-GFP / brm-20 seedling roots under neutral or low pH treatment for 4h;

[0032] Figure 11 Model of BRM-STOP1-NRT1.1 pathway in the present application. DETAILED DESCRIPTION

[0033] The present application provides the application of BRM gene in regulating plant acid resistance, the accession number of the BRM gene in GenBank / EMBL database is AT2G46020. As a specific embodiment, the acid resistance includes at least one of the following effects in the acidic environment: promoting plant root elongation, promoting plant nitrogen utilization rate and promoting plant rhizosphere environment alkalization. As a specific embodiment, the regulation includes inhibiting the expression of the BRM gene or knocking out the BRM gene to improve the acid resistance of the plant; overexpressing the BRM gene to reduce the acid resistance of the plant. In the present application, the plant can include all types of plants, such as Brassicaceae plants, commonly but not limited to Arabidopsis thaliana. The root system in the present application includes the main root.

[0034] The present application also provides a protein encoded by the BRM gene, the amino acid sequence of the protein is shown as SEQ ID NO:1. In the present application, the BRM protein is a sequence composed of 2193 amino acids.

[0035] The present application also provides a primer pair for amplifying the BRM gene, which includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:3; the accession number of the BRM gene in GenBank / EMBL database is AT2G46020.

[0036] The primer pair sequence is as follows:

[0037] The upstream primer (SEQ ID NO: 2) is 5'-CTATGACCATGATTACGAATTCATGCAATCTGGAGGCAGTGGCGGAGG-3';

[0038] The downstream primer (SEQ ID NO: 3) is 5'-AGTGTCGACTCTAGAGGATCCATGGCTAGGCCGTCTTTTACCAGAATC-3'.

[0039] In the present application, the primer is designed according to the Arabidopsis thaliana acid resistance negative regulation gene BRM, and restriction enzyme cutting sites EcoRI and BamHI are designed at both ends to facilitate the preparation of the subsequent vector. The gene number of the BRM gene in the present application is AT2G46020. The primer in the present application can be used for artificial cloning of the complete BRM. The source of the BRM gene in the present application is not particularly limited, and the artificial synthesis method or amplification method known in the art can be used. For example, in the present application, the gene can be obtained by cloning. As a specific embodiment, the primer described in the above technical solution is used for PCR amplification with Arabidopsis thaliana cDNA as a template to obtain the gene BRM containing enzyme cutting sites at both ends. In the present application, the reaction procedure of the PCR amplification can be as follows: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 3 min, 35 cycles; 68℃ final extension for 5 min.

[0040] The present application also provides a biological material capable of adjusting the expression amount of the BRM gene, and the accession number of the BRM gene in the GenBank / EMBL database is AT2G46020.

[0041] As a specific embodiment, the biological material overexpressing the BRM gene and the biological material inhibiting the expression or knocking out the BRM gene are included; the inhibition method includes silencing.

[0042] As a specific embodiment, the biological material overexpressing the BRM gene includes one or more of a recombinant expression vector containing the BRM gene, a recombinant microorganism containing the BRM gene, and a recombinant microorganism containing the recombinant expression vector; as a specific embodiment, the recombinant expression vector containing the BRM gene includes cloning the gene BRM into the plant constitutive overexpression vector 35s-pCAMBIA1300 to obtain the recombinant expression vector 35s-pCAMBIA1300-BRM.

[0043] The application also provides the use of the protein, the primer pair or the biological material in regulating the acid resistance of plants and / or creating plant varieties with target acid resistance.

[0044] The application also provides a method for improving the acid resistance of plants, comprising the following steps: knocking down the expression amount of a BRM gene in a target plant genome or knocking out the BRM gene in a target plant to obtain a plant with improved acid resistance; the BRM gene has the accession number AT2G46020 in the GenBank / EMBL database. The application obtains BRM knockout mutants brm-3 and brm-20 by knocking out the BRM gene, and it is found through experiments that the brm-3 and brm-20 knockout mutants can promote the growth of plant roots and the absorption of nitrogen nutrition under acidic conditions, and at the same time promote the alkalization of the rhizosphere environment of the roots, thereby improving the acid resistance of plants.

[0045] The application also provides a method for improving acid soil, comprising planting the plants obtained by the above method in an acid soil environment. The embodiments of the application show that brm-3 and Col-0 or stop1 can improve the root length of Col-0 or stop1 when they are planted together under low pH conditions. These results show that the deletion of BRM enables plants to create a favorable rhizosphere pH when responding to acid stress. Using pH-sensitive dye bromocresol purple staining and pH measurement, it is observed that the degree of rhizosphere alkalization of the brm-3 mutant is greater than that of Col-0 after 12h of low pH treatment. In summary, these results show that the deletion of BRM increases the rhizosphere pH to enhance H + resistance and has the potential to inhibit soil acidification in acid soil.

[0046] In order to further illustrate the application, the application of the BRM gene and the protein encoded thereby in regulating the acid resistance of plants is described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.

[0047] Unless otherwise specified, the raw materials used in the application have no special requirements and commercially available products known to those skilled in the art can be used.

[0048] Plant materials used in the application: The Arabidopsis plants used in the application are all from the Columbia (Col-0) background. Various mutants include stop1 KO(SLAK_114108, T-DNA insertion mutant of STOP1), nrt1.1-1 (SALK_097431, T-DNA insertion mutant of NRT1.1) (J. Y. Ye, W. H. Tian, M. Zhou, Q. Y. Zhu, W. X. Du, Y. X. Zhu, X. X. Liu, X. Y. Lin, S. J. Zheng, C. W. Jin, STOP1 activates NRT1.1-mediated nitrate uptake to create a favorable rhizospheric pH for plant adaptation to acidity. Plant Cell 33, 3658-3674 (2021).). The brm-3, brm-20, pBRM:BRM-GFP / brm-20 and pBRM:BRM-FLAG / brm-3 transgenic lines used in this study (T. Li, R. Zhang, V. Satheesh, P. Wang, G. Ma, J. Guo, G. Y. An, M. Lei, The chromatin remodeler BRAHMA recruits HISTONE DEACETYLASE6 to regulate root growth inhibition in response to phosphate starvation in Arabidopsis. J. Integr. Plant Biol. 64, 2314-2326 (2022).). The brm stop1 and brm nrt1.1 double mutants were generated by crossing brm-20 with stop1 mutant or nrt1.1-1 mutant, respectively, and homozygous lines were identified by PCR. The pSTOP1:GFP-STOP1 / pBRM:BRM-FLAG (GFP-STOP1 / BRM-FLAG) lines were generated by crossing pSTOP1:GFP-STOP1 / stop1 KO(#B10) Transgenic lines (C. Balzergue, T. Dartevelle, C. Godon, E. Laugier, C. Meisrimler, J. Teulon, A. Creff, M. Bissler, C. Brouchoud, A. Hagege, J. Muller, S. Chiarenza, H. Javot, N. Becuwe-Linka, P. David, B. Peret, E. Delannoy, M. Thibaud, J. Armengaud, S. Abel, J. Pellequer, L. Nussaume, T. Desnos, Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation. Nat. Commun. 8 (2017).) were generated by crossing pBRM:BRM-FLAG / brm-3 and selected for homozygous lines on agar medium containing phosphinothricin and hygromycin.

[0049] The primers used in the present application are shown in Table 1.

[0050] Table 1 Primer information

[0051]

[0052]

[0053] Growth conditions for plants of the present application: Before sowing, seeds were vernalized at 4°C for 2 days. Surface-sterilized Arabidopsis seeds were sown on basal agar medium containing KNO3(6 mM), (NH4)2SO4(1 mM), NaH2PO4(1 mM), MgSO4(500 μM), CaCl2(1 mM), H3BO3(10 μM), MnSO4(0.5 μM), ZnSO4(0.5 μM), CuSO4(0.1 μM), (NH4)6Mo7O 24 (0.1 μM), Fe-EDTA (50 μM), 1% agar (Sigma-Aldrich, A1296) by mass, 1% sucrose by mass, pH 6.5, in a plant growth chamber (BPC600H; Fujian Jiuku Biotechnology, China) at 22-23°C with a 16 h light / 8 h dark photoperiod cycle. Then, 3-day-old plants were used for phenotype analysis in basal agar medium with an initial pH of 4.7 or 6.5, with 0.75 mM Homo-PIPES added for buffer determination. To analyze NO3 -Effect of horizontal positioning on root growth under low-pH treatment, media supplemented with 0.06 mM or 6 mM KNO3, while reducing the concentration of (NH4)2SO4 to 0.5 mM to avoid severe ammonium toxicity, and adjusting the K2SO4 concentration to balance the difference in K concentration.

[0054] Method for measuring root elongation: Seeds of plants were surface-sterilized with 75% ethanol by mass, then washed with sterilized water for 3 times, sowed on the basic solid medium, and placed in a refrigerator at 4°C for 2 days, then placed in a light incubator (light for 16h / dark for 8h) for growth for 3 days; then the seedlings were transferred to the basic solid medium with pH value of 6.5 or 4.7 and the initial root length was marked; after growing for 3 days, the root length of the seedlings of each material was measured with a ruler, and the root elongation of the corresponding material was calculated, that is, the final root length of the plant minus the initial root length.

[0055] Gene expression determination of the application: total RNA was extracted from plant roots using FreeZol reagent (R711-01; Vazyme Biotech Co., Ltd, Nanjing, China), and first-strand cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA remover (TOYOBO, Osaka, Japan). The expression of the corresponding gene was determined using SYBR Green real-time PCR Master Mix (TOYOBO). The expression level of each mRNA was determined by standardizing with the expression level of UBIQUITIN 10.

[0056] Green fluorescent protein (GFP) analysis of the application: after different times of low-pH treatment, the expression of BRM-GFP and GFP-STOP1 in the roots of pBRM:BRM-GFP / brm-20 and pSTOP1:GFP-STOP1 transgenic plants was detected using a confocal laser scanning microscope (LSM880; Zeiss). The excitation and emission wavelengths of the GFP protein were 488nm / 500-530nm. The images were analyzed using ZEN2012 Blue Edition and ImageJ2X software.

[0057] pH analysis and rhizosphere alkalization visualization of the application: 10-day-old Arabidopsis seedlings were pretreated in pH 4.7 agar medium for 2 days before bromocresol purple staining and pH measurement. To visualize rhizosphere acidification, the roots were transferred to the basic agar medium with initial pH of 4.7, and the pH indicator bromocresol purple (0.008%, Sigma-Aldrich) was added. The concentration of NaH2PO4 in the agar medium used was reduced to 0.1 mM. Images were taken after 12h of staining.

[0058] The method for measuring the pH of the agar rooting medium was performed as described in the reference: T. Hachiya, C. K. Watanabe, M. Fujimoto, T. Ishikawa, K. Takahara, M. Kawai-Yamada, H. Uchimiya, Y. Uesono, I. Terashima, K. Noguchi, Nitrate Addition Alleviates Ammonium Toxicity Without Lessening Ammonium Accumulation, Organic Acid Depletion and Inorganic Cation Depletion in Arabidopsis thaliana Shoots. Plant Cell Physiol. 53, 577-591 (2012).

[0059] The application NO3 - Absorption measurements: 7-day-old seedlings were transferred to acid-treated plates or control-treated plates for 1 day of further growth. After a 1 min rinse in 0.05 mM CaSO4solution, seedlings were transferred to a full nutrient solution containing 6 mM KNO3 15 KNO3 15 N atom, 99%) for 5 min; subsequently, the plant roots were rinsed again for 1 min with 0.05 mM CaSO4and the aerial and root parts were harvested separately; samples were thoroughly dried and dry weight was measured, the aerial and subterranean samples of the same plant were mixed and ground, and then the N abundance in the plants was determined by isotope mass spectrometer (Isorime 100; Hanau, Germany). 15

[0060] The application accession numbers: The sequence data herein can be found in the Arabidopsis Genome Initiative or in the GenBank / EMBL databases with the following accession numbers: STOP1 (AT1 G34370), NRT1.1 (AT1 G12110), BRM (AT2G46020), PP2A (AT1 G69960) and UBQ10 (AT4G05320).

[0061] Example 1 BRM physically interacts with STOP1

[0062] To investigate the role of STOP1 in response to H + ​Potential regulators under stress, the present application screened and verified with STOP1 as bait by yeast two-hybrid (Y2H). Y2H amplified BRM and truncated version of BRM [truncated version of BRM refers to N-terminal of BRM (BRMn, amino acid 1 to 976) and C-terminal of BRM (BRMc, amino acid 977 to 2193), as shown in Figure 1 A] using primers with EcorI and BamHI restriction sites and inserted BRM, BRMn and BRMc into pGBKT7 (630443; TaKaRa, Otsu, Japan). The complete CDS of STOP1 was amplified using primers with EcorI and BamHI restriction sites and inserted into pGADT7 (630442; TaKaRa, Otsu, Japan). Specific bait and prey constructs were co-transformed into yeast AH109 cells (STOP1; BRM; STOP1+BRM; BRMn; STOP1+BRMn; BRMc; STOP1+BRMc, respectively). Yeast was grown on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade media (Takara) at 30°C for 4 days and then photographed for observation.

[0063] Co-IP analysis: Total proteins were extracted using RIPA lysis buffer (FD011; Fdbio Science Biotech Co., Ltd.) according to the instructions after 10-day-old seedlings of pSTOP1:GFP-STOP1 (GFP-STOP1) and pSTOP1:GFP-STOP1 / pBRM:BRM-FLAG (GFP-STOP1 / BRM-FLAG) were treated for 6 h under pH 4.7 conditions. The remaining supernatant was then incubated with 30 μL anti-FLAG magnetic beads for 2 h. The eluted proteins and controls were then separated in a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The relevant proteins were detected with a-GFP (1:10,000; ABclonal) and a-FLAG (1:10,000; ABclonal) antibodies.

[0064] The results showed that the core subunit of SWI / SNF complex, BRM, strongly interacted with STOP1 Figure 1 ). Region mapping analysis showed that the N-terminal of BRM was essential for their interaction Figure 1(AC). Given that BRMn is commonly used as a docking site for recruiting transcription factors, it was subsequently used in biochemical experiments. Furthermore, co-immunoprecipitation (Co-IP) assays using transgenic Arabidopsis plants expressing pBRM:BRM-FLAG in the pSTOP1:GFP-STOP1 (GFP-STOP1 / BRM-FLAG) genetic background confirmed that, after 6 h of low pH treatment, GFP-STOP1 and BRM-FLAG specifically co-precipitated (…). Figure 1 (D). These results collectively confirm the interaction between BRM and STOP1 under acid stress conditions.

[0065] Example 2: BRM-deficient enhanced plants in H + Growth under stress and NO3 - Get

[0066] To determine whether BRM participates in H + In response, this invention addresses the BRM deletion mutants (brm-3 and brm-20) and the complementary line pBRM:BRM-FLAG / brm-3(COM) in H + Phenotypic analysis was performed under stress. Three-day-old Arabidopsis seedlings were transferred to neutral (pH 6.5) or low-pH (pH 4.7) media and cultured for 3 days. Root length was measured (pH adjusted with 4M KOH or 2M HCl). The study found that under low-pH conditions, loss of BRM function significantly enhanced root growth; the root elongation of the BRM-deficient mutant was approximately 60% greater than that of the wild-type (Col-0) plants, while BRM complementation completely restored root growth in the brm-3 mutant. Figure 2 China A, Figure 2 (See Table 2). These results indicate that BRM negatively regulates plant tolerance to acid stress.

[0067] Given that STOP1 improves H + Tolerance and nitrogen use efficiency and root NO3 - The increase in acquisition is closely related, and BRM interacts with STOP1 physically ( Figure 1 This invention speculates that BRM may also use NO3. - H is regulated by a dependent method + Tolerance. To verify this view, this invention compared Col-0, brm-3, and stop1 (STOP1 loss-of-function mutant) plants under different concentrations of NO3. - Root elongation effect under supply (low nitrogen: 0.06 mM and normal nitrogen: 6 mM) (using 1 mM NH4) +as an additional nitrogen source). Three-day-old Arabidopsis seedlings were transferred to neutral (pH 6.5) or low pH (pH 4.7) medium containing 0.06 mM or 6 mM KNO3for 3 days. K + concentrations were balanced to 6 mM. It was observed that, under low pH treatment with 0.06 mM NO3 - , root growth of Col-0, brm-3 and stopl mutants was comparable Figure 2 in C, Figure 2 in D). In contrast, under low pH treatment with 6 mM NO3 - , root growth of the brm-3 mutant was significantly better than that of Col-0, while root growth of stopl was reduced Figure 2 in C, Figure 2 in D, Table 3). These results suggest that BRM-regulated H + tolerance is related to the concentration of NO3 - supply.

[0068] Since NRT1.1 plays a major role in root NO3 - uptake under low pH conditions, and low pH-induced NRT1.1 transcription is controlled by STOP1, the inventors analyzed NRT1.1 expression in roots of Col-0, brm (brm-3 and brm-20) and stopl mutants under different pH (neutral pH 6.5 and low pH 4.7) conditions with 6 mM NO3 - supply. As expected, low pH-induced NRT1.1 expression was significantly enhanced in roots of brm mutants compared to Col-0 plants Figure 2 in E, Table 4). To further explore whether BRM and NRT1.1 act in the same pathway in response to H + stress, the inventors generated a brm nrtl.1 double mutant by crossing nrtl.1-1 and brm-20 mutants Figure 3 ). Three-day-old Arabidopsis seedlings were transferred to neutral or low pH (neutral pH 6.5 and low pH 4.7) medium for 5 days. It was found that, in low pH agar medium, root growth of brm nrtl.1 plants was comparable to that of nrtl.1-1 single mutants Figure 2 in F, Figure 2 in G, Table 5), indicating that NRT1.1 is downstream of BRM in H + tolerance. These results suggest that BRM-repressed H + tolerance is related to its repressed NRT1.1 expression.

[0069] Table 2 Root elongation (cm) of wild type, BRM loss-of-function mutants and complemented lines under different pH conditions

[0070] pH Col-0 brm-3 brm-20 COM 6.5 3.62 ± 0.22 a ]] 3.40 ± 0.20 a ]] 3.55 ± 0.21 a ]] 3.57 ± 0.21 a ]] 4.7 1.63 ± 0.21 c ]] 2.72 ± 0.26 b ]] 2.59 ± 0.17 b ]] 1.67 ± 0.24 c ]]

[0071] Table 3 Root elongation (cm) of wild type, BRM deletion mutant and STOP1 deletion mutant under different pH and nitrogen conditions

[0072]

[0073] Table 4 NRT1.1 expression level in wild type, BRM deletion mutant and STOP1 deletion mutant under different pH conditions

[0074] pH Col-0 brm-3 brm-20 stop1 6.5 0.21 ± 0.04 d ]] 0.44 ± 0.08 c ]] 0.40 ± 0.04 c ]] 0.22 ± 0.05 d ]] 4.7 0.66 ± 0.11 b ]] 2.35 ± 0.15 a ]] 1.89 ± 0.32 a ]] 0.24 ± 0.09 d ]]

[0075] Table 5 Root elongation (cm) of wild type, BRM deletion mutant and NRT1.1 deletion mutant and double mutant under different pH conditions

[0076] pH Col-0 brm-20 nrt1.1-1 brm nrt1.1 6.5 4.88 ± 0.27 a ]] 5.12 ± 0.26 a ]] 5.32 ± 0.26 a ]] 5.37 ± 0.25 a ]] 4.7 3.26 ± 0.30 c ]] 4.51 ± 0.27 b ]] 1.54 ± 0.09 d ]] 1.39 ± 0.09 d ]]

[0077] Note: Different letters in Tables 2-5 represent significant differences.

[0078] Example 3 BRM Deletion Enhances STOP1-Activated NRT1.1 Expression and NRT1.1 Chromatin Accessibility

[0079] Next, it was demonstrated how BRM affects NRT1.1 expression in response to H + stress. Since low-pH-induced NRT1.1 expression is controlled by STOP1, it was explored whether BRM affects STOP1 protein accumulation under H + stress. By crossing pSTOP1:GFP-STOP1 (GFP-STOP1) lines with brm-20 mutants to generate pSTOP1:GFP-STOP1 / brm-20 (GFP-STOP1 / brm-20) plants, it was observed that GFP-STOP1 expression was comparable in pSTOP1:GFP-STOP1 / stop1 and pSTOP1:GFP-STOP1 / brm-20 seedlings under low-pH conditions (Table 6), indicating that BRM-repressed NRT1.1 expression is not due to changes in STOP1 protein accumulation. Figure 4

[0080] Table 6 Quantitative analysis of GFP-STOP fluorescence intensity in pSTOP1:GFP-STOP1 / stop1 and pSTOP1:GFP-STOP1 / brm-20 seedlings

[0081] ​ pSTOP1 :GFP-STOP1 / stop1 pSTOP1 :GFP-STOP1 / brm-20 GFP-STOP fluorescence intensity 1.000 ± 0.064 a ]] 1.056 ± 0.059 a ]]

[0082] Next, the present application performed chromatin immunoprecipitation (ChIP)-qPCR experiments to explore whether the BRM-STOP1 module co-regulates the expression of NRT1.1 in vivo. HBT:STOP1-GFP or empty plasmid (HBT:GFP) effector plasmids were transfected [Plasmid transfection used: Plant Protoplast Transfection Kit (C0563S, Biouniquer)] into protoplasts extracted from 3-week-old Col-0 and brm-3 mutant [Protoplast extraction reference done: Yoo, S., Cho, Y. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565-1572 (2007). https: / / doi.org / 10.1038 / nprot.2007.199.] and then immunoprecipitated with anti-GFP antibody at 4°C for 16 h. The present application found that the enrichment of STOP1 on NRT1.1 chromatin was significantly increased in the brm-3 mutant compared to the Col-0 plant (Fig. 7A, Table 7), indicating that BRM inhibits the binding of STOP1 to the NRT1.1 gene region. In addition, when exploring whether BRM alone inhibits NRT1.1 expression using the WashU Epigenome Browser, the present application found that BRM can be associated with the NRT1.1 gene region containing the STOP1 motif (Fig. 7B), indicating that BRM can inhibit the expression of NRT1.1 by inhibiting the binding of STOP1 to the NRT1.1 gene region. Figure 5 Figure 5 ​B), which was further confirmed by ChIP-qPCR with pBRM:BRM-FLAG (performed according to J. Y. Ye, W. H. Tian, M. Zhou, Q. Y. Zhu, W. X. Du, Y. X. Zhu, X. X. Liu, X. Y. Lin, S. J. Zheng, C. W. Jin, STOP1 activates NRT1.1-mediated nitrate uptake to create a favorable rhizospheric pH for plant adaptation to acidity. Plant Cell 33, 3658-3674 (2021).) Figure 5 C, Table 8). Taken together, these findings suggest that BRM antagonizes the regulation of NRT1.1 expression activated by STOP1 in response to H + Stress. Given that BRM can affect the expression of target genes by changing the accessibility of target DNA, the present application then examined the chromatin accessibility of the NRT1.1 gene by FAIRE-qPCR (performed according to M. A. Omidbakhshfard, F. V. Winck, S. Arvidsson, D. M. Riano-Pachon, B. Mueller-Roeber, A step-by-step protocol for formaldehyde-assisted isolation of regulatory elements from Arabidopsis thaliana. J. Integr. Plant Biol. 56, 527-538 (2014). S. Baum, E. Reimer-Michalski, M. R. Jaskiewicz, U. Conrath, Formaldehyde-assisted isolation of regulatory DNA elements from Arabidopsis leaves. Nat. Protoc. 15, 713-733 (2020).). Consistent with the ChIP-qPCR data, the open chromatin of the P1-1 and P2-1 regions of the NRT1.1 gene was significantly higher in the brm mutant than in the Col-0 plants, and a more pronounced effect was observed at the P2-1 region Figure 5 D, Table 9).

[0083] Table 7 ChIP-qPCR detection using Col-0 and brm-3 mutant

[0084]

[0085] Table 8 ChIP-qPCR analysis of Col-0 and pBRM:BRM-FLAG / brm-3 plants with anti-FLAG antibody

[0086]

[0087] Table 9 Open chromatin FAIRE-qPCR analysis of P1-1 and P2-1 regions of NRT1.1 relative to UBQ10 coding sequence

[0088]

[0089] Note: Different letters in Tables 6-9 represent significant differences.

[0090] Example 4 BRM is upstream of STOP1 in regulating NO3 - uptake and H + tolerance

[0091] To clarify the genetic relationship between BRM and STOP1 in response to H + stress, the inventors generated a brm stop1 double mutant by crossing brm-20 and stop1 mutants Figure 6 . Three-day-old plants (Col-0, brm-20, stop1, brm stop1) were transferred to neutral or low pH (neutral pH 6.5 and low pH 4.7) medium for 3 days. In low pH (pH 4.7) agar medium, the root growth of brm stop1 double mutant was similar to that of stop1 mutant, but significantly shorter than that of Col-0 and brm mutant Figure 7 A, Figure 7 B, Table 10). In addition, the NO3 - uptake of brm-20 plants was higher than that of Col-0 Figure 7 C, Table 11), which is consistent with the higher expression of NRT1.1 in the roots of brm-20 mutant Figure 2 E). However, the induction of NO3 - uptake by acid stress in brm-20 plants was completely blocked by the loss of function of STOP1 Figure 7 C). These results indicate that BRM is upstream of STOP1 in regulating NO3 - uptake and H + tolerance

[0092] Table 10 Root elongation (cm) of wild type, BRM loss-of-function mutant, STOP1 loss-of-function mutant and double mutant under different pH conditions

[0093] pH Col-0 brm-20 stop1 brm stop1 6.5 2.147 ± 0.131 a ]] 1.933 ± 0.210 a ]] 2.298 ± 0.241 a ]] 2.140 ± 0.218 a ]] 4.7 1.197 ± 0.136 b’ ]] 1.780 ±0.081 a’ ]] 0.570 ± 0.174 c’ ]] 0.578 ± 0.123 c’ ]]

[0094] Table 11 Root growth of wild type, BRM deletion mutant, STOP1 deletion mutant and double mutant under different pH conditions 15 NO3 - Absorption amount (pmol g -1 DW h -1 )

[0095] pH Col-0 brm-20 stop1 brm stop1 6.5 108.406 ± 14.319 c ]] 150.118 ± 17.197 b ]]> 105.938 ± 15.491 c ]] 105.921 ± 10.549 c ]] 4.7 159.368 ± 15.470 b ]]> 198.600 ± 13.762 a ]] 120.763 ± 15.251 c ]] 125.311 ± 18.378 c ]]>

[0096] Note: Different letters in Tables 10-11 represent significant differences.

[0097] Example 5 BRM deletion enhances the response of H + stress-induced rhizosphere alkalization

[0098] Since the STOP1-NRT1.1 module plays an important role in the induction of root H + coupled NO3 - absorption under low pH conditions, the present application hypothesized that BRM might be an effective gene to create a favorable rhizosphere pH for root growth and inhibit soil acidification when absorbing NO3 - from nitrogen fertilizer through genetic manipulation under low pH conditions. To verify this hypothesis, the present application first studied the effect of mixed co-planting of brm-3 and stop1 on the root growth of stop1 in agar medium. 3-day-old Col-0 and brm-3 seedlings were single-planted or mixed co-planted in neutral pH or low pH (neutral pH 6.5 and low pH 4.7) agar medium, and the plant growth was analyzed after 6 days of treatment. Compared with single-planting treatment, when brm-3 and stop1 mutants were mixed co-planted in agar medium with an initial pH of 4.7, the root growth of stop1 was significantly improved, reaching about 80% of that of the brm-3 mutant, while single-planting was only 57% of that of the brm-3 mutant Figure 9 A, in the neutral pH agar medium Figure 9 B, Table 13). In addition, the present application also studied the effect of mixed co-planting of Col-0 and brm-3 on the root growth of Col-0 in agar medium Figure 8 , Table 12). Considering that the basal expression of the STOP1-NRT1.1 module in Col-0 already has the ability to alkalinize the rhizosphere, more acidic agar medium (initial pH = 4.5) and younger seedlings pre-cultured in agar medium were used in the experiments described in this example. Similarly, mixed co-planting of Col-0 and brm-3 improved the root growth of Col-0 under low pH conditions Figure 8). These results suggest that BRM knock-out enables plants to create a favorable rhizosphere pH for plant growth in response to acidic stress.

[0099] To further determine the effect of BRM on rhizosphere pH regulation, 3-day-old seedlings were grown in acidic agar medium with or without 0.75 mM pH buffer Homo-PIPES for 3 days, which was used to inhibit pH changes. In low-pH agar medium with pH buffer, the root growth of both brm-3 and Col-0 was greatly inhibited, and there was no significant difference between stop1 mutants Figure 9 C, Figure 9 D, Table 14); while in low-pH agar medium without pH buffer, the root growth of the brm-3 mutant was much better than that of Col-0 and stop1 mutants. In addition, by pre-treating 10-day-old Arabidopsis seedlings in pH 4.7 agar medium for 2 days, followed by pH-sensitive dye bromocresol purple staining and pH measurement, the present application observed that the degree of rhizosphere alkalization of the brm-3 mutant was greater than that of Col-0 after 12 h of low-pH (pH 4.7) treatment Figure 9 E, Figure 9 F, Table 15). Taken together, these results suggest that BRM deficiency increases rhizosphere pH to enhance H + tolerance and has the potential to inhibit soil acidification in acidic soils.

[0100] Table 12 Root elongation (cm) of wild type, BRM deficiency mutant, single planting and mixed co-planting under different pH conditions

[0101]

[0102] Table 13 Root elongation (cm) of BRM deficiency mutant, STOP1 deficiency mutant, single planting and mixed planting under different pH conditions

[0103]

[0104] Table 14 Root elongation (cm) of Col-0, brm-3 and stop1 seedlings in agar medium with or without pH buffer

[0105]

[0106] Table 15 pH value in rooting agar medium

[0107] Col-0 brm-3 stop1 pH 5.193 ± 0.045 b ]]> 5.386 ± 0.111 a ]] 4.823 ± 0.088 c ]]

[0108] Note: Different letters in Tables 12-15 represent significant differences.

[0109] Example 6H+ Stress-induced BRM degradation

[0110] Next, this invention investigates how BRM is regulated in the root system to cope with H. + Stress. The transcriptional and protein levels of BRM in the roots of wild-type plants at different time points (2h, 4h, 24h) after low pH (pH 4.7) treatment were detected by RT-qPCR analysis and fluorescence level observation. It was found that although low pH treatment had no significant effect on BRM transcriptional levels... Figure 10 (Table A, 16), but it significantly reduced the nuclear localization of BRM-GFP in the root epidermal cells of pBRM:BRM-GFP / brm-20 transgenic plants (after 4 h of treatment) ( Figure 10 B, Figure 10 (See Table 17, C).

[0111] Table 16. Relative expression levels of BRM at different time points after low pH treatment.

[0112]

[0113]

[0114] Table 17 Effect of pBRM:BRM-GFP accumulation in the roots of pBRM:BRM-GFP / brm-20 seedlings under neutral or low pH treatment for 4 h.

[0115] pH BRM-GFP relative fluorescence intensity 6.5 1.000+0.095 a ]]> 4.7 0.477+0.097 b ]]

[0116] Note: Different letters in Tables 16 and 17 represent significant differences.

[0117] Understanding how plants adapt to H + Stress and nutrient acquisition under acidic conditions are crucial for improving nutrient use efficiency and crop yield in acidic soils. Root H mediated by the STOP1-NRT1.1 module... + The No.3 of the Couple - Absorption is essential for improving plant H2O levels. + Key biological processes of tolerance. Here, the present invention identifies BRM as a pH-dependent inhibitor of the STOP1-NRT1.1 module, revealing the role of plant H... + A new regulatory layer in the adaptation process. In this model ( Figure 11 BRM physically interacts with STOP1 and binds to the NRT1.1 genomic region, inhibiting STOP1's transcriptional activation of NRT1.1. Low pH promotes BRM degradation to alleviate this inhibition, allowing STOP1 to enhance H+ activation by activating NRT1.1. + The No.3 of the Couple -Absorption, and in turn, the promotion of rhizosphere alkalization, establishes a favorable pH environment for root growth under acidic conditions.

[0118] In summary, the research of the present application establishes BRM as a key regulator of the STOP1-NRT1.1 module, which coordinates rhizosphere pH homeostasis and plant adaptation to acidic conditions by regulating H + coupled NO3 - absorption to coordinate rhizosphere pH homeostasis and plant adaptation to acidic conditions, thereby establishing H + stress signals with nutrient acquisition. The elucidation of the BRM-STOP1-NRT1.1 pathway in the present application, combined with future research into BRM degradation mechanisms and its broader regulatory networks, will drive the development of acid-tolerant crops and help address the global challenge of soil acidification.

[0119] As can be seen, the BRM of the present application can physically interact with the core acid-tolerant transcription factor STOP1, inhibit the transcriptional activation activity of STOP1, and in turn affect plant acid tolerance. Therefore, the BRM gene of the present application can be inhibited to enhance plant acid tolerance, promote plant root growth and nitrogen nutrient absorption under acidic conditions, and promote rhizosphere alkalization. The present application provides an effective genetic resource for cultivating acid-tolerant plants and improving acid soils while utilizing acid soils by interpreting the function of the BRM gene.

[0120] Although the above embodiment describes the present application in detail, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. The application of the BRM gene in regulating plant acid resistance, characterized in that, The accession number of the BRM gene in the GenBank / EMB L database is AT2G46020.

2. The application according to claim 1, characterized in that, The acid resistance includes at least one of the following effects in acidic environments: promoting root elongation, promoting nitrogen utilization by plants, and promoting alkalization of the rhizosphere environment of plant roots.

3. The application according to claim 1, characterized in that, The regulation includes inhibiting or knocking out the BRM gene to improve plant acid resistance; and overexpressing the BRM gene to reduce plant acid resistance.

4. The protein encoded by the BRM gene, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

1.

5. Primer pairs for amplifying the BRM gene, characterized in that, The primer pair includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:3; The accession number of the BRM gene in the GenBank / EMBL database is AT2G46020.

6. A biomaterial capable of regulating BRM gene expression, characterized in that, The accession number of the BRM gene in the GenBank / EMBL database is AT2G46020.

7. The biomaterial according to claim 6, characterized in that, This includes biological materials that overexpress the BRM gene and biological materials that inhibit the expression of the BRM gene or knock out the BRM gene; Biological materials that overexpress the BRM gene include one or more of the following: a recombinant expression vector containing the BRM gene, a recombinant microorganism containing the BRM gene, and a recombinant microorganism containing the recombinant expression vector. The suppression methods include silencing.

8. The use of the protein of claim 4, the primer pair of claim 5, or the biomaterial of claim 6 or 7 in regulating plant acid resistance and / or creating plant varieties with target acid resistance.

9. A method for improving the acid resistance of plants, characterized in that, Includes the following steps: By knocking down or eliminating the BRM gene in the genome of target plants, plants with improved acid resistance can be obtained. The accession number of the BRM gene in the GenBank / EMBL database is AT2G46020.

10. A method for improving acidic soil, characterized in that, This includes planting plants obtained using the method of claim 9 in acidic soil environments.

Images