An artificial promoter responsive to salt stress, a reporter system and a method for improving plant salt tolerance

Abstract

The present invention belongs to the field of genetic engineering and relates to a method for monitoring plant salt stress and improving plant salt tolerance. First, a gene containing an AuxRE sequence is added to the gene. SOS1 The first exon of the gene is added to SOS1 In addition, a new artificial promoter fragment SOS1g+SOS1pro was synthesized before the promoter, and the SOS1g+SOS1pro::GUS transgenic GUS reporter strain was created. This strain can more sensitively and accurately monitor the degree of salt stress suffered by plants through simple GUS staining. sos1 The mutant homozygous complementation line proved that the phenotypic analysis of salt stress treatment confirmed that the presence of this sequence can significantly improve the salt resistance of plants; CHIP, EMSA and LUC experiments were also used to analyze the auxin response factor ARF7 binding to the AuxRE sequence on this exon and activating SOS1 expression, thereby significantly enhancing the plant's salt resistance.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to a method for monitoring plant salt stress and improving plant salt tolerance. Background Art

[0002] In recent years, global soil salinization has become increasingly serious. Food crops in many countries and regions have been threatened by salt stress caused by soil salinization, resulting in reduced crop yields or reduced quality. Salt stress refers to the multi-level and multi-faceted harm to plant growth and development caused by a large increase in the soluble salt content in the soil, such as inhibiting the germination of plant seeds and the establishment of seedlings, reducing the absorption of water and nutrients by the roots, and reducing photosynthesis, which ultimately seriously affects the growth and development of plants. With the gradual acceleration of the industrialization process and unreasonable irrigation methods, the area of ​​arable land in my country has been decreasing while the area of ​​saline-alkali land has been increasing. Therefore, the management of saline-alkali land and the cultivation of new varieties of salt-resistant crops are imminent. Traditional management methods consume a lot of manpower and material resources, and the effect is not ideal. In recent years, with the development of molecular biology and the increasing updating of genetic engineering application technology, it has become an effective means to explore the salt-resistant genes of plants themselves and improve the salt tolerance of crops through genetic modification. However, there are still few genes that can improve plant salt resistance.

[0003] The SOS pathway is one of the main salt resistance pathways evolved by plants. SOS1 Encodes Na + / H + Antiporter that transports excess Na + Excreted from the body, it is one of the key salt-resistant genes in plants. Publication No. CN117624317A discloses that mutation of the SOS1 gene can greatly improve the salt tolerance of yeast. When plants are subjected to salt stress, SOS1 The transcription level of SOS1 will be significantly increased, and more SOS1 protein will be synthesized to help plants resist salt stress. SOS1 Monitoring of transcriptional levels can better reflect the extent to which plants are harmed by salt stress, and can also reflect the level of salt resistance of plants to a certain extent. SOS1 Although transgenic materials driven by their own promoters to express GUS have been reported in the literature, their sensitivity and accuracy are low, which is not conducive to breeders and farmers to monitor the degree of salt stress and salt resistance of plants and crops in real time. SOS1 There is also little research on upstream transcription factors of transcription, which greatly limits the improvement and transformation of plant salt resistance. Therefore, discovering new genes that increase SOS1 transcription and enhance plant salt resistance will help cultivate new salt-resistant crops. Summary of the invention

[0004] To solve the above technical problems, the present invention proposes an artificial promoter that responds to salt stress, a reporter system and a method for improving plant salt tolerance, which provides a new approach for breeding new salt-resistant varieties from the perspective of molecular genetic engineering in the future.

[0005] The technical solution of the present invention is achieved in this way:

[0006] This application provides a SOS1 A classic ARF transcription factor binding sequence AuxRE (TGTCTC) on the first exon of the gene can more sensitively and accurately reflect the degree of plant salt stress and help improve the plant's salt resistance.

[0007] Artificial promoters responsive to salt stress, including AuxRE sequences and SOS1 The natural promoter of the gene.

[0008] Also includes SOS1 The first 329 bp of the gene starts from ATG.

[0009] The nucleotide sequence of the artificial promoter ( SOS1g+SOS1pro ) as shown in SEQ ID No.1.

[0010] The reporter system comprising the artificial promoter also comprises a reporter gene connected to the 3' end of the promoter.

[0011] The reporter gene is galactosidase GUS Dual luciferase LUC Gene.

[0012] The preparation method of the above-mentioned reporter system is to insert the above-mentioned artificial promoter before the reporter gene of the intermediate vector containing the reporter gene to obtain the reporter system.

[0013] Specifically: SOS1g+SOS1pro Galactosidase GUS Dual luciferase LUC In front of the gene, SOS1g+SOS1pro::GUS Plasmid or SOS1g+SOS1pro::LUC Plasmid.

[0014] Galactosidase GUS Genes as an example: SOS1g+SOS1pro::GUS The plasmid, i.e., the reporter system, was transformed into Agrobacterium competent GV3101, and then transgenic Arabidopsis plants were obtained by inflorescence infection method to monitor the degree of salt stress in the soil.

[0015] A method for preparing an Arabidopsis reporter strain for monitoring salt stress degree, wherein the above-mentioned reporter system is transferred into Arabidopsis by genetic transformation to obtain the Arabidopsis reporter strain.

[0016] A method for improving the salt tolerance of plants, comprising the following steps:

[0017] (1) Construction of a prokaryotic overexpression vector containing the transcription factor ARF7;

[0018] (2) The above-mentioned reporter system and the prokaryotic overexpression vector in step (1) are simultaneously transfected into the plant to be improved.

[0019] The prokaryotic overexpression vector in the above step (1) is an Escherichia coli expression plasmid.

[0020] The reporter gene in the above reporter system is LUC Gene.

[0021] The present invention has the following beneficial effects:

[0022] The present invention first comprises an AuxRE sequence SOS1 The first exon nucleic acid sequence of the gene (SOS1g, 329 bp in length) was added to SOS1 In addition, a novel artificial promoter fragment SOS1g+SOS1pro was synthesized before the promoter, and a SOS1g+SOS1pro::GUS transgenic GUS reporter strain was created. This strain can more sensitively and accurately monitor the degree of salt stress suffered by plants through simple GUS staining. At the same time, the present invention created a mutant of the AuxRE sequence. sos1 The mutant homozygous complementation strain and salt stress treatment phenotypic analysis confirmed that the presence of the sequence can significantly improve the salt resistance of plants; the present invention analyzed the auxin response factor ARF7 binding to the AuxRE sequence on the exon and activating the SOS1 expression, thereby significantly enhancing the plant's salt resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Picture 1 for SOS1 The structure diagram of the gene, P1 is the atypical ARF transcription factor binding site on the SOS1 promoter, P2 is the SOS1 A typical AuxRE (TGTCTC) sequence on the first exon.

[0025] Picture 2 for SOS1pro::GUSand SOS1g-SOSpro::GUS GUS staining results of homozygous Arabidopsis plants grown on 1 / 2 MS medium and 1 / 2 MS medium containing 100 mM NaCl.

[0026] Picture 3 Wild type (WT), sos1 Mutants, SOS1pro::SOS1 sos1 and SOS1pro::SOS1-mAuxRE sos1 The phenotype results of seed germination greening and seedling swaying under salt treatment of homozygous Arabidopsis plants. Figure A is the germination greening phenotype diagram under different salt concentrations on the 5th day after sowing, Figures B and C are the trend diagrams of seed germination rate and cotyledon greening rate of each material within 1-5 days; Figure D is the phenotype diagram of root length growth 3 days after 4-5 day old seedlings were swung on the salt plate, and Figures E and F are the statistical diagrams of the root length of new primary roots after seedling swaying treatment and the statistical diagrams of fresh weight of each 5 materials.

[0027] Picture 4 ARF7 binds to SOS1 AuxRE sequence consequences on the first exon and ARF7 activation SOS1 Expression results. Figure A shows the CHIP-qPCR results of ARF7 binding to AuxRE sequence; Figure B shows the EMSA results of ARF7 binding to AuxRE sequence; Figure C shows the structure diagram of the reporter gene and effector factor constructed in the dual luciferase reporter gene experiment; Figure D shows the activation of ARF7 through the AuxRE sequence SOS1 Figure 1. Result of dual luciferase reporter gene expression.

[0028] Picture 5 Figure 1 shows the phenotype of seed germination greening and seedling swaying of wild-type overexpression material homozygous Arabidopsis plants under salt treatment. 35S::ARF7 in materials SOS1 Gene expression results; Figure B is WT, 35S:: ARF7 Figure 3 is the germination and greening phenotype of the materials under different salt concentration treatments on the 5th day after sowing. Figure C is the root length growth phenotype of 4-5 day old seedlings 3 days after being placed on the salt board. Figures D and E are the seed germination rate trend charts and cotyledon greening rate trend charts of each material within 1-5 days. Figures F and G are the statistical charts of the root length of the newly born primary roots after the seedling placement treatment and the statistical charts of the fresh weight of every 5 materials, respectively. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] This application is SOS1 Based on the research of key salt resistance genes, the inventors provided SOS1 The new application of the AuxRE sequence in the first exon of the gene in improving the salt resistance of plants provides a new way to breed new salt-resistant varieties through molecular genetic engineering in the future.

[0032] Predictive Control SOS1 By analyzing the upstream regulatory factors of expression SOS1 Based on the possible transcription factor binding sites on the gene sequence, a segment was found in SOS1 A classic ARF transcription factor binding sequence AuxRE (TGTCTC) on the first exon of the gene can more sensitively and accurately reflect the degree of plant salt stress and help improve the salt resistance of plants. SOS1 before its own promoter and with GUS Gene fusion to obtain a new construct; the AuxRE nucleic acid sequence TGTCTG point mutation to TGAGTC, connected with SOS1 After the promoter, create a new construct; ARF7 The coding sequence of the gene is placed after the constitutive overexpression promoter 35S to obtain a new construct; after the above construct is sequenced by a sequencing company and confirmed to be correct, the artificial construct is transformed into Agrobacterium competent cells GV3101, and then the homozygous Arabidopsis plants of the corresponding construct are obtained by the inflorescence infection method;

[0033] The specific operations are as follows:

[0034] Example 1: This example mainly describes SOS1g-SOS1pro::GUS GUS staining experiment under salt treatment of plants.

[0035] (1) Construction of pCAMBIA1381-SOS1g-SOS1pro::GUS recombinant vector.

[0036] The SOS1 genome sequence was searched on the Tair website (http: / / www.arabidopsis.org / ). pro :GUS plasmid, selection EcoR I was used as a multiple cloning site, and specific primers for one-step cloning were designed. The primer sequences were designed as follows:

[0037] SOS1g-1381-F:5'-GACCATGATTACGAATTCATGACGACTGTAATCGACGCG-3';

[0038] SOS1g-1381-R:5'-GACGGATCCCCGGGAATTCctgaaacaccaaacaacatg-3';

[0039] Using Arabidopsis wild-type gDNA as a template, the above primers were used to amplify the SOS1 The 329 bp sequence of the AuxRE sequence on the first exon, i.e. SOS1g ; Then the pCAMBIA1381Z plasmid was EcoR I restriction enzyme cleavage site, enzyme cleavage products and amplified SOS1g The gene sequence was recovered after agarose gel electrophoresis, ligated, and transformed into Escherichia coli DH5a competent cells. The plasmid was extracted and further identified by PCR, enzyme screening, and sequencing to obtain the pCAMBIA1381-SOS1g-SOS1pro::GUS recombinant expression vector.

[0040] (2) Construction of transformants.

[0041] After the obtained recombinant expression vector was sequenced correctly, the constructed plasmid was transferred into Agrobacterium GV3101 to obtain a transformant.

[0042] (3) Acquisition of genetically modified plants.

[0043] The floral dipping method was used to transgenerate Arabidopsis. The specific steps of the floral dipping method are as follows:

[0044] ① Pick a single colony and inoculate it into 5 ml YEB (Rif 50 mg / ml, Kana 50 mg / ml), and culture it at 28℃ overnight. Inoculate it into 100 ml YEB (Rif 50 mg / ml, Kana 50 mg / ml) at a ratio of 1:1000 and expand the culture until OD600 = 1.0-1.2.

[0045] ② Collect bacteria into a 50ml sterile centrifuge tube and centrifuge at 5000rpm at room temperature for 5 minutes.

[0046] ③Discard the supernatant. Resuspend the cells in transformation medium (1 / 2MS, 5% sucrose, adjust pH to 5.4-5.6 with KOH, prepare immediately before use, no need to sterilize), add appropriate amount of transformation medium until OD600 = 0.8-1.0. Add 0.01% silwetL-77.

[0047] ④ When the inflorescence of Arabidopsis wild-type WT seedlings grows to 5-10 cm in height, remove the siliques and opened flowers of the plants, and infect the Arabidopsis inflorescence with Agrobacterium resuspended in the collected bacteria for transformation. The specific process is: immerse the Arabidopsis inflorescence in the bacterial solution, immerse the flowers of each plant for 30 s, place the seedling pots with immersed flowers flat on a tray, cover with plastic film to maintain humidity, place it away from light for 16-24 hours, then take it out and culture it normally; after 5-7 days, repeat the infection once.

[0048] ⑤ Continue to culture the Arabidopsis thaliana infected with Agrobacterium in the above step and harvest the seeds. Sow the harvested seeds on a 1 / 2 MS medium plate containing 25 mg / L hygromycin, vernalize at 4°C for 3 days, and move to the material room for culture for one week.

[0049] ⑥ Positive plants are resistant to hygromycin and can grow normally, while negative plants are sensitive to hygromycin, cannot grow normally and eventually die. Based on this characteristic, positive plants are screened, and the positive plants obtained can be preliminarily considered to have successfully transcribed GUS The transgenic T0 seeds were harvested, sterilized, and planted in 1 / 2MS medium containing 25 mg / L hygromycin for screening to obtain positive seedlings with exogenous transgene insertion.

[0050] ⑦ The positive seedlings screened in the above steps were transplanted into the cultivation soil (the cultivation soil was 3:1 of nutrient soil and vermiculite), cultured at 20℃-22℃, 16 h / 8 h light-dark cycle, and 80% relative humidity. The young leaves of the 2-week-old seedlings were taken for GUS staining detection, and the seedlings that could be stained were continued to be cultured until the seeds matured. The obtained seeds were further screened with hygromycin and GUS staining detection was performed, and the positive seedlings were further propagated.

[0051] ⑧ When the positive seedlings grow to two weeks, the leaves at the rosette are taken for GUS staining. The T1 generation seeds of the positive seedlings that can be stained are collected, disinfected, and then planted in 1 / 2MS medium containing 25 mg / L hygromycin for screening. The resistant plants are planted in cultivation soil (nutrient soil: vermiculite is 3:1). The collected seeds are T2 generation. After disinfection, they are further planted in 1 / 2MS medium containing 25 mg / L hygromycin for screening. This generation has begun to separate homozygous, heterozygous, and wild types (ratio is 1:2:1); homozygous strains are resistant on the resistance plate, all seeds will germinate normally, and the seeds of heterozygous strains will not germinate completely, and finally homozygous transgenic strains are obtained.

[0052] ⑧ In the above Arabidopsis cultivation process, when Arabidopsis seeds are planted in the culture medium, the Arabidopsis seeds must be disinfected. The specific method is: place the Arabidopsis seeds in a 1.5 mL centrifuge tube, disinfect the surface with 84 disinfectant for 5 minutes, rinse with sterile water 5-8 times, and then use a rubber-tipped dropper to suck the seeds for planting. The culture medium used for Arabidopsis seeds is 1 / 2 MS solid culture medium, the formula is: 1 / 2 MS salt + 1% sucrose + 1% agar powder, the pH is adjusted to 5.8-6.0 with KOH, and the culture temperature is 20-22℃.

[0053] (4) Analysis of GUS staining results of transgenic plants under salt stress.

[0054] See also Picture 2 ,Will SOS1pro::GUS Transgenic homozygous plants obtained from plants and constructs SOS1g-SOSpro:: GUS , grown in 1 / 2MS medium for 4-5 days, and then transplanted these seedlings into 1 / 2MS medium containing 100 mM NaCl and continued to grow for 3 days. Then they were stained with GUS dye for 3 h, decolorized with 75% anhydrous ethanol, and finally photographed under a stereo optical microscope after decolorization.

[0055] The results are as follows Picture 2 As shown in Figure 3, there was no obvious difference in GUS staining between the two transgenic plants on 1 / 2 MS medium. However, under the treatment with 100 mM NaCl, SOS1g-SOSpro::GUS Plant GUS staining SOSpro::GUS The plants were obviously darker than those in the SOS1 This AuxRE sequence on the exon can more sensitively and accurately reflect the degree of salt stress in plants.

[0056] Embodiment 2: SOS1 mAuxRE sos1 Analysis of salt stress phenotypes of homozygous lines

[0057] Based on the experimental results of Example 1, it can be seen that the AuxRE sequence on the first exon of SOS1 can more strongly monitor the degree of salt stress suffered by plants. Whether this sequence is related to the salt resistance of plants, the inventors further designed experiments to verify. The relevant process is briefly described as follows.

[0058] (1) Construction of pCAMBIA1300-SOS1pro::GFP recombinant vector.

[0059] The SOS1 gene promoter sequence was searched on the Tair website (http: / / www.arabidopsis.org / ) and the commercial vector pCAMBIA1300-35S-EGFP plasmid was selected. Nco I and Sac I was used as a multiple cloning site, and specific primers for one-step cloning were designed. The primer sequences were designed as follows:

[0060] SOS1pro-1300-F: 5'-CTGAATCAAAGGCCATGGccaaattttttgtacataaaatg-3';

[0061] SOS1pro-1300-R::5'-CCCCGGGTACCGAGCTCtttatttgaatatatctaagaagc-3';

[0062] The Arabidopsis wild-type gDNA was used as a template to amplify the SOS1 promoter sequence using the above primers; and then referring to the vector construction steps in Example 1, the pCAMBIA1300-SOS1pro::GFP recombinant expression vector was obtained.

[0063] (2) Construction of pCAMBIA1300-SOS1pro::SOS1-mAuxRE-GFP recombinant vector.

[0064] Based on the constructed pCAMBIA1300-SOS1pro::GFP recombinant expression vector, BamH I was used as a multiple cloning site, and a one-step cloning specific point primer was designed. The primer sequence was designed as follows:

[0065] SOS1-mAuxRE-F1:

[0066] 5'-GGGGATCCTCTAGAGTCGAATGACGACTGTAATCGACGCG-3';

[0067] SOS1-mAuxRE-R1:

[0068] 5'-CCGAGTACCAGACTCATTCCG-3';

[0069] SOS1-mAuxRE-F2:

[0070] 5'-CGGAATGAGTTCTGGTACTCGG-3';

[0071] SOS1-mAuxRE-R2:

[0072] 5'-CTTGCTCACCATGTCGACTAGATCGTCCTGAAAACG-3';

[0073] The Arabidopsis wild-type gDNA was used as a template to amplify the SOS1-mAuxRE point fragment using the above primers; and then referring to the vector construction steps in Example 1, the pCAMBIA1300-SOS1pro::SOS1-mAuxRE-GFP recombinant expression vector was obtained.

[0074] As described in (2) and (3) of Example 1, obtain pCAMBIA1300-SOS1pro::SOS1-mAuxRE-GFP sos1 Homozygous Arabidopsis lines.

[0075] (3) SOS1 mAuxRE sos1 Phenotypic analysis of materials under salt stress.

[0076] WT, sos1 , 1300-SOS1pro::SOS1-GFP sos1 ( SOS1 / sos1 )and SOS1 mAuxRE sos1 Arabidopsis seeds were sown on 1 / 2 MS and 1 / 2 MS medium containing different concentrations of NaCl, and the seed germination rate and cotyledon greening rate of each material were observed and counted for 1-5 days; similarly, the 4-5 day old seedlings of the above materials were placed on 1 / 2 MS and 1 / 2 MS medium containing 135 mM NaCl and cultured for another 3 days, and the primary root length and fresh weight of every 5 materials were observed and counted.

[0077] The results are as follows Picture 3 As shown in the figure, the results of seed germination rate and cotyledon greening rate under different concentrations of salt stress showed that sos1 The mutant was significantly salt sensitive, while SOS1 / sos1 The strain can recover to a salt stress phenotype similar to WT under salt stress. SOS1 mAuxRE sos1 Homozygous lines could not be recovered, indicating that the AuxRE sequence contributes to plant salt tolerance.

[0078] Example 3: CHIP-qPCR and EMSA to explore the binding of ARF7 to AuxRE

[0079] Based on the fact that AuxRE is essential for plant salt resistance and serves as a binding motif for the transcription factor ARF, the inventors further designed experimental investigations, and the relevant process is described as follows.

[0080] (1) pCAMBIA1300-35S::ARF7-GFP Acquisition of overexpression strains

[0081] The ARF7 (AT5G20730) coding sequence was searched on the Tair website (http: / / www.arabidopsis.org / ) and based on the pCAMBIA1300-35S-EGFP plasmid, the Sac I was used as a multiple cloning site, and specific primers for one-step cloning were designed. The primer sequences were designed as follows:

[0082] SOS1g-1381-F: 5'-CGGGGGACGAGCTCATGAAAGTCCCTTCATCAAATGGAG-3';

[0083] SOS1g-1381-R: 5'-CCCCGGGTACCGAGCTC-CCGGTTAAACGAAGTGGCTGAG-3';

[0084] Using Arabidopsis wild-type cDNA as a template, the ARF7 CDS sequence was amplified with the above primers, and then referring to the vector construction steps in Example 1, the pCAMBIA1300-35S::ARF7-GFP recombinant expression vector was obtained.

[0085] As described in (2) and (3) of Example 1, obtain pCAMBIA1300-35S::ARF7-GFP Homozygous strains.

[0086] (2) Obtain pCAMBIA1300-35S::ARF7-GFP To investigate whether ARF7 can bind to SOS1 Genetically, the inventors first conducted a CHIP-qPCR experiment, and the relevant process is briefly described as follows.

[0087] Will pCAMBIA1300-35S::ARF7-GFPHomozygous Arabidopsis seeds were sown on 1 / 2 MS medium and cultured for 2 weeks. About 300 mg of seedling material was weighed and placed in a 50 mL centrifuge tube with 20 mL fixation buffer (0.4 M Sucrose, 1 M Tris-HCl pH 8.0, 20% Triton X-100, formaldehyde) for vacuum crosslinking for 5 min; 5 mL glycine buffer (2 M Glycine) was added, mixed thoroughly, and vacuumed for 10 min to terminate crosslinking; the samples were carefully washed 3-4 times with distilled water, and excess water was absorbed by absorbent paper; the above samples were quickly ground into powder in a mortar and transferred to a 50 mL centrifuge tube, and 25 mL pre-cooled extraction buffer1 (0.4 M Sucrose, 1 M Tris-HCl pH 8.0, 0.5 M EDTA, 5 mM β-mercaptoethanol, 1 mM PMSF) in a test tube and mix thoroughly; filter through filter cloth (Miracloth, Millipore, cat#475855-1R), collect the filtrate into a 50 mL centrifuge tube, and centrifuge at 2,500 g for 10-15 min at 4°C; discard the supernatant, add 2 mL of pre-cooled extraction buffer 2 (0.25M Sucrose, 0.5 M EDTA, 1 M Tris-HCl pH 8.0, 1 MMgCl2, 20% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF) into a centrifuge tube, resuspend by pipetting, and pipette the resuspended solution into a 2 mL EP tube, and centrifuge at 12,000 g for 10 min at 4°C; discard the supernatant, add 1 mL of pre-cooled nucleilysis buffer (1 M Tris-HCl, 0.5 M EDTA, 10% SDS, 1 mM PMSF) to the precipitate, and mix by pipetting; resuspended solution 1 mL was divided into 4 tubes and broken for 10, 15, 20, and 30 cycles respectively; 16,000 g, centrifuged for 10 min, and 1 mL of supernatant was taken into a new EP tube; 900 μL of pre-cooled ChIP dilution buffer (1 M Tris-HCl pH 8.0, 5 M NaCl, 0.5 M EDTA, 20% Triton X-100, 1 mM PMSF) was added to 100 μL sepharose beads for balance; 4°C, 2,000 g centrifuged for 5 min, and the supernatant was discarded; 900 μL of pre-cooled ChIP dilution buffer, 100 μL of broken chromatin sample, and no (control group) or 2 uL GFP antibody were added, and incubated overnight on a shaker at 4°C.The sections were washed with 900 μL wash buffer (Low-salt: 5 M NaCl, 10% SDS, 20% Triton X-100, 0.5 M EDTA, 1 M Tris-HCl pH 8.0, 1 mM PMSF; High-salt: 5 M NaCl, 10% SDS, 20% Triton X-100 LiCl, 0.5 M EDTA, 1 M Tris-HCl pH 8.0, 1 mM PMSF; LiCl: 0.25 M LiCl, 20% NP-40, Sodium dcoxycholate, 0.5 M EDTA, 1 M Tris-HCl pH 8.0, 1 mM PMSF and TE: 1 M Tris-HCl pH 8.0, 0.5 M EDTA, 1 mM PMSF) in sequence, each wash was performed on a shaker at 4°C for 5 min; the washed samples were centrifuged at 2,000 g for 5 min at 4°C, the supernatant was discarded, and then 300 μL fresh elution buffer was added. Buffer (10% SDS, 0.1 M NaHCO3), vortex briefly to mix, incubate at 65°C metal bath with shaking (0.2 gagitation) for 1 h; centrifuge at 16,000 g for 5 min, transfer the supernatant to a new EP tube, add 13 μL 5 M NaCl and incubate at 65°C overnight to decrosslink; finally, extract DNA to obtain the sample.

[0088] The obtained samples were subjected to fluorescent quantitative PCR, and the CHIP probes were as follows:

[0089] SOS1-P1-F: 5'-ggtaggctagtcgtctaattctaaaatagc-3';

[0090] SOS1-P1-R: 5'-gtaataagtattccaaaacctggtttggcc-3';

[0091] SOS1-P2-F: 5'-CTGGAGGAAGCGACCGATTCG-3';

[0092] SOS1-P2-R: 5'-ggaacgaaatcgaacCGAGAG-3';

[0093] The results are as follows Picture 4 As shown in A, ARF7 can bind to the P2 site, which is SOS1 The AuxRE sequence on the first exon cannot bind to SOS1 on the promoter.

[0094] The inventors further conducted EMSA experiments to verify the binding of ARF7 to the AuxRE, and the relevant steps are briefly described as follows.

[0095] (1) Construction of pGEX4T-GST-ARF7 recombinant vector.

[0096] The ARF7 coding sequence was searched on the Tair website (http: / / www.arabidopsis.org / ) and the commercial plasmid pGEX-4T-1-GST plasmid was selected. Sma I was used as a multiple cloning site, and specific primers for one-step cloning were designed. The primer sequences were designed as follows:

[0097] ARF7-pGEX4T-F:5'-ccggaattcccgggATGAAAGTCCCTTCATCAAATGGAG-3';

[0098] ARF7-pGEX4T-R:5'-ctcgagtcgacccgggCCGGTTAAACGAAGTGGCTGAG-3';

[0099] The Arabidopsis wild-type cDNA was used as a template to amplify the ARF7 CDS sequence using the above primers; and then the vector construction steps in Example 1 were referred to to obtain the pGEX4T-GST-ARF7 recombinant prokaryotic expression vector.

[0100] (2) After obtaining the pGEX4T-GST-ARF7 recombinant prokaryotic expression vector constructed as described above, the ARF7 protein was expressed and purified in prokaryotic cells, and then the inventors conducted an EMSA experiment to verify the binding of ARF7 to the AuxRE (P2) sequence.

[0101] EMSA biotin probe primers are as follows.

[0102] SOS1pro-P2-BioF:

[0103] 5'-Bio-CGTTCTCTTCGTCGGAATGTCTCTGGTACTCGGTATTGCTTC-3';

[0104] SOS1pro-P2-F:

[0105] 5'-CGTTCTCTTCGTCGGAATGTCTCTGGTACTCGGTATTGCTTC-3';

[0106] SOS1pro-P2-R:

[0107] 5'GAAGCAATACCGAGTACCAGAGACATTCCGACGAAGAGAACG-3';

[0108] Anneal the above primers: 95℃-25℃, 0.5℃ / min; prepare 6% EMSA gel; mix the purified protein and probe according to different combinations, incubate at room temperature for 20 min; then perform electrophoresis: pre-run the gel at 100 V for 30 min. After the pre-run, pipette 1 μL of blue loading buffer and mix it with the sample thoroughly. The position of the sample during electrophoresis is judged by observing the position of the blue buffer, 100 V, 60 min, and ice cubes are required for cooling; transfer to the membrane, 100 V, 60 min, and ice cubes are used for cooling; cross-linking: 254 nm ultraviolet wavelength in the ultraviolet cross-linking instrument, irradiate cross-linking for 10 min at a distance of 5-10 cm from the membrane; blocking: after the cross-linking is completed, transfer the membrane to 15mL EMSA blocking solution and incubate for 15-30 min; biotin binding probe: aspirate the blocking solution, incubate with new blocking solution (containing 7.5μL Streptavidin-HRP Conjugate), and shake on a shaker for 15-30 min; washing and exposure: transfer the membrane to another container containing 15-20 mL EMSA washing solution, rinse for 1 min, repeat three times; then add exposure solution to react for 1-2 minutes, and expose for analysis.

[0109] EMSA results Picture 4 As shown in B, ARF7 was confirmed to bind to this AuxRE sequence.

[0110] Example 4: ARF7 Activation SOS1 Expression experiments

[0111] Based on the above binding experiments, it is shown that ARF7 can bind to SOS1 The AuxRE sequence in the first exon cannot bind to SOS1 The present invention is to investigate whether this binding can activate the expression of SOS1, thereby improving the salt resistance of plants. The inventors further designed a dual luciferase reporter gene experiment to explore this issue. The relevant process is briefly described as follows.

[0112] (1) Based on the commercial vector pGreenII 0800-LUC, use Sac I as a multiple cloning site, using the following specific primers, refer to the construction methods (1) and (2) in Example 1 to obtain pGreen0800- SOS1g+SOS1pro::LUC Recombinant expression transformants.

[0113] (2) If Picture 4, collect the Agrobacterium culture liquid of the combination in D by centrifugation and resuspend it in the transformation solution (10 mM MES, 10 mM MgCl2, 0.2 mM acetosyringone, pH 5.6), adjust the OD value to 1.0, and then inject the culture liquid into the tobacco leaves through the back of the tobacco leaves through a 1 mL syringe, the injection volume is about 0.25 mL, and then place the tobacco in a light incubator for 2-3 days, cut about 25 mg of tobacco leaves, freeze them in liquid nitrogen and quickly grind them into powder in a mortar, transfer the powder to an EP tube, add 100-150 μL of cell lysis buffer, 4℃, lyse for at least 30 min; centrifuge at 4℃, 14,000 rpm for 10 min, aspirate 50 μL of protein supernatant into the black microplate reader sample well, and do at least three parallels for each combination, and then add an equal volume of 50 Place 50 μL of firefly luciferase reaction solution in the microplate reader, shake the plate to mix, and detect the LUC fluorescence value at 560 nm; take out the microplate, add 50 μL of Renilla luciferase reaction solution to the sample well, also place it in the microplate reader, shake the plate to mix, and detect the REN fluorescence value at 480 nm; calculate the LUC / REN value.

[0114] The results are as follows Picture 4 As shown, ARF7-GFP did not activate SOS1pro::LUC when co-injected with SOS1pro::LUC. SOS1 expression, and when it is SOS1g+ When SOS1pro::LUC was co-injected, it was significantly activated SOS1 expression.

[0115] Implementation effect analysis: Salt stress phenotype analysis of 35S::ARF7-GFP homozygous materials

[0116] In summary, SOS1 The AuxRE sequence on the first exon of the gene is essential for salt resistance, and ARF7 must bind to this AuxRE sequence to be activated SOS1 Expression of ARF7 in plants, whether this combination can give plants better salt resistance, therefore, the inventors further explored by analyzing the salt stress phenotype of ARF7 overexpression materials, the brief process is as follows.

[0117] (1) The acquired 35S::ARF7-GFP Overexpression homozygous strains were treated with salt stress together with WT, and quantitative qPCR was used for detection SOS1 The results are as follows Picture 5 As shown in A, under salt stress, 35S::ARF7-GFP Overexpression homozygous material SOS1 The expression level of ARF7 was significantly higher than that of WT, which preliminarily indicated that ARF7 can also activate SOS1 Gene expression.

[0118] (2) The inventors further designed an experiment to explore the salt stress phenotype of the overexpression material, and observed and analyzed the seed germination and cotyledon greening, as well as the root length and fresh weight phenotypes of WT and the overexpression material under salt treatment (see Example 2 for the specific process). Picture 5 As shown in BG, the results show 35S::ARF7-GFP The salt resistance of the overexpression homozygous strain was significantly improved compared with that of WT.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An artificial promoter responsive to salt stress, characterized in that: The nucleotide sequence of the artificial promoter is shown in SEQ ID No.

1.

2. A reporter system comprising the artificial promoter according to claim 1, characterized in that: Also included is a reporter gene linked to the 3' end of the promoter.

3. The reporter system of the artificial promoter according to claim 2, characterized in that: The reporter gene is GUS Gene or LUC Gene.

4. The method for preparing the reporter system according to claim 2, characterized in that: The reporter system is obtained by inserting the artificial promoter described in claim 1 before the reporter gene of the intermediate vector containing the reporter gene.

5. A method for preparing an Arabidopsis reporter strain for monitoring salt stress, characterized in that: The reporter system described in claim 3 is introduced into Arabidopsis thaliana by genetic transformation to obtain an Arabidopsis thaliana reporter strain.

6. A method for improving the salt tolerance of Arabidopsis thaliana, characterized in that: The steps are: (1) constructing a prokaryotic overexpression vector containing the transcription factor ARF7; the ID number of the transcription factor ARF7 is AT5G20730; (2) The reporter system in claim 3 and the prokaryotic overexpression vector in step (1) are simultaneously transfected into the Arabidopsis thaliana to be improved.

7. The method for improving the salt tolerance of Arabidopsis thaliana according to claim 6, characterized in that: The prokaryotic overexpression vector in step (1) is an Escherichia coli expression plasmid.

8. The method for improving the salt tolerance of Arabidopsis thaliana according to claim 6, characterized in that: The reporter gene in the reporter system is LUC Gene.

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