Salt-tolerant gene msaH2a.w from miscanthus sinensis and use thereof

Abstract

The application discloses a salt-tolerant gene MsaH2A.W from Miscanthus sacchariflorus and application thereof, and belongs to the technical field of biotechnology.A new salt-tolerant gene MsaH2A.W is cloned and identified from Miscanthus sacchariflorus for the first time.The MsaH2A.W gene is transformed into plants to be overexpressed, so that the salt-tolerant ability of the transgenic plants can be significantly improved, the root growth of the transgenic plants under salt stress is promoted, and then the yield and quality of the transgenic plants are improved.Therefore, the MsaH2A.W gene has very important significance for improving the growth of plants under salt stress.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a salt-tolerant gene MsaH2A.W from Miscanthus sacchariflorus and its application. BACKGROUND

[0002] Salt stress is one of the main abiotic stresses in nature, which seriously affects the ecological environment and agricultural production of human beings. At present, the area of saline-alkali land in the world is about 954 million hectares, about 20%-50% of irrigated farmland is affected by soil salinization, and the saline-alkali land in China is about 99.133 million hectares, accounting for about 4.88% of the available land area in China. The saline-alkali land affects the normal growth of plants due to the presence of a large amount of soluble salt in the soil.

[0003] Histone is one of the basic components of chromatin structure, and histone variants and histone modifications are two basic chromatin structure regulators. Among the four core histones (H2A, H2B, H3, H4) that constitute the nucleosome, H2A has the most variant types and plays an important role in chromatin structure regulation. For example, under salt stress, the enrichment level of H2A.Z in the promoter region of Arabidopsis AtMYB44 gene significantly decreases, which weakens the affinity of AtMYB44 protein binding, leading to a significant increase in the number of AtMYB44 transcripts, thereby regulating plant response to salt stress. Previous studies on H2A and its variants have mainly focused on the regulation of plant growth and development mediated by epigenetics, but the mechanism of histone variants in response to abiotic stress is very complex and has not been well elucidated.

[0004] Miscanthus is a perennial tall grass with high C4 photosynthetic efficiency and high biomass. It has strong stress resistance, such as salt-alkali tolerance, drought tolerance, poor soil tolerance, cold tolerance, and waterlogging tolerance, and can grow in moderate to severe saline-alkali land. It has rough cultivation, high water and fertilizer utilization efficiency, and low production input, and can be harvested continuously for 30 years after planting once. The main species of Miscanthus in China include Miscanthus sinensis, M. sacchariflorus, M. lutariorum, and M. giganteus, etc. There is rich genetic diversity among different species, and the stress resistance varies among different species. Interspecific hybridization exists strong heterosis. Miscanthus has attracted widespread attention from the international community as a new energy source for cellulose biomass, forage, papermaking, edible fungus production, and ornamental plants, and has become one of the hot research fields in biology.

[0005] Different species of Miscanthus have different salt tolerance and waterlogging tolerance. Therefore, Miscanthus sacchariflorus can be used as a source of salt-tolerant genes. Exploring salt-tolerant genes in the genome of M. sacchariflorus has important breeding value and significance for salt-tolerant breeding of food and economic crops. However, there are few reports on salt-tolerant genes derived from M. sacchariflorus. SUMMARY

[0006] In view of the prior art, the purpose of this invention is to provide a salt-tolerant gene MsaH2A.W from the Miscanthus genus and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the invention provides the use of the MsaH2A.W gene in either (1) or (2) below:

[0009] (1) Improve the salt tolerance of plants;

[0010] (2) Promote root growth in plants under salt stress conditions;

[0011] The MsaH2A.W gene is any of the nucleic acid molecules shown in i)-iii) below:

[0012] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0013] ii) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2;

[0014] iii) Nucleic acid molecules that encode the amino acid sequence shown in SEQ ID NO.3, other than i) or ii).

[0015] A second aspect of the present invention provides the use of the protein encoded by the MsaH2A.W gene in either (1) or (2) below:

[0016] (1) Improve the salt tolerance of plants;

[0017] (2) Prepare products that improve the salt tolerance of plants;

[0018] The amino acid sequence of the protein is shown in SEQ ID NO.3.

[0019] A third aspect of the present invention provides the use of a recombinant expression vector containing the MsaH2A.W gene, a transgenic cell line, or an engineered bacterium in any of the following (1)-(3):

[0020] (1) Improve the salt tolerance of plants;

[0021] (2) Promote root growth in plants under salt stress conditions;

[0022] (3) Cultivate plant varieties with improved salt tolerance.

[0023] In the above applications, the recombinant expression vector can be constructed using existing plant expression vectors, such as pCAMBIA3300-35S-3xFlag-Nos, pMal-c2x-mbp, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN, or other derived plant expression vectors.

[0024] The host cells of the engineered bacteria can be Escherichia coli, Agrobacterium, etc.

[0025] In the above applications, the plants include, but are not limited to: reeds, Arabidopsis thaliana, southern reeds, sorghum, rice, corn, and wheat.

[0026] A fourth aspect of the present invention provides a method for improving the salt stress tolerance of plants, comprising the step of overexpressing the MsaH2A.W gene in plants.

[0027] In the above methods, overexpression of the MsaH2A.W gene in plants can be achieved by exogenously transferring the MsaH2A.W gene; or by upregulating the expression of the MsaH2A.W gene or its homologs in the plant genome.

[0028] In a fifth aspect, the invention provides the use of the MsaH2A.W gene or a recombinant expression vector containing the MsaH2A.W gene, transgenic cell lines or engineered bacteria in the cultivation of transgenic plants;

[0029] The MsaH2A.W gene is any of the nucleic acid molecules shown in i)-iii) below:

[0030] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0031] ii) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2;

[0032] iii) Nucleic acid molecules that encode the amino acid sequence shown in SEQ ID NO.3, other than i) or ii).

[0033] In the above applications, the genetically modified plants cultivated showed increased tolerance to salt stress compared to wild-type plants.

[0034] The beneficial effects of this invention are:

[0035] This invention marks the first time a novel salt tolerance gene, MsaH2A.W, has been cloned and identified from the genus *Miscanthus* sacchariflorus. Transforming plants into MsaH2A.W and overexpressing it significantly enhances the salt tolerance of the transgenic plants, promotes root growth under salt stress, and consequently improves yield and quality. Therefore, the MsaH2A.W gene is of great significance for improving plant growth under salt stress. Attached Figure Description

[0036] Figure 1 Phylogenetic analysis of the MsaH2A.W protein.

[0037] Among them, AtH2A.W.6 (Arabidopsis thaliana, NP_200795.1), EsH2A.W.6 (Eutremasalsugineum, XP_006400900.1), MsiH2A.W (Reed, CAD6333595.1), MuH2A.2 (Musaacuminata subsp.Malaccensis, XP_009409009.1), SbH2A.4 (Sorghum, XP_002441223.1), OsH2A.2 (Rice, XP_015640099.1), SiH2A.4 (Setaria italica, XP_004961835.1), and ZmH2A.4 (Maize, ACG38394.1).

[0038] Figure 2 Expression levels of the *MsaH2A.W* gene and its homology AtH2A.W.6 in *Arabidopsis thaliana* under salt stress.

[0039] RNA-seq data of *Reedia spp.* and *Arabidopsis thaliana* under salt treatment conditions were processed to obtain TPM values ​​of gene expression.

[0040] Figure 3 Growth of wild-type and transgenic plants overexpressing *Dioscorea MsaH2A.W* under salt stress.

[0041] A. Semi-quantitative RT-PCR detection of wild-type Arabidopsis and transgenic Arabidopsis lines; B. Phenotypic characteristics of wild-type and overexpressing W6 seedlings after 10 days of culture on medium containing 100 mM NaCl; C. Length of primary roots of wild-type and overexpressing W6 seedlings after 10 days of culture on medium containing 100 mM NaCl. Wherein, WT: wild-type control; OE-2, OE-7: overexpressing transgenic lines. Detailed Implementation

[0042] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0043] As mentioned earlier, Miscanthus (M. sacchariflorus) possesses characteristics such as salt and alkali tolerance, drought tolerance, moisture tolerance, and tolerance to poor soil, making it suitable as a plant for improving saline-alkali land. Therefore, Miscanthus can serve as a source of salt-tolerant genes; however, reports of salt-tolerant genes derived from Miscanthus are currently rare.

[0044] Based on this, this invention conducted in-depth research on the Miscanthus plant *M. sacchariflorus* to uncover potential salt-tolerant genes. The inventors previously analyzed the transcriptional profile of *M. sacchariflorus* under salt stress treatment, identifying many genes potentially related to salt tolerance. Further, by analyzing the expression profiles of highly homologous genes in *Arabidopsis thaliana* and *M. sacchariflorus* under salt treatment, and based on differences in gene expression levels, this invention screened the *MsaH2A.W* gene from *M. sacchariflorus* as a candidate salt-tolerant gene.

[0045] To identify the function of the MsaH2A.W gene, this invention cloned the MsaH2A.W gene from reeds. Total RNA was extracted from reed shoots and reverse transcribed to obtain cDNA. The MsaH2A.W protein sequence was predicted based on RNA-seq Denovo assembly results, primers were designed, and conventional PCR was performed. The PCR product of appropriate size was ligated into the pMD19-T simple vector, transformed into *E. coli* DH5α competent cells, recombinants were screened, and sequencing analysis confirmed the presence of the full-length cDNA sequence of the MsaH2A.W gene.

[0046] Then, a plant overexpression vector containing the MsaH2A.W gene was constructed, and T3 generation transgenic Arabidopsis thaliana was obtained using the inflorescence infection method. Salt tolerance was then analyzed. The results showed that overexpression of the MsaH2A.W gene in transgenic Arabidopsis thaliana significantly improved its salt tolerance.

[0047] The above results indicate that the MsaH2A.W gene is a novel salt tolerance gene in the Miscanthus genus that is associated with resistance to salt stress.

[0048] The cDNA of the MsaH2A.W gene is shown in SEQ ID NO.1, as follows:

[0049]

[0050] Note: Within the transparent box This indicates the start codon, while the area within the gray box... This indicates the stop codon.

[0051] The coding region sequence of the MsaH2A.W gene is shown in SEQ ID NO.2, as follows:

[0052]

[0053] The amino acid sequence of the protein encoded by the MsaH2A.W gene is shown in SEQ ID NO.3, as follows:

[0054]

[0055] Based on the above findings, the scope of protection of this invention also includes the function of DNA fragments homologous to the MsaH2A.W gene, provided that the proteins they encode are functionally equivalent to the protein shown in SEQ ID NO.3. The phrase "functionally equivalent to the protein shown in SEQ ID NO.3" as used herein means that the protein encoded by the target DNA fragment is the same as or similar to the protein shown in SEQ ID NO.3 in this invention in terms of biological function and physiological and biochemical characteristics. The typical biological function of the protein shown in SEQ ID NO.3 is to enhance the plant's tolerance to salt stress.

[0056] These DNA fragments homologous to the MsaH2A.W gene include alleles, homologous genes, mutant genes, and derived genes corresponding to the nucleotide sequence of the present invention (SEQ ID NO.1 or SEQ ID NO.1); the proteins they encode are similar to the protein shown in SEQ ID NO.3 of the present invention, or there are substitutions, deletions, or insertions of one, several, or dozens of amino acids, all of which fall within the scope of the present invention.

[0057] Arabidopsis thaliana is chosen as the transgenic object in this invention because of its rapid growth, short life cycle, simple and easy-to-operate transformation method, and high genetic transformation efficiency. However, the MsaH2A.W gene and the plant expression vector containing this gene can also be used to produce other transgenic plants with improved salt tolerance.

[0058] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0059] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0060] Example 1: Cloning of the *MsaH2A.W* gene

[0061] Total RNA was extracted from the young shoots of reeds using the Trizol method, and cDNA was obtained by reverse transcription. The specific method is as follows:

[0062] (I) Extraction of total RNA

[0063] (1) Weigh about 0.1-0.2g of young shoots of reed, grind them into powder in liquid nitrogen, transfer them into 1mL of Trizol extract pre-cooled at 4℃, vortex and let stand at room temperature for 10min to fully dissolve them;

[0064] (2) After homogenization, centrifuge at 4℃ and 12000rpm for 10min;

[0065] (3) Take the supernatant, add 1 / 5 volume of chloroform, shake vigorously for 15 seconds, mix well, and let stand at room temperature for 2-3 minutes to precipitate the protein.

[0066] (4) Centrifuge at 4℃ and 12000 rpm for 15 min;

[0067] (5) Transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, gently invert and mix, let stand at room temperature for 10 min, centrifuge at 4℃ and 12000 rpm for 10 min.

[0068] (6) Discard the supernatant, add 1 mL of pre-cooled 75% ethanol, shake to resuspend, and centrifuge at 4°C and 7500 rpm for 5 min.

[0069] (7) Rinse 2-3 times with 75% ethanol, centrifuge at 4℃, 7500 rpm for 5 min;

[0070] (8) On a sterile workbench, leave the lid open for 5-10 minutes to dry the RNA. After the ethanol has evaporated completely, rehydrate the RNA with 30-50 μL of water treated with LDPPC.

[0071] (9) Determine the concentration of RNA. The concentration of RNA, and the ratios of A260 / A280 and A260 / A230, were determined using a NanoDrop 2000 micro-spectrophotometer.

[0072] (10) Store at -80℃ or immediately perform the following reverse transcription experiment.

[0073] (II) Synthesis of the first strand of reverse-transcribed cDNA

[0074] (1) Prepare the following mixture in a 0.2 ml RNase-free centrifuge tube (where the RNA is obtained from the extraction in step (I); if the RNA is stored at -80℃, it must be allowed to thaw slowly on ice):

[0075]

[0076] (2) Gently mix with a pipette tip, and place the centrifuge tube in a PCR instrument (65℃ for 5 minutes) for denaturation and annealing reaction;

[0077] Denaturation and annealing reaction conditions:

[0078]

[0079] (3) Prepare the following reverse transcription reaction solution in the centrifuge tubes mentioned above.

[0080]

[0081] (4) Perform reverse transcription reaction on a PCR instrument:

[0082]

[0083] The synthesized reverse transcription product cDNA was used for subsequent related experiments.

[0084] (III) Obtaining the full-length cDNA sequence

[0085] Based on the nucleotide sequence of the gene, specific primers (W6-F / 2R) with restriction enzyme sites were designed, and PCR amplification was performed using the cDNA synthesized by reverse transcription in step (II) as a template.

[0086] H2A.W.6-F;5'- GGATCC ATGGATGCCGGAGCAAAGGT-3'(SEQ.ID.NO.4)

[0087] Note: The underlined part here is the BamHI restriction site;

[0088] H2A.W.6-2R;5'- ACTAGT TGCGACGGCCGCCTTCTTGGG-3'(SEQ.ID.NO.5)

[0089] Note: The underlined part here is the SpeI restriction site.

[0090] PCR amplification system (this system will be used for all subsequent double-primer PCR reactions)

[0091]

[0092] PCR reaction program: 98℃ pre-denaturation for 5 minutes; cycling parameters: 98℃ denaturation for 10 seconds, 58℃ annealing for 5 seconds, 72℃ extension for 30 seconds, for 32 cycles; 72℃ extension for 10 minutes.

[0093] (iv) Transformation and Sequencing

[0094] After the PCR reaction was completed, 1.0% agarose gel electrophoresis was performed to check for bands of appropriate size, and then the following detailed procedures were performed:

[0095] (1) PCR product recovery: The recovery was performed according to the TRAN company's "EasyPure Quick Gel Extraction Kit";

[0096] (2) Vector ligation: Take 4.5 μl of PCR product and ligate it with the pMD19-T vector. Follow the instructions for pMD19-TVector.

[0097] (3) Transformation: The ligation product was transformed into competent Escherichia coli DH5α cells and cultured upside down at 37°C for 12-20 hours on LB agar plates containing ampicillin; single white colonies were picked and cultured overnight in LB liquid medium.

[0098] (4) Plasmid extraction: plasmid DNA of pMD19-T-MsaH2A.W was extracted by alkaline lysis method;

[0099] (5) Enzyme digestion identification: Identification by double enzyme digestion with BamHI and SpeI;

[0100] (6) Sequencing: Take 1 ml of the bacterial solution that was correctly identified by enzyme digestion and put it into a 1.5 ml centrifuge tube, seal it, and send it to Ruiboxingke Biotechnology Co., Ltd. for sequencing;

[0101] After sequencing, nucleotide and amino acid sequences were aligned using DANMAN software to obtain the gene MsaH2A.W, whose nucleotide sequence is shown in SEQ ID NO.1; the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. The plasmid DNA of the correctly sequenced monoclonal pMD19-T-MsaH2A.W was preserved and stored at -20℃ for subsequent functional verification experiments.

[0102] Example 2: Amino acid sequence analysis and cluster analysis of the *MsaH2A.W* protein

[0103] (1) The full-length cDNA of the MsaH2A.W gene is 886 bp, including a 480 bp open reading frame (ORF). Sequence analysis using VectorNTI software revealed that it encodes 159 amino acids, with a predicted molecular weight of approximately 16.57 kDa and an isoelectric point (pI) of 10.68. Analysis of the functional and conserved domains of the MsaH2A.W protein using the InterProScan database and analysis software revealed that the MsaH2A.W protein contains the IPR002119 domain (annotated as Histone_H2A).

[0104] (2) The NCBI database was searched for the following homologous genes of MsaH2A.W (unnamed protein): AtH2A.W.6 (Arabidopsis thaliana, NP_200795.1), EsH2A.W.6 (Eutrema salsugineum, XP_006400900.1), MsiH2A.W (Southern Dichroa, CAD6333595.1), MuH2A.2 (Musa acuminata subsp. Malaccensis, XP_009409009.1), SbH2A.4 (Sorghum, XP_002441223.1), OsH2A.2 (Rice, XP_015640099.1), and SiH2A.4 (Setaria). italica, XP_004961835.1), ZmH2A.4 (maize, ACG38394.1).

[0105] (3) The amino acid sequence of the above sequence was aligned using MUSCLE software (https: / / www.ebi.ac.uk / Tools / msa / muscle / ), and then a phylogenetic tree was constructed for H2A family proteins in different species using the maximum likelihood method using MEGA software (https: / / www.megasoftware.net / ). It was found that MsaH2A.W of Reed is most closely related to SbH2A.4 in sorghum, ZmH2A.4 in maize, MsiH2A.W in Reed spp., and OsH2A.2 in rice. Figure 1 ).

[0106] Example 3: Expression analysis of the *MsaH2A.W* gene and its homolog *AtH2A.W* in *Arabidopsis thaliana* under salt stress.

[0107] (1) The data used in this experiment were downloaded from NCBI, with the accession number SRP133460. The experimental procedure was to treat four-week-old Arabidopsis thaliana and reeds with 100mM NaCl for 3 days, and then collect the samples to extract RNA; RNA library construction and sequencing were performed.

[0108] (2) The sequencing reads were processed using the specialized RNA-seq data analysis software Salmon to obtain the expression level of each gene (expressed as TPM value, TPM stands for Transcripts Per Kilobase Million). Based on the phylogenetic analysis results in Example 2, the expression levels of MsaH2A.W in Reed and AtH2A.W in Arabidopsis were extracted. The results are as follows: Figure 2 As shown.

[0109] Example 4: Construction of the MsaH2A.W gene overexpression vector

[0110] To study the function of the MsaH2A.W gene, a 477bp fragment containing the coding region of the MsaH2A.W gene (as shown in SEQ ID NO.2, excluding the stop codon) was correctly inserted into the expression vector pCAMBIA3300-35S-3xFlag-Nos.

[0111] The expression vector with a 35S promoter is constructed using the following method:

[0112] (1) The plasmid DNA obtained in step four of Example 1 and the pCAMBIA3300-35S-3xFlag-Nos plasmid were simultaneously digested with two restriction enzymes, BamHI and SpeI. The MsaH2A.W fragment and the pCAMBIA3300-35S-3xFlag-Nos vector fragment were recovered and ligated together with T4 ligase. Transformation and positive clone identification were then performed, following the same steps as in step four of Example 1. Positive clones were screened, and the correct recombinant pCAMBIA3300-35S::MsaH2A.W-3xFlag-Nos was selected from them.

[0113] (2) Agrobacterium GV3101 competent cells were transformed with the constructed recombinant pCAMBIA3300-35S::MsaH2A.W-3xFlag-Nos. PCR identification was performed, and positive colonies were picked and preserved. A single clone of the correctly constructed recombinant pCAMBIA3300-35S::MsaH2A.W-3xFlag-Nos was used for subsequent Arabidopsis transformation.

[0114] Example 5: Obtaining Transgenic Arabidopsis

[0115] (1) Arabidopsis thaliana is of the Columbia ecotype. When the transplanted Arabidopsis thaliana seedlings grow to the flowering period, they can be transformed by inflorescence infection.

[0116] (2) Select the correct Agrobacterium monoclonal colony and inoculate it into 5 mL of YEP liquid medium (containing 50 mg / L kanamycin and 100 mg / L rifampin), and incubate at 28℃ and 200 rpm with shaking until the OD600 is 0.6-0.8 (about 48 hours).

[0117] (3) Take 1 mL of the bacterial culture and add it to 20 mL of fresh YEP liquid culture medium. Incubate at 28°C and 200 rpm with shaking until the OD600 is 0.6-0.8 (about 5 hours).

[0118] (4) Pour the bacterial cells into a large centrifuge tube and centrifuge at 5000 rpm for 5 min at room temperature. Discard the supernatant, collect the bacterial cells, and resuspend them in an appropriate amount of infection solution containing 5% sucrose (w / v) and 0.04% Silwet-L77 (v / v). Adjust the bacterial concentration to OD600 = 0.5-1.0 and use it for Arabidopsis inflorescence infection.

[0119] (5) Select wild-type Arabidopsis thaliana in the flowering period, water it thoroughly with 1 / 3 Hongland nutrient solution one day in advance, and remove the flowers and pods that have already opened, in preparation for infection the next day;

[0120] (6) Immerse the Arabidopsis inflorescence in the staining solution for about 15 seconds, then remove the flower from the liquid surface and absorb the excess staining solution with absorbent paper.

[0121] (7) The infected Arabidopsis thaliana was cultured in the dark in a greenhouse for 24 hours, and then transferred to a long-day environment for normal growth. The culture room was managed normally. After one week, it could be infected again, and the seeds were harvested for screening of transgenic seedlings.

[0122] Example 6: Molecular identification of transgenic Arabidopsis thaliana genomic DNA

[0123] Genomic DNA was extracted from different lines of transgenic plants and wild-type plants using the CTAB method. Using this DNA as a template, PCR amplification was performed using the upstream primer H2A.W.6-F of MsaH2A.W and the primer 3xFlag-R on the 3xFlag tag sequence. Transgenic plants were identified by the ability to amplify clear bands.

[0124] 3xFlag-R:5'-GTCATCATCGTCTTTGTAGTC-3'(SEQ ID NO.6)

[0125] Example 7: Salt tolerance analysis of transgenic Arabidopsis thaliana

[0126] To determine the function of the transgenic plants, we analyzed the salt tolerance of the T3 generation transgenic Arabidopsis thaliana lines.

[0127] (1) Relative expression levels of the MsaH2A.W gene in different T3 generation transgenic Arabidopsis thaliana lines.

[0128] After screening with antibiotics (containing 50 mg / L hygromycin), Arabidopsis thaliana transformant lines underwent genomic DNA molecular identification according to the method in Example 5. Seven T2 generation lines were randomly selected from the obtained transgenic Arabidopsis thaliana lines, and the corresponding RNA was extracted and reverse transcribed into cDNA, following the same method as step two in Example 1. Specific primers W6-F and W6-R and Arabidopsis thaliana internal reference primers EF1α-F and EF1α-R were designed in the non-conserved region of the MsaH2A.W gene.

[0129] EF1α-F:5'-GTATGGTTGTTACCTTTGCTCCCACAG-3'(SEQ ID NO.7)

[0130] EF1α-R:5'-CATCATTTGGCACCCTTCTTCACTGC-3'(SEQ ID NO.8)

[0131] The cDNA templates were adjusted using Arabidopsis thaliana internal reference primers EF1α-F and EF1α-R to ensure consistent cDNA template concentrations, and then semi-quantitative RT-PCR was performed.

[0132] The reaction program was as follows: pre-denaturation at 98℃ for 5 minutes; cycling parameters were: denaturation at 98℃ for 30 seconds, annealing at 58℃ for 30 seconds, extension at 72℃ for 30 seconds, for 20-25 cycles; followed by a 10-minute extension at 72℃. The amplified products were analyzed by electrophoresis on a 1% agarose gel, and band brightness was detected using a BIO-RAD Gel Doc XR gel imaging system to determine the expression level of the MsaH2A.W gene in these transgenic lines. The results showed that the expression level of the MsaH2A.W gene varied among different lines, with the highest expression level observed in OE-2. Figure 3 A) Select two lines with high expression levels, OE-2 and OE-7, and collect seeds from individual plants to obtain the corresponding T3 generation seeds for subsequent transgenic function verification experiments.

[0133] (2) Growth of Arabidopsis thaliana 5 days after seed germination of generation T3 after treatment with 100mM NaCl:

[0134] T3 generation transgenic Arabidopsis thaliana seeds OE-2 / 7 and wild-type control WT seeds were sterilized and spread on sterilized soil. Planted on MS medium, vernalized at 4°C in the dark for 5 days. Then, they were removed and placed in a 22°C short-day incubator for 5 days of upright cultivation. Seedlings with uniform growth were then transplanted separately. PNS medium and 100mM NaCl added The roots were cultured vertically with their roots facing down on PNS medium for 10 days. Phenotypic differences were observed and root length was measured.

[0135] MS medium and The raw material composition of PNS culture medium is the same as that of the corresponding culture medium described in Example 7 of Patent CN114214334A.

[0136] The results showed that: under normal conditions On PNS medium, the growth of transgenic lines and wild-type seedlings was largely consistent. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) On PNS medium, the root length of transgenic seedlings was longer than that of wild-type plants. Figure 3 B). The above results indicate that the transfer of the MsaH2A.W gene into Arabidopsis thaliana improves the salt tolerance of transgenic Arabidopsis plants.

[0137] In summary, this invention isolated a histone H2A gene, MsaH2A.W, from the Miscanthus sinensis plant. Functional analysis of the gene in Arabidopsis thaliana revealed that it plays a crucial role in resisting high salt stress, making it a novel salt-tolerant gene. This gene can be transformed into annual crops such as wheat, maize, and rice, or perennial woody plants such as apples and pears, to enhance their salt stress resistance, thereby increasing their yield and quality, and generating significant economic and social benefits.

[0138] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. MsaH2A.W Application of genes in promoting root growth in Arabidopsis thaliana under salt stress conditions; MsaH2A.W Genes are any of the nucleic acid molecules shown in i)-iii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; iii) Nucleic acid molecules that encode the amino acid sequence shown in SEQ ID NO.3, other than i) or ii).

2. MsaH2A.W Application of a gene-encoded protein in promoting root growth in Arabidopsis thaliana under salt stress; the amino acid sequence of the protein is shown in SEQ ID NO.

3.

3. Contains MsaH2A.W The application of recombinant gene expression vectors, transgenic cell lines, or engineered bacteria in promoting root growth in Arabidopsis thaliana under salt stress; characterized in that, The MsaH2A.W Genes are any of the nucleic acid molecules shown in i)-iii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; iii) Nucleic acid molecules that encode the amino acid sequence shown in SEQ ID NO.3, other than i) or ii).

4. The application according to claim 3, characterized in that, The host cells of the engineered bacteria are selected from Escherichia coli or Agrobacterium.

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