Wheat TaRVE8-4A gene and application thereof

Abstract

The invention discloses a wheat TaRVE8-4A gene and application of the wheat TaRVE8-4A gene in regulating and controlling the tolerance of wheat to salt stress and / or high temperature stress, and belongs to the technical field of gene engineering. The nucleotide sequence of the CDS region of the TaRVE8-4A gene is as shown in SEQ ID NO. 1. According to the invention, by cloning and analyzing the wheat TaRVE8-4A gene, silencing the wheat TaRVE8-4A gene by combining a BSMV-VIGS technology and verifying the function of the gene by a wheat transgenic overexpression technology, the expression of the TaRVE8-4A gene is found to be closely related to the salt tolerance and high temperature resistance of wheat. The invention provides a theoretical basis and related genes for salt-resistant and high-temperature-resistant breeding and production of wheat.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to wheat. TaRVE8-4A Genes and their use in regulating wheat tolerance to salt and / or heat stress. Background Technology

[0002] Soil salinization, an increasingly serious global agricultural problem, has become one of the key factors limiting global crop yields and quality.

[0003] In recent years, global warming has led to frequent extreme heat events, significantly impacting crop production and threatening food security and sustainable agricultural development (Challinor et al., 2014). Studies show that for every 1°C increase in global temperature, the average yield of the four major crops—wheat, rice, corn, and soybeans—will decrease by approximately 6.0%, 3.2%, 7.4%, and 3.1%, respectively (Zhao et al., 2017). Against this backdrop, improving the heat resistance and climate adaptability of crops is particularly urgent.

[0004] Salt and heat tolerance in plants are complex quantitative traits involving a series of physiological and biochemical reactions. To cope with high salt and high temperature stress, plants have evolved mechanisms including reactive oxygen species scavenging, stomatal behavior regulation, and sodium ion efflux mechanisms. In actual production, high temperature and salt stress often occur simultaneously; therefore, breeding new crop varieties with both salt and heat tolerance is of great significance.

[0005] wheat( Triticum aestivum Wheat (L.) is one of the most important food crops in the world. After thousands of years of hybridization and domestication, the common wheat that is now widely planted is an allohexaploid (AABBDD). Its complex genome structure, while giving it excellent agronomic traits such as high yield and quality, may also weaken some stress resistance traits present in wild relatives, including salt tolerance and heat tolerance.

[0006] Currently, several salt tolerance or heat tolerance-related genes have been identified in wheat using forward and reverse genetics methods. However, compared with major grains such as rice and maize, the number of key wheat genes whose in situ functional verification has been completed remains limited. Therefore, identifying key genetic loci / genes for salt and heat tolerance in wheat and elucidating new mechanisms of salt tolerance are fundamental to breeding superior wheat materials with strong salt and heat tolerance.

[0007] The RVE (REVEILLE) family is an important subfamily of the MYB-related (1R-MYB) subfamily within the MYB superfamily. It includes CCA1 (CIRCADIAN CLOCK ASSOCIATED 1), LHY (LATE ELONGATED HYPOCOTYL 1), RVE1-RVE8, and RVE7-like, all containing the conserved sequence SHAQK(Y / F)F. Except for RVE5 and RVE6, the expression of the remaining members in seedlings is regulated by the biological clock (Liu et al., 2023). A few studies have shown that RVEs participate in abiotic stress responses. For example, Shan et al. (2021) found that the soybean gene GmMYB133 is homologous to the Arabidopsis RVE8 branch gene. Under salt stress, overexpression of GmMYB133 in Arabidopsis promoted seed germination and plant growth, increased chlorophyll content, and decreased malondialdehyde (MDA) content. Consistent with this, overexpression of GmMYB133 significantly upregulated the expression of four positive regulators of salt tolerance, suggesting that GmMYB133 may enhance the salt stress tolerance of plants. Bao et al. (2024) constructed transgenic Arabidopsis lines overexpressing GmRVE8a. The results showed that under salt and drought stress, the transgenic seedlings exhibited better growth than the wild type, and the malondialdehyde (MDA) content in the transgenic lines was significantly lower than that in the wild type, indicating that GmRVE8a may be a positive regulator of salt and drought stress responses. Currently, there are no reported studies on the function of RVE family genes in wheat. Summary of the Invention

[0008] The purpose of this invention is to provide a method for processing wheat ( Triticum aestivum Genes involved in wheat salt stress and / or heat stress response, cloned from L., were applied to the breeding and production of salt-tolerant and heat-tolerant wheat germplasm.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention identifies a gene involved in wheat salt tolerance response based on bioinformatics analysis and gene expression analysis. Using the salt-tolerant germplasm X118 previously identified by our research group as material, the full-length CDS sequence of this gene was cloned. The corresponding nucleotide sequence is shown in SEQ ID NO.1, and it is named... TaRVE8-4A. Gene sequence analysis results show that TaRVE8- 4A It contains 8 exons and 7 introns, with a total CDS length of 828 bp.

[0010] The TaRVE8-4AThe protein encoded by the gene consists of 275 amino acid residues, and its amino acid sequence is shown in SEQ ID NO. 2. Analysis of the protein sequence using the InterPro website revealed that the protein contains a Myb-like DNA-binding domain, indicating that this gene belongs to the MYB-related subfamily.

[0011] TaRVE8-4A Expression pattern analysis: Quantitative real-time PCR analysis revealed that under salt stress conditions, the sensitive variety X276 showed expression patterns within 48 hours. TaRVE8-4A Expression levels continued to rise. Subcellular localization analysis showed that TaRVE8-4A was located in the cell nucleus.

[0012] This invention utilizes barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) technology and wheat overexpression technology to... TaRVE8-4A Gene validation showed that under salt stress conditions, compared to the control group, TaRVE8-4A Gene-silenced plants exhibit enhanced salt tolerance; TaRVE8-4A Gene overexpression lines exhibited significantly enhanced salt sensitivity. Under high-temperature stress, compared to the control group, TaRVE8-4A The heat tolerance of the gene-overexpressing lines was significantly reduced. This indicates that the... TaRVE8-4A Genes negatively regulate wheat's tolerance to salt stress and / or high temperature stress.

[0013] Therefore, the present invention provides the aforementioned TaRVE8-4A Application of genes in regulating wheat tolerance to salt and / or heat stress.

[0014] Furthermore, the aforementioned TaRVE8-4A Gene silencing significantly enhanced wheat tolerance to salt and / or heat stress. Studies have shown that under salt stress conditions, compared to the control group, TaRVE8-4A Gene-silenced plants showed significantly increased fresh and dry weight in both aboveground and belowground parts. Mechanistic studies revealed that gene silencing significantly increased the aboveground and belowground parts of wheat plants. TaRVE8-4A Gene silencing reduces sodium ion absorption, indicating... TaRVE8-4A Genes may be involved in regulating Na + The transfer process.

[0015] Furthermore, the application includes: using biological techniques to induce... TaRVE8-4A Gene downregulation or loss of function can improve plant tolerance to salt stress and / or heat stress.

[0016] Furthermore, the application includes: utilizing gene mutation, gene knockout, or gene silencing technologies to cause the... TaRVE8-4AGene loss of function, thereby obtaining mutant plants with enhanced tolerance to salt stress and / or heat stress.

[0017] As a specific embodiment of the present invention, the BSMV-VIGS method is used to silence wheat. TaRVE8-4A Genes. Research shows that silencing... TaRVE8-4A The gene does not affect the normal growth of wheat, but can significantly improve tolerance to salt stress and / or high temperature stress.

[0018] Specifically, this invention provides a breeding method for improving wheat's tolerance to salt stress and / or high temperature stress, comprising the following steps: (1) The nucleotide sequence is as shown in SEQ ID NO.3. TaRVE8-4A Gene fragments were inserted into the NheI site of the BSMV:γ vector to construct the recombinant vector RNAγ:TaRVE8-4A; (2) After linearizing the vectors RNAα, RNAβ and RNAγ: TaRVE8-4A, the RNAβ and RNAγ were transcribed in vitro and then infected into wheat seedlings to obtain mutant plants with enhanced tolerance to salt stress and / or high temperature stress.

[0019] The beneficial effects of this invention are as follows: This invention utilizes wheat TaRVE8-4A Gene cloning and analysis, combined with BSMV-VIGS technology to silence wheat TaRVE8-4A Gene and wheat transgenic overexpression technology were used to verify the function of this gene, and it was found that... TaRVE8-4A Gene expression is closely related to the salt and heat tolerance of wheat. This invention provides a theoretical basis and related genes for wheat salt and heat tolerance breeding and production. Attached Figure Description

[0020] Figure 1 for TaRVE8-4A Bioinformatics analysis. Where (a) is... TaRVE8-4A Gene structure; (b) is TaRVE8- 4A Domain prediction.

[0021] Figure 2 for TaRVE8-4A Expression levels at different time points after salt treatment.

[0022] Figure 3 for TaRVE8-4A Natural variations are associated with wheat salt tolerance. Among them, (a) shows the results identified based on 125 genotypes. TaRVE8-4A (a) Two haplotypes (Hap I and Hap II); (b) Salt damage index of the two haplotypes. The smaller the salt damage index, the stronger the salt tolerance. * indicates a significant difference. p <0.05.

[0023] Figure 4 Subcellular localization of transient expression of TaRVE8-4A protein in tobacco. sGFP is a green fluorescent protein; the markers used are located in the nucleus and cell membrane, with a scale bar of 10 μm.

[0024] Figure 5 For silence TaRVE8-4A Effects on salt tolerance of wheat seedlings. (a) represents the BSMV. TaRVE8-4A With BSMV: γ Salt treatment phenotypes of plants, scale bar: 2.5 cm; (b) fresh weight of aboveground and underground parts; (c) dry weight of aboveground and underground parts; (d) Na+ of aboveground and underground parts. + Content; (e) is K content in the aboveground and underground parts. + Content. * and ** respectively indicate content in p <0.05 and p The difference was statistically significant at levels <0.01.

[0025] Figure 6 for TaRVE8-4A Soil culture experiment to identify salt tolerance in overexpressing plants. (a) shows the ratio of wild-type (WT) to... TaRVE8-4A The salt stress phenotype of overexpressing lines (L2, L7) was determined by experiments conducted in plastic pots filled with cultivation substrate. Three-leaf stage seedlings were treated with 200 mM NaCl for 10 days; (b) survival rate after 30 days of salt stress; (c) fresh and dry weight of aboveground parts after 30 days of salt stress; (d) NaCl concentration of aboveground parts after 30 days of salt stress. + and K + Content. * and ** respectively indicate content in p <0.05 and p The difference was statistically significant at levels <0.01.

[0026] Figure 7 High temperature treatment during seedling stage on WT and TaRVE8-4A The effects of overexpression on the growth of the strains are shown in the left image (photograph) and the right image (statistical chart of mortality rate).

[0027] Figure 8 Wild type and after high temperature treatment during the grouting period TaRVE8-4A The effect of overexpression on the strains; the left figure shows the thousand-grain weight; the right figure shows the yield per plant. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0030] This invention uses wheat salt-tolerant germplasm X118, sensitive genotypes X276, and X133 (Lu et al., 2025) previously identified by our research group as materials to clone and analyze genes involved in wheat salt tolerance. TaRVE8-4A This study is of great significance for elucidating the molecular mechanisms of wheat response to high temperature and salt stress, as well as for breeding and production.

[0031] Example 1: TaRVE8-4A Cloning and analysis of gene CDS regions 1. TaRVE8-4A Cloning of gene CDS region This embodiment identifies a gene involved in wheat salt tolerance based on bioinformatics analysis and gene expression analysis. Using salt-tolerant germplasm X118 as material, the full-length CDS sequence of this gene was cloned, and the corresponding nucleotide sequence is shown in SEQ ID NO.1. The gene was named... TaRVE8-4A .

[0032] Total RNA was extracted from X118 leaves using a total RNA extraction kit. Genomic DNA contamination was removed using DNase I, followed by reverse transcription into single-stranded cDNA using a PrimeScripffM II 1st Strand cDNA Synthesis Kit. Primers were designed based on the BLAST sequence, and amplification was performed using KOD ONE high-fidelity enzyme. The primer sequences are as follows: TaRVE8-4A -CDS-F: 5′-ATGGCGGCGGTGATGGAGTCGT-3′; TaRVE8-4A -CDS-R: 5′-TCAGGGATGCGTATGCTCAGGTGT-3′.

[0033] The amplified product was ligated into the pMD18-T vector, transformed into E. coli DH5α, and single clones were selected for sequencing.

[0034] 2. TaRVE8-4A Gene sequence analysis Gene sequence analysis results show that TaRVE8-4A It contains 8 exons and 7 introns, with a full-length CDS of 828 bp, encoding 275 amino acids. Figure 1 (a) Protein sequence analysis was performed using the InterPro website. Figure 1Figure b shows the structural domain of this protein: the Myb-like DNA-binding domain (33-78 aa), which has the ability to bind DNA, indicating that the gene belongs to the MYB-related subfamily.

[0035] 3. TaRVE8-4A Expression pattern analysis Seeds of X118 (tolerant genotype) and X276 (sensitive genotype) were sterilized by soaking in 2% H2O2 for 20 minutes, rinsed thoroughly with distilled water, and whole, healthy seeds were sown in germination boxes at 22ºC / 18ºC. Healthy, uniformly growing wheat seedlings at the two-leaf stage were transplanted into 1 / 5 Hogland nutrient solution. After 5 days of pre-culture, salt treatment was performed, with a control (basal culture medium) and a salt treatment (150 mM NaCl). Samples were taken at 3 h, 6 h, 12 h, and 48 h for gene expression analysis.

[0036] Total RNA was extracted from different samples, reverse transcribed into cDNA, and then analyzed using the SYBR green fluorescent enzyme complex and a LightCycler 480 PCR instrument. TaRVE8-4A Expression level change (qRT-PCR), primer sequences are: TaRVE8-4A -qRT-PCR-F: 5′-GCACACCCGTACCCACATAA-3′; TaRVE8-4A -qRT-PCR-R: 5′-TTGTTCCACGACATGAGGGG-3′; TaActin -F: 5′-AGTTCACGGCCATGTTCA-3′; TaActin -R: 5′-ACGAGGTCGTTCATGTTGCT-3′.

[0037] The results of expression level measurement are as follows Figure 2 As shown, in the salt-tolerant variety X118, gene expression levels did not change significantly from 3 to 12 hours, but slightly increased from 12 to 48 hours. In the sensitive variety X276, gene expression levels remained elevated for 48 hours. These results indicate that salt stress can induce changes in wheat gene expression levels. TaRVE8-4A The expression.

[0038] In order to study TaRVE8-4A To investigate whether differences exist between different wheat genotypes, wheat was cloned from 125 wheat materials using PCR technology. TaRVE8-4A The CDS sequence and primer sequences are: TaRVE8-4A-CDS-F: 5′-ATGGCGGCGGTGATGGAGTCGT-3′; TaRVE8-4A -CDS-R: 5′-TCAGGGATGCGTATGCTCAGGTGT-3′.

[0039] A total of 34 sequence variation sites were identified in these materials. Correlation analysis using salt tolerance phenotypic data revealed 13 variation sites significantly associated with salt tolerance (Figure 3a). Based on these variation sites, the 125 wheat accessions were divided into two haplotypes (Hap I and Hap II). The majority of genotypes (87.2%) belonged to Hap II. Statistical analysis of salt tolerance data for the two haplotypes showed that the Hap I genotype exhibited stronger salt tolerance than the Hap II genotype (Figure 3b). These results indicate that the genetic variation sites contained in Hap I have significant application value for the genetic improvement of wheat salt tolerance.

[0040] 4. Subcellular localization of TaRVE8-4A With saved pMD18-T- TaRVE8-4A - Using CDS plasmid as a template, primers were designed to amplify the complete plasmid. TaRVE8-4A The CDS fragment, after removing the stop codon, has the following primer sequences (lowercase letters represent homologous arm sequences): TaRVE8-4A -YXB-F: 5′-gggacgagctcggtacccgggATGGCGGCGGTGATGGAG-3′; TaRVE8-4A -YXB-R: 5′-gctcaccatgtcgactctagaGGGATGCTTGTATGCTCAGGTG-3′.

[0041] XbaI and SmaI were selected as restriction enzyme sites to digest the SGFP vector. After purification, the SGFP vector was ligated with the target gene fragment. TaRVE8-4A The ligation product of the gene fragment was transformed into *E. coli* DH5α (Takara, 9057) and sequenced. Single clones with correct sequencing were selected, and plasmids were extracted and stored. The subcellular localization vector, nuclear marker, membrane marker, and SGFP plasmid were then transformed into *Agrobacterium* EHA105. After the bacterial culture became turbid, PCR verification was performed. Positive single clones were selected for testing, and bacterial cultures with correct sequencing were stored.

[0042] 100 μL of subcellular localization vector, nuclear marker, membrane marker, and SGFP were added to 15 mL of LB containing kanamycin and rifampin, respectively. The mixture was shaken at 200 rpm at 28 °C until the OD (λ = 600) reached approximately 1.0. After centrifugation at 4000 g for 5 min, the mixture was resuspended in infection buffer (containing 10 mM MES, 150 mM AS, and 10 mM MgCl2), and centrifuged again. The resuscitation was repeated until the OD (λ = 600) reached approximately 0.8. The mixture was allowed to stand for 4 h, and tobacco (pre-dried for 48 h) was injected using a 1 mL syringe with the needle removed. The mixture was then stored in the dark for 24 h. 48 h after injection, the subcellular localization signals of GFP and mCherry fluorescence were observed using a laser scanning confocal microscope (FV3000, Japan). The excitation wavelengths for GFP and mCherry were 488 nm and 561 nm, respectively.

[0043] The results are as follows Figure 4 As shown, TaRVE8-4A is located in the cell nucleus, which is consistent with its predicted location as a transcription factor.

[0044] Example 2: Validation of the BSMV-VIGS method TaRVE8-4A Gene function 1. BSMV: TaRVE8-4A Carrier construction Using the pMD18-T-TaRVE8-4A-CDS plasmid preserved in the laboratory as a template, primers were designed to amplify a 205 bp TaRVE8-4A gene fragment (nucleotide sequence shown in SEQ ID NO.3), and the target gene fragment did not contain NheI (GCTAGC), MluI (ACGCGT), or SpeI (ACTAGT) restriction sites. The primer sequences are as follows: TaRVE8-4A -BSMV-F: 5′-gtgatgattcttcttccgttGCTAGCGGGCCTAGGTGGTGGCAGATGT-3′; TaRVE8-4A -BSMV-R: 5′-gatcaaacatttttttttttttttagctagcTCGTCGGTACCCGGCGTGGA-3′.

[0045] The γ-vector was digested with NheI and purified. The γ-vector was then ligated with the target gene fragment. The ligation product of the TaRVE8-4A gene fragment was transformed into DH5α competent cells (Qingke Biotechnology). Sequencing was performed using the primer γ-stain-F on the γ-vector. Single clones with the gene fragment reverse-inserted into the γ-vector were selected, and the plasmid was extracted, named γ:TaRVE8-4A, and stored. The primer sequences are as follows: γ-stain-F: 5′-CAACTGCCAATCGTGAGTAGG-3′.

[0046] 2. Linearization and in vitro transcription of BSMV vector RNAα, RNAγ, γ:TaPDS, and γ:TaRVE8-4A were digested with restriction endonuclease MluI, respectively; RNAβ was digested with restriction endonuclease SpeI. The digestion products were then used for in vitro transcription using the Ribo MAX™ Large Scale RNA Production System-T7 kit (Promega, USA) and the Ribom 7G Cap Analog kit (Promega, USA) according to the manufacturer's instructions. One μL of the transcribed product was analyzed by 1% (w / v) agarose gel electrophoresis to confirm clear and non-diffuse bands. The remaining product was stored at -80°C for later use and inoculated with viruses as soon as possible.

[0047] 3. BSMV vaccination verification TaRVE8-4A Gene function Hydroponic experiments were conducted using X133 as the experimental material. Germination and cultivation methods were as described above. Inoculation was prepared when wheat seedlings reached the two-leaf-one-heart stage. In vitro transcribed RNAα, RNAβ, and RNAγ:GFP / γ:TaPDS / γ:TaRVE8-4A were mixed in a 1:1:1 volume ratio, diluted with twice the volume of RNase-free water, and then an equal volume of 2×GKP buffer (1% bentonite, 1% diatomaceous earth 545, 50 mM glycine, 30 mM dipotassium hydrogen phosphate, pH adjusted to 9.2) was added and thoroughly mixed. The mixtures were named BSMV:γ, BSMV:TaPDS, and BSMV:TaRVE8-4A for subsequent inoculation. The second fully expanded leaf of the wheat was selected for inoculation. 10 μL of the virus mixture was taken from each leaf and gently rubbed. Immediately after inoculation, the plants were sprayed with a small amount of DEPC-treated water and covered with a transparent plastic cover to provide a high-humidity environment. The cover was removed after 3 days. The entire operation was performed while maintaining an RNase-free state.

[0048] When the new leaves of wheat plants inoculated with the BSMV:TaPDS system show whitening symptoms, it proves that the BSMV-VIGS system was successfully inoculated. Salt stress treatment was applied to wheat plants inoculated with other virus systems to determine the function of candidate genes. A total of 4 treatments were set up: (1) leaf inoculation with BSMV:γ; (2) leaf inoculation with BSMV:γ and 150 mM NaCl salt stress treatment; (3) leaf inoculation with BSMV:TaRVE8-4A; (4) leaf inoculation with BSMV:TaRVE8-4A and 150 mM NaCl salt stress treatment.

[0049] After salt stress treatment, the growth of the plants was observed. When obvious phenotypic differences appeared among wheat seedlings of different treatments, seedlings with the same growth were photographed. The aboveground and underground parts of each line were taken, rinsed with deionized water and dried, and the fresh weight was measured. After drying in an oven at 80℃ for 48 h, the dry weight and ion content were measured.

[0050] The results are as follows Figure 5 As shown, under normal conditions, no significant phenotypic differences were observed between plants inoculated with BSMV:γ and BSMV:TaRVE8-4A, indicating that silencing TaRVE8-4A under normal conditions does not affect the normal growth of wheat. Under 150 mM salt treatment, BSMV:TaRVE8-4A plants exhibited enhanced salt tolerance: compared with control plants, their aboveground and belowground fresh weights increased significantly by 21.7% and 43.3%, respectively, and their dry weights increased significantly by 11.8% and 18.0%, respectively. The Na+ content in the aboveground parts of the silenced plants... + The content decreased significantly by 21.6%, but the Na content in the underground part... + Content, aboveground and underground K + There was no significant difference in content. These results indicate that TaRVE8-4A negatively regulates salt tolerance in wheat and may be involved in regulating Na+ content. + The transfer process.

[0051] Example 3: Wheat Expression Validation TaRVE8-4A Gene function Based on the previously cloned sequences, specific primers were designed. The primer sequences are as follows. TaRVE8-4A -OE-F:5′-GTTGGGCGGTCGTTCATTC-3′; TaRVE8-4A -OE-R: 5′-AGAACATCGTCCCAGGAAGG-3′.

[0052] Amplification using KOD One DNA polymerase TaRVE8-4AThe CDS sequence was ligated into the pLGY-OE3 vector using the Novita-Tex source recombination method. The constructed overexpression recombinant plasmid was transformed into Agrobacterium EHA105 via heat shock and plated onto LB solid medium containing rifampicin and kanamycin resistance. The medium was incubated overnight at 28°C, and single colonies were picked for expansion. Next, 200 μL of the bacterial culture was added to 15 mL of liquid medium (Kan+Rif) and incubated on a shaker at 28°C until the OD value reached approximately 0.2. After centrifugation, the supernatant was discarded, and the prepared infection solution was added to adjust the OD value to 0.6-0.8.

[0053] Agrobacterium-mediated transformation was used to inoculate wheat embryonic callus tissue after induced differentiation. After co-culture, the callus tissue was transferred to a selective medium containing screening agents for selection. Identified callus tissue was then transferred to differentiation and rooting media, respectively, and cultured until the transgenic seedlings developed well. Afterward, the seedlings were transplanted into a greenhouse to complete the creation of overexpression materials. Based on the vector sequence and the CDS sequence of TaRVE8-4A, positive seedling verification primers were designed. PCR detection was performed on the extracted tissue culture seedling DNA, and transgenic plants were screened by sequence alignment. Two transgenic lines, L2 and L7, were selected for salt and heat tolerance assessment.

[0054] A. Salt tolerance assessment Wild-type (WT), overexpression lines L2 and L7 seeds were disinfected by soaking in 2% H2O2 for 20 minutes and then rinsed thoroughly with distilled water. Intact and healthy seeds were selected and sown in nutrient soil. Salt stress treatment was carried out at the two-leaf-one-heart stage. Two treatments were set up: (1) control group; (2) experimental group: 200 mM NaCl salt stress treatment.

[0055] The results are as follows Figure 6 As shown, under normal growth conditions, no significant phenotypic differences were observed between the wild-type and overexpression lines (OE) in the soil culture experiment. However, under salt stress, after 10 days of salt treatment, both L2 and L7 showed significantly enhanced salt sensitivity compared to the wild-type. Figure 6 (a). After 30 days of salt treatment, the survival rate of the overexpression lines was significantly reduced: 30.0% for L2, 26.7% for L7, and 53.3% for the wild type. Figure 6 (b). After 30 days of treatment, compared with the wild type, the aboveground fresh weight of L2 and L7 was significantly reduced by 38.4% and 36.3%, respectively, and the aboveground dry weight was significantly reduced by 24.5% and 33.3%, respectively. Figure 6 (c). Ion homeostasis is also disrupted: compared to the wild type, the aboveground Na+ of L2 is significantly higher. + and K + The content of all three increased significantly, while there was no significant difference in L7. Figure 6 d).

[0056] B. High-Temperature Resistance Functional Assessment Using the above planting method, WT and overexpression lines were planted in nutrient soil. Seedlings that grew to two leaves and one bud were treated with a high temperature of 42℃ for 8 days. After 5 days of recovery, the mortality rate of wild-type and TaRVE8-4A overexpression lines was measured.

[0057] The results are as follows Figure 7 As shown, the overexpression lines L2 and L7 exhibited significantly reduced high-temperature tolerance and significantly higher mortality rates than the wild-type WT.

[0058] Ten days after wheat flowering, the plants were placed in a high-temperature incubator for 16 hours during the day and 8 hours at night. The thousand-grain weight and yield per plant were measured after 5 days of treatment.

[0059] The results are as follows Figure 8 As shown, after high-temperature treatment, the thousand-grain weight and yield per plant of the overexpression lines L2 and L7 were significantly reduced compared with the wild type.

[0060] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. TaRVE8-4A The application of genes in regulating wheat tolerance to salt and / or heat stress is characterized by, The TaRVE8-4A The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The TaRVE8-4A The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The application as described in claim 1 or 2, characterized in that, The TaRVE8-4A Genes negatively regulate wheat's tolerance to salt stress and / or high temperature stress.

4. The application as described in claim 3, characterized in that, The TaRVE8-4A Gene silencing significantly enhanced wheat's tolerance to salt and / or heat stress.

5. The application as described in claim 4, characterized in that, wheat plants TaRVE8-4A After gene silencing, the absorption of sodium ions is reduced.

6. The application as described in claim 1, characterized in that, The applications include: using biological techniques to induce [something] in wheat plants. TaRVE8-4A Gene downregulation or loss of function can improve plant tolerance to salt stress and / or heat stress.

7. The application as described in claim 6, characterized in that, The use of gene mutation, gene knockout, or gene silencing techniques to cause the above TaRVE8-4A Gene loss of function, thereby obtaining mutant plants with enhanced tolerance to salt stress and / or heat stress.

8. A breeding method for improving wheat's tolerance to salt stress and / or high temperature stress, characterized in that, Includes the following steps: (1) The nucleotide sequence is as shown in SEQ ID NO.

3. TaRVE8-4A Gene fragments were inserted into the NheI site of the BSMV:γ vector to construct the recombinant vector RNAγ:TaRVE8-4A; (2) After linearizing the vectors RNAα, RNAβ and RNAγ: TaRVE8-4A, the RNAβ and RNAγ were transcribed in vitro and then infected into wheat seedlings to obtain mutant plants with enhanced tolerance to salt stress and / or high temperature stress.

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