Long-chain non-coding RNA SUR1 and application thereof in regulation and control of wheat salt stress tolerance

Abstract

The invention discloses a long-chain non-coding RNA (Ribonucleic Acid) SUR1 and an application thereof in regulating and controlling salt stress tolerance of wheat. The SUR1 in the plant is knocked out to enhance the salt stress tolerance of the plant, and the gene can be fully utilized to improve the wheat variety subsequently, so that the yield of the wheat can be increased.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a long non-coding RNA SUR1 and an application thereof in regulating wheat salt stress tolerance. Background Art

[0002] As an important food crop in my country, wheat has to maintain stable yield in salinized soil environment, which has become a major scientific proposition for sustainable agricultural development. In recent years, with the rapid development of molecular biology technology, researchers have made important progress in analyzing the molecular mechanism of wheat salt tolerance and have successfully cloned several key genes. For example, the research results of Byrt et al. showed that the high-affinity potassium ion transporter gene TaHKT1, the gene related to ion transport 5-D, and the gene encoding the plasma membrane Na + / H + The antiporters Nax1 and Nax2 act by limiting Na + transported into cells to maintain a high K + / Na + ratio to regulate the salt tolerance of wheat (Byrt et al., 2014); the results of Jin et al. showed that under high salinity conditions, overexpression of calcium-dependent protein kinase TaCIPK25 led to hypersensitivity and Na +Excessive accumulation of H3K9 negatively regulates the salt tolerance of wheat (Jin et al., 2016); Zheng et al.'s research results indicate that wheat histone acetyltransferase TaHAG1 regulates gene expression by increasing the acetylation levels of H3K9 and H3K14, and TaHAG1 can directly bind to the promoter region of the Rboh gene to enhance its transcriptional activity, thereby promoting the production of H2O2 and improving wheat salt tolerance (Zheng et al., 2021); Yang et al.'s research results show that under salt stress, the BZR1 family TaBZR1 gene promotes ABA synthesis by directly activating the expression of TaNCED3, a key gene for ABA (abscisic acid) biosynthesis, and at the same time binds to the promoter of TaGPX2 / 3 genes encoding glutathione peroxidase (GPX) to activate their expression, enhance ROS scavenging ability, and thus improve wheat salt tolerance (Yang et al. al., 2023); Xiao et al.'s research results showed that under salt stress, the SUMO protease TaDSU reduces the SUMOylation level of MYC2 through SUMO protease activity, enhances MYC2 transcriptional activity, and promotes the expression of downstream target genes such as TaRD26. At the same time, MYC2 binds to the TaDSU gene promoter to enhance its expression. The two form a positive feedback regulatory loop, enabling wheat to maintain ion balance, enhance antioxidant and osmotic regulation capabilities under salt stress during germination, thereby enhancing salt tolerance and increasing wheat yield under saline-alkali soil conditions (Xiao et al., 2024); Yang et al.'s research results showed that the wheat domestication gene AP2-type transcription factor Q can inhibit the transcription of TaSOS1-3B and reactive oxygen species (ROS) scavenging genes at the transcriptional level to regulate wheat Na + and ROS homeostasis, negatively regulating wheat salt tolerance (Yang et al., 2024); Wei et al.'s research results showed that the WRKY family transcription factor TaWRKY55 activates the expression of the PLATZ family transcription factor TaPLATZ2, thereby inhibiting the proton pump (H + The WRKY family transcription factor TaWRKY76 negatively regulates the expression of the WRKY-ATPase gene TaHA2 and the SOS family gene TaSOS3, negatively regulating wheat tolerance to saline-alkali stress (Wei et al., 2024). Hou et al.'s research results show that the WRKY family transcription factor TaWRKY76 interacts with the wheat MYC transcription factor TaMYC2 to form a heterodimer, jointly regulating the plant's osmotic stress response. Overexpression of TaWRKY76 in response to salt stress results in higher osmotic regulatory substance content, faster stomatal closure, larger root surface area, higher photosynthetic efficiency, and improved ROS balance (Hou et al., 2024).

[0003] At present, research on wheat salt tolerance mainly focuses on the seedling stage or vegetative growth stage, and there is little research on the regulatory mechanism of salt tolerance during the germination stage. For example, the research results of Yue et al. showed that under salt stress, the wheat calcium-dependent protein kinase gene TaCDPK27 regulates Ca 2+ Salt stress can mitigate damage to wheat seedlings through signal transduction and changes in subcellular localization, enhance antioxidant capacity, reduce ROS accumulation, and protect the photosynthetic system, positively regulating wheat salt tolerance (Yue et al., 2022). However, Peng Zhi et al. used 321 wheat germplasm accessions to comprehensively evaluate wheat salt tolerance by treating wheat seeds and seedlings with varying concentrations of NaCl. Their study showed no significant correlation between salt tolerance at the germination and seedling stages, suggesting that the salt tolerance mechanisms at these two stages are not identical (Peng Zhi, Li Long, Liu Yuping, Liu Huimin, Jing Ruilian, 2017). Wheat seed tolerance to salt stress during germination is fundamental to its ability to successfully grow in salinized soils. Therefore, research on salt stress during wheat germination has gradually become a hot topic in agricultural research. In recent years, wheat researchers have identified several genes that regulate salt tolerance during germination. For example, the research results of Feng et al. showed that the SnRK2 family protein kinase TaSnRK2.9 gene heterologously expressed in tobacco can participate in the ABA-dependent signal transduction pathway through specific interaction with ABF transcription factors, thereby forming a fine regulatory network in the plant body, enhancing the ROS detoxification ability, and improving the tolerance of tobacco to salt and drought stress during the germination period (Feng et al., 2019); the research results of Wang et al. showed that under salt and drought stress, the water channel protein family gene TaTIP4;1 can stimulate stress response genes to affect water relations, ROS balance, proline and Na + Accumulation is a key positive regulatory factor in wheat seedling growth and seed germination (Wang et al., 2022); Chen et al.'s results showed that under salt stress conditions, overexpression of TaSDIR1 of the TaRING-H2 gene family upregulated the expression of genes TaSOD and TaPOD related to ROS scavenging and ion transport-related genes TaHKT1;5, while downregulating the expression of the BZIP transcription factor TaGBF. TaGBF improves the salt tolerance of wheat during seed germination and seedling stages by regulating the expression of downstream genes and participating in the ABA signaling pathway (Chen et al., 2023); Jia et al.'s results showed that under salt stress conditions, the WRKY transcription factor TaWRKY44 increases its own protein stability by interacting with TaWRKY17, thereby promoting the expression of TaDHN7, which helps maintain intracellular Na + / K +Balance and enhance antioxidant enzyme activity to scavenge ROS, ultimately improving wheat salt tolerance during seed germination and early seedling development (Jia et al., 2024). Wang et al.'s research results indicate that TaABI5, a key transcription factor in ABA signaling, acts as an upstream regulator of the wheat glycerol-3-phosphate acyltransferase TaGPAT6. By regulating TaGPAT6 expression, it influences cutin and suberin synthesis, thereby enhancing salt tolerance during wheat germination (Wang et al., 2024b). Therefore, further exploring the genetic factors involved in salt tolerance during wheat germination, analyzing their detailed regulatory networks, and using scientific methods to improve crop salt tolerance are of great significance for cultivating high-quality salt-tolerant varieties throughout their growth period and ensuring national food security.

[0004] As novel regulatory elements, lncRNAs can regulate biological processes such as stress responses, protein modification, protein folding, hormone responses, and various metabolic and biosynthetic processes, playing a crucial role in plant responses to environmental stress during growth and development (Table 1.1) (Babaei et al., 2024; Guan et al., 2024). Furthermore, plant lncRNAs are highly responsive to environmental stresses in terms of expression and evolve rapidly compared to protein-coding genes. Therefore, they serve as suitable environmental sensors or effectors, helping plants adapt to changing environments (Yu et al., 2019). Under environmental stress, lncRNAs can influence gene expression through mechanisms such as target mimicry, transcriptional interference, and DNA methylation (Zheng Jiaqiu, Wu Yongcheng, Wang Weiwei, Mei Yi, Zu Yanxia, ​​Guo Jun, and Liu Yunfen, 2020). In recent years, researchers have identified lncRNAs that regulate plant salt tolerance. For example, Qin et al. demonstrated that the lncRNA DRIR in Arabidopsis thaliana is induced by ABA, drought, and salt stress, is more sensitive to ABA-induced stomatal closure, and enhances stress tolerance through the ABA signaling pathway, positively regulating plant tolerance to drought and salt stress (Qin et al., 2017). Zhang et al. demonstrated that under salt stress conditions in cotton, the expression of lncRNA354 is reduced, reducing its binding to miR160b. Increased miR160b inhibits the expression of the ARF transcription factors GhARF17 / 18, thereby enhancing resistance to salt stress (Zhang et al., 2021). Cui et al. demonstrated that TRABA in cotton participates in the salt stress response by repressing the promoter activity of the gene encoding the endoplasmic reticulum-localized β-glucosidase GhBGLU24-A (Cui et al., 2023).

[0005] In recent years, studies on the functions of lncRNA in wheat in response to abiotic stress have been reported one after another. For example, Lu et al. showed that under low temperature stress, lncR117, lncR9A, and lncR616 can reduce the efficiency of microRNA tae-miR398 in cleaving the target gene copper-zinc superoxide dismutase (Cu / Zn-SOD) TaCSD1 transcript, which can significantly increase the expression of TaCSD1 gene, thereby improving the cold resistance of winter wheat (Lu et al., 2020); Lin et al.'s research results showed that L7519 was upregulated under alkaline stress conditions, and by promoting PM The transcription of the H+-ATPase-encoding gene M9172M9172 enhances wheat tolerance to alkaline stress (Lin Wei, 2022). Xu et al.'s research results indicate that the endogenous target mimic (eTM) lncRNA35557 of miRNAtae-miR6206, by preventing tae-miR6206 from cleaving the NAC transcription factor gene TaNAC018, increases the transcript abundance of TaNAC018 and enhances wheat drought tolerance (Xu et al., 2024b). Currently, little research has been conducted on lncRNAs that function in response to salt stress during wheat germination. Therefore, identifying lncRNAs that function during the germination stage under salt stress is crucial. This can provide a theoretical basis and genetic resources for breeding new salt-tolerant wheat varieties, contributing to food security and sustainable agricultural development. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a long-chain non-coding RNA SUR1 and an application thereof in regulating wheat salt stress tolerance.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0008] A kit for regulating the ability of wheat to tolerate salt stress, comprising molecular biological elements capable of regulating the expression of specific genes; the specific genes are genes related to the ability of wheat to tolerate salt stress.

[0009] As a preferred technical solution of the present invention, the molecular biological elements may optionally include: an element combination for overexpressing the specific gene, and / or an element combination for inhibiting or reducing the expression level of the specific gene, and / or an element combination for silencing the expression of the specific gene, and / or an element combination for knocking out the expression of the specific gene.

[0010] As a preferred technical solution of the present invention, the molecular biological element is a combination of elements for knocking out the expression of the specific gene.

[0011] As a preferred technical solution of the present application, the specific gene is long-chain non-coding RNA SUR1, the cDNA sequence of which is shown in SEQ ID NO: 1, or an equivalent gene having an equivalent physiological function in wheat.

[0012] The present application also includes the following technical solution: a recombinant expression vector containing the long-chain non-coding RNA SUR1 gene or its homologous gene.

[0013] As a preferred technical solution of the present application, the recombinant expression vector is a gene knockout vector.

[0014] A method for enhancing the salt stress tolerance of wheat, which blocks the expression of long-chain non-coding RNA SUR1 in the wheat to enhance the salt stress tolerance of the wheat.

[0015] A method for improving the yield of wheat, which blocks the expression of long-chain non-coding RNA SUR1 in the wheat to enhance the salt stress tolerance of the wheat, thereby improving the yield of the corresponding wheat.

[0016] The present application also includes the use of long-chain non-coding RNA SUR1 for regulating the salt tolerance of wheat.

[0017] The beneficial effects produced by the above technical solution are as follows: in the present application, we have created and screened SUR1 overexpression and CRISPR-Cas9 mediated mutant materials. The germination rate and seedling growth vigor of the SUR1 overexpression material under normal conditions are significantly lower than those of the wild type Fielder. The SUR1 in the mutant material is deleted by 389 bp, and the germination rate under normal conditions has no obvious difference with the wild type, but is higher than the wild type under salt stress. This shows that the deletion of SUR1 increases the tolerance of wheat to salt stress during germination to a certain extent. Therefore, knocking out or blocking the expression of the SUR1 gene in the plant can achieve the purpose of enhancing the salt stress tolerance of the plant, and the gene can be fully utilized for the improvement of wheat varieties in the subsequent process, which helps to improve the yield of wheat. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1Figure 3 is a phenotype diagram of wheat germination and seedling growth under salt stress conditions, where A is a schematic diagram of the effect of salt stress on wheat germination phenotype and its temporal dynamic characteristics. The materials are the grain germination phenotypes of wheat under salt stress at 10 h, 14 h, 18 h and 7 days. The red box is the magnified germination phenotype diagram, and the scale bar in the figure is 2 cm. Figure 3 is a schematic diagram of wheat germination T50 under salt stress. T50 refers to the time required from the beginning of germination to reaching the final germination rate of 50%. It is an important indicator for measuring seed germination rate. The error value represents the standard error SD. The statistical analysis used Student's t-test, **P<0.01; C is a statistical diagram of wheat germination rate within seven days under salt stress, the error value represents the standard error SD; D is a statistical diagram of root length and aboveground part length of wheat germination 7 days under salt stress, the error value represents the standard error SD. The statistical analysis used Student's t-test, ****P<0.0001. The concentration of NaCl solution in A~D is 180mmol / L.

[0019] Figure 2 Figure 2 is the expression pattern of SUR1 during wheat seed development and germination, where A is a schematic diagram of the changes in SUR1 expression patterns in wheat at different growth stages and tissues detected by RT-qPCR. The data are derived from high-throughput sequencing (RNA-seq) analysis. The relative values ​​of gene expression were obtained through transcriptome sequencing and bioinformatics analysis. Columns of different colors represent the expression levels of SUR1 in different growth stages and tissues. Z represents the abbreviation of the Zadoks grading standard for different wheat growth stages, where Z10 and Z13 represent the seedling growth stage, Z 32 and Z 39 represent the jointing stage, Z 65 represents the flowering stage, Z 71 and Z 75 represent the milky stage 2 and 14 days after pollination, respectively. 85 is the waxy stage 30 days after pollination; B is a schematic diagram of the expression pattern of SUR1 in wheat grains at different days after pollination, and the materials are wheat seeds and their embryos and endosperms at different days after pollination; C is a schematic diagram of the changes in the expression pattern of SUR1 at different water absorption times during the grain germination stage, and the materials are the embryo, endosperm, coleoptile, and radicle of wheat grains at different times after water absorption.

[0020] Figure 3 Figure 2 Schematic diagram of SUR1 expression in germinating embryos of different wheat varieties. The materials were embryos and endosperms of dry seeds of Heng 7228, Kenong 9204, Fielder, and Jimai 5265 treated with water and 180 mM NaCl for 6, 9, 12, and 18 h, respectively. The internal reference gene was TaActin. Error values ​​represent standard deviation (SD). Two-way ANOVA was used for statistical analysis with Tukey's test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0021] Figure 4 Schematic diagram of the effect of different NaCl concentrations on SUR1 expression in wheat seeds. Embryos and endosperms of wheat dry seeds were treated with water and 100 mM, 130 mM, 180 mM, and 205 mM NaCl solutions for 9 and 12 hours, respectively. Statistical analysis was performed using the least significant difference (LSD) method after one-way ANOVA. Error values ​​represent standard deviations (SD). The internal reference gene was TaActin. Different lowercase letters in the figure indicate significant differences at the α = 0.05 level. Groups with the same letter showed no significant differences. Colors from light to dark indicate increasing NaCl concentrations.

[0022] Figure 5 Schematic diagram of SUR1 expression pattern analysis under different stress treatment conditions during the germination period. A to E are schematic diagrams of the changes in embryo and endosperm expression patterns of wheat dry seeds 9 h and 12 h after treatment with water, 125 mM NaHCO3 and Na2CO3 solutions, 0.5% salt-alkali solution, 20% PEG6000, 300 mM mannitol solution, and SMG (Submerged Growth), respectively. 0.5% salt-alkali solution is a chemical reagent that simulates the salinity of coastal saline-alkali land in the natural environment; F is a schematic diagram of the changes in whole seed expression pattern after treatment with 10% H2O2 for 9 h and 12 h. The internal reference gene is TaActin. Error values ​​represent standard deviation (SD). Two-way ANOVA was used for statistical analysis with Tukey's test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0023] Figure 6Schematic diagram of identification of SUR1 overexpressing wheat, where A is a schematic diagram of construction of wheat SUR1 overexpression vector, the blue background box is Ubiquitin promoter, the red background box is the full-length SUR1 gene, Ubi-F and q-SUR1-R are primers on Ubiquitin promoter and identification primers on SUR1 gene, respectively; B is a schematic diagram of identification of DNA level of SUR1 overexpressing material, comparison of band size between SUR1 overexpressing wheat material and Fielder, where Fielder is negative control, and the SUR1 overexpression identification band size is 1277bp; C is a schematic diagram of RT-qPCR detection of SUR1 overexpressing material expression, the error value represents standard error SD, and the internal reference gene is TaActin; D is a schematic diagram of phenotypic observation of SUR1 overexpressing wheat material and Fielder under normal conditions for 1, 3 and 7 days of imbibition, the scale bar in the figure is 1 cm; E is a schematic diagram of statistical results of germination rate of SUR1 overexpressing wheat material and Fielder at different days of imbibition, the error value represents standard error SD, and significance is determined by student's t-test, where **** indicates P < 0.0001.

[0024] Figure 7 Schematic diagram of identification of SUR1 wheat mutants. A is a schematic diagram of CRISPR / Cas9-mediated targeted knockout of wheat SUR1. The gray background boxes represent exons, the solid lines represent introns, the dark red boxes represent the target sites, the black base sequences corresponding to the target sites represent the PAM region, and the red base sequences represent the target sequences. B and C are schematic diagrams of the comparison of the PCR amplification products spanning two target sites in CRISPR mutants of UR1 (T2 generation) and the wild-type band size and sequencing results. In B, the Fielder target band is 671 bp, and the mutant band is 282 bp. In C, CRISPR-SUR1-F and CRISPR-SUR1-R are the positions of the primers for identification of SUR1 across two target sites. The solid red area corresponds to the Fielder sequence, and the hollow red area indicates the mutation site, which is 389 bp in size. D shows the germination phenotype of SUR1-overexpressing transgenic wheat and wild-type wheat under salt stress. The scale bar is 1 cm.

[0025] Figure 8Schematic diagram of transient transformation experiment to detect whether SUR1 functions in the form of small peptides, where A is a schematic diagram of the wheat SUR1 gene structure and the location of its open reading frames ORF1 and ORF2, with gray background boxes representing exons, solid lines representing introns, and pink background boxes representing the full-length ORF1 and ORF2 sequences; B is a schematic diagram of the observation of GFP fluorescence signals of different fusion proteins in tobacco cells, with the EGEP channel showing green fluorescence, the Bright channel showing the bright field image of the cells, Merge being the superimposed image of fluorescence and bright field, and the scale bar being 50 μm; C is a schematic diagram of the detection results of EGFP expression levels in tobacco leaves after transformation with the corresponding type of fusion expression vector, where Control refers to tobacco leaves that have not been transformed with Agrobacterium, the error value represents the standard error SD, and tobacco NtEF-1α is used as the internal reference gene. The lower part is a Western blot of tobacco leaves expressing the corresponding proteins using GFP-specific antibodies. Blotting detection results, asterisks indicate the target bands in the samples expressing the corresponding proteins; D is the GEP fluorescence signal observation result of SpORF1-GFP fusion protein in wheat protoplasts, where EGEP represents green fluorescence, Auto represents chloroplast autofluorescence, Bright channel represents bright field, Merge represents superimposed image, and the scale bar is 50 μm.

[0026] Figure 9Schematic diagram of transcriptome analysis of wheat SUR1 mutant, where A is a schematic diagram of the expression level of SUR1 gene under different treatments, the horizontal axis represents the treatment conditions (control and NaCl treatment), and the vertical axis represents the gene expression level (TPM); B is a principal component analysis (PCA) diagram, the horizontal axis is PC1 (explains 36.82% of the variance), the vertical axis is PC2 (explains 19.09% of the variance), and the points of different colors in the figure represent different sample combinations; C is a schematic diagram of the number of differentially expressed genes in different comparison groups, the vertical axis is the number of genes, which are divided into up-regulated and down-regulated categories, showing the specific number of up-regulated and down-regulated genes in each comparison group; D is a schematic diagram of the analysis of differentially expressed genes, the horizontal axis in the above figure is different comparison groups, and the vertical axis is the intersection size, where group I and group II represents the SUR1-dependent up-regulated and down-regulated gene sets under salt stress, respectively. The figure below is a dot plot of differentially expressed genes, with the horizontal axis representing the comparison group and the vertical axis representing the changes in gene expression. The green and purple dots represent the up-regulated and down-regulated genes in the corresponding comparison groups, respectively. E is a GO enrichment analysis heat map of differentially expressed genes, with the vertical axis representing different GO terms and the horizontal axis representing the degree of enrichment (-Log10(FDR)). The depth of color indicates the degree of enrichment, with red indicating a higher degree of enrichment. F is a clustered heat map of the expression levels of 138 Pentatricopeptide repeat-containing protein-encoding genes in different samples. The colors in the figure range from purple to yellow, indicating changes in gene expression levels, with purple representing low expression and yellow representing high expression. DETAILED DESCRIPTION

[0027] The following examples illustrate the present invention in detail. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly obtained by purchasing from the market. In the description of the following examples, for the purpose of illustration rather than limitation, specific details such as specific system structures and technologies are proposed to provide a thorough understanding of the present application embodiments. However, it should be clear to those skilled in the art that the present application can also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices and methods are omitted to prevent unnecessary details from obstructing the description of the present application.

[0028] It should be understood that when used in this specification and the appended claims, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their collections. It should also be understood that the term "and / or" used in this specification and the appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations. As used in this specification and the appended claims, the term "if" can be interpreted as "when..." or "upon..." or "in response to determining..." or "in response to detecting..." depending on the context. Similarly, the phrase "if it is determined" or "if [the described condition or event] is detected" can be interpreted as meaning "upon determination" or "in response to determining" or "upon detecting [the described condition or event]" or "in response to detecting [the described condition or event]" depending on the context.

[0029] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the description and are not to be understood as indicating or implying relative importance. References to "one embodiment" or "some embodiments" etc. described in the present application specification mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized.

[0030] Example 1. Experimental Materials and Methods

[0031] 1.1 Experimental Materials

[0032] Plant materials: The wheat varieties used in this study were Jimai 5265, Kenong 9204, Fielder, and Heng 7228. The tobacco variety was Nicotiana Benthamiana.

[0033] Plasmids and strains: (1) Entry vector: pCR8T (available in the laboratory); Tobacco transient transformation experiment: pCAMBIA1300 (available in the laboratory); Wheat transgenic vector: Overexpression 110 (available in the laboratory). (2) Escherichia coli: Trans5α; Agrobacterium: GV3101, EHA105.

[0034] Primer sequences: Detailed primer sequences are shown in Table 1.

[0035] Table 1 Primer sequence list

[0036]

[0037]

[0038] Experimental reagents: Commonly used drugs and reagents in the experiment include sucrose, NaCl, agar powder, yeast extract, tryptone, isopropanol, ethanol, and chloroform.

[0039] RNA extraction: Plant seed RNA extraction kit.

[0040] Reverse transcription and quantitative PCR: Reverse transcription kit IIQ RT SuperMix for qPCR (+gDNAwiper), quantitative PCR reagent ChamQ Universal SYBR qPCR MasterMix.

[0041] DNA amplification: Phanta enzyme (Phanta Super-Fidelity DNA Polymerase) and 2× Rapid Taq Master Mix, EasyTaq enzyme.

[0042] T4 DNA ligase and DNA restriction endonuclease fast digester.

[0043] LR (Gateway system) reaction enzyme kit; RNase inhibitor RiboLock RNase Inhibitor.

[0044] 1.2 Experimental methods

[0045] (1) Treatment of plant materials: Wheat seed disinfection, normal germination or stress treatment of wheat seeds, and collection of wheat roots, stems, and leaves all adopt conventional standard biochemical treatment methods.

[0046] (2) RNA extraction was carried out according to the instructions of the plant seed RNA extraction kit.

[0047] (3) RNA reverse transcription: Reference for reverse transcription of plant total RNA IIQ RT SuperMix forqPCR (+gDNAwiper) kit instructions;

[0048] (4) Real-time quantitative PCR: The reaction system was prepared according to the instructions of the ChamQ Universal SYBR qPCR MasterMix kit: RNase-free ddH2O to 10 μL; 2× ChamQ Universal SYBR qPCR MasterMix 5 μL; F primer (10 μM) 0.2 μL; R primer (10 μM) 0.2 μL; HiScript II Enzyme Mix 2 μL.

[0049] (5) PCR amplification of target DNA fragment: according to The Vazyme PCR reaction system was configured according to the instructions of the Max Super-Fidelity DNA Polymerase kit: ddH2O to 50 μL; 2×Phanta Max Buffer 25 μL; dNTP Mix (10 mM) 1 μL; F primer (10 μM) 2 μL; R primer (10 μM) 2 μL; PhantaMax Super-Fidelity DNA Polymerase 1 μL; template x μL.

[0050] Vazyme PCR reaction protocol: initial denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at X°C for 15 seconds, extension at 72°C for 30 cycles (30-60 seconds / kb); final extension at 72°C for 5 minutes. The annealing temperature is selected based on the primer TM, and the extension time is determined by the length of the amplified product.

[0051] (6) Enzyme digestion and ligation: 1) Enzyme digestion: Select appropriate enzyme digestion sites based on the sequences of the gene and vector. The amount of gene or vector to be digested should be sufficient so that the ratio of the amount of gene to the amount of vector during the ligation reaction is controlled at (5-10):1. Thermo enzyme digestion reaction system configuration: ddH2O to 50μL; gene / vector xμL; DNA restriction endonuclease 11μL; DNA restriction endonuclease 21μL; 10×FastDigest Buffer 5μL. After gently mixing the above reaction solution, incubate at 37℃ for 30 minutes. 2) Thermo DNA ligation reaction system: ddH2O to 20μL; gene xμL; vector yμL; T4DNALigase 1μL; 10×T4 DNALigase Buffer 2μL; After gently mixing the above reaction solution, incubate at 16℃ for 8 hours.

[0052] (7) Heat shock method to transform Escherichia coli DH5α: using the existing mature technology route.

[0053] (8) Heat shock method to transform Agrobacterium EHA105: using the existing mature technology route.

[0054] (9) Tobacco transient transformation experiment and observation of GFP fluorescence signal:

[0055] 1) Inoculate the Agrobacterium strain carrying the target vector into 10 mL of liquid culture medium containing the corresponding antibiotics, and culture with shaking at 250 rpm at 30°C overnight; 2) Incubate at 4000 rpm for 10 min at room temperature to collect the bacteria; 3) Prepare transformation buffer (prepare immediately): ddH2O to 100 mL, MgCl2 0.1M, AS1M, MES1M; 4) Resuspend the bacteria with an appropriate amount of transformation buffer and adjust the OD600 of the mixture to 0.5-0.6; 5) Use a sterile syringe to inject the mixture in (4) into the lower epidermis of a well-grown tobacco leaf; 6) Place the injected tobacco leaf at 22°C under long-day conditions for 36-48 hours and observe; 7) Carefully tear off a piece of about 1 cm thick stalk with tweezers. 2 The lower epidermis of tobacco was placed on a glass slide, an appropriate amount of ultrapure water was added, and the cover glass was covered, and the GFP fluorescence signal was detected under a confocal microscope.

[0056] (10) Western blot experimental process: adopt the existing mature technology route.

[0057] (11) Extraction, transformation, culture and collection of wheat protoplasts: refer to the instructions of the wheat protoplast extraction kit.

[0058] (12) Identification of long noncoding RNAs and analysis of differentially expressed genes in response to salt stress during germination:

[0059] 1) Low-quality reads in the sequencing data of germinating embryos treated with normal and salt stress for 10, 14, and 18 hours were filtered using sickle software, and high-quality reads were aligned with the Chinese spring wheat reference genome using Tophat2 software; 2) Transcripts were spliced ​​using cufflinks and merged using cuffmerge; 3) Transcripts obtained in cuffmerge were compared with reference transcripts using cuffcompare, and transcripts with a length of at least 200 bp were retained; 4) The coding capacity of retained transcripts was predicted using CPC2, and transcripts with a score > 0 and those that could be aligned with the PfamA and Swiss-Prot databases were discarded; 5) The number of reads of lncRNAs and protein-coding genes was counted using HTSeq-Count, and lncRNAs and genes differentially expressed between salt stress and normal conditions at different time points were identified using edgeR, using an absolute value of the expression change greater than or equal to 1 and an FDR < 0.05 as the threshold.

[0060] Example 2, experimental results analysis

[0061] 2.1 Effects of salt stress on wheat seed germination

[0062] To investigate the effects of NaCl treatment on wheat seed germination, we first observed and statistically analyzed the germination phenotype of wheat seeds treated with 180 mM NaCl. The results of the phenotypic observations showed that NaCl treatment significantly inhibited plant growth and development compared with normal H2O treatment, with the main manifestations being delayed seed germination and decreased germination rate, stagnant growth of the seedling coleoptile, and hindered radicle development ( Figure 1 A). The results of statistical analysis of the T50 value, which characterizes the germination rate, showed that the time required for 50% of the seeds to turn white under NaCl treatment was about 31 hours, which was significantly longer than that under normal conditions (about 13 hours) ( Figure 1 B). Statistical analysis of seed germination rates showed that the germination rates of seeds under normal conditions at 1, 2, 4, and 7 days of imbibition were 62%, 82.5%, 92.5%, and 97%, respectively, while the germination rates under NaCl treatment were only 7.9%, 42%, 61.5%, and 72.5%. Figure 1 C). In addition, the results of measuring the radicle length of the seedlings 7 days after water absorption showed that the radicle length under NaCl treatment was about 2.5 cm, which was significantly lower than that under normal conditions (7.9 cm), a decrease of 68.35%. The results of statistical analysis of the aboveground length showed that compared with the normal conditions (9.9 cm), the length under NaCl treatment (0.35 cm) was reduced by about 96.46% ( Figure 1 D) The results of phenotypic observation and statistical analysis showed that salt stress could significantly prolong germination time, reduce seed germination rate, and severely inhibit subsequent seedling growth and development.

[0063] 2.2 Identification and expression pattern analysis of lncRNA SUR1 in response to salt stress during germination

[0064] 2.2.1 SUR1 is highly expressed during seed development and germination

[0065] Overall, lncRNA expression levels showed a downward trend after salt stress treatment, but we identified a lncRNA that was upregulated after 14 and 18 hours of water absorption. For ease of description, we named this lncRNA SUR1 (Salt-Upregulate lncRNA 1).

[0066] Next, we analyzed the expression pattern of SUR1. First, we used Kallisto software to quantitatively analyze the expression level (TPM value) of SUR1 based on the transcriptome data of different tissues and organs at different developmental stages of Chinese spring wheat and germinating embryos of wheat variety Jimai 5265 at different water absorption times collected from the NCBI SRA database. The results showed that ( Figure 2 A) SUR1 expression was undetectable in roots, stems, and leaves at different stages, as well as in ears at stages Z32 (early jointing) and Z65 (flowering), but weak expression was detected in ears at stage Z39 (late jointing). During wheat seed development, SUR1 expression was low at the milky stage (Z71 and Z75), but its expression increased significantly at the waxy stage (Z85). Furthermore, during wheat seed germination, SUR1 expression levels decreased with prolonged water absorption.

[0067] To further verify the results obtained from the high-throughput sequencing data analysis, we used RT-qPCR technology to accurately detect the expression level of the SUR1 gene. The experimental results showed that as the number of days after pollination increased, that is, during the development of wheat seeds, the expression level of the SUR1 gene showed a gradual upward trend ( Figure 2 B). In addition, during the wheat seed germination stage, as the water absorption time increased, the expression level of SUR1 showed a trend of gradual decrease ( Figure 2 C). At the same time, the expression level of SUR1 in the germination stage after water absorption was significantly higher than that in the grain filling stage after pollination. This may be closely related to the changes in cellular physiological activities during seed water absorption. In addition, in the late stage of seed development and germination, the expression level of SUR1 in the endosperm was higher than that in the embryo, and the expression level was not detected in the plumule and radicle after 48 and 72 hours of water absorption. The above results are consistent with the conclusions drawn from high-throughput sequencing data analysis ( Figure 2 A), further confirming that the SUR1 gene is specifically expressed only in wheat grains.

[0068] 2.2.2 Expression of SUR1 in response to salt stress

[0069] High-throughput sequencing showed that the expression level of SUR1 in germinating embryos showed an upward trend after salt stress treatment. To verify this result, we first used RT-qPCR technology to detect the expression level of SUR1 in wheat seeds germinated at different time points after treatment with 7228180mM NaCl and H2O. The results showed that under normal water absorption conditions, the expression level of the SUR1 gene in embryos and endosperm showed a gradually decreasing trend with the gradual extension of water absorption time. At the same time, the expression level of the SUR1 gene in germinating embryos treated with NaCl for 6 hours was about 1.38 times that of the control group, and in the endosperm it was about 1.67 times that of the control group; in the endosperm after 12 hours, the expression level of the SUR1 gene was about 3 times that of the control group ( Figure 3 ). This result shows that salt stress treatment can upregulate the expression level of SUR1. Subsequently, in order to explore the conservation of the changing trend of SUR1 expression levels in different wheat varieties under salt stress during the germination period, we used RT-qPCR to detect the expression level of SUR1 in wheat varieties Kenong 9204, Fielder and Jimai 5265 before and after salt stress treatment ( Figure 3 ). The results showed that in Kenong 9204, the expression levels of SUR1 at the four time points of NaCl treatment were significantly higher than those in the control group; in the Fielder endosperm treated with NaCl for 6h and 9h, the expression levels of SUR1 were 1.07 times and 1.65 times that of the control group, respectively, while there was no significant difference in the expression level of SUR1 in the embryo; on the contrary, in the germinating embryos treated with NaCl for 12h and 18h, the expression levels of the SUR1 gene were 5 times and 7 times that of the control group, respectively, while no significant changes were detected in the endosperm. In Jimai 5265, the expression levels of SUR1 in the embryos at different time points of NaCl treatment were 2.9 times, 9.28 times, 4.48 times and 9.5 times that of the control group, respectively. The expression levels of SUR1 in the endosperm treated for 9, 12 and 18 hours were 6.7 times, 356 times and 1320 times that of the control group. Overall, the expression levels of SUR1 in the three wheat varieties showed an increasing trend after salt stress treatment ( Figure 3 This result indicates that the phenomenon of salt stress-induced upregulation of SUR1 gene expression is conserved among different wheat varieties.

[0070] 2.2.3 Effects of different NaCl concentrations on SUR1 expression

[0071] High-throughput sequencing and RT-qPCR quantitative analysis revealed that SUR1 responds to salt stress. Based on these results, we further investigated whether SUR1 exhibits a consistent response pattern to different NaCl concentrations. To this end, we treated germinating seeds with NaCl solutions at four concentrations: 100 mM, 130 mM, 180 mM, and 205 mM, and measured SUR1 expression levels in embryos and endosperms at different time points. RT-qPCR results showed that treatment with the lower NaCl concentration (100 mM) significantly upregulated SUR1 expression in both embryos and endosperms after 9 and 12 hours of treatment. Furthermore, in embryos, SUR1 showed significant differences in its response to different NaCl concentrations, with the strongest responses to the lower (100 mM) and higher (205 mM) concentrations. Furthermore, in the endosperm, SUR1 only responded to the lower (100 mM) NaCl concentration after 9 hours of treatment. As the NaCl treatment time increases, SUR1 in the endosperm responds to different concentrations of NaCl, and the expression levels show significant differences ( Figure 4 This result indicates that the response of SUR1 to salt stress is not concentration-dependent and its expression can be upregulated by low concentrations.

[0072] 2.2.4 Analysis of SUR1 expression patterns after other stress treatments

[0073] In addition to salt stress, we also analyzed the expression changes of SUR1 in germinating seeds under alkali (125mM NaHCO3 / Na2CO3), complex salt (0.5%), drought (20% PEG6000 and 300mM mannitol), flooding, and oxidative stress (10% H2O2). RT-qPCR results showed that compared with the control, SUR1 expression levels showed a significant increase after different stress treatments ( Figure 5 Under alkaline stress conditions, the expression levels of SUR1 in the embryo and endosperm at 9 hours and in the endosperm at 12 hours were 1.68, 1.65, and 1.97 times that of the control, respectively ( Figure 5 A). The expression pattern of SUR1 after compound salt treatment was similar to that after alkaline stress, and the expression level in embryo and endosperm at 9 hours and endosperm at 12 hours was significantly increased, which were 1.43 times, 3.32 times and 1.43 times that of the control, respectively ( Figure 5 B); Under the drought stress condition simulated by 20% PEG6000, the expression levels of SUR1 in embryo and endosperm at 9 hours and embryo and endosperm at 12 hours were 3.13 times, 3.42 times, 3.01 times and 3.37 times that of the control, respectively ( Figure 5C); The expression pattern of SUR1 after flooding treatment was similar to that of drought stress simulated by 20% PEG6000, and the expression levels in embryo and endosperm at 9 hours and 12 hours were significantly increased by 3.84 times, 4.77 times, 7.67 times and 2.49 times, respectively ( Figure 5 E); Under mannitol-simulated drought stress, the expression levels of SUR1 in embryo and endosperm at 9 hours were 3.63 times and 2.69 times that of the control, respectively ( Figure 5 D); Under oxidative stress, the expression levels of SUR1 in whole seeds at 9 hours were 2.33 times that of the control ( Figure 5 F) Overall, SUR1's response to 20% PEG6000-simulated drought stress was relatively stable. SUR1 showed the highest level of response, and its expression in both embryos and endosperms increased significantly at different treatment times. Based on these results, we speculate that SUR1 may be a multi-stress response factor.

[0074] 2.3 Analysis of the biological function of SUR1 during wheat seed germination

[0075] 2.3.1 Creation and phenotypic analysis of SUR1-overexpressing wheat

[0076] To clarify the biological function of SUR1 during wheat seed germination, we overexpressed SUR1 in wild-type Fielder wheat using Agrobacterium-mediated genetic transformation. In the T1 generation, we identified a positive strain using pUbi:SUR1 vector-specific primers. PCR amplification of genomic DNA followed by electrophoresis revealed that a 1277-bp target band was specifically amplified in all plants of this strain, whereas this band was absent in wild-type Fielder ( Figure 6 B). For ease of description, we named this line SUR1-OE. At the same time, the results of RT-qPCR detection showed that the transcription level of SUR1 in SUR1-OE overexpressing wheat materials was significantly higher than that in wild-type Fielder ( Figure 6 C). The expression abundance of SUR1 in different plants of this line was upregulated by more than 79 times, indicating that the gene overexpression method is highly efficient and can significantly change the expression level of SUR1. Subsequently, we observed and analyzed the germination phenotype of SUR1-OE under normal conditions. The results showed that the germination rate of SUR1-OE at different time points of imbibition was significantly lower than that of wild-type Fielder, and the aboveground length of the seedlings was also shorter than that of the wild-type 7 days after imbibition ( Figure 6 D). Specific statistical results show that the proportion of seeds with whitening after one day of imbibition in wild Fielder can reach about 60%, while the proportion of seeds with whitening in the SUR1-OE strain is only 7% ( Figure 6E). As the imbibition time progressed, the germination rate of the SUR1-OE strain gradually increased, reaching 50% and 68%, respectively, but was still lower than that of the wild type (100%) ( Figure 6 E). Based on the above results, we speculate that SUR1 negatively regulates wheat seed germination under normal conditions.

[0077] 2.3.2 Creation and phenotypic observation of wheat SUR1 gene deletion mutants

[0078] To further explore the biological function of SUR1 in wheat seed germination, we constructed a SUR1 loss-of-function mutant in the wheat variety "Fielder" by CRISPR / Cas9-mediated gene editing. The cDNA of SUR1 is 2128 bp long and contains 3 exons ( Figure 7 A). We designed two guide RNAs (sgRNAs) targeting specific target sites, both located in the first exon of SUR1 and 575 bp apart ( Figure 7 A). In order to accurately analyze the editing type of the positive mutant materials obtained by resistance screening, we designed a pair of specific primers spanning two target sites and performed PCR amplification of wild-type Fielder and mutants ( Figure 7 B). The results of agarose gel electrophoresis showed that we amplified a band of about 600 bp in the wild type, but only a band of about 350 bp was amplified in different individuals of the mutant ( Figure 7 B). The results of Sanger sequencing showed that the PCR product obtained from the wild-type Fielder was 671 bp in length, while the length of the mutant was consistent across individuals, averaging only 282 bp, with a deletion of 389 bp ( Figure 7 C), and the deleted region contains the ORF1 region of SUR1. For the convenience of subsequent description, we named this homozygous mutant strain SUR1-cr. Since the expression level of SUR1 showed an upward trend after salt stress treatment during the germination period, we subsequently observed the germination phenotype of SUR1-cr under normal and salt stress conditions. Under normal conditions, the germination rate and growth status of SUR1-cr after 7 days of water absorption were not significantly different from those of wild-type Fielder ( Figure 7 D). Under 180 mM NaCl treatment, although both SUR1-cr and wild-type Fielder showed stagnant coleoptile elongation and a slowed germination rate, the germination rate was significantly higher than that of the wild-type, and its radicle growth showed stronger vitality ( Figure 7 D) This result suggests that SUR1 deficiency increases wheat tolerance to salt stress during germination. Therefore, we speculate that SUR1 may negatively regulate salt tolerance during wheat germination.

[0079] 2.4.1 SUR1 may function through encoding small peptides

[0080] 2.4.1 SUR1 may function through encoding small peptides

[0081] Recent studies have shown that some lncRNAs can regulate biological processes through encoding small peptides (Xing et al., 2021). To investigate whether SUR1 has the ability to encode small peptides, we first used the ORF finder online tool (https: / / www.ncbi.nlm.nih.gov / orffinder / ) to predict potential open reading frames (ORFs) in the full-length sequence of SUR1. The results showed that there were two ORFs with a length of 270 nt and 234 nt, respectively, starting with the codon ATG at 33 bp-303 bp and 579 bp-813 bp of the SUR1 transcript, which we named ORF1 and ORF2, respectively (Fig. 2A). Subsequently, to determine whether the predicted ORFs can encode polypeptides, we transiently overexpressed SpORF1-GFP and ORF2-GFP fusion proteins (Sp represents Small peptide) in N. benthamiana by Agrobacterium-mediated transformation. With the ORF3 in PILNCR1, which is known to have no protein-coding ability, and GFP fusion protein and empty GFP as controls, we observed the green fluorescent protein (GFP) signal 36 hours after injecting the tobacco. The results showed that we only observed the GFP green fluorescent signal in tobacco leaves expressing SpORF1-GFP fusion protein, while no fluorescent signal was detected in tobacco leaves expressing ORF2-GFP and PILNCR1-ORF3-GFP fusion protein (Fig. 2B). At the same time, we used RT-qPCR and Western blotting to detect the transcription and protein expression levels of GFP in the above transformed N. benthamiana leaves. The results of RT-qPCR quantitative detection showed that the expression of GFP gene was detected in different groups of transformed materials, and was significantly higher than the background expression level of untransformed tobacco, indicating that the transformation process successfully introduced the GFP expression vector into tobacco cells and achieved effective expression at the transcription level (Fig. 2C). Figure 8 Figure 8 Figure 8 ​​C). The results of Western blotting using GFP antibodies showed that the size of the protein detected in the transformed 35S::GFP tobacco (26.9kD) was consistent with expectations, indicating that the antibody used was appropriate and could successfully detect the GFP protein. SpORF1 encodes 89 amino acids, and the NovoPro protein molecular weight calculation tool (https: / / www.novopro.cn / tools / protein_mw.htmL) predicted that the protein size was approximately 9.16kD. Therefore, we speculated that the size of the SpORF1-GFP fusion protein was approximately 36.06kD. In tobacco leaves expressing the SpORF1-GFP fusion protein, we detected a GFP band at the expected protein molecular weight ( Figure 8 C), which also indicates that ORF1 of SUR1 can encode a small peptide. Subsequently, we transiently transformed wheat protoplasts with ORF1-GFP and ORF2-GFP vectors, and observed the GFP fluorescence signal. The results showed that we only observed the fluorescence signal in the protoplasts expressing the SpORF1-GFP fusion protein ( Figure 8 D) Based on the above results, we speculate that SpORF1 on SUR1 has the ability to encode small peptides.

[0082] In addition, we also analyzed the subcellular localization of SpORF1. The results of transient transformation of tobacco showed that 35S::GFP could detect the fluorescence of unfused GFP throughout the cell, and the GFP protein was localized in the nucleus, cell membrane and cytoplasm. However, the fluorescence of SpORF1-GFP fusion protein was only detected in the cytoplasm ( Figure 8 B). The results of fluorescence signal detection after transient transformation of wheat protoplasts showed that SpORF1 could be normally fused with GFP and expressed to emit fluorescence, and the fluorescence signal was mainly detected in the cytoplasm ( Figure 8 D). Interestingly, we also observed that the fluorescence signal of the SpORF1-GFP fusion protein partially overlapped with the autofluorescence of chloroplasts ( Figure 8 D) Considering that SUR1 is specifically expressed in seeds, we speculate that SpORF1 may be localized in plastids. Taken together, we hypothesize that SUR1 may exert its biological function by encoding small peptides that are localized in the cytoplasm and plastids.

[0083] 2.4.2 Analysis of biological processes regulated by SUR1

[0084] To investigate the biological processes that SUR1 regulates during the salt stress response during wheat germination, we used RNA-seq technology to perform high-throughput transcriptome sequencing on three biological replicates of wild-type Fielder and SUR1-cr mutant seeds that had been watered for 6 hours under normal and salt stress conditions. The results of the transcriptome expression level (TPM) analysis of SUR1 itself in the data showed that the expression level of SUR1 was significantly increased after salt treatment compared with normal conditions, which is consistent with the results of the previous analysis of SUR1 expression patterns, indicating that the salt stress treatment used in the experiment was effective ( Figure 9 A). At the same time, the SUR1 expression level in the SUR1-cr mutant was lower than that in the wild type under both normal and salt stress conditions, and the degree of reduction under salt stress was significantly higher than that under normal conditions ( Figure 9 A). This result indicates that the sequence deletion in the SUR1-cr mutant leads to a decrease in SUR1 expression levels. Subsequently, to clarify the relationship between the transcriptional expression profiles of each sample, we performed principal component analysis (PCA) on the expression levels of all expressed genes. The PCA results showed that the three biological replicates of each sequencing sample clustered together, indicating a good correlation between the replicates ( Figure 9 B). Furthermore, based on the first principal component (PC1), the wild-type Fielder sequencing samples under salt stress conditions were clearly divided into two clusters compared to the other samples. This result indicates that the SUR1-cr mutant is less sensitive to salt stress during seed germination than the wild-type Fielder, and that the difference between the SUR1-cr mutant and the wild-type Fielder is much greater under salt stress than under normal conditions.

[0085] Next, we used |log2(fold change)|>=1 and false discovery rate (FDR)<0.05 as the screening threshold to identify differentially expressed genes between salt stress treatments and controls of different materials, as well as between SUR1-cr mutants and wild-type Fielder under different conditions. The results showed that in wild-type Fielder, 17,985 genes were differentially expressed under salt stress compared with normal conditions, of which the number of downregulated genes (16,305) was much greater than the number of upregulated genes (1,680). In the SUR1-cr mutant, we only identified 2,372 differentially expressed genes, of which 1,743 genes had suppressed expression levels and 629 genes had an increasing expression level ( Figure 9C). This result is consistent with the results of PCA analysis, both indicating that the SUR1-cr mutant is insensitive to salt stress treatment. At the same time, under salt stress conditions, we identified 13,611 genes with differential expression between the SUR1-cr mutant and wild-type Fielder, which was significantly more than under normal conditions (2,553), indicating that salt stress treatment exacerbated the differences between the SUR1-cr mutant and wild-type Fielder. In addition, under normal conditions, the number of downregulated genes in the SUR1-cr mutant (1,869) was higher than the number of upregulated genes (684). In contrast, under salt stress conditions, the SUR1-cr mutant showed a trend of having more upregulated genes (11,141) than downregulated genes (2,470). This result suggests that SUR1 may play different regulatory functions under normal and salt stress conditions.

[0086] Subsequently, by comparative analysis of the identified differentially expressed genes, we identified 677 (group I) genes that were specifically upregulated in wild-type Fielder under salt stress and downregulated in SUR1-cr mutants under salt stress, and 8,325 (group II) genes that were specifically downregulated in wild-type Fielder under salt stress and upregulated in SUR1-cr mutants under salt stress. Figure 9 D). These two categories of salt stress response genes, which are considered to be regulated by SUR1, account for 40.30% and 51.06% of the salt stress up-regulated (1,680) and down-regulated (16,305) genes in wild-type Fielder, respectively. Gene ontology (GO) enrichment analysis of these two categories of genes showed that group I, i.e., SUR1-dependent salt stress up-regulated genes, were mainly enriched in stress-related biological processes such as protein folding, response to heat shock, response to hydrogen peroxide, cellular response to calcium ions, and response to osmotic stress. Figure 9E). Group II, i.e., SUR1-dependent salt stress-downregulated genes, were enriched in GO terms related to chloroplast and plastid biological processes, including chloroplast RNA processing, chloroplast relocation, establishment of plastid localization, plastid organization, and plastid membrane organization. In addition, group II genes were also significantly enriched in biological processes related to the cell cycle, including DNA replication, nucleic acid metabolic process, cell division, meiotic cell cycle, and microtubule-based process ( Figure 9 E), which is consistent with the functional analysis results of the negatively co-expressed SUR1 genes mentioned above. At the same time, group II genes are also enriched in seed germination and brassinosteroid mediated signaling. It is worth noting that among the GO conditions enriched in group II genes, chloroplast RNA processing is the most enriched. By reviewing the literature, we found that the pentatricopeptide repeat (PPR) protein family is mainly located in the mitochondria and chloroplasts of terrestrial plants, and can participate in the post-transcriptional processing of plant organelle mRNA as sequence-specific RNA binding proteins (Shijing, 2023). For example, Lee et al. showed that OsPPR19 is a mitochondrial-targeted PPR protein that participates in the splicing of introns of multiple mitochondrial genes. The seed germination of the osppr19 mutant was delayed and the grain weight and fruit setting rate were reduced (Lee et al., 2024). Among the group II genes, we identified 138 members of the PPR protein family ( Figure 9 F). These PPR genes all exhibited a significant decrease in expression after salt stress in wild-type Fielder, but the SUR1-cr mutant showed no significant change. Based on these results, we hypothesize that SUR1 participates in the salt stress response during germination primarily by inhibiting PPR gene expression.

[0087] In summary, we have created and screened SUR1 overexpression and CRISPR-Cas9-mediated mutant materials. The germination rate and seedling growth vigor of the SUR1 overexpression material under normal conditions were significantly lower than those of the wild-type Fielder. The SUR1 in the mutant material was deleted by 389bp, and its germination rate was not significantly different from that of the wild type under normal conditions, but was higher than that of the wild type under salt stress. This shows that the absence of SUR1 increases the tolerance of wheat to salt stress during the germination period to a certain extent. Therefore, knocking out or blocking the expression of the SUR1 gene in the plant can achieve the purpose of enhancing the plant's tolerance to salt stress, and the gene can be fully utilized in the subsequent improvement of wheat varieties, which helps to increase wheat yield.

[0088] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0089] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention, and should all be included in the scope of protection of the present invention.

Claims

1. A kit for regulating the ability of wheat to tolerate salt stress, characterized in that: The kit contains molecular biological elements capable of regulating the expression of specific genes; the specific genes are genes related to the ability of wheat to respond to salt stress and tolerate.

2. The kit according to claim 1, wherein: The molecular biological elements may optionally include: an element combination for overexpressing the specific gene, and / or an element combination for inhibiting or reducing the expression of the specific gene, and / or an element combination for silencing the expression of the specific gene, and / or an element combination for knocking out the expression of the specific gene.

3. The kit according to claim 1, wherein: The molecular biological elements are a combination of elements for knocking out the expression of the specific gene.

4. The kit according to claim 1, wherein: The specific gene is a long non-coding RNA SUR1 The cDNA sequence is shown in SEQ ID NO: 1, or an equivalent gene having the same physiological function as wheat.

5. Contains long non-coding RNA SUR1 Or a recombinant expression vector of a homologous gene thereof.

6. The recombinant expression vector according to claim 5, characterized in that: The recombinant expression vector is a gene knockout vector.

7. A method for enhancing salt stress tolerance in wheat, characterized in that: Blocking the long non-coding RNA in wheat SUR1 to enhance salt stress tolerance in wheat.

8. A method for increasing wheat yield, characterized in that: Blocking the long non-coding RNA in wheat SUR1 expression to enhance the salt stress tolerance of wheat, thereby increasing the yield of the corresponding wheat.

9. Long noncoding RNA SUR1 It is used to regulate the salt tolerance of wheat.

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