Salt stress resistant gene and application thereof

Abstract

The invention relates to the technical field of plant molecular biology and genetic engineering, and discloses a salt stress resistant gene and application thereof. The salt stress resistant gene is SICA1, and the nucleotide sequence of the salt stress resistant gene is as shown in SEQ ID No.1. The expression quantity of the gene is related to the salt tolerance of the plant. The embodiment verifies that after normal expression or overexpression of the SICA1 gene, calcium signal transduction is normal, and the salt tolerance of the plant is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular biology and genetic engineering, and particularly relates to a salt stress resistance gene and application thereof. BACKGROUND

[0002] With global climate change, plants need to adjust through complex signal pathways to adapt to environmental changes. Calcium signaling plays an important role in the response of plants to environmental changes, especially in the response to salt stress. External salt stress enters cells through calcium ion channels, generates calcium signals, and regulates plant cell activities.

[0003] High-concentration salt ions can cause ion toxicity to plant cells, leading to osmotic stress and oxidative stress, thereby limiting the growth and development of plants.

[0004] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a salt stress resistance gene and application thereof, so as to improve the salt tolerance of plants.

[0006] The technical scheme of the present application is as follows: In a first aspect of the present application, a salt stress resistance gene is provided, wherein the salt stress resistance gene is a SICA1 gene, and the nucleotide sequence is shown as SEQ ID No. 1.

[0007] In a second aspect of the present application, a recombinant expression vector is provided, wherein the recombinant expression vector comprises a basic vector and the above-mentioned SICA1 gene inserted into the basic vector.

[0008] In a third aspect of the present application, a protein encoded by the above-mentioned SICA1 gene is provided, wherein the amino acid sequence of the protein comprises or consists of: an amino acid sequence shown as SEQ ID No. 2; or, an amino acid sequence obtained by adding, deleting or replacing one or more amino acids in the amino acid sequence shown as SEQ ID No. 2 and having the same protein activity.

[0009] In a fourth aspect of the present application, the above-mentioned SICA1 gene, complementation and overexpression vector or amino acid sequence is applied in one or more of the following applications: (1) application in regulating the salt tolerance of plants; (2) application in cultivating salt-tolerant plants; (3) application in preparing products for improving the salt tolerance of plants.

[0010] Optionally, the plant comprises Arabidopsis thaliana.

[0011] In a fifth aspect, the present application provides a method for cultivating a salt-tolerant plant, comprising the step of: allowing the SICA1 gene in the plant to be normally expressed or overexpressed.

[0012] In a sixth aspect, the present application provides a method for improving the salt tolerance of a plant, comprising the step of: allowing the SICA1 gene in the plant to be normally expressed or overexpressed.

[0013] In a seventh aspect, the present application provides a method for judging the salt tolerance of a plant, comprising the step of: detecting the expression amount of the SICA1 gene in the plant, wherein if the expression amount is low or no expression, the plant is a salt-sensitive phenotype, and if the expression amount is normal or overexpression, the plant is a salt-tolerant phenotype.

[0014] In an eighth aspect, the present application provides a method for detecting the calcium signal in a plant, comprising the step of: detecting the expression amount of the SICA1 gene in the plant.

[0015] In a ninth aspect, the present application provides a method for detecting a salt-tolerant plant, comprising the step of: detecting the expression amount of the SICA1 gene in the plant, wherein if the expression amount is low or no expression, the plant is a salt-sensitive plant, and if the expression amount is normal or overexpression, the plant is a salt-tolerant plant.

[0016] The present application has the following advantages: The present application provides a salt stress-resistant gene and application thereof, wherein the salt stress-resistant gene is SICA1 gene, the nucleotide sequence of which is shown as SEQ ID No. 1, and the expression amount of the gene is related to the salt tolerance of a plant. Through implementation verification, after the SICA1 gene is normally expressed or overexpressed, the calcium signal conduction is normal, and the salt tolerance of the plant is improved. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 FIG. 1 is a diagram for the separation and identification of sica1 mutants in the present application under salt stress to induce the absence of calcium signals, wherein A is a water-luminescent protein imaging diagram of the increase of calcium ion concentration in plant cells induced by NaCl under water or 200 mM NaCl treatment, and the calcium ion concentration is shown by a pseudo-color scale (middle and bottom); B is a quantitative analysis diagram of the increase of calcium ion concentration in leaves; C is a quantitative analysis diagram of the increase of calcium ion concentration in roots; D is a time course analysis diagram of the increase of calcium ion concentration in plant cells induced by NaCl, the plants are treated with 200 mM NaCl, and the fluorescence luminescence is recorded every 2 seconds; and E is an average value line graph of the increase of calcium ion concentration induced by different concentration gradients of NaCl.

[0018] Figure 2This is an identification diagram of the SICA1 gene in an embodiment of the present invention: where A is a calcium signal diagram of seedlings treated with 200mM NaCl, and the seedlings express either the SICA1 autopromoter (SICA1 sica1) or the 35S promoter (SICA1 sica1). OX A) Reinforcement of the calcium signaling phenotype in the sica1 mutant; B) Treatment of the same seedlings (right) in the left image with water (left) or solutions containing 0.9 M CaCl2 and 10% (v / v) ethanol, respectively, to measure the total amount of residual jellyfish luminescent protein; C) ... Figure 2 The increase in calcium ion concentration caused by experiment A in section A is presented as a quantitative graph; D is a graph representing the increase in calcium ion concentration caused by experiment A in section B in section C. Figure 2 A quantitative graph was generated to quantify the total amount of jellyfish luminescent protein remaining in B.

[0019] Figure 3 This is a phenotypic diagram of the sica1 mutant's sensitivity to salt stress in an embodiment of the present invention: where A represents the plant's response to salt stress in the presence of 0.2 mM CaCl2. Phenotypic diagrams of growth on culture media are shown. Figure A shows seedlings cultured for 12 days. Figure B shows the phenotypic diagram of plants grown on culture media containing 50 mM NaCl for 12 days. Figures C and D show the quantitative analysis of materials from the same experiments as A and B, where fresh weight (FW; C) and root length (RL; D) were calculated, respectively.

[0020] Figure 4 This is a diagram illustrating the SICA1-regulated intracellular membrane potential in an embodiment of the present invention: where A is a confocal micrograph of charged fluorescently labeled root epidermal meristem; B is... Figure 4 A. Fluorescence intensity of charged fluorescent markers at the underlined positions in the confocal image; C. Quantitative ratio of charged fluorescent markers in the plasma membrane and cytoplasm (Ratio PM / cytosol); D. Change curve of zeta potential in mesophyll protoplasts after NaCl application; E. Quantitative ratio of fluorescence intensity in the plasma membrane and cytoplasm in root epidermal meristem after NaCl application (Ratio PM / cytosol). Detailed Implementation

[0021] This invention provides a salt stress resistance gene and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0023] Although previous studies have revealed how plants decode calcium signals and initiate adaptive mechanisms, the specific mechanisms by which plants perceive salt stress and activate calcium signals remain unclear. In recent years, using calcium imaging technology, researchers have discovered the MOCA1 gene and GIPC molecules through genetic screening, revealing the role of GIPC molecules in plant salt perception: through the binding of sodium ions to GIPC molecules on the plasma membrane, calcium ion channels are activated to regulate salt discharge. This provides a new approach for studying plant perception of salt stress. However, to date, it is still unclear how GIPC regulates calcium ion channels to produce calcium signals under salt stress.

[0024] Based on this, an embodiment of the present application provides an anti-salt stress gene, which is a SICA1 gene, and the nucleotide sequence is shown in SEQ ID No. 1.

[0025] The protein encoded by the SICA1 gene is a core regulatory factor for maintaining the negative charge density on the inside of the plant plasma membrane (membrane potential). Under salt stress (NaCl), the function of this gene is crucial for maintaining or adjusting the potential on the inside of the plasma membrane to perceive stress signals. When the function of this gene is lost, the potential on the inside of the plasma membrane abnormally decreases, causing the cell to be unable to effectively perceive the membrane potential changes induced by salt stress, and thus unable to activate downstream calcium ion channels, resulting in a significant weakening or absence of calcium signals ([Ca 2+ ] cyt ) in the cell. As an important second messenger, the absence of calcium signals directly affects the expression of downstream stress response genes and the initiation of physiological and biochemical reactions (such as ion homeostasis reconstruction, reactive oxygen species scavenging, and osmotic adjustment substance synthesis), ultimately leading to severe growth restriction of mutant plants under salt stress, exhibiting a salt-sensitive phenotype. Conversely, restoring or enhancing the function of this gene through genetic means (back complementation, overexpression) can effectively restore the regulatory ability of the potential on the inside of the plasma membrane, reconstruct calcium signal transduction under salt stress, and significantly improve the salt stress tolerance and growth recovery ability of plants. Therefore, this gene is a key upstream regulatory factor and perception element in the plant anti-salt stress pathway, and manipulating this gene provides a new important target for cultivating salt-tolerant crops.

[0026] Novelty: This gene is a gene that has been proven to regulate calcium signal generation and salt stress response by maintaining the potential on the inside of the plasma membrane under salt stress (different from known genes that regulate the potential on the outside of the plasma membrane, such as MOCA1).

[0027] Invention: A brand new key molecular mechanism of plant sensing salt stress and initiating calcium signal (membrane potential inside as sensing switch) is disclosed.

[0028] Utility: Direct evidence shows manipulating this gene (mutation, back-complementation, over-expression) can significantly change plant calcium signal, membrane potential, growth phenotype under salt stress, with clear anti-salt breeding application value.

[0029] Data support: All the research results (mutant phenotype, back-complementation / over-expression verification, membrane potential measurement, fluorescence localization analysis, zeta potential comparison) obtained are important experimental evidence supporting the claims.

[0030] The following will be described in detail through specific examples.

[0031] Examples This example establishes an EMS (ethyl methane sulfonate) mutagenesis mutant library of Arabidopsis thaliana stably expressing Aequorin (an intracellular calcium ion indicator, a luminescent protein of a jellyfish), uses an imaging system that can capture transient calcium signals, and determines the change in intracellular calcium ion concentration of Arabidopsis thaliana as the starting point. The mutant sica1 screened is studied, and it is found that its calcium signal under salt stress is significantly reduced, and the reduction of its calcium signal is only specific to salt stress, indicating that the SICA1 gene has an important function in the process of Arabidopsis thaliana sensing external salt stress. Further, the function of the SICA1 gene and its molecular mechanism of mediating calcium signal generation under salt stress are further explored through various technical means. This includes the following parts: 1) Isolation and identification of SICA1 gene and bioinformatics analysis The mutant gene of mutant sica1 is cloned and identified by whole genome resequencing and MutMap method. Further analysis of SICA1 gene information is combined with bioinformatics; the self promoter and overexpression vector are constructed for plant transformation, and the method of gene back-complementation is used to verify whether the intracellular calcium ion concentration increase defect phenotype caused by SICA1 gene under salt stress is restored to wild type; in addition, a Crispr / Cas9 gene editing vector is designed according to the SICA1 gene sequence, and sica1-1, sica1-2 mutants with base editing in the key region are obtained by transformation, and whether the calcium signal phenotype of the gene editing material under salt stress is consistent with that of sica1 mutant is recorded and analyzed.

[0032] 2) Effect of SICA1 on Arabidopsis growth and development under salt stress The sica1 mutant, transgenic and gene editing materials obtained above are observed and counted in detail for physiological phenotype, especially the root length and fresh weight of plants under salt stress are recorded and analyzed, providing evidence on phenotype for subsequent functional analysis of SICA1.

[0033] 3) SICA1 regulates calcium signal in Arabidopsis under salt stress Based on the calcium signal imaging system, through fluorescence resonance energy transfer (FRET) experiment, using calcium ion indicator Yellow Cameleon 3.6 (YC 3.6), it is determined whether SICA1 has a significant impact on long-distance transmission of calcium signal in Arabidopsis roots under salt stress; at the same time, protoplasts of Arabidopsis wild type (WT) and mutant (sica1) are prepared, and the activity of calcium ion channel in WT and sica1 is detected by whole-cell voltage clamp, to further analyze whether SICA1 affects the increase of cell calcium ion concentration and affects the activity of channel under salt stress.

[0034] 4) Biological function research of SICA1 The expression pattern of SICA1 gene is analyzed to determine the subcellular localization of SICA1; the sodium and potassium contents in WT and sica1 mutant plants are determined by ICP-MS to analyze whether SICA1 affects the sodium and potassium balance in Arabidopsis cells; the surface zeta potential of the outer side of the cell membrane of WT and sica1 protoplasts is detected, and the surface potential change of the inner side of the cell membrane is detected by charged fluorescent markers to analyze the effect of SICA1 on the surface potential of the inner and outer sides of the cell membrane. The cell membranes of WT and sica1 are extracted to detect the activity of Na + / H + reverse transport protein (SOS1) under salt stress, to further determine whether SICA1 affects the SOS1 signal transduction pathway through calcium signal.

[0035] 5) Correlation analysis of SICA1 and plant salt receptor GIPC sphingolipid Since the previous pre-experiment found that SICA1 can maintain the inner side potential of the cell membrane, in order to determine whether SICA1 affects the synthesis of GIPC molecules, thereby leading to the reduction of calcium signal in Arabidopsis under salt stress, lipidomics analysis is performed. Sphingolipids are extracted from WT and sica1 mutant plants to detect and compare the content of GIPC sphingolipids in sica1; at the same time, it is verified whether SICA1 interacts with GIPC to affect calcium signal, and the binding of SICA1 and Na + and GIPC is analyzed by isothermal titration calorimetry (ITC) to explore the relationship between SICA1 and plant salt receptor GIPC, and comprehensively analyze the molecular action mechanism of SICA1 in plant perception of external salt stress.

[0036] The results of the above experiments are as follows: Figure 1The following are images illustrating the isolation and identification of calcium signal loss induced by the sica1 mutant under salt stress in this invention: A. Imaging of jellyfish luminescent protein induced by NaCl treatment in water or 200 mM NaCl, with calcium ion concentration displayed using a pseudo-color scale (middle and bottom); B. Quantitative analysis of increased calcium ion concentration in leaves; C. Quantitative analysis of increased calcium ion concentration in roots; D. Time-series analysis of NaCl-induced increase in intracellular calcium ion concentration in plant cells, with fluorescence emission recorded every 2 seconds after treatment with 200 mM NaCl; E. Average increase in calcium ion concentration induced by different NaCl concentration gradients.

[0037] Observations of WT and sica1 mutants showed that sica1 seedlings did not exhibit any growth, morphological, or developmental phenotypes. Figure 1 (A). The changes in intracellular calcium ion concentration in sica1 plants after water treatment were similar to those in WT plants, but after treatment with 200 mM NaCl, sica1 plants showed a lower level of intracellular calcium ion concentration. Figure 1 (A). Calculations and analysis of calcium signal intensity revealed that the effect of 200 mM NaCl on leaves of the sica1 plant was lower than that reported in moca1 in previous studies, but similar in the root system. The different calcium signal intensities produced in sica1 and moca1 plants under salt stress suggest that SICA1 and MOCA1 may have different regulatory mechanisms. Furthermore, compared to leaves, the effect of 200 mM NaCl on the roots of the sica1 mutant was more significant. Figure 1 (B, C). Further examination of the changes in calcium signal over time revealed that sica1 plants under salt stress produced a lower peak calcium signal (B, C). Figure 1 (D). Simultaneously, treatment with different NaCl concentration gradients revealed a significant decrease in intracellular calcium ion concentration in sica1 plants (D). Figure 1 (E). The above results indicate that SICA1 is involved in regulating intracellular calcium signaling under salt stress.

[0038] Figure 2 The diagram shows the identification of the SICA1 gene in this embodiment of the invention: A. By expressing the SICA1 self-promoter (SICA1sica1) or the 35S promoter (SICA1... OX A) Reinforcement of the calcium signaling phenotype in the sica1 mutant, calcium signal images of seedlings treated with 200 mM NaCl; B) Treatment with a solution containing 0.9 M CaCl2 and 10% (v / v) ethanol. Figure 2 In B, the same seedlings were used to measure the total amount of remaining jellyfish luminescent protein; no difference was observed between WT and sica1 seedlings; C. Figure 2The increase in calcium ion concentration caused by experiment A in section A was quantified; D. [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] Figure 2 The total amount of remaining jellyfish luminescent protein in B was quantified. In WT and sica1 seedlings, the total amount of remaining jellyfish luminescent protein was similar.

[0039] To confirm that SICA1 is the gene causing the calcium signaling deficiency phenotype in Arabidopsis under salt stress, this example constructed complement (pSICA1::SICA1) and overexpression vector (p35S::SICA1) based on the SICA1 sequence and obtained transformed plants. It was found that SICA1 can restore the calcium signaling enhancement defect phenotype of sica1. Figure 2 (A, C). Simultaneously, the reliability of the luminescence was verified by discharging the total jellyfish luminescent protein from the plant. Figure 2 (B, D)

[0040] Figure 3 Phenotypic diagram of the sica1 mutant's sensitivity to salt stress in this embodiment of the invention: A. Plants in a solution containing 0.2 mM CaCl2 A. Plants were grown in a culture medium for 12 days; B. Plants were grown in a culture medium containing 50 mM NaCl for 12 days; C and D. Quantitative analysis was performed on the materials from the same experiments as A and B, and the fresh weight (FW; C) and root length (RL; D) were calculated, respectively. To investigate the biological functions of SICA1, physiological phenotypic analysis of sica1 plants under salt stress was performed. In low-calcium culture medium, there were no significant phenotypic differences between WT and sica1. Figure 3 When subjected to mild salt stress with 50 mM NaCl, it was found that salt treatment inhibited both the fresh weight and root growth of Sica 1 plants. Figure 3 To further verify the physiological sensitivity phenotype of plants to salt environments caused by SICA1, this example analyzed the constructed SICA1-replenished homozygous lines. The results showed that the fresh weight and root length of the SICA1-replenished homozygous lines recovered to the wild-type state under salt stress. Figure 3 These results provide strong genetic evidence for revealing the important biological function of SICA1 in the regulation of calcium signaling under salt stress in Arabidopsis thaliana.

[0041] Figure 4 The following is a diagram illustrating the SICA1-regulated intracellular membrane potential in an embodiment of the present invention: A. Confocal micrograph of charged fluorescently labeled root epidermal meristem; B. Figure 4Fig. 6 shows the results of the experiments. A. Confocal images of the plasma membrane in WT, moca1 and sica1. The dashed line indicates the position of the line profile. B. The fluorescence intensity of the charged fluorescent marker at the position of the dashed line in A. C. The quantified ratio of the fluorescence intensity of the charged fluorescent marker in the plasma membrane and cytosol (Ratio PM / cytosol). D. The change of the ζ potential of the protoplasts of leaf mesophyll cells after the addition of NaCl. E. The quantified ratio of the fluorescence intensity of the charged fluorescent marker in the plasma membrane and cytosol (Ratio PM / cytosol) in the root epidermis meristem after the addition of NaCl.

[0042] The changes of the inner surface potential of sica1 were detected using WT and moca1 as controls. By observing the fluorescent molecular marker representing the changes of the electric charge in the root meristem of Arabidopsis, it was found that the relocalization of the fluorescent marker from the plasma membrane to the cytosol was increased in sica1 Figure 4 A). The fluorescence intensity of the plasma membrane region and the cytosol region of the cells was further calculated Figure 4 B) and the ratio Figure 4 C) in A, which proved that the charged amount in the inner region of the plasma membrane of sica1 was significantly reduced compared to WT and moca1 Figure 4 A-C). Using NaCl for treatment, the migration of the protoplasts under the stable electric field was analyzed using a microscope, and the ζ potential representing the outer potential of the plasma membrane was measured. It was found that unlike moca1 (MOCA1 reduced the outer surface potential of the plasma membrane), the outer surface potential of the plasma membrane of sica1 was similar to that of WT, with no significant difference Figure 4 D). However, after NaCl treatment, the number of charges in the inner membrane of the WT plasma membrane was significantly reduced, while sica1 remained at a low level Figure 4 E). The above results revealed that SICA1 is crucial for maintaining the inner membrane potential of the plasma membrane, and the inner membrane potential of the plasma membrane plays a key role in the perception of salt stress and the generation of calcium signals in plants.

[0043] It should be understood that the application of the present application is not limited to the above examples, and can be improved or changed by those of ordinary skill in the art according to the above description, and all such improvements and changes shall fall within the scope of protection of the appended claims of the present application.

Claims

1. Application of a salt stress resistance gene in one or more of the following: (1) application in regulating plant salt tolerance; (2) application in breeding salt-tolerant plants; (3) application in preparing products for improving plant salt tolerance; wherein The nucleotide sequence of the salt stress resistance gene is shown as SEQ ID No.

1.

2. Use according to claim 1, characterized in that, The plant includes Arabidopsis thaliana.

3. A method for breeding salt-tolerant plants, characterized in that, The method comprises the following steps: Normal expression or overexpression of the salt stress resistance gene in the plant, the nucleotide sequence of the salt stress resistance gene is shown as SEQ ID No.

1.

4. A method for increasing salt tolerance in plants, characterized by, The method comprises the following steps: Normal expression or overexpression of the salt stress resistance gene in the plant, the nucleotide sequence of the salt stress resistance gene is shown as SEQ ID No.

1.

5. A method for determining salt tolerance of a plant, characterized by, Detection of the expression amount of the salt stress resistance gene in the plant body, if it is low expression or no expression, it is a salt-sensitive phenotype, if it is normal expression or overexpression, it is a salt-tolerant phenotype, the nucleotide sequence of the salt stress resistance gene is shown as SEQ ID No.

1.

6. A method for detecting a salt-tolerant plant, characterized by, The detection method comprises: detecting the expression amount of the salt stress resistance gene in the plant body, if it is low expression or no expression, it is a salt-sensitive plant, if it is normal expression or overexpression, it is a salt-tolerant plant, the nucleotide sequence of the salt stress resistance gene is shown as SEQ ID No. 1.

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