Application of MOS3 gene in regulating plant salt tolerance

Abstract

The present invention discloses the application of the MOS3 gene in regulating plant salt tolerance, belonging to the field of biotechnology. Through transgenic experiments, the present invention proves that MOS3 is involved in salt tolerance, expands our understanding of the generation of plant salt tolerance, provides a theoretical basis for obtaining plants with high salt tolerance, and has great application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the application of the MOS3 gene in regulating plant salt tolerance. Background Art

[0002] During grape cultivation, the problem of soil salinization is becoming increasingly serious, mainly due to high concentrations of Na + and Cl - irrigation with water, and the accumulation of salts accompanied by the evaporation of soil moisture, which cannot be effectively leached. The grape cultivars / varieties used for the production of wine, raisins, and table grapes are mainly Vitis vinifera. Cultivated varieties grown in salt-affected soils are very likely to accumulate salts, change the fruit development process and its biochemical components, and ultimately affect grape yield and the sensory characteristics of wine. In addition, if the soil salinity exceeds the maximum level that grapevines can tolerate, it will lead to the death of grapevines.

[0003] The bidirectional transport of mRNA between the nucleus and the cytoplasm is controlled by the nuclear pore complex (NPC). In yeast cells, the NPC is composed of 35 - 50 proteins. The NPC in mammalian cells is a larger complex composed of 80 - 100 proteins. In higher plants, the NPC contains at least 30 nucleoproteins. The nuclear pore proteins in Arabidopsis thaliana are involved in various biological processes, such as pathogen interaction, cold stress response, flowering, etc.

[0004] The MOS3 gene encodes a protein belonging to the nuclear pore complex (NPC) and plays an important role in plant disease resistance and hormone signal transduction. Patent CN 111304240 A discloses that the grape MOS3 gene is involved in regulating the normal growth and development of grapes. The deletion of the function of this gene will cause obvious dwarfing of plants. However, there is no report on the related research of the MOS3 gene from grapes in regulating plant salt stress resistance. Summary of the Invention

[0005] Aiming at the above-mentioned prior art, the purpose of the present invention is to provide the application of the grape MOS3 gene in regulating plant salt tolerance.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided the application of the grape MOS3 gene in the following (1) or (2):

[0008] (1) Improving the salt tolerance of plants;

[0009] (2) Cultivating plant varieties with improved salt tolerance;

[0010] The grape MOS3 gene is a nucleic acid molecule as shown in the following (i) or (ii):

[0011] (i) A nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO.1;

[0012] (ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than (i).

[0013] In the second aspect of the present invention, there is provided the use of the protein encoded by the grape MOS3 gene in the following (1) or (2):

[0014] (1) Improving the salt tolerance of plants;

[0015] (2) Preparing a product for improving the salt tolerance of plants.

[0016] Furthermore, the amino acid sequence of the protein encoded by the grape MOS3 gene is as shown in SEQ ID NO.2.

[0017] In the above applications, the grape MOS3 gene or the protein encoded by the grape MOS3 gene is used as a target. By overexpressing the grape MOS3 gene in plants, or increasing the expression level or activity of the MOS3 protein, the resistance of plants to salt stress can be positively regulated.

[0018] In the third aspect of the present invention, there is provided the use of a recombinant expression vector or engineering bacteria containing the grape MOS3 gene in the following (1) or (2):

[0019] (1) Improving the salt tolerance of plants;

[0020] (2) Cultivating plant varieties with improved salt tolerance.

[0021] In the fourth aspect of the present invention, there is provided a method for improving the tolerance of plants to salt stress, including: the step of overexpressing the grape MOS3 gene in plants.

[0022] In the above method, the grape MOS3 gene can be overexpressed through the following ways:

[0023] Exogenous transfer of the grape MOS3 gene;

[0024] Or, upregulating the expression of the grape MOS3 gene in the plant genome.

[0025] In the fifth aspect of the present invention, there is provided a method for cultivating salt-resistant plant varieties, including the following steps:

[0026] Transferring the grape MOS3 gene into a wild-type plant to overexpress the grape MOS3 gene and obtaining a transgenic plant.

[0027] In the above method, the transgenic plants have higher resistance to salt stress than wild-type plants.

[0028] In the above method, the method for transferring the grape MOS3 gene into wild-type plants includes, but is not limited to: polyethylene glycol method, Agrobacterium-mediated method, gene gun bombardment method, etc.

[0029] Advantages of the present invention:

[0030] In view of the current weak research on the salt tolerance function of plant nuclear pore complex proteins, the present invention cloned a nuclear pore complex protein gene, MOS3, from grapes. Through transgenic experiments, it was confirmed that MOS3 is involved in salt tolerance, expanding our understanding of the generation of plant salt tolerance and providing a theoretical basis for obtaining highly salt-tolerant plants, with great application value. Description of the drawings

[0031] Figure 1 It shows the expression of the grape MOS3 gene of the present invention after salt stress.

[0032] Figure 2 It shows the growth of tobacco under salt stress after transferring the grape MOS3 gene; in the figure, #1 and #2 respectively represent two transgenic tobacco lines overexpressing MOS3. Detailed implementation manners

[0033] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0034] As mentioned above, the physiological disorder problems of grapes caused by saline-alkali soil are becoming more and more obvious, seriously affecting the normal growth and development of grapes, fruit yield and quality. However, little is known about how salt stress affects the expression pattern of nuclear pore protein genes in plants, and there is still a lack of transgenic evidence to support whether nuclear pore protein genes play a role in plant salt stress response.

[0035] The inventors found in previous studies that the grape MOS3 gene is involved in regulating plant height, plant width, leaf length and leaf width. However, whether the grape MOS3 gene plays a role in plant salt stress response has not been reported yet.

[0036] In view of this, the present invention has carried out research on the cloning and function of the nuclear pore protein gene MOS3 gene derived from grapes. First, the present invention cloned the grape MOS3 gene from grape tissue culture seedlings; then detected the expression of the grape MOS3 gene at different salt treatment time points, and found that the grape MOS3 gene could differentially express in response to salt stress; further, the present invention introduced the grape MOS3 gene into tobacco, and found that the growth state of tobacco plants overexpressing the MOS3 gene under salt-containing conditions was significantly higher than that of the wild type.

[0037] The above results indicate that: the grape MOS3 gene can play a salt-resistant function in plants, and it is a new salt-tolerant gene related to salt stress resistance in grapes.

[0038] The sequence of the grape MOS3 gene is shown in SEQ ID NO.1, as follows:

[0039]

[0040] The amino acid sequence of the protein encoded by the grape MOS3 gene is shown in SEQ ID NO.2, as follows:

[0041]

[0042] In the present invention, the expression pattern of grape MOS3 can be analyzed by real-time fluorescence quantitative PCR, that is, to analyze the presence and quantity of grape MOS3 RNA transcripts in cell tissues.

[0043] Based on the discovered MOS3 gene, the protection scope of the present invention also includes DNA fragments homologous to the MOS3 gene.

[0044] These DNA fragments homologous to the MOS3 gene include alleles, homologous genes, mutant genes, and derivative genes corresponding to the nucleotide sequence (SEQ ID NO.1) of the present invention, and all belong to the content protected by the present invention.

[0045] Those skilled in the art can easily mutate the nucleotide sequence of the MOS3 gene of the present invention by using known methods, such as directed evolution and site-directed mutagenesis. Those artificially modified nucleotides with 70% or higher identity to the nucleotide sequence of the MOS3 gene of the present invention, as long as their functions are equivalent to the function of the nucleotide sequence shown in SEQ ID NO.1, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0046] The term "identity" used herein refers to the sequence similarity to the natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity to the nucleotide sequence shown in SEQ ID NO.1 of the present invention. The identity rate of amino acid or nucleotide sequences can be determined by the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215: 403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90: 5873-5877).

[0047] The above-mentioned identity of 70% or more can be 70%, 75%, 80%, 85%, 90% or 95% or more identity.

[0048] Since tobacco has a fast growth rate, a short life cycle, a simple and easy-to-operate transformation method, and a high genetic transformation efficiency, the present invention selects tobacco as the object of genetic transformation. However, the MOS3 gene and the plant expression vector containing this gene in the present invention can also be used to produce other transgenic plants with improved salt tolerance.

[0049] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below in conjunction with specific embodiments.

[0050] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The experimental methods without specified detailed conditions are carried out according to conventional test methods or according to the operation manuals recommended by the suppliers.

[0051] Example 1: Cloning of Grape MOS3

[0052] 1. Plant materials:

[0053] The plant materials used in the present invention are tissue culture seedlings of 'Crimson Seedless' grapes, and the culture medium is MS medium containing sucrose (30 g / L) and plant agar (6 g / L).

[0054] 2. RNA extraction and reverse transcription:

[0055] Grape leaves were collected for RNA extraction. The total RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) according to the instructions, and 1 μg of RNA was reverse transcribed into cDNA using the TaKaRa PrimeScript TM RT reagent Kit (PerfectReal Time) kit.

[0056] 3. Cloning and verification of MOS3:

[0057] According to the accession number XP_002271967.2 on NCBI, primers were designed:

[0058] MOS3-F: 5′-ATGGCATCTGCTTCTCCCTTT-3′ (SEQ ID NO.3);

[0059] MOS3-F: 5′-CTACACCTCCAACAAAGAGCACGT-3′ (SEQ ID NO.4).

[0060] The PCR reaction was carried out using PrimeSTAR Max Premix high-fidelity enzyme according to the instructions. The PCR product was ligated to the -T1Simple Cloning Vector (Beijing TransGen Biotech Co., Ltd.) vector, transformed into Escherichia coli DH5α, and positive single colonies were screened by colony PCR and sent to Sangon Biotech Co., Ltd. for sequencing. The nucleotide sequence of the cloned grape MOS3 gene is shown in SEQ ID NO.1.

[0061] Example 2: Expression of Grape MOS3 Gene at Different Salt Treatment Time Points

[0062] 1. Plant Materials:

[0063] The tissue culture seedlings of 'Crimson Seedless' grapes that had been subcultured for 1 month and had consistent growth were used. The roots of the tissue culture seedlings were immersed in 200 mM NaCl solution, and then the roots were taken at different time points and immediately frozen in liquid nitrogen for storage.

[0064] 2. RNA Extraction and Reverse Transcription:

[0065] Same as Example 1.

[0066] 3. Changes in the Expression Level of MOS3 Gene at Different Salt Treatment Time Points:

[0067] Specific primers were designed to perform real-time fluorescence quantitative PCR to analyze the change rule of the expression level of MOS3 gene at different salt treatment time points. The designed primers are as follows:

[0068] qMOS3-F: 5′-CTCACAAATTGCACTGCATCAA-3′ (SEQ ID NO.5);

[0069] qMOS3-R: 5′-GCCAACTCCTTCAAGCAAGGT-3′ (SEQ ID NO.6).

[0070] The internal reference gene is ACTIN, and the primers are:

[0071] ACTIN-F: 5′-TCCGTTGTCCAGAAGTCCTCTT-3′ (SEQ ID NO.7);

[0072] ACTIN-R: 5′-GTCAGCAATACCAGGGAACATG-3′ (SEQ ID NO.8).

[0073] 4. Real-time Fluorescence Quantitative Analysis of MOS3 in Test Samples:

[0074] Using cDNA as a template, fluorescence quantitative analysis was performed with the specific primers of MOS3 and ACTIN respectively. The reaction was carried out on a real-time fluorescence quantitative PCR instrument (CFX connect Real Time PCR Detection System, Bio-Rad). A 20 μL reaction system was used (10 μL SYBR Premix Ex Taq, 1 μL each of upstream and downstream primers (10 μM), 1 μL of cDNA template, and made up to 20 μL with water). The program was as follows: 95°C for 30 s; 95°C for 5 s, 60°C for 10 s, for 40 cycles.

[0075] 5. Use 2 -△△Ct methods for relative quantitative analysis of MOS3:

[0076] The results showed that: with the extension of salt treatment time, the expression level of MOS3 showed a trend of first decreasing and then increasing, and increased by 1.5 times at 24 h, indicating that the MOS3 gene could respond to salt stress and showed differential expression( Figure 1 ).

[0077] Example 3: Detection of salt tolerance function of grape MOS3 transgenic tobacco

[0078] Using the pHB-gfp vector (described in the literature "MicroRNA171c-targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis, doi.org / 10.1093 / mp / ssq042"), an overexpression vector of the MOS3 gene was constructed, and the primers were as follows:

[0079] pHBMOS3-F: 5′-accagtctctctctcaagcttATGGCATCTGCTTCTCCCTTT-3′ (SEQ ID NO.9);

[0080] pHBMOS3-R: 5′-gcccttgctcaccatggatccCTCAATGAAATTGAAGTCCAGCC-3′ (SEQ ID NO.10).

[0081] Use PrimeSTAR Max Premix high-fidelity enzyme to perform PCR reaction (in a 20 μL reaction system, 10 μL PrimeSTAR Master Mix, 1 μL each of upstream and downstream primers (10 μM), 1 μL template, and make up to 20 μL with water).

[0082] The procedure was as follows: 98 °C for 10 s, 55 °C for 5 s, 72 °C for 10 s for 34 cycles; 72 °C for 5 mins. The PCR products were separated by 1.5% agarose gel electrophoresis and purified using the SanPrep column DNA gel recovery kit (B518131, Sangon Biotech) according to the standard operating steps.

[0083] The pHB-gfp vector plasmid was digested with enzymes, and the enzyme digestion reaction system was as follows:

[0084]

[0085] The above reaction solution was incubated at 37 °C for 30 minutes, and then subjected to agarose gel electrophoresis followed by gel cutting and recovery.

[0086] The digested pHB-gfp and MOS3 genes were ligated according to the standard operating procedures of the ClonExpress II One Step Cloning Kit. The recombinant plasmid was transferred into Escherichia coli DH5α. Positive clones were screened by colony PCR and sent to Sangon for sequencing. The single colonies with correct sequencing were cultured overnight in a shaker, and the plasmid was extracted according to the standard instructions of the SanPrep Column Plasmid DNA Mini-Preps Kit (Sangon Biotech). Thus, the overexpression vector plasmid containing MOS3 was obtained.

[0087] The MOS3 overexpression vector was transferred into Nicotiana benthamiana using the leaf disc method. The method is as follows: After culturing in LB medium, the bacteria were collected and placed in 50 ml of sterile water containing 75 μmol / L acetosyringone, and the OD value was adjusted to 0.6. The sterile tobacco leaves were cut into small pieces and put into the suspension. After 8 minutes of infection, they were inoculated onto the co-cultivation medium (MS + 6 g / L agar + 30 g / L sucrose + 2 mg / L 6-BA + 0.5 mg / L NAA). After 2 - 3 days of dark culture, they were transferred to the selection medium (MS + 6 g / L agar + 30 g / L sucrose + 2 mg / L 6-BA + 0.5 mg / L NAA + 15 mg / L Hyg). The medium was changed every half month until resistant buds differentiated. When the resistant buds grew to 1 - 2 cm, they were transferred to the selection medium. After the plants grew stably, they were transferred to the rooting medium (1 / 2MS + 6 g / L agar + 30 g / L sucrose + 1 mg / L IBA + 15 mg / L Hyg) for rooting. The rooted seedlings were transferred to soil and seeds were harvested. Positive transgenic lines were screened by glufosinate until homozygous, and the seeds of the homozygous lines were used for salt tolerance identification.

[0088] Seeds of the homozygous transgenic tobacco were used for experiments, with seeds of wild-type tobacco as the control. The seeds were sown in the soil and transplanted into flower pots after germination. 200 mM NaCl solution was irrigated, and the growth and development of transgenic plants and wild-type plants were observed.

[0089] The results are as Figure 2 shown. The results indicate that under salt stress conditions, the growth status of MOS3 overexpressing tobacco (#1, #2) is significantly higher than that of the wild type (WT), that is, the MOS3 gene can positively regulate the salt tolerance of plants.

[0090] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of the grape MOS3 gene in the following (1) or (2): (1) Improving the salt tolerance of plants; (2) Cultivating plant varieties with improved salt tolerance; The grape MOS3 gene is a nucleic acid molecule as shown in the following i) or ii): i) A nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i); The plants and plant varieties are grapes or tobacco.

2. Use of the protein encoded by the grape MOS3 gene in the following (1) or (2): (1) Improving the salt tolerance of plants; (2) Preparing a product for improving the salt tolerance of plants; The amino acid sequence of the protein encoded by the grape MOS3 gene is as shown in SEQ ID NO.2; The plant is grape or tobacco.

3. Use of a recombinant expression vector or engineered bacteria containing the grape MOS3 gene in the following (1) or (2): (1) Improving the salt tolerance of plants; (2) Cultivating plant varieties with improved salt tolerance; The grape MOS3 gene is a nucleic acid molecule as shown in the following i) or ii): i) A nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i); The plants and plant varieties are grapes or tobacco.

4. A method for improving the salt stress tolerance of plants, characterized in that, it includes: a step of overexpressing the grape MOS3 gene in the plant; The grape MOS3 gene is a nucleic acid molecule as shown in the following i) or ii): i) A nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i); The plant is grape or tobacco.

5. The method according to claim 4, characterized in that, the grape MOS3 gene is overexpressed through the following means: exogenous transfer of the grape MOS3 gene; or, up-regulating the expression of the grape MOS3 gene in the plant genome.

6. A method for cultivating salt-resistant plant varieties, characterized in that, it includes the following steps: transferring the grape MOS3 gene into a wild-type plant to overexpress the grape MOS3 gene to obtain a transgenic plant; The grape MOS3 gene is a nucleic acid molecule as shown in the following i) or ii): i) A nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i); The salt-resistant plant variety is grape or tobacco.

7. The method according to claim 6, characterized in that, the transgenic plant has higher resistance to salt stress than the wild-type plant.

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