Application of osnsun5 protein and its coding gene in regulating salt tolerance of rice

Abstract

The application discloses application of Osnsun5 protein and a coding gene thereof in regulating salt tolerance of rice, and belongs to the technical field of biotechnology, and particularly relates to application of Osnsun5 protein and the coding gene thereof in regulating salt tolerance of rice. The protein provided by the application is Osnsun5 protein, which is a protein as shown in sequence 1 in the sequence listing. By introducing a substance for inhibiting expression of the Osnsun5 gene into a receptor plant, a gene editing transformed plant is obtained. Compared with a target plant, the salt stress resistance of the Osnsun5 gene editing transformed plant is improved, and the application has great application and popularization value for regulating salt tolerance of plants and crop breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the Osnsun5 protein and its encoding gene in regulating the salt tolerance of rice. Background Technology

[0002] Rice originated in China and India. It is one of the world's major food crops. In China, rice planting area accounts for one-quarter of the country's total food crop area, while its output accounts for more than half. Its cultivation history dates back 14,000 to 18,000 years. It is an important food crop; besides the edible caryopsis, it can be used to make starch, brew wine, and produce vinegar. Rice bran can be used to make sugar, extract oil, and extract furfural for industrial and pharmaceutical uses. Rice straw is a good feed, papermaking raw material, and weaving material; rice sprouts and roots can be used medicinally.

[0003] In recent years, the rapid development of biotechnology has greatly promoted the innovation of plant breeding research methods and the continuous improvement of research levels. Plant disease and pest resistance and herbicide resistance biotechnology breeding has begun to enter the practical application stage. By using biotechnology to introduce exogenous insecticidal and herbicide-resistant genes into the plant genome, the natural barrier that makes hybridization difficult between plant species has been broken down. This achieves the transfer of insect-resistant and herbicide-resistant genes, enabling plants to quickly and directionally acquire insect resistance and mechanized weeding capabilities while retaining their original good agronomic traits. Because each transgenic corn plant possesses a considerable degree of resistance, its insect and herbicide resistance is better and more stable than manual control, and it also saves manpower and material resources, effectively conserving social resources. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to improve the salt tolerance of plants in order to increase plant yield.

[0005] To address the problems existing in the prior art, the present invention provides the application of proteins or gene expression substances or substances that regulate the activity or content of said proteins in regulating the salt tolerance of plants.

[0006] The use of the protein or gene expression regulator or substance regulating the activity or content of said protein provided by this invention in any of the following:

[0007] 1) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in regulating plant salt tolerance.

[0008] 2) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the preparation of products that regulate plant salt tolerance.

[0009] 3) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the cultivation of plants with altered salt tolerance.

[0010] 4) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the preparation of products that cultivate plants with altered salt tolerance.

[0011] 5) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in plant breeding;

[0012] The protein is any of the following proteins:

[0013] a1) A protein with the amino acid sequence SEQ ID No. 1;

[0014] a2) A protein having the same function as the amino acid sequence shown in SEQ ID No. 1, by substitution and / or deletion and / or addition of one or more amino acid residues;

[0015] Proteins that have more than 75% identity with the amino acid sequence defined in (a3), (a1), or (a2) and have the same function;

[0016] The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in (a4), (a1), and (a3).

[0017] The protein described in a1) above is named Osnsun5.

[0018] To facilitate the purification or detection of the protein in a1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

[0019] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0020] The tagged proteins include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein.

[0021] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein Osnsun5 of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the protein Osnsun5 isolated in this invention, provided they encode and function as protein Osnsun5, are derived from and equivalent to the nucleotide sequence of this invention.

[0022] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0023] In this article, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid or nucleotide sequences, then the identity value (%) can be obtained.

[0024] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0025] In this document, the 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0026] In the above applications, the protein is derived from rice (Oryza sativa L.).

[0027] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein Osnsun5.

[0028] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation:

[0029] 1) Regulation occurring at the transcriptional level of the aforementioned gene;

[0030] 2) Regulation that occurs after the gene is transcribed (i.e., regulation of the splicing or processing of the primary transcript of the gene);

[0031] 3) Regulation of RNA transport of the gene (that is, regulation of the transport of mRNA of the gene from the nucleus to the cytoplasm);

[0032] 4) Regulation of the translation of the aforementioned genes;

[0033] 5) Regulation of mRNA degradation of the aforementioned gene;

[0034] 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0035] In this invention, the regulation can be increased, enhanced, or raised. The regulation can also be decreased, weakened, or reduced.

[0036] In this article, the enhancement, increase or upregulation of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0037] In this article, regulating the expression of the gene encoding the protein can be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0038] In the above applications, the substance regulating gene expression or the substance regulating protein activity or content can be a biological material related to the protein described above, and the biological material can be any of the following:

[0039] c1) The nucleic acid molecule that encodes the protein described above;

[0040] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0041] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0042] c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3);

[0043] c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0044] c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2);

[0045] c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2);

[0046] e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding genes mentioned above;

[0047] e2) An expression cassette containing the nucleic acid molecule described in e1);

[0048] e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0049] e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3);

[0050] e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2);

[0051] e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2);

[0052] e7) A transgenic plant organ containing the nucleic acid molecule described in e1), or a transgenic plant organ containing the expression cassette described in e2).

[0053] In the above applications, the nucleic acid molecule described in c1) can be any of the following DNA molecules:

[0054] d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3;

[0055] d2) The coding sequence is the DNA molecule shown in SEQ ID No. 2;

[0056] d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein described above;

[0057] d4) Hybridizes under strict conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above.

[0058] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0059] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the vector pSG2027.

[0060] Recombinant expression vectors containing the Osnsun5 gene can be constructed using existing plant expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmids (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0061] When constructing a recombinant plant expression vector using the Osnsun5 gene, any enhancing or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing a plant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are broad, and they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0062] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0063] By using any vector capable of guiding the expression of exogenous genes in plants, the Osnsun5 gene or gene fragments provided in this invention can be introduced into plant cells or recipient plants to obtain transgenic cell lines and transgenic plants with altered salt tolerance. The expression vector carrying the Osnsun5 gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0064] Optionally, the expression cassette described in e2) is an expression cassette having the DNA molecule shown in SEQ ID No. 274-616.

[0065] In the above applications, the recombinant vector described in e3) can be a DNA molecule with the nucleotide sequence shown in SEQ ID No. 4.

[0066] Optionally, e3) the recombinant vector contains an expression cassette sequence of sgRNA, where nucleotides 274-518 of SEQ ID No. 4 is the promoter for initiating sgRNA gene transcription, the sgRNA gene is located at positions 519-614 of SEQ ID No. 4 in the sequence listing, and nucleotides 542-616 are the terminator for terminating sgRNA gene transcription.

[0067] The present invention also provides a method for improving the salt tolerance of plants, the method comprising step M, wherein step M is to inhibit or reduce or silence the activity and / or content of the aforementioned proteins in the target plant, or / and, inhibit or reduce or silence the expression level of the encoding gene of the aforementioned proteins, so as to improve the salt tolerance of plants.

[0068] The present invention also provides a method for reducing the salt tolerance of plants, the method comprising step P, wherein step P is to enhance, increase or upregulate the activity and / or content of the aforementioned proteins in the target plant, or / and enhance, increase or upregulate the expression level of the encoding genes of the aforementioned proteins, thereby reducing the salt tolerance of plants.

[0069] In the above method, reducing the expression level and / or activity of the gene encoding the protein Osnsun5 in the target plant can be achieved by using gene mutation, gene knockout, gene editing or gene knockdown techniques to reduce or inactivate the gene encoding the protein Osnsun5 in the genome of the target plant.

[0070] Gene knockout refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knockout inactivates a specific target gene by altering its DNA sequence.

[0071] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0072] The present invention provides a method for cultivating plants with enhanced salt tolerance, comprising inhibiting or reducing or silencing the expression and / or content and / or activity of the coding gene of the aforementioned protein in the target plant, or / and inhibiting or reducing or silencing the activity and / or content of the coding gene of the aforementioned protein, thereby obtaining a plant with enhanced salt tolerance.

[0073] In one embodiment of the present invention, the breeding method for cultivating plants with enhanced salt tolerance includes the following steps:

[0074] (1) Construct recombinant expression vectors that suppress, reduce, or silence the proteins described above;

[0075] (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant with stronger salt tolerance than the recipient plant.

[0076] In this invention, the purpose of plant breeding may include cultivating plants with enhanced salt tolerance.

[0077] In this invention, the plant may be one of the following:

[0078] N1) Monocotyledonous or dicotyledonous plants;

[0079] N2) Plants of the order Poales;

[0080] N3) Gramineae plants;

[0081] N4) Rice plants;

[0082] N5) rice.

[0083] This invention discloses the regulatory role of the Osnsun5 protein and its encoding gene in plant stress tolerance, growth performance, and production performance. This invention protects the negative regulation of Osnsun5 protein in regulating plant stress tolerance, i.e., decreased Osnsun5 protein levels lead to increased salt tolerance in plants. This invention also protects the application of Osnsun5 protein in regulating plant growth performance and / or production performance. This invention can be used for improving plant stress tolerance and has significant application and promotion value for plant breeding, especially rice breeding. Attached Figure Description

[0084] Figure 1 This is a schematic diagram of the recombinant plasmid structure.

[0085] Figure 2 Sequencing results for the Osnsun5#11-1, Osnsun5#12-1 and Osnsun5#12-1 mutation sites and their surrounding nucleotides.

[0086] Figure 3 Phenotypes of wild-type and Osnsun5 mutant under salt stress

[0087] Figure 4 A statistical chart showing the survival rates of wild-type and Osnsun5 mutants under salt stress.

[0088] Figure 5 RNA m 5 Results of C modification level detection. Detailed Implementation

[0089] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0090] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0091] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0092] The rice variety Nipponbare described in the following examples is described in: Zhang Q, Liang Z, Cui X, et al. N6-methyladenine DNA methylation in Japonica and Indica rice genomes and its association with gene expression, plant development, and stress responses. Molecular Plant, 2018, 11(12):1492-1508. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention; it may not be used for any other purpose.

[0093] The following examples use EXCEL to process the data. The experimental results are expressed as mean ± standard deviation. The TTEST test is used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0094] Example 1: Preparation of gene-edited plants

[0095] The genomic DNA of rice Nipponbare contains the genomic sequence encoding the Osnsun5 protein, as shown in SEQ ID No. 3 of the sequence listing, and the protein encoding the amino acid sequence, as shown in SEQ ID No. 1 of the sequence listing. The CDS encoding the Osnsun5 protein is shown in SEQ ID No. 2 of the sequence listing.

[0096] I. Construction of recombinant plasmids

[0097] A recombinant plasmid (circular plasmid) SG2027 was prepared. The nucleotide sequence of the recombinant plasmid SG2027-Osnsun5 is shown in SEQ ID No. 4. The recombinant plasmid can express Cas9 protein and specific sgRNA. In SEQ ID No. 4, nucleotides 2697-6968 encode the Cas9 protein.

[0098] The recombinant plasmid SG2027-Osnsun5 contains an sgRNA expression cassette (nucleotide sequence of the cassette is positions 274-616 of SEQ ID No. 4). Nucleotides 274-518 of SEQ ID No. 4 are the promoter for initiating sgRNA gene transcription, the sgRNA gene is located at positions 519-541 of SEQ ID No. 4, and nucleotides 542-616 are the terminator for terminating sgRNA gene transcription. The specific sgRNA is 5'- CGTCGTCACTACTTCCAGCGAGG-3' (positions 519-541 of SEQ ID No. 4), the target sequence binding region in the sgRNA is positions 438-460 of SEQ ID No. 3 (corresponding to positions 351-373 of SEQ ID No. 2). A schematic diagram of the recombinant plasmid structure is shown below. Figure 1 .

[0099] II. Genetic transformation and obtaining regenerated plants

[0100] The recombinant plasmid SG2027-Osnsun5 prepared in step one was introduced into Agrobacterium EHA105 (Novozymes) to obtain recombinant Agrobacterium EHA105-Osnsun5. Using the Agrobacterium infection method, recombinant Agrobacterium EHA105-Osnsun5 was genetically transformed into embryogenic callus tissue of Nipponbare rice. Resistant callus tissue was then screened (resistance screening used 100 mg / L hygromycin), followed by differentiation and regeneration culture, and then rooting culture to obtain regenerated plants.

[0101] III. Obtaining Gene-Edited Plants and Their Progeny

[0102] The regenerated plants obtained in step two were identified as follows: leaves of the regenerated plants were taken, genomic DNA was extracted, and PCR amplification was performed using primer pairs consisting of primers F and R. The PCR amplification products were then sequenced.

[0103] F: 5'-CTTGGAGGGAGGAGACAG-3';

[0104] R: 5'-GCATCGCTTCCACATTCT-3'.

[0105] Based on the above identification, three homozygous mutant plants (homozygous mutants are those in which the mutations of a pair of homologous chromosomes are identical) were screened from the regenerated plants obtained in step two and named Osnsun5#11-1 gene-edited plant, Osnsun5#12-1 gene-edited plant and Osnsun5#12-2 gene-edited plant, respectively.

[0106] Sequencing analysis revealed that, compared to the genomic DNA of Nipponbare rice (denoted as Nip), the genes encoding the Osnsun5 protein in both homologous chromosomes of the Osnsun5#11-1 plant underwent the following mutation: a deletion of nucleotides "5'-CGCTGG-3'" (positions 441-446 of sequence 3, corresponding to positions 354-359 of sequence 2), causing a frameshift mutation that knocked out the gene encoding the Osnsun5 protein. Sequencing results of this mutation site and its surrounding nucleotides are shown in [link to sequencing data]. Figure 2 .

[0107] Sequencing analysis revealed that, compared to the genomic DNA of Nipponbare rice (denoted as Nip), the genes encoding the Osnsun5 protein in both homologous chromosomes of the Osnsun5#12-1 plant underwent the following mutation: a deletion of nucleotides at positions 443-444 of sequence 3, corresponding to positions 357-358 of sequence 2. This frameshift mutation resulted in the knockout of the gene encoding the Osnsun5 protein. Sequencing results of this mutation site and its surrounding nucleotides are shown below. Figure 2 .

[0108] Sequencing analysis revealed that the gene encoding the Osnsun5 protein in both homologous chromosomes of the Osnsun5#12-2 plant underwent the following mutation: "5'-GCTGGAAGTAG" -3 The deletion of nucleotides '(442-452 of sequence 3, corresponding to 355-365 of sequence 2) caused a frameshift mutation, thereby knocking out the gene encoding the Osnsun5 protein. Sequencing results of this mutation site and its surrounding nucleotides are shown in [link to sequencing data]. Figure 2 .

[0109] Osnsun5#11-1 plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. Osnsun5#11-1 plants and their self-pollinated offspring are called the Osnsun5#11-1 line.

[0110] Osnsun5#12-1 plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. Osnsun5#12-1 plants and their self-pollinated offspring are called the Osnsun5#12-1 line.

[0111] Osnsun5#12-2 plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. Osnsun5#12-2 plants and their self-pollinated offspring are called the Osnsun5#12-2 line.

[0112] Example 2: Study on the salt tolerance of gene-edited plants

[0113] The tested seeds were: Nipponbare rice seeds, T2 generation seeds of the gene-edited plant line Osnsun5#11-1, T2 generation seeds of the Osnsun5#12-1, and T2 generation seeds of the Osnsun5#12-2.

[0114] The test plants were cultured under parallel conditions, specifically as follows: Rice seeds were germinated and seedlings were raised in a greenhouse (timed from the emergence of white shoots, for a total of 3 weeks), resulting in 3-week-old seedlings, which were then photographed. The 3-week-old seedlings were then treated with 150 mM NaCl for 5 days, and photographed again. Phenotypic and survival rate statistics are shown in the table below. Figure 3 and Figure 4 .

[0115] Depend on Figure 3 and Figure 4 It can be seen that after salt stress treatment, the survival rate of mutant seedlings with different editing methods of Osnsun5 was significantly increased by 1-1.5 times compared with wild type, indicating that Osnsun5 negatively regulates the salt stress response of rice.

[0116] Example 3, RNA m 5 C modification level

[0117] The tested seeds were: Nipponbare rice seeds, T2 generation seeds of the gene-edited plant line Osnsun5#11-1, T2 generation seeds of the Osnsun5#12-1, and T2 generation seeds of the Osnsun5#12-2.

[0118] The test plants were cultured under parallel conditions, specifically: the test rice seeds were taken, germinated in a greenhouse and cultured until the three-leaf stage, the above-ground parts were taken, and total RNA of the test rice was extracted.

[0119] Total RNA from the tested rice samples was quantified and subjected to RNA Dot-blot hybridization. The specific antibody used was anti-m... 5 C antibody (Diagenode, C15200081). RNA concentrations were set to 50, 100, or 200 ng / μl, respectively.

[0120] Figure 5 The results showed that, compared with Nipponbare, the RNA m of the Osnsun5#11-1, Osnsun5#12-1, and Osnsun5#12-2 lines was significantly lower. 5 C modification levels increased.

[0121] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of substances that knock out protein-coding genes in any of the following: 1) Application in improving the salt tolerance of plants; 2) Application in the preparation of products that improve the salt tolerance of plants; 3) Application in cultivating plants with enhanced salt tolerance; 4) Application in the preparation of products that cultivate plants with enhanced salt tolerance; The protein is the protein with the amino acid sequence SEQ ID No. 1; The substance is a biomaterial related to the protein, and the biomaterial is any one of the following: e1) Knock out the nucleic acid molecule encoding the protein; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) Transgenic plant organs containing the nucleic acid molecules described in e1), or transgenic plant organs containing the expression cassette described in e2); The plant in question is rice.

2. The application according to claim 1, characterized in that: The protein is derived from rice.

3. A method for improving the salt tolerance of plants, characterized in that: The method includes step M, which involves knocking out the gene encoding the protein described in claim 1 or 2 to improve the salt tolerance of the plant; the plant is rice.

4. A breeding method for cultivating plants with enhanced salt tolerance, characterized in that: The method involves knocking out the gene encoding the protein described in claim 1 or 2 in a target plant to obtain a plant with enhanced salt tolerance, wherein the salt tolerance of the enhanced salt-tolerant plant is stronger than that of the target plant; the plant is rice.

5. The method according to claim 4, characterized in that: Includes the following steps: (1) A recombinant expression vector that knocks out the gene encoding the protein described in claim 1 or 2; (2) The recombinant expression vector constructed in step (1) is transferred into the target plant to obtain a plant with stronger salt tolerance than the target plant.

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