Application of Ossun5 protein and coding gene thereof in regulation and control of salt tolerance of rice

Abstract

The invention discloses an application of an Ossun5 protein and a coding gene thereof in regulating and controlling the salt tolerance of rice. The invention belongs to the technical field of biology, and particularly relates to an application of an Ossun5 protein and a coding gene thereof in regulating and controlling the salt tolerance of rice. The protein provided by the invention is an Osnun5 protein, and is a protein shown as a sequence 1 in a sequence table. According to the present invention, the substance for inhibiting the expression of the Ossun5 gene is introduced into the receptor plant to obtain the gene edited and transformed plant, and the salt stress resistance of the Ossun5 gene edited and transformed plant is improved compared with the target plant, such that the important application promotion value is provided for the regulation of the plant salt tolerance and the crop breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of Osnsun5 protein and its encoding gene in regulating the salt tolerance of rice. Background Art

[0002] Rice is native to China and India. It is one of the world's major grain crops. In China, rice accounts for a quarter of the country's total grain crop area and over half of its production. Its cultivation history dates back 14,000 to 18,000 years. It is an important food crop. Besides its edible caryopsis, rice can be used to make starch, wine, and vinegar. Rice bran can be used to make sugar, oil, and furfural for industrial and pharmaceutical uses. Rice stalks are a good feed, a raw material for papermaking, and a weaving material. Rice sprouts and roots are used medicinally.

[0003] In recent years, the rapid development of biotechnology has significantly driven innovation in plant breeding research methods and the continuous improvement of research capabilities. Plant breeding for pest and disease resistance and herbicide tolerance has entered the practical stage. Using biotechnology to introduce exogenous insecticide and herbicide resistance genes into plant genomes breaks down the natural barriers that prevent hybridization between plant genera and even species, enabling the transfer of insect- and herbicide-resistant genes. This allows plants to rapidly and specifically acquire insect resistance and mechanical weed control while retaining their original favorable agronomic traits. Because each genetically modified corn plant possesses a considerable degree of resistance, its insect and herbicide resistance is superior and more stable than manual control. It also saves manpower and material resources, effectively conserving social resources. Summary of the Invention

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

[0005] In order to solve the problems existing in the prior art, the present invention provides the use of a protein or a substance that regulates the expression of a gene or a substance that regulates the activity or content of the protein in regulating the salt tolerance of a plant.

[0006] Use of the protein or gene expression regulating substance provided by the present invention or the substance regulating the activity or content of the protein in any of the following:

[0007] 1) Use of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in regulating plant salt tolerance;

[0008] 2) Use of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in the preparation of products regulating plant salt tolerance;

[0009] 3) Use of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in cultivating plants with altered salt tolerance;

[0010] 4) Use of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in the preparation of products for cultivating plants with altered salt tolerance;

[0011] 5) Application of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in plant breeding;

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

[0013] a1) a protein having the amino acid sequence of SEQ ID No. 1;

[0014] a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 1 after one or more amino acid residues are substituted and / or deleted and / or added;

[0015] a3) a protein having an amino acid sequence of at least 75% identity with any of the amino acids specified in a1) or (a2) and having the same function;

[0016] a4) A fusion protein obtained by ligating a tag to the end of the protein defined in any one of a1) to (a3).

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

[0018] In order to facilitate purification or detection of the protein in a1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

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

[0020] The tag protein includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0021] Those skilled in the art can readily mutate the nucleotide sequence encoding the Osnsun5 protein of the present invention using known methods, such as directed evolution or point mutagenesis. Artificially modified nucleotide sequences that are 75% or more identical to the nucleotide sequence of the isolated Osnsun5 protein of the present invention are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the Osnsun5 protein and possess the function of the Osnsun5 protein.

[0022] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0023] As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences or nucleotide sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The value (%) of identity can then be obtained.

[0024] Herein, the greater than 80% identity may 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] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

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

[0027] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein Osnsun5.

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

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

[0030] 2) post-transcriptional regulation of the gene (i.e., regulation of the splicing or processing of the primary transcript of the gene);

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

[0032] 4) regulation of the translation of the gene;

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

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

[0035] In the present invention, the regulation can be up-regulation, enhancement or increase. The regulation can also be down-regulation, attenuation or reduction.

[0036] Herein, the enhancement, increase or upregulation of the expression level of the gene encoding the protein mentioned above in the recipient plant, or / and the enhancement, increase or upregulation of the activity and / or content of the gene encoding the above protein is achieved by introducing the gene encoding the above protein into the recipient plant.

[0037] Herein, the regulation of the expression of the gene encoding the protein may be inhibition, reduction or down-regulation of the expression of the gene encoding the protein. Inhibition, reduction or down-regulation of the expression of the gene encoding the protein may be achieved by gene knockout or gene silencing.

[0038] In the above applications, the substance that regulates gene expression or the substance that regulates the activity or content of the protein may be a biological material related to the protein mentioned above, and the biological material may be any of the following:

[0039] c1) a nucleic acid molecule encoding 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) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism 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 molecule 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) a nucleic acid molecule that inhibits, reduces or silences the expression of the protein encoding gene 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) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism 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 molecule 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 application, the nucleic acid molecule 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) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) or d2) and encodes the protein described above;

[0057] d4) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in d1) or d2) and encodes the protein described above.

[0058] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule 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, including but not limited to plasmids, phages (such as lambda phage or M13 filamentous phage), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. Specifically, the vector may be pSG2027.

[0060] Existing plant expression vectors can be used to construct a recombinant expression vector containing the Osnsun5 gene. Such plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. Such plant expression vectors may also contain the 3' untranslated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor. Examples include, but are not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the rouge synthase Nos gene) and the 3' untranslated region transcribed from plant genes (such as the soybean storage protein gene), all of which have similar functions.

[0061] When using the Osnsun5 gene to construct a recombinant plant expression vector, any enhanced promoter 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 ubiquitin promoter of corn. These promoters can be used alone or in combination with other plant promoters. In addition, when using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

[0062] To facilitate identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified to include, but are not limited to, genes encoding enzymes or luminescent compounds that can be expressed in plants (such as the GUS gene or luciferase gene), antibiotic resistance markers (such as gentamicin and kanamycin), or chemical resistance marker genes (such as herbicide resistance genes). For safety reasons, it is possible to omit any selectable marker genes and directly screen for transformed plants using stress.

[0063] By introducing the Osnsun5 gene or gene fragment provided by the present invention into plant cells or recipient plants using any vector capable of directing exogenous gene expression in plants, transgenic cell lines and transgenic plants with altered salt tolerance can be obtained. Expression vectors carrying the Osnsun5 gene can be transformed into plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transfection, and the transformed plant tissues can be cultivated into plants.

[0064] Optionally, the expression cassette described in e2) is an expression cassette having the DNA molecule shown in Nos. 274-616 of SEQ ID No. 4.

[0065] In the above application, the recombinant vector described in e3) may be a DNA molecule having a nucleotide sequence shown as SEQ ID No. 4.

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

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

[0068] The present invention also provides a method for reducing plant salt tolerance, which comprises step P, wherein step P is to enhance, increase or upregulate the activity and / or content of the protein described above in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein described above, so as to reduce the plant salt tolerance.

[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: using gene mutation, gene knockout, gene editing or gene knockdown technology to reduce or inactivate the activity of the gene encoding the protein Osnsun5 in the target plant genome.

[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 changing its DNA sequence.

[0071] The gene silencing refers to the phenomenon of not expressing or underexpressing a gene without damaging the original DNA. Gene silencing is based on the premise that the DNA sequence is not changed, so that the gene is not expressed or underexpressed. Gene silencing can occur at two levels. One is gene silencing at the transcriptional level due to DNA methylation, heterochromatinization and position effects, and the other is post-transcriptional gene silencing, that is, gene inactivation by specifically inhibiting the target RNA at the level after gene transcription, including antisense RNA, co-suppression, gene repression (quelling), RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0072] The present invention provides a method for cultivating plants with enhanced salt tolerance, comprising inhibiting, reducing or silencing the expression of the gene encoding the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the target plant, or / and inhibiting, reducing or silencing the activity and / or content of the gene encoding the above-mentioned protein, to obtain plants with enhanced salt tolerance.

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

[0074] (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein described above;

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

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

[0077] In the present invention, the plant may be as follows:

[0078] N1) Monocotyledonous or dicotyledonous plants;

[0079] N2) Gramineae;

[0080] N3) Grasses;

[0081] N4) Oryza plants;

[0082] N5) Rice.

[0083] The present invention discloses the regulatory effects of the Osnsun5 protein and its encoding gene on plant stress tolerance, growth performance, and production performance. The present invention protects the Osnsun5 protein in regulating plant stress tolerance as negative regulation, that is, the Osnsun5 protein content is reduced and the plant's salt tolerance is enhanced. The present invention also protects the use of the Osnsun5 protein in regulating plant growth performance and / or production performance. The present invention can be used to improve plant stress tolerance and has significant application and promotion value for plant breeding, especially rice breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Schematic diagram of the structure of the recombinant plasmid.

[0085] Figure 2 These are the sequencing results of the mutation sites and surrounding nucleotides of Osnsun5#11-1, Osnsun5#12-1 and Osnsun5#12-1.

[0086] Figure 3 Salt stress phenotypes of wild type and Osnsun5 mutant

[0087] Figure 4 Statistical graph of the survival rate of wild type and Osnsun5 mutant under salt stress.

[0088] Figure 5 RNA m 5 Results of C modification level detection. DETAILED DESCRIPTION

[0089] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

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

[0091] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0092] The rice Nipponbare described in the following examples has been 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. The public may obtain the biological material from the applicant for use only in repeating the experiments of the present invention and may not be used for other purposes.

[0093] The following examples were processed using EXCEL to process the data. The experimental results were expressed as mean ± standard deviation and tested using TTEST. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

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

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

[0096] 1. Construction of recombinant plasmid

[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 the Cas9 protein and a specific sgRNA. In SEQ ID No. 4, nucleotides 2697-6968 encode the Cas9 protein.

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

[0099] 2. Genetic transformation and obtaining regenerated plants

[0100] The recombinant plasmid SG2027-Osnsun5 prepared in step 1 was introduced into Agrobacterium tumefaciens EHA105 (NoviZan) to obtain recombinant Agrobacterium tumefaciens EHA105-Osnsun5. Embryogenic calli of rice Nipponbare were genetically transformed with recombinant Agrobacterium tumefaciens EHA105-Osnsun5 using the Agrobacterium infection method. Resistant calli were then screened (resistance screening was performed using 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 2 were identified as follows: leaves of the regenerated plants were taken, genomic DNA was extracted, PCR amplification was performed using a primer pair consisting of primer F and primer R, and the PCR amplification products were sequenced.

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

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

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

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

[0107] Sequencing revealed that, compared to the genomic DNA of rice Nipponbare (denoted by Nip), the genes encoding the Osnsun5 protein in both homologous chromosomes of the Osnsun5#12-1 plant had the following mutation: a "5'-TG-3'" (nucleotides 443-444 of sequence 3, corresponding to nucleotides 357-358 of sequence 2) deletion, causing a frameshift mutation, thereby knocking out the gene encoding the Osnsun5 protein. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 2 .

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

[0109] Osnsun5#11-1 plants were self-pollinated and seeds were harvested. These seeds were then grown into plants, which became the T1 generation. T1 generation plants were self-pollinated and seeds were harvested, which became the T2 generation. Osnsun5#11-1 plants and their self-pollinated offspring are referred to as the Osnsun5#11-1 line.

[0110] Osnsun5#12-1 plants were self-pollinated and seeds were harvested. These seeds were then grown into plants, which became the T1 generation. T1 generation plants were self-pollinated and seeds were harvested, which became the T2 generation. Osnsun5#12-1 plants and their self-pollinated offspring are referred to as the Osnsun5#12-1 line.

[0111] Osnsun5#12-2 plants were self-pollinated and seeds were harvested. These seeds were then grown into plants, which became the T1 generation. T1 generation plants were self-pollinated and seeds were harvested, which became the T2 generation. Osnsun5#12-2 plants and their self-pollinated offspring are referred to as the Osnsun5#12-2 line.

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

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

[0114] The test plants were cultured under parallel conditions, specifically: rice seeds were germinated and raised in a greenhouse (starting from the time of whitening, for a total of 3 weeks), 3-week seedlings were obtained and photographed; 3-week seedlings were treated with 150 mM NaCl for 5 days and photographed for statistical phenotype and survival rate statistics. 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 seedlings of mutants with different editing methods of Osnsun5 was significantly increased by 1-1.5 times compared with the wild type, indicating that Osnsun5 negatively regulates the salt stress response of rice.

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

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

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

[0119] The total RNA of the test rice was collected and RNA Dot-blot was performed. The specific antibody used was anti-m 5 C antibody (Diagenode, C15200081). The RNA amount was set to 50, 100, or 200 ng / μl.

[0120] Figure 5 The results showed that compared with Nipponbare, the RNA m 5 C modification levels increased.

[0121] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates gene expression or a substance that regulates the activity or content of the protein in any of the following: 1) Use of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in regulating plant salt tolerance; 2) Use of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in the preparation of products regulating plant salt tolerance; 3) Use of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in cultivating plants with altered salt tolerance; 4) Use of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in the preparation of products for cultivating plants with altered salt tolerance; 5) Application of proteins or substances regulating gene expression or substances regulating the activity or content of said proteins in plant breeding; The protein is any one of the following proteins: a1) a protein having the amino acid sequence of SEQ ID No. 1; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 1 after one or more amino acid residues are substituted and / or deleted and / or added; a3) a protein having an amino acid sequence identity of 80% or more to any of a1) to a2) and having the same function; a4) A fusion protein obtained by ligating a tag to the end of the protein defined in any one of a1) to a3).

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

3. The use according to claim 1 or 2, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application according to claim 1 or 2, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); 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); c6) transgenic plant tissue containing the nucleic acid molecule described in c1), or transgenic plant tissue containing the expression cassette described in c2); 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); e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the protein encoding gene; 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) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism 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 molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); 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).

4. The use according to claim 3, characterized in that: c1) The nucleic acid molecule is a DNA molecule as shown below, d1) the nucleotide sequence is the DNA molecule shown in SEQ ID No. 3; d2) the coding sequence is the DNA molecule shown in SEQ ID No. 2; d3) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) or d2) and encodes the protein of claim 1; d4) A DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in d1) or d2) and encodes the protein of claim 1.

5. A method for improving plant salt tolerance, characterized by: The method includes step M, which is to inhibit, reduce or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and, inhibit, reduce or silence the expression level of the gene encoding the protein described in claim 1 or 2, so as to improve the salt tolerance of the plant.

6. A method for reducing salt tolerance of plants, characterized by: The method includes step P, which is to enhance, increase or upregulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein described in claim 1 or 2 to reduce the salt tolerance of the plant.

7. A method for cultivating plants with enhanced salt tolerance, characterized in that: The method comprises inhibiting, reducing or silencing the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant, and / or the activity and / or content of the protein, thereby obtaining a plant with enhanced salt tolerance, wherein the plant with enhanced salt tolerance has stronger salt tolerance than the recipient plant.

8. The method according to claim 7, wherein: The steps include: (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein of claim 1 or 2; (2) The recombinant expression vector constructed in step (1) is transferred into a recipient plant to obtain a plant having a salt tolerance stronger than that of the recipient plant.

9. The protein according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4.

10. The use according to any one of claims 1 to 4, and / or the method according to any one of claims 5 to 8, characterized in that: The plant is any one of the following: N1) Monocotyledonous or dicotyledonous plants; N2) Gramineae; N3) Grasses; N4) Oryza plants; N5) Rice.

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