Application of OsRBP22 gene in regulating salt tolerance in rice

Abstract

The present invention belongs to the field of modern agricultural technology and specifically relates to OsRBP22 The application of genes in regulating rice salt tolerance. OsRBP22 The application of genes in regulating salt tolerance in rice. OsRBP22 Overexpression of genes can significantly improve the salt tolerance of rice, providing a simple and effective technical means for the rapid creation of new salt-tolerant rice varieties.

Description

Technical Field

[0001] The present invention belongs to the field of modern agricultural technology and specifically relates to OsRBP22 Application of genes in regulating salt tolerance in rice. Background Art

[0002] Salt stress is one of the major abiotic stresses that limits plant growth and crop yield. The global area of ​​saline-alkali land is approximately 800 million hectares, threatening global food security. Oryza sativa Rice (L.) is one of the world's most important food crops, but it is very sensitive to salt stress. Saline-alkali land resources in rice-growing areas around the world have enormous development potential.

[0003] Therefore, improving rice salt tolerance is of great practical significance for promoting agricultural utilization of salinized land, increasing rice yields, and ensuring food security. Further research on the regulatory genes involved in rice salt tolerance is necessary to develop new methods for breeding salt-tolerant rice varieties. Summary of the Invention

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0005] The first aspect of the present invention provides the use of an OsRBP22 protein promoter in improving salt tolerance in rice.

[0006] In some embodiments of the present invention, the OsRBP22 protein promoter includes an agent that targets and increases the expression level of the OsRBP22 protein.

[0007] In some embodiments of the present invention, the amino acid sequence of the OsRBP22 protein is shown in SEQ ID NO: 3.

[0008] The second aspect of the present invention provides overexpression OsRBP22 Application of genes in improving salt tolerance in rice.

[0009] In some embodiments of the present invention, the OsRBP22 The genomic nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0010] In some embodiments of the present invention, the OsRBP22 The CDS nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0011] The third aspect of the present invention provides the use of biological materials related to overexpression of OsRBP22 protein in breeding rice varieties with improved salt tolerance.

[0012] In some embodiments of the present invention, the amino acid sequence of the OsRBP22 protein is shown in SEQ ID NO: 3.

[0013] In some embodiments of the invention, the biological material does not include propagation material.

[0014] In some embodiments of the present invention, the biological material comprises a nucleic acid molecule, a vector, or a cell.

[0015] In some embodiments of the present invention, the nucleic acid molecule includes a nucleic acid molecule encoding the protein shown in SEQ ID NO:3.

[0016] In some embodiments of the present invention, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:2.

[0017] In some embodiments of the present invention, the vector includes an overexpression vector, including but not limited to pC1300-35S-GFP and other conventional overexpression vector tools in the art.

[0018] In some embodiments of the present invention, the cell comprises at least one of Escherichia coli and Agrobacterium tumefaciens, wherein Escherichia coli is a common host cell for constructing vectors and plasmids in the art, and Agrobacterium tumefaciens is a common tool for delivering DNA molecules to plants in the art.

[0019] In the third aspect of the present invention, those skilled in the art will appreciate that due to the degeneracy of codons, other OsRBP22 The technical solution of modifying the gene but still encoding the protein with the amino acid sequence as shown in SEQ ID NO: 3 can also achieve the same or similar technical effects as the present invention and still fall within the scope of protection of the present invention.

[0020] A fourth aspect of the present invention provides a method for cultivating a rice variety with improved salt tolerance, comprising increasing the salt tolerance of the rice. OsRBP22 The step of measuring the expression level and / or activity of a gene.

[0021] In some embodiments of the present invention, the method for increasing the yield of rice OsRBP22 The step of increasing the expression level and / or activity of the gene is to introduce the biological material related to overexpression of OsRBP22 protein described in the third aspect of the present invention into rice tissues or rice cells.

[0022] In some embodiments of the invention, the biological material does not include propagation material.

[0023] In some embodiments of the present invention, the introduction method comprises using at least one of Ti plasmid, Ri plasmid, plant virus vector, microinjection, and electroporation.

[0024] In some embodiments of the present invention, increasing OsRBP22 The steps of measuring the expression level and / or activity of a gene are as follows:

[0025] 1) The sequence shown in SEQ ID NO: 2 is OsRBP22 The full-length CDS sequence of the gene was designed and amplified OsRBP22 Primer sequences of the full-length CDS sequence of the gene;

[0026] 2) Using rice cDNA as a template, perform PCR amplification to obtain PCR products;

[0027] 3) After the PCR product is purified, homologous recombination is used to reconstruct the overexpression vector;

[0028] 4) The overexpression vector was transformed into rice through Agrobacterium-mediated transformation.

[0029] In some embodiments of the present invention, the rice variety includes Zhonghua 11.

[0030] In some embodiments of the present invention, the sequences of the primers are shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0031] The beneficial effects of the present invention are:

[0032] The present invention first discovered OsRBP22 The application of genes in regulating salt tolerance in rice. OsRBP22 Overexpression of genes can significantly improve the salt tolerance of rice, providing a simple and effective technical means for the rapid creation of new salt-tolerant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0034] Figure 1 for OsRBP22 Identification of overexpressing material.

[0035] Figure 2 for OsRBP22 Salt tolerance phenotypic results of overexpression materials, where A is the phenotypic result and B is the survival rate statistical result. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0037] Example 1 OsRBP22 Construction of overexpression vector and acquisition and identification of transgenic plants

[0038] The present invention provides RNA binding protein gene OsRBP22 (LOC_Os07g18050) in rice salt tolerance, OsRBP22 The genome sequence is as follows:

[0039]

[0040] Its CDS sequence is:

[0041]

[0042] Its protein sequence is:

[0043] N-terminal-MAHRLLRDAQADGWERSDFPIICESCLGDNPYVRMLRAEYDKECKICARPFTV FRWRPGRDARYKKTEICQTCCKLKNVCQVCLLDLEYGLPVQVRDTALSTNSNDAIPRSDVNREYFAEEHDRRARAGIDYDSSNGKARANDTILKLQRTAPYYKRNRAHVC SFYVRGECTRGAECPYRHEMPETGELSQQNIKDRYYGVNDPVALKLLSKAGEMPSLTPPDDESIRTLYIGGLDSRVTEQDLRDQFYAHGEIETIRMVLQRACAFVTYTTR EGAEKAAEELANKLVIKGVRLKLMWGKPQAPKPEEDEAGRQGHVAHGGMLPRAVISQQQSGDQPQPPGMEGQQQPASASYYFNIPAPPAAERTLYPSMDPQRMGALVESQ EGDGKPGPQQAGQGQASSSSGQSYPEPPPPYYHGGQYPPYYPPYGGYMPPPRMPYQQPPQYPAYQPMLAPPAQSQASSLQQPAPATQQLGQGPQQQTTQNGMT-C terminal (SEQ ID NO: 3).

[0044] The specific steps include:

[0045] 1. Extraction of Total RNA

[0046] The rice variety Zhonghua 11 (ZH11) was used as the material. Seeds were sterilized with 30% NaClO solution for 10 minutes, rinsed, germinated, and cultured to the two-leaf, one-heart stage. Plants with consistent growth were selected, their endosperms removed, and transferred to 1 / 2 IRRI nutrient solution at pH 5.5. At the four-leaf, one-heart stage, the solution was switched to full-strength IRRI nutrient solution and cultured for another week. Leaves and roots were collected and quickly frozen in liquid nitrogen for storage. Approximately 0.1 g of sample was weighed, thoroughly ground, and placed in a 1.5 mL centrifuge tube. 1 mL of Trizol reagent was quickly added, mixed thoroughly, and 0.2 mL of chloroform was added. The supernatant was extracted by centrifugation, and RNA was precipitated with 0.5 mL of isopropanol. The RNA was washed with 70% ethanol and finally dissolved in DEPC water. RNA quality was assessed by 1.7% agarose gel electrophoresis, and concentration and purity were determined spectrophotometrically.

[0047] 2. Total cDNA Synthesis

[0048] Using 5 μL RNA (approximately 2 μg) of each sample as a template, 5×g DNA wiper mix (2 μL) and RNase-free ddH2O (3 μL) were added. After reacting at 42°C for 2 minutes, 4×HiScript IV qRT SuperMix (5 μL) and ddH2O were added to make up to 10 μL. The reaction conditions were 37°C for 15 minutes and 85°C for 15 seconds.

[0049] 3. Amplification of the full-length OsRBP22 CDS sequence

[0050] Using the synthesized cDNA as a template, PCR amplification was performed using the following primers:

[0051] OsRBP22-F: ATGGCCCACCGGCTGCTG (SEQ ID NO: 4);

[0052] OsRBP22-R:TTAAGTCATTCCGTTCTGGGT (SEQ ID NO: 5).

[0053] PCR reaction conditions were: initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 15 s, 56°C for 15 s, and extension at 72°C for 50 s, with a final extension at 72°C for 5 min. The amplified product was examined by 1% agarose gel electrophoresis, revealing a target fragment length of 1461 bp. This product was sequenced and subsequently used for future reference.

[0054] 4. Construction of overexpression vector p35S-OsRBP22

[0055] Using pC1300-35S-GFP (KpnI and SalI restriction sites) as the backbone vector, design recombination primers with homology arms:

[0056] 35S-OsRBP22-F: tttggagaggacagggtaccatggcccaccggctgctgcg (SEQ ID NO: 6);

[0057] 35S-OsRBP22-R:accatggtactagtgtcgacagtcattccgttctgggttg (SEQ ID NO: 7).

[0058] Amplification protocol: 95°C initial denaturation for 1 min, 95°C for 15 s, 56°C for 15 s, and 72°C extension for 1 min 20 s, 30 cycles, with a final extension at 72°C for 7 min. The target fragment was confirmed to be approximately 1500 bp by electrophoresis and recovered from the gel.

[0059] The recovered product was homologously recombined with the pC1300-35S-GFP linearized vector, which had been double-digested with KpnI and SpeI. The reaction system consisted of 2 μL of the linearized vector, 3 μL of the recovered target fragment, and 5 μL of the homologous recombination mix. The reaction was carried out at 37°C for 30 minutes. The recombinant product was transformed into competent Escherichia coli DH5α and cultured overnight on LB plates containing 50 μg / mL kanamycin.

[0060] Positive clones were picked for bacterial liquid PCR and sequencing verification. The clones with correct sequencing were added with an equal volume of 30% glycerol solution and stored at -70°C and named p35S-OsRBP22.

[0061] 5. Obtaining transgenic plants

[0062] Competent cells of Agrobacterium tumefaciens EHA105 containing the p35S-OsRBP22 plasmid were transformed by electroporation and inoculated into YEP solid medium supplemented with kanamycin and streptomycin (50 μg / mL) for 48 hours. Positive colonies were selected for PCR verification and stored in glycerol.

[0063] Rice callus was infected with the Agrobacterium and cultured for 60 hours before selection, differentiation, and rooting to obtain T0 transgenic plants. The transgenic lines were propagated for two generations, and high-expressing, homozygous overexpression materials were screened.

[0064] 6. Identification of overexpression effect (qPCR analysis)

[0065] Total RNA was extracted and reverse transcribed to synthesize cDNA, and qRT-PCR was performed using the following primers:

[0066] Internal reference gene (OsActin):

[0067] OsActinqF: TTATGGTTGGGATGGGACA (SEQ ID NO: 8);

[0068] OsActinqR: AGCACGGCTTGAATAGCG (SEQ ID NO: 9).

[0069] Target gene (OsRBP22):

[0070] OsRBP22qF:CGTGCTTGTGCATTTGTGAC (SEQ ID NO: 10);

[0071] OsRBP22qR: ACGCACACCCTTGATGACTA (SEQ ID NO: 11).

[0072] The total volume of the qPCR reaction system was 10 μL, and the reaction conditions were: 95°C for 5 s, 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s, for a total of 40 cycles, with a final extension at 72°C for 5 min. Data were calculated using the ΔΔCt method.

[0073] Finally, the overexpression strain OE24 with significantly increased expression level and homozygous genetic background was obtained for subsequent functional verification experiments.

[0074] Example 2 OsRBP22 Functional verification of salt tolerance of overexpression lines

[0075] Analyzed under greenhouse conditions using hydroponics OsRBP22 Responses of the overexpression line (OE24) and the wild type (ZH11) to salt stress.

[0076] 1. Experimental design and treatment conditions

[0077] Seeds of ZH11 and OE24 were sterilized with 10% H₂O₂ for 10 minutes, washed, and placed in a Petri dish containing sterilized filter paper. They were incubated in the dark at 37°C for 2 days and then germinated at 28°C for 1 day. After germination, the seeds were transferred to a nutrient solution hydroponic incubator and cultured for 7 days, with the nutrient solution replaced every 2 days.

[0078] The culture environment was set at 28 / 25°C (day / night), relative humidity of approximately 70%, a photoperiod of 14 / 10 h, and a light intensity of 400 μmol·m -2 ·s -1 .

[0079] Salt stress treatment with 100 mM NaCl was applied at the 3-leaf stage. Obvious phenotypic differences were observed 7 days after treatment, and OE24 plants showed obvious salt tolerance.

[0080] The formula of the hydroponic nutrient solution for rice seedlings is shown in Table 1.

[0081] Table 1

[0082]

[0083] The working solution was prepared immediately for use. 1 mL of each mother solution was required for each liter of working solution, and the pH was adjusted to 5.5.

[0084] 2. Observation of salt tolerance phenotype

[0085] After 15 days of treatment, ZH11 showed typical stress phenotypes such as wilting, water loss, and leaf curling, while OE24 maintained relatively good growth. Statistical analysis of the survival rate of OE24 showed a 42.1%–47.7% increase compared to ZH11, indicating that overexpressing OsRBP22 significantly enhances rice tolerance to salt stress.

[0086] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Overexpression OsRBP22 The application of a gene reagent in improving salt tolerance of rice is characterized by: described OsRBP22 The nucleotide sequence of the gene is shown in SEQ ID NO: 2; The reagents are: 1) Overexpression vector; The overexpression vector comprises the nucleotide sequence shown in SEQ ID NO: 2; or 2) cells; The cell comprises the overexpression vector described in 1); The cells include Agrobacterium.

2. A method for cultivating rice varieties with improved salt tolerance, characterized by: Including increasing rice OsRBP22 Steps for determining gene expression levels; The method for increasing the OsRBP22 The step of increasing the expression level of the gene is to overexpress the gene in claim 1 OsRBP22 Gene reagents are introduced into rice tissues or rice cells.

3. The method according to claim 2, wherein: The introduction method includes using at least one of Ti plasmid, Ri plasmid, plant virus vector, microinjection, and electroporation.

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