Application of rice OsRMT1 gene in improvement of salt tolerance of rice

Abstract

The invention provides an application of a rice OsRMT1 gene in improving the salt tolerance of rice, and belongs to the technical field of plant genetic engineering.According to the invention, the OsRMT1 gene in rice is obtained through a designed primer, and by constructing an overexpression positive material and a knockout mutant material of OsRMT1, it is proved that OsRMT1 can negatively regulate the salt tolerance of rice, and the OsRMT1 gene can be used for improving the salt tolerance of rice. The method can be used for cultivating and screening salt-tolerant rice varieties. The OsRMT1 gene in the rice is knocked out, so that the content of OsMBL1 in the rice can be increased, and the salt tolerance of the rice is improved by increasing the content of OsMBL1.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering and relates to an application of a rice OsRMT1 gene in improving the salt tolerance of rice. Background Art

[0002] There are many environmental stress factors in the process of plant growth and development, and high salt is one of them. High salt stress usually induces plants to produce ion stress, osmotic stress and oxidative stress accompanied by the generation of ROS (reactive oxygen species), which eventually leads to the degradation of chlorophyll in the plant body and reduced photosynthesis efficiency. At the same time, it will seriously damage the cell structure and macromolecules, causing abnormal plant growth and development. Rice (Oryza sativa L.) is an important food crop in the world, but soil salinization has seriously threatened the cultivated area and final yield of rice. As a salt-sensitive crop, rice is extremely susceptible to salt stress at all stages from seed germination to harvest. Therefore, it is urgent to study the key factors of rice high salt tolerance and cultivate salt-adapted rice varieties with stable yield potential.

[0003] As a sedentary crop, plants cannot actively escape from adverse factors like animals, so they have evolved a series of molecular and physiological mechanisms to cope with various adverse environments in the face of various abiotic stresses, and ubiquitination-mediated 26S proteasome degradation is an important mechanism for plants to respond to various stresses. Ubiquitin-mediated 26S proteasome degradation works through an important, universal, and complex regulatory pathway. During this degradation process, three enzymes, E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase), participate in connecting ubiquitin chains to target proteins and forming ubiquitin chains. The general process is as follows: First, E1 activates ubiquitin so that it can be accepted by E2 to form an E2-ubiquitin complex, and then E3 transfers the ubiquitin chain to its specific substrate. E3 proteins are divided into four subfamilies based on their structure: HECT, RING, U-Box, and CRLE3 ligases. As the last enzyme in the first stage of the ubiquitin proteasome pathway, E3 plays a very important function in the specific recognition of target substrates. In recent years, a large number of E3 ligases in plants have been shown to play a key role in responding to various abiotic stresses, including tolerance to low temperature environments, tolerance to high temperatures and drought, and salt tolerance.

[0004] Therefore, studying E3 ligase to improve rice's tolerance to high salt not only provides a new genetic resource approach for enhancing rice's tolerance to high salt using genetic means, but also provides a very important idea for the future breeding of new varieties of crops such as rice that can withstand high-salt adversity and maintain high yield and high quality. It has important theoretical guiding significance and practical application value. Summary of the invention

[0005] In order to improve the high salt tolerance of rice, the present invention proposes an application of rice OsRMT1 gene in improving the salt tolerance of rice.

[0006] OsRMT1 is a RING-type E3 ubiquitin ligase in rice. The present invention uses genetic transformation to overexpress the OsRMT1 gene, explores the relationship between the gene and the salt tolerance of rice, and knocks out the gene by CRISPR-Cas9, thereby improving the salt tolerance of rice. The present invention also uses yeast two-hybrid screening library technology to screen for proteins that interact with OsRMT1, namely the rice lectin protein OsMBL1, and further verifies through yeast two-hybrid experiments and bimolecular fluorescence complementation experiments that OsMBL1 interacts with OsRMT1; finally, the role of OsRMT1 in the ubiquitination and degradation of OsMBL1 is verified through in vitro ubiquitination experiments, proving that OsRMT1 can ubiquitinate and degrade OsMBL1, thereby reducing the salt tolerance of rice.

[0007] The protein encoded by the OsRMT1 gene is OsRMT1, the amino acid sequence of the protein OsRMT1 is shown in SEQ ID NO.1, the nucleotide sequence of the OsRMT1 gene is shown in SEQ ID NO.2, and the CDS sequence in the OsRMT1 gene has a nucleic acid sequence shown in SEQ ID NO.3.

[0008] The present invention obtains the OsRMT1 gene of rice by designing primers such as SEQ ID NO.7 and SEQ ID NO.8.

[0009] The OsRMT1 gene and the protein it encodes can be used as negative regulatory factors to regulate rice salt tolerance. The OsRMT1 gene can be used to cultivate and screen salt-tolerant rice varieties, specifically by knocking out the OsRMT1 gene through CRISPR-Cas9 gene editing technology to obtain salt-tolerant rice varieties. The specific operations are:

[0010] 1) Designing a target sequence of the OsRMT1 gene, wherein the target sequence includes sgRNA-1 and sgRNA-2;

[0011] The sequence of sgRNA-1 is shown in SEQ ID NO.13, and the sequence of sgRNA-2 is shown in SEQ ID NO.14.

[0012] 2) synthesizing upstream and downstream primers for the target sequence of the OsRMT1 gene, and using the synthesized upstream and downstream primers to prepare oligo dimers;

[0013] For sgRNA-1, the synthesized upstream primer sequence is shown in SEQ ID NO.15, and the downstream primer sequence is shown in SEQ ID NO.16; for sgRNA-2, the synthesized upstream primer sequence is shown in SEQ ID NO.17, and the downstream primer sequence is shown in SEQ ID NO.18.

[0014] 3) Connect the oligo dimer to the vector BGK015 to obtain a knockout vector; transform the knockout vector into competent Escherichia coli cells and extract the knockout vector plasmid.

[0015] 4) The knockout vector plasmid is introduced into rice plants through genetic transformation, and the plants with the OsRMT1 gene knocked out are selected as salt-tolerant rice varieties.

[0016] The OsRMT1 gene is knocked out by CRISPR / Cas9 gene editing technology, thereby reducing the expression level of its encoded protein and improving the salt tolerance of rice plants.

[0017] By knocking out the OsRMT1 gene in rice, the content of OsMBL1 in rice can also be increased. OsMBL1 is a protein that interacts with OsRMT1. Its function in rice is to reduce oxidative damage caused by salt stress. The present invention found through yeast two-hybrid experiments and bimolecular fluorescence complementation experiments that OsMBL1 interacts with OsRMT1, and found in an in vitro ubiquitination experiment that OsMBL1 can be ubiquitinated and degraded by OsRMT1. The amino acid sequence of protein OsMBL1 is shown in SEQ ID NO.4, the nucleotide sequence of the OsMBL1 gene encoding protein OsMBL1 is shown in SEQ ID NO.5, and the CDS sequence in the OsMBL1 gene is shown in SEQ ID NO.6.

[0018] Beneficial effects:

[0019] (1) The present invention obtains the OsRMT1 gene in rice through designed primers, successfully constructs OsRMT1 overexpressing transgenic rice materials, and uses CRISPR-Cas9 gene editing technology to accurately obtain mutant plants of the OsRMT1 gene. Through salt tolerance evaluation, including but not limited to plant phenotype observation in a high-salt environment, statistical analysis of rice plant survival rate, and DAB staining experiments of rice leaves, it is strongly proved that the OsRMT1 gene plays a key role in significantly improving the salt tolerance of rice.

[0020] (2) The present invention screened the OsRMT1-interacting protein rice lectin protein OsMBL1 through yeast two-hybrid screening library technology, and further verified the interaction between OsRMT1 and OsMBL1 through yeast two-hybrid experiments, bimolecular fluorescence complementation experiments, and in vitro ubiquitination experiments, and confirmed that OsRMT1 can ubiquitinate OsMBL1.

[0021] (3) The present invention not only enriches the understanding of the salt tolerance mechanism of plants, but also opens up a new genetic resource approach for the future breeding of new rice varieties that can withstand high-salt stress and maintain high yield and high quality, which has important theoretical guidance significance and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the plasmid map of the pBI121-OsRMT1 overexpression vector.

[0023] Figure 2 This is the plasmid map of the pGADT7-OsRMT1 yeast two-hybrid vector.

[0024] Figure 3 This is the plasmid map of the pGBKT7-OsMBL1 yeast two-hybrid vector.

[0025] Figure 4 This is the plasmid map of the pSPYNE-OsRMT1 bimolecular fluorescence complementation vector.

[0026] Figure 5 This is the plasmid map of the pSPYCE-OsMBL1 bimolecular fluorescence complementation vector.

[0027] Figure 6 This is the plasmid map of the pET-30a-OsRMT1 ubiquitination vector.

[0028] Figure 7 This is the plasmid map of the pGEX-4T-OsMBL1 ubiquitination vector.

[0029] Figure 8 The relative expression level determination results of three strains (OE-126, OE-131 and OE-132) of OsRMT1 overexpressing rice plants (difference analysis: n=3).

[0030] Fig. 9 Figure 2 shows the growth phenotype of wild-type (WT) and OsRMT1 overexpressing (OE-126, OE-131 and OE-132) rice plants under medium- and long-term high-salt stress (scale bar, 5 cm).

[0031] Fig.10The figure shows the statistical analysis results of the survival rate of wild-type (WT) and OsRMT1 overexpressing (OE-126, OE-131 and OE-132) rice plants under medium- and long-term high-salt stress (data are mean ± standard deviation, difference analysis: n≥3; * represents P<0.05).

[0032] Fig.11 The figure shows the results of DAB staining experiment of wild type (WT) and OsRMT1-OE (OE-126, OE-131 and OE-132) rice leaves under high salt stress.

[0033] Fig.12 Diagram for OsRMT1 gene knockout target design and homozygous mutant material identification.

[0034] Fig.13 Figure 2 shows the growth phenotype of wild-type (WT) and OsRMT1 knockout mutants (KO-120 and KO-123) rice plants under medium- and long-term high-salt stress (scale bar, 5 cm).

[0035] Fig.14 The figure shows the statistical analysis results of the survival rate of wild-type (WT) and OsRMT1 knockout mutants (KO-120 and KO-123) rice plants under medium- and long-term high-salt stress (data are mean ± standard deviation, difference analysis: n≥3; * represents P<0.05).

[0036] Fig.15 The figure shows the results of DAB staining experiment on wild-type (WT) and OsRMT1 knockout mutant (KO-120 and KO-123) rice leaves under high salt stress.

[0037] Fig.16 This is a diagram verifying the interaction between OsMBL1 and OsRMT1 using yeast two-hybrid assay and bimolecular fluorescence complementation assay.

[0038] Fig.17 Analysis diagram for in vitro ubiquitination experiment to verify the role of OsRMT1 in OsMBL1 ubiquitination.

[0039] Fig.18 This is a diagram analyzing the role of OsRMT1 in the degradation of OsMBL1 in rice leaves with OsRMT1 knockout mutant (KO-123) and OsRMT1 overexpression (OE-126, OE-131). DETAILED DESCRIPTION

[0040] The technical solution of the present invention is described in detail below through embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0041] Example 1: Obtaining OsRMT1 overexpressing rice plants

[0042] Amino acid sequence of OsRMT1 (SEQ ID NO.1):

[0043] MDDHMGRRTVGGLLFTKGGSILLFREDSARHKATNCCTRHGCSSKHLAGKDKQTHRAATAAKEASETPRRSQIFRKPSTRTPQGSTATDNISRNAASSYSENDNRPRETPGRDLIARLKERVNASRKRSLNRENSPSSPNGLSATSSSSSRTVSRPSHRAASRIRKADEGANAGAVNVRRDSSGDTRRNSDRDVDDFLLVEQAARDSTEGFISGFLARYRSNHQGLLSSLDDSIEDANGYWRFNMEGSEELENYFIFNDRYRGMRMDIDGMSYEELLALGDRIGTVSTGLSEDALSKCLDRSMYMATTSGTHEDCERKCSICQEEYSDGEEVGKMVCKHYYHFSCIKNWLRQKNWCPICKSVALNTN* (* represents the stop codon TAG).

[0044] OsRMT1 gene sequence (SEQ ID NO.2):

[0045]

[0046] CDS sequence of OsRMT1 gene (SEQ ID NO.3):

[0047]

[0048] The specific operation method for obtaining OsRMT1 overexpressing rice plants is as follows:

[0049] (1) According to the ID of OsRMT1 gene (LOC_Os04g51400), the corresponding CDS sequence information was found on the website of National Rice Data Center (https: / / www.ricedata.cn / ). Full-length sequence primers were designed using Primer 5.0. BamH I and Xho I were used as restriction sites at the 5' end of the upstream and downstream primers, respectively, and homology arms were added (the lowercase letters in the primer sequence are the vector homology arms). The specific sequences of the upstream and downstream primers are as follows:

[0050] OsRMT1-pBI121-F:

[0051] 5'-gatgacgatgacaagggatccATGGATGATCACATGGGAAGACG-3' (SEQ ID NO. 7); OsRMT1-pBI121-R:

[0052] 5'-gaaatcatagagctcctcgagCTAGTTGGTATTCAGAGCGACGG-3' (SEQ ID NO. 8);

[0053] (2) After the primers were designed, they were compared with the NCBI database to determine their specificity. The cDNA of Zhonghan 3 rice was used as a template, and the high-fidelity amplification system shown in Table 1 and the PCR amplification program shown in Table 2 were used to amplify the target fragment to obtain the complete gene of OsRMT1.

[0054] Table 1 High-fidelity amplification system

[0055]

[0056] Table 2 PCR amplification program

[0057]

[0058] (3) Overexpression vector construction: pBI121 (overexpression vector) was double-digested with BamH I and Xho I restriction endonucleases. The double-digestion system is shown in Table 3. After electrophoresis gel recovery, it was homologously recombined with the OsRMT1 gene. The recombination connection reaction system is shown in Table 4. The connection was carried out at 37°C for 30 minutes. After the connection is completed, the Escherichia coli competent cells DH5α (Nanjing Qingke Biological Co., Ltd.) were transformed, coated on the plate, and grown overnight. The next day, single clones were picked and the bacterial solution was identified. The positive bacterial solution that was successfully identified was sequenced. After the bacterial solution sequencing was confirmed to be correct, the plasmid extracted after the bacterial solution was amplified and shaken, which is the pBI121-OsRMT1 recombinant plasmid. The map of the pBI121-OsRMT1 recombinant plasmid is shown in Figure 1 shown.

[0059] Table 3 Double enzyme digestion reaction system

[0060] Components Dosage <![CDATA[ddH 2 The]]> 23μl Plasmids 20μl Cutsmart buffer 5μl BamH 1μl Sac I 1μl Total volume 50μl

[0061] Table 4 Recombination ligation reaction system

[0062] Components Dosage Expression vector fragment 4μL Target gene fragment 3μL 5XCEII buffer 2μL Recombinase 1μL Total volume 10μL

[0063] (4) Agrobacterium-mediated genetic transformation of rice: The pBI121-OsRMT1 recombinant plasmid was transformed into Agrobacterium competent cells GV3101 (Nanjing Novozyme Biotechnology Co., Ltd.), spread on the plate, wait for bacterial growth, pick single clone colonies on the third to fourth day, identify the bacterial solution, and successfully identify the positive bacterial solution as the Agrobacterium species for subsequent experiments. The specific genetic transformation operation of rice was completed by Wuhan Boyuan Biotechnology Co., Ltd., and OsRMT1 overexpression plants OE-126, OE-131 and OE-132 were obtained.

[0064] Example 2: Analysis of relative expression levels in OsRMT1 overexpressing rice plants.

[0065] The specific operation method is as follows:

[0066] i1) Collect leaf samples from rice OsRMT1 overexpressing plants, cut them, wrap them in tin foil, mark them, quickly cool them in liquid nitrogen and store them at -80°C. Extract total RNA from the samples (extract total RNA from rice samples according to the instructions of RNA isolater Total RNA Extraction Reagent, Novozymes).

[0067] (2) Reverse transcription synthesis: In the first step, extract RNA from the leaves of the overexpressing plants. Prepare the mixture in an RNase-free centrifuge tube according to Table 5. After preparation, gently blow and mix with a pipette, and react at 42°C for 2 minutes. In the second step, prepare the reverse transcription reaction system: add 4μl of 5×HisScriptIIIQrt SuperMix to the reaction solution in the first step. Perform the reverse transcription reaction at 37°C for 15 minutes and 85°C for 5 minutes. In the third step, obtain cDNA.

[0068] Table 5 Genomic DNA removal

[0069] Components Dosage <![CDATA[RNase-free ddH 2 The]]> To 16μL 4×gDNA wiper Mix 4μL Template RNA 2μl

[0070] (3) According to the rice qRT-PCR primers in Table 6, the qRT-PCR amplification system in Table 7 and the qRT-PCR amplification program in Table 8, qRT-PCR was performed to detect the relative expression levels of the three strains OE-126, OE-131 and OE-132 in the OsRMT1 overexpressing plants. The results are as follows: Figure 8 shown.

[0071] Table 6 Rice qRT-PCR primer design

[0072] Gene name Primer sequences OsRMT1-F 5′-CCTCAAGTAGTAGCCGCACA-3′(SEQ ID NO.9) OsRMT1-R 5′-TCTTGCTGCCTGCTCAACTA-3′(SEQ ID NO.10) OsUBQ5-F 5′-ACCACTTCGACCGCCACTACT-3′(SEQ ID NO.11) OsUBQ5-R 5′-ACGCCTAAGCCTGCTGGTT-3′(SEQ ID NO.12)

[0073] Table 7 qRT-PCR amplification system

[0074]

[0075] Table 8 qRT-PCR amplification procedure

[0076]

[0077] (4) The relative expression results showed that the expression levels in the overexpression positive plants were significantly higher than those in the wild-type plants. The results showed that the OsRMT1 overexpression rice materials were successfully constructed.

[0078] Example 3: Analysis of salt tolerance of OsRMT1 overexpressing rice plants.

[0079] The specific operation method is as follows:

[0080] (1) Rice hydroponic phenotype: Two-week-old wild-type (WT) and OsRMT1 overexpressing (OE-126, OE-131, and OE-132) rice seedlings with the same growth were cultured in 175 mM NaCl medium for 5 days and then recovered in NaCl-free medium for 5 days. The phenotypes were observed. Fig. 9The results show that the growth of OsRMT1 overexpressing plants is worse than that of wild-type plants, whether after high salt stress treatment or after recovery.

[0081] (2) Statistical analysis of rice plant survival rate: Two-week-old wild-type rice (WT) and OsRMT1 overexpressing (OE-126, OE-131, and OE-132) seedlings with the same growth were cultured in 175 mM NaCl medium for 5 days and then recovered in NaCl-free medium for 5 days. Finally, the survival rate of each strain after recovery from high salt treatment was calculated. Fig.10 As shown in the results, it can be seen that the survival rate of OsRMT1 overexpressing plants (OE-126, OE-131 and OE-132) was significantly lower than that of the wild type (WT) after high salt stress.

[0082] (3) DAB staining of rice leaves: The leaves of two-week-old wild-type rice (WT) and OsRMT1 overexpressing (OE-126, OE-131, and OE-132) seedlings with the same growth were subjected to DAB staining. The experiment was divided into two groups: a control group and a high-salt treatment group, with three biological replicates in each group. Fig.11 As shown: The DAB staining results showed that after high salt treatment, the leaves of OsRMT1 overexpressing (OE-126, OE-131 and OE-132) plants had more stained areas than those of wild-type plants, and the staining was deeper.

[0083] (4) Analysis of the salt tolerance of OsRMT1-overexpressing rice plants revealed that under high salt stress, OsRMT1-overexpressing rice plants were less tolerant to high salt stress than wild-type plants and suffered more severe oxidative damage, indicating that OsRMT1 negatively regulates the salt tolerance of rice.

[0084] Example 4: Obtaining OsRMT1 gene knockout rice plants

[0085] The specific operation method is as follows:

[0086] (1) Design of sgRNA for OsRMT1 and construction of knockout vector: The target sequence sgRNA for OsRMT1 gene was designed using the CRISPR-P2 website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR), including target sequence 1 and target sequence 2. The knockout target site of OsRMT1 gene was designed as follows: Fig.12 shown.

[0087] Target sequence 1, sgRNA-1: 5′-AGAAGACAGTGCGCGTCACAAGG-3′ (SEQ ID NO. 13);

[0088] Target sequence 2, sgRNA-2: 5′-CCTCGGAGATCACAGATTTTCAG-3′ (SEQ ID NO. 14).

[0089] (2) Use the kit to synthesize oligo first, then prepare oligo dimer: Buffer Anneal 18μL, UPOligo 1μL, Low Oligo 1μL, H 2 O to 20 μL; after mixing, heat at 95°C for 3 minutes, and then slowly decrease the temperature to 20°C at a rate of about 0.2°C / second.

[0090] The primer sequences for sgRNA-1 synthesis are:

[0091] UP-1Oligo: 5'-gtttAGAAGACAGTGCGCGTCACAAGG-3' (SEQ ID NO. 15);

[0092] Low-1 Oligo: 5'-aaacCCTTGTGACGCGCACTGTCTTCT-3' (SEQ ID NO. 16);

[0093] The primer sequences for sgRNA-2 synthesis are:

[0094] UP-2Oligo: 5'-gtttCCTCGGAGATCACAGATTTTCAG-3' (SEQ ID NO. 17);

[0095] Low-2 Oligo: 5'-aaacCTGAAAATCTGTGATCTCCGAGG-3' (SEQ ID NO. 18).

[0096] (3) Connect the oligo dimer to the vector BGK015 to obtain the knockout vector: CRISPR / Cas Vector 2 μL, oligo dimer 1 μL, Enzyme Mix 1 μL, H 2 O was added to 10 μL; each component was mixed on ice, and then reacted at room temperature for 1 hour at 20°C. The ligation product was then transformed into E. coli competent cells DH5α (Nanjing Qingke Biotechnology Co., Ltd.). After the ligation was completed, the E. coli competent cells DH5α (Nanjing Qingke Biotechnology Co., Ltd.) were transformed, spread on the plate, and grown overnight. The next day, a single clone colony was picked for bacterial liquid identification, and the positive bacterial liquid that was successfully identified was sequenced. After the bacterial liquid sequencing was confirmed to be correct, the bacterial liquid was amplified and the plasmid was extracted.

[0097] (4) Genetic transformation of OsRMT1 knockout rice plants The method was the same as in Example 1, and OsRMT1 knockout mutant plants KO-120 and KO-123 were obtained. Fig.12 shown.

[0098] Example 5: Analysis of salt tolerance of OsRMT1 gene knockout rice plants.

[0099] (1) Rice hydroponic phenotype: Two-week-old wild-type (WT) and OsRMT1 knockout mutant (KO-120 and KO-123) rice seedlings with the same growth were cultured in 175 mM NaCl medium for 5 days and then recovered in NaCl-free medium for 5 days. The phenotypes were observed. Fig.13 As shown in the results, it can be seen that the OsRMT1 knockout mutant plants grew better than the wild-type plants, both after high salt stress treatment and after recovery.

[0100] (2) Statistical analysis of rice plant survival rate: Two-week-old wild-type rice (WT) and OsRMT1 knockout mutant (KO-120 and KO-123) seedlings with the same growth were cultured in 175 mM NaCl medium for 5 days and then recovered in NaCl-free medium for 5 days. Finally, the survival rate of each strain after recovery from high salt treatment was calculated. Fig.14 As shown in the results, it can be seen that the survival rate of OsRMT1 knockout mutants (KO-120 and KO-123) plants was significantly higher than that of the wild type (WT) after high salt stress.

[0101] (3) DAB staining of rice leaves: The leaves of two-week-old wild-type rice (WT) and OsRMT1 knockout mutants (KO-120 and KO-123) with the same growth were subjected to DAB staining. The experiment was divided into two groups: a control group and a high-salt treatment group, with 3 biological replicates in each group. Fig.15 As shown: The DAB staining results showed that after high salt treatment, the leaves of OsRMT1 knockout mutants (KO-120 and KO-123) had fewer stained areas than those of wild-type plants and the staining was lighter.

[0102] (4) Analysis of the salt tolerance of OsRMT1 knockout rice plants revealed that under high salt stress, OsRMT1 knockout rice plants were more tolerant to high salt stress than wild-type plants, and suffered less oxidative damage. This further demonstrated that OsRMT1 plays a negative regulatory role in rice salt tolerance, and strongly proved that knocking out the OsRMT1 gene can significantly improve rice tolerance to high salt environments.

[0103] Example 6: Yeast two-hybrid assay to verify and analyze the interaction between OsRMT1 and OsMBL1.

[0104] Amino acid sequence of OsMBL1 (SEQ ID NO.4):

[0105] MTLVKIGPWGGNGGSAQDISVPPKKLLGVTIYSSDAIRSIAFNYIGVDGQEYAIGPWGGGEGTSTEIKLGSSEQIKEISGTHGPVYDLADIVTYLKIVTSANNTYEAGVPNGKEFSIPLQDSGHVVGFFGRSGTLIDAIGIYVHP* (* represents the stop codon TAG)

[0106] OsMBL1 gene sequence (SEQ ID NO.5):

[0107]

[0108] CDS sequence of OsMBL1 gene (SEQ ID NO.6):

[0109] ATGACGCTGGTGAAGATTGGTCCGTGGGGCGGAAATGGAGGGTCAGCTCAGGACATCAGTGTGCCACCCAAGAAGCTGTTAGGCGTGACAATCTACAGCTCAGATGCAATCAGATCCATTGCCTTCAACTACATCGGTGTGGATGGACAGGAATATGCCATTGGTCCATGGGGTGGGGGCGAAGGCACCTCTACAGAGATTAAACTGGGCTCTCTGAG CAGATCAAGGAGATTTCTGGAACCCATGGCCCAGTCTATGATCTGGCTGACATTGTCACCTATCTTAAGATTGTGACAAGTGCTAATAATACATACGAGGCTGGAGTCCCAAATGGAAAGGAATTCAGCATTCCACTGCAAGACTCTGGCCATGTCGTTGGATTCTTTGGAAGGTCTGGAACGCTTATCGACGCAATTGGCATCTACGTCCACCCTTGA

[0110] Construct the fusion vector required for yeast two-hybrid and verify the analysis. The specific operations are as follows:

[0111] (1) Based on the CDS sequence information of the OsRMT1 gene and OsMBL1 gene, full-length sequence primers were designed using Primer 5.0, and EcoR I and BamH I were used as restriction sites at the 5' end of the upstream and downstream primers, respectively, and homology arms were added (the lowercase letters in the primer sequence are the vector homology arms), specifically:

[0112] OsRMT1-pGADT7-F:

[0113] 5'-gccatggaggccagtgaattcATGATCACATGGGAAGACG-3' (SEQ ID NO. 19);

[0114] OsRMT1-pGADT7-R:

[0115] 5'-cagctcgagctcgatggatccTCAGTTGGTATTCAGAGCGACGGAT-3' (SEQ ID NO. 20); OsMBL1-pGBKT7-F:

[0116] 5'-tggccatggaggccgaattcATGACGCTGGTGAAGATTGG-3' (SEQ ID NO. 21);

[0117] OsMBL1-pGBKT7-R:

[0118] 5'-gatgacgatgacaagggatccATGGATGATCACATGGGAAGACG-3' (SEQ ID NO. 22);

[0119] (2) After the primers were designed, the specificity of the primers was confirmed by comparing them in the NCBI database. The cDNA of Zhonghan 3 rice was used as a template, and the high-fidelity amplification system shown in Table 1 and the PCR amplification program shown in Table 2 were used to amplify the target fragments to obtain the complete genes of OsRMT1 and OsMBL1.

[0120] (3) Construction of the fusion vector required for yeast two-hybrid: pGADT7 and pGBKT7 (yeast vector) were double-digested with EcoR I and BamHI restriction enzymes. The double-digestion system is shown in Table 3. After electrophoresis gel recovery, they were homologously recombined with OsRMT1 and OsMBL1 genes. The recombination connection reaction system is shown in Table 4. The connection was carried out at 37°C for 30 minutes. After the connection was completed, the Escherichia coli competent cells DH5α (Nanjing Qingke Biological Co., Ltd.) were transformed, spread on the plates, and grown overnight. On the second day, single clones were picked and the bacterial solution was identified. The positive bacterial solution that was successfully identified was sequenced. After the bacterial solution sequencing was confirmed to be correct, the plasmids extracted after the bacterial solution was amplified, namely the target plasmids pGBKT7-OsMBL1 and pGADT7-OsRMT1. The map of the pGADT7-OsRMT1 plasmid is shown in Figure 2 The map of the pGBKT7-OsMBL1 plasmid is shown in Figure 3 shown.

[0121] (4) After centrifugation of the yeast (1000 g for 5 min at room temperature, remove the supernatant), resuspend it in 1 ml of TEB / LiAc 1:1 mixture, add 100 μL of fish sperm DNA, and dispense into 100 μL tubes.

[0122] (5) Add 5 μL of the target plasmid and 600 μL of PEG / LiAc to each tube, vortex for 10 seconds, and incubate at 30°C and 250 rpm for 1 hour.

[0123] (6) After gentle mixing, heat shock at 42°C for 15 min. After ice bath for 1-2 min, centrifuge at 14,000 rpm for 5 s, remove the supernatant, and resuspend in 500 μL 0.9% NaCl. Take 100-150 μL and apply to SD / -Trp / -Leu medium plates, and culture at 30°C for 3-5 days.

[0124] (7) Pick a single clone with a diameter of 2-3 mm and evenly spot it on SD / -Trp / -Leu / -His / -Ade and SD / -Trp / -Leu / plates in 0.9% NaCl to observe the interaction phenomenon. Fig.16 As shown in (a) and (b), (a) shows the growth of colonies on SD / -Trp / -Leu / plates, and (b) shows the growth of colonies on SD / -Trp / -Leu / -His / -Ade plates. It can be seen from the figure that pGBKT7-OsMBL1 interacts with pGADT7-OsRMT1.

[0125] Example 7: Bimolecular fluorescence complementation experiment to verify and analyze the interaction relationship between OsRMT1 and OsMBL1.

[0126] The fusion vector required for bimolecular fluorescence complementation was constructed and verified and analyzed. The specific operations are as follows:

[0127] (1) Based on the CDS sequence information obtained when constructing the OsRMT1-pBI121 vector, full-length sequence primers were designed using Primer 5.0, and BamH I and Sal I were used as restriction sites at the 5' end of the upstream and downstream primers, respectively, and homology arms were added (the lowercase letters in the primer sequence are the vector homology arms), specifically:

[0128] OsRMT1-SPYNE-F:

[0129] 5'-attacaggtacccggggatccATGACGCTGGTGAAGATTGGTCC-3' (SEQ ID NO. 23);

[0130] OsRMT1-SPYNE-R:

[0131] 5'-cacgctgccaccgccgtcgacAGGGTGGACGTAGATGCCAATT-3' (SEQ ID NO. 24);

[0132] OsMBL1-SPYCE-F:

[0133] 5'-attacaggtacccggggatccATGGATGATCACATGGGAAGACG-3' (SEQ ID NO. 25);

[0134] OsMBL1-SPYCE-R:

[0135] 5'-gctcaccataccgccgtcgacGTTGGTATTCAGAGCGACGGAT-3' (SEQ ID NO. 26);

[0136] (2) After the primers were designed, the specificity of the primers was confirmed by comparing them in the NCBI database. The cDNA of Zhonghan 3 rice was used as a template, and the high-fidelity amplification system shown in Table 1 and the PCR amplification program shown in Table 2 were used to amplify the target fragments to obtain the complete genes of OsRMT1 and OsMBL1.

[0137] (3) Construction of the fusion vector required for bimolecular fluorescence complementation: pSPYNE and pSPYCE (bimolecular fluorescence complementation vector) were double-digested with BamH I and Sa1 I restriction endonucleases. The double-digestion system is shown in Table 3. After electrophoresis gel recovery, the cells were homologously recombined with the OsRMT1 and OsMBL1 genes. The recombination and ligation reaction system is shown in Table 4. The cells were connected at 37°C for 30 minutes. After the connection was completed, the competent Escherichia coli cells DH5α (Nanjing Qingke Biotechnology Co., Ltd.) were transformed, spread on the plates, and grown overnight. On the second day, single clones were picked and the bacterial solution was identified. The positive bacterial solution that was successfully identified was sequenced. After the bacterial solution was sequenced and confirmed to be correct, the plasmids extracted after the bacterial solution was amplified, namely the target plasmids pSPYCE-OsMBL1 and pSPYNE-OsRMT1. The map of the pSPYNE-OsRMT1 plasmid is shown in Figure 4 The map of pSPYCE-OsMBL1 plasmid is shown in Figure 5 shown.

[0138] (4) The target plasmids pSPYCE-OsMBL1 and pSPYNE-OsRMT1 were transformed into Agrobacterium GV3101 (Nanjing Qingke Biotechnology Co., Ltd.).

[0139] (5) Mix the Agrobacterium culture solution containing the N-fragment and the C-fragment in a ratio of 1:1 and inject them into tobacco leaves together.

[0140] (6) Laser confocal microscope was used to observe the experimental results and take photos. Fig.16 As shown in (c), it can be seen from the figure that OsMBL1 interacts with OsRMT1.

[0141] Example 8: Analysis of the role of OsRMT1 in the ubiquitination and degradation of OsMBL1.

[0142] The construction and verification analysis of the fusion vector required for ubiquitination are as follows:

[0143] (1) Based on the CDS sequence information obtained when constructing the OsRMT1-pBI121 vector, full-length sequence primers were designed using Primer 5.0, and BamH I and Sal I were used as restriction sites at the 5' end of the upstream and downstream primers, respectively, and homology arms were added (the lowercase letters in the primer sequence are the vector homology arms), specifically:

[0144] OsRMT1-pET-30a-F:

[0145] 5'-gccatggctgatatcggatccATGGATGATCACATGGGAAGACG-3' (SEQ ID NO. 27);

[0146] OsRMT1-pET-30a-R:

[0147] 5'-tgcggccgcaagcttgtcgacGTTGGTATTCAGAGCGACGGAT-3' (SEQ ID NO. 28);

[0148] OsMBL1-pGEX-4T-F:

[0149] 5'-gatctggttccgcgtggatccATGACGCTGGTGAAGATTGGTCC-3' (SEQ ID NO. 29);

[0150] OsMBL1-pGEX-4T-R:

[0151] 5'-gatgcggccgctcgagtcgacTCAAGGGTGGACGTAGATGCC-3' (SEQ ID NO. 30);

[0152] (2) After the primers were designed, the specificity of the primers was confirmed by comparing them in the NCBI database. The cDNA of Zhonghan 3 rice was used as a template, and the high-fidelity amplification system shown in Table 1 and the PCR amplification program shown in Table 2 were used to amplify the target fragments to obtain the complete genes of OsRMT1 and OsMBL1.

[0153] (3) Construction of the fusion vector required for ubiquitination: pET-30a and pGEX-4T (protein expression vector) were double-digested with BamH I and Sa1 I restriction endonucleases. The double-digestion system is shown in Table 3. After electrophoresis gel recovery, homologous recombination ligation with OsRMT1 and OsMBL1 genes was performed. The recombination ligation reaction system is shown in Table 4. The ligation was carried out at 37°C for 30 minutes. After the ligation is completed, Escherichia coli competent cells DH5α (Nanjing Qingke Biological Co., Ltd.) were transformed, coated on plates, and grown overnight. On the second day, single clones were picked and the bacterial solution was identified. The positive bacterial solution that was successfully identified was sequenced. After the bacterial solution sequencing was confirmed to be correct, the plasmids extracted after the bacterial solution was amplified, namely the target plasmids pGEX-4T-OsMBL1 and pET-30a-OsRMT1. The map of the pET-30a-OsRMT1 plasmid is shown in Figure 6 As shown, pGEX-4T-OsMBL1 Figure 7 shown.

[0154] (4) The target plasmids pGEX-4T-OsMBL1 and pET-30a-OsRMT1 were transformed into the competent DE3 medium for large intestine (Nanjing Qingke Biotechnology Co., Ltd.). The plates were spread and grown overnight. The single clone colonies were picked on the second day for bacterial liquid identification. After successful identification, the bacterial liquid was propagated and the proteins of GST-OsMBL1 and His-OsRMT1 were purified.

[0155] (5) Analysis of the role of OsRMT1 in OsMBL1 ubiquitination:

[0156] Prepare the ubiquitination reaction system: including purified ubiquitin, E1 ubiquitin activating enzyme, and E2 ubiquitin conjugating enzyme. Mix them in a buffer to simulate the intracellular ubiquitination process. Add GST-OsMBL1 and His-OsRMT1 proteins to the reaction system for ubiquitination reaction. Incubate at 30°C for 6 hours to allow the substrate protein to covalently link with ubiquitin. Use Western Blot to detect and analyze the ubiquitinated substrate protein. The results are shown in Figure 2. Fig.17 As shown, the results show that OsRMT1 can ubiquitinate OsMBL1 in vitro.

[0157] (6) Analysis of the role of OsRMT1 in OsMBL1 degradation:

[0158] Proteins from OsRMT1 knockout mutant (KO-123) and OsRMT1 overexpression (OE-126, OE-131) plants were extracted, and OsMBL1 protein was detected and analyzed using Western Blot. Fig.18The results show that the OsMBL1 protein content in the OsRMT1 knockout mutant (KO-123) is higher than that in the wild type, and the OsMBL1 protein content in the OsRMT1 overexpression (OE-126, OE-131) plants is lower than that in the wild type. The results show that OsRMT1 can ubiquitinate OsMBL1 in vivo.

[0159] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to the form and details without departing from the spirit and scope of the present invention.

Claims

1. An application of rice OsRMT1 gene in improving rice salt tolerance, characterized in that: The protein encoded by the OsRMT1 gene is OsRMT1, the amino acid sequence of the protein OsRMT1 is shown in SEQ ID NO.1, the nucleotide sequence of the OsRMT1 gene is shown in SEQ ID NO.2, and the CDS sequence in the OsRMT1 gene is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: Improving rice salt tolerance by increasing the content of OsMBL1, an interacting protein of OsRMT1; The amino acid sequence of protein OsMBL1 is shown in SEQ ID NO.4; the nucleotide sequence of OsMBL1 gene encoding protein OsMBL1 is shown in SEQ ID NO.5, and the CDS sequence in OsMBL1 gene is shown in SEQ ID NO.

6.

3. The use according to claim 1, characterized in that: The OsRMT1 gene and the protein it encodes act as negative regulatory factors to regulate rice salt tolerance.

4. The use according to claim 1, characterized in that: The OsRMT1 gene is used for breeding and screening salt-tolerant rice varieties.

5. The use according to claim 1, characterized in that: The method for obtaining the OsRMT1 gene is to obtain the OsRMT1 gene of rice by designing primers such as SEQ ID NO.7 and SEQ ID NO.

8.

6. The use according to claim 1, characterized in that: The OsRMT1 gene was knocked out by CRISPR-Cas9 gene editing technology to obtain a salt-tolerant rice variety.

7. The use according to claim 6, characterized in that: The specific operation method of knocking out the OsRMT1 gene by CRISPR-Cas9 gene editing technology to obtain a salt-tolerant rice variety is as follows: 1) Designing a target sequence of the OsRMT1 gene, wherein the target sequence includes sgRNA-1 and sgRNA-2; The sequence of sgRNA-1 is shown in SEQ ID NO.13, and the sequence of sgRNA-2 is shown in SEQ ID NO.14; 2) synthesizing upstream and downstream primers for the target sequence of the OsRMT1 gene, and using the synthesized upstream and downstream primers to prepare oligo dimers; For sgRNA-1, the synthesized upstream primer sequence is shown in SEQ ID NO.15, and the downstream primer sequence is shown in SEQ ID NO.16; For sgRNA-2, the synthesized upstream primer sequence is shown in SEQ ID NO.17, and the downstream primer sequence is shown in SEQ ID NO.18; 3) Connecting the oligo dimer to the vector BGK015 to obtain a knockout vector; transforming Escherichia coli competent cells with the knockout vector to extract the knockout vector plasmid; 4) The knockout vector plasmid is introduced into rice plants through genetic transformation, and the plants with the OsRMT1 gene knocked out are selected as salt-tolerant rice varieties.

8. The use according to claim 2, characterized in that: By knocking out the OsRMT1 gene in rice, the content of OsMBL1 in rice is increased.

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