Rice OsbZIP52 mutant gene, protein and application thereof

Abstract

The invention relates to the technical field of agriculture, in particular to a rice OsbZIP52 mutant gene, a protein and application of the rice OsbZIP52 mutant gene. The rice OsbZIP52 mutant gene is a wild rice OsbZIP52 gene which is mutated from C to G at the 118th site of a nucleotide sequence. The rice OsbZIP52 mutant gene provided by the invention is a wild rice OsbZIP52 gene which is mutated from C to G at the 118th site of the nucleotide sequence, so that the salt tolerance of rice can be improved, a new gene resource is provided for breeding salt-tolerant rice varieties, and the rice OsbZIP52 mutant gene has great application value.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a rice OsbZIP52 mutant gene, protein, and its applications. Background Technology

[0002] Currently, over 20% of global agricultural land is affected by salt stress, and this percentage is increasing year by year. Excessive accumulation of salt in the soil can cause osmotic stress, ion stress, and oxidative stress, damaging cell structure and components, affecting various physiological and biochemical reactions and metabolic balance in plants, severely inhibiting crop growth and development, and ultimately leading to reduced food yields.

[0003] Rice (Oryza sativa L.) is one of the most important food crops, serving as the primary food source for more than half of the world's population. In my country, the annual rice planting area has consistently remained above 450 million mu (approximately 30 million hectares), while the yield has remained stable at over 200 million tons annually, providing staple food for two-thirds of the Chinese population. However, the increasing area of ​​land affected by salinity is impacting rice production and yield to varying degrees. Improving rice's salt tolerance through genetic modification is one of the most effective ways to address this issue. Therefore, identifying and utilizing superior salt-tolerant genes in rice and cultivating salt-tolerant varieties is of great significance.

[0004] Prime editing, as a next-generation gene editing technology, can precisely locate and perform base substitutions and sequence insertions or deletions in the genome without causing DNA double-strand breaks, providing new technologies and tools for crop genetic improvement and variety breeding. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a rice OsbZIP52 mutant gene, protein, and its application. The rice OsbZIP52 mutant gene is a mutation from C to G at position 118 of the wild-type rice OsbZIP52 gene, which can improve the salt tolerance of rice and provides a new gene resource for the breeding of salt-tolerant rice varieties, thus having significant application value.

[0006] Therefore, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a rice OsbZIP52 mutant gene in an optional embodiment, wherein the rice OsbZIP52 mutant gene is a mutation from C to G at position 118 of the nucleotide sequence of the wild-type rice OsbZIP52 gene.

[0008] Preferably, the nucleotide sequence of the rice OsbZIP52 mutant gene is shown in SEQ ID NO.1. The nucleotide sequence of the wild-type rice OsbZIP52 gene is shown in SEQ ID NO.3.

[0009] Secondly, in an optional embodiment, the present invention provides a rice OsbZIP52 mutant protein, wherein the rice OsbZIP52 mutant protein is a wild-type rice OsbZIP52 protein in which leucine is replaced by valine at position 40 of the amino acid sequence.

[0010] Preferably, the amino acid sequence of the rice mutant protein is shown in SEQ ID NO.2. The amino acid sequence of the wild-type rice OsbZIP52 protein is shown in SEQ ID NO.4.

[0011] Thirdly, in an optional embodiment, the present invention provides a plant expression vector containing the above-mentioned rice OsbZIP52 mutant gene.

[0012] Fourthly, in an optional embodiment, the present invention provides a recombinant protein comprising the above-mentioned rice OsbZIP52 mutant gene.

[0013] Fifthly, in an optional embodiment, the present invention provides an engineered enzyme comprising the above-mentioned rice OsbZIP52 mutant gene.

[0014] In a sixth aspect, the present invention provides, in optional embodiments, the application of the above-mentioned rice OsbZIP52 mutant gene or the above-mentioned rice OsbZIP52 mutant protein in making rice salt-tolerant.

[0015] In a seventh aspect, the present invention provides a method for obtaining salt-tolerant rice in an optional embodiment, comprising the following steps:

[0016] Salt-tolerant rice can be obtained by using gene editing, hybridization, backcrossing, self-pollination, or asexual reproduction to make plants contain the above-mentioned rice OsbZIP52 mutant gene or the above-mentioned rice OsbZIP52 mutant protein.

[0017] Eighthly, in an optional embodiment, the present invention provides a method for improving the salt tolerance of rice, comprising the following steps:

[0018] By using gene editing methods, the OsbZIP52 gene in wild-type rice was edited into the aforementioned OsbZIP52 mutant gene, thereby improving the salt tolerance of rice.

[0019] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0020] ATGATGAAGAAGTGCCCGTCGGAGCTGCAGCTGGAGGCGTTCATCCGGGAGGAGGCCGGCGCCGGCGACCGCAAGCCCGGCGTGTTATCTCCCGGCGACGGCGCGCGTAAGTCCGGCGTGTTCTCTCCCGGCGACGGCGAGATGTCCGTGTTGGATCAGAGTACACTGGACGGAAGCGGCGGCGGCCACCAGCTGTGGTGGCCGGAGAGCGTCCGTACGCCGCCGCGCGCCGCCGCCGCCTTCTCGGCCACGGCCGACGAGCGGACGCCGGCGTCCATCTCCGATGACCCCAAACCAACCACCTCAGCGAACCACGCGCCTGAAAGCGACTCGGACTCCGATTGCGATTCGCTGTTAGAAGCAGAGAGGAGTCCACGCCTGCGTGGCACGAAATCCACAGAAACAAAGCGAATAAGAAGGATGGTGTCCAACAGGGAGTCCGCTCGACGATCCAGGAGGAGAAAGCAGGCACAGTTATCTGAACTCGAATCACAGGTCGAGCAACTCAAAGGCGAAAACTCATCCCTCTTCAAGCAGCTCACAGAGTCCAGCCAGCAGTTCAATACAGCGGTCACGGACAACAGGATCCTCAAATCGGATGTAGAGGCCTTAAGAGTCAAGGTCAAGATGGCTGAAGACATGGTCGCGAGGGCCGCGATGTCGTGTGGCCTGGGCCAGCTCGGGCTGGCGCCATTGCTCAGCTCCAGGAAGATGTGCCAAGCTTTGGATATGCTCAGTTTACCACGGAACGATGCCTGTGGTTTCAAAGGCTTGAACCTGGGTCGACAGGTTCAGAACTCACCGGTTCAAAGCGCTGCAAGCCTAGAGAGCCTGGACAACCGGATATCCAGCGAGGTGACCAGCTGCTCGGCTGATGTGTGGCCTTAA。

[0021] The amino acid sequence shown in SEQ ID NO.2 is as follows:

[0022] MMKKCPSELQLEAFIREEAGAGDRKPGVLSPGDGARKSGVFSPGDGEMSVLDQSTLDGSGGGHQLWWPESVRTPPRAAAAFSATADERTPASISDDPKPTTSANHAPESDSDSDCDSLLEAERSPRLRGTKSTETKRIRRMVSNRESARRSRRRKQAQLSELESQVEQLKGENSSLFKQLTESSQQFNTAVTDNRILKSDVEALRVKVKMAEDMVARAAMSCGLGQLGLAPLLSSRKMCQALDMLSLPRNDACGFKGLNLGRQVQNSPVQSAASLESLDNRISSEVTSCSADVWP。

[0023] The nucleotide sequence of SEQ ID NO.3 is shown below:

[0024] ATGATGAAGAAGTGCCCGTCGGAGCTGCAGCTGGAGGCGTTCATCCGGGAGGAGGCCGGCGCCGGCGACCGCAAGCCCGGCGTGTTATCTCCCGGCGACGGCGCGCGTAAGTCCGGCCTGTTCTCTCCCGGCGACGGCGAGATGTCCGTGTTGGATCAGAGTACACTGGACGGAAGCGGCGGCGGCCACCAGCTGTGGTGGCCGGAGAGCGTCCGTACGCCGCCGCGCGCCGCCGCCGCCTTCTCGGCCACGGCCGACGAGCGGACGCCGGCGTCCATCTCCGATGACCCCAAACCAACCACCTCAGCGAACCACGCGCCTGAAAGCGACTCGGACTCCGATTGCGATTCGCTGTTAGAAGCAGAGAGGAGTCCACGCCTGCGTGGCACGAAATCCACAGAAACAAAGCGAATAAGAAGGATGGTGTCCAACAGGGAGTCCGCTCGACGATCCAGGAGGAGAAAGCAGGCACAGTTATCTGAACTCGAATCACAGGTCGAGCAACTCAAAGGCGAAAACTCATCCCTCTTCAAGCAGCTCACAGAGTCCAGCCAGCAGTTCAATACAGCGGTCACGGACAACAGGATCCTCAAATCGGATGTAGAGGCCTTAAGAGTCAAGGTCAAGATGGCTGAAGACATGGTCGCGAGGGCCGCGATGTCGTGTGGCCTGGGCCAGCTCGGGCTGGCGCCATTGCTCAGCTCCAGGAAGATGTGCCAAGCTTTGGATATGCTCAGTTTACCACGGAACGATGCCTGTGGTTTCAAAGGCTTGAACCTGGGTCGACAGGTTCAGAACTCACCGGTTCAAAGCGCTGCAAGCCTAGAGAGCCTGGACAACCGGATATCCAGCGAGGTGACCAGCTGCTCGGCTGATGTGTGGCCTTAA。

[0025] The amino acid sequence of SEQ ID NO. 4 is as follows:

[0026] MMKKCPSELQLEAFIREEAGAGDRKPGVLSPGDGARKSGLFSPGDGEMSVLDQSTLDGSGGGHQLWWPESVRTPPRAAAAFSATADERTPASISDDPKPTTSANHAPESDSDSDCDSLLEAERSPRLRGTKSTETKRIRRMVSNRESA RRSRRRKQAQLSELESQVEQLKGENSSLFKQLTESSQQFNTAVTDNRILKSDVEALRVKVKMAEDMVARAAMSCGLGQLGLAPLLSSRKMCQALDMLSLPRNDACGFKGLNLGRQVQNSPVQSAASLESLDNRISSEVTSCSADVWP.

[0027] Compared with the prior art, the present invention has one of the following beneficial effects:

[0028] 1. The rice OsbZIP52 mutant gene provided by this invention is a mutation from C to G at position 118 of the wild-type rice OsbZIP52 gene, which can improve the salt tolerance of rice and provide a new gene resource for the breeding of salt-tolerant rice varieties, and has significant application value. Attached Figure Description

[0029] Figure 1 This is a phenotypic diagram and survival rate statistical analysis result of wild-type and salt-tolerant rice mutant rts1 after treatment with 140 mM NaCl salt concentration in Example 1 of this invention;

[0030] Figure 2 This is a comparison of the nucleotide and amino acid sequence mutation sites of the OsbZIP52 gene in wild-type and salt-tolerant rice mutant rts1 in Example 1 of this invention;

[0031] Figure 3 This is a comparison of the target sequences of OsbZIP52 site-edited plants and wild-type plants in Example 3 of this invention;

[0032] Figure 4 This is a phenotypic diagram and survival rate statistical analysis result of OsbZIP52 site-edited plants and wild types after treatment with 140 mM NaCl salt concentration in Example 4 of this invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Example 1

[0037] Salt tolerance is beneficial for obtaining haplotypes.

[0038] Using the rice variety Zhonghua 11 as a background, a salt-tolerant rice mutant rts1 was obtained through ethyl methanesulfonate (EMS) mutagenesis and salt stress treatment (see [link to relevant documentation]). Figure 1 After performing whole-genome sequencing and localization on the salt-tolerant rice mutant rts1, the applicant discovered that the mutant had only one C-to-G mutation at position 118 of the nucleotide sequence of the gene OsbZIP52 (gene number Os06g0662200), resulting in a change from leucine to valine at position 40 (see...). Figure 2 Since the mutant contains only one mutation in this gene, it indicates that the mutation is associated with salt tolerance in rice. Therefore, gene editing technology can be used to introduce this variation into salt-sensitive varieties to create salt-tolerant rice materials.

[0039] Example 2

[0040] Construction of the OsbZIP52 gene editing vector PE-OsbZIP52-L40V

[0041] Vector primers were designed to target the C-to-G mutation at position 118 of the OsbZIP52 gene, and the relevant sequences are shown in Table 1. The primers were annealed to obtain the target sgRNA fragment and RTPBS fragment corresponding to the OsbZIP52 gene. The reaction mixture consisted of 10 μL each of forward and reverse primers, 5 μL of 10×T4 ligation buffer, 1 μL of T4 PNK, and 24 μL of ddH2O.

[0042] The Phuc422-PEmax-NC vector was digested with enzymes in the following reaction mixture: 15 μL plasmid, 4 μL CutSmart buffer, 1 μL BasI enzyme, and 20 μL ddH2O. The mixture was incubated overnight at 37°C, and the digested product was then inactivated at 65°C for 20 min.

[0043] The inactivated PE vector and target fragment were ligated using Goldengate technology to form a complete recombinant vector. The reaction mixture consisted of 1 μL of inactivated vector, 1 μL of sg2.0 fragment, 1 μL of evopreQ1 fragment, 1 μL of each of the two target fragments, 1 μL of T4 buffer, 1 μL of T4 ligase, 1 μL of CutSmart, 1 μL of BasI enzyme, and 1 μL of ddH2O. After mixing, the mixture was placed in a PCR instrument. The Goldengate program was: 37℃ for 5 min, 25℃ for 5 min, 25 cycles; 80℃ for 10 min.

[0044] The recombinant PE vector was transformed into E. coli and cultured overnight on Kanamycin-resistant medium. After single-clon sequencing verification, the PE-OsbZIP52-L40V positive plasmid was obtained.

[0045]

[0046] Example 3

[0047] Obtaining OsbZIP52-based targeted editing plants

[0048] The PE-OsbZIP52-L40V vector plasmid was transformed into Agrobacterium and used to infect the callus tissue of the rice variety Zhonghua 11. After co-culture, recovery, screening, differentiation, and rooting, rice seedlings were obtained.

[0049] Genomic DNA was extracted from the obtained rice seedlings, and LP / RP primer pairs were designed flanking the mutation sites for amplification. The primers are shown in Table 2. The PCR reaction system consisted of: 1 μL DNA template, 1 μL each of forward and reverse primers, 10 μL 2×Phanta Mix Master Mix, and 7 μL ddH2O. The PCR amplification program was: 95 ℃ pre-denaturation for 5 min, 95 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 30 s, for 35 cycles; 72 ℃ extension for 5 min.

[0050] After sequencing verification of the amplified products, the positive strain OsbZIP52, which was homozygous edited at the target site, was obtained. L40V Medium Flower 11 Wild Type (WT) and OsbZIP52 L40V Targeted editing of the nucleotide and amino acid sequences of rice materials, such as Figure 3 As shown.

[0051]

[0052] Example 4

[0053] OsbZIP52 Targeted Editing of Salt Tolerance Analysis of Rice Materials

[0054] To further analyze the role of the OsbZIP52 gene in rice salt tolerance, the phenotype under salt stress was identified. Seeds of OsbZIP52-edited rice and wild-type rice were disinfected with 10% hydrogen peroxide solution for one hour and then rinsed with distilled water. After soaking for two days, the seeds were germinated at 37℃. Seeds with uniform germination were selected and sown in 96-well hydroponic boxes for hydroponics. The cultivation conditions were 28℃ for 14 hours of light, 25℃ for 10 hours of darkness, and a relative humidity of 70%. The seedlings were cultured in pure water until they reached the one-leaf-one-heart stage, then cultured in Yashida nutrient solution, with the nutrient solution changed every two days. When the rice seedlings reached the two-leaf-one-heart stage, they were subjected to a 140 mM NaCl salt solution for stress treatment. Phenotypic observation and plant survival rate were recorded one week later. The results showed that under 140 mM NaCl salt stress, wild-type leaves suffered severe withering, and most seedlings died, while OsbZIP52-edited plants showed significantly higher phenotypes. L40V Only a few seedlings turned yellow and wilted, but their growth and survival rate were significantly better than the wild type; statistical analysis showed that under 140 mM NaCl salt stress, OsbZIP52-edited plants... L40V The survival rate of the OsbZIP52-edited plants was significantly higher than that of the wild type, which had a survival rate of 10%. L40V The survival rate can reach 50-60% (see...) Figure 4 ).

[0055] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. A rice OsbZIP52 mutant gene, characterized in that, The mutant rice OsbZIP52 gene is a mutation from C to G at position 118 of the wild-type rice OsbZIP52 gene.

2. The rice OsbZIP52 mutant gene according to claim 1, characterized in that, The nucleotide sequence of the rice OsbZIP52 mutant gene is shown in SEQ ID NO.

1.

3. A mutant rice OsbZIP52 protein, characterized in that, The mutant rice OsbZIP52 protein is a wild-type rice OsbZIP52 protein in which leucine is replaced by valine at position 40 of the amino acid sequence.

4. The rice OsbZIP52 mutant protein according to claim 3, characterized in that, The amino acid sequence of the rice mutant protein is shown in SEQ ID NO.

2.

5. A plant expression vector comprising the rice OsbZIP52 mutant gene as described in claim 1 or 2.

6. A recombinant protein comprising the rice OsbZIP52 mutant gene as described in claim 1 or 2.

7. An engineered enzyme comprising the rice OsbZIP52 mutant gene as described in claim 1 or 2.

8. The use of the rice OsbZIP52 mutant gene as described in claim 1 or 2, or the rice OsbZIP52 mutant protein as described in claim 3 or 4, in imparting salt tolerance to rice.

9. A method for obtaining salt-tolerant rice, characterized in that, Includes the following steps: Salt-tolerant rice is obtained by using gene editing, hybridization, backcrossing, self-pollination, or asexual reproduction to make plants contain the rice OsbZIP52 mutant gene as described in claim 1 or 2 or the rice OsbZIP52 mutant protein as described in claim 3 or 4.

10. A method for improving the salt tolerance of rice, characterized in that, Includes the following steps: By using gene editing methods, the OsbZIP52 gene in wild-type rice is edited into the rice OsbZIP52 mutant gene as described in claim 1 or 2, thereby improving the salt tolerance of rice.

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