Ehd2 gene for improving salt tolerance of rice, gene mutant, method and application

Abstract

This invention discloses an Ehd2 gene, gene mutants, methods, and applications for improving salt tolerance in rice. Ehd2 is a key flowering factor in rice flowering transition. This invention identifies a series of new Ehd2 allelic mutants using map-based cloning and Mutmap methods. Further research revealed that, in addition to an extremely late heading phenotype, the loss of Ehd2 function in these mutants enhances the resistance of rice seedlings to salt stress. This invention, through functional interpretation of Ehd2 salt tolerance, further elucidates the genetic mechanism of rice salt tolerance and provides a theoretical basis and technical support for improving rice salt tolerance and genetic improvement of rice varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, and more particularly to an Ehd2 gene, gene mutant, method, and application for improving salt tolerance in rice. Background Technology

[0002] Soil salinization severely impacts crop yield (Munns et al., 2008; Deinlein et al., 2014). Rice is a crop sensitive to salt stress, and excessive sodium ion accumulation can seriously impair its growth and development, especially during the seedling and reproductive growth stages (Liu et al., 2022). To survive under salt stress, plants have evolved a variety of adaptive mechanisms, mainly including osmotic homeostasis (such as proline, polyol, and sugar accumulation), ion balance (Na+ / K+ homeostasis), and antioxidants (such as CAT, APX, GST, and NAGPH activities) (Zhang et al., 2013; Tang et al., 2015; Fu et al., 2018).

[0003] In functional genomics research, many transcription factors are involved in regulating salt tolerance in rice. The WRKY family transcription factor OsWRKY53 negatively regulates salt stress by directly inhibiting the expression of OsMKK10.2 and OsHKT1 (Yu et al., 2023). R2R3-MYBs are the most common MYB subfamily in plants, with 117 R2R3-OsMYB genes asymmetrically distributed across 12 chromosomes in rice. Genome-wide identification of R2R3-MYB transcription factor genes under salt stress revealed that OsMYB2-115 may be an important gene related to salt stress in rice (Zhang et al., 2024). In addition, other MYB family transcription factors such as MYB3R, OsMYB36a / b / c, OsMYB39a, OsMYB41, OsMYB92a / b, and OsMYBc are also involved in the regulation of salt stress response (Yuan et al., 2024; Chen et al., 2024). The bZIP family transcription factor OsbZIP72 participates in salt stress response by activating the expression of the high-affinity potassium transporter OsHKT1 (Wang et al., 2021). OsbZIP23 directly binds to the UGT2 promoter, activating its expression to enhance salt stress tolerance (Wang et al., 2023). GPX1 mediates the oxidative modification of bZIP68 to enhance the transcriptional activity of COR413-TM1, OsDREB1A, and OsDREB1B, and positively regulates the ABA-independent salt stress response (Zhou et al., 2022). Plant-specific IDD domain (Cys-2 / His-2 type zinc finger) transcription factors play important roles in plant flowering time, seed and root development, hormone signaling, and biotic and abiotic stresses. Ehd2 / OsID1 / RID1 / Ghd10, an ortholog of maize ZmID1, binds to the “TTTGTC” core motif in the Hd3a or RFT1 promoter, along with OsIDD4, to regulate flowering in rice (Deng et al., 2017). Previous studies have shown that OsIDD10 activates the expression of AMT1;2, ​​GDH2, CIPK9, and CIPK14 to participate in the regulation of ammonium uptake and nitrogen metabolism in roots (Xuan et al., 2013; Xuan et al., 2019). OsIDD3 has been reported to regulate cold stress response by directly binding to the CBF1 promoter (Dou et al., 2016). Furthermore, the OsIDD3-OsIDD13-OsIDD14 / LPA1 transcriptional regulatory complex enhances rice's defense against sheath blight by regulating the expression of PIN1a and PIN1b (Sun et al., 2021; Sun et al., 2020; Wang et al., 2020).However, the mechanism by which IDD domain transcription factors regulate salt tolerance remains unclear. Summary of the Invention

[0004] In view of this, the present invention provides a gene, gene mutant, method and application for improving salt tolerance in rice.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A gene for improving salt tolerance in rice, said gene being the Ehd2 gene, which is a gene encoding protein (a) or (b), wherein,

[0007] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or

[0008] (b) Proteins derived from (a) with the sequence shown in SEQ ID NO:9 substituted, deleted or added with one or more amino acids and having the same function.

[0009] A method to improve the salt tolerance of rice involves using genetic engineering to modify the Ehd2 gene in rice to obtain transgenic rice with the gene function missing.

[0010] A gene mutant that enhances salt tolerance in rice, wherein the genotypes of mutants of the gene Ehd2 (elh5~elh12) are ehd2-1~ehd2-8; wherein,

[0011] Compared to the Ehd2 gene, the CDS sequence of the ehd2-1 (elh5) gene has a mutation from C to G at position 490.

[0012] Compared with the Ehd2 gene, the gene ehd2-2 / 3 (elh6 / 7) has a mutation from G to A at the junction of the first exon and intron.

[0013] Compared to the Ehd2 gene, the CDS sequence of the ehd2-4 (elh8) gene has a mutation from C to T at position 547.

[0014] Compared to the Ehd2 gene, the CDS sequence of the ehd2-5 (elh9) gene has a mutation from C to T at position 473.

[0015] Compared with gene Ehd2, gene ehd2-6 / 7 / 8 (elh10 / 11 / 12) has a TGC deletion at positions 655-657 of its CDS sequence.

[0016] Preferably, the breeding method includes transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0017] The application of a gene mutant that enhances salt tolerance in rice in biomaterials, wherein the biomaterials are expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0018] The preferred and specific application method is as follows:

[0019] 1) Used to regulate the salt tolerance of rice;

[0020] 2) Used for plant breeding;

[0021] 3) Used to prepare transgenic plants.

[0022] Preferably, the plant is a plant of the genus Oryza in the family Poaceae.

[0023] The present invention achieves the following technical effects compared to the prior art:

[0024] (1) This invention identified a series of new Ehd2 allelic mutants by map-based cloning and Mutmap method. Further research found that, in addition to the extreme late heading phenotype, the loss of Ehd2 function in this series of mutants enhanced the resistance of rice seedlings to salt stress.

[0025] (2) This invention further elucidates the genetic mechanism of rice salt tolerance by interpreting the function of Ehd2 salt tolerance, and also provides a theoretical basis and technical support for improving rice salt tolerance and genetic improvement of varieties. Attached Figure Description

[0026] Figure 1 This is a phenotypic and map-based cloning diagram of the ehd2-1(elh5) mutant in an embodiment of the present invention;

[0027] In this table, A represents the phenotypic and heading date statistics of the ehd2-1(elh5) mutant; B represents the identification of ehd2-1 using map-based cloning; DG represents the verification of Ehd2 function using the CRISPR system and genetic complementation; NLD and NSD represent natural long-day and natural short-day conditions, respectively; *** indicates that the differences between different treatment groups are statistically significant, and *** indicates P<0.001;

[0028] Figure 2 The figures show the phenotype of the ehd2-2(elh6) mutant and the Mutmap gene cloning diagram in this embodiment of the invention; AB shows the phenotype and heading date statistics of the ehd2-2(elh6) mutant; C shows the cloning of ehd2-1 using the Mutmap method; DE shows the alternative splicing of the ehd2-2(elh6) mutant; FG shows the function of Ehd2 verified using genetic complementation; where *** indicates that the differences between different treatment groups are statistically significant, and *** indicates P<0.001;

[0029] Figure 3 The figures show the phenotypes of the ehd2-3 to ehd2-8 (elh7 to elh12) mutants and the Mutmap gene cloning diagrams in the embodiments of the present invention; AH represents the phenotypes and heading dates of the ehd2 series alleles; I represents the cloning of ehd2-8 using the Mutmap method; where *** indicates that the differences between different treatment groups are statistically significant, and *** indicates P < 0.001;

[0030] Figure 4 The image shows the salt tolerance phenotype of the ehd2-6 / 7 (elh10 / 11) mutant in this embodiment of the invention; A shows the phenotypes of the ehd2-6 / 7 mutant and wild-type ZH8015 before and after salt treatment; B shows the survival rate statistics of ZH8015 and ehd2-6 / 7 mutant after salt treatment recovery; * indicates that the difference between different treatment groups is statistically significant, * indicates P<0.05;

[0031] Figure 5 The diagram shows the distribution and GO enrichment analysis of differentially expressed genes in the ehd2-6(elh10) mutant after salt treatment in this embodiment of the invention; A shows the statistical analysis of differentially expressed genes between the ehd2-6 mutant and wild-type ZH8015 after salt treatment; B shows the GO enrichment analysis of differentially expressed genes.

[0032] Figure 6 This is an RT-qPCR verification diagram of the differentially expressed genes after salt treatment of the ehd2-6(elh10) mutant in an embodiment of the present invention. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] like Figure 1-6 As shown:

[0035] This invention discloses a gene for improving salt tolerance in rice. The gene is the Ehd2 gene, which encodes protein (a) or (b), wherein...

[0036] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or

[0037] (b) A protein derived from (a) with the sequence shown in SEQ ID NO 9 substituted, deleted or added with one or more amino acids and having the same function.

[0038] This invention also discloses a method for improving the salt tolerance of rice, which involves using genetic engineering techniques to modify the rice Ehd2 gene to obtain transgenic rice with the gene function missing.

[0039] This invention also discloses a gene mutant that enhances salt tolerance in rice, wherein the genotypes of mutants of the Ehd2 gene (elh5~elh12) are ehd2-1~ehd2-8; wherein,

[0040] Compared to the Ehd2 gene, the CDS sequence of the ehd2-1 (elh5) gene has a mutation from C to G at position 490.

[0041] Compared with the Ehd2 gene, the gene ehd2-2 / 3 (elh6 / 7) has a mutation from G to A at the junction of the first exon and intron.

[0042] Compared to the Ehd2 gene, the CDS sequence of the ehd2-4 (elh8) gene has a mutation from C to T at position 547.

[0043] Compared to the Ehd2 gene, the CDS sequence of the ehd2-5 (elh9) gene has a mutation from C to T at position 473.

[0044] Compared with gene Ehd2, gene ehd2-6 / 7 / 8 (elh10 / 11 / 12) has a TGC deletion at positions 655-657 of its CDS sequence.

[0045] The application of a method to improve the salt tolerance of rice in plant breeding, including transgenic, hybridization, backcrossing, self-pollination or asexual reproduction.

[0046] Application of a gene mutant that enhances salt tolerance in rice in biomaterials, where the biomaterials are expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0047] The specific application method is as follows:

[0048] 1) Used to regulate the salt tolerance of rice;

[0049] 2) Used for plant breeding;

[0050] 3) Used to prepare transgenic plants.

[0051] The plant belongs to the genus Oryza in the family Poaceae.

[0052] The best rice variety is selected.

[0053] Example 1

[0054] Phenotypic and map-based cloning of the ehd2-1(elh5) mutant.

[0055] 1. Phenotypic identification of the ehd2-1(elh5) mutant

[0056] EMS mutagenesis was performed on the japonica rice variety Nipponbare, and an extremely late heading single plant 5 (elh5) was found in the mutant library. After years of planting, it was found that this mutant showed a stable extremely late heading phenotype under both natural long-day and natural short-day conditions.

[0057] 2. ehd2-1 map-based cloning.

[0058] The ehd2-1(elh5) mutant was crossed with the indica rice variety Nanjing 11. The F1 generation exhibited a normal heading phenotype. Extremely late-heading single plants were selected from the F2 generation for map-based cloning. Based on molecular marker linkage analysis, ehd2-1 was located in the 94-kb region of chromosome 10. Sequencing revealed Ehd2 as the target gene, and its coding region (CDS) sequence is shown in SEQ ID NO:1. This gene encodes a protein with an IDD domain of 475 amino acids (amino acid sequence shown in SEQ ID NO:2). The ehd2-1(elh5) mutant has a C-to-G mutation at position 490 of the Ehd2 coding region, and its CDS sequence is shown in SEQ ID NO:3.

[0059] 3. CRISPR / Cas9 knockout of Ehd2.

[0060] To verify the function of Ehd2 in rice, its gene was knocked out. In this embodiment, a CRISPR / Cas9 knockout vector was constructed. The specific construction method is as follows:

[0061] Based on the gRNA-Ehd2 target sequence (SEQ ID NO:4) designed from the database, GGCA and AAAC were added before the forward and reverse complementary sequences of the target sequence, respectively, to form gEhd2-F and gEhd2-R construction primers. The gEhd2-F and gEhd2-R primers were annealed to form double strands, and homologous recombination was performed with the AarI-digested intermediate vector K-gRNA. The T7 primer was used to check the correctness of the construction. The correctly constructed SK-gRNA-Ehd2 recombinant plasmid was digested with restriction endonucleases KpnI and BglII, and the fragment was recovered for ligation with the KpnI and BamHI-digested pC1300-Cas9 linear vector. The pC1300-F primer was used again to check the success of the construction.

[0062] 4. Genetic complementation of the ehd2-1(elh5) mutant.

[0063] To verify the function of Ehd2, a genomic fragment of Ehd2 (containing the promoter, coding, and terminator regions) from the Nipponbare background was introduced into the ehd2-1 (elh5) mutant. In this embodiment, a genetic complementation vector was constructed. The specific construction method is as follows:

[0064] Primers were designed based on the Ehd2 gene sequence published in the Rice Genome Annotation Project. Using the genome of wild-type Nipponbare as a template, EcoRI restriction sites were designed on the primers to amplify a 7.29-kb genomic fragment from 2.31-kb upstream of the start codon to 1.94-kb downstream of the stop codon. The expression vector pCAMBIA1300 was digested with EcoRI, and the recovered product was ligated with homologous recombinase at 50°C for 20 min. The product was then transformed into E. coli DH5α competent cells. After single colonies grew, positive bacteria were identified and screened. Plasmids were extracted, and after correct sequencing, the pCAMBIA1300-Ehd2 complementary vector was obtained.

[0065] 5. Agrobacterium-mediated genetic transformation of rice

[0066] The recombinant plasmid with the correct sequence was transferred into the callus of Nipponbare rice using the Agrobacterium strain EHA105-mediated rice genetic transformation system. After callus induction, subculture, pre-culture, infection, co-culture, screening for hygromycin-resistant callus, differentiation, rooting, hardening, and transplantation, transgenic plants were obtained.

[0067] Example 2

[0068] Phenotypic and Mutmap gene cloning of the ehd2-2(elh6) mutant.

[0069] 1. Phenotypic identification of the ehd2-2(elh6) mutant

[0070] EMS mutagenesis was performed on the japonica rice variety Nipponbare, and an extremely late-heading single plant elh6 was found in the mutant library. After years of planting, it was found that this mutant showed a stable extremely late-heading phenotype under both natural long-day and natural short-day conditions.

[0071] 2. Mutmap clone of ehd2-2.

[0072] The ehd2-2(elh6) mutant was backcrossed with Nipponbare. The F1 generation showed a normal heading phenotype. Forty extremely late-heading single plants were selected from the F2 generation for Mutmap cloning. Based on the Mutmap results, the SNP sites on chromosome 10 with an SNP index of 1 were analyzed. Sequencing revealed that the G mutation at the junction of the first exon and intron of Ehd2 was changed to A. Its CDS sequence is shown in SEQ ID NO:5.

[0073] 3. Genetic complementation of the ehd2-2(elh6) mutant.

[0074] To verify the function of ehd2-2, genetic complementation was performed on it. The specific implementation method is the same as in Example 1.

[0075] Example 3

[0076] Phenotypic and Mutmap clones of ehd2-3~ehd2-8 (elh7~elh12) mutants.

[0077] 1. Phenotype of ehd2-3~ehd2-8 (elh7~elh12) mutants.

[0078] 2. Subsequently, we identified six other late-heading mutants of ehd2-3 to ehd2-8 (elh7 to elh12) in the EMS mutant library of Nipponbare and ZH8015. Among them, (elh7 to elh9 are Nip background, and elh10 to elh12 are ZH8015 background). The mutation mode of ehd2-3 (elh7) is the same as that of ehd2-2 (elh6), and its CDS sequence is shown in SEQ ID NO:5. The CDS sequences of ehd2-4 (elh8) and ehd2-5 (elh9) are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0079] 3. Cloning of the ehd2-8 gene.

[0080] The ehd2-8(elh12) mutant was crossed with Nipponbare rice. The F1 generation showed a normal heading phenotype. Extremely late-heading single plants were selected from the F2 generation for Mutmap cloning. Based on the Mutmap results, the InDel site on chromosome 10 with an InDel index of 1 was analyzed. Sequencing revealed that Ehd2 was the target gene. The coding region (CDS) sequence of Ehd2 from the indica rice variety ZH8015 is shown in SEQ ID NO:8. This gene encodes a protein with an IDD domain of 474 amino acids (amino acid sequence shown in SEQ ID NO:9). The coding region of ehd2-6 to ehd2-8 (elh10 to elh12) has a deletion of three TGC bases at positions 655-657. Its CDS sequence is shown in SEQ ID NO:10.

[0081] Example 4

[0082] Salt tolerance phenotype of ehd2-6 / 7(elh10 / 11) mutants.

[0083] Seeds of ZH8015, elh10, and elh11 were first placed in a drying oven at 50°C for two days. Subsequently, they were surface sterilized with 75% ethanol for 5 minutes, rinsed three times with sterile water, then shaken with 40% sodium hypochlorite solution for 30 minutes, and finally rinsed three times with sterile water to break dormancy. The seeds were then stored in filter paper soaked at 37°C for several days until germination. Germinated seedlings were transferred to a 96-well hydroponic incubator containing Yoshida solution. Seedlings were grown under long-day conditions (14 hours light, 30°C / 10 hours darkness, 25°C) in a light incubator for 3 weeks, with the nutrient solution changed every 5 days. After three weeks, the seedlings were treated with 150 mM NaCl solution for 5 days, then cultured in Yoshida nutrient solution for 5 days, followed by phenotypic observation and photography. Before salt stress treatment, there were no significant phenotypic differences between the ZH8015 and elh10 / 11 mutants. However, after 24 hours of salt stress treatment, most ZH8015 seedlings began to curl their leaves, and after 5 days of salt treatment, the leaves showed obvious wilting, drying, and chlorosis. Conversely, the elh10 / 11 mutant (8.5 / 24, 35.4%; 11.1 / 24, 46.3%) showed a higher survival rate than ZH8015 (4.2 / 24, 17.5%). Figure 4 The results showed that Ehd2 is a negative regulator of salt signaling in rice seedlings.

[0084] Example 5

[0085] RNA-Seq analysis of ehd2-6 / 7 (elh10 / 11) mutants after salt treatment.

[0086] Three-week-old ZH8015 and elh10 mutant seedlings were treated with 150 mM NaCl for 24 h, and total RNA was extracted from their leaves. Each sample was isolated three times using the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Novozymes Biotechnology Co., Ltd.), following the kit instructions. The purified total mRNA was used to construct a library, which was then sequenced using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Library sequencing was performed by Beijing Novogene Co., Ltd. The raw sequencing reads were filtered and aligned to the rice reference genome using HISAT2 to obtain the location information of the filtered reads on the genome.

[0087] RNA-Seq analysis identified 5150 differentially expressed genes (DEGs). Compared with ZH8015, the elh10 mutant showed 3509 and 1641 DEGs upregulated and downregulated, respectively. Figure 5Ehd2 may participate in the regulation of salt tolerance in rice through these DEGs. GO enrichment analysis of the genes showed that these DEGs were mainly enriched in the stress response, consistent with Ehd2's negative regulation of salt stress. Furthermore, DEGs were also significantly enriched in other pathways, including oxidoreductase activity, antioxidant activity, peroxidase activity, transmembrane transport protein activity, and double-stranded DNA binding activity. Figure 5 This suggests that Ehd2 regulation of salt stress in rice may involve multiple metabolic processes.

[0088] Example 6

[0089] RT-qPCR validation of differentially expressed genes in ehd2-6(elh10) mutant after salt treatment.

[0090] To verify the expression levels of these DEGs under salt stress, we used RT-qPCR to investigate eight genes associated with ion transporters, enzyme activity regulators, antioxidants, and nucleic acid-binding transcription factors. The transcriptional levels of OsHKT15 (sodium ion transporter), OsHAK22 (potassium ion transporter), OsZHD8 (zinc finger transcription factor), and OsERF115 (AP2 / EREBP family transcription factor) showed no significant difference between elh10 and ZH8015 before salt stress. However, after 24 hours of salt stress treatment, their expression was significantly induced in the elh10 mutant. Figure 6 Furthermore, in the elh10 mutant, the peroxidase family gene OsPrx128 showed upregulated expression levels at 0 and 24 hours under salt stress. Figure 6 Furthermore, genes associated with xyloglucan endotransferases, such as OsXTH27, OsXTH21, and OsXTR1, also showed higher transcriptional levels in the elh10 mutant than in the ZH8015 mutant after 24 hours of salt stress. Figure 6 These results are consistent with our RNA-Seq data and demonstrate the negative regulatory role of Ehd2 in the aforementioned differentially expressed genes.

[0091] The sequence lists are as follows:

[0092] SEQ ID NO:1

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for improving the salt tolerance of rice, characterized in that, The rice Ehd2 gene was modified using genetic engineering techniques to obtain transgenic rice with the gene function missing. The amino acid sequence encoded by the Ehd2 gene is shown in SEQ ID NO:

2.

2. The application of the method for improving salt tolerance of rice according to claim 1 in plant breeding, characterized in that, The breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction, and the application is to improve the salt tolerance of rice.

3. The application of modifying the rice Ehd2 gene in improving rice salt tolerance, characterized in that... The rice Ehd2 gene was modified using genetic engineering techniques to obtain transgenic rice with the gene function missing. The amino acid sequence encoded by the Ehd2 gene is shown in SEQ ID NO:2.

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