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

Abstract

The invention discloses an Ehd2 gene, a gene mutant, a method and application for improving the salt tolerance of rice, Ehd2 is a key flowering factor for rice flowering transformation, a series of Ehd2 new allelic mutants are identified through map-based cloning and Mutmap methods, and further research finds that the series of mutants have the advantages that besides an extremely late heading phenotype, the Ehd2 new allelic mutants can be used for improving the salt tolerance of the rice; the Ehd2 function deficiency enhances the resistance of rice seedlings to salt stress. Through function interpretation of the salt tolerance of the Ehd2, the genetic mechanism of the salt tolerance of the rice is further clarified, and meanwhile, a theoretical basis and a technical support are provided for improvement of the salt tolerance of the rice and genetic improvement of varieties of the rice.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to an Ehd2 gene, a gene mutant, a method and an application thereof for improving the salt tolerance of rice. Background Art

[0002] Soil salinization seriously affects crop yields (Munns et al., 2008; Deinlein et al., 2014). Rice is a crop that is sensitive to salt stress. Excessive sodium ion accumulation can seriously damage the growth and development of rice, especially in the seedling and reproductive growth stages (Liu et al., 2022). In order 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 antioxidant (such as CAT, APX, GST and NAGPH activity) (Zhang et al., 2013; Tang et al., 2015; Fu et al., 2018).

[0003] In the process of functional genomics research, many transcription factors are involved in regulating rice salt tolerance. 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 is the most common MYB subfamily in plants. In rice, there are 117 R2R3-OsMYB genes asymmetrically distributed on 12 chromosomes. Whole-genome identification of R2R3-MYB transcription factor genes under salt stress found that OsMYB2-115 may be an important gene related to rice salt stress (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 the 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 and activates 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 together with OsIDD4 to regulate rice flowering (Deng et al., 2017). Previous studies have shown that OsIDD10 activates the expression of AMT1;2, ​​GDH2, CIPK9, and CIPK14 to regulate ammonium uptake and nitrogen metabolism in roots (Xuan et al., 2013; Xuan et al., 2019). OsIDD3 has been reported to be involved in regulating cold stress responses by directly binding to the CBF1 promoter (Dou et al., 2016). In addition, the OsIDD3-OsIDD13-OsIDD14 / LPA1 transcriptional regulatory complex enhances rice 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, a gene mutant, a method and an application thereof for improving the salt tolerance of rice.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A gene for improving salt tolerance of rice, the gene is 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) A protein derived from (a) with equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO:9.

[0009] A method for improving the salt tolerance of rice, using genetic engineering means to transform the rice Ehd2 gene to obtain transgenic rice with the gene function missing.

[0010] A gene mutant for improving salt tolerance of rice, wherein the genotype of the mutant (elh5 to elh12) of the gene Ehd2 is ehd2-1 to ehd2-8; wherein,

[0011] Compared with gene Ehd2, the 490th base of the CDS sequence of gene ehd2-1 (elh5) mutated from C to G;

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

[0013] Compared with gene Ehd2, the 547th base of the CDS sequence of gene ehd2-4 (elh8) mutated from C to T;

[0014] Compared with gene Ehd2, the 473rd base of the CDS sequence of gene ehd2-5 (elh9) mutated from C to T;

[0015] Compared with gene Ehd2, the CDS sequence of gene ehd2-6 / 7 / 8 (elh10 / 11 / 12) lacks TGC at base 655-657.

[0016] Preferably, the breeding method comprises transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0017] The invention discloses an application of a gene mutant for improving the salt tolerance of rice in a biological material. The biological material is an expression cassette, a transposon, a plasmid vector, a virus vector or an engineered bacterium.

[0018] Preferably, the specific application method is:

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

[0020] 2) Used in 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] Compared with the prior art, the present invention has achieved the following technical effects:

[0024] (1) The present invention identified a series of new Ehd2 allele mutants by using positional cloning and Mutmap methods. Further studies found that in addition to the extremely late heading phenotype, the Ehd2 functional deficiency of this series of mutants enhanced the resistance of rice seedlings to salt stress;

[0025] (2) The present invention further clarifies 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 its varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The phenotype and positional cloning diagram of the ehd2-1(elh5) mutant in the embodiment of the present invention;

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

[0028] Figure 2 Figure 1 is the phenotype of the ehd2-2 (elh6) mutant and the Mutmap gene cloning diagram in the embodiment of the present invention; AB is the phenotype and heading period statistics of the ehd2-2 (elh6) mutant; C is the cloning of ehd2-1 by the Mutmap method; DE is the variable splicing of the ehd2-2 (elh6) mutant; FG verifies the function of Ehd2 by genetic complementation; *** indicates that the difference between different treatment groups is statistically significant, and *** indicates P < 0.001;

[0029] Figure 3 Figure 1 is the phenotype and Mutmap gene cloning diagram of the mutants of ehd2-3 to ehd2-8 (elh7 to elh12) in the embodiments of the present invention; AH is the phenotype and heading period statistics of the ehd2 series alleles; I is the cloning of ehd2-8 by the Mutmap method; *** indicates that the differences between different treatment groups are statistically significant, and *** indicates P < 0.001;

[0030] Figure 4 Figure 2 is a diagram of the salt tolerance phenotype of the ehd2-6 / 7 (elh10 / 11) mutant in the embodiment of the present invention; A is the phenotype of the ehd2-6 / 7 mutant and the wild type ZH8015 before and after salt treatment; B is the survival rate statistics of ZH8015 and ehd2-6 / 7 mutant after recovery from salt treatment; * indicates that the difference between different treatment groups is statistically significant, and * indicates P<0.05;

[0031] Figure 5 Figure 1 is a graph showing the distribution of differentially expressed genes and GO enrichment analysis of the ehd2-6 (elh10) mutant after salt treatment in the embodiment of the present invention; A is a statistical graph showing the differentially expressed genes between the ehd2-6 mutant and the wild type ZH8015 after salt treatment; B is a GO enrichment analysis of the differentially expressed genes;

[0032] Figure 6 This is a RT-qPCR verification diagram of the differentially expressed genes in the ehd2-6 (elh10) mutant after salt treatment in the example of the present invention. DETAILED DESCRIPTION

[0033] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] like Figure 1-6 As shown:

[0035] The present invention discloses a gene for improving salt tolerance of rice, wherein the gene is the Ehd2 gene, which is a gene encoding 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 equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO 9.

[0038] The invention also discloses a method for improving the salt tolerance of rice, which comprises transforming the rice Ehd2 gene by genetic engineering means to obtain transgenic rice with the gene function missing.

[0039] The present invention also discloses a gene mutant for improving the salt tolerance of rice, wherein the genotype of the mutant (elh5 to elh12) of the gene Ehd2 is ehd2-1 to ehd2-8; wherein,

[0040] Compared with gene Ehd2, the 490th base of the CDS sequence of gene ehd2-1 (elh5) mutated from C to G;

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

[0042] Compared with gene Ehd2, the 547th base of the CDS sequence of gene ehd2-4 (elh8) mutated from C to T;

[0043] Compared with gene Ehd2, the 473rd base of the CDS sequence of gene ehd2-5 (elh9) mutated from C to T;

[0044] Compared with gene Ehd2, the CDS sequence of gene ehd2-6 / 7 / 8 (elh10 / 11 / 12) lacks TGC at base 655-657.

[0045] A method for improving salt tolerance of rice is used in plant breeding. The breeding methods include transgenic, hybridization, backcrossing, self-pollination or asexual reproduction.

[0046] The invention discloses an application of a gene mutant for improving the salt tolerance of rice in a biological material. The biological material is an expression cassette, a transposon, a plasmid vector, a virus vector or an engineering bacterium.

[0047] The specific application method is:

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

[0049] 2) Used in plant breeding;

[0050] 3) Used to prepare transgenic plants.

[0051] The plant is a rice plant of the Poaceae family.

[0052] Rice is most preferred.

[0053] Example 1

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

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

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

[0057] 2. Positional cloning of ehd2-1.

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

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

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

[0061] The gRNA-Ehd2 target sequence (SEQ ID NO: 4) was designed according to the database, and GGCA and AAAC were added to the forward and reverse complementary sequences of the target sequence to form gEhd2-F and gEhd2-R construction primers; the gEhd2-F and gEhd2-R forward and reverse primers were annealed to form double strands, and homologous recombination was performed with the intermediate vector K-gRNA digested with AarI, and the T7 primer was used to detect whether the construction was correct; the above-mentioned correctly constructed SK-gRNA-Ehd2 recombinant plasmid was digested with restriction endonucleases KpnI and BglII and the fragments were recovered for connection with the pC1300-Cas9 linear vector digested with KpnI and BamHI. The pC1300-F primer was used to detect whether the construction was successful again.

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

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

[0064] Primers were designed based on the Ehd2 gene sequence published on the Rice Genome Annotation Project. The genome of the wild-type Nipponbare was used as a template, and 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 cut with EcoRI, and the recovered product was ligated with a homologous recombinase at 50°C for 20 min and then transformed into competent Escherichia coli DH5α. After a single clone was grown, the positive bacteria were identified and screened, and the plasmid was extracted. After correct sequencing, the pCAMBIA1300-Ehd2 complementary vector was obtained.

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

[0066] The above-mentioned correctly sequenced recombinant plasmid was transferred into the callus tissue of rice Nipponbare through the rice genetic transformation system mediated by Agrobacterium strain EHA105; after callus induction, subculture, pre-culture, infection, co-culture, screening of callus tissue with hygromycin resistance, differentiation, rooting, seedling hardening and transplanting, transgenic plants were obtained.

[0067] Example 2

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

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

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

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

[0072] The ehd2-2(elh6) mutant was backcrossed with Nipponbare, and the F1 generation showed a normal heading phenotype. 40 extremely late heading plants were selected from the F2 generation for Mutmap cloning. According to the Mutmap results, the SNP sites with a SNPindex of 1 on chromosome 10 were analyzed, and sequencing revealed that the G at the junction of the first exon and intron of Ehd2 mutated to A. The 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.

[0075] Example 3

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

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

[0078] 2. We subsequently 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 which (elh7 to elh9 were in the Nip background, and elh10 to elh12 were in the ZH8015 background). Among them, ehd2-3 (elh7) and ehd2-2 (elh6) had the same mutation mode, and the 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 ehd2-8 gene.

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

[0081] Example 4

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

[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 min, washed three times with sterile water, then shaken with 40% sodium hypochlorite solution for 30 min, and finally washed three times with sterile water to break dormancy. The seeds were stored in soaked filter paper at 37°C for several days until the seeds germinated. The germinated seedlings were transferred to a 96-well hydroponic box filled with Yoshida solution. The seedlings were grown under long-day conditions (14 h light, 30°C / 10 h dark, 25°C) in a light incubator for 3 weeks, and the nutrient solution was changed every 5 days. After three weeks, the seedlings were treated with 150 mM NaCl solution for 5 days and then cultured in Yoshida nutrient solution for 5 days, followed by phenotypic observation and photography. Before salt stress treatment, there was no obvious phenotypic difference between ZH8015 and elh10 / 11 mutants. However, after 24 h 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. In contrast, 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 their leaves were taken for total RNA extraction. Each sample was repeated three times and operated using the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Novogene Biotechnology Co., Ltd.) according to the instructions of the kit. The purified total mRNA was used to construct a library and sequenced using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina). The library sequencing was performed by Beijing Novogene Technology Co., Ltd. The raw sequencing reads were filtered and aligned with the rice reference genome through HISAT2, and the position information of the filtered reads on the genome was finally obtained.

[0087] By RNA-Seq analysis, we identified 5150 differentially expressed genes (DEGs), of which 3509 and 1641 were up-regulated and down-regulated in the elh10 mutant compared with ZH8015 ( Figure 5). Ehd2 may be involved in the regulation of rice salt tolerance through these DEGs. Gene GO enrichment analysis showed that these DEGs were mainly enriched in stress response, which was consistent with the negative regulation of salt stress by Ehd2. In addition, DEGs were 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 rice salt stress may involve multiple metabolic processes.

[0088] Example 6

[0089] RT-qPCR verification of differentially expressed genes in the 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 related to ion transporters, enzyme activity regulators, antioxidants, and nucleic acid binding transcription factors. The transcript levels of OsHKT15 (sodium ion transporter), OsHAK22 (potassium ion transporter), OsZHD8 (zinc finger transcription factor), and OsERF115 (AP2 / EREBP family transcription factor) between elh10 and ZH8015 were not significantly different before salt stress. However, their expressions were significantly induced in the elh10 mutant after 24 h of salt stress treatment ( Figure 6 ). In addition, in the elh10 mutant, the peroxidase family gene OsPrx128 showed upregulated expression levels at 0 h and 24 h under salt stress ( Figure 6 In addition, genes related to xyloglucan endotransglycosylase, such as OsXTH27, OsXTH21, and OsXTR1, also showed higher transcription levels in the elh10 mutant than in ZH8015 after 24 h under salt stress ( Figure 6 The above results are consistent with our RNA-Seq data and prove the negative regulatory effect of Ehd2 on the above differentially expressed genes.

[0091] Among them, the sequence listings are:

[0092] SEQ ID NO:1

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An Ehd2 gene for improving salt tolerance in rice, characterized in that: The gene is a gene encoding protein (a) or (b), wherein: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; or (b) A protein derived from (a) with equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO:

9.

2. A method for improving salt tolerance of rice, characterized in that: Using genetic engineering methods, the rice Ehd2 gene was modified to obtain transgenic rice with missing gene function.

3. A gene mutant for improving salt tolerance of rice, characterized in that: The genotypes of the mutants elh5 to elh12 of the gene Ehd2 are ehd2-1 to ehd2-8; wherein, Compared with gene Ehd2, the 490th base of the CDS sequence of gene ehd2-1 mutated from C to G; Compared with the gene Ehd2, the base at the junction of the first exon and intron of the gene ehd2-2 / 3 mutated from G to A; Compared with gene Ehd2, the 547th base of the CDS sequence of gene ehd2-4 mutated from C to T; Compared with gene Ehd2, the 473rd base of the CDS sequence of gene ehd2-5 mutated from C to T; Compared with gene Ehd2, the CDS sequence of gene ehd2-6 / 7 / 8 lacks TGC at base 655-657.

4. Application of the method for improving salt tolerance of rice according to claim 2 in plant breeding, characterized in that: The breeding methods include transgenic, hybridization, backcrossing, selfing or asexual reproduction.

5. The use of a gene mutant for improving salt tolerance of rice in biomaterials according to claim 3, characterized in that: The biological material is an expression cassette, a transposon, a plasmid vector, a virus vector or an engineered bacterium.

6. The mutant according to claim 3 or the use according to claim 4 or claim 5, characterized in that: The specific application method is: 1) Used to regulate rice salt tolerance; 2) Used in plant breeding; 3) Used for preparing transgenic plants.

7. The use according to claim 6, characterized in that: The plant is a plant of the genus Oryza in the family Gramineae.

Images