TaCBL4-mediated TaSOS1 phosphorylation regulation site in wheat and application of TaCBL4-mediated TaSOS1 phosphorylation regulation site

Abstract

The invention relates to a TaCBL4 mediated TaSOS1 phosphorylation regulation and control site in wheat and an application of the TaCBL4 mediated TaSOS1 phosphorylation regulation and control site. The TaSOS1 phosphorylation regulation and control site is Ser978-Ser1126 / Ser1128 (S978-DSPS); the phosphorylation regulation and control site of the TaSOS1 is S978-DSPS, and serine at the site 978 of the TaSOS1-DAA (S1126AS1128A) is mutated into alanine. The TaSOS1 in the wheat is from a chromosome LOC778390, NC057800.1 (13146069..13157356) of the 3A of the common wheat, and the TaSOS1 in the wheat is from a chromosome NC057800.1 (13146069..13157356). The invention also relates to an application of the TaCBL4 mediated TaSOS1 phosphorylation regulation site Ser978-Ser126 / Ser128 (S978-DSPS) in the wheat as a molecular marker for detecting the salt tolerance of the wheat, and also relates to an application of the TaCBL4 mediated TaSOS1 phosphorylation regulation site. The site is applied to the aspects of wheat salt tolerance improvement and assisted breeding.

Description

Technical Field

[0001] The present invention relates to the field of TaCBL4-mediated TaSOS1 phosphorylation regulation, and particularly relates to TaCBL4-mediated TaSOS1 phosphorylation regulation sites in wheat and their applications. Background Art

[0002] The SOS salt tolerance pathway in the model plant Arabidopsis thaliana has been relatively well studied, and the salt tolerance mechanisms in other plants, especially crops, have also been a research hotspot in recent years. According to previous studies on the regulation mechanism of the SOS pathway in wheat, it was found that TaCIPK24 alone or in combination with the TaCBL4 protein can activate the salt tolerance activity of the Na+ / H+ antiporter TaSOS1, which is different from the regulation mechanism in Arabidopsis thaliana where SOS1 cannot be activated solely by the CIPK24 protein kinase. In the previous research of the experimental team, a super-resistant mutant TaSOS1-Δ974 of wheat SOS1 was obtained, and by comparing the phosphorylation sites at which the SOS1 protein in Arabidopsis thaliana is regulated, it was verified that the sites at which TaSOS1 is phosphorylated by the TaCIPK24 protein kinase are Ser1126 / Ser1128 (TaSOS1-DSPS), but the sites at which the TaCIPK24 / TaCBL4 protein complex regulates TaSOS1 are unknown. At the same time, even though the conserved phosphorylation regulation site DSPS of the wheat SOS1 protein has been mutated to DAPA, it still has the ability to be activated by the complex protein CIPK24 / CBL4. In order to study the sites of TaCBL4-mediated TaSOS1 phosphorylation, it is necessary to further analyze the proteins interacting with TaSOS1, and by studying further deletions and site-directed mutations of the TaSOS1-DAPA mutant, find other possible phosphorylation sites of the TaSOS1 protein and analyze the structural and functional changes when SOS1 is regulated. Summary of the Invention

[0003] To solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide TaCBL4-mediated TaSOS1 phosphorylation regulation sites in wheat and their applications.

[0004] To achieve the above purpose, the technical solution of the present invention is as follows:

[0005] TaCBL4-mediated TaSOS1 phosphorylation regulation sites in wheat, wherein the TaSOS1 phosphorylation regulation site is S978-DSPS;

[0006] Further, in the TaSOS1 phosphorylation regulation site of S978-DSPS, the serine at position 978 of TaSOS1-DAPA (where the serine at the DSPS site is mutated to alanine) is mutated to alanine again.

[0007] Furthermore, the application of the TaCBL4-mediated TaSOS1 phosphorylation regulatory site in wheat as a molecular marker for detecting wheat salt tolerance;

[0008] Furthermore, the application of this site in improving wheat salt tolerance and assisting breeding;

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0010] First, CBL4 is involved in another important specific regulatory site S978 outside the conserved phosphorylation site (DSPS), which is located upstream of the C-terminus of SOS1. In the yeast functional phenotype verification of our truncated mutants, it was found that when truncated to the length of Δ983, the salt tolerance phenotype of yeast co-transformed with CIPK24 could reach above 400 mM NaCl, while the single-transformed Δ983 protein mutant could only reach 100 mM NaCl. This indicates that when TaSOS1 is truncated to the length of 983, there are still regulatory sites that can replace the conserved phosphorylation site DSPS and be activated by the protein kinase CIPK24 complex mediated by CBL4 in the full-length SOS1 protein sequence, enabling the truncated TaSOS1 protein to still retain the ability to regulate the transport of Na+ activity, and the specific target S978 of TaSOS1 regulated by CBL4-CIPK24 was found through single mutation.

[0011] Second, when the wheat SOS1 protein responds to salt stress, the way its protein activity is activated may be more efficient than that of Arabidopsis thaliana. It has been reported that the site in Arabidopsis thaliana that can be regulated by phosphorylation is only the conserved DSPS at its C-terminus, and it must be recognized and activated after the calmodulin CBL4 and the protein kinase CIPK24 form a complex. In this study, it was found that the conserved DSPS regulatory site of wheat SOS1 can also be mediated by CIPK24 in a CBL4-independent activation manner, that is, TaCIPK24 alone can regulate the DSPS phosphorylation site, and the protein complex CBL4-CIPK24 not only recognizes DSPS but also specifically recognizes the S978 target. This indicates that under salt stress, wheat can release the auto-inhibited state of TaSOS1 at both the DSPS and S978 targets simultaneously to activate its active region for reverse transporting Na+.

[0012] Third, in the same yeast expression system, the SOS-pathway of wheat and Arabidopsis thaliana was functionally verified, and it was found that the wheat salt tolerance pathway has obvious salt tolerance advantages ( Figure 23) This indicates that the activity of TaSOS1 can be more activated in transgenic yeast, that is, TaSOS1-DSPS can be phosphorylated by TaCIPK24 in a manner independent of CBL4-mediated, resulting in more TaSOS1 in wheat being released from the auto-inhibited state. That is, under salt stress, wheat CBL4 recruits TaCIPK24 to the plasma membrane, without the need to fully form a complex, and participates in the regulation process of recognizing the target DSPS-S978 for the release of TaSOS1 auto-inhibition in both individual and CBL4-complexed forms. Therefore, there are more activated TaSOS1 with the function of reverse transporting Na+ in the wheat SOS pathway in transgenic yeast, which is very likely the main reason why the wheat SOS pathway is more salt-tolerant than Arabidopsis in evolution. Brief Description of the Drawings

[0013] Figure 1 Phylogenetic tree analysis of TaCBL4 with AtCBLs and OsCBLs;

[0014] Figure 2 Phylogenetic tree analysis of TaCIPK24 with AtCIPKs and OsCIPKs;

[0015] Figure 3 Yeast two-hybrid analysis of TaCBL4 with TaSOS1-C;

[0016] Figure 3 A: Enzyme digestion identification of the recombinant plasmid of the pGADT7 yeast expression vector. 1: Double enzyme digestion detection of the AD-TaSOS1-C168 recombinant plasmid; 2: Double enzyme digestion detection of the AD-TaSOS1-DAPA-C168 recombinant plasmid; M: DL10000 marker. Figure 3 B: Yeast two-hybrid experiment to verify the interaction between TaCBL4 and the C-terminal of TaSOS1 and the C-terminal mutant TaSOS-DAPA. BD-TaCBL4 was used as a bait and co-transformed with 168 amino acid fragments at the C-terminal of TaSOS1 on the AD vector into Y2H yeast cells, and co-transforming the AD empty vector was used as a control at the same time. The initial sample loading amount of the yeast was OD600 = 1, 7 uL, and the sample was loaded once every 10-fold dilution. Figure 3 C: Yeast two-hybrid experiment to verify the interaction between TaCBL4 and TaCIPK24. -Leu / -Trp: SD yeast double-deficient medium lacking Leu and Trp, -Leu / -Trp / -Ade / -His: SD yeast quadruple-deficient medium lacking Leu, Trp, Ade, and His.

[0017] Figure 4 Interaction between TaCBL4 and TaCIPK24 in vitro;

[0018] Figure 4A: Enzyme digestion identification of prokaryotic expression vectors. 1: Double enzyme digestion detection of recombinant plasmid TaCBL4-pET-30α; 2: Double enzyme digestion detection of recombinant plasmid TaCIPK24-pGEX-6p-1; M: DL10000 marker. Figure 4 B: GST-pull down assay of TaCBL4 and TaCIPK24. The result shown by CBB is the experimental result of continued Coomassie staining of the gel after WB transfer.

[0019] Figure 5 Functional verification of TaSOS1-DAPA transgenic yeast;

[0020] Figure 6 Salt tolerance analysis of TaSOS1-DAPA transgenic Arabidopsis thaliana;

[0021] Figure 7 : Subcellular localization analysis in TaSOS1-DAPA

[0022] Figure 7 A: Enzyme digestion detection of transient transformation expression vectors. 1: Enzyme digestion of pCAMBIA1300-eGFP empty vector; 2: Enzyme digestion of pCAMBIA1300-TaSOS1-eGFP expression vector; 3: Enzyme digestion of pCAMBIA1300-TaSOS1-DAPA-eGFP expression vector; M: DL10000 marker. Figure 7 B: Subcellular localization of TaSOS1-WT and TaSOS-DAPA proteins. The scale bar is 25 μm.

[0023] Figure 8 Sequence division of TaSOS1;

[0024] Figure 9 Functional verification of transgenic yeast with TaSOS1-C truncated mutant

[0025] A: Salt tolerance phenotype of yeast single-transformed with TaSOS1 C-terminal truncated mutant; B: Functional verification of yeast co-transformed with TaCIPK24 / TaCBL4 and AXT3K. 0 - 400 mM refers to the NaCl concentration of salt treatment. Δ + number: represents the amino acid length after truncating the C-terminus of TaSOS1.

[0026] Figure 10 PCR detection of TaSOS1 mutant transgenic yeast

[0027] 1: Δ1122; 2: Δ1102; 3: Δ1022; 4: Δ997; 5: Δ993; 6: Δ987; 7: TaSOS1; M: DL10000 marker;

[0028] Figure 11 TaSOS1 C-terminal amino acid sequence;

[0029] Figure 12 Functional verification of TaSOS1-C segment deletion mutants in yeast

[0030] A: Alignment results of the base sequences of the recombinant plasmids with deletion mutations after sequencing by DNAMan; B-C: Functional verification of six deletion mutants co-transformed with p414-TaCIPK24 and p414-TaCIPK24-TaCBL4 plasmids into AXT3K yeast. 0 - 300 mM refers to the NaCl concentration for salt treatment. δ + number represents the amino acid sequence deleted from TaSOS1;

[0031] Figure 13 TaSOS1 C-terminal 974 - 997 amino acid sequence;

[0032] Figure 14 Functional verification of TaSOS1-DAPA transgenic yeast with five Ser site-directed mutations

[0033] A: 23 amino acid sequence deleted from TaSOS1-δ974 - 997-DAPA; B: Functional verification of five site-directed mutants of TaSOS1-DAPA, S974A-DAPA, S978A-DAPA, S990A-DAPA, S994A-DAPA, S995A-DAPA, co-transformed with expression vectors p414-TaCIPK24 and p414-TaCIPK24-TaCBL4 plasmids into AXT3K yeast. 0 - 300 mM refers to the NaCl concentration for salt treatment. The lower right corner is the corresponding legend for the positions of yeast spotting plates at the above gradients. After serial dilution by 10-fold gradient, 7 μL is taken to spot yeast colonies.

[0034] Figure 15 Salt tolerance analysis of TaSOS1-S978A-DAPA transgenic Arabidopsis thaliana;

[0035] Figure 16 Functional verification of TaSOS1 transgenic yeast with site-directed mutation S978A;

[0036] Figure 17 Salt tolerance analysis of TaSOS1-S978A transgenic Arabidopsis thaliana;

[0037] Figure 18 Functional verification of TaSOS1 mutant transgenic yeast

[0038] TaSOS1: Yeast transformed with the expression vector pYPGE15-TaSOS1; TaSOS1-DAPA: Yeast transformed with the expression vector pYPGE15-TaSOS1-DAPA, where the conserved serine phosphorylation site DSPS in the original TaSOS1 gene sequence was mutated to Ala; S978A-DAPA: Yeast transformed with the expression vector pYPGE15-TaSOS1-S978A-DAPA, where all three conserved and specific phosphorylation sites in the gene were mutated to Ala; S978A: Yeast transformed with the expression vector pYPGE15-TaSOS1-S978A-DSPS, where the conserved phosphorylation site was restored to Ser and only the specific phosphorylation site 978 was mutated to Ala. +TaCIPK24: Indicates yeast AXT3K co-transformed with the expression vector p414-TaCIPK24; +TaCIPK24 / TaCBL4: Indicates AXT3K co-transformed with the expression vector p414-TaCIPK24-TaCBL4. 0-1000 mM indicates the NaCl concentration for treatment.

[0039] Figure 19 Determination of ion content in transgenic yeast of TaSOS1 mutants;

[0040] Figure 20 Analysis of salt tolerance of transgenic Arabidopsis thaliana with TaSOS1 phosphorylation sites;

[0041] Figure 21 Analysis of salt tolerance of transgenic Arabidopsis thaliana with TaSOS1 phosphorylation sites;

[0042] Figure 22 Model representing the SOS1 activation mechanism;

[0043] Figure 23 Comparison of salt tolerance activities between AtSOS1 and TaSOS1 in yeast. Detailed implementation manners

[0044] The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners:

[0045] As Figures 1 - 23 shown,

[0046] Experimental examples:

[0047] 1 Phylogenetic tree analysis of TaCIPK24 and TaCBL4 proteins

[0048] Phylogenetic tree analysis was performed on TaCBL4 and TaCIPK24 with the family proteins of model plants rice and Arabidopsis thaliana, and the graph was drawn using the software MEGA. The results are as Figure 1 、 2 shown.

[0049] Figure 1 The comparison results showed that, as monocotyledonous plants, wheat and rice, two crops, have a relatively close genetic relationship in CBL4.

[0050] The results showed that the similarity of the wheat TaCIPK24 gene in the protein families of the model plants rice and Arabidopsis thaliana is very high, indicating that the regulatory mechanism involved in this protein gene in wheat may be similar to that of the model plants.

[0051] 2 Interaction between TaCBL4 and TaCIPK24 proteins

[0052] TaCIPK24 can independently participate in the salt tolerance pathway of TaSOS1, and it has been verified in the yeast expression system that the binding site for TaCIPK24 to regulate the salt tolerance activity of TaSOS1 is S1126S1128. To further study whether there are also regulatory sites for complex proteins to activate TaSOS1 in the wheat SOS pathway, we first verified the interaction between CIPK24 and CBL4 in wheat.

[0053] 2.1 Yeast two-hybrid analysis experiment

[0054] To more intuitively verify whether TaCBL4 directly interacts with TaSOS1-DAPA in yeast, and to screen the regions that can be regulated by TaSOS1-DAPA using the yeast function verification system in the follow-up, the yeast two-hybrid method was used to verify the proteins that interact with TaCBL4. TaSOS1 encodes a total of 1142 amino acids. The super-resistant mutant of TaSOS1 obtained in the laboratory was Δ974 in the early stage. Therefore, the 168 amino acid residues deleted by Δ974 were used as the length of the C-terminus. Using the yeast expression vectors pYPGE15-TaSOS1 and pYPGE15-TaSOS1-DAPA as templates respectively, the 504bp TaSOS1-C168 and TaSOS1-DAPA-C168 gene fragments were amplified and constructed on the yeast expression vector pGADT7. The enzyme digestion identification of the recombinant plasmid with correct sequencing is as Figure 3 shown in A. After TaSOS1-C168 and TaSOS1-DAPA-C168 were correctly sequenced and ligated to the AD vector, they were co-transformed with the plasmid BD-TaCBL4 into the Y2H yeast strain for yeast two-hybrid experiments. The results are as Figure 3 shown in B. The co-transformed yeast could not grow normally in the quadruple-deficient medium with the defect type of HLWT. This indicates that whether the C-terminus of TaSOS1 is phosphorylated to DAPA or not, it cannot directly interact with TaCBL4. And Figure 3The results in C showed that when AD-TaCIPK24 and BD-TaCBL4 were co-transformed into Y2H yeast, they could grow normally on the quadruple-deficient medium, which verified that in yeast, TaCBL4 did not directly interact with the C-terminus of TaSOS1, but first interacted with TaCIPK24 and bound together before it had the ability to activate TaSOS1.

[0055] 2.2 GST-pull down in vitro interaction experiment

[0056] By subcloning, the prokaryotic expression vectors, TaCBL4-pET-30α with his tag and TaCIPK24-pGEX-6p-1 with GST tag, were constructed. The results of double digestion detection of the recombinant plasmids are as Figure 4 shown in A. The two recombinant plasmids with correct sequences verified by sequencing and the empty vector pGEX-6p-1 were respectively transformed into BL21 Escherichia coli. TaCBL4-his and TaCIPK24-GST were induced to express under appropriate conditions. After collecting the bacteria and lysing the proteins for crude extraction, using the empty GST as a control, the in vitro interaction experiment of TaCIPK24 and TaCBL4 proteins was carried out by the GST pull-down method with the mixed solutions of His-TaCBL4 / GST-TaCIPK24 and His-TaCBL4 / GST. The results are as Figure 4 shown in B.

[0057] As seen from Figure 4 A, the constructed prokaryotic expression vectors could be double digested to obtain bands with the correct sizes, and the sequencing results were in agreement. The size of the TaCBL4 gene sequence was 657 bp, and the theoretical molecular weight of the protein without the tag was 24.77 KDa. The size of the TaCIPK24 gene sequence was 1341 bp, and the theoretical molecular weight of the protein without the tag was 49.89 KDa. The mixed solutions of the experimental group and the control group were bound to the column through GST beads, then eluted and loaded. After separating the protein bands by SDS-PAGE electrophoresis and performing WB immunoblotting, the results of color development and photography are shown in Figure WB, as Figure 4As shown in Figure B, from the experimental results of pull-down in Figure B, it can be seen that in the lanes of the experimental group, the protein solution after mixing His-TaCBL4 and GST-TaCIPK24 can be specifically bound and eluted by the GST antibody, and the protein band size is the same as that of His-TaCBL4 in the pull-in lane, which is approximately the size of the TaCBL4+his-tagged protein. The protein mixed solution of GST and His-TaCBL4 in the control group was not pulled down by GST, indicating that the protein interaction occurs between TaCIPK24 and TaCBL4. And after Coomassie staining of the PAGE gel, there is a protein band of about 75KDa in the lanes of His-TaCBL4 and GST-TaCIPK24, which is approximately the theoretical molecular weight of the complex of these two proteins. The above evidence demonstrates the interaction between TaCBL4 and TaCIPK24 proteins in wheat.

[0058] The CBL-CIPK signaling system is of great significance in many regulatory pathways. In previous experiments in the laboratory, it was known that TaCBL4 could not directly interact with TaSOS1 to activate the activity of TaSOS1 transporting Na + And it was found that after the conservative phosphorylation site DSPS of TaSOS1 was mutated, it could still participate in the regulation of the CBL-CIPK signal, indicating that other undiscovered interaction regions might be the specific binding sites for the CBL4-CIPK24 complex protein. Therefore, after TaCBL4 binds to TaCIPK24, it is necessary to continue to explore the regulatory sites for activating the TaSOS1-DAPA protein. Therefore, next, we used the mutant TaSOS1-DAPA with the conservative phosphorylation site mutated in TaSOS1 as the target protein for research, and used the yeast functional verification system to analyze the specific sites where TaSOS1-DAPA is regulated by the TaCIPK24 / TaCBL4 protein complex.

[0059] 2.3 Regulation of TaSOS1 salt stress by the TaCIPK24 protein complex mediated by TaCBL4

[0060] In the above experiment, we saw that regardless of whether the regulatory site DSPS exists at the C-terminus of TaSOS1, TaCBL4 could not interact with the C-terminus of TaSOS1 and directly participate in the regulation of TaSOS1 protein. Next, we verified in the yeast expression system the salt tolerance of TaSOS1-DAPA when it is regulated by the protein complex mediated by TaCBL4 after mutating the serine phosphorylation site DSPS directly regulated by TaCIPK24 at the C-terminus of TaSOS1.

[0061] In this part of the experiment, the yeast expression vectors pYPGE15-TaSOS1, pYPGE15-TaSOS1-DAPA, p414-TaCIPK24, p414-TaCBL4 and p414-TaCIPK24-TaCBL4 were all stored in the laboratory. Since the AXT3K yeast strain lacks the endogenous salt-tolerant gene in yeast and has no sodium ion transport function, we co-transformed TaSOS1 or TaSOS1-DAPA with the regulatory genes TaCIPK24 or TaCIPK24 / TaCBL4 respectively in the salt-sensitive yeast mutant AXT3K strain, and used the exogenous gene regulatory network established by the yeast mutant expression system to verify the salt tolerance of the co-expressing yeast.

[0062] TaSOS1: AXT3K yeast strain transformed with plasmid pYPGE15-TaSOS1; DAPA: Yeast strain transformed with single plasmid pYPGE15-TaSOS1-DAPA; +TaCIPK24: AXT3K yeast strain co-transformed with p414-TaCIPK24 and pYPGE15-TaSOS1 or pYPGE15-TaSOS1-DAPA; +TaCIPK24 / TaCBL4: AXT3K yeast strain co-transformed with p414-TaCIPK24-TaCBL4 and pYPGE15-TaSOS1 or pYPGE15-TaSOS1-DAPA; AXT3K: Un-transgenic yeast mutant empty strain. 0-400: The content of NaCl in the salt gradient medium, unit mM. The spotting was diluted in a 10-fold gradient, and 7 μL was spotted for each colony. After growing in each gradient medium at 28 °C for 3 d, observations and photographs were taken. Three independent repeated experiments were carried out, and the results were similar.

[0063] Figure 5Among them, every three lines were used as a group for comparative observation. After the TaSOS1 gene was transferred into mutant yeast, the salt tolerance of AXT3K yeast could be complemented to 100 mM. In contrast, after the TaSOS1-DSPS mutation to TaSOS1-DAPA, the salt tolerance of SOS1 did not change. Taking TaSOS1 as the control, after co-transforming TaCIPK24 or the TaCIPK24 / TaCBL4 complex protein, the salt tolerance of yeast was significantly activated and could withstand more than 400 mM of NaCl content. Taking TaSOS1+TaCIPK24 as the control, the salt tolerance of yeast after co-transforming TaSOS1-DAPA and TaCIPK24 decreased significantly to the tolerance of 150 mM NaCl, similar to the salt tolerance level of yeast transformed with TaSOS1-DAPA alone. This proved that these two serine sites in DSPS were exactly the phosphorylation binding sites activated by TaCIPK24. Comparing the salt tolerance of yeast co-expressing TaSOS1 and TaCIPK24 / TaCBL4, we can see that the mutation of DSPS to DAPA had almost no effect on the salt tolerance level of yeast, and both could grow normally on the medium with more than 400 mM NaCl. This indicated that the regulatory site of the TaCIPK24 / TaCBL4 complex protein was not the conserved DSPS phosphorylation site, and TaSOS1-DAPA could no longer be regulated by TaCIPK24, which also indicated that in addition to DAPA, there were other unknown regulatory regions or sites that could bind to the protein complex TaCIPK24 / TaCBL4 and activate the salt tolerance function of TaSOS1.

[0064] Analysis of Salt Tolerance of 3TaSOS1-DAPA Transgenic Arabidopsis

[0065] As can be seen in the yeast experiment, TaSOS1-DAPA could still have good salt tolerance under the complex regulation pathway of TaCIPK24 / TaCBL4. To confirm in plants that TaSOS1 still had a certain salt tolerance after lacking the conserved phosphorylation site of DSPS, we used the mutant of Arabidopsis SOS1 protein lacking as the background material, transformed the plant expression vector pCAMBIA-1300-TaSOS1-DAPA into Agrobacterium and infected Arabidopsis, and at the same time transformed the vector pCAMBIA-1300-TaSOS1 of the complete complementation group as a control. In the experiment, the transgenic Arabidopsis were all screened to the T3 generation homozygotes, and two lines were selected for salt tolerance analysis. The results are as Figure 6 shown.

[0066] As can be seen from the plant phenotype and data analysis in the above figure, the salt tolerance of TaSOS1-DAPA in Arabidopsis thaliana is consistent with the results verified in the yeast system. Mutants completely lacking the SOS1 protein in plants cannot grow at a concentration of 100 mM NaCl, while transgenic Arabidopsis thaliana complemented with TaSOS1-DAPA can tolerate more than 100 mM. This indicates that TaSOS1 lacking the DSPS regulatory site still has salt tolerance activity, that is, there are still unrecognized phosphorylation sites in the TaSOS1-DAPA sequence, which can be regulated by the TaCIPK24 / TaCBL4 complex.

[0067] 4 Subcellular localization of TaSOS1-DAPA protein

[0068] The accurate localization of a protein is a prerequisite for it to perform its physiological functions normally. The N-terminus of SOS1 has a transmembrane region. Theoretically, mutating the two phosphorylation sites at the C-terminus will not cause a change in the localization of the entire protein. To verify that TaSOS1-DAPA is still localized on the plasma membrane, the localization of TaSOS1 was used as a control in the experiment. TaSOS1-WT and TaSOS1-DAPA were subcloned from the yeast expression vector pYPGE15 into the modified plant expression vector pCAMBIA1300-eGFP respectively and expressed as a fusion with GFP. By using the method of Agrobacterium-mediated transient transformation of tobacco, the plant expression vectors were transformed into Agrobacterium tumefaciens GV3101 respectively and injected into the epidermal cells of young tobacco leaves, and a confocal fluorescence microscope was used to observe whether the subcellular localization shown by the fluorescence of TaSOS1-GFP and TaSOS1-DAPA-GFP was the same.

[0069] The results showed that after transient expression of TaSOS1-DAPA and TaSOS1 in tobacco, GFP fluorescence signals could be detected at the leaf injection site, and the fluorescence signals of the gene fusion GFP expression were located on the plasma membrane in the same way.

[0070] 5 Sequence analysis of TaSOS1-DAPA

[0071] The TaSOS1 gene of wheat encodes a polypeptide of 1,142 amino acids. The hydrophobic N-terminus contains 12 transmembrane regions and is composed of a polypeptide of 428 amino acids. In the early stage of the experiment, a series of mutants with different lengths of deletion at the C-terminus of the TaSOS1 gene were cloned. In the yeast expression system, a super-resistant mutant Δ974 of TaSOS1 was found, that is, TaSOS1 lacks the amino acid residues at positions 975-1,142. The AXT3K yeast with the single-transferred TaSOS1-Δ974 mutant gene has extremely strong salt tolerance activity and can grow under salt stress containing 1 M NaCl, while TaSOS1-WT can only reach a tolerance of 400 mM NaCl after being activated by TaCIPK24 or TaCIPK24 / TaCBL4. This indicates that the 974-amino acid polypeptide chain in the gene sequence of TaSOS1 includes the complete protein functional region and can independently perform the function of the Na + / H + antiporter. Since the SOS1 protein in the super-resistant state does not exist in plants, the expression and regulation of genes will form their own homeostasis and balance during the evolution of various plants. Therefore, based on the experimental results of previous studies, we believe that the region of TaSOS1 that can be regulated is at least the C-terminal region starting from 974, as Figure 8 shown.

[0072] 6. Yeast functional verification of TaSOS1-C-terminal truncated mutants

[0073] To find the active region where TaCIPK24 / TaCBL4 regulates TaSOS1, we co-transformed a series of TaSOS1-C-terminal truncated mutants constructed in the early stage of the laboratory with the p414-TaCIPK24-TaCBL4 plasmid into AXT3K yeast. The gene sequence of the truncated mutant TaSOS1 is a deletion mutant with a gradually decreasing C-terminal length by about 20 amino acids upstream. After co-transforming the salt-sensitive mutant yeast AXT3K with the complex protein, since the transferred TaSOS1 is a deletion mutant protein of different genotypes and can complement the salt tolerance regulatory regions to different degrees, differences in yeast salt tolerance function will be shown through the regulation of CIPK24-CBL4, thereby inferring whether the deleted C-terminal region is the key regulatory region. The results of yeast functional verification are as Figure 9 shown.

[0074] As can be seen from the figure, the salt tolerance phenotype of yeast with the single-transferred truncated TaSOS1 mutant is basically consistent with the previous results. The yeast phenotype with the full-length TaSOS1 single-transferred can tolerate a concentration of 100 mM NaCl, and the truncated mutant Δ1122 lacking the DAPA regulatory region also has a tolerance of 100 mM NaCl. However, the salt tolerance concentration of yeast with each mutant single-transferred after further truncation upstream is 400 mM NaCl, and when co-transferred with TaCIPK24 / TaCBL4, there is no significant difference in the salt tolerance intensity after complementing yeast, which is 400 mM NaCl. To verify that the expression vector construction of the truncated TaSOS1 gene is correct, the above vectors were sent to Sangon for base sequence determination. Figure 10 To detect the electrophoresis results of plasmid PCR.

[0075] The above experimental results show that it is ineffective to screen the regulatory region at the C-terminus of TaSOS1 by continuous truncation. We speculate that this is probably because during the process of gradually truncating the TaSOS1 gene, the terminal sequence of the gene is severely lost, resulting in the inability of its C-terminus to fold normally or the loss of the most important regulatory sequence at the very end, leading to abnormal SOS1 protein sequence or structural variation in the C-terminal auto-inhibitory region. Therefore, it is impossible to screen the site where SOS1 is regulated by the TaCIPK24 / TaCBL4 complex protein from the gene sequence of the primary structure by the way of truncating the peptide chain length.

[0076] Functional verification of yeast with the TaSOS1-DAPA deletion mutant

[0077] As can be seen from the yeast phenotypes of the truncated mutants, the deletion of only 20 amino acids is sufficient to affect the protein structure. Therefore, we next tried to retain most of the intact C-terminal sequence of TaSOS1-DAPA. On the basis that the conserved phosphorylation sites 1126S / 1128S have been mutated to alanine DAPA, starting from the SOS1 super-resistant mutant Δ974 site, a series of mutants with sequential deletions at the C-terminus were constructed, with a 20-amino acid gap every 20 amino acids towards the C-terminus. Each deletion mutant has the same remaining sequence except for the sequential deletion of non-repeating 20 amino acids.

[0078] As Figure 11 shown, there are 148 amino acid residues at the C-terminus of the TaSOS1 protein from site 974 to 1122. Deletion mutants of δ974-997, δ997-1022, δ1022-1042, δ1042-1062, δ1062-1082, δ1082-1102, and δ1102-1122 were constructed by deleting about 20 amino acids each time. Among them, "δ number" represents that this amino acid region of the TaSOS1 mutant is deleted. Figure 11Different amino acid peptide chains missing each time are marked with different background colors. Since there are only 40 amino acids left from the end for the amino acids downstream of 1102, there is no need to construct a vector with deletion mutations anymore, and in Figure 9 the results, the salt tolerance phenotype of yeast has been verified by truncation. Yeast expression vectors of the above six deletion mutants were constructed by overlapping PCR. Subcloning was performed using the plasmid of TaSOS1 as a template and ligated to the vector pYPGE15, and then sent to Sangon for sequencing. The alignment results are as Figure 12 shown in A. After the sequence was correct, it was transformed into AXT3K, co-expressed with TaCIPK24 / TaCBL4, and yeast function verification was carried out, as Figure 12 shown in B-C.

[0079] From Figure 12 B, it can be seen that when the amino acid sequence between 974-997 of TaSOS1-DAPA is missing, the salt tolerance of yeast with single transfection of δ974-997-DAPA is similar to that of single transfection of TaSOS1 and can grow to a NaCl concentration of 100 mM. The yeast co-transfected with TaCIPK24 cannot grow under salt stress of 200 mM NaCl, which is in line with the salt tolerance phenotype of yeast after the DSPS mutation to DAPA. After co-transfection with TaCIPK24 / TaCBL4, the salt tolerance of yeast has not been improved either and cannot grow under salt stress of 200 mM NaCl. This shows that after the amino acid sequence of 974-997 is missing, δ974-997-DAPA has almost lost all the action sites that can be activated by TaCIPK24 and TaCIPK24 / TaCBL4.

[0080] Yeast function verification of five deletion mutants TaSOS1-DAPA of δ997-1022, δ1022-1042, δ1042-1062, δ1062-1082, δ1082-1102 is as Figure 12 shown in C. It can be seen that the salt tolerance phenotypes of these five mutants are basically the same. When they are co-transfected with TaCIPK24 or TaCIPK24 / TaCBL4, the salt tolerance activity of yeast can reach a concentration of 400 mM NaCl, and this performance is more similar to the state where SOS1 is in an uncontrollable protein state at this time.

[0081] 8 Site-directed mutagenesis of TaSOS1-DAPA

[0082] 8.1 Yeast function verification of TaSOS1-DAPA site-directed mutants

[0083] According to the above prediction, site-directed mutagenesis of all Ser or Thr in the regulatory region needs to be carried out and the phenotypes verified to finally confirm whether it is a regulatory site. The results in 3-12B prove that the regulatory site is locked within the 23-amino acid region from 974 to 997, as Figure 13 shown.

[0084] As shown in the figure above, among these 23 amino acids, there are five serine sites and one threonine site. We first carried out site-directed mutagenesis of a single base for each of these five serines, namely S974A-DAPA, S978A-DAPA, S990A-DAPA, S994A-DAPA, S995A-DAPA, constructed the vector onto pYPGE15, and after correct sequencing, transferred it into AXT3K for yeast function verification. The results are as Figure 14 shown.

[0085] From the experimental results in the figure above, the salt tolerance phenotypes of the TaSOS1-DAPA protein co-expressed with TaCIPK24 or with TaCIPK24 / TaCBL4 after five site-directed mutageneses can be visually compared. Regions 1 and 2 are controls, which are the salt tolerance phenotypes regulated when the TaSOS1 gene sequence has no phosphorylation site deletion, and the salt tolerance phenotype when DSPS is mutated to DAPA, respectively. Regions 3-7 are the phenotypic results of five single Ser mutations on the premise of DAPA mutation. By comparison, it can be found that only after the S978A site-directed mutation in region 4, the salt tolerance ability of the yeast co-transformed with TaCIPK24 / TaCBL4 is significantly reduced and can only grow to a NaCl concentration of 2 mM. While the transgenic yeasts co-expressed with TaCIPK24 / TaCBL4 after the other four groups of site-directed mutageneses can continue to grow normally to a concentration above 400 mM NaCl. These results indicate that S978 is a key binding site. After the S978A mutation, the TaSOS1-S978A-DAPA mutant is in an inhibitory state without an activated target, and TaCIPK24 / TaCBL4 cannot regulate the activity of TaSOS1-S978A-DAPA, indicating that the mutated amino acid is exactly the phosphorylation site for the interaction between the two. The above results also indicate that there is likely no regulatory site in TaSOS1-S978A-DAPA that can be activated by the CIPK24-CBL4 signal.

[0086] 8.2 Salt tolerance analysis of transgenic Arabidopsis thaliana of TaSOS1-S978A-DAPA

[0087] To verify the plant phenotype after serine at the re-mutated 978 site of TaSOS1-DAPA was changed to alanine in plants, a plant expression vector pCAMBIA1300-TaSOS1-S978A-DAPA was then constructed and transformed into Agrobacterium. The mutant sos1-1 was infected, and after screening for homozygosity in the T3 generation, two lines with high expression levels were selected for salt tolerance analysis. The results are as Figure 15 shown.

[0088] In the phenotypic experiment of the transgenic plants in the figure above, the sos1-1 mutant was used as the blank control. Two homozygous lines of DAPA, DAPA-5 and DAPA-9, were compared with two homozygous lines of S978A-DAPA, S978A-DAPA-3 and S978A-DAPA-6, to analyze the growth phenotypes with all three phosphorylation sites deleted under 0 mM, 40 mM, 75 mM, and 100 mM salt treatments. As can be seen from the results, the salt tolerance of the two lines of S978A-DAPA decreased significantly compared with DAPA, and there were also significant differences in fresh weight. The growth phenotypes at a NaCl concentration of 100 mM were close to the salt tolerance level of sos1-1.

[0089] 9 Salt tolerance analysis of TaSOS1-S978A-DSPS restorer line

[0090] 9.1 Functional verification of TaSOS1-S978A-DSPS in yeast

[0091] Through a series of deletions and site-directed mutations of the TaSOS1-DAPA sequence, we obtained that a specific phosphorylation site outside the DSPS site where TaCIPK24-TaCBL4 conservatively binds is S978. To verify the functional activity of the TaSOS1 protein when this site is inactivated alone, the TaSOS1-S978A-DAPA protein that lost its regulatory function was restored to its conserved phosphorylation site DSPS, and the yeast function of the protein TaSOS1-S978A-DSPS with only the site-directed mutation S978A in TaSOS1 was verified, denoted as S978A. The results are as Figure 16 shown.

[0092] As shown in the figure above, the salt tolerance phenotype of the transgenic yeast TaSOSl-S978A-DAPA was stable, consistent with the results during the above screening. When co-expressed with the regulatory complex protein, it could only grow to a concentration of 200 mM NaCl. When DAPA was restored to the wild-type DSPS, the yeast with only the mutation S978A almost restored the salt tolerance phenotype of TaSOS1, similar to the salt tolerance phenotypes of the control group TaSOS1 and the transgenic yeast when it was regulated, and could grow at a concentration of 400 mM NaCl.

[0093] 9.2 Salt tolerance analysis of TaSOS1-S978A-DSPS in transgenic plants

[0094] To verify the salt tolerance ability of TaSOS1-S978A-DSPS, a partial restoration line of TaSOS1-S978A-DAPA, in plants, the vector pCAMBIA1300-TaSOS1-S978A was transferred into Agrobacterium and used to infect Arabidopsis thaliana. The T3 generation was screened for homozygosity, and two lines with high expression levels were selected by semi-quantitative screening for salt tolerance analysis. The results are as Figure 17 shown.

[0095] In the experiment in the figure, col-0, sos1-1, TaSOS1, and TaSOS1-DAPA were used as controls, and NaCl treatment gradients of 0 mM, 40 mM, 75 mM, and 100 mM were set. The focus was on comparing how much the salt tolerance ability of plants would be affected when DAPA was restored to DSPS and only one serine site, S978, was mutated to Ala. From the results, it was seen that when growing at a NaCl concentration of 75 mM, both DAPA and S978A could still survive normally, and the difference was not significant. When the concentration reached 100 mM, the growth of the seedlings was significantly damaged by salt stress. The phenotype of DAPA was closer to that of sos1-1, and the growth of S978A was slightly better than that of DAPA.

[0096] The above salt tolerance phenotypes in plants and yeast indicate that in the wheat SOS1 salt tolerance regulatory pathway, the conserved phosphorylation site DSPS still plays a dominant role. And since S978A single mutation can also be activated by TaCIPK24 / TaCBL4, it shows that TaCIPK24 alone or the TaCIPK24 / TaCBL4 protein complex can recognize the site DSPS and activate the salt tolerance ability of TaSOS1, while the S978 specific phosphorylation site can only be regulated by the TaCIPK24 / TaCBL4 protein complex.

[0097] 10 Determination of ion content in transgenic yeast of TaSOS1 mutants

[0098] The TaSOS1 gene and three genotypes of TaSOS1 with site-directed mutations in phosphorylation sites, TaSOS1-DAPA, S978A-DAPA, and S978A-DSPS, were respectively transformed into yeast AXT3K and co-transformed with two regulatable protein combinations. The salt tolerance functions of these four groups of transgenic yeast were compared, and the phenotypic results are as Figure 18 shown in A, and the relative positions of each group of yeast spotting plates are marked in Figure B. At the same time, transgenic yeast in the blank control group and the 30 mM NaCl treatment group were selected for the determination of ion content in transgenic yeast cells, and the quantitative analysis was used to compare the K in yeast cells when TaSOS1 mutants were transfected alone and co-expressed with regulatory proteins +Content and Na + levels, and the results are as shown.

[0099] From Figure 19 the quantitative analysis data, it can be seen that the K of yeast expressing single TaSOS1 under 30 mM NaCl treatment + / Na + showed little difference. When co-expressed with TaCIPK24, S978A had a higher K + / Na + . When co-expressed with TaCIPK24-TaCBL4, the K of TaSOS1-DAPA and TaSOS1-S978A + / Na + were both relatively high. And the higher the ratio of K + / Na + , the stronger the salt stress resistance of the transgenic yeast. The above experimental results also confirmed our conclusion.

[0100] 11 Salt tolerance analysis of TaSOS1 phosphorylation sites in transgenic Arabidopsis

[0101] Through step-by-step screening and verification in the yeast expression system and plant expression system in the above experiments, it was determined that there are three phosphorylated sites in TaSOS1 that can be activated. Under the signal pathway of the TaCIPK24 and TaCBL4 complex proteins, the third regulatory site S978 of TaSOS1 is only activated by the TaCIPK24 / TaCBL4 protein complex, while the conserved phosphorylated site DAPA can not only be regulated by the TaCIPK24 protein kinase alone, but also be activated by the TaCBL4 / TaCIPK24 complex under the mediation of TaCBL4. In order to compare the salt tolerance phenotypes of the three phosphorylated sites of TaSOS1 in transgenic plants, the homozygous lines of complementary TaSOS1 and TaSOS1 mutants TaSOS1-DAPA, TaSOS1-S978A-DAPA, and TaSOS1-S978A-DSPS in transgenic Arabidopsis were quantitatively analyzed and compared. At the same time, the wild type col-0 and mutants were used as controls, and the results are as Figure 20 shown.

[0102] Figure 20 The phenotypes of four transgenic Arabidopsis, wild type and mutants after salt treatment in nutrient soil for more than 3 weeks are shown. It can be seen that the salt tolerance phenotypes of the adult seedlings treated in the soil are basically the same as those of the salt stress phenotypes at the seedling stage. The results of WB showed the protein expression levels of TaSOS1 and its mutants in transgenic Arabidopsis. The data analyzed the fresh weight of the above-ground leaves and K of the adult seedlings of each transgenic Arabidopsis after 150 mM NaCl treatment + / Na +, and K in the underground part + / Na + . Consistently, as the phosphorylation sites of TaSOS1 were mutated one by one, the resistance of plants to salt stress became weaker and weaker. In addition, we also measured the Na + flux of five groups of plants at the seedling stage: sos1-1, TaSOS1, TaSOS1-DAPA, S978A-DAPA, and S978A-DSPS.

[0103] As known from Figure 21 , sos1-1 became extremely sensitive to salt stress due to the lack of SOS1 gene expression and had the lowest Na + flux. In the sos1-1 mutant, due to the complementation of the exogenous TaSOS1 gene, the TaSOS1 recovery line obtained an ideal salt tolerance level and had the highest Na + flux. The Na + flux in the TaSOS1 mutants with sequential phosphorylation site deletions was in the range of 400 - 600 pmol / cm -2 s -1 , and the difference was not significant. However, it could still be seen that S978A-DAPA had the lowest Na + flux among these three mutants, approaching the flux level of sos1-1. The Na + flux in the two incomplete mutant experimental groups of S978A and DAPA was not much different, and only S978A-DSPS had a more obvious advantage at the beginning of the test. At the same time, it could also be seen that no matter which regulatory site was mutated, the salt tolerance of TaSOS1 would be significantly affected. The above experimental results were consistent with the phenotypic results.

[0104] 12 Conclusions

[0105] The salt stress response mechanism of plants is an abiotic stress signaling pathway that has been studied earlier, and the SOS salt tolerance pathway is a classic regulatory pathway in plants. During our efforts in crop salt tolerance and stress resistance breeding, we have made some new discoveries regarding the regulation of the wheat SOS salt tolerance pathway. Figure 22 This paper compared the differences between the wheat SOS pathway and the Arabidopsis SOS pathway. Generally speaking, there are the following points.

[0106] First, under normal conditions, the SOS1 protein in plants is in a self-inhibited state, and the self-inhibited patterns are similar. In both cases, its C-terminal folds on its own to avoid the exposure of regulatory sites and keep the protein in a silent state. When plants are transferred to a salt-stressed environment for treatment, as the gradually accumulated salt stress pressure reaches the threshold, the Ca 2+When the signal accumulates and is recognized and analyzed by CBL4, the plant SOS salt tolerance pathway is triggered. In Arabidopsis, CBL4 needs to be captured by CIPK24 first and recruited to the plasma membrane to activate the silent SOS1, open its folded area, change its spatial conformation to release the regulatory sites and regulatory regions, and thus start it to perform reverse transport of Na + / H + Ion's mission.

[0107] However, in wheat, we discovered that, first, CBL4 participates in the regulation of another key specific regulatory site, S978, located upstream of the conserved phosphorylation site (DAPA), located in addition to the conserved phosphorylation site (DAPA). Our functional phenotyping of truncation mutants in yeast revealed that when the truncation length was Δ983, co-transfection with CIPK24 resulted in a salt tolerance phenotype exceeding 400 mM NaCl, while transfection with the Δ983 protein alone only reached 100 mM NaCl. This suggests that when TaSOS1 is truncated to 983, the terminal S978 regulatory site likely replaces the conserved phosphorylation site DAPA within the full SOS1 protein sequence for activation by CIPK24, ensuring continued protein function. In this respect, wheat may possess a potential alternative regulatory pathway for salt tolerance compared to Arabidopsis.

[0108] Second, the way wheat SOS1 silenced state is activated may be more efficient than that of Arabidopsis. It has been reported that the only regulatory site that can be phosphorylated in Arabidopsis is DAPA, and it must be activated by the CIPK24-CBL4 protein complex to be regulated. In other words, the phosphorylation site involved in CBL4 is likely to be the key to unlocking autoinhibition. However, this study found that the conserved phosphorylation site of wheat SOS1 does not require the mediation of CBL4, and Ser978 may be the key to unlocking the autoinhibition of TaSOS1 protein. Figure 21 The schematic diagram also shows that activation is more efficient near the folded region than at the C-terminus. Furthermore, the unfolded TaSOS1 reveals two phosphorylation sites for regulation. TaCIPK24 alone can bind to DSPS, while the protein complex is responsible for activating S978 and rapidly releasing the active region. This suggests that the wheat SOS1 gene has a more mature phosphorylation regulatory sequence.

[0109] Thirdly, according to the research reports on the SOS pathway in Arabidopsis thaliana, it may not be accurate that the activation of SOS1 activity requires the prior formation of the CIPK24-CBL4 protein complex to activate the function of SOS1. In Arabidopsis thaliana, the activation of silent SOS1 does require the mediation of CBL4, which forms CIPK24-CBL4 with a protein kinase to complete the process collaboratively. However, the phosphorylation site for activating wheat SOS1 no longer necessarily requires the participation of CBL4. Finally, we also compared the yeast salt tolerance phenotypes of Arabidopsis thaliana SOS1 and wheat SOS1. As Figure 23 shown, it can be seen that the functional verification of the SOS1 genes of wheat and Arabidopsis thaliana in the same yeast expression system also indicates that the TaSOS1 protein has a more obvious salt tolerance advantage. In summary, wheat has a more refined SOS salt tolerance regulatory network during the evolution process, which also conforms to the results of biological evolution.

[0110] As described above, the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be thought of without creative efforts should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.

Claims

1. TaCBL4-mediated TaSOS1 phosphorylation regulatory sites in wheat, characterized in that, The TaSOS1 phosphorylation regulatory site is S978A-DAPA.

2. The regulatory site according to claim 1, characterized in that, In the TaSOS1 phosphorylation regulatory site S978A-DAPA, the serine at position 978 of TaSOS1-DAPA is mutated again to alanine.

3. Application of the regulatory site described in claim 1 or 2 as a molecular marker for detecting wheat salt tolerance.

4. Application of the regulatory site described in claim 1 or 2 in wheat salt tolerance improvement and assisted breeding.

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