Receptor-like protein kinase STRK2 protein, gene and application regulating rice salt tolerance

Abstract

The present invention belongs to the field of plant genetic engineering, and discloses a coding gene capable of regulating rice receptor cytoplasmic kinase STRK2, and its application in regulating plant salt stress response and salt tolerance breeding. STRK2 The study found that STRK2 In response to salt stress signals, STRK2 Overexpression rice lines are more sensitive to salt stress; constructed using CRISPR / Cas9 gene editing system STRK2 Knockout strains strk2 It exhibits strong salt tolerance to salt stress. STRK2 The gene encodes a receptor-like kinase STRK2 protein, which can negatively regulate ascorbic acid synthesis by phosphorylating its interacting protein LRRK1, thereby affecting the accumulation of reactive oxygen species and ultimately negatively regulating the salt tolerance of rice. STRK2 Genes and their knockout mutants have important application value in the breeding of salt-tolerant rice varieties and the improvement of rice agronomic traits.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and specifically relates to a receptor-like protein kinase STRK2 protein and gene for regulating rice salt tolerance and its application in salt-tolerant rice cultivation. Background Art

[0002] Rice ( Oryza sativa L.) is the staple food of more than half of the world's population, and China is also the world's largest rice producer. In recent years, soil salinization has become a major factor threatening and restricting rice production. my country currently has nearly 100 million hm2 of rice. 2 The inland saline-alkali area is about 2.4 million hm2 2 Coastal tidal flats, if these salinized lands can be utilized, they will be a potential reserve of available land. Developing and creating new salt-tolerant rice varieties is the most effective solution to agricultural problems such as increasing rice yields and effectively utilizing saline-alkali land. The most important basis for judging soil salinization is whether it contains high concentrations of salt, among which NaCl, which is the most widely distributed and most soluble, is the most common. The salt accumulated in saline-alkali soils mainly comes from the accumulation of NaCl in seawater and irrigation water. Rice is a moderately salt-sensitive crop. In production, varieties that can grow in land with a salinity of more than 0.3% are defined as rice varieties that can tolerate salt stress and alkali stress. Therefore, in the application of transforming saline-alkali land as cultivated land, rice is the preferred food crop for the improvement of coastal tidal flats and saline-alkali land. Studying the salt stress tolerance mechanism of rice, screening, cultivating and promoting new salt-alkali tolerant varieties are important measures to ensure the area of ​​arable land and maintain food security. To enhance rice's tolerance to salt stress through genetic improvement, it is necessary to discover more potential salt-tolerant genes, analyze the specific mechanisms of action from the levels of gene function and physiological and biochemical mechanisms, and enrich the candidate genes for genetic improvement of rice salt tolerance.

[0003] A high salt concentration growth environment will inhibit the normal growth and development of plants, and in severe cases even cause plant death, leading to ion poisoning (mainly Na + ) and hyperosmotic stress, accompanied by some secondary stresses such as oxidative damage. At the same time, the effects of salt stress on the normal physiological activities and metabolism of plants are complex and multidimensional. From a physiological perspective, the effects mainly include changes in ion balance, osmotic balance, enzyme activity, redox balance, and corresponding signal transduction and gene expression. In addition, high concentrations of Na + It also has a certain degree of impact on cell membrane integrity and protein function. Plants will also offset the negative effects by adjusting their own mechanisms, improving cell adaptability and enhancing tolerance to salt stress.

[0004] High salt stress can cause severe oxidative stress. Under salt stress conditions, the reduction in photosynthetic rate can activate the Mehler reaction and produce a large amount of reactive oxygen species (ROS). Among them, hydrogen peroxide (H2O2) is a very important ROS molecule, which can act as a signal molecule to participate in the growth and development regulation of plants and respond to biotic and abiotic stresses (Zhou Genyou, Zhai Caijiao, Deng Xianliang, Zhang Jiao, Zhang Zhenliang, Dai Qigen, Cui Shiyou. Effects of salt stress on rice yield, photosynthetic characteristics and quality. Chinese Journal of Rice Science, 2018, 32(2), 146–154). However, excessive accumulation of ROS will oxidize and destroy cell components. If not removed in time, it will cause serious harm to plants. Therefore, timely removal of excessive ROS in the body can significantly improve the tolerance of plants to salt-alkali and other stresses. In order to eliminate the toxicity of ROS, plants have evolved effective enzyme and non-enzyme scavenging mechanisms to remove ROS (Xu J., Duan X., Yang J., Beeching JR, Zhang P. Enhanced reactive oxygen species scavenging by overproduction of superoxidedismutase and catalase delays postharvest physiological deterioration ofcassava storage roots. Plant Physiology. 2013, 161, 1517–1528). For example, catalase (CAT) and ascorbic acid (AsA) are important intracellular ROS scavenging enzymes and non-enzyme scavenging antioxidant molecules, respectively. These enzymes and non-enzymes work synergistically to keep ROS in a steady state and maintain the normal level of ROS required in cell signal transduction (Liu Cong, Dong La'ai, Lin Jianzhong, Liu Xuanming. Research progress on reactive oxygen metabolism and regulatory mechanism in plants under adverse stress. Life Science Research, 2019, 23, 253–258).

[0005] The internal changes of plants in response to external stimuli depend on a complex signaling system. Receptor-like protein kinases (RLKs) on the cell surface initiate the cell signaling pathway mechanism by sensing changes in external signals and transmitting signals. A large number of research results show that RLKs and receptor-like cytoplasmic kinases (RLCKs) have synergistic effects in the same signaling pathway, and this synergistic pattern is common in plants. The structure of a protein determines its function. As a type of protein in the receptor-like protein kinase family that lacks an extracellular ligand-binding domain, RLCK cannot complete the biological function of receiving external signals and participating in cell transmembrane signal transduction alone. Therefore, it needs to interact with an RLK with an extracellular domain to transmit signals through a cascade reaction of protein phosphorylation and activate downstream effector molecules (Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1and modulates brassinosteroid signaling. Cell, 2002, 110, 213–22). RLK and RLCK proteins interact with each other and participate in signal transduction pathways that are widely involved in the regulation of plant growth and development, immunity, reproduction, hormone signaling, stress response and other life processes.

[23] For example, the receptor-like cytoplasmic tyrosine kinase STRK1 phosphorylates catalase CatC under salt stress conditions to increase CatC activity and thus improve rice salt tolerance (Zhou Y, Liu C, Tang D, et al. The receptor-like cytoplasmic kinase STRK1 phosphorylates and activates CatC, thereby regulating H2O2 homeostasis and improving salt tolerance in rice. The Plant cell, 2018, 30(5), 1100-1118). Therefore, discovering the RLKs and RLCKs involved in salt stress response and analyzing the molecular mechanisms of salt stress signal transduction in which they participate will help enrich rice salt-tolerant gene resources and analyze the regulatory network of salt stress response, while providing high-quality gene resources and theoretical guidance for the breeding of salt-tolerant rice varieties. Summary of the invention

[0006] The present invention provides a gene encoding a receptor-like protein kinase capable of regulating rice salt tolerance ( Salt tolerance receptor-like kinase 2 , STRK2 ), the protein encoded by this gene regulates the salt stress response of rice through phosphorylation. Under NaCl treatment conditions, STRK2 The seedling length and root length of the overexpression strain during the seed germination period were lower than those of the wild type, and the survival rate of the seedling stage was also lower than that of the wild type, indicating that STRK2 Overexpression lines are sensitive to salt stress. Construction using CRISPR / Cas9 gene editing system STRK2 Knockout strains, transformed by Agrobacterium-mediated transformation, and sequenced to obtain homozygous knockout strains strk2 Under NaCl treatment, the knockout strain strk2 The seedling length and root length at the seed germination stage were significantly higher than those of the wild type, and the survival rate at the seedling stage was also significantly higher than that of the wild type, indicating that the knockout STRK2 It can significantly improve the salt tolerance of rice and has important application value in the breeding of salt-tolerant rice varieties. Subsequently, through mass spectrometry differential expression protein identification, a STRK2 interacting protein LRRK1 was screened, and it was found that STRK2 can phosphorylate LRRK1 and achieve response to salt stress by regulating the phosphorylation of LRRK1 under salt stress conditions. The present invention provides a new gene that negatively regulates rice salt tolerance STRK2 , which provides high-quality candidate genes for breeding salt-tolerant rice varieties and has broad application prospects in the agricultural field.

[0007] Rice salt stress response regulatory gene provided by the present invention STRK2 From rice ( Oryza sativa L.), is a protein having one of the following amino acid residue sequences:

[0008] 1) SEQ ID No: 3 in the sequence listing;

[0009] 2) A protein having the function of regulating the negative regulation of rice response to salt stress by replacing, deleting or adding one to ten amino acid residues in the amino acid residue sequence of SEQ ID No: 3 in the sequence list. Sequence 3 in the sequence list consists of 606 amino acid residues.

[0010] At the same time, the present invention also provides a coding gene of STRK2, a cDNA gene of STRK2, which may have one of the following nucleotide sequences:

[0011] 1) The DNA sequence of SEQ ID No: 2 in the sequence listing;

[0012] 2) a polynucleotide encoding the protein sequence of SEQ ID No: 3 in the sequence list;

[0013] 3) A nucleotide sequence that can hybridize with the DNA sequence defined by SEQ ID No: 2 in the sequence listing under highly stringent conditions;

[0014] 4) A DNA sequence that has more than 70% homology with the DNA sequence defined in SEQ ID No: 2 in the sequence listing and encodes a protein with the same function.

[0015] Sequence 2 in the sequence list consists of 121 bases, and its open reading frame (ORF) is from base 1 to base 1821 at the 5' end.

[0016] The genomic gene of STRK2 may have one of the following nucleotide sequences:

[0017] 1) The DNA sequence of SEQ ID No: 1 in the sequence listing;

[0018] 2) a polynucleotide encoding the protein sequence of SEQ ID No: 3 in the sequence list;

[0019] 3) A nucleotide sequence that can hybridize with the DNA sequence defined by SEQ ID No: 1 in the sequence listing under highly stringent conditions;

[0020] 4) A DNA sequence that has more than 70% homology with the DNA sequence defined in SEQ ID No: 1 in the sequence listing and encodes a protein with the same function.

[0021] Sequence 1 in the sequence listing is STRK2 The genomic sequence of contains 3729 bases. The gene contains 2 exons (from the 5' end of sequence 1: 872-1695, 2222-3219) and 3 introns (from the 5' end of sequence 1: 1-871, 1695-2221, 3220-3729).

[0022] The high stringency condition may be hybridization and membrane washing in a solution of 0.1×SSPE (or 0.1×SS), 0.1×SDS at 65°C.

[0023] contain STRK2 The expression vector, transgenic cell line and host bacteria all belong to the protection scope of the present invention. Specifically, STRK2 The gene expression is constitutive.

[0024] The present invention also provides a method for improving the salt tolerance of rice: using gene editing such as CRISPR / Cas9 gene editing system to knock out STRK2 , obtaining rice knockout lines with significantly improved salt tolerance strk1 Specifically, the CRISPR / Cas9 -STRK2 The vector is introduced into rice tissues or cells through Agrobacterium-mediated method to obtain salt-tolerant rice.

[0025] The rice salt stress negative regulation control gene STRK2By containing STRK2 The plant expression vector is introduced into the explant; the starting vector used to construct the plant expression vector can be any binary Agrobacterium vector or a vector that can be used for plant gene gun bombardment, such as pCAMBIA1301-UbiN (GenBank No.: AF234296), pCAMBIA2301, pCAMBIA1300 or other derived plant expression vectors.

[0026] use STRK2 When constructing plant expression vector, any enhancing, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters. In addition, when using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive, and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0027] In order to facilitate the identification and screening of transgenic plants or transgenic plant cells, the vectors used can be processed, such as adding selective markers (GUS gene, GFP, YFP, As-Red and luciferase gene, etc.) or antibiotic marker genes with resistance (hygromycin, kanamycin, ampicillin, bleomycin, etc.). For the safety of transgenic plant release, it is also possible not to carry any marker genes when constructing plant expression vectors, and to perform specific PCR molecular marker screening at the seedling stage.

[0028] Containing the present invention STRK2 The plant expression vector can be transformed into rice cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated or gene gun, and the transformed rice tissues can be cultivated into plants. The transformed plant host can be either monocotyledonous plants or dicotyledonous plants, such as rice, wheat, corn, cucumber, tomato, poplar, lawn grass, alfalfa, etc.

[0029] The present invention has the following beneficial effects:

[0030] Overexpression in rice STRK2 gene, salt-sensitive phenotypes appeared in both the seedling and seedling stages, and the survival rate was lower than that of the wild type. STRK1 Afterwards, the knockout mutant strain strk1The salt tolerance of rice was significantly improved, and the survival rate and growth were significantly higher than those of the wild type. Further studies found that under salt stress conditions, after STRK2 senses the signal, it participates in the transduction of salt stress signals by phosphorylating its interacting protein LRRK1, and ultimately negatively regulates the salt tolerance of rice. In short, STRK2 It negatively regulates the salt tolerance of rice, and its knockout mutant has good application prospects in the breeding of salt-tolerant rice.

[0031] The present invention STRK2 The discovery of the mechanism of action and the analysis of its action provide molecular markers and theoretical guidance for the breeding of salt-tolerant rice varieties, especially STRK2 The creation of knockout lines is an effective way to increase rice yield in saline-alkali soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 STRK2 Construction of knockout strains and overexpression strains. A. The structure of the pYLCRISPR / Cas9 binary vector. B. STRK2 Primer design for CRISPR / Cas9 knockout target of gene. C. STRK2 Sequencing of the edited region of the CRISPR / Cas9 knockout strain of the gene. D. STRK2 Sequence alignment of gene knockout strains and wild type. E. STRK2 Western blot detection of overexpression lines. Anti-Flag antibody was used to immunodetect the fusion protein Flag-STRK2.

[0033] Figure 2 Analysis of STRK2 protein structure and subcellular localization. A. Structural domain prediction analysis of STRK2. B. 3D structure prediction of STRK2. C. STRK2 Tissue-specific expression analysis of STRK2. D. STRK2 is localized on the cell membrane.

[0034] Figure 3 STRK2 Identification of salt stress phenotypes of overexpression and knockout strains. STRK2 Phenotypic identification of overexpression and knockout strains at the seedling stage. B. Statistics of survival rate after 5 days of recovery from salt stress. 15-day-old seedlings were treated with 140 mM NaCl for 15 days and then recovered in normal hydroponic culture for 5 days. C. Under salt stress conditions STRK2 Phenotypic identification of early seedlings of overexpression and knockout lines. D. Seedling length statistics of early seedlings after salt stress treatment. The effects of salt stress on seedling length were determined under normal conditions and 250 mM NaCl conditions for newly germinated seeds. Values ​​represent mean ± standard deviation (t test, * P ≤ 0.05; ** P ≤ 0.01).

[0035] Figure 4 Verification of the interaction between LRRK1 and STRK2. A. Yeast two-hybrid analysis of the interaction between LRRK1 and STRK2. B. Yeast two-hybrid segmented analysis of the interaction between LRRK1 and STRK2. LRRK1 is divided into 5 segments, LRRK1a is the N-terminus, LRRK1e is the C-terminus, and LRRK1b-d is the kinase domain, which is divided into 3 segments. C. Analysis of the interaction between LRRK1 and STRK2 by bimolecular fluorescence complementation.

[0036] Figure 5 STRK2 phosphorylates LRRK1. A. In vitro phosphorylation detection of LRRK1 kinase inactivated protein. B. In vitro phosphorylation detection of STRK2 kinase inactivated protein. C. Phosphorylation antibody detection of LRRK1 and STRK2 in vitro phosphorylation.

[0037] Figure 6 After NaCl treatment, the level of STRK2 phosphorylated LRRK1 increased. Among them, NaCl treatment enhanced the phosphorylation level of LRRK1 in vivo. Flag-LRRK1 is treated with 100 mM NaCl for different time periods. LRRK1 Fusion proteins obtained by IP in overexpression lines.

[0038] Figure 7 Salt stress LRRK1 Effects of overexpression and knockout strains on ROS and AsA content. A. Reactive oxygen fluorescence probe was used to detect the accumulation of ROS in roots. B. Salt stress and normal conditions in wild type, LRRK1 Determination of the contents of H2O2 (B), superoxide anion (C) and AsA (D) in the overexpression and knockout strains. DETAILED DESCRIPTION

[0039] The present invention is further described below with reference to specific examples. The experimental methods mentioned in the following examples are conventional methods unless otherwise specified.

[0040] 1. STRK2 Structural analysis and construction of overexpression and knockout strains

[0041] (1) STRK2 Structural analysis of genes

[0042] Download from the Rice Database (http: / / rice.plantbiology.msu.edu) STRK2 (LOC_Os11g14420), and compared with the japonica rice variety Nipponbare ( Oryza sativa L. japonica cv. Nipponbare )of STRK2cDNA and genomic DNA sequences, and found STRK2 There are 2 exons (from the 5' end of SEQ ID No: 1: 872-1694, 2222-3219), 3 introns (from the 5' end of SEQ ID No: 1: 1-871, 1695-2221, 3220-3729); the full length of its genome is 3729 bp (SEQ ID No: 1), the full length of its cDNA is 1821 bp (SEQ ID No: 2), the open reading frame is SEQ ID No: 2 from the 1st to 1821st position from the 5' end, with a total of 1821 bases; the length of its encoded protein is 607 amino acids. The primer design software Primer Premier 5 was used to design the amplification STRK2 The full-length specific forward primer STRK2-F: 5'-ATGTCTGTCAAGTGTTCC-3' and reverse primer STRK2-R: 5'-CTAGTTTGCTACAATAA-3' were used as templates to clone the full-length STRK2 Gene.

[0043] (2) STRK2 Construction of overexpression lines

[0044] Through homologous recombination STRK2 The gene was cloned into the GATEWAY entry vector PGWC and sequenced. After sequencing was correct, it was recombined into the modified pCAMBIA1301GW vector through LR reaction. The modified pCAMBIA1301GW vector contains the hygromycin resistance selection gene HPT and red fluorescent protein selection marker gene AsRed Through Agrobacterium-mediated transformation, the ubiquitin gene of maize Ubiquitin Promoter (pUbi) drives the target gene STRK2 Overexpression or constitutive expression, and the N-terminus of STRK2 is fused with the tag protein Flag. Then, the japonica rice variety is infected by Agrobacterium-mediated Kitaake The callus tissue was screened, differentiated, rooted, and hardened to obtain regenerated plants, and positive overexpression lines were obtained after identification. STRK2-4 and STRK2-8 ( Figure 1 Middle E).

[0045] (3) Construction through CRISPR / Cas9 technology STRK2 Knockout strains

[0046] Build STRK2The knockout strain CRISPR / Cas9 vector is divided into two steps, namely the construction of the sgRNA expression cassette and cloning the sgRNA expression cassette into the pYLCRISPR / Cas9 vector by cutting and ligating. After the preparation of the target double-stranded adapter, the target adapter is ligated and amplified, and the sgRNA expression cassette is constructed after two rounds of PCR reaction, and then the sgRNA expression cassette is cloned into the pYLCRISPR / Cas9 vector by cutting and ligating. See the primers for designing the CRISPR / Cas9 vector backbone and editing site for details. Figure 1 B. Construction using pYLCRISPR / Cas9 binary vector STRK2 CRISPR / cas9 knockout strains. STRK2 The CRISPR / Cas9 editing target was designed in the 5′ region of the open reading frame (ORF) and the pYLCRISPR / Cas9 binary vector ( Figure 1 A) Build STRK2 CRISPR / Cas9 knockout strains. STRK2 Design of CRISPR-Cas9 editing target site in the 5′ region of ORF ( Figure 1 In the middle B, the target primers of CRISPR / Cas9 system were designed on the CRISPR-GE website. According to the target position, GC content, potential off-target position and valuation, the target primers with low GC base content (off-target efficiency is low between 45%-70%) and meeting the requirements were selected. The successfully constructed expression cassette was connected to the pYLCRISPR / Cas9 binary vector and transferred into Agrobacterium EHA105, and transformed into rice by Agrobacterium-mediated infection Kitaake Callus tissue was used to obtain T0 seedlings of the knockout strains. DNA from leaves of each knockout strain was extracted, and a region containing the editing site was amplified by PCR, and the sequence was compared with that of the wild type to identify the STRK2 Editing status of target sites. Two types of editing were identified in the T1 generation. STRK2 Knockout strains were named strk2-25 and strk2-29 ( Figure 1 Medium CD).

[0047] 2. Analysis of STRK2 protein structure and subcellular localization

[0048] Structural analysis shows that STRK2 is a typical leucine repeat receptor protein kinase (LRR-RLK), which has an extracellular ligand binding domain, a transmembrane domain and an intracellular kinase domain ( Figure 2 According to the SWISS-MODEL database, the three-dimensional structure of STRK2 is shown in Figure 2. Figure 2B. STRK2 is expressed in rice organs at all stages, and its expression level is relatively high in young leaves, roots and seeds ( Figure 2 C). STRK2 The full-length CDS sequence was constructed into the 1300-YFP subcellular localization vector, and the green fluorescent protein (GFP)-STRK2 fusion protein was expressed in rice protoplasts. Laser confocal microscopy revealed that GFP-STRK2 was localized on the cell membrane of the protoplasts, and the fluorescence signal was highly overlapped with that of the specific cell membrane localization dye FM4-64 ( Figure 2 (D) indicates that STRK2 is localized in the cell membrane, which is consistent with its protein structure with extracellular domain, transmembrane domain and intracellular domain.

[0049] 3. STRK2 Identification of salt stress phenotypes of overexpression plants and CRISPR / Cas9 knockout lines

[0050] The wild type, STRK2 The 15-day-old seedlings of the overexpression and knockout lines were treated with 140 mM NaCl for 15 days, and it was found that compared with the wild type (WT), STRK2 Overexpression strains STRK2-4 and STRK2-8 The knockout strains showed a phenotype sensitive to salt stress. strk2-25 and strk2-29 showed significant tolerance to salt stress ( Figure 3 A). After 5 days of normal hydroponic culture, the survival rate of the overexpression lines was statistically analyzed. STRK2-4 and STRK2-8 The survival rate of the knockout strain was significantly lower than that of the wild type. strk2-25 and strk2-29 The survival rate of the strain was significantly higher than that of the wild type, showing a higher salt tolerance ( Figure 3 (A and B). The newly germinated rice seeds were treated with 250 mM NaCl for 5 days, and then the phenotypes of the seedlings were observed under salt stress. Under normal growth conditions, there was no significant difference in seedling growth between the wild type and all transgenic materials; under 250 mM NaCl treatment, the overexpression lines showed a significant difference in seedling growth compared with the wild type. STRK2-4 and STRK2-8 The growth of strk2-25 and strk2-29 The seedling length of the plants increased significantly, showing a higher tolerance to salt stress ( Figure 3 Therefore, under salt stress conditions at the seedling stage, STRK2 The overexpression strain showed a salt-sensitive phenotype, while the knockout strain showed a higher salt tolerance.

[0051] 4 .STRK2 interacts with the downstream protein LRRK1

[0052] It is common for RLK-RLCK to interact with each other in plants and transduce stress signals through phosphorylation. We speculate that STRK2, as an RLK, can sense Na under salt stress conditions. + Signals are transmitted through the membrane and then transmitted to related effector protein molecules through downstream RLCK. To further reveal the mechanism of STRK2 regulating salt stress response, we isolated the protein complex of STRK2 from rice by co-immunoprecipitation (Co-IP) and performed mass spectrometry identification. As a result, an RLCK protein LRRK1 was screened and identified from the complex, and the interaction between LRRK1 and STRK2 was verified. Yeast two-hybrid experiments showed that LRRK1 can interact with STRK2 ( Figure 4 Further segmental interaction analysis revealed that the direct interaction between LRRK1 and STRK2 occurred in the LRRK1a segment, i.e., the N-terminus of LRRK1 ( Figure 4 Middle B). Subsequent bimolecular fluorescence complementation (BiFC) experiments also verified that LRRK1 and STRK2 interact with each other on the cell membrane of rice protoplasts ( Figure 4 Middle C).

[0053] 5. Receptor-like kinase STRK2 can phosphorylate downstream LRRK1

[0054] First, in Escherichia coli ( E. coli ) LRRK1 protein (His-LRRK1 and GST-LRRK1) and STRK2 protein (His-STRK2 and GST-STRK2) were expressed and purified in prokaryotes of BL21, and then incubated in an in vitro phosphorylation reaction system. The system after the phosphorylation reaction was subjected to 10% separation gel SDS-PAGE, and then immunoblot analysis was performed with antibodies against His, GST and phosphorylated serine (pSer). The results showed that when STRK2 was co-incubated with LRRK1, the kinase-inactive mutant of LRRK1 (LRRK1KE) lost its self-phosphorylation activity and kinase activity, but STRK2 could still phosphorylate LRRK1, and the phosphorylation level of LRRK1KE was still significantly increased, indicating that STRK2 can phosphorylate LRRK1 ( Figure 5 Middle A). When STRK2 and LRRK1 were co-incubated, the phosphorylation level of LRRK1 was significantly increased; when the kinase-inactivated protein STRK2 (GST-STRK2KE) was phosphorylated with LRRK1, the phosphorylation level of GST-STRK2KE did not increase, indicating that LRRK1 cannot phosphorylate STRK2 ( Figure 5 Middle B). When STRK2 and LRRK1 were incubated separately, the phosphorylation level of LRRK1 was significantly increased by detecting with phosphorylated serine antibody ( Figure 5 (C) Thus, it can be seen that STRK2 can phosphorylate LRRK1 and transmit the signal to downstream effector molecules through phosphorylation.

[0055] 6. Phosphorylation of LRRK1 is induced by salt stress

[0056] Constructed Flag-LRRK1 The overexpression transgenic rice was used to study the effect of salt stress on the phosphorylation level of LRRK1. LRRK1 The seedlings of the overexpression strain were treated with 100 mM NaCl for 0, 20, 40 and 60 min, respectively. After grinding in liquid nitrogen, the protein was extracted with IP buffer, and then the fusion protein Flag-LRRK1 was immunoprecipitated with Flag beads. After SDS-PAGE of Flag-LRRK1 protein, the phosphorylation level was detected with anti-pSer antibody. The results showed that with the extension of NaCl treatment time, the phosphorylation level of LRRK1 gradually increased ( Figure 6 ), indicating that the phosphorylation degree of LRRK1 by STRK2 is related to the NaCl treatment time. We speculate that STRK2 participates in the regulation of salt stress response by phosphorylating LRRK1.

[0057] 7. LRRK1 promotes the accumulation of reactive oxygen species (ROS) and the decrease of ascorbic acid (AsA) content in rice under salt stress

[0058] When plants are subjected to abiotic stress, the outbreak of ROS is a hallmark event. Using the ROS fluorescent probe DCFH-DA, it was found that under normal conditions, the wild type and LRRK1 There was no significant difference in the ROS fluorescence signal intensity among the transgenic lines; after salt stress treatment, LRRK1 The ROS fluorescence signal intensity in the overexpression strain was significantly higher than that in the wild type, while the knockout strain lrrk1-1 The ROS fluorescence signal intensity of Figure 7 A). The most stable H2O2 in ROS was measured and it was found that under normal conditions, wild type and LRRK1 There was no significant difference in H2O2 content in the transgenic lines; after salt stress treatment, LRRK1 The H2O2 content in the overexpression strain was higher than that in the wild type, while that in the knockout strain lrrk1-1 is lower than the wild type ( Figure 7 In addition, superoxide anion (O2· - The results of the determination of the content of ) are consistent with the trend of the determination of the content of H2O2 ( Figure 7AsA is one of the important antioxidants for removing ROS in plants and plays an important role in the removal mechanism of ROS. To explore the effect of LRRK1 on AsA in rice, we LRRK1 The AsA content of transgenic lines under salt stress was determined. The results showed that under normal growth conditions, LRRK1 Overexpression and knockout strains lrrk1-1 The AsA content of the wild type was not significantly different; after salt stress treatment, lrrk1-1 The AsA content of the overexpression strain was significantly higher than that of the wild type, while that of the overexpression strain was significantly lower than that of the wild type ( Figure 7 These results suggest that LRRK1 participates in the salt stress response of rice by negatively regulating the biosynthesis of AsA.

[0059] It can be inferred that STRK2 negatively regulates the synthesis of AsA in rice by phosphorylating LRRK1, thereby ultimately participating in the regulation of rice salt stress response. <110> Hunan University <120> Receptor-like protein kinase STRK2 protein, gene and application regulating rice salt tolerance <130> <160> 3 <210> 1 <211> 3729 <212> DNA <213> Rice (Oryza sativa L.) <400> 1 acctcaccgc cgccggacgg cgacccctcc ccggccggga tcggcggcca ggcgagctcg 60 ggcgcccacg gctagccccg atcttcgggg atccacgcgc ggctcggctg cagccggcga 120 gcgcgaggtc gactgcgggg gaggccgcgg cctaggtgta cgggatcggg cggcggccgc 180 tgcgacggcg agctctctcg gctgcgtgac cgccggcgtg gtgcggggga ctgtggtatt 240 ctctttacct ccaccggcct ctcgcgacct agctgtttct tggtcaaagt caaacctcct 300 cctgattagc tctgtattta gctgttcata gtgatgtcga tttccgcttt tcccctgatt 360 tgctctgctg ctagtatcgg atcgaattcc tgctgacggg gatgctcctg ttttgctcgt 420 taggtgatcc aggttgcatg tcctagtgct agttggttgt tgaccattgg tgagagcttt 480 ttcatgttaa gctgtggtct ggtcgatgac caaaaaaaaa agaactcttg ctgttgttat 540 ctttcggttc cagcatttat tttgtataga tccatggtgc atttaatcca cttgccttca tgccttgtat atatcttaaa tagtttggc tags gatgtacttg atgcctcgat gagccacagt atcaatgtgt gtgttacgtg tgatttagct atagtctgat gtgctacaaa 720 tctaaacgtt agttcttatg cattggtaaa ctctgtatga gtattcttgt gaatgttcac actatttct cttatgtttc catgtaggca tccaagagct tcattctcaa gtttggatgt 840. tgaaggggtt caggaagtag agcacaccag gatgtctgtc aagtgttcca ttactattat catccaactt ctcttctgct atatgctttg tcagccatgc tatggcacat tagtgacat ccaatgccta aagagactga aggaatcagt tgacccaaac aataattgg aatggacatt tactaacact actgagggat ctatatgcgg attcaatggc gtggagtgct ggcatcctaa 1080 tgaaaacaag attctttctc ttcatcttgg cagcatgggt cttaagggcc atttccctga 1140 tgggcttgag aattgtagta gcatgacttc actggatctc tcgagcaaca gcctttccgg 1200 tccaatccca gctgacatct caaaacagct gccattcatt acaaaccttg atctatctta 1260 taatagtttc tcaggggaga ttccagaatc cctagctaat tgtacttatc taaatattgt 1320 caatttgcaa aataacaaat taactggagc aatcccaggg cagcttggta ttctatctcg 1380 cctaagccag tttaatgttg ccaacaatca actgtcaggg ccgatacctt catctttcgg 1440 caaattcgcg tcatccaatt ttgcaaatca agacctctgt gggagacctc tgagcaatga 1500 ttgcactgcc acttcaagca gccgtacagg ggtcattatt ggttctgctg ttggtggtgc 1560 agttataatg tttaataatag ttggtgttat cctgttcatt ttcttgcgga aaatgcctgc 1620 caagaagaag gaaaaggatt tggaagagaa taagtgggca aagaatatca agagtgcaaa 1680 aggagcgaag gtatacttct cattttggaa ttgtggtgta tttttgagcg tgcacatcta 1740 1800 tggcagttat tgtctttcc aatcacgctt tgttctgg gatttagaa ttaataggtg 1860 cctgtacttg ttcagatgtt caaaattgt gggtattggt taactcgcta agcatactaa 1920 catatttca tgccacgaaaaccactta aaagattt tattgttata caatctctct 1980 ttcgtaattg atgtatatt aatgtccaaa gcatgttttct atttcccaa tctgttatca 2040 agtggcaccc tctcccagt tcccacct ctctaaagaa acttaattgt atattttatc 2100 agtacagcgc ttaggttt atttttagca tcagacctga taatatttga ttagtcagca 2160 cattcaccta cagaaacagt attgttgat ggattttca ccttttttt tattcaggta 2220 tccatgtttg agaatcagt tgcaagatg aagttaatg atctgatgaa ggcaacaggt 2280 gattttacta agatatat tattggatct ggtcgatcag gaactatgta caagctaca 2340 ctcccagatg gttcattcct tgctatcag aggctacagg attackcaca tccgagt 2400 caatttgcat ctgagatgtc aacattggga agtgtaggc agcgcaactt acttccctt 2460 ttaggctatt gtattgctaa gaaagagagg ctcttggtat acaagtatat gcccaagggt 2520 tcactctatg atcagctaca ccaacagact agtgagaaaa aggcattgga gtggccattg 2580 aggctcaaaa ttgccattgg gtctgctaaa ggtttggcat ggcttcacca cagttgcaat 2640 ccccgcatcc ttcaccggaa cattagttcg aagtgcatat tactcgatga tgactatgac 2700 cctaaaattt ctgattttgg gttggcaagg cttatgaacc ccatcgacac ccacctgagc 2760 acatttgtca acggtgaatt tggagacttg gggtatgtag ctcctgagta tgcacgtacc 2820 ctcgtggcca ctccaaaagg ggatgtttat agctttggag ttgtgttgct tgaacttgtc 2880 actggtgaag agcccacaca ggtgaaaaat gccccggaaa acttcaaagg aagtttggtg 2940 gattggataa catacctgtc aaacaatgct atccttcagg atgcagttga taagtccttg 3000 attggaaagg atcatgatgc tgagctgctt caattcatga aggttgcatg ctcgtgtgtg 3060 ctttccgctc caaaggaaag accgacaatg tttgaggttt accagctaat gagagctatt 3120 ggagagaaat accattttag cgctgcagac gatgaattga cgatgcagcc gcagaatgct 3180 gaagccgaaa agcttgatga gcttattgta gcaaactaga tgagggtgat gaatggatgt 3240 tgctcagatg cagtagctac tgtgaagctt ctagataatc agggtgatcc gaactgaatg 3300 gaatcctgct cagtatattt tttccagcaa ttgcactgct gagcaacact aggtaaagct 3360 gtaagctcca agatatgcat gccatttggt ttgtaagatc tgtatcaatt agccatgtta 3420 aaactttttc tgtacattct tgccttgctg tgtactatct ctaggattgg catgtcctgc 3480 tgttgtaaga tggatcaact gtatcagcat ccgaatttag ttgtagtagc tcttagattt 3540 agcaatgttg aacagtacta ctgggttgtt gatcctccac atttttttcg gtgcgttcct 3600 ttgtcatttt tgttagttaa tgtggagtga gcccctacaa taatctgatg agatgtggga 3660 ttactggttt ctttggtttc ttggtgagtt tcagctaccg agttggtagt tatcaccctt 3720 gtgaaaaga 3729 <210> 2 <211> 1821 <212> DNA <213> Rice (Oryza sativa L.) <400> 2 atgtctgtca agtgttccat tactattatc atccaacttc tcttctgcta tatgctttgt 60 cagccatgct atggcacatt aagtgacatc caatgcctaa agagactgaa ggaatcagtt 120 gacccaaaca ataaattgga atggacattt actaacacta ctgagggatc tatatgcgga 180 ttcaatggcg tggagtgctg gcatcctaat gaaaacaaga ttctttctct tcatcttggc 240 agcatgggtc ttaagggcca tttccctgat gggcttgaga attgtagtag catgacttca 300 ctggatctct cgagcaacag cctttccggt ccaatcccag ctgacatctc aaaacagctg 360 ccattcatta caaaccttga tctatcttat aatagtttct caggggagat tccagaatcc 420 ctagctaatt gtacttatct aaatattgtc aatttgcaaa ataacaaatt aactggagca 480 atcccagggc agcttggtat tctatctcgc ctaagccagt ttaatgttgc caacaatcaa 540 ctgtcagggc cgataccttc atctttcggc aaattcgcgt catccaattt tgcaaatcaa 600 gacctctgtg ggagacctct gagcaatgat tgcactgcca cttcaagcag ccgtacaggg 660 gtcattattg gttctgctgt tggtggtgca gttataatgt ttataatagt tggtgttatc 720 ctgttcattt tcttgcggaa aatgcctgcc aagaagaagg aaaaggattt ggaagagaat 780 aagtgggcaa agaatatcaa gagtgcaaaa ggagcgaagg tatccatgtt tgagaaatca 840 gttgcaaaga tgaagttaaa tgatctgatg aaggcaacag gtgattttac taaggataat 900 attattggat ctggtcgatc aggaactatg tacaaagcta cactcccaga tggttcattc 960 cttgctatca agaggctaca ggatactcaa cattccgaga gtcaatttgc atctgagatg 1020 tcaacattgg gaagtgtaag gcagcgcaac ttacttcctc ttttaggcta ttgtattgct 1080 aagaaagaga ggctcttggt atacaagtat atgcccaagg gttcactcta tgatcagcta 1140 caccaacaga ctagtgagaa aaaggcattg gagtggccat tgaggctcaa aattgccatt 1200 gggtctgcta aaggtttggc atggcttcac cacagttgca atccccgcat ccttcaccgg 1260 aacattagtt cgaagtgcat attactcgat gatgactatg accctaaaat ttctgattttt 1320 gggttggcaa ggcttatgaa ccccatcgac acccacctga gcacatttgt caacggtgaa 1380 tttggagact tggggtatgt agctcctgag tatgcacgta ccctcgtggc cactccaaaa 1440 ggggatgttt atagctttgg agttgtgttg cttgaacttg tcactggtga agagcccaca 1500 caggtgaaaa atgccccgga aaacttcaaa ggaagtttgg tggattggat aacatacctg 1560 tcaaacaatg ctatccttca ggatgcagtt gataagtcct tgattggaaa ggatcatgat 1620 gctgagctgc ttcaattcat gaaggttgca tgctcgtgtg tgctttccgc tccaaaggaa 1680 agaccgacaa tgtttgaggt ttaccagcta atgagagcta ttggagagaa ataccatttt 1740 agcgctgcag acgatgaatt gacgatgcag ccgcagaatg ctgaagccga aaagcttgat 1800 gagcttattg tagcaaacta g 1821 <210> 3 <211> 606 <212> PRT <213> Rice (Oryza sativa L.) <400> 3 MSVKCSITII IQLLFCYMLC QPCYGTLSDI QCLKRLKESV DPNNKLEWTF TNTTEGSICG 60 FNGVECWHPN ENKILSLHLG SMGLKGHFPD GLENCSSMTS LDLSSNSLSG PIPADISKQL 120 PFITNLDLSY NSFSGEIPES LANCTYLNIV NLQNNKLTGA IPGQLGILSR LSQFNVANNQ 180 LSGPIPSSFG KFASSNFANQ DLCGRPLSND CTATSSSRTG VIIGSAVGGA VIMFIIVGVI 240 LFIFLRKMPA KKKEKDLEEN KWAKNIKSAK GAKVSMFEKS VAKMKLNDLM KATGDFTKDN 300 IIGSGRSGTM YKATLPDGSF LAIKRLQDTQ HSESQFASEM STLGSVRQRN LLPLLGYCIA 360 KKERLLVYKY MPKGSLYDQL HQQTSEKKAL EWPLRLKIAI GSAKGLAWLH HSCNPRILHR 420 NISSKCILLD DDYDPKISDF GLARLMNPID THLSTFVNGE FGDLGYVAPE YARTLVATPK 480 GDVYSFGVVL LELVTGEEPT QVKNAPENFK GSLVDWITYL SNNAILQDAV DKSLIGKDHD 540 AELLQFMKVA CSCVLSAPKE RPTMFEVYQL MRAIGEKYHF SAADDELTMQ PQNAEAEKLD 600 ELIVAN 606

Claims

1. Use of a gene encoding a rice negative regulation salt stress response protein in breeding rice varieties, wherein the encoding gene encodes a protein with an amino acid sequence as shown in SEQ ID No:

3.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown as SEQ ID No: 1 or SEQ ID No:

2.

3. The use according to claim 1 or 2, characterized in that: The coding gene is introduced into the target rice and overexpressed to obtain a negative regulatory response of the rice under salt stress, showing a sensitive phenotype; or the expression or activity of the coding gene in the target rice is inhibited or reduced to obtain a genetically modified rice with improved salt tolerance.

4. The use according to claim 3, characterized in that: The introduction is carried out by introducing the plant expression vector containing the coding gene into the explant; the inhibition or reduction of the gene expression can be achieved by gene knockout or gene silencing.

5. The use according to claim 4, characterized in that: The starting vector used to construct the plant expression vector is a binary Agrobacterium vector, or a vector suitable for plant gene gun bombardment.

6. The use according to claim 5, characterized in that: The plant expression vector contains an enhanced or inducible promoter.

7. The use according to claim 4, characterized in that: The gene knockout refers to inactivating the endogenous coding gene of rice through homologous recombination.

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