阅读说明：本技术 调控植物耐盐性的方法及耐盐相关蛋白 (Method for regulating and controlling salt tolerance of plants and salt tolerance related protein ) 是由 王雷 魏华 王希岭 于 2020-06-16 设计创作，主要内容包括：本发明公开了一种调控植物耐盐性的方法及耐盐相关蛋白,本发明提供了一种培调控植物耐盐性的方法,包括调控目的植物中耐盐相关蛋白质的编码基因的表达,其中,所述耐盐相关蛋白质是来源于水稻的耐盐性相关蛋白,名称为OsPRR73,来源于水稻日本晴。本发明利用CRISPR/Cas9技术获得了OsPRR73功能缺失的突变体,进行180mM NaCl模拟盐胁迫处理,观察表型并统计恢复处理后的存活率,最终确定OsPRR73在响应水稻盐胁迫的过程中发挥正调控作用。本发明的OsPRR73可作为一个耐盐基因,在水稻抗盐性方面发挥重要作用。(The invention discloses a method for regulating and controlling plant salt tolerance and salt tolerance related protein, and provides a method for regulating and controlling plant salt tolerance, which comprises regulating and controlling the expression of coding genes of salt tolerance related protein in a target plant, wherein the salt tolerance related protein is derived from rice, is named as OsPRR73 and is derived from rice Nipponbare. The invention obtains the OsPRR73 function-deficient mutant by using CRISPR/Cas9 technology, carries out 180mM NaCl salt stress simulation treatment, observes phenotype and counts the survival rate after recovery treatment, and finally determines that the OsPRR73 plays a positive regulation role in the process of responding to rice salt stress. The OsPRR73 of the invention can be used as a salt-tolerant gene and plays an important role in the salt tolerance of rice.)

1. A method for regulating and controlling the salt tolerance of plants is characterized in that the expression of a coding gene of a salt tolerance related protein in a target plant is regulated and controlled, wherein the salt tolerance related protein is the protein of A1), A2) or A3):

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90 percent of identity with the protein shown in A1), and is related to the salt tolerance of plants;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

2. The salt tolerance-associated protein of claim 1.

3. The biomaterial related to the protein of claim 2, which is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein of claim 2;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule that reduces the expression of the protein of claim 2;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

4. The related biological material according to claim 3, wherein: B1) the nucleic acid molecule is a coding gene of the protein shown in the following b1) or b 2):

b1) the coding sequence is cDNA molecule or DNA molecule of 1 st-2304 th nucleotide of sequence 1 in the sequence table;

b2) the nucleotide is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table.

5. The plant salt-resistant agent is characterized in that: the plant salt-resistant agent contains the protein of claim 2, or/and the biological material of claim 3 or 4.

6. The protein of claim 2, or the biomaterial of claim 3 or 4 for use in any one of the following P1-P4:

use of P1, the protein of claim 2, or the biomaterial of claim 3 or 4 for modulating salt tolerance in a plant;

use of P2, the protein of claim 2, or the biomaterial of claim 3 or 4 for growing salt tolerant/non-salt tolerant plants;

use of P3, a protein according to claim 2, or a biomaterial according to claim 3 or 4 for the manufacture of a product for increasing/decreasing salt tolerance in plants;

use of P4, the protein of claim 2, or the biological material of claim 3 or 4 in plant breeding.

7. A method for cultivating salt-tolerant plants, which comprises increasing the expression level of the protein of claim 2 or its coding gene in the target plants to obtain salt-tolerant plants; the salt tolerance of the salt tolerant plant is higher than that of the target seed plant.

8. A method for producing a transgenic plant with reduced salt tolerance, comprising reducing the expression of a gene encoding the protein of claim 2 in a plant of interest, resulting in a transgenic plant with lower salt tolerance than the plant of interest.

9. The method of claim 8, wherein: the reduction of the expression of the gene encoding the protein of claim 2 in the plant of interest is achieved by inhibiting the content and/or activity of the protein of claim 2 in the plant of interest using the CRISPR/Cas9 gene editing system.

10. The salt-tolerant agent of claim 5, or the use of claim 6, or the method of claim 1, 7 or 8, wherein: the plant according to claim 5 or 6, the plant of interest according to claim 1 or 7 or 8, or a monocotyledonous or dicotyledonous plant.

Technical Field

The invention relates to a salt-tolerant associated protein OsPRR73 and a related biological material thereof in the technical field of biology and a method for cultivating salt-tolerant plants.

Background

Rice (Oryza sativa L.) is one of the most important food crops in China, and the rice yield is greatly improved along with the development of modern agriculture. However, the development of modern agriculture is seriously hindered by the negative effects caused by the deterioration of the environment. Among them, soil salinization is one of the major stresses faced by plants, and has become a serious problem threatening the development of agriculture worldwide. It is estimated that about 6% of the land area worldwide is affected by salinization, including nearly 4500 kilo hectares of irrigation land (Munnsand Tester, 2008).

Gramineous crops are extremely sensitive to abiotic stress, especially rice and wheat, with the most marked decrease in yield in drought and saline environments (Lobell et al, 2014; Landi et al, 2017). Salt stress is a common abiotic stress, and high concentrations of salt mainly cause osmotic stress and ionic toxicity in plants. Plants inhibit the growth of young leaves of plants in response to salt stress primarily in the early stages of osmotic stress, while in the later stages, ion imbalance accelerates leaf senescence (Urano et al, 2014). The influence of salt stress mainly has the following two aspects: on the one hand, Na affecting the plant body⁺/K⁺Ions are in steady balance, so that ion toxicity is generated; on the other hand, salt stress induces the accumulation of Reactive Oxygen Species (ROS) in plants, which in turn generates oxidative stress (Mittler, 2017). During the process of responding to salt stress of plants, a small amount of ROS can enhance the adaptability of plants; excessive ROS accumulation directly causes apoptosis and tissue necrosis. Na (Na)⁺Is the most critical factor causing ion poisoning, mainly because of Na⁺Accumulation in leaves accelerates necrosis of the leaves, resulting in a decrease in crop yield (Munns, 1993). In plants K⁺Is involved in many important metabolic processes, while excess Na⁺Capable of competitively binding to K⁺Sites, e.g. more than 50 biological enzymes, require K⁺Is activated, and this is Na⁺Cannot be replaced, so high concentration of Na⁺It disrupts the activity of various enzymes in cells, leading to metabolic disturbances in plants (Bhadal and Malik, 1988).

Salt stress also has an effect on plant photosynthesis. The carotenoid and chlorophyll content of plant leaves is generally reduced under the salt stress. As the salt stress time increases, the leaves begin to wither away (Hernandez et al, 1999; Ramanjulu et al, 1998). Studies have found that the total chlorophyll, chlorophyll alpha and beta-carotene content in tomato leaves under salt stress conditions decreases gradually as a result of prolonged salt stress time (Kurth et al, 1986). It has also been found in studies on rice of different genotypes that photosynthetic pigments generally decrease with increasing salt stress time (Yu et al, 2012). The influence of salt stress on photosynthesis is divided into short-term effect and long-term effect, the short-term effect occurs within hours or 1 day after the salt stress starts, the effect has great influence on plants, and the carbon assimilation is directly stopped; long-term effects occur after several days in salt stress environments, resulting in reduced carbon assimilation and hence slow plant growth or death due to salt accumulation in the developing leaves (Ramanjulu et al, 1998).

Salt stress can affect each stage of plant growth and development, and ion imbalance caused by the salt stress directly affects seed germination and seedling growth; reduced sensitivity during vegetative growth; but salt stress will directly affect plant seed filling during flowering. Currently, much research on this aspect focuses on the model plant Arabidopsis thaliana, such as the more thorough SOS (salt Over sensitive) pathway, and the important physiological role is homeostatic regulation when Arabidopsis thaliana is subjected to salt stress. Calcium Binding proteins of EF-hand structure SOS3(Salt excess Sensitive 3) and SCaBP8(SOS3-Like calcium Binding Protein 8)/CBL10 (calcium B-Like Protein 10) are capable of sensing and decoding calcium signals (Zhu,2016) generated by Salt stress stimulation, and further activate serine/threonine Protein kinase SOS2(Salt excess Sensitive 2) (Lin et al, 2009). The activated SOS2 can interact with SOS1 on the plasma membrane of the cell and phosphorylate SOS1, and then activate Na⁺/H⁺Exchange channels for Na accumulated in cytoplasm⁺To the outside of the cell (Shi et al, 2000). In addition, numerous studies have shown that plants interact through a variety of complex signaling pathways, such as the mediation of Ca, in response to external stresses such as salt stress₂ ⁺Signal transport of ions (Yuan et al, 2014), accumulation of ABA (Ma et al, 2009), ion transport and transcription factor activity (Vahisalu et al, 2008; Fujita et al)al, 2011) and active oxygen steady state equilibrium, etc.

However, at present, salt stress regulation and control networks in crop rice are relatively rarely researched, and although some salt-tolerant germplasm resources are preliminarily accumulated, the understanding of main key genes or major QTLs for controlling salt tolerance is relatively little. Earlier studies mapped by forward genetics to clone SKC1, OsHKT8, a transporter belonging to the HKT family, located on chromosome 1 by maintenance of K in roots⁺Ion content, thereby retaining Na in the plant body⁺/K⁺The steady state balance of (a), makes plants more tolerant to salt stress (Ren et al, 2005). Subsequently, the population mutagenized by EMS is screened and identified to obtain a Drought Salt Tolerance (DST), which belongs to a transcription factor of a zinc finger structure, and the Drought Salt Tolerance (DST) is found to regulate and control the adaptability of rice to adverse environments mainly by controlling the density and the openness of stomata (Huang et al, 2009). Subsequently, the researchers screened DCA1, a transcription factor belonging to CHY-type zinc finger structure, interacting with DST by using yeast two-hybrid technology, and found that it can interact with DST to form a transcription complex of heterotetramer, and the expression of the transcription factor is controlled by a promoter combined with peroxisome peroxidase24 precorsor (Prx 24). Prx 24 belongs to H₂O₂The scavenger of (1) is mainly expressed in guard cells, and controls the opening and closing of stomata and the response of rice to salt stress by controlling the amount of hydrogen peroxide in guard cells (Cui et al, 2015).

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control the salt tolerance of plants.

In order to solve the technical problems, the invention provides a method for regulating and controlling the salt tolerance of plants, which comprises the step of regulating and controlling the expression of a coding gene of a salt tolerance related protein in a target plant, wherein the salt tolerance related protein is the protein of the following A1), A2) or A3):

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90 percent of identity with the protein shown in A1), and is related to the salt tolerance of plants;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

The invention provides a salt tolerance related protein from rice, which is named as OsPRR73 and is from Nipponbare of rice, and is protein A1), A2) or A3):

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90 percent of identity with the protein shown in A1), and is related to the salt tolerance of plants;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

In the above protein, sequence 2 in the sequence table is composed of 767 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Of the above proteins, OsPRR73 may be derived from rice.

Biomaterials associated with OsPRR73 are also within the scope of the invention.

The biomaterial related to OsPRR73 provided by the invention is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding OsPRR 73;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B1);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule of B1);

B6) a nucleic acid molecule that reduces expression of OsPRR 73;

B7) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule according to B6).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biological material, the nucleic acid molecule according to B1) may specifically be a gene represented by 1) or 2) below:

1) the coding sequence (CDS) is a DNA molecule of 1 st-2304 th nucleotides of a sequence 1 in a sequence table;

2) the nucleotide sequence is a DNA molecule of a sequence 1 in a sequence table.

In the above biological material, the nucleic acid molecule of B6) may specifically be a DNA molecule reverse-complementary to any fragment of the DNA molecule represented by nucleotides 1 to 2304 of sequence 1 in the sequence table.

In the above biological material, B7) the recombinant vector may be a CRISPR/Cas9 recombinant expression vector designed for the OsPRR73 coding sequence (CDS).

Wherein, the sequence 1 in the sequence table is composed of 2304 nucleotides, and the coding sequence is the protein shown by the sequence 1 and the sequence 2 in the sequence table.

In the above-mentioned biological materials, the expression cassette containing a nucleic acid molecule encoding OsPRR73 (OsPRR73 gene expression cassette) described in B2) means a DNA capable of expressing OsPRR73 in a host cell, which may include not only a promoter that initiates transcription of OsPRR73 gene but also a terminator that terminates transcription of OsPRR 73. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiology120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beach et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcSE9 terminator and nopaline and octopine synthase terminatorsSee, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al GenesDev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant expression vector containing the OsPRR73 gene expression cassette can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), marker genes for antibiotics which are expressible in plants (e.g., nptII gene which confers resistance to kanamycin and related antibiotics, bar gene which confers resistance to phosphinothricin which is a herbicide, hph gene which confers resistance to hygromycin which is an antibiotic, dhS gene which confers resistance to methatrexate, EPSPS gene which confers resistance to glyphosate), or marker genes for chemical resistance (e.g., herbicide resistance), mannose-6-phosphate isomerase gene which provides the ability to metabolize mannose, etc. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above biological material, the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

In order to solve the technical problems, the invention also provides a plant salt-resistant agent.

The plant salt-tolerant agent provided by the invention contains the protein or/and biological materials related to the protein.

The active component of the plant salt tolerance agent can be the protein or biological materials related to the protein, the active component of the plant salt tolerance agent can also contain other biological components or/and non-biological components, and other active components of the plant salt tolerance agent can be determined by a person skilled in the art according to the salt tolerance effect of plants.

The protein or the biological material can be applied to any one of the following P1-P4:

use of P1, the protein of claim 1, or the biomaterial of claim 2 or 3 for modulating salt tolerance in a plant;

use of P2, the protein of claim 1, or the biomaterial of claim 2 or 3 for growing salt tolerant/non-salt tolerant plants;

use of P3, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for the manufacture of a product for increasing/decreasing salt tolerance in plants;

use of P4, the protein of claim 1, or the biological material of claim 2 or 3 in plant breeding.

In order to solve the technical problems, the invention also provides a method for cultivating salt-tolerant plants.

The method for cultivating the salt-tolerant plant comprises the steps of improving the expression level of the protein or the coding gene thereof in a target plant to obtain a salt-tolerant plant; the salt tolerance of the salt tolerant plant is higher than that of the target seed plant.

In the above method, the improvement of the expression level of the protein or the gene encoding the protein in the target plant can be achieved by introducing the gene encoding the protein into the target plant.

In the method, the coding gene of the protein can be modified as follows and then introduced into a target plant to achieve better expression effect:

1) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

2) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

3) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

4) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding the protein can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition).

In the method, the salt-tolerant plant can be a transgenic plant or a plant obtained by conventional breeding technologies such as hybridization and the like.

In order to solve the technical problems, the invention also provides a method for cultivating the transgenic plant with reduced salt tolerance.

The method for cultivating the transgenic plant with reduced salt tolerance provided by the invention comprises the step of reducing the expression of the coding gene of the protein in a target plant to obtain the transgenic plant with lower salt tolerance than the target plant.

In the above method, the reduction of the expression of the gene encoding the protein in the plant of interest is achieved by inhibiting the content and/or activity of the protein in the plant of interest using the CRISPR/Cas9 gene editing system.

In the above methods, the transgenic plant is understood to include not only the first to second generation transgenic plants but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

As described above, the plant and the plant of interest are both monocotyledonous or dicotyledonous plants. The plant may be rice.

The invention obtains the OsPRR73 function-deficient mutant by using CRISPR/Cas9 technology, carries out 180mM NaCl salt stress simulation treatment, observes phenotype and counts the survival rate after recovery treatment, and finally determines that the OsPRR73 plays a positive regulation role in the process of responding to rice salt stress. The OsPRR73 of the invention can be used as a salt-tolerant gene and plays an important role in the salt tolerance of rice.

Drawings

FIG. 1 is a graph of 180mM NaCl mimicking salt stress treatment, detecting changes in OsPRR73 expression in response to salt stress. The material was wild type two week old seedlings, treated with 180mM NaCl. The treatment is carried out by adding the liquid nutrient solution of Mucun B into the nutrient solution containing salt and the nutrient solution containing no salt, and collecting materials (aerial parts) during the treatment, wherein three biological processes are repeated at each time point. The histogram in FIG. 1 shows the relative expression of OsPRR73 under normal and salt stress conditions of 4 hours.

Fig. 2 is an osprr73 mutant in the NIP background obtained using CRISPR/Cas9 technology, designated osprr73-C1 and osprr73-C2, respectively, both of which are loss-of-function mutants.

FIG. 3 is a graph of salt stress tolerance analysis of osprr73-C1 and osprr73-C2, homozygous mutant osprr73-C1, osprr73-C2, and wild-type NIP four-week-old seedling material treated for salt stress. Photographing was performed before treatment, 180mM NaCl was added to the nutrient solution at the time of treatment, photographing was performed 21 days after treatment, then salt-free normal nutrient solution was added at the time of recovery, photographing was performed 9 days after recovery, and the survival rate was counted (A is the state of the material without NaCl treatment, 21 days after treatment, and 9 days after recovery; B is the survival rate counted after recovery of growth).

FIG. 4 shows the results of measurements of chlorophyll content and ion leakage rate in NIP and osprr73-C1, osprr73-C2 leaves 7 days after normal and salt stress treatment. (the chlorophyll content measurement results are shown in the A diagram; and the ion leakage rate results are shown in the B diagram).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The experimental material used in the research is Nipponbare (NIP for short) which is a commonly used japonica rice variety.

The CRISPR/Cas9 vector systems in the following examples are described in the following documents (Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, ChenY, Guo J, Chen L, ZHao X, Dong Z, Liu Y-G (2015) A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in monomer and DicotPlants. mol Plant 8: 1274. 1284) available from the university of south China agricultural Hui Liu light laboratories, publicly available from the university of south China agricultural university to replicate the experiments of the present application, and are not available for other uses.

Agrobacterium EHA105 competent cells, Beijing Bomaide, in the examples described below.

The nutrient solution Mucun B formula comprises: the feed additive is prepared by mixing mother liquor A, mother liquor B, EDTA-Fe mother liquor and trace element mother liquor according to the ratio of 5:5:1:1 in each liter of nutrient solution. Wherein 1L (200X) of the A mother liquor contains (NH4)₂SO₄9.64g、KNO₃3.7g、KH₂PO₄4.96g、K₂SO₄3.18g and MgSO₄.7H₂O29.965 g; 1L (200X) of B mother liquor contains Ca (NO3)₂.4H₂O17.235 g; 1L (1000X) of EDTA-Fe mother liquor: 5.57g of FeSO were first dissolved₄.7H₂O in 200mL of distilled water, followed by heating to dissolve 7.45g of Na₂EDTA in 200mL distilled water, FeSO₄.7H₂O solution and Na₂And continuously stirring and mixing the EDTA solution, and cooling the mixture to a constant volume of 1L. 1L (1000X) of the microelement mother liquor contains: h₃BO₄2.86g、CuSO₄.H₂O 0.08g、ZnSO₄.7H₂O 0.22g、MnC_l2.4H₂O1.81 g and NaMO₄.H₂O0.09 g. Adding 100-300 ng of sodium silicate into each liter of nutrient solution, and then adjusting the pH value to 5.8 by using concentrated hydrochloric acid.

NB2 medium: NB Basal Medium powder (brand: Phototech, available from Western Mejie technologies, Inc. of Beijing) (4.1g/L) was dissolved in distilled water, and then hydrolyzed casein (300 mg/L), proline (500 mg/L), glutamine (500 mg/L), sucrose (30 g/L) and 2 mg/L2, 4-D (2 mg/L) were added, respectively.

NB1 medium: on the basis of the NB2 medium formula, 0.5mg/L of 2,4-D was added.

NB1S1 medium: after sterilization of NB1 medium, 600mg/L of cefuroxime and 25mg/L of Hyg were added.

NB1S2 medium: after sterilization of NB1 medium, 300mg/L of cefuroxime and 50mg/L of Hyg were added.

RE1 differentiation medium: MS culture medium powder (4.5g/L) is dissolved in distilled water, and then 300mg/L hydrolyzed casein, 1 mg/L6-BA, 0.5mg/L KT, 0.2mg/L ZT, 0.25mg/L NAA, 30g/L sucrose and 30g/L sorbitol are respectively added.

RE2 differentiation medium: MS culture medium powder (4.5g/L) is dissolved in distilled water, and then 300mg/L hydrolyzed casein, 50mg/L hygromycin, 1 mg/L6-BA, 0.5mg/L KT, 0.2mg/L ZT, 0.5mg/L NAA, 30g/L sucrose and 20g/L sorbitol are respectively added.

1/2MS culture medium: MS medium powder (brand: Caisson, purchased from commercial Co., Ltd. of Beijing Lanbobedded, N) (2.25g/L) was dissolved in distilled water, and 30g/L of sucrose was added. Adjusting pH of all culture media to 5.8, adding agar 8g/L, sterilizing at 120 deg.C under high pressure for 15min

The rice material culture method used in the following examples of the present invention was: well-developed and plump seeds are selected, soaked in distilled water in an incubator at 37 ℃ for 48 hours, then the distilled water is poured off, and the pregermination is continued for 24 hours in a humid environment. And selecting the seeds with consistent germination states, placing the seeds into a bottomless 96-well plate, and placing the seeds into a vessel containing the nutrient solution of the Mucun B for growth. The greenhouse growth conditions were 12 hours light/12 hours dark, the temperature was 30 + -2 deg.C. The fresh nutrient solution was replaced every 3 days during the culture.