Application of rice OsKEA1 gene in regulation and control of rice salt tolerance

Abstract

The invention discloses application of a rice OsKEA1 gene in regulation and control of rice salt tolerance. Belongs to the field of agricultural biotechnology. In order to excavate a new rice salt stress related gene, a mutant oskea1 sensitive to salt stress is screened from an EMS mutagen library taking a japonica rice variety Wuyunjing No.7 as a background, and phenotypic analysis and physiological detection are carried out on the oskea1 mutant and allelic knockout mutants cas9-3 and cas9-8 of the oskea1 mutant. The OsKEA1 gene provided by the invention has deep mechanism research in the aspect of influencing the salt tolerance of the rice.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and more particularly to the application of the rice OsKEA1 gene in regulating rice salt tolerance. Background Art

[0002] Soil salinization is a major problem in global environmental governance, seriously threatening the growth and yield of crops and hindering the sustainable development of modern agriculture. Approximately one-third of the irrigated land in the world is affected by salinization, and this problem is gradually worsening due to climate change, industrial activities, and human intervention. Salt stress continuously disrupts plant growth and development throughout the plant life cycle through oxidative stress, ion toxicity, and osmotic stress. Therefore, improving crop tolerance to salt stress is crucial for environmental governance, plant protection, and food security. Among them, analyzing the plant salt stress response mechanism and exploring salt-tolerant genetic resources have become the key to improving crop salt tolerance.

[0003] Salt stress response involves a series of complex physiological and biochemical processes. It mainly includes synthesizing osmotic adjustment substances to balance osmotic stress, regulating ion transport and absorption to reduce ion toxicity, scavenging reactive oxygen species to eliminate oxidative stress, as well as hormone regulation and transcriptional rearrangement of salt-tolerant genes, etc. For example, proline, as a highly water-soluble amino acid, can rapidly accumulate under salt stress and then reduce water loss in cells by binding to water molecules to regulate the osmotic pressure inside and outside cells. In addition, salt stress can lead to the production of excessive reactive oxygen species (ROS) in plants. Although low levels of ROS can act as signaling molecules, for example, in the early stage of salt stress, SALT-RESPONSIVE ERF1 (SERF1) and GmNTL1 can respond to ROS and salt stress and transmit high-salt toxicity signals. However, continuous salt stress will cause excessive accumulation of ROS in plants, triggering oxidative toxicity, thereby increasing cell membrane permeability, causing DNA damage, and protein degradation. However, multiple pieces of evidence indicate that catalase (CAT) in plants can catalyze the decomposition of H 2 O 2 to improve the tolerance of plants to oxidative stress and salt stress.

[0004] The main cause of salt stress is the excessive content of Na + and Cl - in the soil. Na + in the soil is absorbed by plant roots, loaded and transported to the above-ground parts through the xylem, and then redistributed within plant cells through various channels and transporters. These channels and transporters are crucial for maintaining Na + / K + homeostasis to mitigate salt stress. For example, under high-salt conditions, HKT1 can reduce Na +Accumulation in the xylem and utilization of phloem recycling to re-isolate Na in the roots to mitigate the harm of Na toxicity to the above-ground parts and improve the salt tolerance of plants. Na / H exchangers (NHXs) and K-efflux antiporters (KEAs) are part of the monovalent cation-proton antiporter family and can promote the transmembrane exchange of Na or K for protons (H). OsNHX1 enhances the salt tolerance of rice by transporting Na from the cytoplasm to the vacuole, achieving the compartmentalization of Na in the vacuole. GmNHX5 plays a positive role under salt stress conditions by regulating the accumulation of osmoregulatory substances in soybeans and the Na / K balance. The plasma membrane-localized SOS1, as a member of the NHX family of proteins, has been shown to be involved in the salt stress response of various plants. The SOS1 protein actively excretes Na to the extracellular space, reducing the accumulation of intracellular Na and the toxicity of salt stress to plants. And its function is regulated by the SOS signaling pathway and acts synergistically with SOS2 and SOS3 to maintain ion balance and enhance the salt tolerance of plants. In addition, the KEA protein family has also been widely reported to be involved in various physiological responses and abiotic stress resistance of plants. The loss of function of KEA1 and KEA2 seriously affects the plant development and abiotic stress tolerance of Arabidopsis thaliana. KEA4, KEA5, and KEA6 synergistically support the K / Na and pH homeostasis in the endosomal compartments of Arabidopsis thaliana, which is crucial for plants to alleviate salt stress. + Re-isolation in the roots to mitigate the harm of Na toxicity to the above-ground parts + toxicity and improve the salt tolerance of plants. Na + / H + exchangers (Na + / H + exchangers, NHXs) and K + -efflux antiporters (K + -efflux antiporters, KEAs) are part of the monovalent cation-proton antiporter family and can promote the transmembrane exchange of Na + or K + for protons (H + ). OsNHX1 enhances the salt tolerance of rice by transporting Na + from the cytoplasm to the vacuole, achieving the compartmentalization of Na + in the vacuole. GmNHX5 plays a positive role under salt stress conditions by regulating the accumulation of osmoregulatory substances in soybeans and the Na + / K + balance. The plasma membrane-localized SOS1, as a member of the NHX family of proteins, has been shown to be involved in the salt stress response of various plants. The SOS1 protein actively excretes Na + to the extracellular space, reducing the accumulation of intracellular Na + and the toxicity of salt stress to plants. And its function is regulated by the SOS signaling pathway and acts synergistically with SOS2 and SOS3 to maintain ion balance and enhance the salt tolerance of plants. In addition, the KEA protein family has also been widely reported to be involved in various physiological responses and abiotic stress resistance of plants. The loss of function of KEA1 and KEA2 seriously affects the plant development and abiotic stress tolerance of Arabidopsis thaliana. KEA4, KEA5, and KEA6 synergistically support the K + / Na + and pH homeostasis in the endosomal compartments of Arabidopsis thaliana, which is crucial for plants to alleviate salt stress.

[0005] In summary, how to provide a gene for regulating rice salt stress and explore its salt tolerance mechanism is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the application of the rice OsKEA1 gene in regulating the salt tolerance of rice.

[0007] The purpose of the present invention is to provide the role of the OsKEA1 gene in rice salt stress, which is of great significance for exploring the salt tolerance mechanism of plants and improving the salt tolerance of rice.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] Use of rice OsKEA1 gene in regulating rice salt tolerance, wherein the nucleotide sequence of the rice OsKEA1 gene is as shown in SEQ ID NO:1.

[0010] Furthermore, the regulation is positive regulation.

[0011] Furthermore, it is used to regulate rice Na + / K + homeostasis, expression of salt stress-related genes, and chloroplast development.

[0012] Furthermore, the salt stress-related genes include NHX2, SOS2, OsHKT4, SIT1, SIT2, and DST1 genes.

[0013] Use of knocking out rice OsKEA1 gene in constructing a rice model with reduced salt tolerance.

[0014] Use of a mutant of rice OsKEA1 gene in constructing a rice model with reduced salt tolerance, wherein the nucleotide sequence of the mutant is as shown in SEQ ID NO:3, and the encoded amino acid sequence is as shown in SEQ ID NO:4.

[0015] From the above technical solutions, it can be seen that compared with the prior art, the beneficial effects obtained by the present invention are:

[0016] In order to explore new rice salt stress-related genes, the present invention screened a salt stress-sensitive mutant osken1 from an EMS mutant library with the japonica rice variety Wuyunjing 7 as the background, and conducted phenotypic analysis and physiological detection on the osken1 mutant and its allelic knockout mutants cas9-3 and cas9-8. The OsKEA1 gene of the present invention has an in-depth mechanism exploration in affecting rice salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0018] Figure 1Sequencing analysis of the oskea1 mutant in Example 1 of the present invention. Among them, A represents the schematic diagrams of two independent CRISPR-Cas9 knockout lines (cas9-3, cas9-8); B represents the schematic diagram of the fine mapping of the OsKEA1 gene; C represents the sequencing analysis of the oskea1 mutant. The mutated base in the oskea1 mutant is shown within the red box. The mutation from G to A causes the conversion of glycine to arginine.

[0019] Figure 2 Results of salt treatment of the wild type WT and three oskea1 mutant lines in Example 2 of the present invention. Among them, A represents the phenotypic characteristics of different plants under normal conditions; B represents the phenotypic characteristics of different plants under the condition of 180 mM NaCl treatment for 6 days; C represents the fresh weight of different plants after 6 days of treatment with 180 mM NaCl; D represents the dry weight of different plants after 6 days of treatment with 180 mM NaCl; E represents the survival rate of different plants after 7 days of rehydration; ** indicates significant difference at the 0.01 level (t-test).

[0020] Figure 3 Determination of potassium and sodium ion contents of the wild type WT and three oskea1 mutant lines in Example 3 of the present invention. Among them, A is the Na + content; B is the K + content; C is the Na + / K + value; ** indicates significant difference at the 0.01 level (t-test).

[0021] Figure 4 Expression levels of salt stress-related genes of the wild type WT and three oskea1 mutant lines in Example 4 of the present invention. Among them, A represents the result of NHX2; B represents the result of SOS2; C represents the result of OsHKT4; D represents the result of SIT1; E represents the result of SIT2; F represents the result of DST1; ** indicates significant difference at the 0.01 level (t-test).

[0022] Figure 5 Observation of the chloroplast structure of the wild type WT and three oskea1 mutant lines in Example 5 of the present invention. Among them, Og represents osmiophilic granules; Dc represents degraded chloroplasts. Detailed implementation manners

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0024] The reagents required for this invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods, which will not be elaborated here one by one.

[0025] Example 1

[0026] Obtaining of mutant materials

[0027] This invention chemically mutates the japonica rice variety Wuyunjing 7 with EMS (EthylmethaneSulphonate):

[0028] Soak the seeds of Wuyunjing 7 in water for 10 hours, then transfer the soaked seeds into a 0.8% EMS solution and soak for 10 hours. During this period, it is necessary to carry out in a low-speed shaker to ensure uniform mixing. After mutagenesis, the seeds need to be rinsed overnight with running tap water to remove the residual EMS solution, and then the seeds are germinated at 30°C for 36h and then sown. The growth period is managed according to the conventional field management, and the seeds are harvested by individual plants after maturity. Finally, through the reproduction and segregation of the offspring, a stably inherited salt-sensitive mutant is screened and named oskea1.

[0029] Population construction and genetic analysis:

[0030] Reciprocal crosses are made between the mutant oskea1 and the indica rice TN1. The F 1 plants of both combinations show normal phenotypes, but the F 1 plants show trait segregation after self-crossing. The traits of the F 2 segregation population are statistically analyzed. The results show that the segregation ratio of plants with normal phenotypes to plants with mutant phenotypes is close to 3:1 after chi-square test verification, indicating that the mutant phenotype of the mutant oskea1 is controlled by a single recessive nuclear gene. 2 This invention locates the OsKEA1 gene by map-based cloning:

[0031] Table 1 Genetic analysis of the F 2 population from the cross between the mutant oskea1 and the indica rice TN1

[0032]

[0033] Design 229 pairs of SSR primers evenly distributed on 12 chromosomes of rice to conduct preliminary mapping of the OsKEA1 gene. The OsKEA1 gene is preliminarily located between the M1 and M2 markers on chromosome 4.

[0034] Genomic DNA is extracted by the CTAB method. The specific steps are as follows:

[0035] Genomic DNA is extracted by the CTAB method. The specific steps are as follows:

[0036] ① Add an appropriate amount of pre - shredded rice leaves into a 2.0 mL Eppendorf tube. After adding steel beads to each tube, pre - cool with liquid nitrogen, break the tissue with a high - throughput tissue grinder, and then add 750 μL of CTAB buffer. Place the sample in an oven at 65 °C for 60 minutes, and shake the sample during this period to fully mix it with the CTAB buffer.

[0037] ② After the lysis is completed, add 500 μL of chloroform to each tube, mix well, centrifuge at 10000 rpm for 8 minutes, and transfer an appropriate amount of the supernatant to a new centrifuge tube.

[0038] ③ Add an equal volume of pre - cooled absolute ethanol to the supernatant obtained in step ② above. After mixing well, place the sample in a - 20 °C refrigerator until DNA precipitates. Then centrifuge the sample at 10000 rpm for 10 minutes, pour out the supernatant, and air - dry the precipitate overnight to volatilize the remaining alcohol.

[0039] ④ Dissolve the air - dried DNA in 300 μL of pure water, which can be used for downstream experiments.

[0040] The PCR reaction system (10 μL) is as follows: 1 μL of DNA template, 5 μL of 2×PCR mix buffer, 0.5 μL each of forward and reverse primers (10 μmol / L), and add ddH 2 O to make up to 10 μL.

[0041] The PCR amplification program is as follows: pre - denaturation at 98 °C for 2 min; denaturation at 94 °C for 15 s, annealing at 56 °C for 15 s, extension at 72 °C for 15 s, for 35 cycles; finally, extension at 72 °C for 5 min.

[0042] The PCR products were separated by 4% agarose gel electrophoresis. After the electrophoresis was completed, they were scanned and recorded with a gel imager. Using the 229 pairs of SSR primers mentioned above for linkage analysis of the OsKEA1 gene, it was found that it showed a linkage phenomenon between the M1 and M2 markers on chromosome 4. Then new InDel markers were designed in this interval, and finally the mapping interval of the target gene was locked within an approximately 64 kb interval between the InDel molecular markers L3 and L4 ( Figure 1 B). The primer sequences for map - based cloning are shown in Table 2.

[0043] Table 2 Primer sequences for map - based cloning of the OsKEA1 gene

[0044]

[0045] After sequencing and alignment of the open reading frame in this interval, it was found that the 2572nd nucleotide G in the coding region of the LOC_Os04g58620 gene in the oskea1 mutant was changed to A, resulting in the corresponding amino acid residue changing from glycine to arginine ( Figure 1C). Therefore, it was set as a candidate gene for OsKEA1.

[0046] The cDNA sequence of the OsKEA1 gene in Wuyunjing 7 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0047] In the rice salt-sensitive mutant, the 2572nd nucleotide in the coding region of the OsKEA1 gene was changed from G to A. The mutated cDNA sequence is shown in SEQ ID NO: 3, resulting in the 858th amino acid of the encoded protein being changed from glycine to arginine. The mutated amino acid sequence is shown in SEQ ID NO: 4.

[0048]

[0049]

[0050]

[0051]

[0052] Generation of oskea1 knockout mutants via the CRISPR-Cas9 system:

[0053] Two target sites were selected in the first exon sequence of the OsKEA1 gene (i.e., target site 1: TAGCCTCTTCTACTTGGCT, SEQ ID NO: 21, target site 2: GCCCTTGCGTCCGCGCAAG, SEQ ID NO: 22) to construct a CRISPR-Cas9 knockout plasmid. The constructed plasmid was transformed into competent cells of Agrobacterium tumefaciens EHA105, and it was transferred into the calli of wild-type Wuyunjing 7 via Agrobacterium-mediated transformation to knockout the wild-type OsKEA1 gene. Then, through hygromycin screening and differentiation for rooting, T 0 -generation positive plants were obtained. After subsequent self-crossing and segregation, T 1 -generation seeds were harvested. After sowing, DNA of individual plants was extracted, and then the gDNA of OsKEA1 at the target site was sequenced by PCR. Homozygous mutants were selected based on the sequencing results and continuously self-crossed to the T 3 -generation to obtain a genetically stable oskea1 homozygous mutant line (as shown in Figure 1 A).

[0054] All rice materials were planted in the experimental field of the China National Rice Research Institute in Fuyang District, Hangzhou City, Zhejiang Province, with conventional management.

[0055] Example 2

[0056] Phenotypic analysis of salt treatment of plants

[0057] Under the salt treatment condition of 180 mM concentration, compared with the wild-type WT, the leaves of cas9-3, cas9-8, and oskea1 mutants were more shriveled and curled. Therefore, we statistically analyzed the fresh weight and dry weight of each line 6 days after salt treatment and found that the fresh weight and dry weight of cas9-3, cas9-8, and oskea1 mutants were significantly lower than those of the wild-type. And by counting their survival rates after 7 days of rehydration recovery, it was found that the survival rates of cas9-3, cas9-8, and oskea1 mutants were significantly reduced. On the contrary, under normal culture conditions, there were no obvious differences in the growth states of each line, indicating that the loss of function of the OsKEA1 gene would lead to a decrease in its salt tolerance ( Figure 2 ).

[0058] Example 3

[0059] Analysis of potassium and sodium ion contents

[0060] Under the salt treatment condition, we measured and analyzed the potassium and sodium ion contents of each line to reflect the dynamic balance of ions in the plants.

[0061] The specific steps are as follows:

[0062] ① Collect leaf samples from plants treated with 180 mM NaCl for 6 days or untreated plants. Take 3 parallel samples for each group, dry and weigh them, and record the weight of each sample.

[0063] ② Digest all samples with 4 mL of nitric acid and 2 mL of hydrogen peroxide at 200 °C for about 4 hours.

[0064] ③ After the samples are cooled, dilute all samples with ultrapure water and make the volume up to 50 mL.

[0065] ④ Vortex and mix the solution in each sample, then filter it through two layers of Miracloth into a 50 mL centrifuge tube. This is used as the sample to be measured.

[0066] ⑤ Use a flame spectrophotometer (Sherwood Model 410) to measure the content of Na + and K + in the solution. Then use the prepared Na + and K + standard curves to calculate the content of Na + , K + in each sample respectively, and calculate the corresponding Na + / K + value.

[0067] Ion content analysis showed that the cas9-3, cas9-8, and oskea1 mutants disrupted the Na + / K + homeostasis under salt stress. Specifically, compared with the wild type WT, the Na + level and the Na + / K + ratio both increased ( Figure 3 ).

[0068] Example 4

[0069] Expression levels of salt stress-related genes

[0070] As can be known from Figure 2 , the loss of function of the OsKEA1 gene leads to more sensitivity of plants to salt stress. To further explore the regulatory molecular mechanism of the weakened salt tolerance of the oskea1 mutant in rice, we used qRT-PCR to detect the expression levels of salt stress-related genes in each line before and after salt treatment.

[0071] Table 3 Quantitative primers related to salt stress

[0072]

[0073] The results showed that under salt ion stress, compared with the wild type, the transcriptional levels of the salt stress positive regulatory genes NHX2, SOS2 and OsHKT4 were significantly decreased in the oskea1 and cas9 knockout mutants, while the expression levels of the salt stress negative regulatory genes SIT1, SIT2 and DST1 were significantly increased in the oskea1 and cas9 knockout mutants( Figure 4 ).

[0074] Example 5

[0075] Chloroplast development under salt stress

[0076] Chloroplast observation: After 6 days of 180 mM salt stress treatment, leaf samples of each line were cut and placed into the electron microscope fixative (2.5% glutaraldehyde sodium phosphate buffer, pH 7.2). Vacuum was applied until the samples sank to the bottom of the fixative, and then they were stored at 4°C for subsequent transmission electron microscope observation.

[0077] The results obtained by transmission electron microscope observation are as Figure 5 shown. After salt treatment, the chloroplast structure of the wild type was relatively complete and plump, the starch grains developed normally, and the grana lamellae were relatively clear, showing no obvious difference from the chloroplast morphology developed under normal conditions. On the contrary, the number of chloroplasts in the oskea1 and cas9 knockout mutants decreased sharply under salt stress, the morphology was blurred and irregular, the structure of the thylakoid membrane was severely damaged, and the stacking of the grana lamellae was disordered. The above results indicate that the loss of function of OsKEA1 causes abnormal chloroplast development in rice under salt stress.

[0078] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same and similar parts among the various embodiments, reference can be made to each other.

[0079] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of rice OsKEA1 gene in regulating rice salt tolerance, characterized in that: The nucleotide sequence of the rice OsKEA1 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The regulation is positive regulation.

3. The use according to claim 1, characterized in that For regulating rice Na + / K + Homeostasis, salt stress-related gene expression, and chloroplast development.

4. The use according to claim 3, characterized in that The salt stress-related genes include NHX2, SOS2, OsHKT4, SIT1, SIT2 and DST1 genes.

5. Application of knocking out the rice OsKEA1 gene in constructing a rice model with reduced salt tolerance.

6. Use of a mutant of the rice OsKEA1 gene in constructing a rice model with reduced salt tolerance, characterized in that: The nucleotide sequence of the mutant is shown in SEQ ID NO:3, and the encoded amino acid sequence is shown in SEQ ID NO:4.

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