Application of oscpk12 gene in regulating drought tolerance of rice

Abstract

The application discloses application of an OsCPK12 gene in regulating drought resistance of rice and belongs to the technical field of genetic engineering. The accession number of the OsCPK12 gene is LOC_Os04g47300, and the nucleotide sequence of the OsCPK12 gene is shown as SEQ ID NO. 5. The application proves through experiments that the OsCPK12 is a gene responding to drought, in order to verify the influence of the OsCPK12 on drought, the wild type ZH8015, a knockout mutant oscpk12-cr and an overexpression family OsCPK12-OE are subjected to drought treatment, and the result shows that the overexpression family OsCPK12-OE is more drought-resistant.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the OsCPK12 gene in regulating drought resistance in rice. Background Technology

[0002] Approximately 90% of crop dry weight comes from organic matter produced by photosynthesis, making improved photosynthetic efficiency a key objective for increasing crop yield (Gibson et al., 2011). Light is a crucial environmental determinant of photosynthesis. In nature, plants experience fluctuating light intensities; both excessively low and high light levels affect photosynthetic efficiency. Therefore, plants have evolved unique physiological mechanisms to adapt to varying external light conditions, ensuring optimal and efficient photosynthesis. Chloroplast development is controlled by genes in both the plastids and the nucleus (Jarvis and Lopez-Juez, 2013). In rice, over 95% of the genes responsible for chloroplast development are encoded by nuclear genes (Reumann et al., 2005). Chloroplast proteins synthesized by these nuclear genes are produced in the cytoplasm, and subsequently, these precursor proteins are transported to designated locations within the chloroplast via transmembrane transport complexes to perform specific functions. The sequences responsible for introducing these proteins into the chloroplast are called chloroplast transport peptides (cTPs). These cTPs are protein sequences essential for targeting proteins to chloroplasts (Li et al., 2013). Nuclear genes encoding chloroplast proteins are typically activated by light-responsive transcription factors such as HY5 (Han et al., 2020). Following transcription and translation in the cytoplasm, these prechloroplast proteins undergo post-translational modifications (PTM). Subsequently, they are introduced into the chloroplast stroma via two translocation codon complexes: translocation codons in the outer chloroplast membrane (TOC) and translocation codons in the inner chloroplast membrane (TIC) (Chen et al., 2018). The phosphorylation state of chloroplast transport peptides plays a crucial role in regulating the input of preproteins (Martinet et al., 2006). Fluctuating light conditions in the natural environment often lead to dynamic photosynthesis in plant leaves. To maintain high photosynthetic efficiency and prevent damage to photosynthetic structures under such conditions, plants have evolved various strategies (Morales and Kaiser, 2020). The adaptive mechanisms of plants to fluctuating light mainly include state transitions, cyclic electron transport, and other pathways independent of these two processes. Reversible phosphorylation of proteins in chloroplasts plays a crucial role in state transitions. When PSII is overstimulated relative to PSI, thylakoid protein kinases such as STATE TRANSITION7 (SNT7) are activated, phosphorylating LHCII (pLHCII). This leads to pLHCII binding to PSI instead of PSII, enhancing the absorption and utilization of light by PSI (Bellafiore et al., 2005; Grieco et al., 2015).Furthermore, SNT7-mediated pLHCII phosphorylation translates changes in plastoquinone libraries and matrix redox status into signals regulating the expression of photosynthetic-related genes, fine-tuning the photosynthetic apparatus in response to rapid light changes; this process corresponds to state 2 (Pesaresi et al., 2010). However, when energy is in excess at PSI, PQ libraries are oxidized, leading to SNT7 inactivation. This inactivation, via PROTEIN PHOSPHATASE1 / THYLAKOID-ASSOCIATED PHOSPHATASE 38 (PPH1 / TAP38), dephosphorylates pLHCII, separating LHCII from PSI and binding it to PSII to form state 1, which facilitates the redistribution of energy to PSII (Pribil et al., 2010; Shapiguzov et al., 2010). Currently, little is known about the precise role of chloroplast protein shuttle mechanisms in plant light adaptation regulation.

[0003] Plants are subjected to various adverse environmental stimuli during their growth and development, which negatively impact their growth, development, and crop yield (Munns and Millar, 2023). With the increasing severity of global warming, drought is becoming a major limiting factor for plant growth and crop production (Wilschut et al., 2022). Under drought stress, plant morphology and physiological processes are affected to varying degrees. In particular, drought affects photosynthetic characteristics, chlorophyll accumulation, reactive oxygen species (ROS) metabolism, and the antioxidant system (Li et al., 2022a, Li et al., 2022b; Ahuja et al., 2010; Ahammed et al., 2019). Therefore, elucidating the mechanisms of drought stress is a prerequisite for breeding drought-resistant and improved crop germplasm. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the OsCPK12 gene in regulating drought resistance in rice, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] One of the technical solutions of this invention is the application of the OsCPK12 gene in regulating drought resistance in rice. The accession number of the OsCPK12 gene is LOC_Os04g47300, and its nucleotide sequence is shown in SEQ ID NO.5.

[0007] The second technical solution of the present invention is a method for regulating the drought resistance of rice by silencing or knocking out the OsCPK12 gene to reduce the drought resistance of rice; and by overexpressing the OsCPK12 gene to improve the drought resistance of rice.

[0008] The third technical solution of the present invention is the application of the method in drought-resistant rice breeding or drought-resistant rice variety improvement.

[0009] The fourth technical solution of the present invention is the application of OsCPK12 protein in regulating drought resistance in rice, and its amino acid sequence is shown in SEQ ID NO.6.

[0010] Based on the above technical solution, the present invention has the following technical effects:

[0011] This invention provides the application of the OsCPK12 gene in the process of rice photosynthesis. The accession number for the OsCPK12 gene is LOC_Os04g47300. In this embodiment, an OsCPK12 knockout mutant plant, oscpk12-cr, was constructed, and its agronomic traits and photosynthetic-related physiological indicators were measured. Compared with the wild type, oscpk12-cr exhibited shorter plant height, reduced photosynthetic efficiency, decreased photosynthetic pigment content, reduced expression levels of photosynthetic-related proteins and chlorophyll synthesis-related proteins in leaves, and a premature senescence phenotype. Under low light conditions, the premature senescence phenotype of the mutant oscpk12-cr was suppressed; under continuous light conditions, the premature senescence phenotype of the mutant oscpk12-cr occurred earlier. Overexpression of OsCPK12 can improve the photosynthetic efficiency and drought resistance of rice. Subcellular localization results and protein-protein interaction experiments confirmed that OsCPK12 interacts with and phosphorylates OsAtpD1, thereby mediating the transport of OsAtpD1 to chloroplasts. In this embodiment of the invention, light-induced OsCPK12-mediated phosphorylation of OsAtpD1 promotes its introduction into chloroplasts, participating in ATP synthase, directly affecting ATPase activity, and indirectly affecting the expression of chlorophyll synthesis-related genes and related proteins in the photosynthetic electron transport chain, thus participating in the dynamic regulation of photosynthesis. Overexpression of OsCPK12 can improve the photosynthetic efficiency and drought resistance of rice, providing a theoretical basis for high photosynthetic efficiency breeding and drought-resistant breeding of rice. OsCPK12 affects ATP synthase activity by influencing ATP synthase assembly, and affects the expression of photosynthetic respiratory chain-related proteins, thereby affecting the net photosynthetic rate and photosynthetic pigment content of rice, ultimately affecting the photosynthetic efficiency of rice and participating in the regulation of the dynamic photosynthetic process of rice. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1Compared with ZH8015, most photosynthetic system-related proteins in oscpk12-cr leaves were reduced.

[0014] Figure 2 Compared with ZH8015, most of the proteins related to chlorophyll synthesis in oscpk12-cr leaves were reduced.

[0015] Figure 3 The net photosynthetic rate in the leaves of oscpk12-cr decreased compared to ZH8015.

[0016] Figure 4 Compared with ZH8015, overexpression of OsCPK12 can increase the photosynthetic pigment content and net photosynthetic rate of rice leaves.

[0017] Figure 5 The expression levels of OsCPK12 were measured under normal and low light conditions. In this figure, A represents the light intensity measurement under normal and low light conditions; B represents the expression level measurement of OsCPK12 under normal and low light conditions.

[0018] Figure 6 The etiolation phenotype of the oscpk12-cr knockout transgene was suppressed under low light conditions. A represents the phenotypes of ZH8015 and oscpk12-cr knockout transgenes under normal and low light conditions; B represents the chlorophyll a content in leaves of ZH8015 and oscpk12-cr knockout transgenes under normal and low light conditions; C represents the chlorophyll b content in leaves of ZH8015 and oscpk12-cr knockout transgenes under normal and low light conditions; D represents the carotenoid content in leaves of ZH8015 and oscpk12-cr knockout transgenes under normal and low light conditions. Data in B and D are expressed as mean ± standard deviation. Student's t-test was used to calculate significance, **: phenotype is highly significant at the P < 0.01 level.

[0019] Figure 7 Phenotypes of ZH8015 and oscpk12-cr knockout transgenes 5 days after being transferred from low light to normal light conditions.

[0020] Figure 8This study aimed to promote the etiolation phenotype of the oscpk12-cr knockout transgenic seedlings under continuous light treatment. Specifically, AC represents the phenotype of ZH8015 and oscpk12-cr knockout transgenic seedlings after 15 days of normal culture, followed by 7 days of continuous light treatment. D shows the chlorophyll a content in the leaves of ZH8015 and oscpk12-cr knockout transgenic seedlings after 7 days of continuous light treatment; E shows the chlorophyll b content in the leaves of ZH8015 and oscpk12-cr knockout transgenic seedlings after 7 days of continuous light treatment; and F shows the carotenoid content in the leaves of ZH8015 and oscpk12-cr knockout transgenic seedlings after 7 days of continuous light treatment. Data in DF are expressed as mean ± standard deviation. Student's t-test was used to calculate significance, with ** indicating highly significant phenotypes at the P < 0.01 level.

[0021] Figure 9 To confirm the interaction between OsCPK12 and OsAtpD1 using LCI (LUC complementation imaging). A shows the co-transformation of OsCPK12-CLuc and NLuc-AtpD1 into tobacco. B shows NLuc and CLuc as negative controls. C shows the capture of luminescence signals using a low-light-cooled CCD imaging system 2 days after transformation. D shows the quantitative analysis of LUC activity in leaves. Error bars represent standard deviation (SD), n=3; (D) Immunoprecipitation (Co-IP) assay to verify the interaction between OsCPK12 and OsAtpD1. Total protein was extracted from rice protoplasts transiently transformed with OsCPK12-Myc / Myc-Cluc and OsAtpD1-GFP. Immunoprecipitation was performed using anti-GFP agarose beads. Crude lysate proteins and immunoprecipitated proteins were detected using anti-GFP and anti-Myc antibodies.

[0022] Figure 10 OsCPK12 phosphorylates OsAtpD1. OsCPK12 phosphorylates OsAtpD1 in vitro. The sampled proteins His-TF-OsCPK12 and GST-OsAtpD1 were visualized using Coomassie Brilliant Blue (CBB) staining. The phosphorylation status of GST-OsAtpD1 was analyzed by Western blotting using 7% 50 μM Phos-TagSDS-PAGE.

[0023] Figure 11 The images show the subcellular localization results of OsAtpD1 in ZH8015 and oscpk12-cr rice protoplasts. In A, the image shows the subcellular localization results of OsAtpD1 in ZH8015 rice protoplasts, with the GFP and RFP fluorescence intensity distribution shown in the right box. In B, the image shows the subcellular localization results of OsAtpD1 in oscpk12-cr rice protoplasts, with the GFP and RFP fluorescence intensity distribution shown in the right box.

[0024] Figure 12 The expression levels of OsAtpD1 in ZH8015 leaves under normal and low light conditions.

[0025] Figure 13 This study investigated how light-induced phosphorylation of OsCPK12 into OsAtpD1 promotes chloroplast translocation. Figure A shows the abundance of OsCPK12 protein in total protein from ZH8015 leaves at different time points throughout the day; Figure B shows the abundance of OsAtpD1 protein in total protein from ZH8015 leaves at different time points throughout the day. The measurements in the figure are relative levels of target proteins normalized to the corresponding actin bands; Figure C shows the abundance of OsAtpD1 protein in chloroplast proteins from ZH8015 leaves at different time points throughout the day. The measurements in the figure are relative levels of target proteins normalized to the corresponding RbcL bands; Figure D shows the abundance of OsAtpD1 protein in chloroplast proteins from oscpk12-cr leaves at different time points throughout the day. The measurements in the figure are relative levels of target proteins normalized to the corresponding RbcL bands.

[0026] Figure 14 To analyze the expression levels of OsCPK12 under normal and drought conditions.

[0027] Figure 15 To improve the drought resistance of rice by overexpressing OsCPK12.

[0028] Figure 16 Analysis of different haplotypes corresponding to the coding region of gene OsCPK12. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0035] This invention provides the application of the OsCPK12 gene in regulating drought resistance in rice. The accession number of the OsCPK12 gene is LOC_Os04g47300, and its nucleotide sequence is shown in SEQ ID NO.5.

[0036] In some specific implementation schemes, silencing or knocking out the OsCPK12 gene reduces the drought resistance of rice; overexpressing the OsCPK12 gene increases the drought resistance of rice.

[0037] This invention also provides a method for regulating the drought resistance of rice: silencing or knocking out the OsCPK12 gene to reduce the drought resistance of rice; and overexpressing the OsCPK12 gene to improve the drought resistance of rice.

[0038] In some specific embodiments, the method for overexpressing the OsCPK12 gene is selected from at least one of the following methods:

[0039] 1) By importing a plasmid containing the gene;

[0040] 2) By increasing the copy number of the aforementioned genes on plant chromosomes;

[0041] 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes;

[0042] 4) By operatively linking a strong promoter to the gene;

[0043] 5) By introducing enhancers.

[0044] In some specific implementation schemes, Agrobacterium-mediated transformation is used to transfer a recombinant expression vector carrying the CDS sequence of the OsCPK12 gene into rice callus tissue. The transformed material undergoes co-culture screening, differentiation, rooting, hardening-up of transgenic seedlings, and transplanting to screen for transgenic positive rice plants.

[0045] In some specific implementations, the recombinant expression vector is pCAMBIA2300 or pCAMBIA1300-GFP-flag.

[0046] In some specific implementations, the method for preparing the recombinant vector includes: using the gene OsCPK12 as the target gene, designing a CRISPR / Cas9-based sgRNA sequence, ligating a DNA fragment containing the encoding the sgRNA sequence into a vector carrying CRISPR / Cas9, transforming rice, and thus obtaining transgenic rice with the gene function lost.

[0047] In some specific implementations, the nucleotide sequence of the sgRNA action site is 5'-GTTGTCGAAGAACTCGGCG-3.

[0048] In some specific implementation schemes, the OsCPK12 gene is constructed into the vector pCAMBIA2300 to obtain a recombinant vector, which is then transformed into the indica rice variety ZH8015. Positive transgenic rice plants are then screened to obtain OsCPK12 overexpression families.

[0049] The specific method is as follows: Using primers OsCPK12-OE-F and OsCPK12-OE-R, PCR amplification was performed using the cDNA of indica rice variety ZH8015 as a template to obtain the full-length cDNA sequence of OsCPK12 as shown in SEQ ID NO.5. Then, the fragment was recombined into the XmaI / XbaI site of the pCAMBIA2300 vector through homologous recombination. The result was transferred into the indica rice variety ZH8015 through Agrobacterium EHA105-mediated genetic transformation. Later, transgenic families overexpressing OsCPK12 were obtained through hygromycin screening and quantitative analysis.

[0050] In some specific implementations, the Agrobacterium is EHA105.

[0051] The present invention also provides the application of the method in drought-resistant rice breeding or drought-resistant rice variety improvement.

[0052] In some specific implementation plans, breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0053] This invention also provides the application of OsCPK12 protein in regulating drought resistance in rice, the amino acid sequence of which is shown in SEQ ID NO.6.

[0054] Knocking out the OsCPK12 gene in rice leads to stunted plant height, reduced expression of photosynthetic-related proteins in leaves, and the emergence of a premature senescence phenotype. Under low light conditions, the premature senescence phenotype in the mutant is suppressed; under continuous light conditions, the premature senescence phenotype occurs earlier. OsCPK12 participates in the dynamic regulation of photosynthesis by interacting with and phosphorylating OsAtpD1, thus mediating the transport of OsAtpD1 to chloroplasts. Overexpression of OsCPK12 can improve the photosynthetic efficiency and drought resistance of rice.

[0055] The OsCPK12 protein is localized in the nucleus, cytoplasm, and cell membrane, and its expression level is high in green tissues. In this invention, by using CRISPR / Cas9 gene editing technology to mutate the OsCPK12 gene, a mutant oscpk12-cr was obtained, which resulted in stunted growth and reduced photosynthesis. This invention found that the yellowing phenotype of the mutant oscpk12-cr is light-induced; weak light inhibits the occurrence of the phenotype, while strong light promotes its occurrence. The expression of chlorophyll synthesis-related genes and related proteins in the photosynthetic electron transport chain is decreased in the leaves of oscpk12-cr plants. Further research revealed that OsCPK12 interacts with and phosphorylates OsAtpD1, thereby mediating the transport of OsAtpD1 to chloroplasts. Furthermore, light can induce the expression of OsCPK12 and promote the transport of OsCPK12 phosphorylated into OsAtpD1 to chloroplasts; overexpression of OsCPK12 can improve the photosynthetic efficiency of rice, which can provide a theoretical basis for high photosynthetic efficiency breeding and drought-resistant breeding of rice.

[0056] The wild-type ZH8015 used in this embodiment of the invention is a restorer line bred by our research group using Zhonghui 218, a self-bred restorer line resistant to bacterial blight, as the female parent and Zhonghui 8006 as the male parent, through multiple generations of selection and identification of resistance to bacterial blight and rice blast.

[0057] Example 1

[0058] Gene selection and material creation

[0059] Approximately 90% of a crop's dry weight comes from organic matter produced through photosynthesis; therefore, the strength and efficiency of crop photosynthesis are crucial factors determining crop yield. Researchers aim to improve crop photosynthetic efficiency by modifying its photosynthetic characteristics to cultivate high-yielding and ultra-high-yielding varieties—a process known as High Photosynthesis Efficiency (HPE) breeding. Currently, even high-yielding rice has a light energy utilization rate of only 1.5%-2.0%. If rice light energy utilization could reach the ideal level of 3%-5%, rice yield would increase significantly. This fully demonstrates the enormous potential of improving rice yield through enhanced photosynthetic efficiency in ensuring food security and promoting sustainable agricultural development.

[0060] Overexpression of OsCPK12 can significantly improve the photosynthetic efficiency and drought resistance of rice. The results of this study show that OsCPK12 participates in regulating the dynamic photosynthetic process of rice. To determine the function of the rice gene OsCPK12, knockout and overexpression plants were constructed in the ZH8015 background to reveal the function of this gene.

[0061] The primer sequences used to construct the knockout transgenic vector are (5′-3′):

[0062] OsCPK12-Cas9-F (SEQ ID NO.1): AGATGATCCGTGGCATCGACCGCATCACGGCCAAGGGGGTTTTAGAGCTATGC;

[0063] OsCPK12-Cas9-R (SEQ ID NO. 2): GCATAGCTCTAAAACCCCCTTGGCCGTGATGCGGTCGATGCCACGGATCATCT.

[0064] The primer sequences used to construct the overexpression transgenic vector are (5′-3′):

[0065] OsCPK12-OE-F (SEQ ID NO.3): GTAGAAGAGGTACCCGGGCACCATTCCAC CCTCGCTT;

[0066] OsCPK12-OE-R (SEQ ID NO. 4): GCAGGTCGACTCTAGATAACTTTTTTGCGTTTCATCTGC.

[0067] Example 2

[0068] OsCPK12 knockout plants exhibit a yellowing phenotype, with reduced photosynthetic rate and decreased photosynthetic pigment content in leaves, and downregulated expression of most related proteins in the photosynthetic electron transport chain.

[0069] The vector used for knockout was pCas9-AarI, with prokaryotic resistance of spectinomycin and eukaryotic resistance of hygromycin. The target sequence for the OsCPK12 gene was designed using an online website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR). A sequence located in the CDS region and with a high score close to the ATG start site was selected as the sgRNA. Corresponding adapters were added to form F primers, and the F primer sequences were reverse-complemented to form R primers. Primers F and R were mixed and reacted at 94℃ for 10 min, then annealed at 0.1℃ / s to 15℃, and held at 15℃ for 10 min to complete annealing and form double strands. The pCas9 vector was digested overnight with AarI, and the target band was recovered by gel extraction. 1 μL of the double-stranded product was recombined with the recovered pCas9-AarI linear vector, transformed into DH5α, plated on spectinomycin-resistant plates, and incubated overnight at 37℃. The next day, single clones were selected and sequenced at Shangya Biotechnology Co., Ltd. Finally, the successfully sequenced plasmid was sent to Wuhan Boyuan Biotechnology Co., Ltd. for genetic transformation in the background of indica rice ZH8015. The T0 generation transgenic plants were sequenced, and the homozygous mutant was selected and named oscpk12-cr for subsequent research.

[0070] The knockout mutant oscpk12-cr exhibited a yellowing phenotype. To investigate the reason for the yellowing phenotype after OsCPK12 knockout, leaves from wild-type ZH8015 and the knockout mutant were collected before the phenotype appeared for TMT proteomics analysis. The results showed that, compared with ZH8015, the downregulated proteins in oscpk12-cr leaves mainly included photosystem II, photosystem I, cytochrome b6 / f complex, and components of photosynthetic electron transport (…). Figure 1 Most chlorophyll synthesis-related proteins were downregulated. Figure 2 ).

[0071] 2. Net photosynthetic rate was measured using a LI-6400XT portable photosynthesis meter. On a sunny morning between 9:30 and 11:00 during the heading stage, the third leaf from the bottom of wild-type ZH8015 and oscpk12-cr transgenic families was selected for photosynthetic rate measurement.

[0072] Parameter settings: Photon density 1200 μmol·m -2 ·s -1 Flow rate 500 μmol·s -1Three measurements of every three leaves were considered as one replicate, and statistical analysis was performed on the three replicates. Photosynthetic pigment and chlorophyll content were determined according to the methods of ARNON and PORRA et al. (Arnon, 1949; Porra et al., 2010).

[0073] Net photosynthetic rate measurements showed that, compared with the wild type, the net photosynthetic rate in the leaves of the OsCPK12 knockout transgenic plant oscpk12-cr was significantly reduced. Figure 3 ).

[0074] 3. Obtain transgenic plants overexpressing OsCPK12

[0075] Using primers OsCPK12-OE-F and OsCPK12-OE-R, PCR amplification was performed using cDNA from the indica rice variety ZH8015 as a template to obtain the full-length cDNA sequence of OsCPK12 as shown in SEQ ID NO. 5. This fragment was then recombined into the XmaI / XbaI site of the pCAMBIA2300 vector via homologous recombination. The resulting DNA was then transferred into the indica rice variety ZH8015 via Agrobacterium EHA105-mediated genetic transformation. Overexpression of OsCPK12 increased the photosynthetic pigment content and net photosynthetic rate in rice plant leaves. Figure 4 ).

[0076]

[0077] The protein sequence encoded by OsCPK12 is shown in SEQ ID NO.6.

[0078] SEQ ID NO.6: MGNCFTKTYEIPITSGTMRRPASTAERSKARGGDEPGTWRRPSF PRHGAPPHRPPTGSSSAAGALSRRASGGGGEMGPVLQRAMVSVRSLYQLDRKLGSGQFGTTYLCTERATGNRYACKSVSKRKLVRRTDVDDVRREITILQHLSGQPNIAEFRGAYEDNDHVH LVMEFCSGGELFDRITAKGSYSERQAAAVCRDILTVVHVCHFMGVIHRDLKPENFLLASADDDAPLKAIDFGLSVFIEEGKVYKDIVGSAYYVAPEVLQRNYGKEADIWSAGVILYILLCGTP PFWAETEKGIFDAILVNQVDFSTSPWPSISESAKDLIRQMLHRDPQKRITASQALEHRWLKEGGASDRPIDSAVLSRMKQFKAMNKLKQLALKVIAENLSPEEIKGLKQMFNNMDTDRSGTIT VEELKVGLTKLGSRISEAEVQKLMEAVDVDKGSSIDYSEFLTAMINKHKLEKEEDLLRAFQHFDKDNSGYITRDELEQAMAEYGMGDEANIKQVLDEVDKDKDGRIDYEEFVEMMRKGIQT*.

[0079] Example 3

[0080] The yellowing phenotype of oscpk12-cr knockout plants is light-induced. To verify that the yellowing phenotype of oscpk12-cr is light-induced, yellowing was observed under natural light (normal light) and under conditions covered with black netting (low light). Figure 5(A) Observe the phenotypes of wild-type ZH8015 and the mutant oscpk12-cr. To detect whether OsCPK12 is light-induced, total RNA was extracted from leaf samples of ZH8015 at different time points throughout the day using Tiangen's plant RNA extraction kit. The total RNA was then reversed to cDNA using Toyobo's ReverTra-Ace qPCR-RT-Master Mix quantitative reverse transcription kit. qRT-PCR was then performed using Takala's ExTaqII quantitative kit in a Light Cycler 480II instrument, following the manufacturer's instructions. UBQ10 was used as an internal control. The expression level of OsCPK12 was analyzed using OsCPK12-qRTF / OsCPK12-qRTR as shown in Table 1. The quantitative analysis results showed that light does indeed induce the expression of OsCPK12. Figure 5 (B) Under natural light conditions, the mutant oscpk12-cr exhibits a premature aging phenotype. Figure 6 In the mutant (A), the content of photosynthetic pigments in the leaves was significantly lower than that in the wild type (A). Figure 6 (B~D), but under the condition of covering with black netting, the mutant oscpk12-cr did not show the premature aging phenotype. Five days after the black netting was removed, the premature aging phenotype of the mutant oscpk12-cr reappeared. Figure 7 Wild-type ZH8015 and the mutant oscpk12-cr were cultured in a normal hydroponic incubator for 15 days. A portion of these were then transferred to continuous light conditions. It was found that after 7 days of continuous light exposure, the mutant exhibited a premature aging phenotype. Figure 8 In the mutant (A-C), the content of photosynthetic pigments in the leaves was significantly lower than that in the wild type. Figure 8 The yellowing phenotype of wild-type ZH8015 and the mutant oscpk12-cr under normal light conditions was light-induced (D-F), while the phenotypes of wild-type ZH8015 and the mutant oscpk12-cr under normal light conditions were not significantly different. These results indicate that the yellowing phenotype of oscpk12-cr knockout plants is light-induced, and light induces the expression of OsCPK12.

[0081] Table 1 RT-qPCR primers

[0082]

[0083] Example 4

[0084] OsCPK12 and OsAtpD1 interact

[0085] The CDS sequence of OsCPK12 (with terminator removed) was amplified and ligated to the KpnI / SalI site of the CLuc vector, and the CDS sequence of OsAtpD1 was amplified and ligated to the SalI site of the NLuc vector. The constructed vectors were transformed into Agrobacterium GV3101, and different combinations of Agrobacterium competent cells were transiently expressed in tobacco leaves. After 24-48 h of culture, the leaves were cut off, sprayed with luciferase substrate, and treated in the dark for 10 min. The leaves were then placed on the black panel of an imaging device to detect fluorescence signals. Additionally, samples were taken near the injection site and immersed in ddH2O in a cell culture plate. After sampling for all combinations, the ddH2O was removed, 200 μL of luciferase substrate was added, and the samples were treated in the dark at room temperature for 15 min. The signal intensity was detected using a Glomax instrument. Amplification primers are shown in Table 2. Luciferase complementation imaging analysis results showed that OsCPK12 may interact with the δ subunit of chloroplast ATP synthase, OsAtpD1. Figure 9 (A-C). Subsequently, yeast two-hybrid point-to-point verification was used to confirm that OsCPK12 interacts with OsAtpD1. To further verify the interaction between OsCPK12 and OsAtpD1, OsCPK12-Myc and OsAtpD1-GFP were constructed in this embodiment. After transient transformation in rice protoplasts for 20 h, total protein was extracted and immunoprecipitated using anti-GFP magnetic beads. Co-IP results showed that OsAtpD1-GFP could pull down OsCPK12-Myc, but could not pull down Myc-Luc (…). Figure 9 (D).

[0086] Table 2 Primers used in protein interaction, in vitro phosphorylation, and subcellular localization experiments.

[0087]

[0088] Example 5

[0089] OsCPK12 phosphorylation of OsAtpD1

[0090] To verify whether OsAtpD1 is a substrate protein of OsCPK12, the CDS sequence encoding OsCPK12 was fused into the pCold-TF vector containing a His tag, and the CDS sequence of OsAtpD1 was fused into the pGEX-4T-1 vector containing a GST tag. After transformation into BL21 bacteria, sequencing was performed. Positive clones were selected and purified after induction with 0.1 mM IPTG. GST-tagged and His-tagged proteins were purified using BeaverBeads GSH (catalog number: 70601-100, XIYAN CO., LTD.) and a His-tag protein purification kit (catalog number: P2226, Beyotime Biotechnology Co., Ltd.), respectively. The purified proteins were then quantified using the NCM BCA protein analysis kit (catalog number: WB6501, New Cell & Molecular Biotech Co., Ltd.).

[0091] Approximately 2 μg of kinase (His-TF-OsCPK12(Dead)) and 1 μg of substrate (OsAtpD1-GST) protein were mixed with kinase buffer (100 mM Tris-HCl [pH 8.0], 5 mM DTT, 5 mM EGTA, 5 mM MgCl2 and 100 μM ATP) to a total volume of 30 μL. The mixture was incubated at 30 °C for 0–30 min, and then heated at 100 °C for 1 min to stop the reaction. Protein buffer was added, and the mixture was boiled for 8 min to denature the protein. The phosphorylation of OsAtpD1 was then detected by immunoblotting using a 7% acrylamide gel containing 50 μM hos-tag (Phos-tag AAL-107; Wako), and the amount of protein loaded was indicated by CBB staining.

[0092] The results showed that after incubation with His-TF-OsCPK12 (Dead) and OsAtpD1-GST, and after incubation of His-TF-OsCPK12 and OsAtpD1-GST in ATP-free kinase buffer for 30 minutes, only one band of OsAtpD1-GST was observed. However, when His-TF-OsCPK12 and OsAtpD1-GST were incubated in normal kinase buffer, a phosphorylated OsAtpD1-GST band appeared, and the phosphorylated OsAtpD1-GST band gradually deepened with increasing incubation time. Figure 10 The above results indicate that OsAtpD1 is a substrate protein of OsCPK12, and that OsCPK12 can phosphorylate OsAtpD1.

[0093] Example 6

[0094] OsCPK12 affects the subcellular localization of OsAtpD1

[0095] To demonstrate the effect of OsCPK12 on OsAtpD1, the CDS of OsAtpD1 without the terminator was ligated into a vector containing an eGFP tag to construct an OsAtpD1-eGFP fusion vector. The vector was then transiently transformed into wild-type ZH8015 and mutant oscpk12-cr protoplasts. Protoplast protein extraction and subcellular localization results were observed 14 h after transformation.

[0096] The results showed no significant difference in OsAtpD1 protein levels between wild-type ZH8015 and the mutant oscpk12-cr background, indicating that OsCPK12 does not affect the protein stability of OsAtpD1. OsAtpD1 is mainly located in chloroplasts in wild-type ZH8015 protoplasts, but mainly in the cytoplasm in oscpk12-cr protoplasts. Figure 11 The above results indicate that OsCPK12-mediated phosphorylation of OsAtpD1 may alter its subcellular localization.

[0097] Example 7

[0098] Photoinduced phosphorylation of OsCPK12 into OsAtpD1 to promote chloroplast translocation

[0099] Light is the primary energy source for plant photosynthesis (Yousef et al., 2021), and the daily fluctuations in plant photosynthesis are closely related to changes in light intensity. This invention demonstrates that light induces OsCPK12 expression. To investigate whether the OsCPK12-OsAtpD1 pathway participates in dynamic photosynthesis, the expression levels of OsAtpD1 were analyzed using the OsAtpD1-qRTF / OsAtpD1-qRTR ratios listed in Table 1, detecting the expression levels of OsAtpD1 at different time points. This invention observed that OsAtpD1 expression is also light-induced, with its peak expression occurring at 8 o'clock, indicating that OsAtpD1 is particularly sensitive to light. Figure 12 Further analysis was conducted on the protein abundance of OsCPK12 and OsAtpD1 in the total protein and chloroplast protein of ZH8015 and oscpk12-cr leaves under different light durations. This invention found that the protein levels of OsCPK12 and OsAtpD1 increased over time, reaching a peak at 10:00 AM. Figure 13 (A and B). However, in the chloroplast proteins of wild-type ZH8015 and oscpk12-cr, the abundance of OsAtpD1 protein peaked at 12 noon ( Figure 13In the chloroplast proteins of oscpk12-cr, the protein abundance of OsAtpD1 was significantly reduced (C), but the protein abundance of OsAtpD1 was significantly reduced (C). Figure 13 (Middle D). These results indicate that increased light intensity promotes the accumulation of OsCPK12 and OsAtpD1, and that OsCPK12 phosphorylation of OsAtpD1 is crucial for chloroplast translocation. These results demonstrate that light-induced OsCPK12 phosphorylation of OsAtpD1 promotes chloroplast translocation and participates in dynamic photosynthesis. This mechanism is essential for protecting rice from light damage under strong light conditions.

[0100] Example 8

[0101] Overexpression of OsCPK12 can improve drought resistance in rice.

[0102] A portion of the ZH8015 seedlings that had been hydroponically cultured for 15 days were removed from the hydroponic box and subjected to drought treatment, while another portion continued hydroponics as a control. Samples were taken from the control group and the treatment group at 0, 0.5, 1, 2, 4 and 8 hours after treatment for quantitative analysis.

[0103] The results showed that drought could induce the expression of OsCPK12. The expression level of OsCPK12 was highest after 1 hour of drought treatment, and then the expression level of OsCPK12 began to decrease. Figure 14 Therefore, OsCPK12 is a drought-responsive gene. To verify the effect of OsCPK12 on drought, this invention subjected wild-type ZH8015, the knockout mutant oscpk12-cr, and the overexpressing family OsCPK12-OE to drought treatment. This invention found that drought can promote the yellowing phenotype of oscpk12-cr, and the OsCPK12-OE family is more drought-tolerant. Figure 15 ).

[0104] Example 9

[0105] Haplotype analysis of gene OsCPK12 in rice genome

[0106] Haplotype analysis of the coding region of gene OsCPK12 was performed using the 3K Rice Genome Database (http: / / www.rmbreeding.cn / Index). The results showed that it could be divided into 6 haplotypes (Hap). Figure 16 ZH8015 belongs to Hap1. Japonica rice varieties and Bas varieties are mainly Hap2 and Hap4, while indica rice varieties are mainly Hap1, Hap3, Hap5, and Hap6. These results indicate that the OsCPK12 gene has been artificially selected in breeding.

[0107] OsCPK12, derived from the rice restorer line Zhonghui 8015, encodes a 534-amino acid protein kinase containing an N-terminal variable region, a kinase domain, an autoinhibition domain, and a calcium-binding domain. Knocking out the OsCPK12 gene using CRISPR / Cas9 gene editing technology yielded the mutant oscpk12-cr. Compared to the wild type, oscpk12-cr exhibited shorter plant height, reduced expression levels of photosynthesis-related proteins and chlorophyll synthesis-related proteins in leaves, and a premature senescence phenotype. Under low light conditions, the premature senescence phenotype in the mutant oscpk12-cr was suppressed; under continuous light conditions, the premature senescence phenotype occurred earlier in the mutant oscpk12-cr. Overexpression of OsCPK12 improved the photosynthetic efficiency and drought resistance of rice. Subcellular localization results and protein-protein interaction experiments confirmed that OsCPK12 interacts with and phosphorylates OsAtpD1, thereby mediating the transport of OsAtpD1 to chloroplasts. The accession number for the OsCPK12 gene described in this invention is LOC_Os04g47300. In this embodiment, the light-induced OsCPK12 mediates OsAtpD1 phosphorylation, promoting its introduction into chloroplasts to participate in ATP synthase, directly affecting ATPase activity, and indirectly affecting the expression of chlorophyll synthesis-related genes and related proteins in the photosynthetic electron transport chain, thereby participating in the dynamic regulation of photosynthesis. This invention can provide candidate genes and theoretical basis for high photosynthetic efficiency breeding and drought-resistant breeding of rice.

[0108] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. OsCPK12 The application of genes in regulating drought resistance in rice is characterized by, The OsCPK12 The gene's accession number is LOC_Os04g47300 Its nucleotide sequence is shown in SEQ ID NO.

5.

2. The application according to claim 1, characterized in that, Silence or Knockout OsCPK12 Genes that reduce drought resistance in rice; overexpression OsCPK12 Genes that improve the drought resistance of rice.

3. A method for regulating drought resistance in rice, characterized in that, Silence or Knockout OsCPK12 Genes that reduce the drought resistance of rice; overexpression OsCPK12 Genes that enhance the drought resistance of rice; The OsCPK12 The gene's accession number is LOC_Os04g47300 Its nucleotide sequence is shown in SEQ ID NO.

5.

4. The method according to claim 3, characterized in that, The overexpression OsCPK12 The gene-based approach is selected from at least one of the following methods: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the genes mentioned on the plant chromosomes.

5. The application of the method as described in claim 3 or 4 in drought-resistant rice breeding or drought-resistant rice variety improvement.

6. The application of OsCPK12 protein in regulating drought resistance in rice, characterized by: Its amino acid sequence is shown in SEQ ID NO. 6.

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