Application of OsCPK12 gene in regulation and control of photosynthesis of rice

Abstract

The invention discloses application of an OsCPK12 gene in regulation and control of photosynthesis of rice, and belongs to the technical field of gene engineering. The login number of the OsCPK12 gene is LOCOs04g47300, and the nucleotide sequence of the OsCPK12 gene is as shown in SEQ ID NO. 5. Photo-induction OsCPK12 mediated OsAtpD1 phosphorylation promotes introduction of OsAtpD1 into chloroplast to participate in ATP synthase, directly affects ATP enzyme activity, and indirectly affects expression of chlorophyll synthesis related genes and related proteins on a photosynthetic electron transport chain so as to participate in a dynamic regulation process of photosynthesis. The invention can provide candidate genes and theoretical basis for high photosynthetic efficiency breeding of rice.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of OsCPK12 gene in regulating rice photosynthesis. Background Art

[0002] Approximately 90% of crop dry weight is derived from organic matter produced by photosynthesis, so improving photosynthetic efficiency is a key goal for increasing crop yields (Gibson et al., 2011). Light is a key environmental determinant of photosynthesis. In nature, plants experience fluctuating light intensities, and both low and high light levels affect photosynthetic efficiency. Therefore, plants have evolved unique physiological mechanisms to adapt to different external light conditions and ensure optimal and efficient maintenance of photosynthesis. Chloroplast development is controlled by genes in both plastids and nuclei (Jarvis and Lopez-Juez, 2013). In rice, more than 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 chloroplasts through transmembrane transport complexes to perform specific functions. The sequence responsible for importing these proteins into the chloroplast is called chloroplast transit peptide (cTP). These cTPs are protein sequences that are required for targeting proteins to chloroplasts (Li et al., 2013). Nuclear genes encoding chloroplast proteins are often activated by light-responsive transcription factors such as HY5 (Han et al., 2020). After transcription and translation in the cytoplasm, these chloroplast preproteins undergo post-translational modifications (PTMs). Subsequently, they are imported into the chloroplast stroma via two translocation codon complexes: the translocation codon of the chloroplast outer membrane (TOC) and the translocation codon of the chloroplast inner membrane (TIC) (Chen et al., 2018). The phosphorylation state of the chloroplast transit peptide plays a key role in regulating the import of preproteins (Martin et al., 2006). Fluctuating light conditions in the natural environment often lead to dynamic photosynthesis in plant leaves. In order to maintain high photosynthetic efficiency and prevent damage to the photosynthetic apparatus under such conditions, plants have evolved various strategies (Morales and Kaiser, 2020). The adaptive mechanisms of fluctuating light evolved by plants mainly include state transition, cyclic electron transfer, and other pathways independent of these two processes. Reversible phosphorylation of proteins in chloroplasts plays an important role in state transition. When PSII is overstimulated relative to PSI, thylakoid protein kinases such as STATE TRANSITION7 (SNT7) are activated and phosphorylate LHCII (pLHCII). This causes pLHCII to bind to PSI instead of PSII, enhancing the absorption and utilization of light by PSI (Bellafiore et al., 2005; Grieco et al., 2015).In addition, SNT7-mediated pLHCII phosphorylation converts changes in the plastoquinone pool and the redox state of the matrix into signals that regulate the expression of photosynthetic-related genes, fine-tuning the photosynthetic machinery for rapid light changes, a process corresponding to state 2 (Pesaresi et al., 2010). However, when energy is in excess to PSI, the PQ pool is oxidized, leading to SNT7 inactivation and dephosphorylation of pLHCII by PROTEIN PHOSPHATASE1 / THYLAKOID-ASSOCIATED PHOSPHATASE 38 (PPH1 / TAP38), which separates LHCII from PSI and binds to PSII to form state 1, which is conducive to the redistribution of energy to PSII (Pribil et al., 2010; Shapiguzov et al., 2010). At present, little is known about the exact role of the shuttling mechanism of chloroplast proteins in the regulation of plant light adaptation.

[0003] Plants are exposed to various adverse environmental stimuli during their growth and development, which have adverse effects on plant growth and crop yield (Munns and Millar, 2023). With the increasing global warming, drought is becoming a major limiting factor for plant growth and crop production (Wilschut et al., 2022). Under drought stress, the morphological structure and physiological processes of plants are affected to varying degrees. In particular, drought affects photosynthetic characteristics, chlorophyll accumulation, reactive oxygen species (ROS) metabolism, and antioxidant systems (Li et al., 2022a, Li et al., 2022b; Ahuja et al., 2010; Ahammed et al., 2019). Therefore, revealing the mechanism of drought stress is a prerequisite for cultivating drought-tolerant improved crop germplasm. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the OsCPK12 gene in regulating rice photosynthesis, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is the application of the OsCPK12 gene in regulating rice photosynthesis. 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 reducing rice photosynthesis, which comprises silencing or knocking out the OsCPK12 gene in the rice plant.

[0008] The third technical solution of the present invention is a method for improving rice photosynthesis, which overexpresses the OsCPK12 gene in the rice plant.

[0009] A fourth technical solution of the present invention is application of the method in rice breeding or rice variety improvement.

[0010] A fifth technical solution of the present invention is the use of OsCPK12 protein in regulating rice photosynthesis, and its amino acid sequence is shown in SEQ ID NO.6.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] The present invention provides an application of the OsCPK12 gene in the process of participating in rice photosynthesis, and the accession number of the OsCPK12 gene is LOC_Os04g47300. In the embodiment of the present invention, a knockout mutant plant oscpk12-cr of OsCPK12 was constructed, and agronomic traits and photosynthesis-related physiological indicators were investigated. Compared with the wild type, the plant height of oscpk12-cr became shorter, the photosynthetic efficiency was reduced, the content of photosynthetic pigments was reduced, the expression of photosynthesis-related proteins and chlorophyll synthesis-related proteins in the leaves was reduced, and a premature senescence phenotype appeared; the premature senescence phenotype of the mutant oscpk12-cr was suppressed under weak light conditions; the premature senescence phenotype of the mutant oscpk12-cr occurred prematurely under continuous light conditions; overexpression of OsCPK12 can improve the photosynthetic efficiency and drought resistance of rice. Subcellular localization results and protein interaction experiments verified that OsCPK12 interacted with OsAtpD1 and phosphorylated it, thereby mediating the transport of OsAtpD1 to chloroplasts. In an embodiment of the present invention, light-induced OsCPK12 mediated the phosphorylation of OsAtpD1 to promote 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 to participate in the dynamic regulation process of photosynthesis. Overexpression of OsCPK12 can improve the photosynthetic efficiency and drought resistance of rice, and can provide a theoretical basis for high-light-efficiency breeding and drought-resistant breeding of rice. OsCPK12 affects ATP synthase activity by affecting the assembly of ATP synthase, affects the expression of photosynthetic respiratory chain-related proteins to affect the net photosynthetic rate and photosynthetic pigment content of rice, and ultimately affects the photosynthetic efficiency of rice to participate in the regulation of the dynamic photosynthesis process of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 Compared with ZH8015, the expression of most photosynthetic system-related proteins in oscpk12-cr leaves was decreased.

[0015] Figure 2 Compared with ZH8015, the expression of most chlorophyll synthesis-related proteins in oscpk12-cr leaves was decreased.

[0016] Figure 3 The net photosynthetic rate in oscpk12-cr leaves decreased compared with ZH8015.

[0017] Figure 4 Compared with ZH8015, overexpression of OsCPK12 can increase the photosynthetic pigment content and net photosynthetic rate in rice leaves.

[0018] Figure 5 is the expression level of OsCPK12 under normal light and weak light conditions. Wherein, A is the illumination measurement under normal light and weak light conditions; B is the expression level of OsCPK12 under normal light and weak light conditions.

[0019] Figure 6 The yellowing phenotype of oscpk12-cr knockout transgenic plants is suppressed under weak light conditions. Among them, A is the phenotype of ZH8015 and oscpk12-cr knockout transgenic plants under normal light and weak light; B is the determination of chlorophyll a content in leaves of ZH8015 and oscpk12-cr knockout transgenic plants under normal light and weak light; C is the determination of chlorophyll b content in leaves of ZH8015 and oscpk12-cr knockout transgenic plants under normal light and weak light; D is the determination of carotenoid content in leaves of ZH8015 and oscpk12-cr knockout transgenic plants under normal light and weak light. Data in BD are expressed as mean ± standard deviation, and the significance of differences is calculated by Student's t-test. **: The phenotype is extremely significant at the P<0.01 level.

[0020] Figure 7 Figure 2 Phenotypes of ZH8015 and oscpk12-cr knockout transgenics 5 days after transfer from low light to normal light conditions.

[0021] Figure 8The continuous light promotes the yellowing phenotype of oscpk12-cr knockout transgenic plants. Among them, AC shows the phenotype of ZH8015 and oscpk12-cr knockout transgenic seedlings that were cultivated normally for 15 days and treated with continuous light for 7 days. D shows the chlorophyll a content in the leaves of ZH8015 and oscpk12-cr knockout transgenic plants after 7 days of continuous light treatment; E shows the chlorophyll b content in the leaves of ZH8015 and oscpk12-cr knockout transgenic plants after 7 days of continuous light treatment; F shows the carotenoid content in the leaves of ZH8015 and oscpk12-cr knockout transgenic plants after 7 days of continuous light treatment. The data in DF are expressed as mean ± standard deviation, and the significance of the difference is calculated by Student's t-test. **: The phenotype is extremely significant at the P<0.01 level.

[0022] Fig. 9 To confirm the interaction between OsCPK12 and OsAtpD1 using LCI (LUC complementation imaging). A shows OsCPK12-CLuc and NLuc-AtpD1 co-transformed into tobacco. B shows NLuc and CLuc as negative controls. C shows the luminescence signal captured 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) Co-immunoprecipitation (Co-IP) assay verifies the interaction between OsCPK12 and OsAtpD1. Total proteins were 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 probed using anti-GFP and anti-Myc antibodies.

[0023] Fig.10 Phosphorylation of OsAtpD1 by OsCPK12. OsCPK12 phosphorylates OsAtpD1 in vitro. The loaded proteins His-TF-OsCPK12 and GST-OsAtpD1 were visualized using Coomassie Brilliant Blue (CBB) staining. GST-OsAtpD1 phosphorylation was analyzed by immunoblotting using 7% 50 μM Phos-Tag SDS-PAGE.

[0024] Fig.11 The results of subcellular localization of OsAtpD1 in ZH8015 and oscpk12-cr rice protoplasts. A is the subcellular localization result of OsAtpD1 in ZH8015 rice protoplasts, and the right side is the distribution of GFP and RFP fluorescence intensity in the box; B is the subcellular localization result of OsAtpD1 in oscpk12-cr rice protoplasts, and the right side is the distribution of GFP and RFP fluorescence intensity in the box.

[0025] Fig.12 is the expression level of OsAtpD1 in ZH8015 leaves under normal light and weak light conditions.

[0026] Fig.13 Light-induced OsCPK12 phosphorylates OsAtpD1 to promote chloroplast translocation. A is the abundance of OsCPK12 protein in total protein in ZH8015 leaves at different time points of the day; B is the abundance of OsAtpD1 protein in total protein in ZH8015 leaves at different time points of the day. The measured values ​​in the figure are the relative levels of target proteins normalized to the corresponding actin bands; C is the abundance of OsAtpD1 protein in chloroplast proteins in ZH8015 leaves at different time points of the day. The measured values ​​in the figure are the relative levels of target proteins normalized to the corresponding RbcL bands; D is the abundance of OsAtpD1 protein in chloroplast proteins in oscpk12-cr leaves at different time points of the day. The measured values ​​in the figure are the relative levels of target proteins normalized to the corresponding RbcL bands.

[0027] Fig.14 Expression analysis of OsCPK12 under normal conditions and drought conditions.

[0028] Fig.15 To overexpress OsCPK12 to improve drought tolerance in rice.

[0029] Fig.16 Analysis of different haplotypes corresponding to the coding region of the gene OsCPK12. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0036] The embodiment of the present invention provides an application of the OsCPK12 gene in regulating rice photosynthesis. The accession number of the OsCPK12 gene is LOC_Os04g47300, and the nucleotide sequence thereof is shown in SEQ ID NO.5.

[0037] In some specific embodiments, silencing or knocking out the OsCPK12 gene reduces the photosynthesis of rice; overexpressing the OsCPK12 gene increases the photosynthesis of rice.

[0038] In some specific embodiments, the regulating rice photosynthesis includes regulating the net photosynthetic rate, the content of photosynthetic pigments and the expression level of related proteins in the photosynthetic respiratory chain.

[0039] In some specific embodiments, the OsCPK12 gene affects the activity of ATP synthase by affecting the assembly of ATP synthase, affects the expression of photosynthetic respiratory chain-related proteins, thereby affecting the net photosynthetic rate and photosynthetic pigment content of rice and ultimately affecting the photosynthetic efficiency of rice.

[0040] Knocking out the OsCPK12 gene in rice leads to shorter plant height, reduced expression of photosynthesis-related proteins in leaves, and premature senescence phenotypes. The premature senescence phenotype of the mutant is suppressed under weak light conditions, and the premature senescence phenotype of the mutant occurs prematurely under continuous light conditions. OsCPK12 participates in dynamic photosynthesis regulation by interacting with OsAtpD1 and phosphorylating it to mediate the transport of OsAtpD1 to chloroplasts. Overexpression of OsCPK12 can improve the photosynthetic efficiency and drought tolerance of rice.

[0041] The embodiment of the present invention also provides a method for reducing rice photosynthesis, which comprises silencing or knocking out the OsCPK12 gene in the rice plant.

[0042] The embodiment of the present invention also provides a method for improving rice photosynthesis, which comprises overexpressing the OsCPK12 gene in the rice plant.

[0043] In some specific embodiments, the overexpression method is selected from at least one of the following methods:

[0044] 1) by introducing a plasmid carrying the gene;

[0045] 2) by increasing the copy number of the gene on the plant chromosome;

[0046] 3) by changing the promoter sequence of the gene on the plant chromosome;

[0047] 4) by operably linking a strong promoter to the gene;

[0048] 5) By introducing enhancers.

[0049] In some specific embodiments, the recombinant expression vector carrying the CDS sequence of the OsCPK12 gene is transferred into rice callus by Agrobacterium-mediated method, and the transformed material is subjected to co-cultivation screening-differentiation-rooting-transgenic seedling training and transplanting to screen transgenic positive rice plants.

[0050] In some specific embodiments, the recombinant expression vector is pCAMBIA2300 or pCAMBIA1300-GFP-flag.

[0051] In some specific embodiments, the method for preparing the recombinant vector includes: taking the gene OsCPK12 as the target gene, designing a sgRNA sequence based on CRISPR / Cas9, connecting a DNA fragment containing the encoding sgRNA sequence to a vector carrying CRISPR / Cas9, transforming rice, and thereby obtaining transgenic rice with a missing gene function.

[0052] In some specific embodiments, the nucleotide sequence of the sgRNA action site is 5'-GTTGTCGAAGAACTCGGCG-3.

[0053] In some specific embodiments, the OsCPK12 gene is constructed into the vector pCAMBIA2300 to obtain a recombinant vector, which is then transformed into the indica rice variety ZH8015, and positive transgenic rice plants are screened to obtain OsCPK12 overexpression lines.

[0054] The specific method is as follows: using primers OsCPK12-OE-F and OsCPK12-OE-R, PCR amplification was performed with the cDNA of the indica rice variety ZH8015 as a template to obtain the full-length cDNA sequence of OsCPK12 as shown in SEQ ID NO.5, and then the fragment was recombined into the XmaI / XbaI site of the pCAMBIA2300 vector by homologous recombination, and transferred into the indica rice variety ZH8015 by Agrobacterium EHA105-mediated rice genetic transformation, and later, the transgenic line with overexpression of OsCPK12 was obtained by hygromycin screening and quantitative analysis.

[0055] In some specific embodiments, the Agrobacterium is EHA105.

[0056] The embodiments of the present invention also provide application of the method in rice breeding or rice variety improvement.

[0057] In some specific embodiments, the purpose of breeding or improvement is to improve the photosynthetic efficiency of rice.

[0058] In some specific embodiments, the breeding method includes transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0059] The embodiment of the present invention also provides the use of OsCPK12 protein in regulating rice photosynthesis, and its amino acid sequence is shown in SEQ ID NO.6.

[0060] The OsCPK12 protein is localized in the nucleus, cytoplasm and cell membrane, and is highly expressed in green tissues. In an embodiment of the present invention, by using CRISPR / Cas9 gene editing technology to mutate the OsCPK12 gene, a mutant oscpk12-cr can be obtained, the plant becomes shorter and the photosynthesis is reduced. The present invention finds that the yellowing phenotype of the mutant oscpk12-cr is light-induced, weak light can inhibit the occurrence of the phenotype, and strong light promotes the occurrence of the yellowing phenotype. The expression of chlorophyll synthesis-related genes and related proteins in the photosynthetic electron transport chain in the leaves of oscpk12-cr plants decreased. Further studies have found that OsCPK12 interacts with OsAtpD1 and phosphorylates it, thereby mediating the transport of OsAtpD1 to chloroplasts. Moreover, light can induce the expression of OsCPK12 and promote the transport of OsAtpD1 to chloroplasts after OsCPK12 phosphorylates OsAtpD1; overexpression of OsCPK12 can improve the photosynthetic efficiency of rice, which can provide a theoretical basis for high light-efficiency rice breeding and drought-resistant breeding.

[0061] The wild type ZH8015 used in the embodiment of the present invention is a restorer line bred by our research group using the self-bred bacterial blight-resistant restorer line Zhonghui 218 as the female parent and Zhonghui 8006 as the male parent through multiple generations of breeding and identification of bacterial blight and rice blast resistance.

[0062] Example 1

[0063] Gene selection and material creation

[0064] About 90% of the dry weight of crops comes from organic matter generated by photosynthesis, so the strength and efficiency of crop photosynthesis are important factors that determine crop yield. People hope to improve the photosynthesis efficiency of crops by modifying the photosynthetic characteristics of crops to cultivate high-yield and super-high-yield varieties, that is, high photosynthesis efficiency breeding (High Photosynthesis Efficiency, HPE). At present, even the light energy utilization rate of high-yield rice is only 1.5%-2.0%. If the light energy utilization rate of rice can reach the ideal level of 3%-5%, then the rice yield will be greatly improved. This fully demonstrates the great application of increasing rice yield by improving the photosynthesis efficiency of rice in ensuring food security and promoting sustainable agricultural development.

[0065] Overexpression of OsCPK12 can significantly improve the photosynthesis efficiency and drought tolerance of rice. The research results of the present invention show that OsCPK12 is involved in regulating the dynamic photosynthesis process of rice. In order to determine the function of the rice gene OsCPK12, knockout and overexpression plants were constructed in the ZH8015 background to reveal the function of the gene.

[0066] The primer sequences used to construct the knockout transgenic vector are (5′-3′):

[0067] OsCPK12-Cas9-F (SEQ ID NO.1): AGATGATCCGTGGCATCGACCGCATCACGGCCAAGGGGGTTTTAGAGCTATGC;

[0068] OsCPK12-Cas9-R (SEQ ID NO. 2): GCATAGCTCTAAAACCCCCTTGGCCGTGATGCGGTCGATGCCACGGATCATCT.

[0069] The primer sequences used to construct the overexpression transgenic vector are (5′-3′):

[0070] OsCPK12-OE-F (SEQ ID NO.3): GTAGAAGAGGTACCCGGGCACCATTCCAC CCTCGCTT;

[0071] OsCPK12-OE-R (SEQ ID NO. 4): GCAGGTCGACTCTAGATAACTTTTTTGCGTTTCATCTGC.

[0072] Example 2

[0073] OsCPK12 knockout plants showed a yellowing phenotype, with reduced photosynthetic rate and photosynthetic pigment content in leaves, and downregulated expression of most related proteins in the photosynthetic electron transport chain.

[0074] 1 The vector used for knockout was pCas9-AarI, and the prokaryotic resistance of the vector was spectinomycin, and the eukaryotic resistance was hygromycin. The target sequence of the target gene OsCPK12 was designed using the online website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), and the sequence with a higher score located in the CDS region and close to the ATG start site was selected as the sgRNA, and the corresponding linker was added to form the F primer, and the F primer sequence was formed by reverse complementation to form the R primer. Mix the F and R primers, react at 94℃ for 10min, anneal to 15℃ at 0.1℃ / s, and then keep at 15℃ for 10min to complete annealing and form double strands; digest the pCas9 vector with AarI overnight and then cut the gel to recover the target band; take 1μL of double-stranded product and recombinant with the recovered pCas9-AarI linear vector, transform DH5α, plate on a plate containing spectinomycin resistance, and culture in a 37℃ incubator overnight; select single clones for sequencing at Shangya Biotechnology Co., Ltd. the next day, and finally send the successfully sequenced plasmid to Wuhan Boyuan Biotechnology Co., Ltd. for genetic transformation in the indica rice ZH8015 background. Sequence the transgenic plants of the T0 generation, select the homozygous mutant and name it oscpk12-cr for subsequent research.

[0075] The knockout mutant oscpk12-cr exhibited a yellowing phenotype. To explore the cause of the yellowing phenotype after OsCPK12 knockout, leaves of wild-type ZH8015 and knockout mutants were taken for TMT proteomics analysis before the phenotype appeared. The results showed that compared with ZH8015, the downregulated proteins in the leaves of oscpk12-cr 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 ).

[0076] 2 The net photosynthetic rate was determined using a LI-6400XT portable photosynthetic meter. The third leaf blade of the wild-type ZH8015 and oscpk12-cr transgenic family plants was selected between 9:30 and 11:00 in the morning on a sunny day during the heading period for photosynthetic rate determination.

[0077] Parameter setting: Photon density 1200 μmol·m -2 ·s -1 , flow rate 500 μmol·s -1 The three measurements of leaves of three plants were taken as one replicate, and the three replicates were used for statistical analysis. The determination of photosynthetic pigment content and chlorophyll content was based on the method of ARNON and PORRA (Arnon, 1949; Porra et al., 2010).

[0078] The results of the net photosynthetic rate measurement 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 ).

[0079] 3 Obtaining transgenic plants overexpressing OsCPK12

[0080] Using primers OsCPK12-OE-F and OsCPK12-OE-R, the cDNA of indica rice variety ZH8015 was used as a template for PCR amplification 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 by homologous recombination, and then transferred into the indica rice variety ZH8015 by Agrobacterium EHA105-mediated rice genetic transformation. Overexpression of OsCPK12 can increase the photosynthetic pigment content and net photosynthetic rate in rice plant leaves ( Figure 4 ).

[0081]

[0082] The protein sequence encoded by OsCPK12 is shown in SEQ ID NO.6.

[0083] SEQ ID NO.6: MGNCFTKTYEIPITSGTMRRPASTAERSKARGGDEPGTWRRPSF PRHGAPPHRPPTGSSSAAGALSRRASGGGGEMGPVLQRAMVSVRSLYQLDRKLGSGQFGTTYLCTERATGNRYACKSVSKRKLVRRTDVDDVRREITILQHLSGQPNIAEFRGAYEDNDHVHLVMEFCSGGELFDRITAKGSYSERQAAAVCRDILTVVHVCHFMGVIHRDLKPENFLLASADDDAPLKAIDFGLSVFI EEGKVYKDIVGSAYYVAPEVLQRNYGKEADIWSAGVILYILLCGTPPFWAETEKGIFDAILVNQVDFSTSPWPSISESAKDLIRQMLHRDPQKRITASQALEHRWLKEGGASDRPIDSAVLSRMKQFKAMNKLKQLALKVIAENLSPEEIKGLKQMFNNMDTDRSGTITVEEKVGLTKLGSRISEAEVQKLMEAVDVD KSGSIDYSEFLTAMINKHKLEKEEDLLRAFQHFDKDNSGYITRDELEQAMAEYGMGDEANIKQVLDEVDKDKDGRIDYEEFVEMMRKGIQT*.

[0084] Example 3

[0085] The etiolation phenotype of oscpk12-cr knockout plants is light-induced

[0086] To verify that the yellowing phenotype of oscpk12-cr is light-induced, the cells were grown under natural light (normal light) and under a black net (weak light condition) ( Figure 5(A) Observe the phenotypes of wild-type ZH8015 and mutant oscpk12-cr. To detect whether OsCPK12 is light-induced, the plant RNA extraction kit of Tiangen was used to extract total RNA from leaf samples of ZH8015 at different time points of the day, and then the total RNA was reversed into cDNA using Toyobo's quantitative reverse transcription kit ReverTra-Ace qPCR-RT-Master Mix. Then, using Takala's quantitative kit ExTaqII, qRT-PCR was performed in a Light Cycler 480II instrument according to the instructions. UBQ10 was used as an internal reference, and the expression level of OsCPK12 was analyzed using OsCPK12-qRTF / OsCPK12-qRTR in Table 1. The quantitative analysis results showed that light can indeed induce the expression of OsCPK12 ( Figure 5 Middle B). Under natural light conditions, the mutant oscpk12-cr showed premature senescence phenotype ( Figure 6 The photosynthetic pigment content in the mutant leaves was significantly lower than that in the wild type ( Figure 6 However, under the condition of covering the black net, the mutant oscpk12-cr has not yet shown the premature aging phenotype. Five days after the black net was removed, the premature aging phenotype of the mutant oscpk12-cr reappeared ( Figure 7 After the wild-type ZH8015 and the mutant oscpk12-cr were cultured in a normal hydroponic culture in an incubator for 15 days, some of them were transferred to continuous light conditions. It was found that after 7 days of continuous light, the mutant showed premature senescence phenotype ( Figure 8 A~C), the photosynthetic pigment content in the mutant leaves was significantly lower than that in the wild type ( Figure 8 D-F), while the phenotypes of wild-type ZH8015 and mutant oscpk12-cr under normal light were not much different. The above results indicate that the yellowing phenotype of oscpk12-cr knockout plants is light-induced, and light induces the expression of OsCPK12.

[0087] Table 1 RT-qPCR primers

[0088]

[0089] Example 4

[0090] OsCPK12 interacts with OsAtpD1

[0091] The CDS sequence of OsCPK12 without terminator was amplified and connected to the KpnI / SalI site of the CLuc vector, and the CDS sequence of OsAtpD1 was amplified and connected to the SalI site of NLuc; the constructed vector was transformed into Agrobacterium GV3101, and different combinations of Agrobacterium competent cells were transiently expressed in tobacco leaves. After that, the leaves were cut after 24-48 hours of culture, and the luciferase substrate was sprayed on the leaves, and the leaves were placed in the dark for 10 minutes. Then the leaves were placed on the black panel in the imaging device to detect the fluorescent signal. In addition, a hole was punched near the injection port area to sample, and the sample was immersed in ddH2O on the cell culture plate; after completing the sampling of all combinations, the ddH2O was aspirated, 200μL of luciferase substrate was added, and the dark treatment was carried out at room temperature for 15 minutes, and the signal intensity was detected using a Glomax instrument. The amplification primers are shown in Table 2. Luciferase complementation imaging analysis results showed that OsCPK12 may interact with the chloroplast ATP synthase delta subunit OsAtpD1 ( Fig. 9 A to C in the figure). Yeast two-hybrid point-to-point was then used to verify that OsCPK12 indeed interacted with OsAtpD1. In order to further verify the interaction between OsCPK12 and OsAtpD1, OsCPK12-Myc and OsAtpD1-GFP were constructed in this example, and total protein was extracted after transient transformation in rice protoplasts for 20 hours, and immunoprecipitation was performed using anti-GFP magnetic beads. The Co-IP results showed that OsAtpD1-GFP could pull down OsCPK12-Myc, but could not pull down Myc-Luc ( Fig. 9 (middle D).

[0092] Table 2 Primers used in protein interaction, in vitro phosphorylation and subcellular localization experiments

[0093]

[0094]

[0095] Example 5

[0096] OsCPK12 phosphorylates OsAtpD1

[0097] In order to verify whether OsAtpD1 is a substrate protein of OsCPK12, the CDS sequence encoding OsCPK12 was fused to the pCold-TF vector containing a His tag, and the CDS sequence of OsAtpD1 was fused to the pGEX-4T-1 vector containing a GST tag. After transformation into BL21 bacteria, sequencing was performed, and positive clones were selected and purified after induction with 0.1 mM IPTG. GST-tagged protein and His-tagged protein were purified using BeaverBeads GSH (Cat. No.: 70601-100, XIYAN CO., LTD.) and His-tag protein purification kit (Cat. No.: P2226, Beyotime Biotechnology Co., Ltd.) respectively. The purified protein was quantitatively analyzed using the NCM BCA protein analysis kit (Cat. No.: WB6501, New Cell & Molecular Biotech Co., Ltd.).

[0098] 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) in a total volume of 30 μL, 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 boiled for 8 min for protein denaturation. The phosphorylation of OsAtpD1 was then detected by immunoblotting using a 7% acrylamide gel containing 50 μM Phos-tag (Phos-tag AAL-107; Wako), and the amount of loaded protein was displayed by CBB staining.

[0099] The results showed that when His-TF-OsCPK12(Dead) was incubated with OsAtpD1-GST, and when His-TF-OsCPK12 and OsAtpD1-GST were incubated in kinase buffer without ATP for 30 minutes, OsAtpD1-GST had only one band. However, when His-TF-OsCPK12 and OsAtpD1-GST were incubated in normal kinase buffer, phosphorylated OsAtpD1-GST bands appeared, and the phosphorylated OsAtpD1-GST bands gradually deepened with the increase of incubation time ( Fig.10 ). The above results indicate that OsAtpD1 is a substrate protein of OsCPK12 and OsCPK12 can phosphorylate OsAtpD1.

[0100] Example 6

[0101] OsCPK12 affects the subcellular localization of OsAtpD1

[0102] To demonstrate the effect of OsCPK12 on OsAtpD1, the CDS of OsAtpD1 without the terminator was ligated to a vector containing an eGFP tag to construct an OsAtpD1-eGFP fusion vector. OsAtpD1-eGFP was transiently transformed into protoplasts of wild-type ZH8015 and mutant oscpk12-cr. Protoplast protein extraction and subcellular localization results were observed 14 h after transformation.

[0103] The results showed that there was no significant difference in the protein amount of OsAtpD1 between the wild-type ZH8015 and the mutant oscpk12-cr background, indicating that OsCPK12 did not affect the protein stability of OsAtpD1. OsAtpD1 was mainly localized in the chloroplasts in the wild-type ZH8015 protoplasts, but was mainly localized in the cytoplasm in the oscpk12-cr protoplasts ( Fig.11 The above results indicate that OsCPK12-mediated phosphorylation of OsAtpD1 may change its subcellular localization.

[0104] Example 7

[0105] Light-induced OsCPK12 phosphorylates OsAtpD1 to promote chloroplast translocation

[0106] Light is the main energy source for plant photosynthesis (Yousef et al., 2021), and the daily fluctuations in plant photosynthesis are closely related to changes in light intensity. The present study shows that light induces OsCPK12 expression. In order to study whether the OsCPK12-OsAtpD1 pathway is involved in dynamic photosynthesis, the expression level of OsAtpD1 was analyzed using OsAtpD1-qRTF / OsAtpD1-qRTR in Table 1, and the expression level of OsAtpD1 at different time points was detected. The present invention observed that the expression of OsAtpD1 is also induced by light, and its expression peak occurs at 8 o'clock, indicating that OsAtpD1 is particularly sensitive to light ( Fig.12 ). The protein abundance of OsCPK12 and OsAtpD1 in the total leaf proteins and chloroplast proteins of ZH8015 and oscpk12-cr at different light exposure times was further analyzed. The present invention found that the protein levels of OsCPK12 and OsAtpD1 increased over time, and the protein abundance of OsCPK12 and OsAtpD1 reached a peak at 10 am ( Fig.13 However, among the chloroplast proteins of wild-type ZH8015 and oscpk12-cr, the abundance of OsAtpD1 protein peaked at 12 noon ( Fig.13C), but the protein abundance of OsAtpD1 in the chloroplast proteins of oscpk12-cr was significantly reduced ( Fig.13 D). These results indicate that increased light intensity promotes the accumulation of OsCPK12 and OsAtpD1, and that phosphorylation of OsAtpD1 by OsCPK12 is essential for its chloroplast translocation. The above results indicate that light-induced OsCPK12 phosphorylates OsAtpD1, promotes its chloroplast translocation, and participates in dynamic photosynthesis. This mechanism is essential for protecting rice from light damage under high light conditions.

[0107] Example 8

[0108] Overexpression of OsCPK12 can improve drought tolerance in rice

[0109] 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 the other portion continued to be hydroponically cultured as a control. Samples were taken from the control and treatment groups at 0, 0.5, 1, 2, 4, and 8 hours after treatment for quantitative analysis.

[0110] The results showed that drought could induce the expression of OsCPK12. The expression level of OsCPK12 was the highest after 1 hour of drought treatment, and then the expression level of OsCPK12 began to decrease ( Fig.14 ). Therefore, OsCPK12 is a gene that responds to drought. In order to verify the effect of OsCPK12 on drought, the present invention subjected wild-type ZH8015, knockout mutant oscpk12-cr and overexpression family OsCPK12-OE to drought treatment. The present invention found that drought can promote the occurrence of oscpk12-cr yellowing phenotype, and the OsCPK12-OE family is more drought-tolerant ( Fig.15 ).

[0111] Example 9

[0112] Haplotype analysis of gene OsCPK12 in rice genome

[0113] The coding region of the gene OsCPK12 was analyzed by haplotype analysis in the 3K rice genome database (http: / / www.rmbreeding.cn / Index). The results showed that it can be divided into 6 haplotypes (Hap) ( Fig.16 ). ZH8015 belongs to Hap1, japonica varieties and Bas varieties are mainly Hap2 and Hap4, and indica varieties are mainly Hap1, Hap3, Hap5 and Hap6. The above results indicate that the gene OsCPK12 has been artificially selected in breeding.

[0114] OsCPK12 is from the indica rice restorer line Zhonghui 8015. The gene encodes a protein kinase with 534 amino acids containing an N-terminal variable region, a kinase region, an autoinhibitory region, and a calcium ion binding region. The mutant oscpk12-cr was obtained by knocking out the OsCPK12 gene using CRISPR / Cas9 gene editing technology. Compared with the wild type, oscpk12-cr had shorter plant height, reduced expression of photosynthesis-related proteins and chlorophyll synthesis-related proteins in leaves, and an early senescence phenotype. The early senescence phenotype of the mutant oscpk12-cr was suppressed under low light conditions. The early senescence phenotype of the mutant oscpk12-cr occurred prematurely under continuous light conditions. Overexpression of OsCPK12 can improve the photosynthetic efficiency and drought tolerance of rice. Subcellular localization results and protein interaction experiments verified that OsCPK12 interacted with OsAtpD1 and phosphorylated it, thereby mediating the transport of OsAtpD1 to chloroplasts. The accession number of the OsCPK12 gene of the present invention is LOC_Os04g47300. In the embodiment of the present invention, the light-induced OsCPK12 mediates the phosphorylation of OsAtpD1 to promote its introduction into the chloroplast to participate in ATP synthase, directly affects the ATPase activity, and indirectly affects the expression of chlorophyll synthesis-related genes and related proteins in the photosynthetic electron transport chain, thereby participating in the dynamic regulation process of photosynthesis. The present invention can provide candidate genes and theoretical basis for rice high light efficiency breeding and drought resistance breeding.

[0115] 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 methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of OsCPK12 gene in regulating rice photosynthesis, characterized in that: The accession number of the OsCPK12 gene is LOC_Os04g47300, and its nucleotide sequence is shown in SEQ ID NO.

5.

2. The use according to claim 1, characterized in that: Silencing or knocking out the OsCPK12 gene reduces rice photosynthesis; overexpressing the OsCPK12 gene increases rice photosynthesis.

3. The use according to claim 1, characterized in that: The regulation of rice photosynthesis includes regulating the net photosynthetic rate, the content of photosynthetic pigments and the expression amount of related proteins in the photosynthetic respiratory chain.

4. A method for reducing rice photosynthesis, characterized in that: The OsCPK12 gene in the rice plant is silenced or knocked out.

5. A method for improving rice photosynthesis, characterized in that: Overexpressing the OsCPK12 gene in the rice plant.

6. The method according to claim 5, characterized in that The method for overexpressing the OsCPK12 gene is selected from at least one of the following methods: 1) by introducing a plasmid carrying the gene; 2) by increasing the copy number of the gene on the plant chromosome; 3) by changing the promoter sequence of the gene on the plant chromosome; 4) by operably linking a strong promoter to the gene; 5) By introducing enhancers.

7. Use of the method according to claim 5 or 6 in rice breeding or rice variety improvement.

8. Application of OsCPK12 protein in regulating rice photosynthesis, characterized in that: Its amino acid sequence is shown in SEQ ID NO.6.

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