Unannotated functional protein cpsa and application thereof

Abstract

The application discloses a functionally unannotated protein CpsA and application thereof, and belongs to the technical field of biochemistry. synpcc7942_1022 The CpsA protein provided by the application is a functionally unannotated thylakoid membrane protein encoded by The protein can be specifically combined with an early PSII assembly factor Psb28, promotes the formation and stability of a final oxygen-releasing complex of PSII, and significantly improves the assembly efficiency of the light system II and the formation of a functional complex. In addition, the CpsA protein is highly conservative in cyanobacteria, microalgae and terrestrial plants, and has wide application potential.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to an unannotated protein CpsA and its applications. Background Technology

[0002] The key protein complex in the photosynthetic reaction center of photosynthetic organisms is photosystem II (PSII), which is responsible for catalyzing the splitting of water and the production of oxygen, and is an important foundation for maintaining life on Earth. Under natural light conditions, PSII is susceptible to damage and requires continuous dissociation and repair processes. Therefore, the biogenesis and assembly of PSII is extremely complex and depends on the coordination of multiple cofactors. However, the specific molecular mechanisms of each cofactor in the biogenesis of PSII are not yet fully elucidated.

[0003] PSII assembly begins with the formation of the reaction center (RC) complex, followed by the gradual integration of subunits with the participation of accessory proteins such as Psb28. However, there are still gaps in our understanding of the binding and release mechanisms of Psb28, especially the fine-tuning mechanism of Psb28 in the early stages of PSII assembly.

[0004] Currently, our understanding of the role of unannotated GreenCut2 proteins in PSII biogenesis is insufficient, limiting further exploration of photosystem assembly and the optimization of photosynthesis. Therefore, it is urgent to identify new cofactors and elucidate their specific functions and mechanisms of action in the early PSII assembly process to advance basic research on photosynthesis and its applications in energy development. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a functionally unannotated protein, CpsA, and its applications. The CpsA (cyanobacterial photosynthesis-associated factor) protein provided by this invention is derived from *Synechococcus elongatus* PCC 7942. synpcc7942_1022 The encoded thylakoid membrane protein has no publicly annotated function. This protein specifically binds to the early PSII assembly factor Psb28, promoting the formation and stability of the final PSII oxygen-evolving complex, significantly improving the assembly efficiency of photosystem II and the formation of functional complexes. Furthermore, CpsA proteins are highly conserved in cyanobacteria, microalgae, and terrestrial plants, and have broad application potential.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a functionally unannotated protein CpsA, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The CpsA amino acid sequence is as follows: MATPLWQTIYNNAVLLPESPKAIVHFLGGAFIAAAPQVTYRRLLEGLGDRGFAVIATPFINTFDHTTLADEVGRSYRLAYERLLALGLISPELPIFGVGHSMGSKLHLLMGCAQTSDRRGNVLLAF NNYSARRSIPLLGQLAPALDVTVEFSPSPARTLRLIRDRYSVANNLLVKFRNDEIDQTRDLIGALRDRFPETSEWLRLSGNHLTPVGQSLPQQTGLAWLDGVSQLASRELGRDLEVVTTAIADWCDRQLTAPATVV (SEQ IDNO.1).

[0008] Furthermore, the functionally unannotated protein CpsA is... synpcc7942_1022 The gene encodes a protein with a molecular weight of 28.8 kDa; furthermore, the functionally unannotated protein CpsA has a DUF1350 domain of unknown function; furthermore, the functionally unannotated protein CpsA has less than 14% similarity to known proteins at the amino acid sequence level.

[0009] Secondly, the present invention provides a gene encoding the above-mentioned protein. The gene sequence is as follows: synpcc7942_ 1022 .

[0010] Thirdly, the present invention provides a biological material comprising the aforementioned unannotated protein CpsA or the aforementioned gene.

[0011] The biological material is one or more of the following: recombinant expression vector, plasmid, expression cassette, transgenic cell line, recombinant bacteria, and recombinant virus.

[0012] CpsA homologs are highly conserved across all green algae (cyanobacteria, microalgae, and plants), but no CpsA homologs have been found in non-photosynthetic organisms. This point can be used for screening, diagnosis, or auxiliary diagnosis of all green algae.

[0013] Fourthly, the present invention provides the application of the above-mentioned unannotated protein CpsA, or the above-mentioned encoding gene, or the above-mentioned biological material in regulating the early photosystem II (PSII) of plants.

[0014] The functionally unannotated protein CpsA specifically binds to the early PSII assembly factor Psb28, and the two have a direct interaction relationship. CpsA can affect the conformational changes of Psb28, thereby regulating its binding to PSII.

[0015] Fifthly, the present invention provides a CpsA-Psb28 complex comprising the aforementioned unannotated protein CpsA or a gene encoding the aforementioned unannotated protein CpsA or the aforementioned biological material, and further comprising the plant early photosystem II assembly factor Psb28.

[0016] CpsA and Psb28 have a direct interaction relationship and are co-located in the synechocybe sink region.

[0017] CpsA and Psb28 can combine to form a CpsA-Psb28 complex. In this complex, hydrogen bonds are formed between Tyr10 in CpsA and Asp37 in Psb28, and between Asn11 in CpsA and Tyr35 in Psb28.

[0018] The identification method for CpsA protein interaction networks integrates the following techniques: A1: STRING database predictive analysis; A2: AP-MS (Affinity Purification Mass Spectrometry) technique; A3: Bacterial adenylate cyclase two-hybrid system (BACTH); A4: Colocalization experiment verification.

[0019] Methods for functional studies of the CpsA-interacting protein complex (CpsA-Psb28 complex) include: A1: Molecular docking simulation; A2: Molecular dynamics simulation.

[0020] Sixthly, the present invention provides the use of the above-mentioned unannotated protein CpsA, or a gene encoding the above-mentioned unannotated protein CpsA, or the above-mentioned biological material in any one or more of the following: (a) Its application in regulating plant chlorophyll, and / or; (b) Its application in regulating plant chloroplast development, and / or; (c) Its application in regulating plant photosynthesis, and / or; (d) Application in regulating the salt tolerance of plants, and / or; (e) Application in plant subcellular localization.

[0021] The chlorophyll includes chlorophyll a.

[0022] Furthermore, the plant includes algae, which includes eukaryotic algae, and more preferably cyanobacteria (blue-green algae).

[0023] The protein's localization and distribution pattern can be determined using techniques such as fluorescent labeling to achieve subcellular localization detection of CpsA protein.

[0024] In a seventh aspect, the present invention provides a method for regulating chlorophyll, chloroplast development and / or photosynthesis in plants, wherein a formulation is used to detect the expression level or activity of the unannotated functional protein CpsA in plants, the formulation comprising the aforementioned unannotated functional protein CpsA or a gene encoding the aforementioned unannotated functional protein CpsA or the aforementioned biological material.

[0025] Eighthly, the present invention provides a method for regulating the salt tolerance of plants, wherein the expression level or activity of the unannotated functional protein CpsA in a plant is detected by means of a formulation, wherein the formulation comprises the unannotated functional protein CpsA or a gene encoding the unannotated functional protein CpsA or the aforementioned biological material.

[0026] Ninthly, the present invention provides a system for verifying the physiological function of the above-mentioned CpsA protein, comprising: A1、 cpsA Gene knockout mutants: mutants that have undergone homologous recombination synpcc7942_1022 The gene was replaced with the kanamycin tag gene; A2. Replacement strains: including those introduced from exogenous sources. cpsA Gene; A3. Verify the function of CpsA by comparing the phenotypic differences between wild-type, knockout mutant and complement mutant.

[0027] The phenotypic differences include: A1, altered photosynthetic oxygen release capacity; A2, altered growth vigor under high light conditions.

[0028] The phenotypic differences of PSII include: A1, changes in PSII abundance; A2, changes in the accumulation levels of various intermediates of photosystem II; A3, changes in the distribution of the D1 / D2 / CP47 / CP43 pigment protein complex.

[0029] A method for screening photosynthetic regulatory elements based on CpsA homologous proteins includes: A1. Identify homologous sequences through phylogenetic tree analysis; A2. Determine functional conservation through domain analysis.

[0030] One or more of the above technical solutions have the following advantages or beneficial effects: (1) This invention provides a functionally unannotated protein CpsA, which participates in the early PSII biosynthesis in the polar region of the thylakoid membrane. CpsA mediates the dissociation of Psb28 from the PSII intermediate by binding to Psb28 and inducing structural changes, thereby promoting the formation of a larger PSII complex to maintain the normal photosynthetic efficiency and salt tolerance of cyanobacteria.

[0031] (2) CpsA and Psb28 have a direct interaction relationship, and both are co-located in the network region of Synechococcus, suggesting that they may interact in a loose or transient manner. Structure prediction using AlphaFold, molecular docking analysis, and molecular dynamics simulations revealed a strong binding between CpsA and Psb28. Molecular dynamics (MD) simulations showed significant fluctuations in the residues at the CpsA-Psb28-PSII complex binding interface. Site-directed mutagenesis analysis indicated that residues such as Tyr10, Asn11, Arg74, and Arg82 play key roles in the interaction between CpsA and Psb28, with mutants exhibiting significantly reduced binding capacity.

[0032] (3) CpsA is present in all forms of the PSII complex, while Psb28 is only detected in the RC47 intermediate and free form. In the knockout mutant, the abundance of PSII monomers and dimers is reduced, with increased accumulation of D1, D2, and CP47 in RC47, but significantly reduced accumulation of CP43 in larger PSII complexes. Meanwhile, the RC47 intermediate is enriched with proteins such as CpcH and PsbO. This indicates that CpsA participates in PSII assembly through interaction with Psb28, and its absence affects the assembly of the RC47 complex into larger PSII complexes, and may hinder the effective binding of CP43 to RC47, thus affecting CP43 binding and conversion to larger complexes.

[0033] (4) CpsA homologs are highly conserved across all green lineages (cyanobacteria, microalgae, and plants), but no CpsA homologs have been found in non-photosynthetic organisms. As a thylakoid membrane protein, CpsA can induce conformational changes in Psb28 protein by binding to it, promoting the assembly of larger PSII complexes and thus maintaining normal photosynthetic efficiency and salt tolerance in cyanobacteria. Furthermore, the high conserved nature of CpsA protein in the green lineage suggests its important functional value in the evolution of photosynthetic organisms. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1This document describes the physiological function and subcellular localization of CpsA in Synechococcus slenderus PCC 7942 in Examples 1 and 2 of this invention. A represents the predicted CpsA domains, B represents the predicted CpsA operon, C represents the analysis of the photosynthetic oxygen-evolving capacity of Synechococcus cells under normal light (GL) and strong light (HL) conditions, D represents the growth status of Synechococcus cells cultured under strong light, E represents the CpsA transmembrane domain predicted using TMHMM, F represents a schematic diagram of the generation of the Synechococcus CpsA:mNG-labeled mutant, and G represents a schematic diagram of the Synechococcus cell sink membrane region. Figure 2 This is a magnified view of the location of CpsA:mNG (green) in Synechococcus cells, the long axis and short axis of the cells, and the relationship between fluorescence intensity and distance in Example 2 of this invention; wherein, A is a location map of CpsA:mNG (green) in Synechococcus cells with a size of 1 micrometer, B is a magnified view (inset) of the short axis of part (a) in Figure A and the corresponding relationship between fluorescence intensity and short axis distance, and C is a magnified view (inset) of the long axis of part (b) in Figure A and the corresponding relationship between fluorescence intensity and long axis distance; Figure 3 This is a study on protein-protein interactions of CpsA in Example 3 of the present invention; wherein, A is the potential interacting protein of CpsA identified in the AP-MS experiment, B is the high confidence protein interaction (WD score > 3) identified by AP-MS analysis of CpsA, C is the in vivo interaction between potential interacting proteins of CpsA analyzed by BACTH, and D is the immunoprecipitation experiment of CpsA and Psb28. Figure 4 This is a diagram showing the subcellular localization of Psb28 in Synechococcus in Example 3 of the present invention, a magnified view of the cell's long and short axes, and a graph showing the relationship between fluorescence intensity and distance. Specifically, A is the subcellular localization of Psb28 in Synechococcus, B is a magnified view (inset) of the short axis of part (a) in Figure A and the corresponding graph showing the relationship between fluorescence intensity and short axis distance, and C is a magnified view (inset) of the long axis of part (b) in Figure A and the corresponding graph showing the relationship between fluorescence intensity and long axis distance. Figure 5 The images shown are: an immunofluorescence co-localization analysis diagram of CpsA and Psb28 in Example 3 of this invention; a magnified view of the cell's long axis and short axis; and a graph showing the relationship between fluorescence intensity and distance. Specifically, A is a subcellular localization diagram of Psb28 in Synechococcus, with a size of 2 micrometers; B is a magnified view (inset) of the short axis of part (a) in Figure A and a graph showing the relationship between fluorescence intensity and short axis distance; and C is a magnified view (inset) of the long axis of part (b) in Figure A and a graph showing the relationship between fluorescence intensity and long axis distance. Figure 6This is a molecular docking model of the CpsA-Psb28 and PSII complex in Example 4 of the present invention; Figure 7 The graphs show the changes in RMSD (root mean square deviation) and RMSF (root mean square fluctuation) of the CpsA-Psb28 complex obtained in a 100 ns molecular dynamics (MD) simulation in Example 4 of this invention; where a is the RMSD graph of the CpsA-Psb28 complex, b is the RMSF change graph of CpsA, and c is the RMSF change graph of Psb28. Figure 8 The diagram shows the overall and partial binding conformation of the CpsA and Psb28 complex obtained by molecular docking in Example 4 of the present invention; wherein, a is a partial magnified view of part a in diagram d, b is a partial magnified view of part b in diagram d, c is a partial magnified view of part c in diagram d, and d is the overall binding conformation of the CpsA and Psb28 complex obtained by molecular docking. Figure 9 These are the key residues mediating the binding of CpsA and Psb28 identified by the bacterial two-hybrid experiment after site-directed mutagenesis in Example 4 of this invention; where a is the TLC result of the bacterial two-hybrid experiment and b is the β-galactosidase activity diagram. Figure 10 This illustrates the functional role of CpsA binding to Psb28 in the early biosynthesis of PSII in Example 5 of this invention; where A represents the localization and distribution of CpsA and Psb28 in the thylakoid membrane complex, and B represents the BN-PAGE analysis. cpsA Effect of gene knockout on the abundance of thylakoid membrane protein complexes, C represents 2D BN / SDS-PAGE analysis. cpsA The impact of knockout on the distribution and abundance of PSII core subunits; Figure 11 This is a mechanism model of CpsA mediating the release of Psb28 from early PSII intermediates during PSII biogenesis in Example 5 of the present invention; wherein, (a) is the case with CpsA present, and (b) is the case without CpsA. Figure 12 In Embodiment 5 of the present invention cpsA Analysis of the impact of knockout on the levels of photosynthetic-related proteins; where A represents the results obtained through proteomics analysis. cpsA B shows the relative protein abundance of photosynthetic complex subunits in the thylakoid membrane of the knockout mutant and the wild type. B shows the protein content of the PSII core subunit in total cells and thylakoid membrane detected by Western blot. C shows the quantitative analysis of protein content using ImageJ software. Figure 13 This is a phylogenetic analysis of CpsA in Embodiment 6 of the present invention. Detailed Implementation

[0036] Explanation of terms involved: CpsA: cyanobacterial photosynthesis-associated factor.

[0037] It should be noted that when CpsA is represented as a protein, it is written in upright font. cpsA When represented as a gene, it is written in italics.

[0038] Cyanobacteria are also known as blue-green algae.

[0039] PSII: Photosystem II. In cyanobacteria, PSII is a large homodimeric complex located on the thylakoid membrane, composed of at least 20 protein subunits and bound to various pigments and cofactors.

[0040] RC: Reaction Center.

[0041] RC47 intermediate, also known as "RC47 complex": one of the key intermediate states in the assembly process of Optical System II.

[0042] Psb28: A co-protein of photosystem II, belonging to the peripheral subunit of the PSII complex.

[0043] CP43: One of the core peripheral antenna pigment proteins of Optical System II, composed of... psbC Gene encoding.

[0044] The thylapse-like region refers to the cellular polarity region of the thylakoid membrane.

[0045] Long axis distance: In a magnified view of the cell's long axis, the distance that varies along the direction indicated by the arrow.

[0046] Minor axis distance: In a magnified view of the cell's minor axis, the distance that varies along the direction indicated by the arrow.

[0047] The "+" sign in the full text of the instruction manual or in the accompanying drawings means "and".

[0048] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0049] Anti-D1, anti-D2, anti-CP47 and anti-CP43 antibodies, secondary antibodies, etc. were all purchased from PhytoAB.

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0051] Example 1 This embodiment provides the physiological function of CpsA in Synechococcus longiflora PCC 7942.

[0052] This embodiment measures cpsA The impact of the absence on the physiological functions of Synechococcus. Specifically, such as... Figure 1 (A)-(B), CpsA possesses a functionally unknown domain 1350 (DUF1350), located in photosynthesis-related genes. psbP ( synpcc7942_1038 ) 16kbp upstream. Among them, the CpsA amino acid sequence is as follows: MATPLWQTIYNNAVLLPESPKAIVHFLGGAFIAAAPQVTYRRLLEGLGDRGFAVIATPFINTFDHTTLADEVGRSYRLAYERLLALGLISPELPIFGVGHSMGSKLHLLMGCAQTSDRRGNVLL AFNNYSARRSIPLLGQLAPALDVTVEFSPSPARTLRLIRDRYSVANNLLVKFRNDEIDQTRDLIGALRDRFPETSEWLRLSGNHLTPVGQSLPQQTGLAWLDGVSQLASRELGRDLEVVTTAIADWCDRQLTAPATVV (SEQ IDNO.1).

[0053] To investigate its physiological functions, knockout and complement mutant strains were constructed. All *Synechococcus* strains were cultured in BG11 medium at pH 8.0 at 30°C and 40 μmol photons m -2 s -1 Cells were cultured under continuous light. In the high-light experiment, cells were cultured at 380 μmol photons m -2 s -1 Cells were cultured and their growth was recorded every 24 hours. Salt stress experiments were conducted using a medium containing 0.3 M NaCl. For pigment content determination, cells with OD730 = 1.0 were collected, extracted with methanol, and chlorophyll a content was calculated using absorbance at 665 nm (Chl a (μg / mL) = OD665 nm × 13.9). Phycocyanin and carotenoid levels were determined using in vivo absorption. Photosynthetic oxygen production rate was measured using a Clark-type oxygen electrode with a 10 mM NaHCO3 suspension as the reaction system, and chlorophyll content and oxygen release rate were recorded simultaneously. Results are as follows: Figure 1 As shown, where Figure 1 (C) Under normal and high light conditions cpsA The effect of gene deletion on the rate of photosynthetic oxygen evolution, the results showed cpsAThe photosynthetic efficiency of the knockout mutant was significantly reduced. Figure 1 (D) shows the growth status of wild type (WT), knockout (KO) and replacement (OE) under high light conditions. The knockout plant shows a yellow phenotype.

[0054] Example 2 This embodiment provides information on the subcellular localization of CpsA.

[0055] To detect the subcellular localization of proteins such as CpsA in *Synechococcus septemlobus* PCC 7942, bioinformatics analysis and prediction were performed using TMHMM-2.0 and TOPCONS. The results are as follows: Figure 1 (E) shows that CpsA is likely a transmembrane protein with two transmembrane regions located at amino acid residues 21-41 and 94-114, respectively. To visualize the subcellular localization of CpsA, as shown... Figure 1 (F) CpsA was fused with the mNeonGreen (mNG) and 3×FLAG tag genes, and fluorescently labeled under the control of its natural promoter. The thylakoid membrane was further separated by sucrose gradient ultracentrifugation and verified by Western blotting analysis. Subsequently, the target ORF gene was cloned into a plasmid backbone containing mNeonGreen (mNG), the 3×FLAG tag, the kanamycin resistance gene, and a negative selection marker. The recombinant plasmid was then transformed into *Synechococcus elongatus* cells, initially screened using kanamycin BG11 agar plates, and the resistance gene was removed in 5-fluorocytosine-containing medium to obtain purified labeled mutants, which were verified by colony PCR. Confocal imaging was performed using a Zeiss 880 (AiryScan plus) microscope. mNG fluorescence signals were acquired at 488 nm excitation and emitted at 420-480 nm and 495-550 nm emission bands, while chlorophyll autofluorescence signals were detected at 561 nm excitation and emitted at 495-550 nm and above 570 nm emission bands. The results are as follows Figure 1 (G) and Figure 2 CpsA is located in the sink membrane region of Synechococcus.

[0056] Example 3 This embodiment provides potential interacting proteins for CpsA.

[0057] This embodiment aims to detect CpsA interacting proteins. Specifically, by... cpsAA FLAG tag was inserted at the 3' end of the gene to construct a mutant strain, which was then cultured under standard conditions. After cell lysis, affinity purification was performed using magnetic beads conjugated with anti-FLAG antibodies, followed by elution with FLAG peptides and mass spectrometry analysis. Data analysis was conducted using CompPASS software to calculate normalized weighted D scores (WD scores) to assess interacting proteins. Furthermore, protein interactions were validated using the bacterial adenylate cyclase two-hybrid system (BACTH). cpsA The target gene was cloned into pKT25 and pUT18C plasmids, respectively, and transformed into *E. coli* strain BTH101. Interaction relationships were screened using MacConkey medium, and β-galactosidase activity was measured in a liquid culture system for quantitative analysis of the interaction. Finally, co-IP was used to further verify the interaction between CpsA and Psb28. Expression strains with FLAG or HA tags were constructed, and cells were collected under the same culture conditions. After lysis, immunoprecipitation was performed using magnetic beads conjugated with anti-FLAG or anti-HA antibodies. The eluted protein samples were analyzed by SDS-PAGE and Western blotting to detect the target protein, further confirming the protein-protein interaction relationship. Results are as follows: Figure 3 (A)-(D) A total of 52 proteins were identified by isolating CpsA-FLAG and its related proteins from cyanobacterial cell lysates, including the photosystem subunits Psb28 and PsbO, and the PSII biogenetic factor FtsH. The in vivo interactions between CpsA and these proteins were verified using the bacterial adenylate cyclase two-hybrid system (BACTH) and co-IP (immunoprecipitation), indicating a direct interaction between CpsA and Psb28.

[0058] Furthermore, in the protein colocalization analysis, Psb28 strains carrying the FLAG tag (CpsA) and the HA tag (HA) were collected for immunofluorescence microscopy analysis. The results are as follows: Figure 4 and Figure 5 As shown, Psb28 is distributed in the thylakoid membrane. CpsA and Psb28 are partially co-localized in the polar region of the thylakoid membrane. Psb28 is mainly free in the thylakoid membrane in the form of dimers, with only a small portion bound to the RC47 intermediate. This suggests that the two may interact in a loose or transient manner rather than forming a stable complex.

[0059] Example 4 This embodiment provides a binding mechanism between CpsA and Psb28.

[0060] This embodiment aims to reveal the interaction mechanism between CpsA and Psb28. Molecular docking and molecular dynamics simulations were used to resolve the binding mode and stability of the CpsA-Psb28 complex. The results are as follows: Figures 6-9As shown, CpsA and Psb28 can bind tightly, with a binding free energy of -48.991 kcal / mol. The binding sites are mainly hydrogen bonds formed between Tyr10 in CpsA and Asp37 in Psb28, and between Asn11 in CpsA and Tyr35 in Psb28. Furthermore, the binding of CpsA leads to significant fluctuations in Psb28 residues 10 to 17 and residues 38 to 53 at the Psb28-PSII complex binding interface, indicating that CpsA can influence the conformational changes of Psb28, thereby regulating its binding with PSII.

[0061] Example 5 This embodiment provides the mechanism by which the interaction between CpsA and Psb28 regulates the early biogenesis of PSII.

[0062] This embodiment aims to reveal the mechanism by which CpsA regulates the assembly of the PSII complex. Specifically, to assess the abundance changes of PSII assembly intermediates induced by CpsA deficiency, thylakoid membrane samples containing 50 μg of chlorophyll were dissolved, loaded onto a gradient acrylamide gel, and separated by BN-PAGE on ice, followed by two-dimensional electrophoresis for protein separation. Silver staining and Western blotting were further used for protein detection. To identify specific proteins, mass spectrometry analysis of the silver-stained gel strips was performed to identify peptides and proteins. The results are as follows: Figure 10 CpsA was present in all forms of the PSII complex, while Psb28 was detected only in the RC47 complex and in its free form. In the knockout mutant, the levels of PSII monomers and dimers were reduced compared to the wild type, while the abundance of D1, D2, and the CP47 subunit in the RC47 complex was significantly increased. Silver staining and mass spectrometry analysis further indicated that the RC47 complex in the knockout mutant was enriched with CpcH, PsbO, and an unannotated protein, Synpcc7942_2469.

[0063] Figure 11 A mechanism model for CpsA mediating the release of Psb28 from early PSII intermediates during PSII biogenesis is proposed, in which (a) CpsA forms droplets and recruits Psb28, promoting the dissociation of Psb28 from the RC47 intermediate, thereby promoting the orderly assembly of the PSII complex; (b) in the absence of CpsA, the release of Psb28 is inhibited, hindering the binding of CP43 and the progress of the RC47 complex into larger PSII assemblies, ultimately leading to a decrease in the photosynthetic oxygen production capacity of PSII.

[0064] Furthermore, thylakoid membrane samples were prepared for thylakoid membrane proteomics analysis. Crude thylakoid membranes were extracted after cell collection. The crude thylakoid membranes were dissolved to obtain membrane proteins. The resulting protein solutions were analyzed and identified using LC-MS / MS. Figure 12 (A) shows the difference between wild type (WT) and Δ cpsA Differences in the expression levels of several photosynthesis-related proteins were observed in the mutant strain. Proteomics analysis was used to compare the expression levels of multiple photosynthetic complexes and related functional proteins at WT and Δ. cpsA The expression changes in protein are shown in the figure. A bar chart is used to present protein abundance, and colors are used to distinguish sample sources (green for WT, red for Δ). cpsA The small color bar on the left side of the graph represents the log2 change factor (Δ). cpsA (v vs WT), with "*" indicating statistically significant differences. As shown in the figure, Δ cpsA In mutant strains, the expression of multiple PSII subunits (such as CP43, FtsH2, and FtsH3) was significantly downregulated; while the expression of multiple core PSI subunits (such as PsaA, PsaB, PsaC, and PsaJ) was significantly upregulated. No significant changes were observed in the expression of ATP synthase subunits. In the NDH-1 complex, subunits such as NdhK and NdhJ showed Δ... cpsA The expression of these proteins was significantly increased, and some subunits of the cytochrome b6f complex also showed an upregulation trend; in contrast, proteins related to photosynthetic pigment synthesis generally showed a downregulation trend. Therefore, the results are as follows: Figure 12 (A), Knockout cpsA This leads to significant changes in the abundance of various photosynthetic membrane proteins.

[0065] To validate the proteomics results, further immunoblotting analysis of the PSII core subunit was performed. Dissolved thylakoid membrane proteins were separated by SDS-PAGE and then immunoblotted with anti-D1, anti-D2, anti-CP47, and anti-CP43 antibodies (1:1000 dilution, PhytoAB). Detection was then performed using secondary antibodies (1:5000 dilution), and signal quantification was performed using ImageJ software. Figure 12 As shown in (B)-(C), the CP43 content in the knockout mutant was reduced by 36.17% compared to the wild-type (WT), and the expression levels of D1 and CP43 proteins in the total protein were also significantly decreased. Based on this, a mechanism of action model for CpsA is proposed, as follows: Figure 11 CpsA participates in the assembly of the PSII complex through its interaction with Psb28. Its absence can affect the assembly of the RC47 complex into the larger PSII complex and may hinder the effective binding of CP43 and RC47. The assembly of the larger PSII complex refers to the complex formed after the RC47 intermediate in the complex assembly process.

[0066] Example 6 This embodiment provides a phylogenetic analysis of CpsA. Homologous proteins of CpsA were screened using BLAST searches in the NCBI and UniProtKB databases. The screening criteria were: e-value ≤ 0.05 and sequence identity ≥ 30%. In the phylogenetic analysis, ClustalW software was used to perform multiple sequence alignment of CpsA-like sequences of representative species, and a maximum likelihood (ML) phylogenetic tree was constructed using MEGA11 software based on the JTT model. Figure 13 As shown, CpsA homologs are mainly distributed in photosynthetic bacteria containing thylakoids and are highly conserved across all green lineages (cyanobacteria, microalgae, and plants), but no CpsA homologs have been found in non-photosynthetic organisms. Furthermore, the copy number of CpsA homologs in eukaryotic algae and plants gradually increases over time, indicating their importance in photosynthetic organisms. Particularly in microalgae, CpsA homologs diverge into two main branches: one similar to proteins derived from cyanobacteria, and the other similar to proteins derived from plants.

[0067] The CpsA protein in this invention participates in early PSII biosynthesis within the polar region of the thylakoid membrane. CpsA mediates the dissociation of Psb28 from the PSII intermediate by binding to Psb28 and inducing structural changes, promoting the formation of a larger PSII complex to maintain normal photosynthetic efficiency and salt tolerance in cyanobacteria. More specifically, this manifests as follows: In this invention, under standard cyanobacterial culture conditions (30°C, light intensity 40 μmol photons m), -2 s -1 At a shaking speed of 120 rpm, cpsA The photosynthetic efficiency of the knockout mutant was significantly reduced, with a 38.8% decrease in photosynthetic oxygen evolution rate, while the replacement mutant recovered to normal levels. Under high light conditions (30℃, light intensity 380 μmol photons / m²), the photosynthetic efficiency was significantly reduced. -2 s -1 (Shaker speed 120 rpm), the knockout mutant exhibited a yellow phenotype, and its photosynthetic oxygen evolution rate decreased by 59.3% compared to the wild type. Under salt stress conditions (0.3 M NaCl), the growth rate of the knockout mutant was significantly reduced by 78.2% compared to the wild type, indicating a decrease in its salt tolerance.

[0068] In this invention, the CpsA protein can be localized to the thylakoid membrane and concentrated in the cell polar region, namely the thylapse-like region.

[0069] In this invention, CpsA and Psb28 have a direct interaction relationship and are co-located in the synechococcus sink membrane region, suggesting that they may interact in a loose or transient manner.

[0070] In this invention, structural and molecular docking analyses using AlphaFold predictions and molecular dynamics simulations revealed a strong binding between CpsA and Psb28. Molecular dynamics (MD) simulations showed significant fluctuations in the residues at the CpsA-Psb28-PSII complex binding interface. Site-directed mutagenesis analysis indicated that residues such as Tyr10, Asn11, Arg74, and Arg82 play crucial roles in the CpsA-Psb28 interaction, with mutants exhibiting significantly reduced binding affinity.

[0071] In this invention, CpsA is present in all forms of the PSII complex, while Psb28 is detected only in the RC47 intermediate and in its free form. In the knockout mutant, the abundance of PSII monomers and dimers is reduced, with increased accumulation of D1, D2, and CP47 in RC47, but significantly reduced accumulation of CP43 in the larger PSII complex. Simultaneously, the RC47 intermediate is enriched with proteins such as CpcH and PsbO. This indicates that CpsA participates in PSII assembly through interaction with Psb28, and its deletion affects CP43 binding and conversion to the larger complex.

[0072] In this invention, analysis revealed that CpsA homologs are highly conserved across all green lineages (cyanobacteria, microalgae, and plants), but no CpsA homologs were found in non-photosynthetic organisms.

[0073] In this invention, the CpsA protein, as a thylakoid membrane protein, can induce conformational changes in the Psb28 protein by binding to it, promoting the assembly of larger PSII complexes and thus maintaining normal photosynthetic efficiency and salt tolerance in cyanobacteria. Furthermore, the CpsA protein is highly conserved in the green spectrum, suggesting its important functional value in the evolution of photosynthetic organisms.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for reducing the photosynthetic efficiency or photosynthetic oxygen release rate of cyanobacteria, characterized in that, The method involves knocking out the CpsA protein in cyanobacteria; The amino acid sequence of the CpsA protein is shown in SEQ ID NO.

1.

2. A method for reducing the salt tolerance of cyanobacteria, characterized in that, The method involves knocking out the CpsA protein in cyanobacteria; The amino acid sequence of the CpsA protein is shown in SEQ ID NO.1.

Images