Method for improving photosynthetic electron transfer activity and photosynthetic rate of plants

Abstract

The invention provides a method for improving photosynthetic electron transfer and carbon dioxide assimilation rate of gramineous plants. Through deep research, the AOC in the gramineous plant has the effects of regulating and controlling linear electron transfer, ring electron transfer activity and photosynthetic rate, and the photosynthetic electron transfer activity and the photosynthetic rate can be improved by increasing the expression quantity of the AOC gene. Therefore, as a positive regulation factor for regulating photosynthetic electron transfer activity, AOC can provide a new way for improving photosynthetic characters and increasing photosynthetic yield through targeted regulation of the gene.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany, and more specifically, this invention relates to a method for improving the photosynthetic electron transport activity and photosynthetic rate of plants. Background Technology

[0002] Against the backdrop of global warming and frequent extreme weather events, food security remains a crucial issue that humanity must address for survival and development. On the one hand, the world's population has surpassed 8 billion, yet approximately one-ninth of the global population still faces food insecurity. On the other hand, crops also face various risks in their growing environment. It is projected that by the end of this century, the global average temperature will rise by 3.3°C, and extreme weather events will become more frequent, posing a severe challenge to stable and increased crop yields. Therefore, under this climate context, improving crop adaptability to various environmental stresses and promoting high and stable crop yields is an important research direction.

[0003] Past yield increases brought about by the Green Revolution were mainly due to improvements in yield traits, such as plant height and economic indices, but the remaining potential is currently limited. Future yield increases may largely stem from improvements in photosynthetic performance. Field crops operating in increasingly frequent extreme climates are highly susceptible to stresses such as high temperatures, fluctuating light levels, and drought. Therefore, optimizing the electron transport chain is fundamental for plants to achieve maximum photosynthetic yield in field environments.

[0004] The photosynthetic electron transport pathway on the thylakoid membrane of higher plant chloroplasts includes linear and cyclic electron transport pathways. The linear electron transport pathway converts light energy into ATP and reduced NADPH; the cyclic electron transport (CET) pathway, centered around photoreactive system I (PSI), produces only ATP and does not accumulate NADPH. In the cyclic electron transport pathway around PSI, electrons pass sequentially through ferrore-reducing proteins (Fd), the p-quinone (PQ) pool, cytochrome b6 / f, and finally return to PSI. It can also pump protons into the thylakoid lumen, forming a proton gradient (ΔpH) across the thylakoid membrane, driving ATP synthase to produce ATP. The cyclic electron transport pathway is widely involved in plant defense and adaptation to various stresses, possessing physiological functions such as promoting carbon assimilation, stress resistance, and protecting photosynthetic structures. Optimizing the photosynthetic electron transport chain in plants by enhancing cyclic electron transport activity is a potential option for improving photosynthetic carbon assimilation and yield.

[0005] The cyclic electron transport pathway is further divided into the NAD(P)H dehydrogenase (NDH) complex-mediated pathway (NDH-CET) and the proton gradient regulator protein PGR5 / PGRL1-mediated pathway, both of which are partially redundant in C3 plants. The NDH complex consists of at least 29 subunits and further forms a supercomplex with PSI, exhibiting a sophisticated structure and complex overall regulatory mechanism. However, to date, no genetic regulatory factors related to the cyclic photosynthetic electron transport pathway have been identified, which significantly limits its targeted application in high-efficiency crop breeding and bioengineering.

[0006] Therefore, there is a need in this field to explore methods to improve the linear electron transport activity, cyclic electron transport activity, and photosynthetic rate of plants, so as to modify the photosynthetic traits of plants with the help of genetic engineering technology and increase the photosynthetic yield of plants. Summary of the Invention

[0007] The purpose of this invention is to provide a method for improving photosynthetic electron transport activity and photosynthetic rate in plants. Through in-depth research, this invention has discovered that AOC in grasses plays a role in regulating linear and cyclic electron transport activity and photosynthetic rate. Increased AOC expression can enhance photosynthetic electron transport activity and increase photosynthetic rate. Therefore, AOC, as a positive regulator of photosynthetic electron transport activity, can provide a new approach for targeting and regulating this gene to improve plant photosynthetic traits and increase photosynthetic yield.

[0008] In a first aspect of the invention, a method is provided to improve the photosynthetic electron transport and carbon dioxide assimilation rate of grass plants, the method comprising: upregulating the expression or activity of AOC in grass plants; wherein improving photosynthetic traits includes: increasing linear electron transport activity and cyclic electron transport activity, thereby increasing the photosynthetic rate.

[0009] In one or more embodiments, the upward adjustment refers to a significant upward adjustment, such as an increase of 20%, 40%, 60%, 80%, 90%, or more.

[0010] In one or more embodiments, upregulating AOC expression or activity includes administering an upregulator of AOC to the plant to increase its expression or activity.

[0011] In one or more embodiments, the upregulator includes: a polynucleotide or construct encoding AOC, an upregulator that enhances AOC gene expression or activity, an upregulator that interacts with the AOC protein to increase its expression or activity, a chemical upregulator of AOC, or a combination thereof.

[0012] In one or more embodiments, the upregulators that enhance AOC gene expression or activity include, but are not limited to, promoters and enhancers.

[0013] In one or more embodiments, the improvement is made when the light intensity is greater than 350 μmol·m -2 ·s -1 Improvements in the environment.

[0014] In one or more embodiments, the improvement is made when the light intensity is greater than 400 μmol·m -2 ·s -1 Greater than 500 μmol·m -2 ·s -1 Greater than 600 μmol·m -2 ·s -1 Greater than 700 μmol·m -2 ·s -1 Greater than 800 μmol·m -2 ·s -1 Improvements in the environment; preferably at greater than 1000 μmol·m -2 ·s -1 Greater than 1200 μmol·m -2 ·s -1 Greater than 1500 μmol·m -2 ·s -1 Greater than 1800 μmol·m -2 ·s -1 Improvements under suitable environmental conditions; for example, at 350–2500 μmol·m -2 ·s -1 The preferred range is 800–2000 μmol·m -2 ·s -1 The range is more preferably 800–1800 μmol·m -2 ·s -1 .

[0015] In one or more embodiments, the grasses include: rice, barley, wheat, oats, rye, corn, sorghum, and short-stalked grass.

[0016] In a second aspect of the invention, there is a use of an AOC protein or an upregulator thereof for: improving photosynthetic traits in grasses; wherein the photosynthetic traits include: increasing linear electron transport activity and cyclic electron transport activity, thereby increasing the photosynthetic rate.

[0017] In one or more embodiments, the upregulation of AOC expression or activity includes administering an AOC upregulator to the plant to increase its expression or activity.

[0018] In one or more embodiments, the upregulator includes: a polynucleotide or construct encoding AOC, an upregulator that enhances AOC gene expression or activity, an upregulator that interacts with the AOC protein to increase its expression or activity, a chemical upregulator of AOC, or a combination thereof.

[0019] In one or more embodiments, the AOC protein includes:

[0020] (a) A protein with the amino acid sequence shown in SEQ ID NO:2; or

[0021] (b) A protein derived from (a) that functions identically to the protein in (a) by substitution, deletion, or addition of one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the amino acid sequence shown in SEQ ID NO:2; or

[0022] (c) A protein derived from (a) whose amino acid sequence is 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; more preferably 98% or 99% or more) identical to the amino acid sequence defined in (a) and has the same function as the protein in (a).

[0023] In a third aspect of the invention, a gramineous plant cell, tissue, or organ is provided, characterized in that the plant is an AOC-expressing plant, wherein it contains an exogenous AOC upregulator.

[0024] In one or more embodiments, the upregulator includes: a polynucleotide or construct encoding AOC, an upregulator that enhances AOC gene expression or activity, an upregulator that interacts with the AOC protein to increase its expression or activity, a chemical upregulator of AOC, or a combination thereof.

[0025] In a fourth aspect of the invention, a method is provided for selecting or identifying grasses with superior photosynthetic traits, the method comprising: identifying the expression or activity of AOC in a test grass, wherein if the test grass has high AOC expression or activity, it is a grass with superior photosynthetic traits; wherein the photosynthetic traits include: linear electron transport activity and cyclic electron transport activity, and photosynthetic rate.

[0026] In one or more embodiments, high expression or high activity means a statistically significant increase in expression or activity compared to the average expression or activity of similar or identical plants, such as an increase of 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0027] In one or more embodiments, the screening method further includes setting up a control group to clearly distinguish the expression or activity of AOC.

[0028] In a fifth aspect of the invention, a method is provided for screening potential substances for improving photosynthetic traits in grasses, wherein the improved photosynthetic traits include: increasing linear electron transport activity and cyclic electron transport activity, and increasing photosynthetic rate; the method includes:

[0029] (1) Treat an expression system expressing AOC with candidate substances; and

[0030] (2) Detect the expression or activity of AOC in the system; if the candidate substance statistically increases the expression or activity of AOC, it indicates that the candidate substance is a potential substance for improving the photosynthetic traits of plants.

[0031] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules, agonists, nucleic acid inhibitors, binding molecules, CRISPR constructs, and small molecule compounds targeting AOC proteins or their encoding genes, or upstream or downstream proteins or genes thereof.

[0032] In one or more embodiments, the expression system includes (but is not limited to): a cell system (such as a cell system expressing AOC and a cell system containing a photosynthetic system, or a cell culture system), a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

[0033] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0034] Figure 1 1. OsAOC overexpression vector and OsAOC overexpression rice lines; (A) Schematic diagram of OsAOC overexpression vector; (B) PCR identification of T1 generation OsAOC overexpression rice lines positive for HYG gene, where ZH11 represents rice that has not been transformed into OsAOC overexpression vector, and the plasmid positive control represents rice with vector plasmid as template; (C) PCR identification of T1 generation OsAOC overexpression rice lines positive for OsAOC gene, where ZH11 represents rice that has not been transformed into OsAOC overexpression vector, and the plasmid positive control represents rice with vector plasmid as template; (D) PCR detection of the relative expression level of OsAOC gene in T2 generation OsAOC overexpression rice lines, where ZH11 represents rice that has not been transformed into OsAOC overexpression vector. Figure 1 35S in B and 1C pro AOC 1-1~35S pro :AOC5-2 indicates an overexpression strain.

[0035] Figure 2The effects of OsAOC overexpression on cyclic electron transport activity and linear electron transport; (A) Effect of OsAOC overexpression on the initial rate of dark reduction of P700+ (indicating cyclic electron transport activity), where ZH11 represents rice without OsAOC overexpression vector; (B) and (C) show the changes in fluorescence parameters ETR(II) and NPQ after OsAOC overexpression, respectively. ETR(II) indicates linear electron transport activity, and NPQ indicates non-photochemical energy dissipation. 35S pro OsAOC-3, 35S pro OsAOC-4 and 35S pro :OsAOC-5 indicates an overexpression strain.

[0036] Figure 3 (A) Illuminance (PAR) is 1200 (μmol m -2 s -1 (A) Results of photosynthetic rate of rice at different times, where ZH11 represents rice without OsAOC overexpression vector; (B) Effect of OsAOC overexpression on leaf ATP content, where ZH11 represents rice without OsAOC overexpression vector; (C) Light intensity (PAR) at 1800, 1200, 800, and 300 μmol / m². -2 s -1 The photosynthetic rate of rice at 35 s is shown in the figure, where ZH11 represents rice that has not been transfected with the OsAOC overexpression vector. pro OsAOC-3, 35S pro OsAOC-4 and 35S pro :OsAOC-5 indicates an overexpression strain. Detailed Implementation

[0037] Through in-depth research, the inventors discovered that AOC in grasses plays a role in regulating photosynthetic electron transport activity and photosynthetic rate. Increased AOC expression can enhance linear and cyclic electron transport activities, as well as photosynthetic rate. Therefore, AOC, as a positive regulator of photosynthetic electron transport activity, can provide a new approach for targeted regulation of this gene to improve photosynthetic traits and increase photosynthetic yield.

[0038] AOC gene

[0039] As used herein, "AOC gene" or "AOC protein" refers to an AOC gene or polypeptide derived from rice that is homologous (with homology exceeding 80%, 85%, 90%, 95%, or 98%) to genes or polypeptides of rice origin, contains substantially the same structural domains, and has substantially the same function. In grasses, AOC genes / AOC proteins exhibit a certain degree of conservation, but this conservation is relatively low compared to other families and genera.

[0040] In this invention, the AOC proteins also include their fragments, derivatives, and analogs. As used herein, the terms "fragment," "derivative," and "analyte" refer to protein fragments that substantially retain the same biological function or activity as the polypeptide, and may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by the fusion of additional amino acid sequences into the protein sequence, etc. These fragments, derivatives, and analogs are well known to those skilled in the art as defined herein. All bioactive fragments of the AOC proteins described herein can be used in this invention.

[0041] In this invention, the term "AOC protein" refers to a protein having the SEQ ID NO:2 sequence with AOC protein activity, and also includes homologs of the protein with the same sequence (homologous proteins).

[0042] The present invention also includes polynucleotides (genes) encoding the protein, such as polynucleotides of the nucleotide sequence shown in SEQ ID NO:1 or degenerate sequences thereof, which may encode the AOC protein of SEQ ID NO:2, and also includes homologs (homologous genes) of proteins of that sequence.

[0043] Vectors containing the said coding sequence, and host cells genetically engineered using the said vector or polypeptide coding sequence, are also included in this invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0044] The host cell can be a plant cell. Transformation of plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, the leaf disc method or rice embryo transformation; Agrobacterium-mediated transformation is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits compared to the wild type.

[0045] As used herein, "plants (including crops)" refers to plants that express an AOC or its homologs. Preferably, the plant is or the AOC is derived from a cereal crop; preferably, the cereal crops include plants of the Poaceae family, such as rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays). Examples include: rice, sorghum, maize, barley, wheat, oats, and rye. It should be understood that the plants applicable to the technical solutions of this invention are not limited to those listed above, and suitable plants can be determined by identifying the presence of AOC homologs.

[0046] In this paper, selecting appropriate "control plants" is a routine part of experimental design and can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same or the same class as the plant being evaluated. Control plants can also be individuals of transgenic plants that have lost their transgenic components due to segregation. As used in this paper, control plants refer not only to whole plants but also to plant parts, including seeds and seed parts.

[0047] AOC upregulators and their applications

[0048] During the process of plant absorption and utilization of light energy, when the absorbed light energy exceeds the plant's capacity, especially under stresses such as high temperature, fluctuating light, and drought, excess light energy is generated. This excess light energy leads to a decrease in the activity of photoreactive system I (PSI), photoreactive system II (PSII), and other thylakoid membrane proteins, resulting in a decline in photosynthetic rate, photoinhibition, and in severe cases, photo-oxidative damage, ultimately reducing plant yield. Cyclic electron transport (CET) is an electron transport pathway on the thylakoid membrane of plant chloroplasts, playing a crucial role in maintaining ATP / NADPH balance, regulating electron transport rates, protecting the photosystem, promoting carbon assimilation, and resisting stress. Therefore, improving the activity of cyclic electron transport in plants can increase the photosynthetic rate, improve photosynthetic traits, and reduce yield reductions caused by photoinhibition and photo-oxidative damage.

[0049] During their research, the inventors discovered that when the expression level of AOC increased, the linear electron transport and cyclic photosynthetic electron transport activities, as well as the photosynthetic rate, of grasses were significantly higher than those of the wild type. These findings of the present invention have not been reported in previous studies in this field.

[0050] Based on the inventor's new discovery, the present invention provides a method for improving the photosynthetic traits of grass plants, the method comprising: upregulating the expression or activity of AOC in plants; wherein the photosynthetic traits include or are selected from the group consisting of: improving the linear electron transport activity, cyclic electron transport activity, and improving the photosynthetic rate of plants.

[0051] This invention also provides the use of AOC protein or its upregulator for improving photosynthetic traits in gramineous plants. In some embodiments, the photosynthetic traits include those selected from the group consisting of increased linear and cyclic electron transport activity, thereby increasing the photosynthetic rate of the plant. Knowing the function of the AOC, various methods well-known to those skilled in the art can be used to overexpress the AOC, thereby improving plant photosynthetic traits. For example, expression units carrying the AOC gene (such as expression vectors or viruses) can be delivered to a target site via methods known to those skilled in the art, thereby expressing the active AOC.

[0052] As a preferred embodiment, the present invention provides a method for preparing transgenic plants, comprising:

[0053] (1) Transferring exogenous AOC-encoding nucleic acids into plant tissues, organs, or seeds to obtain plant tissues, organs, or seeds encoding polynucleotides transformed into AOC; and

[0054] (2) The plant tissues, organs or seeds obtained in step (1) that have been transferred with exogenous coding nucleic acids are regenerated into plant plants.

[0055] Other methods for increasing the expression of the AOC gene or its homologs can also be applied to this invention. For example, the expression of the AOC gene or its homologs can be enhanced by using a strong promoter. Alternatively, the expression of the AOC gene can be enhanced by an enhancer. Strong promoters suitable for this invention include, but are not limited to, the 35S promoter, the Ubi promoter of rice and maize, etc. Enhancers suitable for this invention include, but are not limited to, the 35S enhancer.

[0056] In this invention, the upregulators of AOC or its encoding genes include agonists, promoters, and activators, and these terms are used interchangeably. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of protein / peptide activity or protein / peptide expression, and these are statistically significant "upregulation" and "promotion." Any substance that can increase AOC activity, improve AOC stability, upregulate AOC expression, or increase the effective duration of AOC action can be used in this invention as a useful substance for upregulating AOC. These can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. The AOC or its upregulators are particularly suitable for application to a class of plants whose AOC expression is below the average of that class of plants or whose AOC is not expressed; thus, the application of the AOC protein or its upregulators can revert this class of plants to a wild-type phenotype or a better phenotype. The AOC or its upregulator is also particularly suitable for another type of plant whose photosynthetic traits are impaired, such as by photoinhibition or photooxidative damage. The application of the AOC or its upregulator can improve the photosynthetic traits of this type of plant, restoring it to a wild-type phenotype or a better phenotype.

[0057] As a preferred embodiment, the present invention provides a method for upregulating the expression of AOC protein in plants, the method comprising: transferring an expression construct or vector of AOC protein or its encoded protein into plants.

[0058] The present invention also provides a method for preparing transgenic plants, comprising:

[0059] (a1) The nucleic acid encoding the exogenous AOC protein is transferred into a plant organ or tissue to obtain a plant tissue or organ that has been transformed into the nucleic acid encoding the AOC.

[0060] (a2) Regenerate plant plants from plant tissues or organs that have been transferred with exogenous AOC protein-encoding nucleic acids obtained in step (a1).

[0061] As a preferred example, the method includes the steps of:

[0062] (b1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains nucleic acid encoding the AOC protein of the present invention;

[0063] (b2) Contacting plant tissues or organs with Agrobacterium in step (b1) to transfer the nucleic acid encoding the polypeptide into and integrate it into the chromosome of the plant cell;

[0064] (b3) Selecting plant tissues or organs into which the AOC protein-encoding nucleic acid has been transferred; and

[0065] (b4) Regenerate the plant tissues or organs from step (b3) into a plant.

[0066] The present invention also includes plants obtained using any of the foregoing methods, said plants comprising: transgenic plants into which the encoding nucleic acid of the AOC protein has been transferred.

[0067] filter

[0068] Based on the inventors' new findings, this invention provides molecular markers suitable for identifying photosynthetic traits in grasses, namely, the AOC gene. This invention also relates to specific molecular markers designed for the AOC gene, and identification strategies, thereby enabling early determination of plant photosynthetic performance.

[0069] Therefore, the present invention provides a method for specifically identifying photosynthetic traits of grass plants, comprising: identifying the expression of AOC in the plant to be tested; if the plant has high AOC expression, it is a plant with excellent photosynthetic traits.

[0070] Therefore, the present invention provides a method for targeted selection or identification of plants with regulated agronomic traits, comprising: identifying the expression or activity of AOC protein in a test plant; if the expression or activity of AOC protein in the test plant is higher than the average expression or activity of AOC protein in the same type of plant (control plant), then it is a plant with excellent photosynthetic traits; or, if the expression or activity of AOC protein in the test plant is lower than the average expression or activity of AOC protein in the same type of plant (control plant), then it is a plant with poor photosynthetic traits.

[0071] Based on the novel findings of this invention, those skilled in the art can employ any of the various techniques known in the art or under development to analyze nucleic acid sequences, and these techniques are all included in this invention. The methods described include, but are not limited to: sequencing, PCR amplification, probe methods, hybridization, restriction enzyme digestion analysis, allele polymorphism analysis (such as melting curve analysis) for nucleic acid sequence identification, etc.

[0072] The identification method of this invention only requires PCR reaction and / or agarose gel electrophoresis, and by judging the length of the corresponding PCR product, the phenotype of the sample can be accurately and quickly determined. It is low-cost, suitable for large-scale identification, and requires very little sample. If needed, those skilled in the art can design primers for identifying the molecular markers.

[0073] Methods for obtaining DNA from the sample to be tested are well-known to those skilled in the art, such as the traditional phenol / chloroform / isoamyl alcohol method, or commercially available DNA extraction kits. Polymerase chain reaction (PCR) is also well-known to those skilled in the art; its basic principle is the in vitro enzymatic synthesis of specific DNA fragments. The method of this invention can be performed using conventional PCR techniques.

[0074] This invention has promising applications in molecular design breeding and crop variety improvement using genetic engineering technology.

[0075] After understanding the function of the AOC gene, it can be used as a molecular marker for targeted plant screening. This new discovery can also be used to screen for substances or potential substances that can be targeted to regulate insect resistance by modulating this mechanism.

[0076] This invention provides a method for screening substances (potential substances) that promote the improvement of photosynthetic traits in plants. The improvement of traits includes: increasing the linear electron transport activity and cyclic electron transport activity of plants, and increasing the photosynthetic rate of plants. The method includes: (1) adding the candidate substance to a system expressing AOC protein; (2) detecting the system and observing the expression or activity of AOC protein therein. If its expression or activity is increased, it indicates that the candidate substance is a substance that promotes the photosynthetic traits of plants; conversely, if its expression or activity is decreased, it indicates that the candidate substance is a substance that reduces the photosynthetic traits of plants.

[0077] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0078] The detection of protein-protein interactions and their strength can be achieved using a variety of techniques well-known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation assays, yeast two-hybrid systems, or immunoprecipitation techniques.

[0079] Through large-scale screening, a class of substances that specifically act on AOC proteins or their encoding genes and have a regulatory effect on the improvement of photosynthetic traits can be obtained.

[0080] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0081] Materials and Methods

[0082] 1. Vector construction and transgenic plant production

[0083] RNA was extracted from rice ZH11 and reverse transcribed into cDNA. Using the rice cDNA as a template, the CDS sequence (Os03g0438100) of the rice gene OsAOC was amplified using primers (forward BamHI-AOC-F, SEQ ID NO: 3: GAGATAAACAACGAAA GGATCCATGGCCGCCGCCGCCCCCTCGCGAG); (reverse EcoRI-AOC-R, SEQ ID NO: 4: CAGCTATGACCATGATTACGAATTCCTAGTTGGTGAAGTTGTTGAGGCAG). The full-length CDS sequence was 723 bp. The primers had BamHI and EcoRI restriction endonuclease sequences at both ends. The amplified product was inserted into a modified pCAMBIA1300 vector (with a 35S enhanced promoter preceding the target gene) via homologous recombination. The vector was plant-resistant to hygromycin and bacterial-resistant to kanamycin. The vector was first digested with BamHI and EcoRI restriction endonucleases, recovered via gel electrophoresis, and then reacted with the amplified fragment using homologous recombination. It was then transformed into *E. coli* DH5α. Subsequently, transformation and regeneration of rice variety ZH11 were mediated by *Agrobacterium* strain LBA4404. Hygromycin resistance markers were used for progeny selection to obtain positive seedlings. Finally, gene expression levels in the three transgenic progeny lines were identified by qPCR.

[0084] 2. Growth conditions of transgenic lines

[0085] OsAOC-overexpressing rice lines were planted in artificial climate chambers and at the Songjiang base in Shanghai, and their field performance was evaluated.

[0086] Artificial climate chamber conditions: Rice lines were grown in pots under natural light (with supplemental lighting) and watered twice a week. Cyclic electron transport activity and photosynthetic efficiency were measured 60 days after sowing. The room temperature was controlled at 27℃, light intensity at approximately 600 PPFD, relative humidity at 62–75%, and a 16-hour photoperiod was used. Six biological replicates were performed for each line.

[0087] The Shanghai Songjiang trial, with latitude and longitude information of (121°8′1″E, 30°56′44″N), was planted in June 2023. The average temperature during the growing season was approximately 32°C. Each strain was replicated at least 6 times.

[0088] All transgenic lines underwent PCR testing for the hygromycin resistance gene using primers (HYG-F and HYG-R). The integration of the target gene OsAOC into the transgenic rice genome was also detected by PCR using primers (35S-F, SEQ ID NO: 7 and AOC-R, SEQ ID NO: 8). Lines showing positive results for both PCR tests were selected. For the T2 generation transgenic lines, the expression level of the OsAOC gene was verified by qPCR using primers (qPCR-AOC-F, SEQ ID NO: 5 and qPCR-AOC-R, SEQ ID NO: 6).

[0089] 3. Verify OsAOC gene expression level using qPCR.

[0090] RNA extraction:

[0091] (1) Take the second leaf from the top of the rice plant during the heading stage, put the sample into a grinding tube, add small steel balls, freeze quickly with liquid nitrogen, and grind into powder.

[0092] (2) Add 1 ml RNAiso Plus (Takara) (store at 4℃), vortex to mix, and let stand at room temperature for 5 min.

[0093] (3) Centrifuge at 12000 rpm, 4℃, for 5 min. Transfer 800 μl of the supernatant to a new centrifuge tube and add 1 / 5 volume (160 μl) of chloroform (for RNA) to the fume hood. Vortex until no layering occurs and let stand at room temperature for 5 min.

[0094] (4) Centrifuge at 12000 rpm, 4℃, for 10 min. Transfer 300 μl of the supernatant to a new centrifuge tube and add an equal volume of isopropanol to the fume hood. Vortex to mix and let stand at room temperature for 10-20 min.

[0095] (5) Centrifuge at 12000 rpm, 4℃, for 10 min. The white precipitate is RNA. Discard the supernatant and place the centrifuge tube on ice. Add 0.5 ml of 75% ethanol and mix well. Centrifuge at 12000 rpm, 4℃, for 5 min. Open the cap and let it stand at room temperature for 5 min to allow the ethanol to evaporate completely. Add 20 μl of DEPC water and incubate in a metal bath at 55℃ for 5 min to dissolve the RNA.

[0096] Reverse transcription:

[0097] (6) Place the RNA on ice and measure its concentration using a NanoDrop2000 (diluted to 400-800 ng / μl). Take a PCR tube and add 1000 ng of the corresponding volume of RNA according to the concentration, then add DEPC water (add to 12 μl).

[0098] Using Yeasen's reverse transcription kit ( Ⅲ1st Strand cDNA Synthesis SuperMixfor qPCR)

[0099] (7) Genomic DNA removal reaction. Add on ice:

[0100] 5×gDNA digester mix (with gDNA remover added) 3μL

[0101] 1000ng RNA (the volume corresponding to 1000ng RNA)

[0102] Add 15μl of nuclease-free water (DEPC water is fine).

[0103] Gently shake the reaction solution to mix well, incubate at 42°C for 2 minutes.

[0104] (8) Reverse transcription reaction.

[0105] On ice, add 5 μl of the reaction solution from the previous step. ⅢSuperMix plus, making the system 20μl.

[0106] After gently mixing the reaction solution:

[0107]

[0108] qPCR was performed using the SYBR Green Master Mix reaction system (Yeasen) and the Bio-Rad quantitative PCR instrument.

[0109]

[0110] The amplification reaction program was: pre-denaturation at 95℃ for 2 min + 40 cycles (95℃ for 10 s, 60℃ for 30 s). Housekeeping gene: Osactin. Three cycles.

[0111] Biological replication and three-stage technical replication.

[0112] 4. Photosynthetic efficiency

[0113] A portable photosynthesis system (LICOR-6400XT) was used for the measurements. The leaf chamber temperature was 27℃, and the light intensity PAR was 1200 (μmol / m²).-2 s -1 The CO2 concentration was 400 ppm. When measuring the photosynthetic rate under different light intensities, the light intensity PAR gradient was set to 1800, 1200, 800, and 300 (μmol m²). -2 s -1 ).

[0114] 5. P700 + dark reduction rate

[0115] Using P700 + The principle of determining the activity of cyclic electron transport by dark reduction rate is as follows:

[0116] P700 is the reaction center pigment of photosystem I (PSI), named so because its maximum absorption peak is at 700 nm. Far-red light (700-730 nm) only excites PSI and not PSII, thus not generating linear electron transport from PSII. If the wavelength of the excitation light is less than 700 nm, both PSI and PSII will be excited simultaneously, including cyclic and linear electron transport. The redox of PSI is detected by measuring the change in absorption at 820 nm. On the other hand, the oxidation of P680, the reaction center pigment of PSII, also leads to an increase in absorption at 820 nm. Since the reduction rate of P680+ (ns-level) is much faster than that of P700+ (ms-level), measuring the reduction of P700+ with a time resolution of μs can eliminate the interference of P680+ in both of the above aspects.

[0117] When P700 is irradiated with far-red light, P700 is oxidized to P700. + Electrons are continuously transferred from P700 to electron acceptors such as Fd (ferroredoxin) and NADP. The higher the degree of oxidation of P700, the greater the rise in the signal curve. Under continuous far-red light, the curve gradually stabilizes, and the oxidized state of P700 (P700...) + The system reaches a steady state. At this point, the far-red light is turned off, and the accumulated electrons flow into optical system I (P700) through the ring electron transport pathway. + As the electrons are reduced, the curve descends; the faster the curve descends, the higher the electron transport rate. Therefore, the instantaneous slope of the descending curve can be used to evaluate the electron transport rate of the blade ring.

[0118] The specific measurement method is as follows:

[0119] Using the Dual PAM 100 instrument (Heinz Walz), the P700 was tested with the far-red light off. + Determination of dark reduction rate. Specifically, after rice plants underwent dark adaptation for more than 15 minutes, the leaf was clamped with a probe and oxidized with far-red light (wavelength >705nm) for 30 seconds, then the light was turned off, and the P700 was recorded in the dark. +The re-restoration curve. The slope of the curve's descent when the far-infrared beam is turned off is calculated to represent P700. + The dark reduction rate.

[0120] 6. NPQ and ETR(II)

[0121] NPQ (Non-photochemical quenching coefficient) = Fm / Fm'-1, the proportion of energy absorbed by photosystem II that is dissipated as heat, i.e., the plant's ability to dissipate excess light energy as heat. ETR(II) = PAR*Y(II)*0.84*0.5, reflecting the linear electron flow rate through photosystem II.

[0122] Measurement method: Mature leaves were selected for measurement, and a portable chlorophyll fluorometer PAM-2000 was used. Leaves underwent a 30-minute dark adaptation pretreatment, and the photochemical light intensity was set to 500 μmol / m². -2 s -1 Fine-tune the measurement light to bring Ft between 0.2 and 0.4. First, apply saturated pulsed light and display Fv / Fm, ensuring that Fv / Fm is between 0.8 and 0.85, indicating a healthy plant growth state. Then, turn on the photochemical light and apply multiple saturated pulsed lights continuously. After the curve has basically flattened and stabilized, end the measurement. After measuring all leaves, export NPQ and ETR(II).

[0123] 7. Determine the ATP content in leaves

[0124] ATP was measured using luciferase luminescence assay, employing the BacTiter-Lumi assay from Beyotime Biotechnology. TM luminescence assay kit. The assay method is as follows:

[0125] (1) Add 1 ml of distilled water to a 2 ml centrifuge tube.

[0126] (2) Use a punch to take samples - count the leaf discs, add them to centrifuge tubes, and immerse them in water.

[0127] (3) Immediately place the centrifuge tube in boiling water for 5 minutes. Remove and place on ice. The water at this point is the sample solution (which has dissolved the ATP released from the leaves).

[0128] (4) Prepare ATP standard solutions and ATP reaction solutions (containing luciferin and luciferase, which chemiluminesce in the presence of ATP) with different concentration gradients and place them on ice.

[0129] (5) Take a white ELISA plate and add 50 μL of sample solution / ATP standard solution + 50 μL of reaction solution to each well.

[0130] (6) Detect chemiluminescence using an enzyme-linked immunosorbent assay (ELISA) reader, and perform calculations and

[0131] Note: Leaf discs should be taken separately for fresh weight measurement, and at least 6 biological replicates should be performed.

[0132] 8. Primers

[0133] The primers involved in this invention are shown in Table 1.

[0134] Table 1

[0135]

[0136]

[0137] Example 1: Acquisition of Genes and Their Information

[0138] The gene OsAOC, which is highly associated with cyclic electron transport activity, was identified through genome-wide association studies (GWAS).

[0139] The coding region sequence of OsAOC (723bp, SEQ ID NO:1):

[0140] atggccgccgccgccccctcgcgagtctccgtcagggccgcggcgcccgggcaaacggggggcttcgccaagatccggccgcaggtggtggtggcagcggcggcgaggtcggccggggtgagcggccgcagggcgaggagcgttcgggcgtcgctgttctcgccgaagccggcgacgcccaaggacgcgaggccggccaaggtgcaggagatgttcgtgtacgagatcaacgagcgcgaccgcgagagccccgcctacctccgcctcagcgccaagcagacggagaacgccctcggcgatctcgtccccttcaccaacaagctgtacagcggaagcctggacaagcggctggggatctcggcggggatctgcatcctgatccagcacgtgccggagcgcaacggcgaccgctacgaggccatctacagcttctacttcggcgactacggccacatctccgtgcagggcccgtacctgacctacgaggagtcctacctcgccgtcaccggcggctccggcgtcttcgaaggcgcctacggccaggtcaagctcaaccagatcgtcttccccttcaagatcttctacaccttctacctcaagggcatccccgacctgccgcgggagctgctctgcacgcccgtcccgccgtccccgaccgtcgagccaacgccagccgccaaggccaccgagccccacgcctgcctcaacaacttcaccaactag

[0141] Protein sequence of OsAOC (240 aa, SEQ ID NO: 2):

[0142] MAAAAPSRVSVRAAAPGQTGGFAKIRPQVVVAAAARSAGVSGRRARSVRASLFSPKPATPKDARPAKVQEMFVYEINERDRESPAYLRLSAKQTENALGDLVPFTNKLYSGSLDKRLGIS AGICILIQHVPERNGDRYEAIYSFYFGDYGHISVQGPYLTYEESYLAVTGGSGVFEGAYGQVKLNQIVFPFKIFYTFYLKGIPDLPRELLCTPVPPSPTVEPTPAAKATEPHACLNNNFTN*

[0143] Example 2: OsAOC overexpression vector and OsAOC transgenic lines

[0144] In this embodiment, the full-length CDS sequence of the OsAOC gene (SEQ ID NO:1) was amplified and transformed into rice ZH11 to obtain transgenic rice lines overexpressing OsAOC.

[0145] A schematic diagram of the construction of the OsAOC overexpression vector is shown below. Figure 1 As shown in Figure A, the pCAMBIA1300 vector serves as the backbone. This vector includes: boundary sequences (left boundary LB and right boundary RB), a CaMV 35S promoter and a polyA sequence (CaMV 35S pA), a hygromycin resistance gene coding region sequence (HYG) driven by one 35S-enhance promoter, an OsAOC gene coding region sequence (OsAOC) driven by another 35S-enhance promoter, and a Nos terminator. Rice ZH11 was transformed using this vector to obtain T1 generation OsAOC-overexpressing rice.

[0146] Using HYG-F / R and 35S-F / AOC-R primers respectively, HYG( Figure 1 B) and the 35S-enhance-driven OsAOC gene ( Figure 1 C) Positive T1 generation lines. T1 generation HYG-positive lines were planted and harvested normally to obtain T2 generation transgenic lines. Further, the OsAOC gene was amplified using qPCR with qPCR-AOC-F / R primers, and the expression level of the OsAOC gene in the T2 generation transgenic lines was detected.

[0147] The results showed that, compared to ZH11, OsAOC was significantly upregulated in OsAOC-overexpressing rice. Figure 1 D).

[0148] Example 3: Determination of cyclic electron transport activity in OsAOC-overexpressing rice

[0149] In this embodiment, the T2 generation OsAOC-overexpressing rice obtained in Example 2 was grown in an artificial climate chamber, and the P700 was measured. + The dark reduction rate was investigated to explore the changes in cyclic electron transport activity after OsAOC overexpression.

[0150] The results showed that, compared with ZH11, OsAOC-overexpressing rice (P700) + The increased initial rate of dark reduction indicates higher cyclic electron transport activity. Figure 2 A).

[0151] Example 4: Determination of NPQ and ETR(II) in OsAOC-overexpressing rice

[0152] In this embodiment, the T2 generation OsAOC-overexpressing rice obtained in Example 2 was grown in an artificial climate chamber to investigate the changes in fluorescence parameters NPQ and ETR(II) after OsAOC overexpression. NPQ and ETR(II) were measured using a PAM2000 instrument.

[0153] The results showed that the ETR(II) of OsAOC-overexpressing rice was significantly higher than that of ZH11 ( Figure 2 B); NPQ is significantly lower than ZH11 ( Figure 2 C). This indicates that in a linear electron transport chain, photosystem II absorbs more energy for enhancing the photosynthetic electron transport rate and less for heat dissipation.

[0154] Example 5: Determination of photosynthetic performance of OsAOC-overexpressing rice

[0155] In this embodiment, the T2 generation OsAOC-overexpressing rice obtained in Example 2 was planted in an artificial climate chamber to investigate the changes in photosynthetic efficiency after OsAOC overexpression.

[0156] Using a photosynthesis apparatus (LICOR-6400XT), the concentration of 1200 μmol / m³ was measured. -2 s -1 Photosynthetic rate of rice under light intensity.

[0157] The results showed that the photosynthetic rate of rice overexpressing OsAOC was significantly higher than that of rice overexpressing ZH11 ( Figure 3 A).

[0158] Furthermore, using a photosynthesis analyzer (LICOR-6400XT), light intensities of 1800, 1200, 800, and 300 μmol / m² were measured. -2 s -1 The photosynthetic rate of rice at that time.

[0159] The results showed that the higher the light intensity, the more significant the advantage in photosynthetic rate between OsAOC-overexpressing rice and ZH11. Figure 3 C).

[0160] Example 6: Determination of ATP content in leaves of OsAOC-overexpressing rice

[0161] In this embodiment, the T2 generation OsAOC-overexpressing rice obtained in Example 2 was planted in an artificial climate chamber to investigate the changes in leaf ATP content after OsAOC overexpression.

[0162] The ATP content of leaves was determined using luciferin luminescence method.

[0163] The results showed that the ATP content of OsAOC-overexpressing rice was higher than that of ZH11 ( Figure 3 B).

[0164] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for increasing the photosynthetic electron transport and carbon dioxide assimilation rates of grass plants, the method comprising: Upregulating the expression or activity of AOC in grasses; among which, improving photosynthetic traits includes: increasing linear electron transport activity and cyclic electron transport activity, and increasing photosynthetic rate.

2. The method as described in claim 1, characterized in that, Upregulating AOC expression or activity includes administering AOC upregulators to plants, thereby increasing their expression or activity; Preferably, the upregulator includes: a polynucleotide or construct encoding AOC, an upregulator that enhances AOC gene expression or activity, an upregulator that interacts with the AOC protein to increase its expression or activity, a chemical upregulator of AOC, or a combination thereof.

3. The method as described in claim 1 or 2, characterized in that, The improvement is to be performed when the light intensity is greater than 350 μmol·m -2 ·s -1 Improvements in the environment.

4. The method as described in claim 1 or 2, characterized in that, The aforementioned grasses include: rice, barley, wheat, oats, rye, corn, sorghum, and short-stalked grass.

5. The use of an AOC protein or its upregulator for: improving the photosynthetic traits of gramineous plants; wherein, The photosynthetic traits include: enhanced linear and cyclic electron transport activity, thereby increasing the photosynthetic rate.

6. The use as described in claim 5, characterized in that, The upregulation of AOC expression or activity includes administering an AOC upregulator to the plant to increase its expression or activity.

7. The use as described in claim 5, characterized in that, The upregulators include: polynucleotides or constructs encoding AOC, upregulators that enhance AOC gene expression or activity, upregulators that interact with the AOC protein to increase its expression or activity, chemical upregulators of AOC, or combinations thereof.

8. The method as described in any one of claims 1-4 or the use as described in any one of claims 5-7, characterized in that, The AOC protein includes: (a) A protein with the amino acid sequence shown in SEQ ID NO:2; or (b) A protein derived from (a) that functions identically to the protein in (a) by substitution, deletion, or addition of one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the amino acid sequence shown in SEQ ID NO:2; or (c) A protein derived from (a) whose amino acid sequence is 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; more preferably 98% or 99% or more) identical to the amino acid sequence defined in (a) and has the same function as the protein in (a).

9. A cell, tissue, or organ of a grass family plant, characterized in that, The plant described is an AOC-expressing plant containing an exogenous AOC upregulator; Preferably, the upregulator includes: a polynucleotide or construct encoding AOC, an upregulator that enhances AOC gene expression or activity, an upregulator that interacts with the AOC protein to increase its expression or activity, a chemical upregulator of AOC, or a combination thereof.

10. A method for selecting or identifying grasses with superior photosynthetic traits, the method comprising: The expression or activity of AOC in grass plants is identified and tested. If the expression or activity of AOC in the tested grass plant is high, it is a grass plant with excellent photosynthetic traits. The photosynthetic traits include: linear electron transport activity and cyclic electron transport activity, and photosynthetic rate.

11. A method for screening potential substances for improving photosynthetic traits in grasses, wherein the improved photosynthetic traits include: The method aims to improve linear and cyclic electron transport activity and increase photosynthetic rate. (1) Treat an expression system expressing AOC with candidate substances; and (2) Detect the expression or activity of AOC in the system; if the candidate substance statistically increases the expression or activity of AOC, it indicates that the candidate substance is a potential substance for improving the photosynthetic traits of plants.

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