Application of CsPHO1;H3 gene in improving photosynthetic efficiency and energy metabolism of cucumber seedlings

Abstract

The application discloses CsPHO1;H3 The application relates to the fields of plant genetic engineering and vegetable breeding technology, and discloses application of a gene in improving photosynthetic efficiency and energy metabolism of cucumber seedlings. CsPHO1;H3 The application proves for the first time that overexpression of the gene can positively improve photosystem II efficiency and net photosynthetic rate of cucumber, and can improve TP, ATP and ATP / ADP, and optimize cell energy state, and the photosynthetic improvement is not dependent on stomatal regulation, and does not increase transpiration water consumption, and the plant is more stable and stronger in resistance. The application has clear gene function, and the obtained cucumber seedlings are stable in traits, and can be directly used for cucumber transgenic breeding, gene editing breeding and molecular marker assisted breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and vegetable breeding technology, specifically to... CsPHO1;H3 Application of genes in improving photosynthetic efficiency and energy metabolism in cucumber seedlings. Background Technology

[0002] cucumber( Cucumis sativus Cucumber (L.) is a high-value-added vegetable mainly grown in greenhouses in my country. The quality of seedlings directly determines the survival rate after transplanting, the plant's resistance to adverse conditions, its growth vigor, and its later yield and quality. Photosynthesis is the core basis for the accumulation of substances and growth and development of cucumber seedlings. The incomplete development of the photosynthetic system, low light energy utilization efficiency, and insufficient cellular energy supply in the seedling stage are key physiological bottlenecks that cause cucumber seedlings to be weak, slow to recover, and have limited yield.

[0003] Current research on high-yield cucumber breeding mainly focuses on fruit traits at maturity, stress resistance traits, and yield components. Research on functional gene discovery and molecular mechanisms for improving seedling photosynthetic efficiency and regulating endogenous energy homeostasis is relatively scarce. Existing technologies for improving crop photosynthetic efficiency mostly focus on stomatal regulation pathways, increasing carbon dioxide uptake by altering stomatal conductance, thereby improving the photosynthetic rate. However, this approach is usually accompanied by increased transpiration rates, easily leading to increased water loss from plants, seedling wilting, and decreased drought resistance, exhibiting significant technical shortcomings.

[0004] Phosphorus is an essential macronutrient for plants, participating in photosynthetic phosphorylation, energy metabolism, nutrient synthesis, and signal regulation. The PHO1 family of genes are classic phosphorus transport genes, and current research mainly focuses on phosphorus uptake, transport, and distribution in plant roots, as well as responses to low phosphorus stress. Currently, no research has been published on cucumbers. CsPHO1;H3 The gene is involved in regulating the photochemical efficiency of leaf photosystem II, net photosynthetic rate, and ATP energy metabolism, and there are no reports that it can specifically enhance plant photosynthetic capacity without changing stomatal conductance and transpiration rate.

[0005] Therefore, excavation CsPHO1;H3 Discovering novel biological functions of the gene and developing its applications in enhancing photosynthesis, improving energy efficiency, and molecular breeding during the cucumber seedling stage has significant theoretical value and promising prospects for industrial application. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies in cucumber seedlings, such as low photosynthetic utilization rate, insufficient endogenous energy accumulation, increased transpiration water consumption due to existing photosynthetic improvement techniques, and a lack of usable functional breeding genes. It provides... CsPHO1;H3 The application of genes in improving photosynthetic efficiency and energy metabolism in cucumbers, through overexpression CsPHO1;H3The gene specifically enhances the activity of photosystem II in cucumber seedling leaves, increases the net photosynthetic rate, enriches total phosphorus and ATP in leaves, and optimizes cellular energy status. Moreover, this photosynthetic enhancement effect is independent of the stomatal regulation pathway, does not change the plant's transpiration characteristics, and can stably cultivate robust cucumber seedlings.

[0007] Therefore, in a first aspect, the present invention provides CsPHO1;H3 The application of genes in improving photosynthetic efficiency and energy metabolism in cucumber seedlings, the aforementioned CsPHO1;H3 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0008] Furthermore, the application is achieved by overexpressing the CsPHO1;H3 Genes that improve the photosynthetic efficiency and / or energy metabolism level of cucumber seedlings.

[0009] Furthermore, by overexpressing the aforementioned CsPHO1;H3 Genes that enhance at least one of the maximum photochemical efficiency, actual photochemical efficiency, and net photosynthetic rate of cucumber seedling leaves.

[0010] Furthermore, by overexpressing the aforementioned CsPHO1;H3 Genes that increase at least one of the following in cucumber seedling leaves: total phosphorus content, adenosine triphosphate (ATP) content, and the ATP / DP ratio.

[0011] A second aspect of the present invention provides a molecular breeding method for producing cucumber seedlings with high photosynthetic efficiency and high energy accumulation, the molecular breeding method comprising: overexpressing the [specific ingredient] in the application in cucumber plants. CsPHO1;H3 Gene.

[0012] Furthermore, the overexpression CsPHO1;H3 Genetic methods include: Construct the CsPHO1;H3 Gene overexpression vectors; The overexpression vector was transformed into Agrobacterium, and the cucumber explants were infected by Agrobacterium-mediated infection. Infected explants were cultured and screened by PCR to obtain... CsPHO1;H3 Overexpression lines; Cultivation CsPHO1;H3 Overexpression lines were then harvested and seeds were collected. By planting the aforementioned seeds, cucumber seedlings with high photosynthetic efficiency and high energy accumulation can be cultivated.

[0013] Furthermore, the method for constructing the overexpression vector includes: The amplification of the above using specific primers CsPHO1;H3 The gene is then amplified and the amplified product is linked to a linearized vector via homologous recombination to obtain the overexpression vector.

[0014] Furthermore, the specific primer is CsPHO1;H3 -F and CsPHO1;H3 -R; the CsPHO1;H3 The nucleotide sequence of -F is shown in SEQ ID NO:2. CsPHO1;H3 The nucleotide sequence of -R is shown in SEQ ID NO:3.

[0015] Furthermore, the Agrobacterium is EHA105, and the cucumber explant is a cotyledon node of the cucumber inbred line CCMC.

[0016] Furthermore, the primers used for PCR identification and screening are... CsPHO1;H3 -OE-F and CsPHO1;H3 -OE-R; the CsPHO1;H3 The nucleotide sequence of -OE-F is shown in SEQ ID NO:4. CsPHO1;H3 The nucleotide sequence of -OE-R is shown in SEQ ID NO:5.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides CsPHO1;H3 Application of genes in regulating photosynthetic efficiency and energy metabolism in cucumber seedlings. CsPHO1;H3 The nucleotide sequence of the gene is shown in SEQ ID NO:1. This invention is the first to demonstrate overexpression. CsPHO1;H3 The gene can positively enhance the efficiency of photosystem II and net photosynthetic rate in cucumbers, while simultaneously increasing TP, ATP, and ATP / ADP, optimizing cellular energy status. Furthermore, this photosynthetic enhancement is independent of stomatal regulation and does not increase transpiration water consumption, resulting in more robust plants with stronger stress resistance. This invention has a clearly defined target, and the obtained cucumber seedlings exhibit stable traits, making them directly applicable to cucumber transgenic breeding, gene editing breeding, and marker-assisted breeding. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0019] Figure 1 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Results of qPCR detection on leaves of cucumber seedlings (OE2 and OE5) 20 days after germination; Figure 2 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3Phenotypic and chlorophyll fluorescence images of overexpressing cucumber seedlings (OE2 and OE5) 20 days after germination, showing maximum photochemical efficiency (Fv / Fm). Figure 3 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Quantitative analysis of the maximum photochemical efficiency (Fv / Fm) of cucumber seedlings overexpressing OE2 and OE5 20 days after germination; Figure 4 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Quantitative analysis of actual photochemical efficiency (ΦPSII) of cucumber seedlings overexpressing OE2 and OE5 20 days after germination; Figure 5 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Results of net photosynthetic rate (Pn) of leaves of overexpressing cucumber seedlings (OE2 and OE5) 20 days after germination; Figure 6 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Results of stomatal conductance (gsw) measurement of leaves of overexpressing cucumber seedlings (OE2 and OE5) 20 days after germination; Figure 7 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Results of leaf transpiration rate (Tr) measurement in cucumber seedlings (OE2 and OE5) 20 days after germination; Figure 8 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Results of total phosphorus (TP) content determination in leaves of overexpressing cucumber seedlings (OE2 and OE5) 20 days after germination; Figure 9 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Results of adenosine triphosphate (ATP) content determination in leaves of overexpressing cucumber seedlings (OE2 and OE5) 20 days after germination; Figure 10 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Results of ADP content determination in leaves of overexpressing cucumber seedlings (OE2 and OE5) 20 days after germination; Figure 11 Wild-type cucumber seedlings (WT) provided for embodiments of the present invention and CsPHO1;H3 Results of determination of the ATP / ADP ratio in leaves of overexpressing cucumber seedlings (OE2 and OE5) 20 days after germination. Detailed Implementation

[0020] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.

[0021] A first aspect of the present invention provides CsPHO1;H3 Application of genes in improving photosynthetic efficiency and energy metabolism in cucumber seedlings. CsPHO1;H3 The nucleotide sequence of the gene is shown in SEQ ID NO:1, and its CDS sequence is shown in SEQ ID NO:8.

[0022] In some embodiments, the application is through overexpression CsPHO1;H3 Genes that improve the photosynthetic efficiency and / or energy metabolism level of cucumber seedlings.

[0023] In some embodiments, through overexpression CsPHO1;H3 Genes that enhance at least one of the following in cucumber seedling leaves: maximum photochemical efficiency (Fv / Fm), actual photochemical efficiency (ΦPSII), and net photosynthetic rate (Pn).

[0024] In some embodiments, through overexpression CsPHO1;H3 Genes that increase at least one of the following in cucumber seedling leaves: total phosphorus (TP), ATP content, and ATP / ADP ratio.

[0025] Specifically, this invention is the first to demonstrate overexpression CsPHO1;H3 This gene can positively enhance the efficiency of photosystem II and net photosynthetic rate in cucumber, breaking through the conventional understanding in this field. The mechanism by which the photosynthetic efficiency of cucumber seedlings is enhanced is through optimization of photosystem efficiency within mesophyll cells, a non-stomatal regulation pathway, without increasing transpiration water consumption, resulting in more robust plants and stronger stress resistance. Furthermore, it can simultaneously increase TP, ATP, and ATP / ADP, supporting efficient photosynthesis from an energy metabolism perspective, resulting in significantly stronger seedlings. This invention has a clearly defined target and produces stable cucumber seedlings, which can be directly used for transgenic cucumber breeding, gene editing breeding, and molecular marker-assisted breeding, providing novel gene resources and molecular improvement strategies for cultivating robust cucumber seedlings and high photosynthetic efficiency breeding.

[0026] A second aspect of this invention provides a molecular breeding method for cucumber seedlings with high photosynthetic efficiency and high energy accumulation, comprising: overexpressing [a specific gene] in cucumber plants. CsPHO1;H3 Gene.

[0027] In some embodiments, overexpression CsPHO1;H3 Genetic methods include: Infected explants were cultured and screened by PCR to obtain... CsPHO1;H3 Overexpression lines; Cultivation CsPHO1;H3 Overexpression lines were then harvested and seeds were collected. By planting the aforementioned seeds, cucumber seedlings with high photosynthetic efficiency and high energy accumulation can be cultivated.

[0028] In some embodiments, the method for constructing the overexpression vector includes: Amplification using specific primers CsPHO1;H3 The gene is then amplified and its products are linked to a linearized vector via homologous recombination to obtain an overexpression vector.

[0029] In some embodiments, the specific primers are CsPHO1;H3 -F and CsPHO1;H3 -R; CsPHO1;H3 The nucleotide sequence of -F is shown in SEQ ID NO:2. CsPHO1;H3 The nucleotide sequence of -R is shown in SEQ ID NO:3.

[0030] In some embodiments, Agrobacterium is EHA105, and the cucumber explant is a cotyledon node of the cucumber inbred line CCMC.

[0031] In some embodiments, the primers used for PCR identification and screening are CsPHO1;H3 -OE-F and CsPHO1;H3 -OE-R; the CsPHO1;H3 The nucleotide sequence of -OE-F is shown in SEQ ID NO:4. CsPHO1;H3 The nucleotide sequence of -OE-R is shown in SEQ ID NO:5.

[0032] The background material for this invention is the cucumber variety CCMC, which has been used in the 2024 publication of the article "A genome-wide association study reveals molecular mechanism underlying powdery mildewresistance in cucumber" in the journal Genome Biology. The germplasm resources mentioned above are preserved in the applicant's laboratory germplasm bank.

[0033] Example 1: Construction and Material Breeding of Transgenic Cucumber Lines 1. Positive CsPHO1;H3 Construction of gene overexpression lines p1305.4-3×Flag was used as the backbone vector. The vector was linearized by single-enzyme digestion with the restriction endonuclease BamHI (NEB). The digestion system consisted of: 2 μg vector DNA, 2 μL BamHI, 5 μL 10× FastDigest Buffer, and ddH2O to a final volume of 50 μL. The reaction was carried out at 37°C for 3 hours. After complete digestion was confirmed by 1.0% agarose gel electrophoresis, the linearized vector was purified using an OMEGA gel extraction kit.

[0034] Using cDNA from cucumber "CCMC" leaves as a template, high-fidelity DNA polymerase was used for amplification. CsPHO1;H3 Full-length coding region sequence. Primer pair is... CsPHO1;H3 -F (SEQ ID NO:2) and [[ID=CO1]]CsPHO1;H3 -R (SEQ ID NO:3) introduces sequences homologous to the ends of the linearized vector at the 5' and 3' ends, respectively. PCR reaction system (50 μL): 10 μL 5×Phusion HF Buffer, 1 μL dNTPs (10 mM), 1.5 μL each of forward and reverse primers (10 μM), 2 μL cDNA template, 0.5 μL Phusion DNA polymerase, and ddH2O added to 50 μL. Reaction program: 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 60°C annealing for 20 s, 72°C extension for 1 min (1 kb / min), for a total of 35 cycles; final extension at 72°C for 10 min. PCR products were identified by 1.0% agarose gel electrophoresis, and the target fragment was recovered and purified using a gel extraction kit.

[0035] The purified PCR product was inserted upstream of the 3×Flag tag sequence (N-terminal fusion) using the homologous recombination method (ClonExpress® II One Step Cloning Kit, Vazyme) to construct the recombinant plasmid p1305.4-3×Flag- CsPHO1;H3 H3 Recombinant reaction system (20 μL): linearized vector 50 ng - 100 ng, PCR product (vector to insert molar ratio 1:2), 5×CE II Buffer 4 μL, Exnase® II 2 μL, add ddH2O to 20 μL, and react at 37°C for 30 minutes.

[0036] Transform 10 μL of the recombinant product into *E. coli* DH5α chemocompetent cells (TransGen Biotech), incubate on ice for 30 min, heat shock at 42°C for 45 s, incubate on ice for 2 min, add 800 μL of LB broth, and incubate at 37°C and 200 rpm for 1 h. Spread the mixture onto a substrate containing 50 mg·L⁻¹ of LB broth. -1Incubate kanamycin on LB agar plates (solid) at 37°C (inverted) for 16-18 hours. Pick a single colony and inoculate it onto LB liquid medium (containing 50 mg / L). -1 In kanamycin, culture at 37°C and 200 rpm for 12-16 hours with shaking.

[0037] Initial screening was performed using bacterial culture PCR. The reaction system (20 μL) consisted of: 10 μL of 2×Taq Master Mix, 0.5 μL each of forward and reverse primers (10 μM), 1 μL of bacterial culture, and ddH2O added to a final volume of 20 μL. The reaction program was: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, for a total of 30 cycles; and a final extension at 72°C for 5 minutes. PCR-positive clones were selected, and plasmid DNA was extracted using an OMEGA plasmid miniprep kit and sent to a sequencing company (Sangon Biotech) for Sanger sequencing verification. The sequencing results were compared with the target gene sequence using BLAST. Positive recombinant plasmids with completely correct sequences were named p1305.4-3×Flag- CsPHO1; .

[0038] The correctly sequenced recombinant plasmid was transformed into *Agrobacterium tumefaciens* EHA105 competent cells using a freeze-thaw method. Specific procedures: 2 μg of recombinant plasmid was added to 100 μL of *Agrobacterium tumefaciens* competent cells, mixed thoroughly, incubated on ice for 30 minutes, flash-frozen in liquid nitrogen for 1 minute, heat-shocked in a 37°C water bath for 5 minutes, then 800 μL of LB broth was added, and the cells were thawed at 28°C and 200 rpm for 4 hours. The mixture was then plated onto a substrate containing 50 mg·L⁻¹ of LB broth. -1 Kanamycin and 25 mg / L -1 Incubate rifampicin-containing YEB solid medium at 28°C inverted for 48 hours. Single colonies are picked for colony PCR identification. Positive clones confirmed by successful transformation are stored at -80°C (containing 25% glycerol) for later use. Agrobacterium-mediated transformation is used to transform the cotyledonary nodes of the cucumber inbred line CCMC. For specific genetic transformation and screening procedures, please refer to the paper published in Horticulture Research in 2022: "Targeted creation of new mutants with compactplant architecture using CRISPR / Cas9 genome editing by an optimized genetic transformation procedure in cucumber plants".

[0039] 2. Plant material screening and cultivation Using the DNA from the leaves of the plant to be tested as a template, CsPHO1;H3-OE-F (SEQ ID NO:4) and CsPHO1;H3 PCR was performed using OE-R (SEQ ID NO:5). T0 generation plants with positive PCR and a single band were selected for self-pollination, and T1 generation seeds were harvested from each plant. After sowing the T1 generation, PCR identification was performed again using the above method to screen for positive plants. Wild-type cucumber (WT) seeds and T1 generation positive overexpression lines (OE2 and OE5 lines) were simultaneously sown in an artificial climate chamber under uniform light (16h light / 8h darkness), temperature (28°C / 22°C), humidity (70%~80%), and water and fertilizer conditions, and were cultured normally until seedlings reached 20 days of growth (three-leaf stage). Leaves from WT, OE2, and OE5 seedlings were taken, flash-frozen in liquid nitrogen, and total RNA was extracted and reverse transcribed into cDNA. CsPHO1;H3 qPCR reactions were performed using specific primers qPCR-F (SEQ ID NO:6) and qPCR-R (SEQ ID NO:7), with CsActin as an internal control, and 2 -ΔΔCt The relative expression level was calculated using the method. Results ( CsPHO1;H3 (Displays OE2 and OE5) Figure 1 The expression levels were significantly higher than those of WT, proving that the overexpression line was successfully constructed.

[0040] Primer sequences are shown in Table 1, and the 10 μL qPCR reaction system is shown in Table 2.

[0041] Table 1 Primer Sequences

[0042] Table 2 Reaction System

[0043] The PCR reaction procedure is as follows: Stage 1: Pre-denaturation at 95℃ for 30 seconds; Stage 2: Denaturation at 95℃ for 10 seconds; Annealing at 60℃ for 30 seconds; Extension at 72℃ for 1 minute; 40 cycles.

[0044] The reaction was performed on a Bio-Rad PCR instrument, with each sample tested in triplicate.

[0045] Example 2: Determination of photochemical efficiency index of cucumber seedlings Wild-type cucumber seedlings (WT) grown to 20 days old under uniform cultivation conditions were selected. CsPHO1;H3Overexpression was performed on cucumber seedlings (OE2 and OE5), with three biological replicates for each line. Fully expanded functional leaves from the middle of the plant were selected, and the leaves were subjected to a 20-minute dark adaptation treatment to ensure the photosystem was in its initial state. Chlorophyll fluorescence parameters were measured using a LI-6800 portable photosynthesis measurement system, including maximum photochemical efficiency (Fv / Fm) and actual PSII photochemical efficiency (ΦPSII).

[0046] Test results as follows CsPHO1;H3 , Figure 2 and Figure 3 As shown, compared to wild-type plants, Figure 4 The maximum photochemical efficiency (Fv / Fm) and actual PSII photochemical efficiency (ΦPSII) of leaves in overexpressing plants were significantly increased. These results demonstrate that overexpression... CsPHO1;H3 The gene can effectively activate the photosystem II activity of cucumber seedlings, enhancing the leaves' ability to capture, transfer, and photochemically convert light energy.

[0047] Example 3: Measurement of gas exchange parameters in cucumber seedlings Wild-type cucumber seedlings (WT) grown to 20 days old were used. CsPHO1;H3 Cucumber seedlings (OE2 and OE5) overexpressing the gene were used as experimental materials. Leaf gas exchange parameters were measured using a LI-6800 portable photosynthesis measurement system. The environmental parameters were set as follows: photosynthetic photon flux density 500 μmol·m⁻¹. -2 s -1 The ambient CO2 concentration was 400 μmol·mol⁻¹ -1 The leaf temperature was 28℃.

[0048] The measured parameters included net photosynthetic rate (Pn), stomatal conductance (gsw), intercellular CO2 concentration (Ci), and transpiration rate (Tr). Statistical analysis of the experimental data is as follows: CsPHO1;H3 , Figure 5 and Figure 6 As shown, Figure 7 The net photosynthetic rate (Pn) of the overexpressing plants was significantly higher than that of the wild-type plants; there were no statistically significant differences in stomatal conductance (gsw) and transpiration rate (Tr) between the two groups of plants.

[0049] This result confirms that CsPHO1;H3 The gene-mediated enhancement of cucumber photosynthetic efficiency does not depend on stomatal regulation pathways, but mainly achieves enhanced photosynthetic capacity by optimizing the operation efficiency of the photosystem inside mesophyll cells; the plant's transpiration water consumption remains stable, effectively ensuring the physiological stability and stress resistance of cucumber seedlings.

[0050] Example 4: Detection of Energy and Phosphorus Metabolism Indicators in Cucumber Seedling Leaves 1. Preparation of experimental materials and samples Wild-type cucumber seedlings (WT) cultivated to 20 days old under uniform light, temperature, humidity, and water and fertilizer management conditions were selected. CsPHO1;H3 Cucumber seedlings (OE2 and OE5) with overexpressed genes were used as experimental materials. Each experimental line was set up with 3 independent biological replicates. For each biological replicate, 6 healthy seedlings with uniform growth, free from pests and diseases, and of consistent growth status were randomly selected.

[0051] Healthy, fully expanded functional leaves from the middle of each seedling stem were uniformly picked, the main veins of the leaves were removed, and the leaves were quickly chopped and mixed. After sampling, the samples were immediately placed in liquid nitrogen for rapid freezing, then aliquoted into cryovials and labeled, and transferred to an ultra-low temperature freezer at -80℃ for cryopreservation. The samples were then used to determine physiological indicators such as total phosphorus, ATP, and ADP in the leaves.

[0052] 2. Determination of total phosphorus (TP) content in leaves This experiment used the molybdenum blue colorimetric method to determine the total phosphorus content in leaves. The specific operating steps are as follows: (1) Accurately weigh 0.1g of frozen leaf sample and place it in a pre-cooled mortar. Add an appropriate amount of pre-cooled trichloroacetic acid extract and grind it thoroughly in an ice bath until it becomes a homogeneous slurry. (2) Transfer all the homogenate obtained from grinding to centrifuge tubes, let it stand at room temperature for 20 min, and then centrifuge at 8000 r / min and 4℃ for 15 min using a high-speed refrigerated centrifuge. (3) Carefully aspirate the supernatant after centrifugation as the sample solution to be tested. At the same time, prepare a series of total phosphorus standard solutions with gradient concentrations. Add the color reagents in sequence according to the molybdenum blue color reaction system and develop the color at room temperature in the dark for 15 min. (4) Using a UV-Vis spectrophotometer, the absorbance values ​​of the standard solution and the sample solution to be tested were measured at a wavelength of 700 nm. A standard curve was plotted with the total phosphorus standard concentration as the abscissa and the corresponding absorbance value as the ordinate. (5) The total phosphorus content per unit fresh weight of the sample leaves is calculated by combining the absorbance of the sample to be tested with the standard curve.

[0053] 3. Determination of leaf ATP and ADP content and ATP / ADP ratio The ATP and ADP contents in leaves were determined and the ATP / ADP ratio was calculated using high-performance liquid chromatography (HPLC). The specific steps are as follows: (1) Accurately weigh 0.1g of frozen leaf sample, grind it into powder with liquid nitrogen, add pre-cooled perchloric acid extraction solution for ice bath extraction, vortex mix and let stand for 10min; (2) Place the extract in a high-speed refrigerated centrifuge and centrifuge at 12000 r / min and 4℃ for 20 min. Take the supernatant, adjust the pH value to neutral with an alkaline solution, centrifuge again to remove impurities, and obtain the purified test solution. (3) Chromatographic detection conditions: A reversed-phase C18 column was selected, the detection wavelength was set to 254 nm, the mobile phase was a phosphate buffer system, the column temperature was controlled at 30 °C, and the appropriate flow rate and injection volume were set. (4) Inject ATP and ADP standards and the sample solution to be tested in sequence, record the chromatogram and the corresponding peak area; establish a quantitative standard equation based on the concentration of the standard and the peak area, and calculate the content of ATP and ADP in the sample. (5) Based on the measured ATP and ADP content, calculate the ATP / ADP ratio of each sample group to comprehensively evaluate the overall energy metabolism status of cucumber leaf cells.

[0054] 4. Experimental Results and Mechanism Analysis Test results as follows CsPHO1;H3 , Figure 8 , Figure 9 and Figure 10 As shown, compared to wild-type cucumbers, Figure 11 The total phosphorus (TP) content in the leaves of overexpressing plants was significantly increased; at the same time, the ATP content and ATP / ADP ratio in the leaves were also significantly increased.

[0055] Total phosphorus is a core substrate in plant photosynthetic phosphorylation and energy metabolism. Increased total phosphorus accumulation in leaves provides sufficient raw materials for photosynthetic processes such as light energy conversion and carbon fixation, continuously promoting ATP synthesis and accumulation, and comprehensively improving cellular energy levels. In this invention... CsPHO1;H3 CsPHO1;H3 Overexpression of the gene can effectively improve the problem of insufficient energy supply in cucumber seedlings, promote robust seedling growth, and lay a solid physiological foundation for subsequent vegetative growth and yield formation.

[0056] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. CsPHO1;H3 The application of genes in improving photosynthetic efficiency and energy metabolism in cucumber seedlings is characterized by, The CsPHO1;H3 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The application is achieved by overexpressing the... CsPHO1;H3 Genes that improve the photosynthetic efficiency and / or energy metabolism level of cucumber seedlings.

3. The application according to claim 2, characterized in that, By overexpressing the above CsPHO1;H3 Genes that enhance at least one of the maximum photochemical efficiency, actual photochemical efficiency, and net photosynthetic rate of cucumber seedling leaves.

4. The application according to claim 2, characterized in that, By overexpressing the above CsPHO1;H3 Genes that increase at least one of the following in cucumber seedling leaves: total phosphorus content, adenosine triphosphate (ATP) content, and the ATP / DP ratio.

5. A molecular breeding method for cucumber seedlings with high photosynthetic efficiency and high energy accumulation, characterized in that, The molecular breeding method includes: overexpressing the expression described in claim 1 in cucumber plants. CsPHO1;H3 Gene.

6. The molecular breeding method according to claim 5, characterized in that, The overexpression CsPHO1;H3 Genetic methods include: Construct the CsPHO1;H3 Gene overexpression vectors; The overexpression vector was transformed into Agrobacterium, and the cucumber explants were infected by Agrobacterium-mediated infection. Infected explants were cultured and screened by PCR to obtain... CsPHO1;H3 Overexpression lines; Cultivation CsPHO1;H3 Overexpression lines were then harvested and seeds were collected. By planting the aforementioned seeds, cucumber seedlings with high photosynthetic efficiency and high energy accumulation can be cultivated.

7. The molecular breeding method according to claim 6, characterized in that, The method for constructing the overexpression vector includes: The amplification of the above using specific primers CsPHO1;H3 The gene is then amplified and the amplified product is linked to a linearized vector via homologous recombination to obtain the overexpression vector.

8. The molecular breeding method according to claim 7, characterized in that, The specific primer is CsPHO1;H3 -F and CsPHO1;H3 -R; the CsPHO1;H3 The nucleotide sequence of -F is shown in SEQ ID NO:

2. CsPHO1;H3 The nucleotide sequence of -R is shown in SEQ ID NO:

3.

9. The molecular breeding method according to claim 6, characterized in that, The Agrobacterium is EHA105, and the cucumber explant is the cotyledon node of the cucumber inbred line CCMC.

10. The molecular breeding method according to claim 6, characterized in that, The primers used for PCR identification and screening are: CsPHO1;H3 -OE-F and CsPHO1;H3 -OE-R; the CsPHO1;H3 The nucleotide sequence of -OE-F is shown in SEQ ID NO:

4. CsPHO1;H3 The nucleotide sequence of -OE-R is shown in SEQ ID NO:5.

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