Agrobacterium-mediated transient expression method for spinach

Abstract

An Agrobacterium-mediated transient expression method for spinach. The method comprises the following five steps: preparing a spinach material, constructing a plant expression vector, preparing an Agrobacterium permeation solution, injecting same into spinach leaves, and carrying out fluorescence detection.

Description

A method for transient expression of spinach mediated by Agrobacterium Technical Field

[0001] The invention relates to an Agrobacterium-mediated transient expression method for spinach, and relates to the technical field of plant molecular biotechnology. Background Art

[0002] Spinach (Spinacia oleracea L., 2N=2×=12) is an annual or biennial leafy green vegetable belonging to the dicotyledonous Amaranthaceae family. Spinach is an important economic vegetable crop that contains a large amount of vitamins, minerals, and antioxidants. In the past 20 years, with the rapid expansion of the fresh vegetable market, the demand for spinach has also increased. Typically, spinach leaves are used as fresh vegetables in salads or as an ingredient in cooking and steaming. However, during the cultivation process, spinach is susceptible to biotic stresses such as pathogens and abiotic stresses such as salt, drought, and high temperature, which seriously affect its yield and quality.

[0003] Molecular design breeding can accelerate the breeding process to a certain extent and is one of the ideal and effective breeding strategies for cultivating resistant spinach varieties. However, due to the limitations of the immature spinach genetic transformation system, it is difficult to conduct in-depth research on spinach gene functions and their regulatory pathways. Transient expression technology can be used to introduce target genes into living plants and express them in a short period of time (up to several days), which can accurately reflect the function of the gene. Therefore, transient expression technology is widely used in promoter analysis, protein interaction, and gene function analysis. The transient expression system can overcome the disadvantages of low regeneration efficiency of stable genetic transformation systems and complex tissue culture procedures. It is a fast, convenient and effective gene expression method for conducting gene function research.

[0004] Currently, widely used transient expression methods in plants include Agrobacterium infiltration, gene gun bombardment, and polyethylene glycol (PEG)-mediated protoplast transformation. Among them, gene gun bombardment requires high equipment requirements and yields a small amount of plant tissue each time, making it difficult to meet the needs of subsequent analysis. Protoplast transformation is also limited by the availability of plant material and is primarily used in Arabidopsis leaves, maize protoplasts, and rice suspension cells. Agrobacterium infiltration-mediated transient expression systems offer the advantages of simplicity and high efficiency, and have been widely used for genetic transformation in a variety of plants and tissues. However, the efficiency of transient expression methods is affected by the species and experimental conditions, with different species requiring different optimal experimental conditions. Quinoa and sugar beet, both belonging to the same Amaranthaceae family as spinach, have reported transient expression methods. Although these two species share the same family, they are relatively distantly related and have significantly different biological characteristics. Therefore, transient transformation methods suitable for quinoa and sugar beet cannot be directly applied to spinach, and a mature transient expression system for spinach genes is still lacking. Although spinach leaves are relatively easy to inject, how to improve the efficiency of gene expression, that is, how to determine the optimal transient expression conditions, is a technical problem that needs to be solved urgently by those skilled in the art.

[0005] Summary of the Invention

[0006] The present invention provides a method for Agrobacterium-mediated transient expression in spinach. Based on the characteristics of spinach itself, experimental conditions are optimized and the optimal transient expression conditions suitable for spinach are determined. This method can not only improve transformation efficiency but also be used for subcellular localization analysis of target proteins and protein interaction research, providing an auxiliary means for analyzing spinach gene function.

[0007] The present invention provides a method for Agrobacterium-mediated transient expression in spinach, comprising the following steps:

[0008] Step 1. Prepare spinach materials: Place spinach seeds on filter paper soaked in sterile water for vernalization, and plant the germinated seeds in soil for growth. When the spinach seedlings grow to the four or six true leaf stage, prepare for subsequent experiments.

[0009] Step 2: Constructing a plant expression vector: constructing the target gene into a plant expression vector;

[0010] Step 3: Prepare Agrobacterium infiltration solution: transform the plant expression vector containing the target gene into Agrobacterium GV3101 (pSoup-p19) to obtain Agrobacterium infiltration solution containing the target gene;

[0011] Step 4, injecting spinach leaves: injecting the Agrobacterium infiltration solution into the spinach leaves obtained in step 1 until the injected leaves become translucent and are filled with the Agrobacterium infiltration solution;

[0012] When spinach grows to the stage of four true leaves, the Agrobacterium infiltration solution is injected into the first pair of leaves; when spinach grows to the stage of six true leaves, the Agrobacterium infiltration solution is injected into the first pair of leaves or the second pair of leaves;

[0013] Step 5: Cultivate the spinach injected with the Agrobacterium infiltration solution, perform fluorescence detection, and calculate the transient expression efficiency.

[0014] Furthermore, the spinach seeds were subjected to vernalization treatment under dark shading conditions, the temperature of the vernalization treatment was 4° C., and the time was 7 days.

[0015] Furthermore, the soil includes perlite, vermiculite and peat, and the volume ratio of perlite, vermiculite and peat is 1:2:2.

[0016] Furthermore, in step 1, the spinach planted in the soil is placed in an artificial climate chamber for cultivation, and the artificial climate chamber is operated alternately in a light 10h / dark 14h manner, with the light temperature controlled at 22°C, the dark temperature at 20°C, and the relative humidity at 50%.

[0017] Furthermore, the plant expression vector is pCAMBIA2300.

[0018] Furthermore, the plant expression vector containing the target gene is transformed into Agrobacterium, which specifically includes the following steps:

[0019] The plant expression vector containing the target gene is transformed into Agrobacterium by heat shock method, and a single positive colony is obtained by culture;

[0020] The single positive colony was cultured, and when the OD600 of the bacterial solution was 0.6-0.8, the culture was stopped, and the bacteria were collected by centrifugation;

[0021] The bacteria were resuspended in an infiltration solution and diluted to an OD600 of 0.8 to obtain an Agrobacterium infiltration solution;

[0022] The infiltration solution included 1 / 2 MS medium, 10 mM MgCl 2 , 10 mM MES·H 2 O, and 200 μM acetosyringone.

[0023] Furthermore, the method further comprises: after the spinach leaves are filled with the Agrobacterium infiltration solution, absorbing the Agrobacterium infiltration solution on the surface of the spinach leaves with filter paper, culturing the instantaneously transformed spinach leaves under dark conditions at 20° C. for 12 hours, and then transferring to normal light culture conditions for further culturing for 41 to 60 hours;

[0024] The normal light culture conditions were alternating in a light 12 h / dark 10 h pattern, with the light temperature controlled at 22° C. and the dark temperature at 20° C.

[0025] Furthermore, the spinach seeds are derived from the heat-resistant spinach material Sp75 and the heat-sensitive spinach material Sp73.

[0026] Furthermore, when spinach seeds were derived from the heat-tolerant spinach material Sp75, and the Agrobacterium infiltration solution was injected into the first pair of leaves of spinach grown to the four-true leaf stage, the transiently transformed spinach leaves were cultured for 53 h, the spinach was subjected to fluorescence detection, and the transient expression efficiency was calculated;

[0027] When spinach seeds were derived from the heat-sensitive spinach material Sp73, and the Agrobacterium infiltration solution was injected into the first pair of leaves of spinach grown to the four-true leaf stage, the transiently transformed spinach leaves were cultured for 64 h, the spinach was subjected to fluorescence detection, and the transient expression efficiency was calculated.

[0028] Furthermore, the transient expression efficiency = the number of leaves with fluorescent signals / the number of all injected leaves*100%.

[0029] The present invention establishes an Agrobacterium-mediated transient transformation system for spinach genes, which not only improves the transformation efficiency but also enables the subcellular localization analysis of target proteins and protein interaction studies using the system, providing an auxiliary means for analyzing the functions of spinach genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a schematic diagram of the process of Agrobacterium-mediated transient expression provided by the present invention;

[0031] Figure 2 shows leaves of two spinach materials, Sp75 and Sp73, at the four-true-leaf stage (left) and the six-true-leaf stage (right). The scale bar is 1 cm.

[0032] Figure 3 shows the transient expression of HopF2-GFP in the four-true-leaf stage of spinach material Sp75, wherein A is the GFP fluorescence images obtained by infiltrating the first and second pairs of leaves of spinach Sp75 with Agrobacterium strain GV3101 (pSoup-p19) and AGL1 carrying the pCAMBIA2300-HopF2-GFP vector, and observing the fluorescence signal at 53h, 64h, and 72h after injection, with the scale bar being 50μm; B is the fluorescence intensity and conversion efficiency of HopF2-GFP. The experimental results were repeated three times, and the data provided are the mean ± SD (n≥6);

[0033] Figure 4 shows the transient expression of HopF2-GFP in the four-true-leaf stage of spinach material Sp73, wherein A is the GFP fluorescence image obtained by infiltrating the first and second pairs of leaves of spinach Sp73 with Agrobacterium strain GV3101 (pSoup-p19) and AGL1 carrying the pCAMBIA2300-HopF2-GFP vector, and observing the fluorescence signal at 53 h, 64 h, and 72 h after injection, with the scale bar being 50 μm; B is the fluorescence intensity and conversion efficiency of HopF2-GFP. The experimental results were repeated three times, and the data provided are the mean ± SD (n ≥ 6);

[0034] Figure 5 shows the transient expression of HopF2-GFP in spinach materials Sp75 and Sp73 at the six-true-leaf stage, wherein A is the GFP fluorescence images obtained by infiltrating the first, second, and third pairs of leaves of spinach Sp75 and Sp73 with Agrobacterium strain GV3101 (pSoup-p19) carrying the pCAMBIA2300-HopF2-GFP vector, and observing the fluorescence signal at 53 h, 64 h, and 72 h after injection. The scale bar is 50 μm. B and C are the fluorescence intensity and conversion efficiency of HopF2-GFP. The experimental results were repeated three times, and the data provided are the mean ± SD (n ≥ 6);

[0035] Figure 6 shows the subcellular localization analysis of spinach SoMBF1c and SoRbohF;

[0036] Figure 7 shows the interaction between SoCRK10 and SoRbohB detected by BiFC (the bimolecular fluorescence complementation assay), with SoCRK10-YFPc+YFPn and SoRbohB-YFPn+YFPc as negative controls. The scale bar is 50 μm. DETAILED DESCRIPTION

[0037] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0038] Example 1 Agrobacterium-mediated transient expression method in spinach

[0039] The schematic flow diagram of the Agrobacterium-mediated transient expression method for spinach provided in this embodiment is shown in FIG1 , and each step is described in detail below:

[0040] 1. Prepare spinach materials: Use two spinach materials, Sp75 and Sp73, provided by the Germplasm Resources Development Center of Shanghai Normal University. Sp75 is a heat-resistant spinach material with fleshy leaves, and Sp73 is a heat-sensitive spinach material with waxy leaves.

[0041] Seeds of Sp75 and Sp73 were placed on filter paper soaked in sterile water and vernalized for one week at 4°C in the dark. The germinated seeds were then potted in a soil mixture (perlite:vermiculite:peat soil = 1:2:2, v / v / v) and grown in a climatic chamber at 22°C (light) / 20°C (dark), 50% relative humidity, and a photoperiod of 10 h light / 14 h dark. Spinach seedlings reached the four-true-leaf stage after approximately 28 days and the six-true-leaf stage after approximately 35 days. Images of leaves at the four- and six-true-leaf stages are shown in Figure 2.

[0042] 2. Construction of a plant expression vector: HopF2 is an effector protein produced by Pseudomonas syringae DC3000 and localized to the cytoplasmic membrane. HopF2 was constructed into the plant expression vector pCAMBIA2300-GFP. The HopF2-GFP fusion protein was used as a cytoplasmic membrane marker protein, and the efficiency of transient gene expression was assessed by detecting the fluorescent signal.

[0043] 3. Prepare Agrobacterium infection solution:

[0044] (1) The plant expression vector pCAMBIA2300-HopF2-GFP containing the target gene was transformed into Agrobacterium by heat shock. The transformed Agrobacterium was stored in 50% (v / v) glycerol and placed in a -80°C freezer. The frozen Agrobacterium was inoculated on LB solid plates (25 μg / μL rifampicin + 50 μg / μL kanamycin) and cultured at 28°C for 48 h to obtain a single positive colony.

[0045] (2) Day 1: Agrobacterium activation. A single positive colony obtained in the above step was inoculated into 500 μL LB liquid medium (25 μg / μL rifampicin + 50 μg / μL kanamycin) and cultured in a shaking incubator at 28°C and 200 rpm for 12-18 h.

[0046] (3) Day 2: Pre-culture: Add 200 μL of Agrobacterium cells from the above step to 3 mL of LB liquid medium (25 μg / μL rifampicin + 50 μg / μL kanamycin) and continue to culture in a shaking incubator at 28°C and 200 rpm overnight.

[0047] (4) Day 3: Transfer 200 μL of Agrobacterium cells from the above step into 20 mL of LB liquid medium and culture in a shaking incubator at 28°C and 200 rpm for approximately 2 h until the logarithmic growth phase (OD 600 =0.6-0.8). The cells were centrifuged at 6500 rpm for 10 min at room temperature, and the supernatant was discarded. The collected cells were then resuspended and diluted with infiltration solution (1 / 2 MS medium (pH 5.7-5.8), 10 mM MgCl2, 10 mM MES·H2O, 200 μM acetosyringone) to an OD of 600 =0.8, ready to penetrate.

[0048] 4. Injecting Spinach Leaves: Use a 1ml plastic syringe to infiltrate the resuspended Agrobacterium solution into the spinach leaves. Gently infiltrate until the entire leaf becomes translucent and is completely filled with the Agrobacterium solution. Typically, only one injection is required on both sides of the vein of a single leaf. Inject three plants of each spinach material at a time.

[0049] 5. Fluorescence Observation: Days 4-5: After blotting the bacterial suspension from the leaf surfaces with filter paper, incubate the transiently transformed spinach leaves in the dark at 20°C for 12 hours. Then, transfer to normal conditions (12 hours of light / 10 hours of darkness, 22°C (light) / 20°C (dark)) and continue incubation for 41-60 hours. Observe and photograph using a confocal fluorescence microscope, and calculate the transient transformation efficiency (transient transformation efficiency = number of leaves showing fluorescent signal / total number of leaves injected).

[0050] Example 2 The above method was used to further study the Agrobacterium strain, spinach seedling growth period, spinach leaf position, and gene effective expression time.

[0051] (1) Optimization of experimental conditions for the four-leaf stage of spinach materials Sp75 and Sp73

[0052] Spinach seedlings of Sp75 and Sp73 were cultivated to the four-true-leaf stage, and Agrobacterium strains GV3101 (pSoup-p19) and AGL1 carrying the expression vector pCAMBIA2300-HopF2-GFP were infiltrated into the first and second pairs of leaves of Sp75 and Sp73, and GFP signals were observed.

[0053] The observation results are shown in Figures 3-4. In spinach Sp75, using Agrobacterium strain GV3101 (pSoup-p19), the fluorescence intensity and transformation efficiency of HopF2-GFP decreased significantly with increasing expression time (from 53 to 72 hours) in both the first and second pairs of leaves. At 72 hours of gene expression, very strong GFP fluorescence was observed only in the first pair of leaves, while no GFP signal was detected in the second pair of leaves. Using strain AGL1, GFP signals were detected in the first pair of leaves at all three time points, with peak fluorescence intensity at 72 hours and peak transformation efficiency at 64 hours. However, in the second pair of leaves, GFP signals were only detectable at 72 hours. This suggests that AGL1-mediated gene expression may require more time in the second pair of leaves. Overall, the GFP fluorescence intensity obtained with strain AGL1 was significantly lower than that obtained with strain GV3101 (pSoup-p19).

[0054] In spinach Sp73, using strain GV3101 (pSoup-p19) for infiltration, GFP signals were observed at both 53h and 64h of detection. There was no significant difference in fluorescence intensity and expression efficiency at 53h between the first and second pairs of leaves. At 64h, the fluorescence intensity and conversion efficiency of the first pair of leaves were significantly higher than those of the second pair of leaves. When strain AGL1 was used for infiltration, GFP signals were present in the first pair of leaves at 53h of expression, and in the second pair of leaves at 64h and 72h of expression. Overall, the fluorescence intensity and conversion efficiency of transient expression mediated by strain GV3101 (pSoup-p19) were higher than those of strain AGL1.

[0055] (2) Optimization of experimental conditions for the six-leaf stage of spinach materials Sp75 and Sp73

[0056] Spinach materials Sp75 and Sp73 seedlings were cultivated to the six-true leaf stage, and the strain GV3101 (pSoup-p19) carrying the pCAMBIA2300-HopF2-GFP vector was infiltrated into the first, second and third pairs of leaves of spinach materials Sp75 and Sp73, respectively, and GFP signals were observed.

[0057] The observation results are shown in Figure 5. In both spinach materials, GFP fluorescence was observed in the first and second pairs of leaves at three time points. In the third pair of leaves of both materials, only a weak GFP fluorescence signal was observed at 72 hours.

[0058] In the spinach material Sp75, fluorescence intensity and transformation efficiency followed the order: first pair of leaves > second pair of leaves > third pair of leaves. Both transformation efficiency and fluorescence intensity were high in the first pair of leaves at 64 and 72 hours of expression. This indicates that 64 to 72 hours of expression in the first pair of leaves of the spinach material Sp75 is the optimal condition.

[0059] In the spinach material Sp73, the second pair of leaves showed the highest fluorescence intensity after 72 hours of expression, while the highest transformation efficiency was achieved after 72 hours of expression in the first pair of leaves and 53 hours of expression in the second pair of leaves. Overall, the first and second pairs of leaves of the six-leaf spinach material Sp73 can be used for transient expression.

[0060] Example 3 Subcellular Localization Analysis of SoMBF1c and SoRbohF

[0061] MBF1c is an evolutionarily highly conserved transcriptional coactivator that belongs to the MBF1 (multiprotein bridging factor 1) gene family. Arabidopsis thaliana contains three MBF1s: AtMBF1a, AtMBF1b, and AtMBF1c. AtMBF1c is a key regulator of plant responses to heat stress, regulating the expression of heat-stress-related genes. SoMBF1c is the spinach homolog of AtMBF1c.

[0062] Plant NADPH oxidases, also known as respiratory burst oxidase homologues (Rbohs), can generate reactive oxygen species (ROS). Arabidopsis thaliana has 10 Rboh genes, of which RbohF and RbohD are two members. Both are located at the cytoplasmic membrane and participate in stomatal immunity by regulating ROS and the dynamics of cytoplasmic pH. SoRbohF and SoRbohB are the spinach homologs of Arabidopsis RbohF and RbohD.

[0063] The sequences of the two genes were downloaded from the Spinach database (http: / / www.spinachbase.org / ). The gene number of SoMBF1c in the Spinach database is Spo17527, and the gene number of SoRbohF in the Spinach database is Spo18705.

[0064] The specific analysis steps are as follows:

[0065] 1. Spinach material Sp75 seeds were placed on filter paper soaked in sterile water and vernalized for one week in the dark at 4°C. The germinated seeds were then planted in a soil mixture (perlite:vermiculite:peat soil = 1:2:2, v / v / v) and grown in a culture room at 22°C (light) / 20°C (dark), 50% relative humidity, and a photoperiod of 10 h light / 14 h dark. Spinach seedlings were obtained after 28 days of cultivation at the four-true-leaf stage.

[0066] 2. Construction of plant expression vector: Using homologous recombination, the CDS sequences of SoMBF1c and SoRbohF cloned by PCR were constructed into the plant expression vector pCAMBIA2300-GFP. An empty vector pCAMBIA2300-GFP was used as an experimental control.

[0067] 3. Prepare Agrobacterium infection solution:

[0068] (1) The empty vector pCAMBIA2300-GFP and the plant expression vectors pCAMBIA2300-SoMBF1c-GFP and pCAMBIA2300-SoRbohF-GFP were transformed into Agrobacterium GV3101 (pSoup-p19) by heat shock. The transformed Agrobacterium was stored in 50% (v / v) glycerol and placed in a -80°C freezer. The frozen Agrobacterium was inoculated onto LB solid plates (25 μg / μL rifampicin + 50 μg / μL kanamycin) and cultured at 28°C for 48 h to obtain single colonies.

[0069] (2) Day 1: Agrobacterium activation. Inoculate the corresponding single positive colony obtained in the above step into 500 μL LB liquid medium (25 μg / μL rifampicin + 50 μg / μL kanamycin) and culture in a shaking incubator at 28°C and 200 rpm for 12-18 h.

[0070] (3) Day 2: Pre-culture: Add 200 μL of the corresponding Agrobacterium cells from the above step to 3 mL of LB liquid medium (25 μg / μL rifampicin + 50 μg / μL kanamycin) and continue to culture in a shaking incubator at 28°C and 200 rpm overnight.

[0071] (4) Day 3: Transfer 50 μL of Agrobacterium cells from the above steps into 5 mL of LB liquid medium and culture in a shaking incubator at 28°C and 200 rpm for nearly 2 h until the logarithmic growth phase (OD 600 =0.6~0.8). Centrifuge at 6500 rpm for 10 min at room temperature and discard the supernatant. Then, the collected cells were resuspended in osmotic solution (1 / 2 MS medium (pH 5.7~5.8), 10 mM MgCl2, 10 mM MES·H2O, 200 μM acetosyringone) and diluted to OD 600 =0.8.

[0072] 4. Injecting Spinach Leaves: Using a 1ml plastic syringe, infiltrate the first pair of leaves of Spinach accession Sp75 with resuspended Agrobacterium cells containing the empty vector pCAMBIA2300-GFP and Agrobacterium cells containing pCAMBIA2300-SoMBF1c-GFP and pCAMBIA2300-SoRbohF-GFP. Gently infiltrate until the entire leaf becomes translucent and is completely filled with the Agrobacterium cell suspension. Typically, only one injection is required on each side of the vein of a single leaf. Inject three plants of each spinach accession at a time.

[0073] 5. Fluorescence observation: After absorbing the bacterial liquid on the leaf surface with paper, the transiently transformed spinach leaves were cultured in the dark at 20°C for 12 h. Then, they were transferred back to normal conditions (12 h light / 10 h dark and 22°C (light) / 20°C (dark)) and cultured for another 41-60 h. The leaves were observed and photographed using a confocal fluorescence microscope.

[0074] As shown in Figure 6 , 53 h after Agrobacterium injection, we observed that SoMBF1c-GFP was localized in both the nucleus and cytoplasm, and co-localized with the nuclear dye DAPI in the nucleus; 64 h after injection, SoRbohF-GFP was found to be localized in the cytoplasmic membrane and co-localized with the cytoplasmic membrane dye FM4-64.

[0075] Example 4 BiFC analysis of SoRbohB and SoCRK10

[0076] CRKs (Cysteine-rich receptor-like kinases) belong to the RLK family of receptor-like protein kinases and play key roles in plant immunity, abiotic stress responses, and growth and development. Arabidopsis thaliana contains 44 CRK members. SoCRK10 is the closest homolog to Arabidopsis AtCRK10. In plant immune responses, CRKs mediate ROS production by interacting with RbohF / RbohD.

[0077] The CDS sequence of SoCRK10 and SoRbohB can be found at http: / / www.spinachbase.org / . The gene ID for SoRbohB is Spo06343, and the gene ID for SoCRK10 is Spo14647.

[0078] The analysis method is as follows:

[0079] 1. The process is the same as that of Example 3.

[0080] 2. Construction of plant expression vector: To verify the interaction between SoCRK10 and SoRbohB, the CDS sequence of SoRbohB obtained by PCR cloning was constructed into the plasmid pCAMBIA1300-YFPn using the homologous recombination method, and the CDS sequence of SoCRK10 obtained by PCR cloning was constructed into the plasmid pCAMBIA1300-YFPc.

[0081] Prepare Agrobacterium infection medium:

[0082] (1) The constructed vectors pCAMBIA1300-YFPn, pCAMBIA1300-YFPc, pCAMBIA1300-SoRbohB-YFPn, and pCAMBIA1300-SoCRK10-YFPc were transformed into Agrobacterium tumefaciens GV3101 (pSoup-p19) by heat shock method, and the transformed Agrobacterium were stored in 50% (v / v) glycerol at -80°C. The frozen Agrobacterium was inoculated on LB solid plates (25 μg / μL rifampicin + 50 μg / μL kanamycin) and cultured at 28°C for 48 h to obtain single colonies.

[0083] (2) Day 1: Agrobacterium activation. The corresponding single positive colony obtained in the above step was inoculated into 500 μL LB liquid medium (25 μg / μL rifampicin + 50 μg / μL kanamycin) and cultured in a shaking incubator at 28°C and 200 rpm for 12-18 h.

[0084] (3) Day 2: Pre-culture: Add 30 μL of the corresponding Agrobacterium cells from the above step to 3 mL of LB liquid medium (25 μg / μL rifampicin + 50 μg / μL kanamycin) and continue to culture in a shaking incubator at 28°C and 200 rpm overnight.

[0085] (4) Day 3: Transfer the corresponding 200 μL Agrobacterium cells from the above step into 20 mL LB liquid medium and culture in a shaking incubator at 28°C and 200 rpm for nearly 2 h until the logarithmic growth phase (OD 600 =0.6-0.8). Centrifuge at 5000 rpm for 10 min at room temperature and discard the supernatant. Then, resuspend the corresponding collected cells and dilute them with infiltration solution (1 / 2 MS medium (pH 5.7-5.8), 10 mM MgCl2, 10 mM MES·H2O, 200 μM acetosyringone) to OD 600= 1.0. The four Agrobacterium infiltration solutions were combined in pairs according to pCAMBIA1300-YFPn + pCAMBIA1300-YFPc, pCAMBIA1300-SoRbohB-YFPn + pCAMBIA1300-YFPc, pCAMBIA1300-YFPn + pCAMBIA1300-SoCRK10-YFPc, and pCAMBIA1300-SoRbohB-YFPn + pCAMBIA1300-SoCRK10-YFPc, mixed in a 1:1 (v:v) ratio, and allowed to stand for 3 h before subsequent injection.

[0086] 4. Injecting Spinach Leaves: Use a 1ml plastic syringe to infiltrate the mixed Agrobacterium cells into the first pair of leaves of the spinach material Sp75. Gently infiltrate until the entire leaf becomes translucent and is filled with the Agrobacterium cell suspension. Typically, only one injection is required on each side of the vein of a single leaf. Inject three plants of each spinach material at a time.

[0087] 5. Fluorescence observation: After absorbing the bacterial liquid on the leaf surface with paper, culture the transiently transformed spinach leaves in dark conditions at 20°C for 12 hours, then transfer them back to normal conditions (12 hours of light / 10 hours of darkness and 22°C (light) / 20°C (dark)) and continue to culture for 41 to 60 hours. Observe and photograph using a confocal fluorescence microscope.

[0088] The photographic results are shown in Figure 7. When the first pair of leaves of spinach Sp75 were injected with the GV3101 (pSoup-p19) strain containing pCAMBIA1300-SoCRK10-YFPc and pCAMBIA1300-SoRbohB-YFPn, a significant YFP signal was observed in the leaves 69 hours after injection, whereas no fluorescence signal was observed in leaves co-expressing the pCAMBIA1300-YFPc and pCAMBIA1300-YFPn empty vectors. These experimental results indicate that SoCRK10 can interact with SoRbohB on the cytoplasmic membrane.

[0089] From the results provided in Examples 3-4, it can be seen that the GV3101 (pSoup-p19)-mediated transient transformation of spinach leaves can provide sufficient target protein for functional verification such as protein subcellular localization and BiFC interaction.

[0090] The primers used to amplify the target sequence in Example 3-4 are shown in Table 1:

[0091] Table 1

[0092] In summary, the present invention primarily optimizes the transient expression process by targeting five key factors: different spinach materials, different spinach stages, different leaf positions, different Agrobacterium types, and different gene expression times, to improve transient transformation efficiency. Subcellular localization experiments with SoMBF1c and SoRbohF, as well as BiFC experiments with SoRbohB and SoCRK10, further validated the feasibility of the transient expression method. Therefore, the present invention establishes an Agrobacterium-mediated transient transformation system for spinach genes, which not only improves transformation efficiency but also enables analysis of the subcellular localization of target proteins and protein-protein interaction studies, providing a complementary tool for understanding spinach gene function.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for Agrobacterium-mediated transient expression of spinach, characterized in that: The steps include: Step 1, prepare spinach materials: place spinach seeds on wet filter paper soaked in sterile water for vernalization, and plant the seeds that germinate after vernalization in soil for growth. When the spinach seedlings grow to the stage of four true leaves or six true leaves, prepare for subsequent experiments; Step 2, constructing a plant expression vector: constructing the target gene into a plant expression vector; Step 3, preparing Agrobacterium infiltration solution: transforming the plant expression vector containing the target gene into Agrobacterium GV3101 (pSoup-p19) to obtain Agrobacterium infiltration solution containing the transformed target gene; Step 4, injecting spinach leaves: injecting the Agrobacterium infiltration solution into the spinach leaves obtained in step 1 until the injected leaves become translucent and are filled with the Agrobacterium infiltration solution; When spinach grows to the stage of four true leaves, the Agrobacterium infiltration solution is injected into the first pair of leaves; when spinach grows to the stage of six true leaves, the Agrobacterium infiltration solution is injected into the first pair of leaves or the second pair of leaves; Step 5: Cultivate the spinach injected with the Agrobacterium infiltration solution, perform fluorescence detection, and calculate the transient expression efficiency.

2. The method according to claim 1, characterized in that: The spinach seeds were subjected to vernalization treatment under dark shading conditions, the temperature of the vernalization treatment was 4° C., and the time was 7 days.

3. The method according to claim 1, characterized in that The soil includes perlite, vermiculite and peat soil, and the volume ratio of perlite, vermiculite and peat soil is 1:2:

2.

4. The method according to claim 1, characterized in that: In step 1, the spinach planted in the soil is placed in an artificial climate chamber for cultivation. The artificial climate chamber is operated alternately in a light 10h / dark 14h mode, and the light temperature is controlled at 22°C, the dark temperature is 20°C, and the relative humidity is 50%.

5. The method according to claim 1, characterized in that The plant expression vector is pCAMBIA2300.

6. The method according to claim 1, characterized in that The plant expression vector containing the target gene is transformed into Agrobacterium, which specifically includes the following steps: The plant expression vector containing the target gene is transformed into Agrobacterium by heat shock method, and a single positive colony is obtained by culture; The single positive colony was cultured and the OD of the bacterial solution was 600 When the pH value is 0.6-0.8, the culture is stopped and the bacteria are collected by centrifugation; The cells were resuspended in the infiltration solution and diluted to OD 600 is 0.8, and the Agrobacterium infiltration solution is obtained; The infiltration solution included 1 / 2 MS medium, 10 mM MgCl2, 10 mM MES·H2O and 200 μM acetosyringone.

7. The method according to claim 1, characterized in that The method further comprises: after the spinach leaves are filled with the Agrobacterium infiltration solution, absorbing the Agrobacterium infiltration solution on the surface of the spinach leaves with filter paper, culturing the instantaneously transformed spinach leaves under dark conditions at 20° C. for 12 hours, and then transferring to normal light culture conditions for continued culture for 41 to 60 hours; The normal light culture conditions were carried out in an alternating manner of 12 h light / 10 h dark, with the light temperature controlled at 22° C. and the dark temperature controlled at 20° C.

8. The method according to claim 1, characterized in that The spinach seeds are derived from the heat-resistant spinach material Sp75 and the heat-sensitive spinach material Sp73.

9. The method according to claim 8, characterized in that When spinach seeds were derived from the heat-resistant spinach material Sp75, and the Agrobacterium infiltration solution was injected into the first pair of leaves of spinach grown to the four true leaves stage, the transiently transformed spinach leaves were cultured for 53 h, the spinach was subjected to fluorescence detection, and the transient expression efficiency was counted; When spinach seeds were derived from the thermosensitive spinach material Sp73, and the Agrobacterium infiltration solution was injected into the first pair of leaves of spinach grown to the four true leaves stage, the transiently transformed spinach leaves were cultured for 64 h, the spinach was subjected to fluorescence detection, and the transient expression efficiency was counted.

10. The method according to claim 1, characterized in that The transient expression efficiency = the number of leaves with fluorescent signals / the number of all injected leaves*100%.

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