Rapid detection method for strawberry flower bud differentiation

Abstract

This invention discloses an enzyme-linked immunosorbent assay (ELISA) rapid detection kit based on strawberry FT1 protein and a colloidal gold test strip for rapid detection of strawberry flower bud differentiation. The invention also discloses a method for rapid detection of strawberry flower bud differentiation using the kit and the colloidal gold test strip. This method provides technical support for the rapid detection of strawberry flower bud differentiation status in the future, and has the advantages of being non-destructive, dynamically detecting, simple to operate, rapid and efficient, providing objective results, high sensitivity, low cost, and easy to promote.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology detection, and in particular to a rapid FT1 enzyme-linked immunosorbent assay kit for strawberries and a colloidal gold test strip for rapid detection of strawberry flower bud differentiation based on FT1 protein, as well as a method for rapid detection of strawberry flower bud differentiation using the kit or the colloidal gold test strip. Background Technology

[0002] Cultivated strawberries (Fragaria × ananassa Duch.), a perennial herbaceous plant belonging to the genus Fragaria in the family Rosaceae, are a key industry for rural revitalization in my country. Currently, the main strawberry varieties cultivated in my country are seasonal flowering (SF) strawberries. After being planted in spring, the seedlings undergo vegetative growth. As the days shorten and temperatures drop in autumn, the apical meristem is induced to differentiate into flower buds, a process known as flower bud differentiation. To achieve optimal yield and quality, precise transplanting of strawberry seedlings based on the critical state of flower bud differentiation is necessary in production.

[0003] Currently, the main method for detecting the critical point of flower bud differentiation in strawberry production both domestically and internationally is the dissecting microscope observation method. This method requires manually removing the strawberry leaves and the pubescence on the apical surface, and observing the morphological changes of the apical meristem under a dissecting microscope. This method has many drawbacks: (1) the equipment is expensive and the operation is highly specialized, making it difficult to popularize among ordinary growers; (2) the detection process damages the plants, making it impossible to continuously track the same plant; (3) it can only be evaluated through a small number of samplings, resulting in sampling lag and errors. It is impossible to achieve continuous and dynamic monitoring of large-scale seedlings, causing farmers to often miss the best planting time because they cannot accurately determine the critical point of flower bud differentiation, which has become a bottleneck problem restricting the improvement of quality and efficiency in the strawberry industry. Therefore, there is an urgent need for a rapid detection tool that can be used for large-scale sampling, is easy to operate, and does not damage the plants. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a rapid detection method for flower bud differentiation that is simple in structure, low in cost, and easy to operate.

[0005] Under flowering induction conditions, the expression of the FLOWERING LOCUS T1 (FT1) gene in SF strawberry leaves was significantly suppressed. Specifically, under long-day conditions, FT1 gene expression and FT1 protein levels were high in leaves, inhibiting flower bud differentiation; while under short-day conditions, FT1 expression and protein levels decreased, relieving the inhibition and promoting flower bud differentiation. Therefore, changes in FT1 protein content are significantly correlated with the progress of strawberry flower bud differentiation and can serve as an ideal marker for judging the state of flower bud differentiation.

[0006] To address the issues of existing technologies that rely on microscopes for detecting strawberry flower bud differentiation, are complex to operate, and damage plants, we selected strawberry varieties 'Hongyan' and 'Ningyu' as experimental materials. Using techniques such as quantitative fluorescence, enzyme-linked immunosorbent assay (ELISA) kits, and colloidal gold test strips, we clarified the correlation between FT1 gene expression and protein content in strawberry leaves and flower bud differentiation in the apical meristem, providing theoretical support for the future discovery of more strawberry flower bud differentiation marker genes.

[0007] A monoclonal antibody pair prepared using strawberry FT1 recombinant protein as an antigen, wherein the monoclonal antibody pair is 3G41E10 and 9E11 1H10, and both recognize different epitopes of the FT1 protein; wherein:

[0008] The amino acid sequence of the light chain variable region of 3G4 1E10 is shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6;

[0009] The amino acid sequence of the light chain variable region of 9E11 1H10 is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8.

[0010] The application of FT1 protein as an antigen and the monoclonal antibody prepared using strawberry FT1 recombinant protein as an antigen in the FT1 enzyme-linked immunosorbent assay kit and the colloidal gold test strip for rapid detection of strawberry flower bud differentiation, wherein the amino acid sequence of the FT1 protein is shown in SEQ ID NO:1.

[0011] The nucleotide sequence encoding the monoclonal antibody pair of the present invention, wherein:

[0012] The nucleotide sequence encoding the amino acid of the 3G4 1E10 light chain variable region is shown in SEQ ID NO:9, and the nucleotide sequence encoding the amino acid of the 3G4 1E10 heavy chain variable region is shown in SEQ ID NO:10.

[0013] The nucleotide sequence encoding the amino acid of the 9E11 1H10 light chain variable region is shown in SEQ ID NO:11, and the nucleotide sequence encoding the amino acid of the 9E11 1H10 heavy chain variable region is shown in SEQ ID NO:12.

[0014] An FT1 enzyme-linked immunosorbent assay (ELISA) rapid test kit, wherein the kit comprises an ELISA plate coated with FT1 capture antibody 9E11 1H10, FT1 standard, FT1 biotinylated antibody 3G4 1E10, FT1 biotinylated antibody diluent, horseradish peroxidase-labeled avidin, horseradish peroxidase-labeled avidin diluent, sample diluent, substrate chromogenic solution, stop solution, and concentrated washing solution.

[0015] A colloidal gold test strip for rapid detection of strawberry flower bud differentiation based on FT1 protein includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially overlapped on the base plate. The conjugate pad is coated with colloidal gold-labeled strawberry FT1 monoclonal antibody 3G4 1E10. The nitrocellulose membrane has a detection line and a control line. The detection line is coated with strawberry FT1 monoclonal antibody 9E11 1H10, and the control line is coated with goat anti-mouse IgG antibody.

[0016] The present invention discloses a colloidal gold test strip for rapid detection of strawberry flower bud differentiation based on FT1 protein, wherein: a sample pad, a nitrocellulose membrane, and an absorbent pad are arranged sequentially from front to back on the base plate; a portion of the rear side of the sample pad overlaps a portion of the front side of the nitrocellulose membrane, and a portion of the front side of the absorbent pad overlaps a portion of the rear side of the nitrocellulose membrane; the conjugate pad is divided into upper and lower parts, the lower structure is in contact with the rear end of the sample pad, and the upper structure extends from the front and rear sides of the lower structure, the front end covering the upper rear side of the sample pad, and the rear end covering the nitrocellulose membrane; on the nitrocellulose membrane between the conjugate pad and the absorbent pad, a detection line is provided on the front side and a control line is provided on the rear side; a MAX sample level line is also provided on the conjugate pad.

[0017] The application of the FT1 enzyme-linked immunosorbent assay kit or the FT1 protein-based colloidal gold test strip for rapid detection of strawberry flower bud differentiation in the rapid detection of strawberry flower bud differentiation.

[0018] A rapid detection method for strawberry flower bud differentiation, wherein the method is one of the following two:

[0019] The first type:

[0020] The FT1 enzyme-linked immunosorbent assay (ELISA) rapid test kit was used for detection, and the specific steps included the following:

[0021] (A) Preparation of working solutions: including the preparation of FT1 standard solution, washing working solution, FT1 biotin-labeled antibody working solution and horseradish peroxidase-labeled avidin working solution;

[0022] (B) Sample preparation: Take strawberry leaves, grind them, add phosphate buffer to lyse and shake to mix, centrifuge and take the supernatant as the sample solution to be tested;

[0023] (C) Labeling detection: This includes adding samples to an ELISA plate coated with FT1 capture antibody, adding FT1 biotin-labeled antibody working solution, washing the plate, adding horseradish peroxidase-labeled avidin working solution, washing the plate, adding substrate chromogenic solution, and adding stop solution. After completion, the absorbance of each well at 450 nm wavelength is measured using an ELISA reader. A standard curve is fitted using software with the concentration of FT1 standard as the x-axis and the corresponding OD value as the y-axis.

[0024] Judgment criteria: The state of strawberry flower bud differentiation is identified based on changes in the protein content of FT1 in strawberry leaves.

[0025] If the FT1 protein content in strawberry leaves is > 50 pg / mL, the leaves are considered to be in an undifferentiated state.

[0026] If the FT1 protein content in strawberry leaves is < 25 pg / mL, it is determined that the flower bud has been initiated.

[0027] The second type:

[0028] The detection was performed using a colloidal gold test strip based on the FT1 protein for rapid detection of strawberry flower bud differentiation. The specific steps included:

[0029] (a) Sample preparation: Take strawberry leaves, grind them, add phosphate buffer to lyse and shake to mix, centrifuge and take the supernatant as the sample solution to be tested;

[0030] (b) Detection: Add 100 μL of the sample solution to be tested into the sample pad, let it stand for 5-10 minutes after adding the sample, and observe the color development area;

[0031] Judgment criteria: The differentiation status of strawberry flower buds can be identified based on changes in the protein content of FT1 in strawberry leaves;

[0032] If the FT1 protein content in strawberry leaves is high and the colloidal gold test strip is obvious (i.e., the C line is colored and the T line is visible to the naked eye), regardless of the color intensity, it is positive, indicating that the FT1 protein content is high and the strawberry is in an undifferentiated state.

[0033] If the FT1 protein content in strawberry leaves is significantly reduced and there is no obvious band on the colloidal gold test paper (i.e., the C line is colored and the T line is not colored), it is negative, indicating that the FT1 protein content is extremely low or absent, and the strawberry flower buds have been initiated.

[0034] If line C does not develop color, the test strip is considered invalid regardless of whether line T develops color, and it needs to be tested again.

[0035] The rapid detection method for strawberry flower bud differentiation described in this invention, wherein when using an FT1 enzyme-linked immunosorbent assay (ELISA) kit for detection, the labeling detection method specifically includes the following steps:

[0036] ① Sample addition: Set up standard wells S0-S7 and sample wells in an ELISA plate coated with FT1 capture antibody. There are two standard wells and two sample wells for each concentration of standard solution. Perform double-well assay. Add 100 μL of standard or sample solution to each well, gently shake to mix, cover with plate label, and incubate at 37°C for 2 hours.

[0037] ② Discard the liquid, spin dry, no washing required;

[0038] ③ Add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour;

[0039] ④ Discard the liquid in the wells, spin dry, and wash the plate 3 times; soak for 2 minutes each time, 200μL / well, and spin dry;

[0040] ⑤ Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour;

[0041] ⑥ Discard the liquid in the wells, spin dry, and wash the plate 5 times; soak for 2 minutes each time, 200μL / well, and spin dry;

[0042] ⑦ Add 90 μL of substrate and colorimetric solution to each well in sequence, and develop the color at 37°C in the dark for 15-30 minutes;

[0043] ⑧ Add 50 μL of stop solution to each well in sequence to terminate the reaction;

[0044] ⑨ Within 5 minutes after the reaction is terminated, measure the OD value of each well sequentially at a wavelength of 450 nm using an ELISA reader.

[0045] The rapid detection method for strawberry flower bud differentiation according to the present invention includes the following steps in the sample processing process:

[0046] 1) Take 0.2 g of washed and dried strawberry leaves with filter paper, cut them into small pieces with sterilized scissors, and grind them into a thin paste using a grinding rod in a sample processing tube;

[0047] 2) Add 1 mL of pre-cooled phosphate buffer and vortex for 5 minutes to mix.

[0048] 3) Centrifuge at 4000 rpm for 3 minutes;

[0049] 4) Use a dropper to take 100 μL of the supernatant as the sample solution to be tested, for subsequent detection and analysis.

[0050] The rapid detection method for flower bud differentiation in this invention differs from existing technologies in that:

[0051] The enzyme-linked immunosorbent assay kit and colloidal gold test strip based on strawberry FT1 protein disclosed in the rapid detection method for flower bud differentiation of this invention provide technical support for the rapid detection of strawberry flower bud differentiation status in the future.

[0052] 1) Achieve non-destructive and dynamic detection: Only a small number of leaves need to be collected without damaging the plant. The same plant can be continuously sampled and detected at different stages to grasp the dynamic process of flower bud differentiation, overcoming the shortcomings of traditional microscopic examination methods that damage the plant and can only be sampled.

[0053] 2) Simple, fast and efficient operation: No complicated dissection and microscope operation is required. Only a few simple steps are needed to complete the detection of a large number of samples, which greatly reduces the technical requirements for operators.

[0054] 3) Objective and highly sensitive results: Based on the immunological principle of antigen-antibody specific reaction, the detection results are objective and clear, and can sensitively capture subtle changes in the content of FT1 protein in leaves;

[0055] 4) Low cost and easy to promote: The reagent kit and colloidal gold form are easy to store and transport, and do not require expensive equipment, which significantly reduces the detection cost and is conducive to its promotion and application among strawberry growers and production enterprises.

[0056] The rapid detection method for flower bud differentiation of the present invention will be further described below with reference to the accompanying drawings. Attached Figure Description

[0057] Figure 1 This is a bar chart illustrating the expression patterns of the FT1 gene in 'Hongyan' and 'Ningyu' in different tissues using quantitative real-time PCR (qRT-PCR) according to an embodiment of the present invention.

[0058] Figure 2 This is a bar chart illustrating the expression patterns of the FT1 gene in the leaves of 'Hongyan' and 'Ningyu' plants at different stages of flower bud differentiation, as detected by qRT-PCR in this embodiment of the invention.

[0059] Figure 3 This is the correlation between the FT1 gene expression level and the apical flower bud differentiation status in the leaves of 'Hongyan' and 'Ningyu' in the embodiments of the present invention;

[0060] Figure 4 This invention provides an analysis of the effect of silencing the FT1 gene on the flower bud differentiation rate of 'Hongyan' and 'Ningyu' strawberries.

[0061] Figure 5 This is a schematic diagram illustrating the usage method of the strawberry FT1 enzyme-linked immunosorbent assay kit according to an embodiment of the present invention;

[0062] Figure 6 This is the standard curve of the strawberry FT1 enzyme-linked immunosorbent assay kit according to an embodiment of the present invention;

[0063] Figure 7This is the detection result of the strawberry FT1 enzyme-linked immunosorbent assay kit of the present invention at different stages of strawberry flower bud differentiation.

[0064] Figure 8 This is a schematic diagram of the strawberry FT1 double antibody sandwich colloidal gold test strip structure according to an embodiment of the present invention;

[0065] Figure 9 This is a schematic diagram illustrating the interpretation of the detection results of the strawberry FT1 double antibody sandwich colloidal gold test strip according to an embodiment of the present invention;

[0066] Figure 10 This is the detection result of the strawberry FT1 double antibody sandwich colloidal gold test strip in different stages of strawberry flower bud differentiation, according to an embodiment of the present invention. Detailed Implementation

[0067] This invention, by detecting the expression patterns of the FT1 gene in different tissues and at different flower bud differentiation stages of cultivated strawberry 'Hongyan' and 'Ningyu' varieties, found that FT1 is highly expressed in robust and old leaves of strawberry plants during vegetative growth. Compared to the early-flowering strawberry variety 'Ningyu', the expression level of FT1 in the leaves of 'Hongyan' is higher. Figure 1 During flower bud differentiation, FT1 expression showed a decreasing trend in leaves of both 'Ningyu' and 'Hongyan' varieties. The decrease in FT1 expression in 'Ningyu' leaves occurred earlier than in 'Hongyan' strawberry leaves. Figure 2 ).like Figure 3 As shown, FT1 expression level was significantly correlated with the flower bud differentiation rate of both strawberry varieties, with a more significant negative correlation between FT1 expression level and the flower bud differentiation rate of the 'Hongyan' strawberry variety. The strawberry flower bud differentiation rate refers to the ratio of the number of plants with differentiated flower buds to the total number of plants in the experiment. Figure 4 To investigate the FT1 phenotype, a gene silencing method mediated by TRV (tobacco brittle virus) was used to infect 'Hongyan' and 'Ningyu' plants containing TRV2-FT1 with Agrobacterium tumefaciens during the late vegetative growth stage. After 3 weeks, the apical meristem was observed, and it was found that silencing FT1 significantly improved the strawberry flower bud differentiation rate.

[0068] To address the current difficulties in detecting flower bud differentiation, this invention prepares an FT1 recombinant protein antigen based on the FT1 amino acid sequence, and develops an FT1 enzyme-linked immunosorbent assay kit and an FT1 colloidal gold antibody rapid test strip based on the FT1 antigen.

[0069] I. Preparation of FT1 recombinant protein antigen and monoclonal antibody

[0070] 1. Preparation of FT1 recombinant protein antigen:

[0071] (1) Using leaves from the vegetative growth stage of cultivated strawberry 'Hongyan' as material, total RNA was extracted and then cDNA was synthesized. Using cDNA as a template, the FT1 fragment was cloned by PCR using FT1-pET28a primers with added homologous arms. The PCR reaction program was: 98 ℃, 30 s; (98 ℃, 10 s; 55 ℃, 20 s; 72 ℃, 15 s) 38 cycles; 72 ℃, 5 min. At the same time, pET28a was digested with BamHI and EcoRI restriction enzymes respectively;

[0072] (2) The cloned FT1 fragment was inserted into the cut pET28a vector using homologous recombinase (50 ℃, 10 min), and transformed into BL21(DE3) Escherichia coli. The transformation steps were: on ice for 30 min; 42 ℃ for 90 s; on ice for 2 min. After incubation at 37 ℃ for 1 h, the bacterial culture was evenly spread onto LB agar plates containing Kan antibiotic and incubated overnight at 37 ℃. Positive clones were screened by colony PCR and sequencing.

[0073] (3) Escherichia coli containing the FT1-pET28a recombinant vector was cultured and induced to express the protein by 0.2 M IPTG. After collecting the bacterial cells (5000 rpm, 6 min), the bacterial solution was lysed by sonication to obtain the protein. The FT1 recombinant protein was purified by nickel ion affinity chromatography to obtain FT1 recombinant protein with a purity of over 90% and a concentration of 0.5 mg / mL.

[0074] The amino acid sequence of the FT1 protein is shown in SEQ ID NO:1:

[0075] MPRDRDPLVVGRVIGDVLDPFTKSVSLRVTYTSKEVNNGCELKPSQVVRQPRVDIGGEDLRTFYTLVMVDPDAPSPSDPNLKEYLHWLVTDIPATAGAVFGQEIVCYESPRPTAGIHRYLFVLFRQLGRQTVYAPGWRQNFNTRDFAELYNLGSPVAAVYFNCQRESGSGGRRRSS

[0076] The nucleotide sequence encoding the FT1 protein is shown in SEQ ID NO:2: ATGCCTAGGGACAGGGACCCCCTCGTTGTGGGAAGAGTCATAGGTGATGTTCTGGACCCTTTTACAAAGTCTGTTTCTCTCAGGGTGACTTACACTTCTAAGGAGGTCAACAATGGTTGTGAGCTCAAACCTTCCCAAGTTGTCAGACAACCTCGAGTTGATATAGGAGGGGAGGATCTTAGGACCTTCTACACTCTGGTCATGGTCGATCCTGATGCACCCAGCCCAAGTGATCCCAACCTGAAAGAATATTTGCATTGGT TAGTCACTGATATTCCTGCAACAGCTGGGGCAGTTTTCGGCCAAGAGATTGTGTGTTATGAAAGTCCACGGCCAACAGCGGGGATTCATCGCTACCTTTTTGTGTTGTTTCGGCAGTTGGGAAGGCAAACTGTG TATGCTCCGGGATGGCGCCAAAACTTTAACACCAGAGACTTTGCCGAGCTCTACAATCTTGGATCACCGGTGGCTGCCGTCTACTTTAACTGCCAGAGAGAAAGTGGCTCCGGCGGAAGGAGAAGATCATCGTAA

[0077] The PCR amplification primers are shown in SEQ ID NO:3 and SEQ ID NO:4:

[0078] FT1-pET28a forward primer: 5'-CAAATGGGTCGCGGATCCATGCCTAGGGACAGGGAC-3',

[0079] FT1-pET28a reverse primer: 5'-TCGACGGAGCTCGAATTCCGATGATCTTCTCCTTCCGC-3'.

[0080] 2. Monoclonal antibody preparation and screening:

[0081] (1) Animal immunization: Six 6-8 week old BALB / c mice were immunized multiple times using purified FT1 recombinant protein as the antigen. The first immunization was emulsified with complete Freund's adjuvant, subsequent booster immunizations were emulsified with incomplete Freund's adjuvant, and the final immunization was performed via intraperitoneal pulse immunization. During the period, the immunogenicity was detected by ELISA.

[0082] The specific steps for detecting immunotiter using the above ELISA are as follows: FT1 recombinant protein was coated at a concentration of 1 μg / mL and incubated overnight at 4°C. Mouse serum was blocked with 5% skim milk powder at 37°C (centrifuged at 10000 r / min, 4°C for 5 min, and the supernatant serum was collected) for 2 h. After blocking, the sample was diluted with 1×PBS at dilutions of 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000, and 100 μL / well was added, incubated at 37°C for 1 h. The sample was washed three times with 200 μL / well of TBST, and secondary antibody (goat anti-mouse-HRP) was added, incubated at 37°C for 40 min. The sample was washed five times with 200 μL / well of TBST, and 90 μL / well of single-component TMB chromogenic solution was added for color development for 15 min. Finally, stop solution was added at 50 μL / well, and the OD was measured using a microplate reader. 450 reading.

[0083] (2) Cell fusion and monoclonal antibody screening:

[0084] a. Using a combination of semi-solid and liquid methods, spleen cells from immunized mice were fused with myeloma cells (SP2 / 0) under PEG-mediated fusion. The cell culture supernatant after fusion was detected by indirect ELISA 1 day later.

[0085] b. Select cell lines with high titer and affinity for subcloning, and detect the cell culture supernatant of the first subcloning by indirect ELISA.

[0086] c. Select cell lines with high titers and affinity for a second subcloning, and collect the supernatant for Western blotting. Detect the cell culture supernatant of the second subcloning using indirect ELISA, and expand the culture of all-positive cell lines.

[0087] d. The cell lines of the established strains were passaged and expanded, and the supernatant was collected for subtype identification. All the cells were identified as IgG type.

[0088] e. Collect cells, inject them into mice to prepare ascites fluid, and purify the ascites fluid using a Protein G affinity chromatography column.

[0089] The specific steps for purifying the ascites fluid are as follows: First, the chromatography column is pretreated by washing 3-5 times with 10 times the column bed volume of Protein G deionized water at a flow rate of 1 mL / min. Then, it is washed 3-5 times with 10 times the column volume of 0.02 M PB + 0.3 M NaCl at a flow rate of 1 mL / min. The ascites fluid / serum is diluted with 0.02 M PB and filtered through a 0.22 μm filter membrane at a flow rate of 0.6 mL / min. Washing is continued with 0.02 M PB until no protein is detected (G250 does not turn blue) at a flow rate of 1.5 mL / min. Elution is then performed with 0.1 M H3.0 glycine, and the eluent is collected and detected using a G250 until no blue color is detected. The pH of the eluent is adjusted to neutral using saturated sodium carbonate, and the product is concentrated to approximately 1-5 mL using a 10 kDa ultrafiltration tube. The concentrated product was dialyzed with 0.01M PBS (pH 7.4), and the column was washed with deionized water until neutral. Finally, the column was re-chromatographically analyzed with 5 column volumes of 20% ethanol and stored at 4°C.

[0090] (3) Antibody pairing verification: The obtained monoclonal antibodies were paired and screened by sandwich ELISA to obtain antibody pairs that can better distinguish between positive and negative samples: 3G4 1E10 and 9E11 1H10.

[0091] The amino acid sequence of the light chain variable region of 3G4 1E10 is shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6;

[0092] The amino acid sequence of the light chain variable region of 9E11 1H10 is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8.

[0093] The nucleotide sequence encoding the amino acid of the 3G4 1E10 light chain variable region is shown in SEQ ID NO:9, and the nucleotide sequence encoding the amino acid of the 3G4 1E10 heavy chain variable region is shown in SEQ ID NO:10.

[0094] The nucleotide sequence encoding the amino acid of the 9E11 1H10 light chain variable region is shown in SEQ ID NO:11, and the nucleotide sequence encoding the amino acid of the 9E11 1H10 heavy chain variable region is shown in SEQ ID NO:12.

[0095] SEQ ID NO:5:

[0096] DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYFYMYWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREVPFTFGSGTKLEIK

[0097] SEQ ID NO:6:

[0098] EVQLQQSGTVLARPGTSVKMSCKATGYSFSDYWIHWVTQRPGQGLEWIGSIHPGNSDSNYNQKFKGKATLTVVASANTAYMELSSLTNEDSAVYFCTRNYYGRTSWFAYWGQGTLVTVSA

[0099] SEQ ID NO:7:

[0100] DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLQWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYFCQNGHSFPLTFGAGTKLELK

[0101] SEQ ID NO:8:

[0102] QVQLQQSGPELVKPGTSVRMSCKASGYTFTTYYIHWVKQRPGQGLEWIGWIYPGDVITEYNEKFKGKATLTADKSSSTAYMQLISLTSEDSAVYFCAREGYGRNYYFDYWGQGTTLTVSS

[0103] SEQ ID NO:9:

[0104] GACATTGTGCTGACACAGTCTCCTGCTTCCTTAACTGTATCTCTGGGGCAGAGGGCCACCATCTCCTGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATTTTTATATGTACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAATCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA

[0105] SEQ ID NO:10:

[0106] GAGGTTCAGCTCCAGCAGTCTGGGACTGTGTTGGCAAGGCCTGGGACTTCCGTGAAGATGTCCTGTAAGGCTACTGGCTACAGCTTTTCCGACTACTGGATACACTGGGTCACCCAGAGGCCTGGACAGGGTCTAGAATGGATTGGAAGTATTCATCCTGGAAATAGTGATTCTAACTACAACCAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGTCGCATCCGCCAACACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTTCTGCACAAGAAATTACTACGGAAGAACGTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACAGTCTCTGCA

[0107] SEQ ID NO:11:

[0108] GACATTTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTACAGTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATATGCTTCCCAAT CCATCTCTGGGATCCCCTCCAGGTTTCAGTGGCAGTGGATCAGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTGGAGTGTATTTCTGTCAAAATGGTCACAGCTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0109] SEQ ID NO:12:

[0110] CAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCCTGGGACTTCAGTGAGGATGTCCTGCAAGGCTTCTGGCTACACCTTCACAACCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAGATGTTATTACTGAGTAT AATGAGAAGTTCAAGGGCAAGGCCACCCTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCATCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGAGGGCTACGGTCGTAACTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0111] II. Preparation and Application of FT1 Enzyme-Linked Immunosorbent Assay Kit

[0112] Detection range: 7.8125-500 pg / mL; Precision: intra-assay CV% <10%, inter-assay CV% <10%; Specificity: This kit specifically detects FT1 and has no cross-reactivity with other related proteins.

[0113] Detection Principle: FT1 capture antibody 9E11 1H10 and FT1 detection antibody 3G4 1E10 are a pair of paired antibodies that recognize different sites of the FT1 protein antigen. A microplate is coated with purified FT1 capture antibody to form a solid-phase carrier. The sample or standard, biotin-labeled FT1 detection antibody, and horseradish peroxidase (HRP)-labeled avidin are added sequentially to the wells of the FT1 capture antibody microplate. After thorough washing, the substrate TMB is used for color development. TMB is converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The intensity of the color is positively correlated with the FT1 concentration in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader to calculate the sample concentration.

[0114] The reagent kit components of this invention include:

[0115] ELISA plate coated with FT1 capture antibody: 96T, 12 strips × 8 wells;

[0116] The preparation method of an ELISA plate coated with FT1 capture antibody includes the following steps:

[0117] The FT1 capture antibody (9E11 1H10) was diluted to 1 μg / mL with carbonate coating buffer (CBS) at pH 9.6, and 100 μL was added to each well of a 96-well microplate. The plate was incubated overnight at 4°C. The coating solution was discarded, and the plate was washed three times with PBST (0.01M PBS, pH 7.4, containing 0.05% Tween-20). 200 μL of blocking buffer (PBST containing 2% BSA) was added to each well, and the plate was blocked at 37°C for 2 h. The blocking solution was discarded, the plate was patted dry, and then dried in a 37°C oven for later use.

[0118] FT1 standard: 2 vials (lyophilized). The purified FT1 recombinant protein was diluted to 500 pg / mL with PBS containing 5% trehalose and 1% BSA, aliquoted, and then lyophilized to obtain the lyophilized standard powder.

[0119] FT1 biotin-labeled antibody, i.e., detection antibody (Biotin-antibody): 1×120 uL / vial (100×), the FT1 detection antibody (3G4 1E10) was labeled using the biotinamide hexanoate N-hydroxysuccinimide ester chemical labeling method, and the biotinamide hexanoate N-hydroxysuccinimide ester was purchased from Sigma (B2643).

[0120] FT1 Biotin-labeled Antibody Diluent: 1×15 mL / bottle. Dilute the prepared biotin-labeled FT1 detection antibody with biotin-labeled antibody diluent (purchased from Beyotime, P0269) at a volume ratio of 1:100. Prepare immediately before use.

[0121] Horseradish peroxidase-labeled avidin (HRP-avidin): 1×120 uL / vial (100×), prepared by sodium periodate (NaIO4) oxidation method;

[0122] Horseradish peroxidase-labeled avidin dilution solution: 1×15 mL / bottle. Dilute horseradish peroxidase-labeled avidin with dilution solution (PBS containing 0.2% BSA and 0.05% Tween-20, pH 7.4) at a volume ratio of 1:100. Prepare immediately before use.

[0123] Sample dilution solution: 50 mL / bottle, PBS buffer, pH 7.4;

[0124] Concentrated washing solution; 1×20 mL / bottle (25×), the solution consists of 25×PBS containing 1.25% Tween-20, pH 7.4;

[0125] Substrate and colorimetric solution: TMB solution, 1×10 mL / bottle;

[0126] Stop solution: 2 M H2SO4, 1×10 mL / bottle;

[0127] Board mounting: 4 pieces;

[0128] The testing process is as follows Figure 5 As shown:

[0129] 1. Reagent preparation: Bring all reagents to room temperature (18-25℃) and equilibrate for at least 30 minutes before use.

[0130] (1) FT1 standard

[0131] Remove the FT1 standard from the kit, centrifuge at 6000-10000 rpm for 30 seconds, dissolve in 1 mL of sample diluent, and repeatedly pipette the solution 5 times from the bottom of the cryovial to aid dissolution. Mix thoroughly to obtain standard S7, and set aside for later use.

[0132] Arrange seven 1.5 mL centrifuge tubes (S0-S6) sequentially, and add 250 μL of sample diluent to each. Pipette 250 μL of standard S7 into the first centrifuge tube (S6) and gently mix. Pipette 250 μL of standard S7 into the second EP tube (S5) and gently mix. Continue this serial dilution process for the standards. S0 is the sample diluent. Standard concentrations: S7, 500 pg / mL; S6, 250 pg / mL; S5, 125 pg / mL; S4, 62.5 pg / mL; S3, 31.25 pg / mL; S2, 15.625 pg / mL; S1, 7.8125 pg / mL; S0, 0 pg / mL.

[0133] (2) Washing working solution

[0134] The concentrated washing solution should be diluted with deionized water at a ratio of 1:25. For example, measure 240 mL of deionized water using a graduated cylinder, pour it into a beaker or other clean container, then measure 10 mL of the concentrated washing solution, add it evenly, stir well, and prepare the solution just before use. Salt precipitation may occur when storing the concentrated washing solution at low temperatures; therefore, it can be warmed in a water bath to aid dissolution during dilution.

[0135] (3) FT1 biotinylated antibody working solution

[0136] Dilute the FT1 biotin-labeled antibody solution 1:100 with FT1 biotin-labeled antibody diluent. For example, add 990 μL of FT1 biotin-labeled antibody diluent to 10 μL of FT1 biotin-labeled antibody solution, mix gently, and prepare within 10 minutes before use.

[0137] (4) Horseradish peroxidase labeled avidin working solution

[0138] Horseradish peroxidase-labeled avidin was diluted 1:100 with horseradish peroxidase-labeled avidin diluent. For example, 10 μL of horseradish peroxidase-labeled avidin was added to 990 μL of horseradish peroxidase-labeled avidin diluent, and the mixture was gently mixed and prepared within 10 minutes before use.

[0139] 2. Sample processing methods:

[0140] 1) Take 0.2 g of washed and dried strawberry leaves with filter paper, cut them into small pieces with sterilized scissors, and grind them into a thin paste using a grinding rod in a sample processing tube;

[0141] 2) Add 1 mL of pre-cooled sample diluent (PBS) and vortex for 5 minutes to mix.

[0142] 3) Centrifuge at 4000 rpm for 3 minutes;

[0143] 4) Use a dropper to take 100 μL of the clear liquid as the sample solution to be tested, for subsequent detection and analysis.

[0144] 3. Detection method:

[0145] ① Sample addition: Set up standard wells S0-S7 and sample wells for each concentration of standard solution in an ELISA plate coated with FT1 capture antibody. There are two standard wells and two sample wells for each concentration of standard solution, i.e., perform double-well assay to ensure the accuracy of the results. Add 100 μL of standard or sample solution to each well, gently shake to mix, cover with the plate label, and incubate at 37°C for 2 hours.

[0146] ② Discard the liquid, shake dry, no need to wash.

[0147] ③ Add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour.

[0148] ④ Discard the liquid in the wells, spin dry, and wash the plate 3 times. Soak for 2 minutes each time, 200 μL / well, then spin dry.

[0149] ⑤ Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour.

[0150] ⑥ Discard the liquid in the wells, spin dry, and wash the plate 5 times; soak for 2 minutes each time, 200μL / well, and spin dry.

[0151] ⑦ Add 90 μL of substrate and colorimetric solution to each well in sequence, and develop the color at 37°C in the dark for 15-30 minutes.

[0152] ⑧ Add 50 μL of stop solution to each well in sequence to terminate the reaction.

[0153] ⑨ Within 5 minutes after the reaction is terminated, use an ELISA reader to measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm.

[0154] 4. Establishing the standard curve:

[0155] Plotting the standard concentration on the x-axis and the corresponding OD value on the y-axis, a standard curve is fitted using software. For example... Figure 6 As shown, the standard curve exhibits a good linear relationship, R0 2 =0.9997, detection range is 7.8125-500 pg / mL, suitable for quantitative detection.

[0156] 5. Precautions: To ensure the accuracy of the test results, both standards and samples should be tested in duplicate, and a standard curve should be prepared for each test. If the concentration of the analyte in the sample is too high, dilute it with sample diluent first to make the sample conform to the detection range of the kit, and then multiply by the corresponding dilution factor when calculating the final result. The sample addition time should be controlled within 10 minutes at a time. If there are many samples, use a multi-channel pipette for addition. To prevent sample evaporation or contamination, the ELISA plate must be covered with a plate patch during incubation. The ELISA plate should not be allowed to dry out during the experiment. During incubation, the incubator temperature should be monitored at any time to ensure it remains constant at 37°C and adjusted as needed. The incubator should not be opened too many times during incubation to avoid affecting temperature balance. The washing process is very important; insufficient washing can easily lead to false positives. Manual plate washing method: Aspirate (without touching the walls and bottom of the wells) or shake off the liquid in the ELISA plate; place several layers of absorbent paper on the lab bench, and tap the ELISA plate face down several times; inject 200 μL of washing buffer into each well and soak for 2 minutes. Repeat this process several times as described in the operating instructions, or use an automatic plate washer. During the color development process, to ensure the accuracy of the experimental results, the stop solution should be added as soon as the substrate reaction time is up. The color development can be observed periodically after adding the substrate solution to control the reaction time (e.g., every 10 minutes). When a clear gradient of blue is visible in the first 3-4 wells of the standard, and the color development is not obvious in the last 3-4 wells, the stop solution can be added to terminate the reaction; at this point, the blue will immediately turn yellow. The order of adding the stop solution should be as similar as possible to the order of adding the substrate solution. The substrate solution should be light blue or colorless; if the color darkens significantly, it must be discarded. The substrate solution is easily contaminated and must be stored properly away from light.

[0157] 6. Result determination:

[0158] A curve can be plotted by subtracting the S0 well value from the standard and sample values. If duplicate wells are used, the average value should be taken. Plot the standard curve on logarithmic graph paper with the standard concentration on the ordinate (logarithmic scale) and the OD value on the x-axis (logarithmic scale). The corresponding concentration can be found from the standard curve based on the sample OD value; alternatively, the regression equation of the standard curve can be calculated using the standard concentration and OD value, and the sample OD value can be substituted into the equation to calculate the sample concentration. If the sample was diluted before testing, it needs to be multiplied by the corresponding dilution factor in the final calculation to obtain the actual sample concentration.

[0159] The differentiation status of strawberry flower buds can be identified by the changes in FT1 protein content in strawberry leaves. If the FT1 protein content in strawberry leaves is higher than 50 pg / mL, the strawberry is in an undifferentiated state; if the FT1 protein content in strawberry leaves is lower than 25 pg / mL, the strawberry flower buds have been initiated.

[0160] 7. Actual Sample Detection Results: This invention continuously monitored the changes in FT1 protein content in the leaves of 22 strawberry plants (11 'Hongyan' and 11 'Ningyu') using a kit. The results are as follows... Figure 7 As shown, the mean FT1 protein content in undifferentiated leaves was 76.98 pg / mL (n=22, SD=32.56, range 41.22–154.67 pg / mL); the mean FT1 protein content in leaves after flower bud initiation (differentiation) was 19.00 pg / mL (n=22, SD=4.83, range 10.28–30.17 pg / mL). The two groups showed a highly significant difference according to the t-test (p<0.001).

[0161] Based on the above distribution, the threshold for determining undifferentiated samples was set to > 50 pg / mL (higher than the lowest value of 41.22 pg / mL in the undifferentiated group, and covering the vast majority of undifferentiated samples), and the threshold for determining flower bud initiation was set to < 25 pg / mL (higher than the highest value of 30.17 pg / mL in the differentiated group, but 25 pg / mL was used as the initiation threshold to ensure sensitivity). In practical applications, if the sample concentration is between 25 and 50 pg / mL, it is recommended to repeat the test or combine it with morphological observation.

[0162] III. Colloidal gold test strip for rapid detection of strawberry flower bud differentiation based on FT1 protein and its application

[0163] 1. Detection device, such as Figure 8 As shown, a colloidal gold test strip for rapid detection of strawberry flower bud differentiation based on FT1 protein includes a base plate 1, and a sample pad 2, a conjugate pad 5, a nitrocellulose membrane 3, and an absorbent pad 4 sequentially overlapped on the base plate; the conjugate pad 5 is coated with colloidal gold-labeled strawberry FT1 monoclonal antibody 3G4 1E10; the nitrocellulose membrane 3 has a detection line 6 and a control line 7, the detection line 6 is coated with anti-strawberry FT1 monoclonal antibody 9E11 1H10, and the control line 7 is coated with goat anti-mouse IgG antibody; wherein, the amino acid sequence of the FT1 protein is shown in SEQ ID NO:1.

[0164] Preferably, a sample pad 2, a nitrocellulose membrane 3, and an absorbent pad 4 are arranged sequentially from front to back on the base plate 1. A portion of the rear side of the sample pad 2 overlaps a portion of the front side of the nitrocellulose membrane 3, and a portion of the front side of the absorbent pad 4 overlaps a portion of the rear side of the nitrocellulose membrane 3. The binding pad 5 is divided into upper and lower parts. The lower structure is in contact with the rear end of the sample pad 2, and the upper structure extends from the front and rear sides of the lower structure. The front end covers the upper part of the rear side of the sample pad 2, and the rear end covers the nitrocellulose membrane 3. On the nitrocellulose membrane 3 between the binding pad 5 and the absorbent pad 4, the detection line 6 is arranged on the front side, and the quality control line 7 is arranged on the rear side. A MAX sample level line 8 is also arranged on the binding pad 5.

[0165] 2. Preparation method, including the following steps:

[0166] (1) Soak the sample pad in sample pad pretreatment solution (0.02M PBS with pH=7.4 containing 5 mL / L Tween-20, 8 g / L NaCl and 0.5 g / L ProClin 300 antibacterial agent) for 5 min, and then dry it for later use;

[0167] (2) Immerse the conjugate pad in conjugate pad pretreatment solution (0.02M PBS with pH=7.4 containing 5 mL / L Tween-20, 5 g / L casein, 10 g / L LBSA, 50 g / L trehalose and 0.5 g / L ProClin 300 antibacterial agent) for 5 min, then dry for later use;

[0168] (3) Slowly add 0.1 mol / L K2CO3 to the colloidal gold solution until the pH is 8.5, add FT1 monoclonal antibody 3G41E10, stir for 30 min, add bovine serum albumin (BSA) to a final concentration of 1%, stir for 15 min, centrifuge at 8000 rpm for 15 min, discard the supernatant, and resuspend the resuspension (10 mM Tris-HCl, 1% BSA, 10% sucrose, pH = 7.6) at one-tenth the volume of the original solution to obtain the colloidal gold-labeled FT1 monoclonal antibody. Spray the colloidal gold label onto the conjugate pad and dry at 37°C for 1 h.

[0169] (4) The goat anti-mouse IgG solution and FT1 monoclonal antibody 9E11 1H10 solution were respectively streaked onto the NC membrane and dried to obtain an NC membrane containing C lines and T lines.

[0170] (5) Assemble the processed sample pad, the binding pad coated with gold-labeled FT1 antibody 3G4 1E10, the NC membrane containing C and T lines, the absorbent pad, and the PVC backing in sequence to obtain the colloidal gold antibody test strip.

[0171] 3. Detection Principle: The Strawberry FT1 Colloidal Gold Rapid Detection Card is convenient, rapid, and sensitive, suitable for large-scale on-site sample testing. When the sample contains FT1 protein (antigen), the antigen first binds to the gold-labeled antibody on the binding pad, forming an "antigen-gold-labeled antibody" complex. This complex moves to the T region with liquid chromatography and is captured by the capture antibody immobilized in the T region, forming a double antibody sandwich structure of "capture antibody-antigen-gold-labeled antibody". If the sample is positive, a purple-red band appears in the T region after sample addition; if the sample is negative, no purple-red band appears in the T region. Regardless of the presence of FT1 protein in the sample, a purple-red band will appear in the C region.

[0172] Repeatability: The coefficient of variation (CV, %) should not exceed 15%;

[0173] Inter-batch variation: The relative deviation (R) of the results shall not exceed ±15%;

[0174] 3. Detection method:

[0175] (1) Sample preparation:

[0176] 1) Take 0.2 g of washed and dried strawberry leaves with filter paper, cut them into small pieces with sterilized scissors, and grind them into a thin paste using a grinding rod in a sample processing tube;

[0177] 2) Add 1 mL of pre-cooled phosphate buffer and vortex for 5 minutes to mix.

[0178] 3) Centrifuge at 4000 rpm for 3 minutes;

[0179] 4) Use a dropper to take 100 μL of the supernatant as the sample solution to be tested, for subsequent detection and analysis.

[0180] (2) During testing, use the attached dropper to draw up the supernatant of the sample to be tested and add 100 μL of the sample solution to the sample pad. After adding the sample, let it stand for 5-10 minutes, observe the color development area, and determine the result. Among them, if the C line is colored and the T line is visible to the naked eye (regardless of the color intensity), it is positive, indicating that the FT1 protein content is high; if the C line is colored and the T line is not colored, it is negative, indicating that the FT1 protein content is extremely low or non-existent; if the C line is not colored, regardless of whether the T line is colored, the test strip is considered invalid and needs to be tested again. Figure 9 ).

[0181] 4. Actual Sample Detection Results: In this invention, 'Hongyan' and 'Ningyu' strawberry seedlings with undifferentiated flower buds in the apical meristem were selected. Total protein was extracted from older leaves, and the detection result was positive, with a clear T-line, indicating a high FT1 protein content at this stage. 'Hongyan' and 'Ningyu' strawberry seedlings with differentiated flower buds in the apical meristem were selected, and the detection result was negative, with only the C-line showing color. These detection results are consistent with the results observed under a dissecting microscope. Figure 10 ).

[0182] This invention develops two simple, convenient, and intuitive FT1 protein-based rapid detection technologies for strawberry flower bud differentiation. These technologies offer advantages such as ease of operation, intuitive results, no damage to plants, and low cost. They are suitable for large-scale rapid detection in the field and can effectively replace existing microscopic observation methods, providing reliable technical support for precise strawberry planting.

[0183] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody pair prepared using strawberry FT1 recombinant protein as an antigen, characterized in that: The monoclonal antibody pair is 3G4 1E10 and 9E11 1H10, which recognize different epitopes of the FT1 protein; wherein: The amino acid sequence of the light chain variable region of 3G4 1E10 is shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6; The amino acid sequence of the light chain variable region of 9E11 1H10 is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

8.

2. The application of FT1 protein as an antigen and the monoclonal antibody prepared using recombinant FT1 protein as an antigen as described in claim 1 in the FT1 enzyme-linked immunosorbent assay kit and the colloidal gold test strip for rapid detection of strawberry flower bud differentiation, characterized in that: The amino acid sequence of the FT1 protein is shown in SEQ ID NO:

1.

3. The nucleotide sequence encoding the monoclonal antibody pair of claim 1, characterized in that: The nucleotide sequence encoding the amino acid of the 3G4 1E10 light chain variable region is shown in SEQ ID NO:9, and the nucleotide sequence encoding the amino acid of the 3G41E10 heavy chain variable region is shown in SEQ ID NO:

10. The nucleotide sequence encoding the amino acid of the 9E11 1H10 light chain variable region is shown in SEQ ID NO:11, and the nucleotide sequence encoding the amino acid of the 9E11 1H10 heavy chain variable region is shown in SEQ ID NO:

12.

4. An FT1 enzyme-linked immunosorbent assay (ELISA) rapid detection kit, characterized in that: The kit includes an ELISA plate coated with FT1 capture antibody 9E11 1H10, FT1 standard, FT1 biotinylated antibody 3G4 1E10, FT1 biotinylated antibody diluent, horseradish peroxidase-labeled avidin, horseradish peroxidase-labeled avidin diluent, sample diluent, substrate chromogenic solution, stop solution, and concentrated wash solution.

5. A colloidal gold test strip for rapid detection of strawberry flower bud differentiation based on FT1 protein, characterized in that: It includes a base plate (1), and a sample pad (2), a conjugate pad (5), a nitrocellulose membrane (3), and an absorbent pad (4) sequentially attached to the base plate; the conjugate pad (5) is coated with colloidal gold-labeled strawberry FT1 monoclonal antibody 3G4 1E10; the nitrocellulose membrane (3) is provided with a detection line (6) and a control line (7), the detection line (6) is coated with strawberry FT1 monoclonal antibody 9E11 1H10, and the control line (7) is coated with goat anti-mouse IgG antibody.

6. The colloidal gold test strip for rapid detection of strawberry flower bud differentiation based on FT1 protein according to claim 5, characterized in that: A sample pad (2), a nitrocellulose membrane (3), and an absorbent pad (4) are arranged sequentially from front to back on the base plate (1). A portion of the rear side of the sample pad (2) overlaps a portion of the front side of the nitrocellulose membrane (3), and a portion of the front side of the absorbent pad (4) overlaps a portion of the rear side of the nitrocellulose membrane (3). The conjugate pad (5) is divided into upper and lower parts. The lower structure is in contact with the rear end of the sample pad (2), and the upper structure extends from the front and rear sides of the lower structure. The front end covers the upper part of the rear side of the sample pad (2), and the rear end covers the nitrocellulose membrane (3). On the nitrocellulose membrane (3) between the conjugate pad (5) and the absorbent pad (4), the detection line (6) is arranged on the front side, and the quality control line (7) is arranged on the rear side. A MAX sample level line (8) is also arranged on the conjugate pad (5).

7. The application of the FT1 enzyme-linked immunosorbent assay kit of claim 4 or the colloidal gold test strip based on FT1 protein for rapid detection of strawberry flower bud differentiation of claim 5 in the rapid detection of strawberry flower bud differentiation.

8. A rapid detection method for strawberry flower bud differentiation, characterized by: The method is one of the following two: The first type: The FT1 enzyme-linked immunosorbent assay (ELISA) rapid test kit was used for detection, and the specific steps included the following: (A) Preparation of working solutions: including the preparation of FT1 standard solution, washing working solution, FT1 biotin-labeled antibody working solution and horseradish peroxidase-labeled avidin working solution; (B) Sample preparation: Take strawberry leaves, grind them, add phosphate buffer to lyse and shake to mix, centrifuge and take the supernatant as the sample solution to be tested; (C) Labeling detection: This includes adding samples to an ELISA plate coated with FT1 capture antibody, adding FT1 biotin-labeled antibody working solution, washing the plate, adding horseradish peroxidase-labeled avidin working solution, washing the plate, adding substrate chromogenic solution, and adding stop solution. After completion, the absorbance of each well at 450 nm wavelength is measured using an ELISA reader. A standard curve is fitted using software with the concentration of FT1 standard as the x-axis and the corresponding OD value as the y-axis. Judgment criteria: The state of strawberry flower bud differentiation is identified based on changes in the protein content of FT1 in strawberry leaves. If the FT1 protein content in strawberry leaves is > 50 pg / mL, the leaves are considered to be in an undifferentiated state. If the FT1 protein content in strawberry leaves is < 25 pg / mL, it is determined that the flower bud has been initiated. The second type: The detection was performed using a colloidal gold test strip based on the FT1 protein for rapid detection of strawberry flower bud differentiation. The specific steps included: (a) Sample preparation: Take strawberry leaves, grind them, add phosphate buffer to lyse and shake to mix, centrifuge and take the supernatant as the sample solution to be tested; (b) Detection: Add 100 μL of the sample solution to be tested into the sample pad, let it stand for 5-10 minutes after adding the sample, and observe the color development area; Judgment criteria: The differentiation status of strawberry flower buds can be identified based on changes in the protein content of FT1 in strawberry leaves; If the FT1 protein content in strawberry leaves is high and the colloidal gold test strip is obvious (i.e., the C line is colored and the T line is visible to the naked eye), regardless of the color intensity, it is positive, indicating that the FT1 protein content is high and the strawberry is in an undifferentiated state. If the FT1 protein content in strawberry leaves is significantly reduced and there is no obvious band on the colloidal gold test paper (i.e., the C line is colored and the T line is not colored), it is negative, indicating that the FT1 protein content is extremely low or absent, and the strawberry flower buds have been initiated. If line C does not develop color, the test strip is considered invalid regardless of whether line T develops color, and it needs to be tested again.

9. The rapid detection method for strawberry flower bud differentiation according to claim 8, characterized in that: When using the FT1 enzyme-linked immunosorbent assay kit for detection, the labeling detection method specifically includes the following steps: ① Sample addition: Set up standard wells S0-S7 and sample wells in an ELISA plate coated with FT1 capture antibody. There are two standard wells and two sample wells for each concentration of standard solution. Perform double-well assay. Add 100 μL of standard or sample solution to each well, gently shake to mix, cover with plate label, and incubate at 37°C for 2 hours. ② Discard the liquid, spin dry, no washing required; ③ Add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour; ④ Discard the liquid in the wells, spin dry, and wash the plate 3 times; soak for 2 minutes each time, 200μL / well, and spin dry; ⑤ Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour; ⑥ Discard the liquid in the wells, spin dry, and wash the plate 5 times; soak for 2 minutes each time, 200μL / well, and spin dry; ⑦ Add 90 μL of substrate and colorimetric solution to each well in sequence, and develop the color at 37°C in the dark for 15-30 minutes; ⑧ Add 50 μL of stop solution to each well in sequence to terminate the reaction; ⑨ Within 5 minutes after the reaction is terminated, measure the OD value of each well sequentially at a wavelength of 450 nm using an ELISA reader.

10. The rapid detection method for strawberry flower bud differentiation according to claim 8, characterized in that: The sample processing procedure specifically includes the following steps: 1) Take 0.2 g of washed and dried strawberry leaves with filter paper, cut them into small pieces with sterilized scissors, and grind them into a thin paste using a grinding rod in a sample processing tube; 2) Add 1 mL of pre-cooled phosphate buffer and vortex for 5 minutes to mix. 3) Centrifuge at 4000 rpm for 3 minutes; 4) Use a dropper to take 100 μL of the supernatant as the sample solution to be tested, for subsequent detection and analysis.

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