Preparation of brassinolide-derived carbon quantum dots and method for sensing hormone receptor by using brassinolide-derived carbon quantum dots

Abstract

The invention discloses a preparation method of brassinolide derived carbon quantum dots and application of the brassinolide derived carbon quantum dots in plant hormone receptor detection. According to the method, BRs is used as a precursor, CQDs with a BRs semi-reserved structure are prepared through co-carbonization with citric acid, and the nano material with the particle size of 3.1 + / -0.5 nm and the surface rich in hydroxyl / keto is obtained after dialysis and purification. When the CQDs are combined with the BRs receptors, the fluorescence intensity of the CQDs is linearly enhanced along with the concentration of the receptors, the nano sensor constructed based on the CQDs can realize rapid quantitative detection of the BRs receptors in rice leaves, the linear range is 0.01-4.0 mg / mL, the detection limit is as low as 0.003 mg / mL, and the sensitivity is improved by 10 times compared with that of ELISA (Enzyme-Linked Immunosorbent Assay). By detecting the receptor expression quantity of a wild type and BRs receptor overexpression strain in a germination period, a heading period and a mature period, it is verified that the result of the strain is highly consistent with that of Western Blot, and the detection time is shortened to 30 minutes. The method does not need targeted modification, has the advantages of high specificity, interference resistance and low cost, and provides an efficient detection tool for rice molecular breeding and stress resistance regulation.

Description

Technical Field

[0001] The present invention relates to the preparation of functional nanomaterials, and specifically to a carbon quantum dot synthesis and purification technology based on a brassinolide precursor, and a quantitative analysis method thereof for brassinolide hormone receptors. Background Art

[0002] Brassinosteroids (BRs) are a class of natural plant sterol hormones first isolated from rapeseed pollen in the 1970s. As the sixth largest class of plant hormones after auxins and gibberellins, BRs play a key role in plant growth and development, stress response, and yield regulation. Their chemical structure is centered on a sterol skeleton, and they form a variety of active forms through hydroxylation and side chain modification. They are widely present in higher plants. In rice, BRs significantly promote root development and stem elongation by activating genes related to cell division and elongation, laying the foundation for high yield. At the same time, BRs enhance leaf photosynthetic efficiency and increase dry matter accumulation by regulating chlorophyll synthesis genes. Furthermore, BRs alleviate oxidative damage caused by drought and salt stress by inducing antioxidant enzyme activity. During the reproductive growth period of rice, BRs promote spikelet differentiation and reduce abortion.

[0003] BRs regulates rice growth, development, and yield formation by binding to specific receptors on the cell membrane (such as BRI1 and BAK1) to trigger signal transduction. BRs receptors serve as the core mediator of signal transmission, and their expression directly affects key agronomic traits of rice, such as tillering, panicle development, and stress resistance. When BRs molecules bind to the BRI1 receptor, they form a complex with the co-receptor BAK1, activating the downstream kinase cascade reaction, releasing the transcription factor BZR1 into the cell nucleus, and then regulating the expression of genes related to cell elongation, tillering, and reproduction. Therefore, the dynamic balance of receptor expression is crucial: moderate high receptor expression can enhance the efficiency of BRs signal transduction, promote a 20%-30% increase in the number of rice tillers, thicken the stems, and increase plant height. At the same time, by activating the activity of the panicle meristem, the number of grains per panicle can be increased by 15%-25%, and the expression of endosperm development genes can be upregulated to reduce empty grains. Furthermore, upregulation of receptor expression under adverse conditions can induce a 50% increase in antioxidant enzyme activity or improve water use efficiency by enhancing aquaporin expression, reducing yield losses under drought or salt stress by 30%-50%. However, imbalanced receptor expression can lead to negative effects: insufficient BRI1 expression can cause severe plant dwarfing, a 60%-70% reduction in tillering, and a more than 50% decrease in grain number per panicle; while overexpression can lead to a decrease in stem mechanical strength due to excessive cell elongation, a 2-3-fold increase in lodging rate, or a 10%-15% decrease in grain 1000-grain weight due to an imbalance in photosynthate distribution. Therefore, accurately assessing receptor expression is not only fundamental for understanding the mechanism of action of BRs but also a key prerequisite for optimizing rice plant architecture, stress resistance, and yield.

[0004] Among the methods for evaluating receptor expression, different technical approaches have their own advantages and limitations. Gene expression level detection is mainly achieved through real-time fluorescence quantitative PCR (qRT-PCR) or RNA sequencing (RNA-seq). It has the advantages of high sensitivity (can detect as few copies of transcripts) and is suitable for dynamic analysis (such as expression changes from tillering to heading), but it only reflects the transcription level and cannot be directly associated with protein functional activity, and it relies on the stability of internal reference genes. RNA-seq can comprehensively analyze the transcriptome and identify alternative splicing forms and co-expression networks of receptor genes, but its cost is high and the data analysis is complex, and its applicability to small sample studies is limited. Protein expression level detection often uses Western Blot or enzyme-linked immunosorbent assay (ELISA), but its operation is cumbersome, its sensitivity to low-abundance proteins is insufficient, and it is easily interfered by cross-reactions, resulting in low quantitative accuracy. Research on receptor function and localization requires a combination of genetics and imaging techniques. For example, constructing BRI1-GFP transgenic rice and observing the dynamic distribution of the receptor in tiller buds or panicles using confocal microscopy can directly correlate expression levels with phenotypes. Such methods can visually reveal the temporal and spatial expression patterns of receptors, but the transgenic cycle is long and phenotypic analysis requires validation in multiple environments. Furthermore, emerging technologies such as single-cell sequencing can analyze the heterogeneity of receptor expression in specific cell types (such as panicle primordium cells), but this is expensive and data interpretation relies on bioinformatics support.

[0005] In summary, the assessment of BR receptor expression requires a multi-layered approach. This multidimensional strategy can circumvent the limitations of a single method while providing precise targets for molecular breeding, balancing the conflict between high yield and lodging resistance. With the advancement of nanomaterials and nanotechnology, receptor expression assessment will become more cost-effective, simplified, and highly accurate, providing core data support for the design of stress-tolerant and high-yielding rice varieties. As a zero-dimensional, carbon-based fluorescent material, carbon quantum dots (CQDs) offer numerous advantages, including excellent optical properties, ease of preparation and modification, and easily tunable luminescence intensity. Nanosensors developed using CQDs as fluorophores have attracted widespread attention in the field of hormone receptor labeling and detection. However, the interaction between CQDs and hormone receptors lacks specificity, requiring targeted modification, which increases the complexity and cost of nanosensor construction and can affect the dispersion, chemical structure, and optical properties of CQDs. Summary of the Invention

[0006] To address existing issues, the present invention discloses a method for synthesizing CQDs using BRs as a precursor. Furthermore, by leveraging the semi-conserved nature of the BRs structure within CQDs, CQDs can be used as fluorescent sensors for detecting BRs receptors. CQDs bound to hormone receptors experience an increase in fluorescence intensity due to changes in the surface microenvironment. This nanosensor can be used for highly sensitive and reliable detection of BRs receptors, enabling accurate assessment of their expression levels.

[0007] The technical solutions of the present invention are as follows:

[0008] The first object of the present invention is to provide a method for preparing brassinolide-derived carbon quantum dots (CQDs), comprising the following steps:

[0009] a. Dissolve brassinolide (BRs) powder and organic acid in N,N-dimethylformamide (DMF) to prepare BRs stock solution and organic acid stock solution respectively;

[0010] b. Mixing the BRs stock solution and the organic acid stock solution in a reaction vessel at a volume ratio of 1:1, sealing the vessel, and heating the vessel for co-carbonization to generate BRs-derived CQDs;

[0011] c. After cooling the reaction solution, transfer it to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze it for 24-72 hours using 50 mM PBS buffer (pH 7.4) as the dialysate to remove unreacted precursors and obtain a purified CQDs solution.

[0012] Furthermore, in step a: the concentration of the BRs stock solution is 10-100 mg / mL; the organic acid is citric acid (CTA); and the mass ratio of BRs to CTA is 1:2-1:8.

[0013] In step a, 17.5 mL of each BRs and organic acid stock solution was added to a 50 mL polytetrafluoroethylene reactor.

[0014] Furthermore, in step b: the temperature of the co-carbonization reaction is 160-200° C., preferably 180° C.; and the reaction time is 4-8 hours, preferably 6 hours.

[0015] The second object of the present invention is to provide brassinolide-derived carbon quantum dots, which are prepared using the aforementioned preparation method.

[0016] The third object of the present invention is to provide an application of the brassinosteroid-derived carbon quantum dots in the detection of brassinosteroid sterol receptors (BRs receptors), wherein the detection method comprises the following steps:

[0017] d. Mix the purified CQDs solution with the BRs receptor sample to be tested at a volume ratio of 9:1 and incubate in the dark for 15-25 minutes;

[0018] e. Scan the fluorescence spectrum in the range of 380-550nm with an excitation wavelength of 365nm, and record the fluorescence intensity at 460nm;

[0019] f. Calculate the concentration of BRs receptors in the sample based on the pre-established standard curve equation.

[0020] Furthermore, the method for establishing the standard curve equation is:

[0021] After purifying the BRs receptor protein, a standard solution with a concentration gradient of 0-8 mg / mL and at least 1 concentration was prepared;

[0022] The aforementioned CQDs solution was mixed with each concentration standard solution at a volume ratio of 9:1 and incubated for 20 minutes. The linear regression equation was fitted with the fluorescence intensity as the ordinate and the BRs receptor concentration as the abscissa.

[0023] Furthermore, the concentration of the CQDs solution is 1-10 μg / mL, preferably 5 μg / mL; and the incubation time is 10-30 minutes, preferably 20 minutes.

[0024] In the above-mentioned BRs receptor detection method, the BRs receptor sample to be tested in step d is derived from rice leaves, and the purification steps are: taking fresh rice leaves, quickly freezing them in liquid nitrogen, and grinding the leaves into green powder; adding the powder to pre-cooled washing buffer, vortexing to mix, and then ice bathing; centrifuging at 4°C, discarding the precipitate, transferring the supernatant to a new tube, centrifuging, discarding the precipitate, transferring the supernatant to an ultracentrifuge tube, centrifuging, and precipitating the cell membrane component; resuspending the precipitate in lysis buffer, shaking on a shaker to fully dissolve the membrane protein; ultrasonic treatment in an ice bath to disrupt the membrane structure and release the receptor protein; centrifuging, collecting the supernatant containing the dissolved BRs receptor protein; and measuring the mass concentration of the BRs receptor using Nanodrop2000.

[0025] In a particular embodiment, in step d, the BRs receptor is derived from rice leaves, and the purification steps are as follows: 5 g of fresh rice leaves are quickly frozen in liquid nitrogen, and the leaves are ground into a green powder; the powder is transferred to a 50 mL centrifuge tube, and 20 mL of pre-chilled washing buffer (manufacturer: TermoFisher Sientific, trade name: Brij TM-35), vortex to mix and then ice bath for 30 minutes; centrifuge at 3000g for 10 minutes at 4°C, discard the precipitate, transfer the supernatant to a new tube, centrifuge at 10000g for 20 minutes, discard the precipitate, transfer the supernatant to an ultracentrifuge tube, centrifuge at 100000g for 1 hour, and precipitate as the cell membrane component; resuspend the precipitate in 10 mL of lysis buffer and shake on a low-temperature shaker at 4°C for 2 hours to fully dissolve the membrane protein; ultrasonic treatment in an ice bath (power 200W, pulse 3 seconds / stop 5 seconds, a total of 5 minutes) to break the membrane structure and release the receptor protein; centrifuge at 12000g for 15 minutes, collect the supernatant (containing the dissolved BRs receptor protein); use Nanodrop2000 to measure the mass concentration of BRs receptor.

[0026] The fourth object of the present invention is to provide a kit for quantitative detection of rice BRs receptors, comprising: the aforementioned brassinolide-derived carbon quantum dots.

[0027] Beneficial effects of the present invention:

[0028] This invention, through the design of BRs-derived CQDs and their receptor sensing method, achieves highly sensitive and specific detection of BRs receptor expression. This method combines low cost, rapidity, and ease of operation, providing breakthrough technical support for rice molecular breeding and stress resistance improvement. Its core advantages are reflected in the following aspects:

[0029] (1) Precursor innovation and CQDs function optimization. CQDs prepared by co-carbonization reaction using BRs as precursors retain key active groups such as hydroxyl and ketone groups in BRs molecules. This half-retained structural feature gives CQDs a natural affinity with BRs receptors, allowing specific binding without additional targeted modification, solving the problem of traditional CQDs sensors requiring complex functional transformation. At the same time, the optimized preparation process ensures that CQDs have high quantum yield, excellent water solubility and long-term fluorescence stability, significantly improving detection reliability;

[0030] (2) Highly sensitive and anti-interference receptor detection performance. When CQDs bind to BRs receptors, the hydrophobic microenvironment of the receptor protein changes the electronic transition path of CQDs, and the fluorescence intensity increases linearly with the increase of receptor concentration. This sensing method is 10 times more sensitive than ELISA, with a linear range of 0.01-4 mg / mL and strong anti-interference ability. The signal fluctuation is less than 5% in complex plant crude extracts containing chlorophyll, polysaccharides, etc. The entire detection process takes only 30 minutes, which is much faster than Western Blot or qRT-PCR, and is suitable for rapid screening of field samples;

[0031] (3) Accurately correlate receptor expression with agronomic traits. Verified by the standard curve method and dynamic monitoring, the method of the present invention can accurately quantify the dynamic changes in BRs receptor expression from the tillering stage to the heading stage of rice. The test results are consistent with the trends of Western Blot and qRT-PCR, demonstrating that the data can effectively guide stress resistance breeding and cultivation regulation.

[0032] (4) Low cost and easy promotion. Compared with RNA-seq or single-cell sequencing, the raw materials for preparing CQDs in the present invention are cheap, and the consumption of detection reagents is low. Combined with a portable fluorescence spectrometer, it can achieve real-time detection in the field, providing farmers with scientific guidance on the timing and concentration of exogenous BRs spraying. In addition, this method is compatible with molecular marker-assisted breeding, which can be used for high-throughput screening of receptor-high-expressing germplasm, accelerating the breeding process of high-yield and stress-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 .Transmission electron microscope photograph of the CQDs nanosensor synthesized in Example 1.

[0034] Figure 2 .Absorption spectrum of the CQDs nanosensor synthesized in Example 1.

[0035] Figure 3 . Fluorescence spectrum of the CQDs nanosensor synthesized in Example 1.

[0036] Figure 4 .Infrared spectrum of the CQDs nanosensor synthesized in Example 1.

[0037] Figure 5 . Fluorescence spectra (a) and fluorescence intensity dot-line graph (b) of the nanosensor detecting different concentrations of BRs receptors.

[0038] Figure 6 .The expression levels of BRs receptors in leaves of wild-type (WT) rice and BRs receptor-overexpressing transgenic lines (OE) at the germination, heading and maturity stages were measured by nanosensor and western blot methods. DETAILED DESCRIPTION

[0039] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0040] Technical solution: The present invention involves a variety of correlation technologies.

[0041] Technical Solution 1: Preparation Technology of BRs-derived CQDs. The core of this technical solution is to prepare CQDs with a semi-retained BRs structure through the carbonization reaction of BRs precursors. The specific solution is as follows:

[0042] BRs powder and an organic acid were dissolved in DMF, respectively, to prepare a BRs solution and a CTA solution. 17.5 mL of each solution was added to a 50 mL polytetrafluoroethylene reactor. This ratio ensured that the BRs molecules were partially embedded in the CQD carbon skeleton during carbonization, preserving their hydroxyl (-OH) and keto (C=O) active sites. The reactor was sealed and placed in a thermostatic reactor for carbonization. After cooling, the reaction solution was transferred to a 1000 Da dialysis bag and dialyzed with 50 mM PBS buffer (pH 7.4) under magnetic stirring for 48 hours to completely remove unreacted BRs precursors and small molecule byproducts. The purified CQD solution was freeze-dried and stored at -20°C in the dark.

[0043] Preferred: The organic acid type is determined to be CTA, whose carboxyl and hydroxyl groups can promote the formation of hydrophilic groups on the surface of CQDs and enhance water solubility; the mass ratio of BRs to CTA is 1:4, which ensures that the BRs molecules are partially embedded in the CQDs carbon skeleton during the carbonization process, retaining their active sites; the carbonization temperature is selected to be 180°C and the time is determined to be 6 hours, which can balance the graphitization degree of CQDs and the surface functional group density, avoiding complete destruction of the BRs structure; the molecular retention capacity of the dialysis bag is 1000Da, ensuring the purity of CQDs while avoiding loss.

[0044] Technical Solution 2: Establishment of a quantitative relationship between the fluorescence intensity of CQDs nanosensors and the concentration of BRs receptors. This technical solution establishes a quantitative relationship between the fluorescence intensity of CQDs and the concentration of BRs receptors through the standard curve method. The specific process is as follows: extract BRs receptor protein from rice leaves (see step d in the invention content section for the method), use Nanodrop 2000 to determine its mass concentration, and dilute it with PBS buffer; take 1.8mL of 5μg / mL CQDs solution, mix it with 0.2mL of BRs receptor standards of different concentrations, vortex mix, and incubate; use a fluorescence spectrophotometer to measure the fluorescence spectrum and record the peak fluorescence intensity; use BRs receptor concentration as the horizontal axis (X) and fluorescence intensity as the vertical axis (Y) to obtain the standard curve equation using linear regression fitting.

[0045] Preferably, the concentrations of the BRs receptor standard solution are set to 0, 0.001, 0.01, 0.1, 0.5, 1.0, 4.0, and 8.0 mg / mL; the incubation time is 20 minutes, which is the optimal window for stabilization of the fluorescence signal; and the excitation wavelength of 365 nm can minimize the interference of chlorophyll (absorption peaks 430 nm and 660 nm) in the crude plant extract.

[0046] Technical Solution 3: Quantitative Detection of BRs Receptors at Unknown Concentrations. This technical solution uses a standard curve to rapidly quantify BRs receptors in unknown samples. The steps are as follows: Extract the BRs receptor protein from the rice leaves to be tested according to the method described in Technical Solution 2 and dilute it with PBS buffer to the appropriate concentration range; Mix 1.8 mL of CQDs solution (5 μg / mL) with 0.2 mL of the sample to be tested, incubate in the dark for 20 minutes, and then measure the fluorescence intensity; Substitute the fluorescence intensity into the standard curve equation to infer the BRs receptor concentration. If the sample fluorescence intensity exceeds the linear range, it needs to be diluted proportionally and retested.

[0047] Preferred: For samples with high background interference (such as senescent leaves), an ultrafiltration step (10kDa filter membrane) can be added before detection to remove chlorophyll degradation products; a blank control (CQDs + PBS) and a positive control (known concentration of BRs receptor) can be set during the test to ensure data reliability.

[0048] Example 1

[0049] Take 875 mg of BRs powder and 3.5 g of CTA with a purity of ≥95%, dissolve them in DMF respectively, and ultrasonically treat for 10 minutes until completely dissolved. Transfer the two solutions to a 50 mL polytetrafluoroethylene reactor, seal it, and place it in a program-controlled temperature reactor. Heat it to 180°C at 2°C / min and maintain it for 6 hours. Under this condition, BRs and CTA undergo a co-carbonization reaction to form CQDs with a semi-retained structure of BRs. After the reactor is cooled to room temperature, it is transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed for 48 hours under magnetic stirring using 50 mM PBS buffer (pH 7.4) as the dialysate (replace the dialysate every 6 hours). Figure 1 Transmission electron microscopy images show that CQDs are spherical and monodisperse, with a particle size distribution of 3.1±0.5nm (100 particles) and a lattice spacing of 0.21nm (corresponding to the graphene (100) crystal plane), indicating a highly graphitized structure. UV-visible absorption spectra show ( Figure 2 ), CQDs showed a characteristic absorption peak of BRs at 280nm (attributed to the π-π* transition of the sterol skeleton), and a typical absorption band of CQDs (n-π* transition) appeared at 360nm, proving that the BRs structure was partially retained. When the excitation wavelength was 360nm, CQDs emitted strong blue fluorescence at 460nm ( Figure 3 ). Fourier transform infrared spectroscopy ( Figure 4 ), CQDs at 3400 cm -1 (-OH / NH stretching vibration), 1720cm -1 (C=O stretching vibration), 1620cm -1 (C=C skeleton vibration) and 1050cm -1The presence of a characteristic peak at (COC stretching vibration) indicates that the surface is rich in hydroxyl groups, carboxyl groups and ether bonds, which match the functional groups of BRs molecules and give it the ability to specifically bind to the receptor.

[0050] Example 2

[0051] Extract BRs receptor protein from rice leaves (refer to step d of the invention for the method), use Nanodrop 2000 to determine the concentration of the crude extract, and dilute it with PBS buffer to a series of standard solutions of 0, 0.001, 0.01, 0.1, 0.5, 1.0, 4.0, and 8.0 mg / mL. Use the CQDs solution prepared in Example 1 (5 μg / mL, solvent is 50 mM PBS buffer, pH 7.4) as a nanosensor and operate according to the following steps: Take 1.8 mL of CQDs solution and mix it with 0.2 mL of each concentration of BRs receptor standard, vortex to mix, and incubate in the dark for 20 minutes; use a fluorescence spectrophotometer (excitation wavelength 365 nm, emission wavelength scanning range 380-550 nm) to determine the fluorescence intensity of the mixed solution, and record the peak at 460 nm as the detection signal. Each concentration was measured in parallel 3 times and the average value was taken. Draw a standard curve with the BRs receptor concentration as the horizontal axis and the fluorescence intensity as the vertical axis. As shown Figure 5 As shown in the figure, the fluorescence intensity of the nanosensor continued to increase with the concentration of BRs. In the concentration range of 0.01-4.0 mg / mL, the sensor fluorescence increased linearly, and the regression equation was Y=1.77X+20.69(R 2 =0.998), where Y represents the fluorescence intensity and X is the logarithmic value of the BRs concentration. The detection limit (LOD, 3σ / S) of this method is 0.003 mg / mL and the quantification limit (LOQ, 10σ / S) is 0.01 mg / mL.

[0052] Example 3

[0053] Two rice varieties were selected: the wild type (WT) and the BRs receptor overexpressing transgenic line (OE), and the top fully expanded leaves were collected at the germination stage (7 days after sowing), heading stage (stage when panicle differentiation is completed), and maturity stage (stage when filling is completed). Three rice plants were taken in each period, quick-frozen in liquid nitrogen, and then ground into powder. The BRs receptor protein was extracted according to step d of the invention, and the concentration was adjusted to the detection range with PBS buffer. Western Blot detection was performed simultaneously, using BRs receptor antibody (Agrisera, AS121852) as the primary antibody, β-Actin as the internal reference, and Image J software to quantify the grayscale value of the band. The CQDs solution prepared in Example 1 (5 μg / mL, PBS buffer) was taken and quantified according to the standard curve equation of Example 2. The sample to be tested was mixed with CQDs at a volume of 1:9 (0.2 mL sample + 1.8 mL CQDs), incubated in the dark for 20 minutes, the fluorescence intensity at 460 nm was measured, and the BRs receptor concentration was calculated. The detection was repeated 3 times for each sample. Figure 6 As shown, in both strains, the expression of BRs receptors showed an increasing and then decreasing trend as the plants developed, with the maximum BRs receptor expression in leaves at the heading stage. In the BRs overexpression strain, the expression of BRs was higher than that of BRs receptors in wild-type rice during the rice growth cycle. This example confirms that the nanosensor based on BRs-derived CQDs can accurately quantify the dynamic expression of BRs receptors at different growth stages of rice. The detection results are highly consistent with Western Blot, and have the advantages of rapidity and low sample consumption. This method provides an efficient tool for analyzing the spatiotemporal expression patterns of BRs receptors and optimizing exogenous hormone spraying strategies. It is particularly suitable for high-throughput detection needs of multiple samples in the field during multiple periods.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing brassinolide-derived carbon quantum dots (CQDs), characterized in that: The following steps are involved: a. Dissolve brassinolide (BRs) powder and organic acid in N,N-dimethylformamide (DMF) to prepare BRs stock solution and organic acid stock solution respectively; b. Mixing the BRs stock solution and the organic acid stock solution in a reaction vessel at a volume ratio of 1:1, sealing the vessel, and heating the vessel for co-carbonization to generate BRs-derived CQDs; c. After cooling the reaction solution, transfer it to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze it for 24-72 hours using 50 mM PBS buffer (pH 7.4) as the dialysate to remove unreacted precursors and obtain a purified CQDs solution.

2. The preparation method according to claim 1, characterized in that In step a: The concentration of the BRs stock solution is 10-100 mg / mL; The organic acid is citric acid (CTA); The mass ratio of BRs to CTA is 1:2-1:

8.

3. The preparation method according to claim 1, characterized in that In step b: The temperature of the co-carbonization reaction is 160-200°C, preferably 180°C; The reaction time is 4-8 hours, preferably 6 hours.

4. A brassinolide-derived carbon quantum dot, characterized in that: The product is prepared by the preparation method according to any one of claims 1 to 3.

5. A method for detecting brassinosteroid sterol receptors (BRs receptors) by using the brassinosteroid-derived carbon quantum dots according to claim 4, characterized in that: The detection method comprises the following steps: d. Mix the purified CQDs solution with the BRs receptor sample to be tested at a volume ratio of 9:1 and incubate in the dark for 15-25 minutes; e. Scan the fluorescence spectrum in the range of 380-550nm with an excitation wavelength of 365nm, and record the fluorescence intensity at 460nm; f. Calculate the concentration of BRs receptors in the sample based on the pre-established standard curve equation.

6. The use according to claim 5, characterized in that The method for establishing the standard curve equation is: After purifying the BRs receptor protein, a standard solution with a concentration gradient of 0-8 mg / mL and at least 1 concentration was prepared; The CQDs solution of claim 4 was mixed with each concentration standard solution at a volume ratio of 9:1 and incubated for 20 minutes; The linear regression equation was fitted with the fluorescence intensity as the ordinate and the BRs receptor concentration as the abscissa.

7. The use according to claim 5, characterized in that The concentration of the CQDs solution is 1-10 μg / mL, preferably 5 μg / mL; the incubation time is 10-30 minutes, preferably 20 minutes.

8. The use according to claim 5, characterized in that The BRs receptor sample to be tested in step d is derived from rice leaves, and the purification steps are as follows: taking fresh rice leaves, quickly freezing them in liquid nitrogen, and grinding the leaves into a green powder; adding the powder to pre-chilled washing buffer, vortexing to mix, and then placing it in an ice bath; centrifuging at 4°C, discarding the precipitate, transferring the supernatant to a new tube, centrifuging, discarding the precipitate, transferring the supernatant to an ultracentrifuge tube, and centrifuging to precipitate the cell membrane component; The precipitate was resuspended in lysis buffer and shaken to fully dissolve the membrane protein; ultrasonic treatment was performed in an ice bath to break the membrane structure and release the receptor protein; centrifugation was performed to collect the supernatant containing the dissolved BRs receptor protein; the mass concentration of the BRs receptor was measured using Nanodrop2000.

9. A kit for quantitative detection of rice BRs receptors, characterized in that: Comprising the brassinolide-derived carbon quantum dots according to claim 4.

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