Phosphorylation regulation screening method for kinase recognition and function verification in plant triterpenoid metabolism regulation path
By using site-directed mutagenesis and co-expression systems of the Ser/Thr-Pro sites of rate-limiting enzymes in plant triterpenoid biosynthesis, combined with metabolite detection and reporter genes, the problems of poor species adaptability and weak functional correlation in existing kinase screening technologies have been solved, enabling high-throughput and high-precision kinase screening and functional verification.
Patent Information
- Application Number
- CN202511306553.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2026-01-09
AI Technical Summary
Existing kinase screening technologies suffer from poor species adaptability, weak functional correlation, and insufficient throughput and precision in plant triterpenoid metabolism research, making it difficult to achieve efficient screening and verification of kinase functions.
By site-directed mutagenesis of the conserved Ser/Thr-Pro site of the rate-limiting enzyme in plant triterpenoid biosynthesis, a mutant expression vector was constructed and co-expressed with candidate kinases in plant cells. Combined with triterpenoid metabolite detection and reporter gene systems, multi-dimensional data integration and analysis were performed to achieve high-throughput kinase screening and functional verification.
This method improves the species specificity and functional relevance of kinase screening, increases screening throughput and accuracy, ensures that the screened kinases can actually participate in metabolic regulation in plants, and provides an efficient kinase screening method.
Smart Images
Figure CN121294494A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of plant molecular biology, metabolic engineering and protein function screening, and particularly relates to a phosphorylation regulation screening method for kinase recognition and function verification in a plant triterpenoid metabolic regulation pathway. BACKGROUND
[0002] As an important component of plant secondary metabolic network, triterpenoids have become one of the core directions in the field of natural product research due to their rich biological activities and wide application scenarios. This kind of compounds are widely distributed in Chinese herbal medicines, economic crops and woody oil plants, and have shown irreplaceable value in the fields of anti-tumor drug research and development, functional food formulation and natural cosmetic raw materials, etc. due to their multiple physiological functions such as antioxidant, anti-inflammatory and immune regulation. For example, squalene, as a key intermediate product of triterpenoid synthesis pathway, is not only an important substance for maintaining the stability of cell membrane, but also a core ingredient of high-end skin care products due to its strong antioxidant properties; triterpenoid saponins such as glycyrrhizic acid are the main active substances of Chinese herbal medicine licorice for exerting anti-inflammatory and liver-protecting effects, and have been widely used in clinical treatment of liver diseases.
[0003] The biosynthesis of triterpenoids depends on a complex network of enzymatic reactions. As the first rate-limiting enzyme of the mevalonate pathway, hydroxymethylglutaryl coenzyme A reductase (HMGR) directly determines the synthesis efficiency of triterpenoid precursors by catalyzing the conversion of hydroxymethylglutaryl coenzyme A to mevalonate, and is a key target for regulating triterpenoid accumulation. Studies have shown that the activity of rate-limiting enzymes such as HMGR is not only regulated at the transcriptional level, but also finely regulated by post-translational modification, among which protein phosphorylation modification is the most critical regulatory mode. Specifically, the phosphorylation state of the conserved Ser / Thr-Pro site in the HMGR molecule can significantly affect its catalytic efficiency and intracellular localization by changing the spatial conformation of the enzyme protein - the phosphorylated active site can increase the enzyme activity by several times, driving the metabolic flow to triterpenoid synthesis; while the dephosphorylated inhibited site will cause significant decrease in enzyme activity, causing accumulation of precursor substances to be blocked.
[0004] It is currently known that members of the mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK) families may be involved in the phosphorylation regulation of these sites, but there are still many scientific gaps in terms of which kinases are responsible for the modification of specific sites, the functional differentiation mechanism of different kinases, and how the phosphorylation signal responds to environmental stress to regulate triterpenoid synthesis.
[0005] The existing kinase screening technology cannot meet the specific needs of plant triterpenoid metabolism research, mainly having three limitations: first, the model species adaptability is poor, most of the screening relies on yeast two-hybrid or mammalian cell system, lacking the plant-specific metabolic background and signal network, leading to the screened kinases often cannot play the expected function in plants; second, the functional correlation is weak, the traditional method is based on protein interaction screening, ignoring the functional regulation of kinases on target enzymes, resulting in a large number of candidate kinases cannot be verified by metabolic function; third, the throughput and precision are insufficient, relying on low-throughput detection methods such as Western blot, it is difficult to realize the system screening of large-scale kinase library, and the single signal output cannot distinguish the activation / inhibition effect of kinases, the screening precision is low.
[0006] Therefore, it is urgent to develop a phosphorylated kinase screening system with target enzyme specificity, quantifiable signal response and high screening efficiency, in order to promote the analysis and application development of plant natural product synthesis regulation mechanism. SUMMARY
[0007] In view of the above shortcomings, the present application provides a phosphorylation regulation screening method for kinase identification and function verification in plant triterpenoid metabolism regulation pathway, which solves the problems of poor species adaptation, weak functional correlation and low throughput and precision of the existing kinase screening.
[0008] To achieve the above technical purposes, the present application adopts the following technical solutions:
[0009] A phosphorylation regulation screening method for kinase identification and function verification in plant triterpenoid metabolism regulation pathway, comprising the following steps:
[0010] (1) performing site-directed mutagenesis on the conserved Ser / Thr-Pro site in the plant triterpenoid biosynthesis rate-limiting enzyme to construct a mutant expression vector simulating one or both of phosphorylation and dephosphorylation;
[0011] (2) co-expressing the mutant of step (1) and the candidate kinase in plant cells to screen protein kinases with phosphorylation regulation function;
[0012] (3) detecting the change of triterpenoid target metabolites in the expression system to evaluate the regulation effect of the kinase on the function of the target enzyme;
[0013] (4) rapidly screening the functional response of the regulation factor by means of the reporter gene system;
[0014] (5) performing integrated analysis based on multi-dimensional data to realize high-throughput kinase screening and function classification.
[0015] Preferably, the plant triterpenoid biosynthesis rate-limiting enzyme in step (1) is hydroxymethylglutaryl coenzyme A reductase.
[0016] Preferably, in step (1), pCAMBIA or pBI121 is used as the vector backbone for constructing the mutant expression vector.
[0017] Preferably, in step (1), the conserved Ser / Thr-Pro site includes one or both of Ser47 and Thr208.
[0018] Preferably, in step (1), the site-directed mutation is performed using PCR technology.
[0019] Preferably, in step (2), the candidate kinase includes at least one of MAPK, SnRK, and CDPK.
[0020] Preferably, in step (2), the plant cell includes one or more of plant protoplast, plant callus cell, and plant leaf epidermal cell.
[0021] Preferably, in step (3), the change in the target triterpenoid metabolite in the expression system is detected using one or both of gas chromatography-mass spectrometry analysis and liquid chromatography-mass spectrometry analysis.
[0022] Preferably, in step (4), the reporter gene includes one or more of luciferase gene, green fluorescent protein gene (GFP), and β-galactosidase gene.
[0023] Preferably, in step (5), the multi-dimensional data includes one or both of metabolite level and fluorescence signal.
[0024] The mechanism of each preparation step is as follows:
[0025] (1) Mechanism of site-directed mutation of conserved Ser / Thr-Pro site and construction of mutant expression vector
[0026] The activity regulation of plant triterpene biosynthesis rate-limiting enzyme depends on the dynamic balance of phosphorylation of the conserved Ser / Thr-Pro site. Phosphorylation of Ser / Thr residues can change the protein conformation by introducing a negative charge, enhancing its binding capacity with substrates or coenzymes; and dephosphorylation can cause conformational relaxation through charge neutralization, inhibiting catalytic activity.
[0027] In this step, the site-directed mutation technology is used to mutate Ser to Asp or Ala (simulating dephosphorylation and eliminating the phosphorylation site), and a mutant expression vector is constructed. The vector backbone provides stable replication and expression elements, the 35S / Ubi promoter drives high-efficiency transcription of the mutant, and the FLAG / His tag facilitates protein localization and detection. The core mechanism is to lock the phosphorylation state of the enzyme protein by site-directed mutation, excluding the interference of endogenous kinases, and to lay a controllable target enzyme background for subsequent screening of specific regulatory kinases.
[0028] (2) Mechanism of action of co-expression of mutants and candidate kinases
[0029] The candidate kinase catalyzes phosphorylation modification by recognizing the conserved Ser / Thr-Pro motif of the target enzyme, catalyzing the transfer of the γ-phosphate group of ATP to the target enzyme residue. This step utilizes the transient expression characteristics of plant protoplasts or transient transformation systems to enable the mutant and the candidate kinase to be translated synchronously within the same cell.
[0030] The dual promoter system (35S-driven mutant and Ubi-driven kinase) ensures that the expression sequence of the two is matched, avoiding distortion of regulatory signals due to expression differences. The core principle of the co-expression system is to reconstruct the in vivo interaction environment of the kinase-target enzyme, enabling the candidate kinase to functionally regulate the simulated phosphorylation / dephosphorylation sites of the mutant, thereby distinguishing the regulatory activity of the kinase through subsequent detection.
[0031] (3) Mechanism of action for the detection of triterpenoid metabolites
[0032] The accumulation level of triterpenoid metabolites is a direct phenotypic output of target enzyme activity and kinase regulatory function. After the rate-limiting enzymes such as HMGR are activated, the flux of the mevalonate pathway increases, and squalene is generated by squalene synthase. The changes in its content can be quantified by selected ion monitoring mode of gas chromatography-mass spectrometry (GC-MS) / liquid chromatography-mass spectrometry (LC-MS).
[0033] GC-MS separates different terpenoid compounds using gas chromatography and achieves precise quantification by combining it with characteristic ions from mass spectrometry; LC-MS is suitable for highly polar triterpenoid saponins, and improves detection sensitivity through retention time and multiple reaction monitoring (MRM). The mechanism of this step is based on a cascade response of "enzyme activity-metabolic flux-product accumulation," which converts the phosphorylation regulatory signal of kinases into quantifiable differences in metabolite concentrations, providing objective physiological indicators for evaluating kinase function.
[0034] (4) Mechanism of action of reporter gene system
[0035] Reporter genes translate kinase regulatory effects into visual signals by forming fusion proteins with or co-expressing target enzymes. The fluorescence intensity of GFP is positively correlated with the expression level of the target enzyme, and its nuclear localization signal (NLS) can indicate changes in the intracellular localization of the target enzyme; luciferase produces bioluminescence by catalyzing the oxidation of luciferin, and the signal intensity is linearly related to enzyme activity.
[0036] The core principle of this system is to utilize the signal amplification effect of reporter genes to convert weak phosphorylation regulatory signals into easily detectable optical signals, thereby achieving high-throughput primary screening.
[0037] (5) The mechanism of multi-dimensional data integration and analysis
[0038] Metabolite levels and fluorescence signals form a multi-dimensional chain of evidence that mutually validates each other. Metabolite data reflect long-term regulatory effects, while fluorescence signals reflect transient changes in enzyme activity; correlation analysis between the two can rule out non-specific regulation.
[0039] Data analysis employed Principal component analysis (PCA) using R / Python to differentiate metabolic profiles among different regulatory groups, and combined this with a pre-defined threshold model to achieve automated screening. The principle behind this approach is to reduce the false positive rate of a single indicator through multivariate statistics. For example, kinases with strong fluorescence signals but no change in metabolites may only affect the target enzyme's localization without altering its activity. Therefore, through integrated analysis, precise classification of kinase functions can be achieved.
[0040] Compared with the prior art, the present invention has the following technical advantages:
[0041] I. Targeted Breakthroughs in Plant-Specific Metabolic Regulation Bottlenecks
[0042] Existing kinase screening systems mostly rely on animal or yeast models. Due to the lack of plant-specific metabolic background and signaling networks, the screened kinases often fail to perform their intended functions in plants. This invention uses the rate-limiting enzymes of plant triterpenoid biosynthesis as the core target, focusing on their conserved Ser / Thr-Pro phosphorylation sites. Through site-directed mutagenesis simulating phosphorylation / dephosphorylation states, it precisely constructs plant-specific kinase targets. This design directly links to the core regulatory nodes of plant triterpenoid metabolic pathways, fundamentally solving the problem of poor species adaptability and ensuring that the screened kinases can truly participate in the regulation of endogenous metabolism in plants.
[0043] II. Functional relevance enables a closed loop from "interaction screening" to "metabolic verification".
[0044] Traditional methods often screen kinases based on protein-protein interactions, neglecting the functional regulation of target enzymes by kinases, resulting in many candidate kinases failing metabolic function verification. This invention innovatively combines a mutant-candidate kinase co-expression system, triterpenoid metabolite detection, and reporter gene signal output to form a complete evidence chain of "molecular interaction-enzyme activity regulation-metabolic response." By directly verifying the functional effects of kinases through changes in metabolite levels, rather than relying solely on protein-protein interactions, the functional reliability of screening results is significantly improved, effectively addressing the technical challenge of weak functional correlation.
[0045] III. Synergistic Optimization of High Throughput and Precision Screening
[0046] Existing technologies rely on low-throughput detection methods such as Western blot, making it difficult to achieve systematic screening of large-scale kinase libraries. Furthermore, single signal outputs cannot distinguish between kinase activation and inhibition effects. This invention employs a dual-promoter co-expression system to achieve simultaneous and efficient expression of mutants and candidate kinases. Combined with a high-throughput 96-well plate cell system and multi-dimensional data integration analysis, it significantly improves screening throughput. Simultaneously, through a pre-set threshold model and multivariate statistical analysis, it can accurately distinguish the direction and intensity of kinase regulation, solving the problems of low throughput and insufficient precision in traditional methods.
[0047] IV. Universality and scalability of the technical system
[0048] This invention presents a screening system based on HMGR, which can be extended to other key enzymes in triterpenoid synthesis and phosphorylation regulatory sites, making it suitable for metabolic studies in different plant species. Its modular design (mutant construction - co-expression - signal detection) allows for flexible adaptation to different research needs, providing a general technical framework for elucidating the phosphorylation regulatory network of plant natural product synthesis and promoting the upgrade of metabolic engineering from empirical regulation to precise design. Attached Figure Description
[0049] Figure 1 This is a structural diagram of the optimized expression vector with functional mutation of the present invention;
[0050] Figure 2 This is a diagram illustrating the dual promoter co-expression system and fluorescence feedback mechanism in plant cells according to the present invention.
[0051] Figure 3 This is a diagram illustrating the phosphorylation regulation screening process and platform structure of the present invention;
[0052] Figure 4 This is a quantitative analysis of functional regulation and kinase response diagrams for the present invention. Detailed Implementation
[0053] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.
[0054] A method for screening phosphorylation regulation for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways includes the following steps:
[0055] (1) Site-directed mutagenesis was performed on the conserved Ser / Thr-Pro site in the rate-limiting enzyme of plant triterpenoid biosynthesis to construct expression vectors that mimic one or two of the phosphorylation and dephosphorylation mutants.
[0056] (2) The mutant from step (1) and the candidate kinase were co-expressed in plant cells to screen for protein kinases with phosphorylation regulation function.
[0057] (3) Detect changes in triterpenoid target metabolites in the expression system and evaluate the regulatory effect of kinases on target enzyme function;
[0058] (4) Using reporter gene systems, the functional responses of regulatory factors can be rapidly screened;
[0059] (5) Based on multi-dimensional data integration and analysis, high-throughput kinase screening and functional classification are achieved.
[0060] In step (1), the rate-limiting enzyme for the biosynthesis of plant triterpenoids is hydroxymethylglutaryl-CoA reductase.
[0061] When constructing the mutant expression vector in step (1), one or both of pCAMBIA and pBI121 are used as the vector backbone.
[0062] The conserved Ser / Thr-Pro sites in step (1) include one or both of Ser47 and Thr208.
[0063] In step (1), site-directed mutagenesis was performed using PCR technology.
[0064] The candidate kinases mentioned in step (2) include at least one of MAPK, SnRK, and CDPK.
[0065] The plant cells in step (2) include one or more of the following: plant protoplasts, plant callus cells, and plant leaf epidermal cells.
[0066] When detecting changes in triterpenoid target metabolites in the expression system in step (3), one or two of the following methods may be used: gas chromatography-mass spectrometry (GC-MS) analysis and liquid chromatography-mass spectrometry (LC-MS) analysis.
[0067] The reporter genes in step (4) include one or more of the following: luciferase gene, green fluorescent protein gene, and β-galactosidase gene.
[0068] The multidimensional data in step (5) includes one or both of the following: metabolite levels and fluorescence signals.
[0069] To make the present invention more fully disclosed, more specific embodiments are described below.
[0070] Example 1: Construction of HMGR phosphorylation sites and screening of kinase responses based on site-directed mutagenesis
[0071] This embodiment uses the known HMGR sequence in Camellia oleifera as a basis and employs PCR technology (overlap extension PCR) to perform site-directed mutagenesis on the conserved Ser / Thr-Pro sites (Ser47, Thr208): Ser47→Asp simulates persistent phosphorylation, and Ser47→Ala and Thr208→Ala simulate dephosphorylation. The mutant vectors were cloned into pBI121-GFP and pCAMBIA-His fusion expression vectors, respectively, to construct two mutant expression systems with different backbones (see attached). Figure 1 (As shown in "Optimized expression vector structure with functional mutation").
[0072] The above mutants and candidate kinases (MAPK3, CDPK2, SnRK2.3) were co-expressed in tobacco leaf epidermal cells (transient expression system) using a dual promoter system (Ubi promoter driving the kinases, 35S promoter driving the HMGR mutant and GFP reporter gene). The molecular mechanism of this system is shown in the attached figure. Figure 2 The diagram shows the dual promoter co-expression system and fluorescence feedback mechanism in plant cells of this invention.
[0073] Forty-eight hours later, the phosphorylation status of the target enzyme was detected by Western blotting, and the locational differences of the mutants were analyzed using a dual reporter system of GFP and β-galactosidase. The results showed that MAPK3 exhibited the strongest phosphorylation response to the Ser47 mutant, with a 2.3-fold increase in GFP signal intensity and a 1.8-fold increase in β-galactosidase activity compared to the control group, confirming its positive regulatory effect on this site. The signal change trend was similar to that of the control group. Figure 2 The feedback mechanism shown is consistent.
[0074] Example 2: Construction and Functional Verification of a Coupling Screening System for Squalene Metabolites
[0075] In Camellia oleifera endosperm protoplasts and Arabidopsis thaliana callus cells, the HMGR-Ser47A mutant was co-expressed with candidate kinases (MAPK3, SnRK2, CDPK1). The experimental procedure is detailed in the attached document. Figure 3 The "Transfection → Culture → Detection" step in the "Phosphorylation Regulation Screening Process and Platform Structure Diagram of this Invention".
[0076] Forty-eight hours later, the target triterpenoid metabolites were detected using both GC-MS and LC-MS: GC-MS focused on squalene (characteristic ion m / z 62.1) for quantification, while LC-MS simultaneously analyzed the downstream product 2,3-squalene oxide. The results showed that in the MAPK3 co-expression group, the squalene content was 85% higher than in the non-kinase group (GC-MS data), and the 2,3-squalene oxide content increased by 62% (LC-MS data); while the squalene content in the CDPK1 group decreased by 30%, indicating a negative regulatory effect (metabolite change trends are shown in the attached figure). Figure 4The squalene content curve is shown in the "Quantitative Analysis of Functional Regulation and Kinase Response Diagram of this Invention". The results of the two detection methods showed a consistency of 92%, verifying the reliability of metabolite detection.
[0077] Example 3: An experiment to evaluate the effects of multi-site mutation combinations on triterpenoid metabolism.
[0078] Single mutants (Ser47A, Thr208D) and double mutants (Ser47A+Thr208D) were constructed targeting the Ser47 and Thr208 sites of HMGR. The expression system was constructed using the pBI121 vector backbone, and the vector structure design and association were then implemented. Figure 1 Component layout of the pBI121 skeleton.
[0079] The promoter configuration of the mutant co-expression system with kinase combinations (MAPK3+SnRK1, CDPK2 alone) is associated with the following: Figure 2 The dual promoter design logic monitors transient enzyme activity through a luciferase reporter gene system and combines it with GC-MS to detect squalene accumulation.
[0080] The results showed that when the double mutant was co-expressed with MAPK3+SnRK1, the luciferase activity was 3.1 times that of the control group, and the squalene content was 2.1 times that of the control group. (The data trends are shown in the attached figure.) Figure 4 The multiple comparative curves in the "Quantitative Analysis of Functional Regulation and Kinase Response Map" showed significantly higher regulatory effects than those of single mutants and single kinase combinations, confirming the synergistic regulatory mechanism of multi-site phosphorylation.
[0081] Example 4: Application of High-Throughput Kinase Screening System and Data Analysis Platform
[0082] An expression library containing 50 plant-derived candidate kinases (covering the MAPK, SnRK, and CDPK families) was established. An HMGR-Ser47p-LUC reporter gene system was constructed using pCAMBIA as the vector. Vector element layout association was performed. Figure 1 Design of the pCAMBIA framework. High-throughput screening was performed in a 96-well Arabidopsis protoplast transformation system. The structure and workflow of the screening platform are described in the appendix. Figure 3 "Screen Structure and Flowchart of the Screening System of this Invention", attached Figure 3 The upper part shows the 96-well transfection platform and dual signal acquisition device, while the lower part clearly shows the operation sequence.
[0083] Ten candidate kinases were initially screened using fluorescence signals (luciferase activity), and then squalene levels were detected by GC-MS. The two datasets (fluorescence signal intensity and metabolite content) were integrated for machine learning analysis. The results showed that the fluorescence signals of four kinases (MAPK3, SnRK1, CDPK2, and MAPK6) were significantly positively correlated with squalene content (R0).2 >0.85), the correlation analysis results are attached. Figure 4 The correlation curve between GFP signal and squalene content in the "Quantitative Analysis of Functional Regulation and Kinase Response Diagram of this Invention" shows that MAPK3 has the highest comprehensive regulation score and can be used as a key regulatory factor for squalene synthesis.
[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A method for screening phosphorylation regulation for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways, characterized in that, Includes the following steps: (1) Site-directed mutagenesis was performed on the conserved Ser / Thr-Pro site in the rate-limiting enzyme of plant triterpenoid biosynthesis to construct expression vectors that mimic one or two of the phosphorylation and dephosphorylation mutants. (2) The mutant from step (1) and the candidate kinase were co-expressed in plant cells to screen for protein kinases with phosphorylation regulation function. (3) Detect changes in triterpenoid target metabolites in the expression system and evaluate the regulatory effect of kinases on target enzyme function; (4) Using reporter gene systems, the functional responses of regulatory factors can be rapidly screened; (5) Based on multi-dimensional data integration and analysis, high-throughput kinase screening and functional classification are achieved.
2. The phosphorylation regulation screening method according to claim 1 for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways, characterized in that, In step (1), the rate-limiting enzyme for the biosynthesis of plant triterpenoids is hydroxymethylglutaryl-CoA reductase.
3. The phosphorylation regulation screening method for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways according to claim 1, characterized in that, When constructing the mutant expression vector in step (1), one or both of pCAMBIA and pBI121 are used as the vector backbone.
4. The phosphorylation regulation screening method for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways according to claim 1, characterized in that, The conserved Ser / Thr-Pro sites in step (1) include one or both of Ser47 and Thr208.
5. A phosphorylation regulation screening method for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways according to claim 1, characterized in that, In step (1), site-directed mutagenesis was performed using PCR technology.
6. The phosphorylation regulation screening method for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways according to claim 1, characterized in that, The candidate kinases mentioned in step (2) include at least one of MAPK, SnRK, and CDPK.
7. The phosphorylation regulation screening method for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways according to claim 1, characterized in that, The plant cells in step (2) include one or more of the following: plant protoplasts, plant callus cells, and plant leaf epidermal cells.
8. The phosphorylation regulation screening method for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways according to claim 1, characterized in that, When detecting changes in triterpenoid target metabolites in the expression system in step (3), one or two of the following methods may be used: gas chromatography-mass spectrometry (GC-MS) analysis and liquid chromatography-mass spectrometry (LC-MS) analysis.
9. The phosphorylation regulation screening method for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways according to claim 1, characterized in that, The reporter genes in step (4) include one or more of the following: luciferase gene, green fluorescent protein gene, and β-galactosidase gene.
10. The phosphorylation regulation screening method for kinase recognition and functional verification in plant triterpenoid metabolic regulatory pathways according to claim 1, characterized in that, The multidimensional data in step (5) includes one or both of the following: metabolite levels and fluorescence signals.