Gene detection system for promoting andrographolide synthesis based on biosensor

Abstract

The invention relates to the technical field of gene engineering detection, and particularly discloses a gene detection system for promoting andrographolide synthesis based on a biosensor, a key gene ApDof29 for regulating and controlling andrographolide synthesis is determined through whole genome analysis, gene expression profile research and co-expression network analysis, the core regulation and control effect of ApDof29 is verified, and the gene detection system for promoting andrographolide synthesis based on the biosensor is used for promoting andrographolide synthesis. The invention also develops a real-time dynamic monitoring system, and realizes non-invasive monitoring of andrographolide content accumulation, biosynthesis rate and metabolic intermediate concentration change by utilizing the fusion of a specific response element and a reporter gene. Through comprehensive calculation processing of an accumulation abnormal coefficient, a synthesis rate abnormal coefficient and a concentration change abnormal coefficient, the regulation and control effect of ApDof29 is dynamically evaluated, and a scientific basis is provided for subsequent gene modification.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering detection, and specifically relates to a gene detection system for promoting the synthesis of andrographolide based on a biosensor. Background Art

[0002] Andrographis paniculata is one of the traditional Chinese medicinal materials in China and has effects such as clearing heat and detoxifying. Its main active ingredient, andrographolide, is a structurally complex diterpenoid secondary metabolite with various pharmacological activities such as antibacterial, antiviral, anti-inflammatory, anti-tumor, and immunomodulatory effects, and has important application value in the modern pharmaceutical field, especially in the Chinese patent medicine industry. At present, andrographolide mainly relies on plant extraction. Although there have been research reports on chemical synthesis, due to its complex molecular structure, long synthesis route, and high cost, the artificial synthesis process still faces major challenges in terms of economy and large-scale production. However, the content of andrographolide in wild or cultivated Andrographis paniculata plants is generally low, and problems such as poor extraction efficiency have led to high production costs, seriously restricting its large-scale production and industrial application.

[0003] With the rapid development of molecular biology technology, genetic engineering technology provides a new solution for increasing the content of plant secondary metabolites and reducing production costs. However, the understanding of the regulatory mechanism of the andrographolide biosynthesis pathway is still imperfect, especially the key genes involved in biosynthesis and their regulatory networks have not been fully analyzed, seriously restricting the application prospects of precise regulation and directed modification of andrographolide synthesis. Therefore, in-depth research on the key genes and their regulatory mechanisms of the andrographolide biosynthesis pathway, especially the functional analysis of regulatory elements such as transcription factors, not only helps to clarify the molecular mechanism of andrographolide accumulation, but also provides a theoretical basis and technical support for achieving the directed increase of andrographolide production through means such as molecular breeding and genetic engineering, thus promoting the sustainable development of the andrographolide-related industry. Through molecular biology means such as gene cloning, the present invention found that ApDof29 can regulate the expression of the key enzyme gene ApCPS2 in the andrographolide synthesis pathway, thereby increasing the synthesis and accumulation of andrographolide, indicating the core role of this gene in the andrographolide biosynthesis regulatory network, and providing a key functional gene marker and genetic operation target for Andrographis paniculata molecular breeding.

[0004] The prior art has the following deficiencies: Aiming at the industrial bottleneck of the imperfect transcriptional regulation mechanism of andrographolide biosynthesis and the lack of transcriptional regulatory genes leading to the absence of metabolic engineering transformation targets; The present invention provides a novel ApDof29 transcription factor gene with a significant function of promoting andrographolide accumulation, providing a core gene resource for the directional cultivation of new varieties with high andrographolide content. Summary of the Invention

[0005] The purpose of the present invention is to provide a gene detection system for promoting andrographolide synthesis based on a biosensor to solve the problems in the above background.

[0006] The purpose of the present invention can be achieved by the following technical solutions: A gene detection system for promoting andrographolide synthesis based on a biosensor, comprising: A target gene screening module, which screens and determines the key gene ApDof29 that regulates andrographolide synthesis through whole-genome analysis, gene expression profile research, and co-expression network analysis; A gene function verification module, which constructs overexpression plants and knockout mutants of the ApDof29 gene through transgenic technology, and combines the expression detection of the key enzyme gene ApCPS2 and the determination of andrographolide content to verify whether ApDof29 plays a core role in andrographolide synthesis; A real-time monitoring module, which constructs a dynamic monitoring system based on a biosensor based on the core role verification result, real-time monitors the accumulation of andrographolide content, the biosynthesis rate, and the concentration changes of metabolic intermediates, and dynamically evaluates the regulatory effect of ApDof29 on andrographolide synthesis according to the monitoring results and data analysis; A gene function modification and optimization module, which optimizes the function of the ApDof29 gene through directed evolution methods and increases the content of andrographolide.

[0007] As a further aspect of the present invention: The screening and determination of the key gene ApDof29 that regulates andrographolide synthesis specifically includes: Annotate the whole genome of Andrographis paniculata and manually screen out specific gene family members involved in secondary metabolite synthesis, including cytochrome P450 enzymes and transcription factors, identify sequences highly similar to known andrographolide synthesis-related genes through homology search as candidate genes; screen differentially expressed genes, based on the obtained list of differentially expressed genes, establish a gene co-expression network, calculate the betweenness centrality value and closeness centrality value of nodes, and rank all genes according to the calculated betweenness centrality value and closeness centrality value; select genes with both betweenness centrality value and closeness centrality value higher than the network average as candidates for key regulatory genes, and if the initially screened candidates promote andrographolide accumulation, finally lock in the screened candidate ApDof29 as the key gene that regulates andrographolide synthesis.

[0008] As a further solution of the present invention: the calculation process of the betweenness centrality value and the closeness centrality value is as follows: The calculation process of the betweenness centrality value is as follows: For each node in the constructed gene co-expression network, its betweenness centrality value is calculated by a graph theory algorithm. Specifically: for each pair of nodes and all the shortest paths between them are counted. If the node appears on these paths, the count of its betweenness centrality value is increased. According to the sum of the ratios of the number of paths passing through the node to the number of all shortest paths, the betweenness centrality value is obtained; The calculation process of the closeness centrality value is as follows: Based on the gene co-expression network, a graph theory algorithm is used to calculate the closeness centrality value of each node. Specifically: by calculating the reciprocal of the average shortest path length from the node to all other nodes in the network, the closeness centrality value is obtained.

[0009] As a further solution of the present invention: to verify whether ApDof29 plays a core role in andrographolide synthesis, specifically includes: First, overexpression plants and knockout mutants of the ApDof29 gene are constructed respectively by transgenic technology. In the overexpression plants, the expression of ApDof29 is driven by a strong promoter to increase its level in the plant; while in the knockout mutants, the CRISPR gene editing tool is used to precisely delete the ApDof29 gene. In these two transgenic plants and the wild-type control, the qRT-PCR technology is used to detect the expression level of the key enzyme gene ApCPS2 involved in the andrographolide biosynthesis pathway, and the high performance liquid chromatography method is used to quantitatively determine the content of andrographolide. If an up-regulation of ApCPS2 expression and an increase in andrographolide content are observed in the ApDof29 overexpression plants, while a down-regulation of ApCPS2 expression and a decrease in andrographolide content are observed in the knockout mutants, it is confirmed that ApDof29 has a core regulatory role in andrographolide synthesis.

[0010] As a further solution of the present invention: the dynamic evaluation of the regulatory effect of ApDof29 on andrographolide synthesis specifically includes: According to the dynamic monitoring system of the biosensor, accumulate data on the content of andrographolide, data on the biosynthesis rate, and data on the concentration of metabolic intermediates in real time, analyze the collected real-time data, calculate the accumulation anomaly coefficient, the synthesis rate anomaly coefficient, and the concentration change anomaly coefficient according to the analysis results, perform comprehensive calculation and processing on the accumulation anomaly coefficient, the synthesis rate anomaly coefficient, and the concentration change anomaly coefficient, calculate the synthesis regulation coefficient through the comprehensive calculation expression, and determine whether the synthesis regulation coefficient is greater than or equal to the preset threshold. If so, the regulation effect of ApDof29 on the synthesis of andrographolide is unqualified; if not, the regulation effect of ApDof29 on the synthesis of andrographolide is qualified.

[0011] As a further solution of the present invention: the process for obtaining the accumulation anomaly coefficient is as follows: Accumulate data on the content of andrographolide in real time and organize it into a matrix. Standardize the accumulated data on the content of andrographolide collected in real time to obtain a standardized matrix. Based on the standardized accumulated data on the content of andrographolide, calculate its covariance matrix. Perform eigenvalue decomposition on the covariance matrix to obtain eigenvalues and the corresponding eigenvectors ; sort the eigenvalues from largest to smallest; select the first important eigenvalues and their corresponding eigenvectors as the main components according to the cumulative contribution rate of the eigenvalues; project the accumulated data on the content of andrographolide onto the selected main components to obtain a new coordinate matrix, and calculate the sum of the distances between all the accumulated data on the content of andrographolide and the data center in the principal component space to obtain the accumulation anomaly coefficient.

[0012] As a further solution of the present invention: the process for obtaining the synthesis rate anomaly coefficient is as follows: Obtain the time series data of the biosynthesis rate, preprocess the time series data to ensure that the data is continuous and has no missing values; calculate the fluctuation series between adjacent time points; determine a series of scale parameters, and for each scale parameter, divide the fluctuation series into segments of corresponding lengths and count the number of non-empty boxes; based on the relationship between the number of non-empty boxes and the scale parameters at different scales, estimate the fractal dimension through a linear regression model; obtain the synthesis rate anomaly coefficient according to the absolute value of the difference between the estimated fractal dimension and the fractal dimension in the expected normal state.

[0013] As a further solution of the present invention: the process for obtaining the concentration change anomaly coefficient is as follows: Obtain time series data of metabolite intermediate concentrations; preprocess the time series data to ensure the data is continuous and has no missing values; decompose the preprocessed data using Haar wavelet transform to obtain a series of approximation coefficients and detail coefficients; calculate the energy of the detail coefficients at each Haar wavelet transform level, and calculate the energy ratio of each level based on the sum of the energies of all Haar wavelet transform levels; compare the calculated energy ratio with a preset threshold to determine which fluctuations at the Haar wavelet transform levels are considered abnormal; based on the sum of the ratios exceeding the set threshold, obtain the concentration change anomaly coefficient.

[0014] As a further aspect of the present invention: The directed evolution method optimizes the function of the ApDof29 gene, specifically including: Create a diverse gene library, achieved through saturation mutagenesis technology, aiming to introduce a large number of random mutations to cover a wide sequence space, use high-throughput screening to evaluate the regulatory effect of each variant on andrographolide synthesis, select mutants with performance superior to the wild type from them, perform multiple rounds of iterative optimization on the initially selected high-quality mutants, repeat the processes of mutation and screening until the target variant that meets the expected functional improvement is obtained.

[0015] As a further aspect of the present invention: The increase in andrographolide content specifically includes: Regulate the key steps in the andrographolide biosynthesis pathway through metabolic engineering strategies, specifically including enhancing the precursor supply of isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the acetate-mevalonate pathway, overexpressing the diterpene cyclase gene responsible for diterpene backbone formation to improve the production efficiency of the diterpene backbone; optimizing the expression of key enzyme genes involved in the oxidation modification and subsequent structural modification steps, including P450 monooxygenase and methyltransferase; by overexpressing the ApDof29 gene, enhancing its positive regulatory effect on genes related to andrographolide synthesis, and iteratively optimizing the regulatory strategy to ultimately achieve an increase in andrographolide content.

[0016] Advantages of the present invention: (1) By precisely screening and identifying the key gene ApDof29 that regulates andrographolide synthesis, and optimizing its function using directed evolution methods, the present invention not only significantly enhances the positive regulatory effect of ApDof29 on genes related to andrographolide synthesis, but also comprehensively optimizes the andrographolide biosynthesis pathway through a series of fine-tuning strategies. Specifically, we used metabolic engineering means to overexpress the enzymes responsible for diterpene backbone formation and optimized the expression of key enzyme genes including P450 monooxygenase and methyltransferase. These measures together promoted the supply of andrographolide precursors, improved the conversion efficiency of intermediate products, and increased the accumulation of the final product. In addition, based on the constructed biosensor dynamic monitoring system, real-time monitoring and feedback adjustment of the andrographolide synthesis process were achieved, ensuring the efficient operation of each step. This comprehensive strategy not only greatly increases the yield of andrographolide, but also provides solid technical support for the development of new varieties rich in andrographolide, demonstrating great potential and broad application prospects in the improvement of medicinal plants and the high-efficiency production of natural products.

[0017] (2) The present invention innovatively constructs a dynamic monitoring system based on a biosensor, achieving real-time and precise monitoring of the accumulation of andrographolide content, the biosynthesis rate, and the changes in the concentration of metabolic intermediates. This system ingeniously fuses specific response elements with reporter genes (such as fluorescent proteins), enabling sensitive and continuous tracking of ApDof29 gene expression and its specific impact on the andrographolide biosynthesis pathway under in vivo conditions, providing a non-invasive research method. By calculating and analyzing the abnormal coefficient of accumulation, the abnormal coefficient of synthesis rate, and the abnormal coefficient of concentration change, we can dynamically evaluate the regulatory effect of ApDof29 in andrographolide synthesis and accordingly guide subsequent gene modification and optimization work. This integrated real-time monitoring and precise regulation platform not only significantly improves the experimental efficiency and data accuracy, but also provides strong technical support for in-depth analysis of the molecular mechanism of andrographolide biosynthesis. In addition, the application of this system promotes the transformation from basic research to actual production, demonstrating its great potential and broad application prospects in increasing the yield of active ingredients in medicinal plants, and is of great significance for promoting the development of synthetic biology of natural products. Brief Description of the Drawings

[0018] The present invention will be further described below with reference to the accompanying drawings.

[0019] Figure 1 is the flowchart of the gene detection system for promoting andrographolide synthesis based on a biosensor of the present invention; Figure 2 is the mind map schematic diagram of the gene detection system for promoting andrographolide synthesis based on a biosensor of the present invention. Detailed Embodiments

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the protection scope of the present invention.

[0021] Example 1. Refer to Figure 1 As shown, the present invention is a gene detection system for promoting the synthesis of andrographolide based on a biosensor, including: A screening module for target genes. The screening module for target genes screens and determines the key gene ApDof29 that regulates the synthesis of andrographolide through whole-genome analysis, gene expression profile research, and co-expression network analysis; A gene function verification module. The gene function verification module constructs overexpression plants and knockout mutants of the ApDof29 gene through transgenic technology, and combines the expression detection of the key enzyme gene ApCPS2 and the determination of andrographolide content to verify whether ApDof29 plays a core role in the synthesis of andrographolide; A real-time monitoring module. Based on the core role verification result, the real-time monitoring module constructs a dynamic monitoring system based on a biosensor to monitor the accumulation of andrographolide content, the biosynthesis rate, and the concentration change of metabolic intermediates in real time, and dynamically evaluates the regulatory effect of ApDof29 on the synthesis of andrographolide according to the monitoring results and data analysis; A gene function modification and optimization module. The gene function modification and optimization module optimizes the function of the ApDof29 gene through directed evolution methods and increases the content of andrographolide.

[0022] Example 2. Refer to Figure 2 As shown, in the screening module for target genes, the key gene ApDof29 that regulates the synthesis of andrographolide is screened and determined through whole-genome analysis, gene expression profile research, and co-expression network analysis, specifically including: The whole genome of Andrographis paniculata was exhaustively annotated, and specific gene family members involved in the synthesis of secondary metabolites were manually screened out, including cytochrome P450 enzymes and transcription factors. Through strict homology search (using the BLAST tool with an E-value set less than 1e-10), sequences highly similar to the known genes related to andrographolide synthesis were identified as candidate genes. Experiments were designed to collect samples of Andrographis paniculata at different growth stages for RNA-seq sequencing. The DESeq2 tool was used to set strict statistical thresholds to screen for differentially expressed genes and analyze their temporal dynamics to verify the relevance of candidate genes. Based on the list of differentially expressed genes obtained by screening, the weighted gene co-expression network analysis method was used to construct a gene co-expression network. After optimizing the soft threshold parameter, highly connected modules were identified, and functional enrichment analysis was performed on the genes within each module. The betweenness centrality value and closeness centrality value indicators of the nodes were calculated, and all genes were ranked according to the calculated betweenness centrality value and closeness centrality value. Genes with both betweenness centrality value and closeness centrality value higher than the network average were selected as candidates for key regulatory genes. If the initially screened candidates could promote andrographolide accumulation, ApDof29 was finally locked as the key gene regulating andrographolide synthesis.

[0023] Among them, the network average is the mean of all betweenness centrality values and the mean of all closeness centrality values.

[0024] Among them, the calculation process of the betweenness centrality value is as follows: For each node (i.e., gene) in the constructed gene co-expression network, its betweenness centrality value is calculated through graph theory algorithms, and this value represents the frequency of a node appearing in all shortest paths. The specific calculation method is to count all the shortest paths between each pair of nodes and . If the node appears on these paths, the count of its betweenness centrality value is increased. The calculation expression of the betweenness centrality value is: ; In the formula, represents the betweenness centrality value of node , represents the number of all shortest paths from node to node , represents the number of all shortest paths from node to node that pass through node , , and represent the nodes in the gene co-expression network; Among them, the calculation process of the closeness centrality value is as follows: Based on the gene co-expression network, a graph theory algorithm is used to calculate the closeness centrality value of each node. This value reflects the reciprocal of the average shortest path length from the node to all other nodes in the network and measures the reachability of the node in the network. The calculation expression of the closeness centrality value is: ; In the formula, represents the shortest path distance between node and node , represents the closeness centrality value of node , represents the set of all nodes in the network.

[0025] It should be noted that: the betweenness centrality value measures the importance of a gene as a "bridge" in the network, and genes with high betweenness centrality values are key points on the signal transmission path. The closeness centrality value: reflects the reachability of a gene in the network, and genes with high closeness centrality values can quickly exchange information with other genes or affect other genes. This series of steps not only provides a comprehensive research strategy from the whole genome level to specific functional verification, but also ensures the uniqueness and accuracy of the results through detailed data analysis and the application of bioinformatics tools.

[0026] In the gene function verification module, overexpression plants and knockout mutants of the ApDof29 gene are constructed through transgenic technology, and combined with the detection of the expression of the key enzyme gene ApCPS2 and the determination of the andrographolide content, to verify whether ApDof29 plays a core role in andrographolide synthesis. Specifically, it includes: First, overexpression plants and knockout mutants of the ApDof29 gene are constructed through transgenic technology respectively. In the overexpression plants, the expression of ApDof29 is driven by a strong promoter to increase its level in the plant; while in the knockout mutants, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing tool is used to precisely delete the ApDof29 gene. Subsequently, in these two transgenic plants and the wild-type control, qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction) technology is used to detect the expression level of the key enzyme gene ApCPS2 (involved in the andrographolide biosynthesis pathway), and high performance liquid chromatography is used to quantitatively determine the andrographolide content. If an up-regulation of ApCPS2 expression and an increase in andrographolide content are observed in the ApDof29 overexpression plants, while a down-regulation of ApCPS2 expression and a decrease in andrographolide content are observed in the knockout mutants, then it can be confirmed that ApDof29 has a core regulatory role in andrographolide synthesis.

[0027] It should be noted that: By constructing overexpression plants and knockout mutants of the ApDof29 gene, and using a strong promoter to enhance the expression of ApDof29 and the CRISPR technology to precisely delete this gene, combined with qRT-PCR to detect the change in the expression level of the key enzyme gene ApCPS2 and high-performance liquid chromatography to determine the content of andrographolide, the core regulatory role of ApDof29 in andrographolide synthesis was systematically verified. The experimental results showed that the expression of ApCPS2 was up-regulated and the content of andrographolide increased significantly in the ApDof29 overexpression plants, while the opposite trend was presented in the knockout mutants. This not only revealed the key role of ApDof29 in regulating andrographolide biosynthesis, but also provided a solid theoretical basis and technical support for the development of new varieties with high-yield andrographolide based on this mechanism. This discovery enhanced the understanding of the regulatory network of specific metabolic pathways and demonstrated important technological innovation and application prospects.

[0028] In the real-time monitoring module, based on the core role verification results, the real-time monitoring module constructs a dynamic monitoring system based on biosensors to real-time monitor the accumulation of andrographolide content, the biosynthesis rate and the concentration change of metabolic intermediates, and dynamically evaluate the regulatory effect of ApDof29 on andrographolide synthesis according to the monitoring results and data analysis, specifically including: Based on the confirmation that ApDof29 has a core regulatory role in andrographolide synthesis, a dynamic monitoring system based on biosensors is constructed. Using synthetic biology methods, a biosensor that can real-time and sensitively reflect the expression level of the ApDof29 gene and its impact on andrographolide biosynthesis is designed and constructed. Specifically, by fusing a specific response element with a reporter gene (such as a fluorescent protein) and introducing it into target plant cells, the biosensor can specifically sense ApDof29, and then activate the expression of the reporter gene, generating a quantifiable fluorescent signal or other easily detectable phenotypic changes. In this way, researchers can dynamically track the expression of ApDof29 and the related activities of the andrographolide biosynthesis pathway under in vivo conditions through simple optical measurement means, such as fluorescence microscopy, to achieve non-invasive and continuous monitoring of the target metabolic process.

[0029] It should be noted that: A specific response element refers to a molecular component that can respond to a specific stimulus or condition within a cell and convert this response into a detectable signal. In the construction of a dynamic monitoring system based on biosensors, a specific response element refers to a DNA sequence and protein that can specifically recognize and bind to a target molecule (such as the ApDof29 gene product).

[0030] According to the dynamic monitoring system of biosensors, accumulate data on the content of andrographolide, data on the biosynthesis rate, and data on the concentration of metabolic intermediates in real time. Analyze the real-time data collected. According to the analysis results, calculate the accumulation anomaly coefficient, the synthesis rate anomaly coefficient, and the concentration change anomaly coefficient. Conduct comprehensive calculation and processing on the accumulation anomaly coefficient, the synthesis rate anomaly coefficient, and the concentration change anomaly coefficient. Through the comprehensive calculation expression, calculate the synthesis regulation coefficient. Determine whether the synthesis regulation coefficient is greater than or equal to the preset threshold. If so, the regulation effect of ApDof29 on andrographolide synthesis is unqualified. If not, the regulation effect of ApDof29 on andrographolide synthesis is qualified.

[0031] The process of obtaining the accumulation anomaly coefficient is as follows: Accumulate data on the content of andrographolide in real time and organize it into a matrix. Among them, each row of the matrix represents a sample, and each column represents the content of andrographolide at different time points or under different conditions. Standardize the accumulated data on the content of andrographolide collected in real time so that each feature has a zero mean and a unit variance. Calculate its covariance matrix based on the standardized accumulated data on the content of andrographolide. The calculation expression is: ; In the formula, represents the covariance matrix, represents the number of accumulated data on the content of andrographolide collected, represents the standardized accumulated data on the content of andrographolide, represents the transpose operation of the matrix; perform eigenvalue decomposition on the covariance matrix to obtain eigenvalues and the corresponding eigenvectors ; These eigenvalues represent the variance size of the data in the corresponding directions, while the eigenvectors define these directions. The eigenvalues are sorted from largest to smallest to determine which principal components are the most important; select the first important eigenvalues and their corresponding eigenvectors as the main components. When the cumulative contribution rate reaches 80% - 95%, they are important eigenvalues; project the original data onto the selected main components to obtain a new coordinate matrix. Specifically: Map the data from the original high-dimensional space to the new space composed of the main components, simplifying the data structure while retaining as much information as possible; for each accumulated data on the content of andrographolide, calculate the sum of the distances between all accumulated data on the content of andrographolide and the data center in the principal component space to obtain the accumulation anomaly coefficient. The calculation expression is: ; In the formula, represents the th accumulation anomaly coefficient of the accumulated data on the content of andrographolide, Indicates the number of selected eigenvalue, Indicates the th selected eigenvalue, Indicates the coordinate matrix, Indicates the mean of all coordinate matrices, Indicates the th accumulation data of andrographolide content.

[0032] The process for obtaining the abnormal coefficient of the synthesis rate is as follows: Obtain the time series data of the biosynthesis rate, preprocess the time series data to ensure the data is continuous and has no missing values; calculate the fluctuation series between adjacent time points to capture the changes in the synthesis rate; determine a series of scale parameters, for each scale parameter, divide the fluctuation series into segments of corresponding lengths and count the number of non-empty boxes; based on the relationship between the number of non-empty boxes and the scale parameter at different scales, estimate the fractal dimension through a linear regression model; obtain the abnormal coefficient of the synthesis rate according to the absolute value of the difference between the estimated fractal dimension and the fractal dimension in the expected normal state; Among them, for each selected scale parameter, use the box-counting method to divide the fluctuation series and count the number of non-empty boxes; the calculation logic of the box-counting method is: ; In the formula, Indicates a series of selected scale parameters, Indicates the number of non-empty boxes, Indicates the collected data points, Indicates the total number of collected data points, Indicates the indicator function, which returns 1 when the condition is met and 0 otherwise.

[0033] Among them, the fractal dimension is estimated through the following linear regression model: ; In the formula, Is the constant term, perform a linear regression on and at different scales, and the slope is the fractal dimension.

[0034] The process for obtaining the abnormal coefficient of the concentration change is as follows: Obtain the time series data of the concentration of the metabolic intermediate; preprocess the time series data to ensure the data is continuous and has no missing values; use the Haar wavelet transform to decompose the preprocessed data to obtain a series of approximation coefficients and detail coefficients; calculate the energy of the detail coefficients at each Haar wavelet transform level and calculate the energy ratio of each level according to the sum of the energies of all Haar wavelet transform levels; compare the calculated energy ratio with the preset threshold to determine which fluctuations at the Haar wavelet transform levels are considered abnormal; obtain the abnormal coefficient of the concentration change based on the sum of the ratios exceeding the set threshold.

[0035] Among them, the Haar wavelet transform is used to decompose the time series data into approximation coefficients of different levels through a recursive method. For each Haar wavelet transform level, the energy of the detail coefficients is calculated, and the calculation expression is: ; represents the th Haar wavelet transform level, represents the maximum transform level of the Haar wavelet transform, represents the th detail coefficient of the th metabolite intermediate concentration number at the th Haar wavelet transform level, represents the number of metabolite intermediate concentration data at each Haar wavelet transform level, represents the energy of the

[0036] The comprehensive calculation expression is: ; In the formula, , and are preset proportionality factors, and , and sum to 1, represents the synthetic regulation coefficient, represents the accumulation anomaly coefficient, represents the synthetic rate anomaly coefficient, represents the concentration change anomaly coefficient.

[0037] In the gene function modification and optimization module, the function of the ApDof29 gene is optimized by directed evolution method to increase the content of andrographolide, specifically including: Based on the evaluation result that the regulation effect of the corresponding ApDof29 on andrographolide synthesis is qualified, the function of the ApDof29 gene is optimized by the directed evolution method, specifically including: Directed evolution is a powerful technique that mimics the natural selection process to optimize the function of genes or proteins, especially suitable for improving the activity, stability or other key properties of enzymes. To optimize the regulation effect of the ApDof29 gene in andrographolide synthesis, it includes traditional mutagenesis breeding, chemical mutagenesis, and adjusting the gene expression level by classical genetic methods.

[0038] The specific process of optimizing the function of the ApDof29 gene using directed evolution methods first involves creating a diverse gene library, achieved through saturation mutagenesis techniques, with the aim of introducing a large number of random mutations to cover a wide sequence space. High-throughput screening is used to evaluate the regulatory effect of each variant on andrographolide synthesis, and mutants with performance superior to the wild type are selected. The initially selected high-quality mutants are subjected to one or more rounds of iterative optimization, repeating the processes of mutation and screening until the target variant with the desired functional improvement is obtained. Throughout the process, an efficient screening strategy is relied upon to ensure the rapid and accurate identification of ApDof29 variants with the required characteristics.

[0039] The increase in the content of andrographolide specifically includes: Regulating key steps in the andrographolide biosynthetic pathway through metabolic engineering strategies, specifically including enhancing the supply of precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, in the acetate-mevalonate pathway; overexpressing the diterpene cyclase gene responsible for diterpene backbone formation to improve the production efficiency of the diterpene backbone; optimizing the expression of key enzyme genes involved in oxidation modification and subsequent structural modification steps, including P450 monooxygenase and methyltransferase; enhancing the positive regulatory effect of ApDof29 on genes related to andrographolide synthesis by overexpressing the ApDof29 gene or optimizing its function using directed evolution techniques, or inhibiting its negative regulatory effect through gene editing techniques; combining high-throughput screening techniques to evaluate the improvement effects of each step and iteratively optimizing the above regulatory strategies to ultimately achieve a significant increase in the content of andrographolide; The key steps include: The biosynthesis of andrographolide starts from isopentenyl pyrophosphate and dimethylallyl pyrophosphate produced by the acetate-mevalonate pathway. These are common precursors for the synthesis of all terpenoids. Through a series of condensation reactions, a more complex diterpene backbone is formed. This process is catalyzed by specific diterpene cyclases, which is the starting point for the specific biosynthesis of andrographolide. Optimizing this step can be achieved by overexpressing cyclases with high conversion capabilities. Starting from the initial diterpene backbone, through multiple oxidation, methylation and other modifications, andrographolide is finally formed. This involves the action of various enzymes such as P450 monooxygenase and methyltransferase. These enzymes precisely modify the molecule, increasing chemical diversity and biological activity.

[0040] Using metabolic engineering strategies to adjust the key steps of the andrographolide biosynthetic pathway mainly includes controlling the supply of precursor substances, optimizing the activity of diterpene cyclases and precisely regulating subsequent oxidation modification steps. At the same time, by studying the specific function of ApDof29 in this process, its regulatory effect on target genes can be enhanced or weakened specifically, thereby effectively increasing the content of andrographolide.

[0041] Working principle of the present invention: By means of a series of advanced molecular biology and metabolic engineering techniques, the content of andrographolide is increased. The target gene screening module determines the key gene ApDof29 regulating the synthesis of andrographolide through whole-genome analysis, gene expression profile research and co-expression network analysis. Through strict homology search, RNA-seq sequencing and weighted gene co-expression network analysis, ApDof29 is identified as a key regulatory factor. Then, the gene function verification module constructs overexpression plants and knockout mutants of ApDof29 using transgenic technology and CRISPR technology, and combines the expression detection of the key enzyme gene ApCPS2 and the determination of andrographolide content to verify the core role of ApDof29 in the synthesis of andrographolide. Subsequently, based on the confirmed core regulatory role, the real-time monitoring module constructs a dynamic monitoring system based on a biosensor, which can monitor the accumulation of andrographolide content, the biosynthesis rate and the concentration change of metabolic intermediates in real time. By calculating the abnormal coefficient of accumulation, the abnormal coefficient of synthesis rate and the abnormal coefficient of concentration change, the regulatory effect of ApDof29 on the synthesis of andrographolide is comprehensively evaluated. Finally, in the gene function modification and optimization module, a diverse ApDof29 gene library is created by directed evolution method. After high-throughput screening and multiple rounds of iterative optimization, variants with superior performance are selected, significantly increasing the content of andrographolide. In addition, by enhancing the supply of precursor substances, optimizing the activity of diterpene cyclase and subsequent oxidation modification steps, the biosynthetic pathway of andrographolide is further strengthened. The entire technical solution not only reveals the key role of ApDof29 in the synthesis of andrographolide, but also provides a solid theoretical basis and technical support for developing new varieties with high-efficiency production of andrographolide, demonstrating important technological innovation and application prospects.

[0042] One embodiment of the present invention has been described in detail above, but the content described is only the preferred embodiment of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of the present invention application shall still fall within the scope covered by the patent of the present invention.

Claims

1. A biosensor-based gene detection system for promoting andrographolide synthesis, characterized in that: include: A target gene screening module, wherein the target gene screening module screens and determines the key gene ApDof29 regulating andrographolide synthesis through whole genome analysis, gene expression profile research and co-expression network analysis; A gene function verification module, wherein the gene function verification module constructs overexpression plants and knockout mutants of the ApDof29 gene through transgenic technology, and combines the expression detection of the key enzyme gene ApCPS2 and the determination of andrographolide content to verify whether ApDof29 plays a core role in andrographolide synthesis; A real-time monitoring module, which builds a dynamic monitoring system based on biosensors based on the core effect verification results, monitors the accumulation of andrographolide content, biosynthesis rate and concentration changes of metabolic intermediates in real time, and dynamically evaluates the regulatory effect of ApDof29 on andrographolide synthesis based on the monitoring results and data analysis; A gene function modification and optimization module, wherein the gene function modification and optimization module optimizes the function of the ApDof29 gene through a directed evolution method and increases the content of andrographolide.

2. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 1, characterized in that: The screening and determination of the key gene ApDof29 regulating the synthesis of andrographolide specifically includes: The whole genome of Andrographis paniculata was annotated and specific gene family members involved in the synthesis of secondary metabolites were manually screened out, including cytochrome P450 enzymes and transcription factors. Sequences highly similar to known andrographolide synthesis-related genes were identified as candidate genes through homology search; differentially expressed genes were screened, and a gene co-expression network was established based on the screened differentially expressed gene list. The betweenness centrality value and closeness centrality value of the nodes were calculated, and all genes were ranked according to the calculated betweenness centrality value and closeness centrality value; genes with betweenness centrality value and closeness centrality value higher than the network average were selected as candidates for key regulatory genes. If the initially screened candidates promote the accumulation of andrographolide, the screened candidate ApDof29 was finally locked in as the key gene regulating andrographolide synthesis.

3. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 2, characterized in that: The calculation process of the betweenness centrality value and the closeness centrality value is: The calculation process of betweenness centrality value is: For each node in the constructed gene co-expression network, its betweenness centrality value is calculated by graph theory algorithm. Specifically: for each pair of nodes and All the shortest paths between nodes are counted. Appearing on these paths, the count of its betweenness centrality value is increased, according to the nodes passed The sum of the ratios of the number of and the number of all shortest paths gives the betweenness centrality value; The calculation process of the closeness centrality value is: Based on the gene co-expression network, a graph theory algorithm is used to calculate the closeness centrality value of each node. Specifically, the closeness centrality value is obtained by calculating the inverse of the average shortest path length from the node to all other nodes in the network.

4. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 1, characterized in that: The verification of whether ApDof29 plays a core role in the synthesis of andrographolide specifically includes: First, overexpression plants and knockout mutants of the ApDof29 gene were constructed through transgenic technology. In the overexpression plants, a strong promoter was used to drive the expression of ApDof29 to increase its level in the plant; in the knockout mutant, the CRISPR gene editing tool was used to precisely delete the ApDof29 gene. In these two transgenic plants and the wild-type control, the qRT-PCR technology was used to detect the expression level of the key enzyme gene ApCPS2 involved in the andrographolide biosynthesis pathway, and the high-performance liquid chromatography method was used to quantitatively determine the andrographolide content. If upregulated expression of ApCPS2 and increased andrographolide content were observed in the ApDof29 overexpression plants, while downregulated expression of ApCPS2 and decreased andrographolide content were observed in the knockout mutant, it was confirmed that ApDof29 played a core regulatory role in the synthesis of andrographolide.

5. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 1, characterized in that: The dynamic evaluation of the regulatory effect of ApDof29 on andrographolide synthesis specifically includes: According to the dynamic monitoring system of the biosensor, the accumulation data of andrographolide content, the biosynthesis rate data and the concentration data of metabolic intermediates are collected in real time, and the collected real-time data are analyzed. According to the analysis results, the accumulation abnormality coefficient, the synthesis rate abnormality coefficient and the concentration change abnormality coefficient are calculated, and the accumulation abnormality coefficient, the synthesis rate abnormality coefficient and the concentration change abnormality coefficient are comprehensively calculated and processed. Through the comprehensive calculation expression, the synthesis regulation coefficient is calculated to obtain the synthesis regulation coefficient, and it is determined whether the synthesis regulation coefficient is greater than or equal to the preset threshold value. If so, the corresponding ApDof29 has an unqualified regulatory effect on the synthesis of andrographolide; if not, the corresponding ApDof29 has a qualified regulatory effect on the synthesis of andrographolide.

6. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 5, characterized in that: The process of obtaining the accumulated anomaly coefficient is as follows: The accumulated data of andrographolide content are collected in real time and organized into a matrix. The accumulated data of andrographolide content collected in real time are standardized to obtain a standardized matrix. , the covariance matrix was calculated based on the accumulation data of standardized andrographolide content; Perform eigendecomposition on the covariance matrix to obtain the eigenvalues and the corresponding eigenvector ; The eigenvalues ​​are sorted from large to small; the top ones are selected according to the cumulative contribution rate of the eigenvalues. The important eigenvalues ​​and their corresponding eigenvectors were selected as the main components; the accumulated data of andrographolide content were projected onto the selected main components to obtain a new coordinate matrix, and the sum of the distances between the accumulated data of all andrographolide content and the data center in the main component space was calculated to obtain the accumulated anomaly coefficient.

7. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 5, characterized in that: The acquisition process of the synthetic rate anomaly coefficient is as follows: The time series data of biosynthesis rate are obtained, and the time series data are preprocessed to ensure that the data is continuous and has no missing values; the fluctuation sequence between adjacent time points is calculated; a series of scale parameters are determined, and for each scale parameter, the fluctuation sequence is divided into segments of corresponding lengths, and the number of non-empty boxes is counted; based on the relationship between the number of non-empty boxes and the scale parameter at different scales, the fractal dimension is estimated through a linear regression model; and the synthesis rate anomaly coefficient is obtained according to the absolute value of the difference between the estimated fractal dimension and the fractal dimension under the expected normal state.

8. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 5, characterized in that: The process of obtaining the concentration change anomaly coefficient is as follows: Obtain time series data of metabolic intermediate concentrations; preprocess the time series data to ensure that the data is continuous and has no missing values; use Haar wavelet transform to decompose the preprocessed data to obtain a series of approximation coefficients and detail coefficients; calculate the energy of the detail coefficients of each Haar wavelet transform level, and calculate the energy ratio of each level based on the sum of the energies of all Haar wavelet transform levels; determine which Haar wavelet transform levels are considered abnormal based on the calculated energy ratio compared with the preset threshold; obtain the concentration change abnormality coefficient based on the sum of the ratios exceeding the set threshold.

9. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 1, characterized in that: The directed evolution method optimizes the function of the ApDof29 gene, specifically comprising: Create a diverse gene library through saturation mutagenesis technology, with the aim of introducing a large number of random mutations to cover a wide range of sequence space, use high-throughput screening to evaluate the regulatory effect of each variant on andrographolide synthesis, select mutants with better performance than the wild type, perform multiple rounds of iterative optimization on the initially selected high-quality mutants, and repeat the mutation and screening process until the target variant that meets the expected functional improvement is obtained.

10. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 1, characterized in that: The method of increasing the content of andrographolide specifically comprises: The key steps in the andrographolide biosynthesis pathway were regulated by metabolic engineering strategies, including enhancing the supply of precursors of isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the acetate-mevalonate pathway, overexpressing the diterpene cyclase gene responsible for the formation of the diterpene skeleton, and improving the efficiency of the diterpene skeleton generation; optimizing the expression of key enzyme genes involved in oxidative modification and subsequent structural modification steps, including P450 monooxygenase and methyltransferase; overexpressing the ApDof29 gene to enhance its positive regulatory effect on genes related to andrographolide synthesis, and iteratively optimizing the regulatory strategy to ultimately achieve an increase in the andrographolide content.

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