CN-121975971-A - Gene detection system for promoting andrographolide synthesis based on biosensor

CN121975971ACN 121975971 ACN121975971 ACN 121975971ACN-121975971-A

Abstract

The invention relates to the technical field of genetic engineering detection, and particularly discloses a genetic detection system for promoting andrographolide synthesis based on a biosensor, which determines a key gene ApDof for regulating andrographolide synthesis through whole genome analysis, gene expression profile research and co-expression network analysis, verifies the core regulation effect of ApDof29, and also develops a real-time dynamic monitoring system which utilizes a specific response element to be fused with a reporter gene, realizes non-invasive monitoring on andrographolide content accumulation, biosynthesis rate and metabolic intermediate concentration change, dynamically evaluates the regulation effect of ApDof through comprehensive calculation processing of accumulation anomaly coefficient, synthesis rate anomaly coefficient and concentration change anomaly coefficient, and provides scientific basis for subsequent genetic modification.

Inventors

HUANG MINGKUN
YANG LANG
HU YUFANG
YANG HUA
ZHANG LING
XIONG YING
Lv Xingbin
LIANG QI
ZHANG ZHIYI
TIAN SHUYUN

Assignees

江西省、中国科学院庐山植物园

Dates

Publication Date: 20260505
Application Date: 20260123
Priority Date: 20250326

Claims (10)

1. A biosensor-based gene detection system for promoting andrographolide synthesis, comprising: the screening module of the target genes screens and determines key genes ApDof and ApDof for regulating and controlling andrographolide synthesis through whole genome analysis, gene expression profile research and co-expression network analysis; the screening and determining key genes ApDof and ApDof for regulating and controlling andrographolide synthesis specifically comprises the following steps: The method comprises the steps of annotating the whole genome of andrographis paniculata, manually screening out specific gene family members involved in the synthesis of secondary metabolites, wherein the specific gene family members comprise cytochrome P450 enzyme and transcription factors, identifying sequences which are highly similar to known andrographolide synthesis related genes through homology search to serve as candidate genes, screening differential expression genes, establishing a gene co-expression network based on a screened differential expression gene list, calculating the medium central value and the near central value of nodes, and sequencing all genes according to the calculated medium central value and the near central value, wherein if the preliminarily screened candidates promote andrographolide accumulation, finally locking the screened candidates ApDof to serve as key genes for regulating andrographolide synthesis; The gene function verification module is used for constructing a ApDof gene silencing strain through a VIGS technology, and verifying whether ApDof is a core function in andrographolide synthesis or not by combining expression detection of a key enzyme gene ApCPS2 and andrographolide content measurement; The real-time monitoring module is used for constructing a dynamic monitoring system based on a biosensor based on a core effect verification result, monitoring accumulation of andrographolide content, biosynthesis rate and concentration change of metabolic intermediates in real time, and dynamically evaluating ApDof on regulation and control effects of andrographolide synthesis according to monitoring results and data analysis; the gene function modification and optimization module optimizes the function of ApDof genes by a directed evolution method and improves the content of andrographolide; The method for improving the content of andrographolide comprises the following steps: Regulating and controlling key steps in an andrographolide biosynthesis pathway through a metabolic engineering strategy, specifically comprising the steps of enhancing the supply of precursors of isopentenyl pyrophosphate and dimethylpropenyl pyrophosphate in an acetic acid-mevalonate pathway, overexpressing diterpenoid cyclase genes responsible for diterpenoid skeleton formation, improving the generation efficiency of the diterpenoid skeleton, optimizing the expression of key enzyme genes involved in oxidative modification and subsequent structural modification steps, including P450 monooxygenase and methyltransferase, enhancing the forward regulation and control action of the genes on andrographolide synthesis through over-expression ApDof genes, iterating and optimizing the regulation and control strategy, and finally realizing the improvement of the andrographolide content; The key steps comprise that biosynthesis of andrographolide starts from isopentenyl pyrophosphate and dimethylpropenyl pyrophosphate generated in an acetic acid-mevalonate pathway, the isopentenyl pyrophosphate and the dimethylpropenyl pyrophosphate are common precursors for synthesizing all terpenoids, a more complex diterpene skeleton is formed through a series of condensation reactions, the diterpene skeleton is catalyzed by diterpene cyclase and is a starting point of specific biosynthesis of andrographolide, and andrographolide is finally formed through multiple oxidation, methylation and other modifications from the original diterpene skeleton.
2. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 1, wherein the calculation process of the median centrality value and the near centrality value is as follows: The calculation process of the median centrality value is as follows: For each node in the constructed gene co-expression network, calculating the median centrality value by a graph theory algorithm, specifically for each pair of nodes And Counting all shortest paths among the nodes, if the nodes are On these paths, the count of the median centrality values is incremented according to the transit nodes The sum of the ratio of the number of the shortest paths to the number of all the shortest paths to obtain a median centrality value; The calculation process of the approximate centrality value is as follows: Based on the gene co-expression network, calculating the proximity centrality value of each node by adopting a graph theory algorithm, specifically, calculating the reciprocal of the average shortest path length from the node to all other nodes in the network to obtain the proximity centrality value.
3. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 1, wherein the verification ApDof is a core role in andrographolide synthesis, specifically comprising: Firstly, silencing ApDof genes in andrographis paniculata by a VIGS technology, taking empty-load dip-dyed plants as a control, adopting a qRT-PCR technology to detect the expression level of key enzyme genes ApCPS2 participating in the biosynthesis pathway of andrographolide, and measuring the content of andrographolide by using a high performance liquid chromatography method, and if ApCPS2 expression is down-regulated in the silencing plants and the content of andrographolide is reduced, confirming ApDof that the gene has a core regulation and control effect on andrographolide synthesis.
4. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 1, wherein the dynamic evaluation ApDof of the effect of regulating andrographolide synthesis specifically comprises: According to a dynamic monitoring system of a biosensor, accumulation data of andrographolide content, biosynthesis rate data and concentration data of metabolic intermediates are collected in real time, the collected real-time data are analyzed, accumulation abnormal coefficients, synthesis rate abnormal coefficients and concentration change abnormal coefficients are calculated according to analysis results, comprehensive calculation processing is carried out on the accumulation abnormal coefficients, the synthesis rate abnormal coefficients and the concentration change abnormal coefficients, a synthesis regulation and control coefficient is obtained through calculation through a comprehensive calculation expression, whether the synthesis regulation and control coefficient is larger than or equal to a preset threshold value is judged, if yes, the regulation and control effect of the corresponding ApDof on andrographolide synthesis is not qualified, and if not, the regulation and control effect of the corresponding ApDof on andrographolide synthesis is qualified.
5. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 4, wherein the process of obtaining the accumulated anomaly coefficient is as follows: collecting accumulation data of andrographolide content in real time, organizing into a matrix, and performing standardization processing on the accumulation data of andrographolide content collected in real time to obtain standardized matrix Calculating a covariance matrix based on the accumulated data of the standardized andrographolide content; Performing feature decomposition on the covariance matrix to obtain feature values And corresponding feature vector The characteristic values are sorted from big to small, and before the characteristic value is selected according to the accumulated contribution rate The method comprises the steps of taking important characteristic values and corresponding characteristic vectors as main components, projecting the accumulated data of the andrographolide contents onto the selected main components to obtain a new coordinate matrix, and calculating the sum of the distances between the accumulated data of the andrographolide contents and a data center in a space of the main components to obtain an accumulated abnormal coefficient.
6. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 4, wherein the synthetic rate abnormality coefficient obtaining process is as follows: The method comprises the steps of obtaining time sequence data of a biosynthesis rate, preprocessing the time sequence data to ensure continuous data without missing values, calculating a fluctuation sequence between adjacent time points, determining a series of scale parameters, dividing the fluctuation sequence into segments with corresponding lengths aiming at each scale parameter, counting the number of non-empty boxes, estimating fractal dimension through a linear regression model based on the relation between the number of the non-empty boxes under different scales and the scale parameters, and obtaining a synthesis rate abnormal coefficient according to the absolute value of the difference between the estimated fractal dimension and the fractal dimension under an expected normal state.
7. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 4, wherein the concentration variation abnormality coefficient obtaining process is as follows: The method comprises the steps of obtaining time series data of metabolic intermediate concentration, preprocessing the time series data to ensure that the data are continuous and have no missing values, decomposing the preprocessed data by using Haer wavelet transformation to obtain a series of approximation coefficients and detail coefficients, calculating the energy of the detail coefficients of each Haer wavelet transformation level, calculating the energy proportion of each level according to the energy sum of all Haer wavelet transformation levels, comparing the calculated energy proportion with a preset threshold value to determine which fluctuation of the Haer wavelet transformation levels is considered to be abnormal, and obtaining concentration change abnormal coefficients based on the sum of the proportions exceeding the set threshold value.
8. The biosensor-based gene detection system for promoting andrographolide synthesis according to claim 1, wherein the directed evolution method optimizes ApDof functions of the gene, specifically comprising: Creating a diversified gene library, realizing by a saturation mutation technology, aiming at introducing a large number of random mutations to cover a wide sequence space, evaluating the regulation effect of each variant on andrographolide synthesis by high-throughput screening, selecting mutants with better performance than wild type, performing multiple rounds of iterative optimization on the initially selected high-quality mutants, and repeating the processes of mutation and screening until the target variants meeting the expected functional improvement are obtained.
9. A gene ApDof of an andrographis paniculata transcription factor gene, which is applied to the biosensor-based gene detection system for promoting andrographolide synthesis according to any one of claims 1 to 8, and is characterized in that the CDS sequence of the ApDof gene is shown as SEQ ID NO. 1.
10. A method for regulating andrographolide synthesis using an andrographis paniculata transcription factor gene ApDof gene according to claim 9, by regulating the expression level of ApDof gene, including silencing ApDof gene.

Description

Gene detection system for promoting andrographolide synthesis based on biosensor Technical Field The invention relates to the technical field of genetic engineering detection, in particular to a genetic detection system for promoting andrographolide synthesis based on a biosensor. Background Herba Andrographitis (Andrographis paniculata) is one of the traditional Chinese medicinal materials in China, and has effects of clearing heat and detoxicating. The main active ingredient andrographolide (andrographolide) is a diterpenoid secondary metabolite with a complex structure, has various pharmacological activities such as antibiosis, antivirus, anti-inflammatory, anti-tumor, immunoregulation and the like, and has important application value in the field of modern medicine, in particular in the Chinese patent medicine industry. At present, andrographolide is mainly dependent on plant extraction. Although research on chemical synthesis has been reported, the synthetic process still faces significant challenges in terms of economy and mass production due to its complex molecular structure, long synthetic route and high cost. However, the problems of low content of andrographolide in wild or cultivated andrographis paniculata plants, poor extraction efficiency and the like generally lead to high production cost, and severely restrict the large-scale production and industrial application of the andrographolide. With the rapid development of molecular biology technology, the genetic engineering technology provides a new solution for improving the content of secondary metabolites of plants and reducing the production cost. However, the knowledge of the regulation mechanism of the biosynthesis pathway of andrographolide is still imperfect, and especially the key genes involved in biosynthesis and the regulation network thereof are not fully resolved, thus severely restricting the application prospect of precise regulation and directional modification of andrographolide synthesis. Therefore, the key genes and the regulation and control mechanisms thereof of the andrographolide biosynthesis pathway are studied deeply, and particularly, the functional analysis of the regulation and control elements such as transcription factors is helpful for elucidating the molecular mechanism of andrographolide accumulation, and theoretical basis and technical support are provided for realizing the directional improvement of the andrographolide yield through means such as molecular breeding, genetic engineering and the like, so that the sustainable development of andrographolide related industries is promoted. The invention discovers ApDof that the gene can improve the synthesis and accumulation of andrographolide by regulating and controlling the expression of key enzyme gene ApCPS2 in the synthetic route of andrographolide through molecular biological means such as gene cloning, and the like, which shows the core effect of the gene in an andrographolide biosynthesis regulation network and provides a key functional gene marker and genetic operation target for andrographolide molecular breeding. The prior art has the following defects: aiming at the industrial bottleneck that metabolic engineering modification targets are lost due to imperfect transcriptional regulation mechanism and deficient transcriptional regulation genes of andrographolide biosynthesis; the invention provides a brand new ApDof transcription factor gene with a function of obviously promoting andrographolide accumulation, and provides core gene resources for directionally cultivating new varieties of high andrographolide. Disclosure of Invention The invention aims to provide a biosensor-based gene detection system for promoting andrographolide synthesis, so as to solve the problems in the background. The aim of the invention can be achieved by the following technical scheme: A biosensor-based gene detection system for promoting andrographolide synthesis, comprising: the screening module of the target genes screens and determines key genes ApDof and ApDof for regulating and controlling andrographolide synthesis through whole genome analysis, gene expression profile research and co-expression network analysis; The gene function verification module is used for constructing a ApDof gene silencing strain through a VIGS technology, and verifying whether ApDof is a core function in andrographolide synthesis or not by combining expression detection of a key enzyme gene ApCPS2 and andrographolide content measurement; The real-time monitoring module is used for constructing a dynamic monitoring system based on a biosensor based on a core effect verification result, monitoring accumulation of andrographolide content, biosynthesis rate and concentration change of metabolic intermediates in real time, and dynamically evaluating ApDof on regulation and control effects of andrographolide synthesis according to monitoring results and data analysis; And the gene function modificatio