A method and system for constructing crop drought resistance cis-regulation rules based on comparative genomics and cis-regulation element network analysis

Abstract

This invention discloses a method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis, comprising: screening candidate drought resistance motif subsets from a set of gene regulation-related region sequences using transcription factor binding motifs as reference motifs; calculating the co-occurrence relationship of different drought resistance cis-regulatory elements in the same gene regulation-related region sequence based on the candidate drought resistance motif subsets; constructing a combinatorial regulatory network based on the number of co-occurrences and performing topological feature analysis to obtain network topological features; constructing a cross-species drought resistance cis-regulatory network based on the correspondence between drought resistance cis-regulatory elements and transcription factor binding motifs and performing conservation analysis to obtain cross-species conserved edges and conserved modules; using comparative genomics methods to calculate the evolutionary rate and positional distribution relationship of drought resistance cis-regulatory elements, thereby systematically revealing the spatial organization characteristics, combinatorial regulatory patterns, and evolutionary conservation of drought resistance cis-regulatory elements at the cross-species scale.

Description

Technical Field

[0001] This invention relates to the fields of computational biology and agricultural bioinformatics, specifically to a method and system for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis. Background Technology

[0002] Drought stress is one of the major environmental factors affecting crop growth and development and the stability of agricultural production. Plants respond to drought stress through complex gene regulatory networks, among which cis-regulatory elements, as important sequence units for gene expression regulation, play a crucial role in regulating the expression of drought-resistant genes. Cis-regulatory elements typically exist in promoters and other non-coding regulatory regions in the form of specific motifs, forming regulatory complexes by binding with transcription factors, thereby participating in the regulation of plant responses to drought stress. Therefore, systematically analyzing the combinational relationships, spatial organization patterns, and evolutionary laws among drought-resistant cis-regulatory elements is of great significance for a deeper understanding of plant drought resistance regulation mechanisms and for conducting molecular design breeding.

[0003] Currently, research on cis-regulatory elements largely focuses on single motif identification or single-gene regulatory analysis. Common methods include sequence scanning based on transcription factor binding site databases, prediction based on sequence conservation, and enrichment analysis based on promoter regions of differentially expressed genes. These methods typically only focus on the presence of a single cis-element, making it difficult to systematically analyze the combination relationships and synergistic regulatory patterns among multiple cis-regulatory elements. Furthermore, most studies focus on a single species, lacking cross-species comparative analysis, making it difficult to reveal the conservation of cis-regulatory elements across different species and their evolutionary patterns. In addition, the function of cis-regulatory elements in the genome is not only related to their sequence characteristics but also closely related to their location distribution in different gene regions and the spatial spacing between elements. Existing methods still lack systematic modeling of the spatial organization structure of cis-regulatory elements and their combination regulatory relationships. Summary of the Invention

[0004] To overcome the limitations of existing studies on cis-regulatory elements, which often focus on single motif identification or single-gene regulation analysis and fail to systematically reveal the combinatorial regulatory relationships, spatial organization patterns, and cross-species conservation of cis-regulatory elements, this invention provides a method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis. By systematically revealing the spatial organization characteristics, combinatorial regulatory patterns, and evolutionary conservation of drought resistance cis-regulatory elements at the cross-species scale, this invention provides methods and basis for research on crop drought resistance regulation mechanisms and drought resistance molecular breeding.

[0005] According to one aspect of this invention, a method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis is provided, comprising: constructing a set of gene regulation-related region sequences, using transcription factor binding motifs as reference motifs, and screening candidate drought resistance motif subsets from the set of gene regulation-related region sequences; calculating the co-occurrence relationship of different drought resistance cis-regulatory elements within the same gene regulation-related region sequence based on the candidate drought resistance motif subsets; constructing a combined regulatory network of drought resistance cis-regulatory elements based on the number of co-occurrences of different drought resistance cis-regulatory elements within the same gene regulation-related region sequence and performing topological feature analysis to obtain network topological features; constructing a cross-species drought resistance cis-regulatory network based on the correspondence between drought resistance cis-regulatory elements and transcription factor binding motifs and performing conservation analysis to obtain cross-species conserved edges and conserved modules; and calculating the evolutionary rate and positional distribution relationship of drought resistance cis-regulatory elements using comparative genomics methods.

[0006] Furthermore, a set of gene regulation-related region sequences is constructed, and a subset of candidate drought-resistant motifs is selected from the set of gene regulation-related region sequences, using transcription factor binding motifs as reference motifs. This includes: obtaining reference genome sequences and gene annotation files from genome databases of multiple plant species; extracting gene regulation-related region sequences based on the reference genome sequences and gene annotation files to construct a set of gene regulation-related region sequences; selecting the set of gene regulation-related region sequences based on preset drought-resistant genes to obtain a set of drought-resistant gene regulation-related region sequences; and selecting a subset of candidate drought-resistant motifs from the set of drought-resistant gene regulation-related region sequences, using transcription factor binding motifs as reference motifs.

[0007] Furthermore, using transcription factor binding motifs as reference motifs, a subset of candidate drought-resistant motifs is obtained from the set of sequences related to the regulation of drought-resistant genes. This includes: obtaining transcription factor binding motifs from a publicly available transcription factor binding site database; identifying candidate drought-resistant related motifs in the set of sequences related to the regulation of drought-resistant genes using a motif discovery algorithm; and using the transcription factor binding motifs as reference motifs to screen the candidate drought-resistant related motifs to obtain a subset of candidate drought-resistant motifs.

[0008] Further, the co-occurrence relationship of different drought-resistant cis-regulatory elements within the same gene regulation-related region sequence is calculated based on the candidate drought-resistant motif subset, including: constructing a position weight matrix based on the motif representation in the candidate drought-resistant motif subset; performing a whole-genome scan in each gene regulation-related region sequence using the position weight matrix to obtain the hit sites of drought-resistant cis-regulatory elements; wherein, the hit sites of drought-resistant cis-regulatory elements refer to the location information of drought-resistant cis-regulatory elements; performing regional statistics on the drought-resistant cis-regulatory elements according to the gene regulation-related region type to obtain the distribution ratio of each drought-resistant cis-regulatory element in different gene regulation-related regions; annotating each drought-resistant cis-regulatory element with functional categories based on a functional annotation database, calculating the proportion of each functional category in different gene regulation-related regions to obtain the position preference rules of drought-resistant cis-regulatory elements; calculating the co-occurrence relationship of different drought-resistant cis-regulatory elements within the same gene regulation-related region sequence, and statistically analyzing their relative orientation and spacing distribution.

[0009] Furthermore, based on the co-occurrence frequency of different drought-resistant cis-regulatory elements within the same gene regulation-related region sequence, a combined regulatory network of drought-resistant cis-regulatory elements is constructed and topological feature analysis is performed to obtain network topological features. This includes: using drought-resistant cis-regulatory elements as network nodes, constructing an element co-occurrence relationship matrix of drought-resistant cis-regulatory elements based on their co-occurrence frequency within multiple gene regulation-related region sequences; constructing a combined regulatory network of drought-resistant cis-regulatory elements based on the element co-occurrence relationship matrix; and performing topological feature analysis on the combined regulatory network to obtain network topological features. The network topological features constitute the combined regulatory rules of drought-resistant cis-regulatory elements from the perspective of the combined regulatory network.

[0010] Furthermore, based on the correspondence between drought-resistance cis-regulatory elements and transcription factor binding motifs, a cross-species drought-resistance cis-regulatory network is constructed and its conservation is analyzed to obtain cross-species conserved edges and conserved modules. This includes: associating transcription factors, drought-resistance cis-regulatory elements, and target genes based on the correspondence between drought-resistance cis-regulatory elements and transcription factor binding motifs to construct a cross-species drought-resistance cis-regulatory network; and performing conservation analysis on the cross-species drought-resistance cis-regulatory network to obtain cross-species conserved edges and conserved modules. The cross-species conserved edges and conserved modules constitute the combinatorial regulatory rules of drought-resistance cis-regulatory elements from the perspective of the cis-regulatory network.

[0011] Furthermore, the evolutionary rate and positional distribution relationship of drought-resistant cis-regulatory elements are calculated using comparative genomics methods, including: obtaining homologous genes and their corresponding regulatory-related region sequence pairs through multi-species genome collinearity analysis; performing multiple sequence alignment on the homologous gene regulatory-related region sequences to obtain alignment results; calculating the relative evolutionary rate of non-coding regions and coding regions based on the alignment results; grouping the drought-resistant cis-regulatory element hit sites according to the type of gene regulatory-related region, including coding regions and regulatory regions as well as different segments of the regulatory region; comparing the differences in evolutionary rate distribution under different region types to obtain element position-evolutionary constraint rules.

[0012] According to one aspect of this invention, a system for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis is provided, comprising: a candidate drought resistance motif subset construction module, used to construct a set of drought resistance gene regulation-related region sequences, using transcription factor binding motifs as reference motifs, and screening candidate drought resistance motif subsets from the drought resistance gene regulation-related region sequence set; an element co-occurrence relationship acquisition module, used to calculate the co-occurrence relationship of different drought resistance cis-regulatory elements within the same gene regulation-related region sequence based on the candidate drought resistance motif subset; and a combined regulatory network construction module, used to construct a combined regulatory network based on different drought resistance cis-regulatory elements within the same gene regulation-related region sequence. The co-occurrence frequency within gene regulation-related regions is used to construct a combined regulatory network of drought-resistant cis-regulatory elements and perform topological feature analysis to obtain network topological features. A cis-regulatory network construction module is used to construct a cross-species drought-resistant cis-regulatory network based on the correspondence between drought-resistant cis-regulatory elements and transcription factor binding motifs, and to perform conservation analysis to obtain cross-species conserved edges and conserved modules. The network topological features and cross-species conserved edges and conserved modules together constitute the combined regulatory rules of drought-resistant cis-regulatory elements. An element evolution rate calculation module is used to calculate the evolution rate and positional distribution relationship of drought-resistant cis-regulatory elements using comparative genomics methods.

[0013] According to one aspect of the present invention, an electronic device is provided, including a memory and a processor, the memory storing program instructions executable by the processor, the processor invoking the program instructions to execute the method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis.

[0014] According to one aspect of the present invention, a non-transitory computer-readable storage medium is provided, characterized in that the non-transitory computer-readable storage medium stores computer instructions that cause the computer to execute the method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis.

[0015] The above technical solution first integrates the sequences of drought-resistant gene regulatory regions and information on drought-resistant cis-regulatory elements from multiple species, and systematically analyzes the location distribution characteristics and spatial co-occurrence relationships of drought-resistant cis-regulatory elements in different gene regulatory regions. Based on this, a combinatorial regulatory network of drought-resistant cis-regulatory elements is constructed, and the combinatorial regulatory rules of drought-resistant cis-regulatory elements are obtained through network topology analysis. Furthermore, a "transcription factor—cis-regulatory element—target gene" cis-regulatory network is constructed, and conserved regulatory relationships are identified through cross-species comparative analysis. Simultaneously, comparative genomics methods are used to calculate the evolutionary rates of non-coding and coding regions, and the evolutionary constraints of cis-regulatory elements at different genomic locations are analyzed. This invention can systematically reveal the spatial organization characteristics, combinatorial regulatory patterns, and evolutionary conservation of drought-resistant cis-regulatory elements at a cross-species scale, providing methods and basis for research on crop drought resistance regulation mechanisms and drought-resistant molecular breeding.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] (1) This invention systematically analyzes the location distribution characteristics and spatial co-occurrence relationships of cis-regulatory elements in different gene regions at the cross-species genome scale, which can reveal the location preference and spatial organization pattern of cis-regulatory elements in the genome, and provide a new analytical framework for elucidating the structural rules of drought resistance regulatory elements;

[0018] (2) By constructing a combined control network of cis-regulatory elements and using the network topology characteristics to analyze the combination relationship between cis-regulatory elements, this invention can systematically identify the synergistic control mode between cis-regulatory elements, thereby obtaining the combined control rules of drought-resistant cis-regulatory elements.

[0019] (3) This invention combines comparative genomics methods to calculate the evolutionary rate of non-coding regulatory regions and corresponding coding regions, and analyzes the differences in evolutionary constraints of cis-regulatory elements in different genomic regions, thereby revealing the conservation and selection pressure of drought-resistant cis-regulatory elements at the cross-species scale, and improving the systematicness and reliability of drought-resistant regulatory rule analysis. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The flowchart illustrates the overall implementation of a method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis, as provided in this embodiment of the invention.

[0022] Figure 2 This is a schematic diagram showing the location distribution and spatial interaction spacing calculation results of drought-resistant cis-regulatory elements provided in the embodiments of the present invention, illustrating the location distribution characteristics of cis-regulatory elements in different gene regions and the spatial spacing distribution between elements.

[0023] Figure 3 This is a schematic diagram of the construction and network feature calculation results of the drought-resistant cis-regulatory element combination regulation network provided in the embodiment of the present invention, showing the structure of the cis-regulatory element combination regulation network and its topological features such as node degree and network average degree.

[0024] Figure 4 This is a schematic diagram of the construction of drought-resistant cis-regulatory network and the identification results of cross-species conserved network provided in the embodiments of the present invention, showing the regulatory relationship of "transcription factor-cis-regulatory element-target gene" and its conservation in different species.

[0025] Figure 5 This is a schematic diagram showing the changes in the evolution rate of drought-resistant cis-regulatory elements with their genomic location distribution, as provided in the embodiments of the present invention. It illustrates the differences in the evolution rate of cis-regulatory elements in different gene regions and their evolutionary constraints. Detailed Implementation

[0026] It should be noted that:

[0027] The terms “comprising” and “having”, and any variations thereof, in the specification, claims, and accompanying drawings of this invention are intended to cover a non-exclusive inclusion, such as a process, method, system, product, or apparatus that includes a series of steps or units, not necessarily limited to those explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0028] The block diagrams shown in the accompanying drawings are merely functional entities and do not necessarily correspond to physically independent entities. That is, these functional entities can be implemented in software, in one or more hardware modules or integrated circuits, or in different network and / or processor devices and / or microcontroller devices. The flowcharts shown in the accompanying drawings are merely illustrative and do not necessarily include all content and operations / steps, nor do they necessarily have to be performed in the described order. For example, some operations / steps can be decomposed, while others can be combined or partially combined; therefore, the actual execution order may change depending on the specific circumstances.

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In addition, the technical features of the various embodiments or individual embodiments provided by the present invention can be arbitrarily combined to form new technical solutions. Such combinations are not bound by the order of steps and / or structural composition patterns, but must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0030] Please refer to the appendix. Figure 1 This invention provides a method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis. The overall process includes steps such as acquiring drought resistance cis-regulatory element data, analyzing the spatial distribution of drought resistance cis-regulatory elements, constructing a combined regulatory network of drought resistance cis-regulatory elements, identifying cross-species conserved regulatory relationships, and analyzing the evolutionary rate of drought resistance cis-regulatory elements. Specifically, it includes:

[0031] Step S1 involves constructing a set of gene regulation-related region sequences, using transcription factor binding motifs as reference motifs, and screening from this set to obtain a subset of candidate drought-resistant motifs. Specifically, step S1 includes:

[0032] Step S11: Obtain reference genome sequences and gene annotation files from genome databases of multiple plant species.

[0033] In step S11, the genome includes gene DNA sequences and DNA sequences that regulate the function of genes. Cis-regulatory elements are DNA sequences that regulate the function of genes. Therefore, multi-species drought-resistance cis-regulatory elements contain DNA sequences that regulate the function of drought-resistance genes from multiple species. Each species contains multiple drought-resistance cis-regulatory elements, including their sequence composition and location on the genome. The reference genome sequence refers to the chromosome-level DNA sequence file of the target plant species, used to provide genomic background sequence information. The gene annotation file refers to the annotation file describing the location, strand direction, and structural composition of genes on the reference genome, including coordinate information of elements such as genes, transcripts, exons, introns, UTRs, and CDS, preferably in GFF or GTF format. In this embodiment, 15 representative plant species are preferably selected, including rice, wheat, maize, barley, sorghum, and Arabidopsis thaliana. The reference genome sequence (FASTA format) and gene annotation file (GFF format) can be downloaded from genomic databases (such as the Ensembl Plants database or the NCBI database).

[0034] Step S12: Extract gene regulation-related region sequences based on the reference genome sequence and gene annotation file to construct a set of gene regulation-related region sequences.

[0035] In step S12, the set of gene regulation-related region sequences is a subset of the genome, and it consists of multiple gene regulation-related region sequences. The drought resistance cis-regulatory element is a short fragment subset of the gene regulation-related region sequences. Each gene regulation-related region sequence consists of multiple gene regulation-related regions, and the drought resistance cis-regulatory element is located on each of these regions. These multiple gene regulation-related regions include, but are not limited to, the promoter region (3000 bp), the 5′UTR region, the intron region, the exon region, the 3′UTR region, and the region downstream of the transcription termination site (2000 bp).

[0036] Step S13: Screen the set of sequences related to the regulation of drought-resistant genes according to the preset drought-resistant gene sequence to obtain the set of sequences related to the regulation of drought-resistant genes.

[0037] In step S13, the set of sequences related to drought resistance gene regulation is a subset of the set of sequences related to gene regulation. The above-mentioned preset sources of drought resistance genes include various methods, such as obtaining them from databases (e.g., DroughtDB database) or from transcriptome data analysis.

[0038] Step S14: Using transcription factor binding motifs as reference motifs, a subset of candidate drought-resistant motifs is selected from the set of sequences related to the regulation of drought-resistant genes.

[0039] In step S14, transcription factor binding motifs are obtained from a publicly available transcription factor binding site database. A motif discovery algorithm is then used to identify candidate drought-resistant motifs within a set of sequences representing drought-resistant gene regulatory regions. These transcription factor binding motifs are used as reference motifs to further filter the candidate drought-resistant motifs, resulting in a subset of candidate drought-resistant motifs. The publicly available transcription factor binding site database can be CIS-BP, PLACE, or JASPAR. The set of sequences representing drought-resistant gene regulatory regions is the source sequence set for the candidate drought-resistant motifs. The candidate drought-resistant motif subset is a set of biologically meaningful motifs selected from the candidate drought-resistant motifs, using the transcription factor binding motif as a reference. This subset is used in the subsequent step S21 to construct the position weight matrix.

[0040] Step S2: Calculate the co-occurrence relationships of different drought-resistant cis-regulatory elements within the same gene regulatory region sequence based on the candidate drought-resistant motif subset.

[0041] It should be noted that step S2 aims to utilize the candidate drought-resistant motif subset obtained in step S1 to acquire the co-occurrence relationships of different drought-resistant cis-regulatory elements in the gene regulation-related region sequence set. Specifically, step S2 includes:

[0042] Step S21: Construct a position weight matrix based on the representation of motifs in the candidate drought-resistant motif subset.

[0043] In step S21, the candidate drought-resistant motif subset is a set of biologically meaningful motifs selected from candidate drought-resistant related motifs, using transcription factor binding motifs obtained from public databases as a reference. The motifs are represented as a site frequency matrix (PFM) and a site probability matrix (PPM), which is a probability matrix calculated from multiple sequences indicating the probability of a specific base appearing at each site. When the motifs in the candidate drought-resistant motif subset are represented as a site probability matrix (PPM), the position weight matrix (PWM) is directly constructed using the site probability matrix (PPM). When the motifs in the candidate drought-resistant motif subset are represented as a site frequency matrix (PFM), the site frequency matrix (PFM) is first normalized to obtain the corresponding site probability matrix (PPM), and then the position weight matrix (PWM) is constructed using the site probability matrix (PPM). The calculation formula is as follows:

[0044]

[0045] in, Indicates the base at position j The probabilities of adenine, cytosine, guanine, and thymine, respectively; Indicates background base frequencies; This represents the probability of base i appearing at position j in the site probability matrix.

[0046] Step S22: Use position weight matrix (PWM) to perform whole-genome scanning in the gene regulation-related region sequences to obtain the hit sites of drought resistance cis-regulatory elements.

[0047] In step S22, using the FIMO tool, a whole-genome scan is performed on the sequences of each gene regulatory region using motif models corresponding to drought-resistant cis-regulatory elements (such as scoring models converted from PWM, PPM, or PFM). The specific sequence fragments that meet the preset matching threshold and their position coordinates in the genome are then obtained; these are the drought-resistant cis-regulatory element hit sites. Preferably, the significance threshold for the FIMO scan is set to p < 1 × 10⁻⁶. .

[0048] It should be noted that drought-resistance cis-regulatory elements are abstract cis-regulatory motif patterns or functional element types; the location of a drought-resistance cis-regulatory element is the specific position of this abstract element within the drought-resistance gene regulation-related region sequence (i.e., the location information of the drought-resistance cis-regulatory element). The gene regulation-related region sequences of each species serve as the search space and carrier sequences for drought-resistance cis-regulatory element location sites; these location sites are identified through motif scanning within these drought-resistance gene regulation-related region sequences.

[0049] Furthermore, for any candidate hit site The PWM matching score is defined as:

[0050]

[0051] in, Candidate hit sites The PWM matching score, where L is the sequence length. For the j-th base of the candidate site; when The location is determined to be the hit site of the drought-resistant cis-regulatory element, where T is the preset matching threshold. It should be noted that the preset matching threshold can be set according to actual needs and is not limited here.

[0052] Step S23: Perform regional statistics on the hit sites of drought-resistant cis-regulatory elements according to the types of gene regulation-related regions to obtain the distribution ratio of the hit sites of each drought-resistant cis-regulatory element under different gene regulation-related regions; perform functional category annotation on each drought-resistant cis-regulatory element based on the functional annotation database, and calculate the proportion of each functional category in different gene regulation-related regions.

[0053] In step S23, the different gene regulation-related regions include promoter regions, UTR regions, intron regions, and intergenetic regions. Understandably, the distribution ratio of each drought resistance cis-regulatory element's hit site within different gene regulation-related regions represents the overall positional distribution, while the proportion of each functional category within different gene regulation-related regions represents the local positional distribution. Together, these two constitute the positional preference rule of drought resistance cis-regulatory elements.

[0054] Step S24: Calculate the co-occurrence relationship of different drought resistance cis-regulatory elements in the same gene regulatory region sequence, and statistically analyze their relative orientation and spacing distribution.

[0055] In step S24, co-occurrence refers to the presence of two different drought resistance cis-regulatory elements on the same gene regulatory region sequence (see Appendix for details). Figure 2 By statistically analyzing the spacing distribution of different drought-resistant cis-regulatory element combinations, combinations of drought-resistant cis-regulatory elements with spatial organization patterns can be identified. Spacing is defined as the shortest distance between the start and end coordinates of two hit sites. Preferably, the spacing between drought-resistant cis-regulatory elements is defined as the shortest base distance between the hit sites of two elements.

[0056] Step S3 involves constructing a combined regulatory network of drought-resistance cis-regulatory elements based on the co-occurrence frequency of different drought-resistance cis-regulatory elements within the same gene's regulatory region sequence, and performing topological feature analysis to obtain the network's topological characteristics. Specifically, step S3 includes:

[0057] Step S31: Using drought-resistant cis-regulatory elements as nodes, construct an element co-occurrence relationship matrix of drought-resistant cis-regulatory elements based on their co-occurrence frequency in multiple drought-resistant gene regulatory region sequences.

[0058] In step S31, the co-occurrence count refers to the number of times a co-occurrence relationship occurs, and the component co-occurrence relationship matrix... This refers to a matrix used to characterize the pairwise combinations of drought-resistant cis-regulatory elements, and the corresponding formula is:

[0059]

[0060] in, This represents a combination relation matrix. The Middle Line number Column elements, For components With components The number of times it co-occurs in regions related to gene regulation.

[0061] Step S32: Construct a combined regulation network of drought-resistant cis-regulatory elements based on the element co-occurrence relationship matrix.

[0062] In step S32, the combined control network includes two elements: nodes and edges. In step S33, when calculating the network topology features and the identification module, it is necessary to calculate the node and edge weights of the combined control network, using point mutual information as the edge weights. Point mutual information is defined as:

[0063]

[0064] in, For co-occurrence probability, , For the probability of occurrence alone, when Preserve edges during operation.

[0065] Step S33: Perform topological feature analysis on the combined control network to obtain network topological features; wherein, the network topological features constitute the combined control rules of drought-resistant cis-regulatory elements from the perspective of the combined control network.

[0066] In step S33, topology feature analysis includes extracting feature parameters and identifying the module structure of the combined control network using a module detection algorithm. Correspondingly, the network topology features include topology feature parameters and module partitioning results, which together constitute the combined control rules of drought-resistant cis-regulatory elements from the perspective of the cis-regulatory network. The topology feature parameters include node degree and network average degree, which are defined as follows:

[0067]

[0068]

[0069] in, The node degree of drought-resistant cis-regulatory element i; represents the elements of the connection matrix between drought-resistant cis-regulatory elements i and j. N represents the number of nodes in the combined regulation network of drought-resistant cis-regulatory elements. Let be the node degree of the a-th drought-resistant cis-regulatory element node.

[0070] Figure 3 The results of rule construction for drought-resistant cis-regulatory elements from the perspective of combined regulatory networks are presented. Figure 3 a shows the correlation structure between the frequencies of drought-resistant cis-regulatory elements constructed based on correlation heatmaps. Figure 3 b illustrates a functional interaction network consisting of drought-resistant cis-regulatory elements. Figure 3 c. The average node degree of the combined control network of drought-resistant cis-regulatory elements and its functional subnetworks was compared using bar charts. Figure 3d. Drought-response regulatory elements are ranked according to node degree centrality in the functional interaction network, highlighting the differences in the proportion of pivotal elements (top 5% of node degree centrality) across different functional categories. This invention demonstrates that, at the module level, drought response regulatory networks are not primarily organized around classic ABA-related motifs. Instead, LTR, W-box, MYC, and MYB motifs recur across different species, serving as key pivotal nodes in the network.

[0071] Step S4 involves constructing a cross-species drought resistance cis-regulatory network based on the correspondence between drought resistance cis-regulatory elements and transcription factor binding motifs, and performing conservation analysis to obtain cross-species conserved edges and modules. Specifically, step S4 includes:

[0072] Step S41: Based on the correspondence between drought resistance cis-regulatory elements and transcription factor binding motifs, transcription factors, drought resistance cis-regulatory elements, and target genes are associated to construct a cross-species drought resistance cis-regulatory network.

[0073] In step S41, the target gene is a gene containing the hit site of the cis-regulatory element. By default, the target gene and its corresponding gene regulation-related sequence are obtained from the reference genome and gene annotation file. As mentioned earlier, the drought-resistant cis-regulatory element was obtained through transcription factor binding motifs, thus establishing a correspondence between the two.

[0074] Step S42: Conservatism analysis is performed on the cross-species drought resistance cis-regulatory network to obtain cross-species conservative edges and conservative modules; wherein, the cross-species conservative edges and conservative modules constitute the combined regulatory rules of drought resistance cis-regulatory elements from the perspective of the cis-regulatory network.

[0075] In step S42, cross-species conserved edges are defined as follows: in the homologous genomes corresponding to the same transcription factor family, a "transcription factor family—drought-resistant motif—target gene family" association exists in all species. It should be noted that the cis-regulatory element network of this invention includes the combinatorial regulatory network constructed in step S3 and the cis-regulatory network constructed in step S4. The combinatorial regulatory network in step S3 is constructed based on the quantitative relationship of drought-resistant cis-elements, while the cis-regulatory network in step S4 is constructed based on the association between transcription factors, drought-resistant cis-elements, and target genes. These two networks obtain their combinatorial regulatory rules from two different perspectives. By combining the network topology features obtained in step S3 and the cross-species conserved edges and conserved modules obtained in step S4, a complete combinatorial regulatory rule for drought-resistant cis-regulatory elements can be obtained, thus providing target design ideas for subsequent molecular breeding.

[0076] Figure 4 This is the result of constructing combination rules for drought-resistant cis-regulatory elements from the perspective of cis-regulatory networks. Figure 4 a and Figure 4 b describes a conserved family of transcription factors and their target gene families in the homologous drought-resistance cis-regulatory network. Figure 4 c illustrates the conservative family-level regulatory patterns identified in the homologous drought-resistance cis-regulatory network. Figure 4 d highlights the conservative family-level regulatory pattern in the homologous drought-resistant cis-regulatory network. Figure 4 e presents the conserved drought response PWM (position weight matrix) identified in the homologous drought resistance cis-regulatory network. The results indicate that 51 conserved drought resistance co-regulatory modules were identified, centered on MYB, bHLH, and DOF transcription factors.

[0077] Step S5: Calculate the evolutionary rate and positional distribution relationship of drought resistance cis-regulatory elements. Step S4 specifically includes:

[0078] It should be noted that step S5 aims to construct cis-regulatory evolutionary constraint rules, which are also part of the regulatory rule construction in this application. Drought-resistance cis-regulatory elements are short DNA sequence patterns located in non-coding regulatory regions that participate in drought-related transcriptional regulation. Non-coding regulatory regions correspond to coding regions; the former mainly undertakes expression regulation functions, while the latter mainly undertakes protein coding functions. A coding region refers to the DNA sequence portion of a gene that encodes the amino acid sequence of a protein, i.e., the CDS region. Non-coding regulatory regions refer to DNA sequence regions that do not directly encode proteins but are related to gene expression regulation. These include promoter regions, 5'UTR, 3'UTR, regulatory-related portions of introns, upstream and downstream regulatory regions of transcription termination sites, and other intergene regulatory regions.

[0079] Step S51: Obtain homologous genes and their corresponding regulatory-related region sequence pairs through multi-species genome collinearity analysis.

[0080] In step S51, the multi-species genome refers to the reference genomes of multiple species. Homologous genes are genes that originate from a common ancestor and have significant sequence similarity and comparability across different species. Corresponding gene regulatory region sequence pairs are two one-to-one regulatory region sequences formed by extracting the gene regulatory region sequences of a pair of identified homologous genes in two species.

[0081] Step S52: Perform multiple sequence alignment on the homologous gene regulatory region sequences to obtain alignment results, and calculate the relative evolutionary rate of non-coding regions and coding regions based on the alignment results.

[0082] In step S52, homologous gene regulatory region sequence pairs are mainly used for the conservation and evolutionary rate analysis of non-coding regulatory regions. The alignment result refers to the base correspondence result obtained after performing multiple sequence alignment on the homologous gene regulatory region sequences obtained in step S51. It includes the matching, substitution, insertion, and deletion information of sequences from different species at each alignment site, and serves as the basis for calculating the substitution rate of non-coding regulatory regions.

[0083] Furthermore, based on the alignment results, the substitution rate Kn of the non-coding regulatory region is calculated and normalized using the synonym substitution rate Ks of the coding region to obtain the relative evolution rate of the non-coding regulatory region. The corresponding formula is:

[0084]

[0085]

[0086] in, Replace the distance for non-coded regions. The length of the non-coded region. Synonym substitution rate This represents the relative evolution rate of non-coding regions.

[0087] Furthermore, based on the alignment results, the non-synonymous substitution rate is calculated separately. With synonym substitution rate This is used to characterize the evolution rate of the coding region. The non-synonymous substitution rate and the synonymous substitution rate are calculated as follows:

[0088]

[0089]

[0090] in, The distance between non-synonymous substitutions in the coding region. This represents the number of non-synonymous sites. The distance for synonym substitutions in the encoding region. The number of synonymous sites. This is determined by the non-synonymous substitution rate. With synonym substitution rate The calculation can obtain information on the evolutionary rate of the coding region and compare it with the evolutionary rate of the non-coding regulatory region, thereby assessing the selection pressure in the region where drought-resistant cis-regulatory elements are located and their evolutionary conservation at different genomic locations.

[0091] Step S53: Group the drought resistance cis-regulatory element hit sites according to the type of gene regulatory region, including coding region and regulatory region and different segments of the regulatory region, and compare the differences in evolutionary rate distribution under different region types to obtain the element position-evolutionary constraint rule (i.e., cis-regulatory evolutionary constraint rule).

[0092] Figure 5 The results of constructing evolutionary constraint rules for drought-resistant cis-regulatory elements are presented. Figure 5 a demonstrates the evolutionary rate of homologous gene coding regions across species. Figure 5 b shows the evolutionary rate of non-coding regions of homologous genes across species. Figure 5 c describes the evolution rate of drought-associated k-mers located in the region 0–4,000 bp upstream of the transcription start site (TSS). Figure 5 d describes the evolution rate of drought-related k-mers located 4,000–10,000 bp upstream of the transcription start site (TSS). The results show that the regulatory region evolves faster than the coding sequence, and its evolution rate increases in a distance-dependent manner with increasing distance from the TSS.

[0093] Based on the same technical concept as the foregoing embodiments, this invention also provides a crop drought resistance cis-regulatory rule construction system based on comparative genomics and cis-regulatory element network analysis, comprising: a candidate drought resistance motif subset construction module, used to construct a set of drought resistance gene regulation-related region sequences, using transcription factor binding motifs as reference motifs, and screening candidate drought resistance motif subsets from the drought resistance gene regulation-related region sequence set; an element co-occurrence relationship acquisition module, used to calculate the co-occurrence relationship of different drought resistance cis-regulatory elements within the same gene regulation-related region sequence based on the candidate drought resistance motif subsets; and a combined regulatory network construction module, used to construct a combined regulatory network based on different drought resistance cis-regulatory elements. The co-occurrence frequency of drought-resistant cis-regulatory elements within the same gene regulatory region sequence is used to construct a combined regulatory network of drought-resistant cis-regulatory elements and perform topological feature analysis to obtain network topological features. A cis-regulatory network construction module is used to construct a cross-species drought-resistant cis-regulatory network based on the correspondence between drought-resistant cis-regulatory elements and transcription factor binding motifs, and perform conservation analysis to obtain cross-species conserved edges and conserved modules. The network topological features and cross-species conserved edges and conserved modules together constitute the combined regulatory rules of drought-resistant cis-regulatory elements. An element evolution rate calculation module is used to calculate the evolution rate and positional distribution relationship of drought-resistant cis-regulatory elements using comparative genomics methods.

[0094] Based on the same technical concept as the foregoing embodiments, the present invention also provides an electronic device, including a memory and a processor, wherein the memory stores program instructions that are executed by the processor, and the processor calls the program instructions to execute the method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis.

[0095] Based on the same technical concept as the foregoing embodiments, the present invention also provides a non-transitory computer-readable storage medium, characterized in that the non-transitory computer-readable storage medium stores computer instructions, the computer instructions causing the computer to execute the method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis.

[0096] In summary, this invention first integrates sequences of drought-resistance gene regulatory regions and information on drought-resistance cis-regulatory elements from multiple species, and systematically analyzes the location distribution characteristics and spatial co-occurrence relationships of drought-resistance cis-regulatory elements in different gene regulatory regions. Based on this, a combinatorial regulatory network of drought-resistance cis-regulatory elements is constructed, and the combinatorial regulatory rules of these elements are obtained through network topology analysis. Furthermore, a "transcription factor—cis-regulatory element—target gene" cis-regulatory network is constructed, and conserved regulatory relationships are identified through cross-species comparative analysis. Simultaneously, comparative genomics methods are used to calculate the evolutionary rates of non-coding and coding regions, and the evolutionary constraints of cis-regulatory elements at different genomic locations are analyzed. This invention can systematically reveal the spatial organization characteristics, combinatorial regulatory patterns, and evolutionary conservation of drought-resistance cis-regulatory elements at a cross-species scale, providing methods and basis for research on crop drought resistance regulation mechanisms and drought-resistant molecular breeding.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing cis-regulatory rules for crop drought resistance based on comparative genomics and cis-regulatory element network analysis, characterized in that, include: A set of gene regulation-related region sequences was constructed, and transcription factor binding motifs were used as reference motifs. A subset of candidate drought-resistant motifs was then screened from the set of gene regulation-related region sequences. Based on the candidate drought-resistant motif subset, calculate the co-occurrence relationship of different drought-resistant cis-regulatory elements within the same gene regulatory region sequence; Based on the co-occurrence frequency of different drought-resistance cis-regulatory elements within the same gene's regulatory region sequence, a combined regulatory network of drought-resistance cis-regulatory elements was constructed and its topological features were analyzed to obtain the network's topological characteristics. Based on the correspondence between drought resistance cis-regulatory elements and transcription factor binding motifs, a cross-species drought resistance cis-regulatory network was constructed and its conservation was analyzed to obtain cross-species conserved edges and conserved modules. We used comparative genomics to calculate the evolutionary rate and positional distribution of drought-resistance cis-regulatory elements.

2. The method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis as described in claim 1, characterized in that, A set of gene regulation-related region sequences was constructed, and transcription factor binding motifs were used as reference motifs. A subset of candidate drought-resistant motifs was obtained from this set of gene regulation-related region sequences, including: Reference genome sequences and gene annotation files were obtained from genome databases of multiple plant species; Gene regulation-related region sequences were extracted based on the reference genome sequence and gene annotation file to construct a set of gene regulation-related region sequences; The set of sequences related to the regulation of the genes is screened according to the preset drought-resistant genes to obtain the set of sequences related to the regulation of drought-resistant genes. Using transcription factor binding motifs as reference motifs, a subset of candidate drought-resistant motifs was obtained by screening from the set of sequences related to the regulation of drought-resistant genes.

3. The method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis as described in claim 2, characterized in that, Using transcription factor binding motifs as reference motifs, a subset of candidate drought-resistant motifs was obtained by screening from the set of sequences related to the regulation of drought-resistant genes, including: Obtain transcription factor binding motifs from publicly available transcription factor binding site databases; Candidate drought-related motifs were identified in the set of sequences related to the regulation of drought-resistant genes using a motif discovery algorithm. Using the transcription factor binding motif as a reference motif, the candidate drought-resistant motifs are screened to obtain a subset of candidate drought-resistant motifs.

4. The method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis as described in claim 1, characterized in that, Based on the candidate drought-resistant motif subset, the co-occurrence relationships of different drought-resistant cis-regulatory elements within the same gene regulatory region sequence were calculated, including: A position weight matrix is ​​constructed based on the representation of motifs in the candidate drought-resistant motif subset; The location weight matrix is ​​used to perform a whole-genome scan in the gene regulatory region sequences to obtain the hit sites of drought resistance cis-regulatory elements; wherein, the hit sites of drought resistance cis-regulatory elements refer to the location information of drought resistance cis-regulatory elements. The drought-resistant cis-regulatory elements were statistically analyzed according to the gene regulation-related region type to obtain the distribution ratio of each drought-resistant cis-regulatory element in different gene regulation-related regions; the functional categories of each drought-resistant cis-regulatory element were annotated based on the functional annotation database, and the proportion of each functional category in different gene regulation-related regions was calculated to obtain the positional preference rules of drought-resistant cis-regulatory elements. Calculate the co-occurrence relationships of different drought-resistance cis-regulatory elements within the same gene's regulatory region sequence, and statistically analyze their relative orientation and spacing distribution.

5. The method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis as described in claim 1, characterized in that, Based on the co-occurrence frequency of different drought-resistance cis-regulatory elements within the same gene's regulatory region, a combined regulatory network of drought-resistance cis-regulatory elements was constructed and its topological features were analyzed to obtain the network's topological characteristics, including: Using drought-resistance cis-regulatory elements as network nodes, an element co-occurrence matrix of drought-resistance cis-regulatory elements is constructed based on their co-occurrence frequency in sequences of multiple gene regulatory regions. A combined regulation network of drought-resistant cis-regulatory elements is constructed based on the element co-occurrence relationship matrix. A topological feature analysis is performed on the combined control network to obtain network topological features; wherein, the network topological features constitute the combined control rules of drought-resistant cis-regulatory elements from the perspective of the combined control network.

6. The method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis as described in claim 1, characterized in that, Based on the correspondence between drought resistance cis-regulatory elements and transcription factor binding motifs, a cross-species drought resistance cis-regulatory network was constructed and its conservation was analyzed to obtain cross-species conserved edges and conserved modules, including: Based on the correspondence between drought resistance cis-regulatory elements and transcription factor binding motifs, transcription factors, drought resistance cis-regulatory elements, and target genes are linked to construct a cross-species drought resistance cis-regulatory network. Conservatism analysis was performed on the cross-species drought resistance cis-regulatory network to obtain cross-species conservative edges and conservative modules; wherein, the cross-species conservative edges and conservative modules constitute the combined regulatory rules of drought resistance cis-regulatory elements from the perspective of the cis-regulatory network.

7. The method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis as described in claim 1, characterized in that, The evolutionary rate and positional distribution of drought resistance cis-regulatory elements were calculated using comparative genomics methods, including: Homologous genes and their corresponding regulatory-related sequence pairs were obtained through multi-species genome collinearity analysis; Multiple sequence alignment is performed on the sequences of homologous gene regulatory regions to obtain alignment results, and the relative evolutionary rate of non-coding and coding regions is calculated based on the alignment results. The sites of drought-resistant cis-regulatory elements were grouped according to the type of gene regulatory region they belonged to, including coding regions, regulatory regions, and different segments of the regulatory region. The differences in the distribution of evolutionary rates under different region types were compared to obtain the element location-evolutionary constraint rules.

8. A system for constructing cis-regulatory rules for crop drought resistance based on comparative genomics and cis-regulatory element network analysis, characterized in that, include: The candidate drought-resistant motif subset construction module is used to construct a set of sequences related to drought-resistant gene regulation. The transcription factor binding motif is used as a reference motif to screen and obtain a subset of candidate drought-resistant motifs from the set of sequences related to drought-resistant gene regulation. The co-occurrence relationship acquisition module is used to calculate the co-occurrence relationship of different drought-resistant cis-regulatory elements in the same gene regulatory region sequence based on the candidate drought-resistant motif subset; The combined regulatory network construction module is used to construct a combined regulatory network of drought resistance cis-regulatory elements based on the number of co-occurrences of different drought resistance cis-regulatory elements in the same gene regulatory region sequence and perform topological feature analysis to obtain the network topological features; A cis-regulatory network construction module is used to construct a cross-species drought resistance cis-regulatory network and perform conservation analysis based on the correspondence between drought resistance cis-regulatory elements and transcription factor binding motifs, so as to obtain cross-species conserved edges and conserved modules; wherein, the network topological features and cross-species conserved edges and conserved modules together constitute the combinatorial regulatory rules of drought resistance cis-regulatory elements. The element evolution rate calculation module is used to calculate the relationship between the evolution rate and positional distribution of drought-resistant cis-regulatory elements by combining comparative genomics methods.

9. An electronic device, characterized in that, The system includes a memory and a processor, wherein the memory stores program instructions that are executed by the processor, and the processor invokes the program instructions to execute the method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis as described in any one of claims 1 to 7.

10. A non-transitory computer-readable storage medium, characterized in that, The non-transitory computer-readable storage medium stores computer instructions that cause the computer to execute the method for constructing crop drought resistance cis-regulatory rules based on comparative genomics and cis-regulatory element network analysis as described in any one of claims 1 to 7.

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