Method for comparative evolution analysis based on soybean-related WD40 gene

Abstract

The invention provides a method for comparative evolution analysis based on soybean-related WD40 genes, and relates to the technical field of plant molecular markers. The method for comparative evolution analysis based on the soybean-related WD40 gene specifically comprises the following steps: S1, identifying WD40 gene family members; s2, chromosome distribution characteristics; s4, performing phylogenetic analysis; s5, carrying out Ka / Ks analysis and collinearity analysis; s6, gene structure, Motif and protein structure; and S7, analyzing a cis-regulatory element. The mining and analysis method of the WD40 gene related to the gray mold and the salt stress of the wild soybean and the cultivated soybean is based on a whole genome sequence database, and 497 and 500 WD40 family members are respectively identified by utilizing a bioinformatics method according to the whole genome database of the wild soybean and the cultivated soybean, so that the WD40 gene related to the gray mold and the salt stress of the wild soybean and the cultivated soybean is obtained. Gene characteristics and evolution comparative analysis is carried out on the genes, and the analysis result provides important reference value for improving gray mold resistance and salt tolerance of the cultivated soybeans through genetic engineering means in the future so as to improve the yield of the soybeans and improve the quality of the soybeans.

Description

Technical Field

[0001] The invention relates to the technical field of plant molecular markers, in particular to a method for comparative evolution analysis based on soybean-related WD40 genes. Background Art

[0002] Soybean is an important source of vegetable oil and protein in my country, but the production of soybean is seriously damaged by gray mold and salt stress, and the discovery of excellent germplasm resources is lagging behind. Therefore, the discovery of gray mold resistance and salt tolerance genes is crucial for breeding soybean varieties resistant to gray mold and tolerant to salt.

[0003] Wild soybean is the closest ancestor of cultivated soybean. During the domestication of soybean, cultivated soybean lost a large number of genes, resulting in a decrease in genetic diversity. Wild soybean germplasm resources are rich and contain many potential genes for disease resistance and salt-alkali tolerance, which can help broaden the genetic diversity of cultivated soybean. The WD40 domain is one of the most abundant domains in the eukaryotic genome and one of the most interactive domains. It is a regulatory protein superfamily that is widely present in eukaryotic organisms and plays an important role in regulating plant growth and development. Previous studies have shown that the WD40 domain has the function of improving soybean salt tolerance or resistance to gray mold, but the identification and comparative analysis of the WD40 gene family of wild soybean and cultivated soybean has not yet been reported.

[0004] Therefore, the present invention proposes a method for identification and comparative evolution analysis based on soybean-related WD40 genes, which provides certain support for the breeding of soybean salt-tolerant or gray mold-resistant transgenic plants. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for identification and comparative evolution analysis based on soybean-related WD40 genes, which solves the problem that the identification and comparative analysis of the WD40 gene family of wild soybean and cultivated soybean have not been reported.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for comparative evolution analysis based on soybean-related WD40 genes, specifically comprising the following steps:

[0007] S1. Identification of WD40 gene family members

[0008] The genome sequence files, amino acid protein sequence files, gtf annotation files, gff annotation files, and CDS sequence files of wild soybean and cultivated soybean were downloaded from the Ensembl Plants database and the NCBI database. The accession number PF00400 of the conserved domain of WD40 protein in the Pfam database was obtained from the NCBI website. The hidden Markov model file PF00400.hmm of the conserved domain of WD40 protein was downloaded from the Pfam database. The WD40 protein sequence files of wild soybean and cultivated soybean were screened using the hmmer 3.4 software in the Linux-Ubuntu operating system. The E-value was less than 0.01, and the first WD40 gene sequence was selected from the protein sequence encoded by the same gene.

[0009] S2. Chromosome distribution characteristics

[0010] By downloading the wild soybean and cultivated soybean genome data annotation files, we searched according to the WD40 member gene names, determined the chromosome length and gene location, and mapped them;

[0011] S3. Physicochemical properties and subcellular localization

[0012] TBtools software was used to analyze the physicochemical properties of wild soybean and cultivated soybean WD40 gene proteins, including molecular weight, amino acid length, and isoelectric point. CELLO v.2.5 was used to predict the subcellular localization of wild soybean and cultivated soybean WD40 gene proteins. SignalP 6.0 online tool was used to analyze the signal peptides of wild soybean and cultivated soybean WD40 genes.

[0013] S4. Phylogenetic Analysis

[0014] Multiple sequence alignment of wild soybean and cultivated soybean WD40 gene family protein sequences was performed using MEGA11 software based on MUSCLE. Based on the aligned sequences, the neighbor-joining method was used to construct a phylogenetic tree. The bootstrap value was set to 1000, and other parameters were default. The phylogenetic tree results were saved as nwk files and imported into the iTOL online website for beautification. Collinearity analysis was performed using the jcvi software on the Linux system.

[0015] S5.Ka / Ks analysis and collinearity analysis

[0016] Prepare the whole genome homologous pairs of wild soybean and cultivated soybean, the homologous pairs of WD40 gene family, and the corresponding gff and cds files obtained by colinearity analysis, and use TBtools software to calculate and compare the Ka / Ks of the whole genome homologous pairs of wild soybean and cultivated soybean and the homologous pairs of WD40 gene family;

[0017] S6. Gene structure, motif, protein structure

[0018] The TBtools software was used to analyze the gene structure of the WD40 family of wild soybean and cultivated soybean. The GXF Stat module was used to count the number of exons, gene length and other information. The MEME online tool was used to analyze the conserved motifs. The secondary structure of the protein of the wild soybean and cultivated soybean WD40 family gene members was predicted on the SOPMA online website. The 10 wild soybean and cultivated soybean WD40 family homologous gene pairs were compared in the Phyre2 database to create a model.

[0019] S7. Analysis of cis-regulatory elements

[0020] The wild soybean and cultivated soybean genome sequences and gtf annotation sequences were processed using bedtools software on the Linux system, and the 1000bp upstream and 500bp downstream of the transcription start site of the WD40 gene were extracted as the promoter region. The cis-regulatory elements in the promoters of wild soybean and cultivated soybean WD40 family members were analyzed using the PlantCARE database website, and visual analysis was performed using R software.

[0021] The present invention provides a method for comparative evolution analysis based on soybean-related WD40 genes, which has the following beneficial effects:

[0022] The present invention provides a method for comparative evolution analysis of soybean-related WD40 genes. The present invention screens and identifies all WD40 gene family members contained in wild soybean and cultivated soybean based on protein sequences at the whole genome level of wild soybean and cultivated soybean. Compared with other databases such as transcriptome analysis, more and more complete WD40 gene family members can be obtained.

[0023] The present invention provides a method for comparative evolution analysis based on soybean-related WD40 genes. Based on bioinformatics analysis technology, the present invention compares and analyzes the chromosome location, physicochemical properties and subcellular localization, gene structure, motif, protein structure and cis-acting element characteristics of all WD40 gene family members in wild soybean and cultivated soybean, providing valuable information for verifying the functions of wild soybean and cultivated soybean WD40 genes in the process of plant development.

[0024] The present invention provides a method for comparative evolution analysis based on soybean-related WD40 genes. Based on the WD40 genes screened from wild soybean and cultivated soybean genomes, the present invention further performs systematic evolution and colinearity analysis, and analyzes the selection pressures occurring in the evolution of wild soybean to cultivated soybean from the perspectives of the whole genome and a single gene family, which is of great significance for analyzing the species evolution of wild soybean and cultivated soybean.

[0025] The present invention provides a method for comparative evolution analysis based on soybean-related WD40 genes. The present invention relates to a bioinformatics analysis method for screening WD40 gene families based on wild soybean and cultivated soybean genome sequences, which is universal for comparative analysis of other soybean-related gene families. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram comparing the chromosome density and chromosome distribution characteristics of the WD40 gene family in wild soybean and cultivated soybean of the present invention;

[0027] Figure 2 This is a schematic diagram of the evolutionary tree analysis of the wild soybean and cultivated soybean WD40 genes of the present invention;

[0028] Figure 3 This is a schematic diagram of the synteny points between wild soybean and cultivated soybean proteins of the present invention;

[0029] Figure 4 It is a histogram of the synlinear depth of wild soybean and cultivated soybean of the present invention;

[0030] Figure 5 This is a schematic diagram of the colinearity analysis of the protein sequences of the wild soybean and cultivated soybean WD40 gene family of the present invention;

[0031] Figure 6 It is a schematic diagram of the Ka / Ks selection pressure of all genes and WD40 gene homologous genes of wild soybean and cultivated soybean of the present invention;

[0032] Figure 7 It is a schematic diagram of the correlation analysis of the number and length of exons of 835 homologous gene pairs of the wild soybean and cultivated soybean WD40 gene families of the present invention;

[0033] Figure 8 This is a schematic diagram of the analysis of the number of exons of the wild soybean and cultivated soybean WD40 gene family genes of the present invention;

[0034] Fig. 9 This is a schematic diagram of gene length analysis of wild soybean and cultivated soybean WD40 gene family of the present invention;

[0035] Fig.10It is a schematic diagram of correlation analysis of the number of genes with different exon numbers and different lengths in the wild soybean and cultivated soybean WD40 gene families of the present invention;

[0036] Fig.11 This is a schematic diagram of the Motif analysis of the wild soybean and cultivated soybean WD40 gene family of the present invention;

[0037] Fig.12 It is a schematic diagram of the correlation analysis of the contents of four structural elements, namely, random coil, extended chain, α-helix and β-turn, in 10 homologous gene pairs of WD40 genes in wild and cultivated soybeans of the present invention;

[0038] Fig.13 Schematic diagram of comparative analysis of protein tertiary structures of 9 wild and cultivated soybean WD40 gene homologous genes of the present invention;

[0039] Fig.14 This is a schematic diagram of the analysis of cis-regulatory elements of the promoter of homologous genes of the wild soybean WD40 gene family of the present invention;

[0040] Fig.15 This is a schematic diagram of the analysis of cis-regulatory elements of the promoter of homologous genes of the cultivated soybean WD40 gene family of the present invention;

[0041] Fig.16 The figure is a schematic diagram showing the relationship between the numbers of 17 cis-acting elements in the wild soybean and cultivated soybean WD40 gene promoters of the present invention. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] This example provides an analysis method for downloading wild soybean and cultivated soybean genome data and screening and identifying the WD40 gene family:

[0045] 1. Download and obtain wild soybean and cultivated soybean genome data files

[0046] The full-length DNA fasta sequence files, amino acid protein fasta sequence files, gtf annotation files, gff annotation files, and CDS sequence files of wild soybean and cultivated soybean were downloaded from the Ensembl Plants database (https: / / plants.ensembl.org / index.html) and the NCBI database (https: / / ftp.ncbi.nlm.nih.gov / genomes / genbank / ).

[0047] 2. Download and obtain the hidden Markov model file of wild soybean and cultivated soybean WD40 protein

[0048] Obtain the accession number PF00400 of the conserved domain of WD40 protein in the Pfam database from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and download the hidden Markov model file of the conserved domain of WD40 protein from the Pfam database (http: / / pfam-legacy.xfam.org / ).

[0049] 3. Screening and obtaining the names and protein sequences of the wild soybean and cultivated soybean WD40 gene family

[0050] Install the Linux-Ubuntu virtual machine operating system under the Windows system, install the Conda23.5.0 software in the virtual machine, and install the hmmer 3.4, seqtk 1.4, and seqkit 2.7.0 software through the bioconda channel.

[0051] After the files were processed by seqtk 1.4 and seqkit 2.7.0 software, the WD40 protein sequences of wild soybean and cultivated soybean were screened according to the Pfam file using hmmer 3.4 software. The IDs of wild soybean and cultivated soybean WD40 gene family proteins were screened with an E-value less than 0.01. According to the ID, the fasta sequence corresponding to the ID was extracted from the protein sequence file.

[0052] Example 2

[0053] This example provides a method for analyzing the chromosome location, physicochemical properties, and subcellular localization of the WD40 gene family in wild soybean and cultivated soybean:

[0054] 1. Chromosome localization analysis method of WD40 gene family in wild soybean and cultivated soybean

[0055] By downloading the wild soybean and cultivated soybean genome data annotation files, we searched according to the names of WD40 member genes, determined the length of the chromosome and the location of the chromosome where the gene was located, and mapped it.

[0056] 2. Physicochemical properties and subcellular localization analysis methods of candidate genes of WD40 family in wild and cultivated soybeans

[0057] TBtools software (https: / / tbtools.cowtransfer.com / s / 0a9cbf41b47b4a) was used to analyze the physicochemical properties of wild soybean and cultivated soybean WD40 gene proteins, such as molecular weight, amino acid length, and isoelectric point. CELLOv.2.5 (http: / / cello.life.nctu.edu.tw / ) was used to predict the subcellular localization of wild soybean and cultivated soybean WD40 gene proteins. The signal peptides of wild soybean and cultivated soybean WD40 genes were analyzed using the SignalP 6.0 online tool (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ).

[0058] Example 3

[0059] This example provides a method for phylogenetic analysis, colinearity, and evolutionary analysis of the WD40 gene family of wild soybean and cultivated soybean:

[0060] 1. Phylogenetic analysis of the WD40 gene family in wild and cultivated soybeans

[0061] The multiple sequence alignment of the wild soybean and cultivated soybean WD40 gene family protein sequences was performed using MEGA11 software based on MUSCLE. Based on the aligned sequences, the neighbor-joining method was used to construct a phylogenetic tree. The bootstrap value was set to 1000, and other parameters were default. The phylogenetic tree results were saved as nwk files and imported into the iTOL online website (https: / / itol.embl.de / ) for beautification.

[0062] 2. Collinearity analysis of the WD40 gene family between wild soybean and cultivated soybean

[0063] Prepare the wild soybean and cultivated soybean WD40 protein and site files required by the jcvi software, perform collinearity analysis using the jcvi software on the Linux system, and draw the synlinearity dot map and synlinearity depth histogram of wild soybean and cultivated soybean proteins.

[0064] 3. Ka / Ks evolution analysis of WD40 gene family in wild and cultivated soybean

[0065] The whole genome homologous pairs of wild soybean and cultivated soybean, the homologous pairs of WD40 gene family, and the corresponding gff and cds files obtained by colinearity analysis were prepared, and the Ka / Ks of the homologous pairs in the whole genome of wild soybean and cultivated soybean and the homologous pairs in the WD40 gene family were calculated and compared using TBtools software.

[0066] Example 4

[0067] This example provides a method for analyzing the gene structure, motif, protein structure, and cis-acting elements of the WD40 family of wild soybean and cultivated soybean:

[0068] 1. Analysis of gene structure, motif and protein structure of WD40 family in wild soybean and cultivated soybean

[0069] The gene structure of the WD40 family of wild soybean and cultivated soybean was analyzed using TBtools software (https: / / www.yuque.com / cjchen / hirv8i), and the GXF Stat module was used to count the number of exons, gene length and other information. The MEME (https: / / meme-suite.org / meme / tools / meme) online tool was used for conservative motif analysis. The secondary structure of the protein of the wild soybean and cultivated soybean WD40 family gene members was predicted on the SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa%20_sopma.html) online website. The 10 wild soybean and cultivated soybean WD40 family homologous gene pairs were compared in the Phyre2 database (http: / / www.sbg.bio.ic.ac.uk / phyre2 / html / page.cgi?id=index) to create a model.

[0070] 2. Analysis of cis-regulatory elements of WD40 family genes in wild and cultivated soybeans

[0071] The wild soybean and cultivated soybean genome sequences and gtf annotation sequences were processed using bedtools software on the Linux system, and the 1000bp upstream and 500bp downstream of the transcription start site of the WD40 gene were extracted as the promoter region. The cis-regulatory elements in the promoters of wild soybean and cultivated soybean WD40 family members were analyzed using the PlantCARE database website, and visual analysis was performed using R software.

[0072] Example 5

[0073] This example provides the results of screening wild and cultivated soybean WD40 families based on genomic analysis technology and analyzing their genetic characteristics and phylogenetic comparative analysis:

[0074] 1. Identification of WD40 family gene members in wild and cultivated soybeans

[0075] Based on the protein sequences of wild soybean and cultivated soybean, 497 (Table 1) and 500 (Table 2) WD40 gene family members were identified, respectively, and the gene names were unified according to the gff annotation file.

[0076] Table 1 Names of 497 wild soybean WD40 gene family proteins

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Table 2 Names of 500 cultivated soybean WD40 gene family proteins

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] 2. Chromosome localization analysis of the WD40 gene family in wild and cultivated soybeans

[0095] There are 15, 25, 18, 30, 27, 28, 25, 34, 22, 38, 18, 25, 34, 17, 34, 12, 26, 18, 28, and 23 WD40 gene family members on chromosomes 1-20 of wild soybean, and 13, 27, 19, 28, 29, 28, 22, 36, 24, 37, 17, 21, 36, 14, 41, 12, 29, 19, 24, and 24 WD40 gene family members on chromosomes 1-20 of cultivated soybean, respectively. Figure 1 WD40 gene family members are unevenly distributed on different chromosomes of wild soybean and cultivated soybean. The distribution densities on chromosomes 1-20 of wild soybean are 0.26, 0.51, 0.37, 0.57, 0.65, 0.56, 0.55, 0.70, 0.47, 0.71, 0.46, 0.61, 0.75, 0.34, 0.65, 0.32, 0.63, 0.30, 0.53, and 0.47 Mb, respectively, and on chromosomes 1-20 of cultivated soybean, the distribution densities on chromosomes 1-20 of wild soybean are 0.26, 0.51, 0.37, 0.57, 0.65, 0.56, 0.55, 0.70, 0.47, 0.71, 0.46, 0.61, 0.75, 0.34, 0.65, 0.32, 0.63, 0.30, 0.53, and 0.47 Mb, respectively. The distribution densities on chromosomes 1-20 were 0.23, 0.56, 0.42, 0.53, 0.69, 0.54, 0.49, 0.75, 0.48, 0.72, 0.49, 0.52, 0.78, 0.29, 0.79, 0.32, 0.70, 0.33, 0.47, and 0.50 Mb, respectively; however, the distribution densities on the same chromosome of wild soybean and cultivated soybean were highly positively correlated (R = 0.95, p = 1.6e -10 ).

[0096] 3. Physicochemical properties and subcellular localization of WD40 family genes in wild and cultivated soybeans

[0097] There is diversity in the number of amino acid residues, molecular weight, isoelectric point, instability coefficient, fat solubility index and hydrophilicity index of each member of the WD40 gene family of wild soybean and cultivated soybean. The maximum, minimum and average values ​​are listed in Table 3, respectively. From the isoelectric point, it can be seen that the WD40 gene family members of wild soybean and cultivated soybean include both acidic proteins (PI>0) and alkaline proteins (PI<0); from the instability coefficient, lipid solubility index and total average hydrophilicity (hydrophilicity index), it can be seen that the WD40 gene family members of wild soybean and cultivated soybean have both stable hydrophilic and hydrophobic proteins and unstable hydrophilic and hydrophobic proteins. From the perspective of stability, most of the WD40 gene family members of wild soybean (295) are stable proteins (instability coefficient <40), and most of the WD40 gene family members of cultivated soybean (289) are unstable proteins (instability coefficient >40); from the perspective of hydrophilicity, most of the WD40 gene family members of wild soybean and cultivated soybean (470, 479) are hydrophilic proteins (hydrophilicity index <0).

[0098] Table 3 Physicochemical properties of wild soybean and cultivated soybean WD40 gene proteins

[0099]

[0100]

[0101] The subcellular localization of wild soybean WD40 gene family members is predicted to be nucleus, plasma membrane, cytoplasm, extracellular, mitochondria, and Golgi apparatus (Table 4). In addition, cultivated soybean WD40 gene family members are also present in chloroplasts.

[0102] Table 4 Results of subcellular localization analysis of WD40 gene protein in wild soybean and cultivated soybean

[0103]

[0104] In the wild soybean and cultivated soybean WD40 gene families, there are 7 genes with signal peptides, the type is Sec / SPISignal Peptide (Table 5), and the remaining WD40 genes do not contain signal peptides.

[0105] Table 5 Prediction results of protein signal peptides of wild soybean and cultivated soybean WD40 gene

[0106]

[0107]

[0108] 4. Phylogenetic analysis, colinearity and evolutionary analysis results of the WD40 gene family in wild soybean and cultivated soybean

[0109] (1) Phylogenetic analysis of WD40 gene family members in wild and cultivated soybeans

[0110] Phylogenetic analysis of the protein sequences of the WD40 gene family in wild and cultivated soybean revealed that 497, 500 WD40 proteins were divided into five subfamilies ( Figure 2 ), containing 27, 33, 106, 38, 293; 18, 128, 36, 81, 237 members respectively.

[0111] (2) Colinearity and evolution analysis of WD40 gene family members between wild soybean and cultivated soybean

[0112] The WD40 gene family members of wild soybean and the WD40 gene family members of the closely related species cultivated soybean were analyzed for colinearity ( Figure 3 to Figure 5), it was found that 43,276 and 44,115 genes in the genomes of wild soybean and cultivated soybean formed 1,444 collinear segments (the number of homologous gene pairs ≥ 4), and 819 collinear segments containing at least 10 homologous gene pairs were drawn into a collinearity map ( Figure 3 ).

[0113] In wild soybean and cultivated soybean, 79,742 homologous gene pairs were screened out from 43,276 and 44,115 genes respectively. Further, it was found that 835 homologous gene pairs were formed pairwise between the WD40 genes of 432 wild soybeans and 438 cultivated soybeans. The ratio range of Ka / Ks (ratio analysis of non-synonymous substitution rate Ka and synonymous substitution rate Ks) of all homologous gene pairs in the genomes of wild soybean and cultivated soybean was 0 - 19.98. Among them, 58,793 homologous genes evolved by purifying selection (Ka / Ks < 1), 3 homologous genes did not receive natural selection pressure during evolution (Ka / Ks = 1), 2,437 homologous genes were under weak positive selection (1 < Ka / Ks < 10), and 3 homologous genes were under strong positive selection (10 < Ka / Ks < 20). These genes may be rapidly evolving genes recently and are of great significance for the evolution of the species. The ratio range of Ka / Ks of WD40 homologous gene pairs in wild soybean and cultivated soybean was 0 - 1.73. Among them, 698 homologous genes evolved by purifying selection (Ka / Ks < 1), and 25 homologous genes were under weak positive selection (1 < Ka / Ks < 2)( Figure 6 ).

[0114] 5. Gene Structure and Motif Analysis of WD40 Gene Family in Wild Soybean and Cultivated Soybean

[0115] The number of exons in the WD40 gene family of wild soybean and cultivated soybean was 1 - 42 and 1 - 40 respectively, and their distribution characteristics were similar. The number of genes containing different numbers of exons was positively correlated (R = 0.85, p = 2.5e -08 )( Figure 7 ). The number of genes with 7 exons in the WD40 gene family of wild soybean and cultivated soybean was the largest, which were 39 and 53 respectively( Figure 8 ). The gene lengths of the WD40 gene family in wild soybean and cultivated soybean both showed a normal distribution, and their distribution characteristics were similar. The number of genes with different gene lengths was positively correlated (R = 0.98, p = 2.2e -16 )( Figure 7 ). The gene lengths were 200 - 58,638 bp and 200 - 64,006 bp respectively. Most of the WD40 genes in wild soybean (414) and cultivated soybean (429) were 1,000 - 11,000 bp long( Fig. 9The number of exons of 835 WD40 gene family homologous gene pairs between wild soybean and cultivated soybean (R = 0.95, p < 2.2e -16 ) and gene length (R = 0.86, p < 2.2e -16 ) are significantly positively correlated ( Fig.10 ).

[0116] In the wild soybean WD40 gene family, 492, 425, 414, 430, and 382 members contain Motif1, Motif2, Motif3, Motif4, and Motif5, respectively; in the cultivated soybean WD40 gene family, 481, 454, 469, 451, and 395 members contain Motif1, Motif2, Motif3, Motif4, and Motif5, respectively (Table 6, Fig.11 ).

[0117] Table 6 Conservative Motif sequences of WD40 gene proteins between wild soybean and cultivated soybean

[0118]

[0119]

[0120] 6. Analysis of protein structure of WD40 gene family in wild and cultivated soybean

[0121] The expression of 10 pairs of homologous genes (SoyGsojaF_08G195200, GLYMA_08G195200; SoyGsojaF_07G270300, GLYMA_07G270300; SoyGsojaF_12G182700, GLYMA_12G182700; SoyGsojaF_10G051600, GLYMA_10G051600; SoyGsojaF_20G235800, GLYMA_20G23 5800; SoyGsojaF_08G354500, GLYMA_08G354500; SoyGsojaF_04G228000, GLYMA_04G228000; SoyGsojaF_19G005200, GLYMA_19G005200; SoyGsojaF_10G258300, GLYMA_10G258300; SoyGsojaF_06G131400, GLYMA_06G131400) protein secondary and tertiary structures.

[0122] (1) Prediction of secondary structures of proteins encoded by the WD40 gene family in wild and cultivated soybeans

[0123] The secondary structures of the WD40 gene homologous gene pairs in the 10 wild and cultivated soybeans all included random coils (36.93-55.93%; 35.99-54.94%), extended chains (18.08-37.35%; 18.59-39.51%), α-helices (5.59-28.97%; 5.59-28.85%), and β-turns (3.58-9.02%; 4.18-9.28%), and the main structural element was random coils (Table 7). The correlation between the contents of the four structural elements of random coils, extended chains, α-helices, and β-turns in the WD40 gene homologous gene pairs in the wild and cultivated soybeans was 0.94 (p=5.1e -05 ), 0.97(p=2.7e -06 ), 0.98(p=1.1e -06 ), 0.8(p=0.55e -02 )( Fig.12 ).

[0124] Table 7 Secondary structure composition of proteins encoded by the WD40 gene family in wild soybean and cultivated soybean

[0125]

[0126] (2) Prediction of tertiary structures of proteins encoded by the WD40 gene family in wild and cultivated soybeans

[0127] The confidence of the model created by comparing the protein structures of WD40 gene homologous gene pairs in 10 wild and cultivated soybeans with other homologous protein structures in the Phyre2 database was 99.9-100%. Except for SoyGsojaF_04G228000 and GLYMA_04G228000 (RMSD=7.975), the tertiary structures of the other 9 homologous gene pairs were very similar (RMSD was 0-0.231) ( Fig.13 ).

[0128] 7. Analysis results of cis-regulatory elements of the WD40 gene family in wild and cultivated soybeans

[0129] Screening and identification revealed that the WD40 gene family members of both wild soybean and cultivated soybean had abscisic acid responsiveness, auxin responsiveness, cell cycle regulation, defense and stress responsiveness, drought-inducibility, endosperm expression, endosperm-specific negative expression, flavonoid biosynthetic genes regulation, gibberellin-responsiveness, light responsiveness, low-temperature responsiveness, MeJA-responsiveness, meristem expression, root specificity, salicylic acid responsiveness, seed-specific regulation, and wound-responsiveness. There are 8110 17 cis-acting elements in 497 wild soybean WD40 genes ( Fig.14 ), there are 8151 17 cis-acting elements in 500 cultivated soybean WD40 genes ( Fig.15 The most abundant light-responsive elements in the promoters of the WD40 gene of wild soybean and cultivated soybean were light-responsive elements (4338, 4464), followed by methyl jasmonate response (1184, 1186) and abscisic acid response (735, 701). There was a highly significant positive correlation between the number of 17 cis-acting elements in the promoters of the WD40 gene of wild soybean and cultivated soybean (R=1, p<2.2e -16 )( Fig.16 ).

[0130] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for comparative evolution analysis based on soybean-related WD40 genes, characterized in that: The specific steps include: S1. Identification of WD40 gene family members Download the data files of wild soybean and cultivated soybean from Ensembl Plants database and NCBI database, obtain the accession number PF00400 of the conserved domain of WD40 protein in Pfam database from NCBI website, download the hidden Markov model file PF00400.hmm of the conserved domain of WD40 protein from Pfam database, use hmmer3.4 software to screen the WD40 protein sequence files of wild soybean and cultivated soybean in Linux-Ubuntu operating system, and screen with E-value less than 0.

01. The first WD40 gene sequence is selected from the protein sequence encoded by the same gene; S2. Chromosome distribution characteristics By downloading the wild soybean and cultivated soybean genome data annotation files, we searched according to the WD40 member gene names, determined the chromosome length and gene location, and mapped them; S3. Physicochemical properties and subcellular localization TBtools software was used to analyze the physicochemical properties of wild soybean and cultivated soybean WD40 gene proteins, CELLOv.2.5 was used to predict the subcellular localization of wild soybean and cultivated soybean WD40 gene proteins, and the signal peptides of wild soybean and cultivated soybean WD40 genes were analyzed using the SignalP 6.0 online tool; S4. Phylogenetic Analysis Multiple sequence alignment of wild soybean and cultivated soybean WD40 gene family protein sequences was performed using MEGA11 software based on MUSCLE. Based on the aligned sequences, a phylogenetic tree was constructed using the neighbor-joining method. The phylogenetic tree results were saved as nwk files, imported into the iTOL online website for beautification, and collinearity analysis was performed using the jcvi software on the Linux system. S5.Ka / Ks analysis and collinearity analysis Prepare the whole genome homologous pairs of wild soybean and cultivated soybean, the homologous pairs of WD40 gene family, and the corresponding gff and cds files obtained by colinearity analysis, and use TBtools software to calculate and compare the Ka / Ks of the whole genome homologous pairs of wild soybean and cultivated soybean and the homologous pairs of WD40 gene family; S6. Gene structure, motif, protein structure The TBtools software was used to analyze the gene structure of the WD40 family of wild soybean and cultivated soybean. The GXF Stat module was used to analyze the statistical information. The MEME online tool was used to analyze the conserved motifs. The secondary structure of the protein of the wild soybean and cultivated soybean WD40 family gene members was predicted on the SOPMA online website. The proteins of 10 wild soybean and cultivated soybean WD40 family homologous gene pairs were compared in the Phyre2 database and the model was created. S7. Analysis of cis-regulatory elements The wild soybean and cultivated soybean genome sequences and gtf annotation sequences were processed using bedtools software on the Linux system, and the 1000bp upstream and 500bp downstream of the WD40 gene transcription start site were extracted as the promoter region. The cis-regulatory elements in the promoters of wild soybean and cultivated soybean WD40 family members were analyzed using the PlantCARE database website.

2. The method for comparative evolution analysis based on soybean-related WD40 genes according to claim 1, characterized in that: The wild soybean and cultivated soybean data downloaded in the identification of the WD40 gene family members in step S1 are: genome sequence file, amino acid protein sequence file, gtf annotation file, gff annotation file, and CDS sequence file.

3. The method for comparative evolution analysis based on soybean-related WD40 genes according to claim 1, characterized in that: The physicochemical properties of wild soybean and cultivated soybean WD40 gene proteins in the physicochemical property analysis and subcellular localization in step S3 include molecular weight, amino acid length and isoelectric point.

4. The method for comparative evolution analysis based on soybean-related WD40 genes according to claim 1, characterized in that: In the phylogenetic analysis of step S4, the bootstrap value of the phylogenetic tree was set to 1000, and the other parameters were default.

5. The method for comparative evolution analysis based on soybean-related WD40 genes according to claim 1, characterized in that: In step S6, the GXF Stat module is used to count the number of exons and gene length information in the gene structure, motif, and protein structure.

6. The method for comparative evolution analysis based on soybean-related WD40 genes according to claim 1, characterized in that: In the cis-regulatory element analysis in step S7, R software can be used for visual analysis.

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