Method for carrying out screening identification and functional analysis on poplar latex protein family at whole genome level by utilizing multiple bioinformatics methods

Abstract

The invention discloses a method for screening, identifying and functionally analyzing a poplar latex protein family at a whole genome level by using multiple bioinformatics methods, and relates to the field of plant gene identification and screening. The method comprises the following steps: performing preliminary screening, re-modeling, re-screening and identification on an obtained poplar whole genome file, an annotation information file, a protein sequence file and an MLP protein family structure file to obtain a final poplar MLP gene family member; on-line physicochemical property analysis software is used for carrying out predictive analysis on physicochemical properties, and predictive software is used for carrying out possible function analysis on a gene structure, a conservative structural domain, a promoter region cis-acting element and protein subcellular localization; evolutionary trees of the poplar MLP family members and the arabidopsis MLP family members are constructed through evolutionary tree software, and evolution analysis and classification of the poplar MLP family members and the arabidopsis MLP family members are analyzed. According to the method, the interested poplar MLP family gene can be effectively and quickly screened, and the time and cost of molecular means screening are greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of plant gene identification and screening, and in particular to the use of a variety of bioinformatics methods to identify poplar MLP family genes at the whole genome level and to perform physicochemical property analysis, evolutionary analysis and functional analysis. Background Art

[0002] In 1985, latex protein was first discovered in opium poppy, with a molecular weight of about 20kDa. The protein is distributed in the mature latex ducts of opium poppy and is secreted to the damaged surface when the poppy is mechanically damaged. Subsequently, MLP was found in plants such as Arabidopsis, soybean, tobacco, apple, and grape. Studies have shown that MLP is closely related to stress responses to drought, salt, plant hormones, and pathogen infection. Other studies have shown that the number of MLP genes varies from species to species but is not related to genome size. For example, the size of the Arabidopsis genome is 135M, and 25 MLP genes have been identified. The size of the soybean genome is 978M, but only 9 MLP genes have been identified. The size of the poplar genome is 480M, and the MLP gene in the whole genome has not been identified. MLP protein homologs can be divided into three categories: MLPs, Bet v1, and pathogenesis-related protein 10 (PR-10). The research on Bet v 1 and PR-10 started earlier and was mainly from a medical perspective, because both are allergens in some plants. Some PR-10 proteins show allergenicity and can cause oral allergy syndrome and other conditions through cross-reaction with homologous Bet v 1-specific IgE. Therefore, people have studied Bet v 1 and PR-10 more, but less on MLP. However, compared with the other two homologous proteins, MLP has received increasing attention in recent years because it has a wider range of biological functions. Members of the MLP family can respond to biotic and abiotic stresses and play an important role in the growth and development of plants. At present, the biological function of MLP protein is not very clear, but the study of the expression pattern of MLP under biotic and abiotic stresses shows that it may play an important role in defense or stress response.

[0003] Existing studies have shown that pathogens that invade plants can induce the expression of MLP family proteins, which mainly use innate immunity and acquired resistance signals (SAR) to help plants defend themselves. Yang et al. found that GhMLP28 was expressed in the roots, stems and leaves of upland cotton and was induced by exogenous Verticillium dahliae, indicating that GhMLP28 is involved in the defense process of Verticillium dahliae. Further studies have shown that GhMLP28 interacts with cotton ethylene response factor 6 (GhERF6) both in vivo and in vitro. GhMLP28 can enhance the binding of GhERF6 to the GCC-box recognition site in the upstream region of the PR gene, thereby enhancing the expression of the PR gene and making cotton disease-resistant. MLP is also believed to be related to plant SAR signals. In the mlp mutant of Arabidopsis, leaves farther from the inoculation point lost resistance, indicating that MLP is involved in plant SAR; previous transcriptome analysis showed that the MLP gene is induced in the phloem by pathogen infection and SAR inducers BRs. Therefore, MLPs transmit SAR signals through phloem vessels and play an important role in the SAR signaling pathway, thereby enabling plants to acquire disease resistance. Flavonoids in plants act as a barrier to pathogen infection and have antibacterial activity as phytoalexins. In addition to inducing the expression of disease resistance genes and participating in plant acquired resistance, MLP can also increase plant disease resistance by inducing the formation of flavonoids. In summary, it can be seen that MLP may not have direct antibacterial activity, but can induce PR genes, participate in plant SAR, and biosynthesize substances such as flavonoids, and has indirect antibacterial activity.

[0004] When plants are subjected to abiotic stresses such as drought, low temperature, and salinity, their photosynthesis will be limited and their metabolism will be disordered, resulting in slow growth and development, and even death in severe cases. Plants can participate in abiotic stress responses through effector molecules directly involved in metabolism and their upstream regulatory factors. There are few studies on the effects of MLP protein on abiotic stress in plants, but in recent years, more and more studies have shown that MLP protein may be involved in the abiotic stress response of plants. The cis-elements in the promoter region of genes play a vital role in gene expression. The cis-acting elements in the MLP gene were first identified in Gh-MLP of upland cotton. It was found that the promoter region of Gh-MLP contained cold response elements, SA-induced elements, salt stress response elements, and damage response elements; subsequently, abiotic stress response elements were identified in the promoter regions of tobacco MLP28 and grape VvMLP, including low temperature, light, dehydration, and plant hormone-related response elements such as abscisic acid (ABA), auxin, ethylene (ET), gibberellin (GA), and jasmonic acid (JA), etc., which indicates that MLP may be involved in the response of plants to abiotic stress. In 2016, Wang et al. showed that Arabidopsis MLP43, as a positive regulatory factor, is located in the upstream region of the SnRK2 gene, and can bind to SnRK2 and ABA response element binding factor (ABF1), participating in ABA signaling to regulate plant drought resistance; in addition, the expression of some MLP homologous genes has been shown to be induced by salt and enhance the salt tolerance of plants. In 2021, Li Xinxin et al. found that some MLP genes in apple are induced by ABA, NaCl and drought stress. Protein database interaction prediction shows that apple MLP genes may participate in apple's response to abiotic stress through PRSP, SNRK1 / 2, bHLH, etc.

[0005] In summary, although people have cloned and identified MLP genes from a variety of dicotyledonous plants, and have preliminarily proved that the MLP gene family is involved in plant growth and development and various biotic and abiotic stress responses, MLP genes in poplar, a model plant for woody plant research, have not been reported, and the function of poplar MLP genes in responding to biotic / abiotic stresses is also unclear.

[0006] Nowadays, bioinformatics technology is developing rapidly. Compared with the traditional method of gene screening and identification that only relies on molecular experimental means, bioinformatics methods can quickly, efficiently and conveniently screen and preliminarily analyze the entire gene family. The completion of the whole genome sequencing of poplar also makes it possible to use bioinformatics methods to identify and analyze genes. The use of bioinformatics methods to make preliminary predictions and analyses of the functions of the target gene family helps to quickly understand the gene family that has not been studied in a species and obtain the target gene of interest, thereby greatly reducing time and economic costs.

[0007] In the study of poplar MLP family genes, it is urgently needed to use a variety of bioinformatics methods to screen and identify poplar MLP family genes and explore the possible functions of poplar MLP genes. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using a variety of bioinformatics methods.

[0009] The present invention uses a variety of bioinformatics methods to screen, identify and analyze the functions of the poplar latex protein family at the whole genome level, and is characterized in that the method is as follows:

[0010] 1) Obtain the whole genome file, annotation information file, protein sequence file and MLP protein family structure file Bet_v_1.hmm of poplar, conduct preliminary screening, remodeling and re-screening of poplar MLP gene family members to obtain preliminary poplar MLP gene family members; use the protein conserved domain detection website to identify the poplar MLP gene family members obtained by the preliminary screening to obtain the final poplar MLP gene family members;

[0011] 2) using online physicochemical property analysis software to predict and analyze the physicochemical properties of the screened poplar MLP family member proteins, and using the prediction software to analyze the possible functions of the gene structure, conserved domain, promoter region cis-acting elements and protein subcellular localization of the poplar MLP gene family members in step (1);

[0012] 3) Use phylogenetic tree software to construct phylogenetic trees of poplar MLP family members and Arabidopsis MLP family members, and analyze their evolutionary analysis and classification.

[0013] Further, the poplar MLP gene family members described in step 1) were initially screened as follows:

[0014] HMMER3.0 was used to screen the MLP protein family structure file Bet_v_1.hmm to obtain the initially screened poplar MLP family members; the line number where the ID of the initially screened poplar MLP family member was located was obtained, the line was intercepted using the sed command, and then the ID number was matched and redirected using the grep command, and the sequence was extracted using the samtools tool to obtain the protein sequence file.

[0015] Furthermore, the E-value of hmmsearch in HMMER3.0 is set to 10 -20 .

[0016] Further, the poplar MLP gene family members described in step 1) are remodeled as follows:

[0017] ClustalW software was used to perform multiple sequence alignment of the MLP proteins in the protein sequence files obtained in the preliminary screening. Based on the alignment results, the hidden Markov model was constructed using the hmmbuild algorithm.

[0018] Furthermore, the poplar MLP gene family members described in step 1) were screened again as follows:

[0019] The hidden Markov model constructed in the remodeling was used to repeat the initial screening and perform a secondary screening of the poplar MLP family members to obtain the final gene ID and protein sequence.

[0020] Furthermore, the protein conserved domain detection website was used to identify the poplar MLP gene family members obtained by preliminary screening to obtain the final poplar MLP gene family members, which are as follows:

[0021] The candidate MLP protein sequences were identified by domain identification using SMART and InterPro online software, and proteins with the Bet_v_1 domain were screened out, which were the final members of the poplar MLP gene family.

[0022] Furthermore, in step 2), the online physicochemical property analysis software is used to predict and analyze the physicochemical properties of the screened poplar MLP family member proteins, as follows:

[0023] ExPASy online software was used to analyze the molecular weight, isoelectric point and instability coefficient of poplar MLP family members; MEME online software was used to perform conservative motif analysis on MLP family members.

[0024] Furthermore, in step 2), prediction software is used to analyze the possible functions of the gene structure, conserved domain, cis-acting elements in the promoter region, and protein subcellular localization of the poplar MLP gene family members in step (1), as follows:

[0025] The MLP gene structure was analyzed using TBtools software, and the 2000bp sequences of all family member promoters were integrated into one file. The cis-acting elements of the MLP promoter were predicted and analyzed using PlantCARE online software. The results were visualized using Simple BioSequence Viewer, and the subcellular localization of the poplar MLP protein was predicted using the Wolf PSORT tool. The possible functions of the poplar MLP gene family members were analyzed by analyzing the physicochemical properties, conserved motifs, cis-acting elements, and protein localization.

[0026] Furthermore, the evolutionary tree of the poplar MLP family members and the Arabidopsis MLP family members was constructed using the evolutionary tree software in step 3), and the evolutionary analysis and classification of the two were analyzed as follows:

[0027] MEGA 7.0 software was used to perform multiple sequence alignment of poplar and Arabidopsis MLP proteins, and the nearest neighbor method was used to construct a phylogenetic tree. The GFF file, FASTA file and protein file of the poplar genome were obtained from JGI. The poplar genome was processed by BLAST and GFF files using Tbtools to obtain BLAST results and processed GFF files. The BLAST alignment results and the processed GFF format files were self-aligned using MCScanX software to obtain collinear genes. The collinearity file was processed to obtain the homologous gene pairs of the link. According to the poplar MLP gene family ID selected in the screening and identification of the first poplar MLP gene family members, MLP genes with homology were selected in the homologous gene pair link file.

[0028] Furthermore, the phylogenetic tree was constructed using the nearest neighbor method, and the Bootstrap value was set to 1000.

[0029] The present invention has the following beneficial effects:

[0030] The present invention uses a variety of bioinformatics methods to screen and identify poplar MLP family genes at the whole genome level, predicts and analyzes their evolutionary relationships, gene structures, protein localization and functions, etc., so as to explore the possible functions of poplar MLP genes. The present invention can effectively and quickly screen the poplar MLP family genes of interest, greatly reducing the time and cost of molecular screening. Research and analysis of the entire gene family can lay a foundation for further revealing the regulatory mechanism of the poplar latex protein family involved in abiotic stress and its application in poplar stress resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the chromosome location map of the poplar MLP gene;

[0032] Figure 2 This is the conservative structure and gene structure analysis diagram of the poplar MLP gene family;

[0033] Figure 3 This is the predicted subcellular localization map of the poplar MLP family protein;

[0034] Figure 4This is the evolutionary tree of the MLP genes of poplar and Arabidopsis; the red asterisks represent members of the poplar MLP family, the green spheres represent members of the Arabidopsis MLP family, the red ones represent subfamily I, the blue ones represent subfamily II, and the green ones represent subfamily III;

[0035] Figure 5 This is a diagram of the colinearity analysis within the poplar MLP family genome; the gray lines represent gene pairs with colinearity, and the red lines represent poplar MLP gene pairs with segmental duplications. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0037] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0038] Example

[0039] The method of screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using a variety of bioinformatics methods in this embodiment is as follows:

[0040] 1. Screening and identification of members of the poplar MLP gene family

[0041] The whole genome file, annotation information file, and protein sequence file of Populus trichocarpa were downloaded from the JGI website (https: / / phytozome.jgi.doe.gov / pz / portal.html), and the plant MLP protein family structure file Bet_v_1.hmm was obtained from the Pfam database (https: / / pfam.xfam.org / ). The hmmsearch function in HMMER3.0 was used, and the E-value of the screening was set to 10 -20, and the members of the poplar MLP gene family were preliminarily screened. After obtaining the preliminarily screened poplar MLP family members, the line number of the preliminarily screened result ID was obtained, the line was intercepted using the sed command, and then the ID number was matched and redirected using the grep command. The sequence was extracted using the samtools tool to obtain the protein sequence file. The MLP protein in the protein sequence file was multiple sequence aligned using the ClustalW software. According to the alignment results, the hidden Markov model was constructed using the hmmbuild algorithm. The constructed hidden Markov model was used to repeat the previous "matching and redirecting the ID number using the grep command. Sequence extraction using the samtools tool to obtain the protein sequence file" operation, and the poplar MLP family members were screened for the second time to obtain the final gene ID and protein sequence. The candidate MLP protein sequences were identified by domain structure using the SMART (http: / / smart.embl-heidelberg.de / ) and InterPro (https: / / www.interproinc.com / ) online software, and proteins with the Bet_v_1 domain were screened. Chromosome information and gene density information were extracted from the poplar genome file, and the protein of Bet_v_1 domain of poplar MLP gene family was localized on chromosome using MG2C tool.

[0042] A total of 20 poplar MLP genes were obtained by preliminary screening using the Bet_v_1 structure file, and 46 poplar MLP genes were screened after sequence alignment and remodeling. SMART and Interpro were used to verify that all proteins encoded by the identified genes had Bet_v_1 domains. Finally, a total of 43 poplar MLP genes were screened at the genome level and named PtMLP1-PtMLP43. Chromosome localization analysis showed that PtMLP1-PtMLP43 were distributed on 12 different chromosomes of poplar, among which chromosome 8 had the most poplar MLP genes, with 14, while chromosomes 2, 5, 7, 9, 12, 15, and 19 had no MLP genes. Most genes were distributed at the front and end of the chromosomes, with more genes distributed at the front of chromosome 4 and the end of chromosome 8 ( Figure 1 ).

[0043] 2. Analysis of the physicochemical properties and functions of poplar MLP family members

[0044] The protein sequences of the Bet_v_1 domain encoded by the identified poplar MLP family members were integrated into Fasta sequence files. The molecular weight, isoelectric point and instability coefficient of the poplar MLP family members were analyzed using the ExPASy online software (https: / / web.expasy.org / protparam / ), and the conservative motif analysis of the MLP family members was performed using the MEME online software (http: / / meme-suite.org / tools / meme). The MLP gene structure was analyzed using the TBtools software, and the 2000bp sequence of the promoter of all family members was selected and integrated into a file. The MLP promoter cis-acting element prediction analysis was performed using the PlantCARE online software (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ), and the results were visualized using the Simple BioSequence Viewer. The subcellular localization of the poplar MLP protein was predicted using the Wolf PSORT (https: / / www.genscript.com / wolf-psort / html) tool. The possible functions of the poplar MLP gene family were analyzed by analyzing the physicochemical properties, conserved motifs, cis-acting elements and protein localization.

[0045] Genes in the same family are divided into different subfamilies and may have different functions, while genes with similar structures have similar functions and physicochemical properties, and are clustered together into a subfamily with similar protein localization. Cis-acting element analysis can predict gene function. The above-mentioned analysis of the physicochemical properties and functions of the poplar MLP family members is to classify the genes of the entire family and predict gene functions through multiple results.

[0046] The physicochemical property analysis showed that the physicochemical properties of the proteins encoded by the MLP family genes in poplar trees were different. The protein length (Number of amino acids) was 79 (PtMLP21) to 221 (PtMLP41) amino acids, the predicted protein molecular weight (Molecular Weight) was 8471.61 (PtMLP21) to 24059.16 (PtMLP9) Da, the theoretical isoelectric point (Theoretical PI) was 4.65 (PtMLP7) to 8.73 (PtMLP9), the instability coefficient (Instability Index) was 16.92 (PtMLP14) to 54.69 (PtMLP31), the aliphatic amino acid index (Aliphatic Index) was 74.9 (PtMLP38) to 109.21 (PtMLP37), and the average hydrophilicity (Grand Average of Hydropathicity) was -0.522 (PtMLP38) ~ 0.028 (PtMLP37) (Table 1).

[0047] Table 1 Physicochemical properties and predicted subcellular localization of members of the poplar MLP gene family

[0048]

[0049]

[0050]

[0051]

[0052] MEME analysis showed that the poplar MLP family has three conserved motifs. Class I subfamily members have two conserved motifs (motif1 and motif3). Except for PtMLP3 (which has two conserved motifs), Class II subfamily members have only one conserved motif (motif3), and Class III subfamily members have three conserved motifs. In order to gain a deeper understanding of the structure of the poplar MLP gene family, we conducted an intron and exon distribution analysis. The results showed that most PtMLP genes have two exons, and a small number have only one exon, and all belong to the Class III subfamily. The results show that family members with similar gene structures can be clustered into one category. Genes with closer genetic relationships have higher similarity in gene structure, but there are certain differences between individual genes ( Figure 2 ).

[0053] The 2000bp region upstream of the start codon of the PtMLP gene was selected for cis-acting element analysis. The cis-acting elements were divided into three main categories according to their functions: hormone response elements, biotic and abiotic stress response elements, and growth and development related elements. PtMLP30 had the most cis-acting elements, with 24, while PtMLP19 and PtMLP42 had the least cis-acting elements (2). Hormone response elements were the most common elements in subfamilies I and II, accounting for 44.2% and 46.3%, respectively, while the most common elements in subfamily III were biotic and abiotic stress response elements (45.9%). In the three subfamilies, the least common elements were all growth and development related elements. The most common elements in Class I subfamily were ARE elements and TGACG elements (methyl jasmonate response elements). The most common hormone response element was TGACG element, with 18, and the most common stress response element was ARE element, with 19. The most common hormone response element in the Class II subfamily is the ABRE (abscisic acid response element) element, with 33 elements; the most common stress response element is the ARE element, with 17 elements, and most genes have ABRE elements. The most common hormone response element in the Class III subfamily is the TGACG element, with 64 elements, and the most common stress response element is the ARE element, with 47 elements. Most family genes have TGACG elements and W-box elements. The most common growth and development-related elements in the three subfamilies are CAT-box, and only Class I family genes contain AACA elements. Protein subcellular localization prediction analysis showed that most poplar MLP proteins are localized in the cytoplasm (79.1%), and very few are localized in the extracellular space (11.6%), the nucleus (6.98%), and organelles such as chloroplasts (6.98%) and mitochondria (2.33%) ( Figure 3 ).

[0054] 3. Analysis of evolutionary relationships among members of the poplar MLP family

[0055] All Arabidopsis MLP protein sequences were obtained from the TAIR website. MEGA 7.0 software was used to perform multiple sequence alignment of poplar and Arabidopsis MLP proteins. The nearest neighbor method was used to construct a phylogenetic tree, and the Bootstrap value was set to 1000. The GFF file, FASTA file, and protein file of the poplar genome were obtained from JGI. Tbtools was used to process the poplar genome with BLAST and GFF files to obtain BLAST results and processed GFF files. MCScanX software was used to self-align the BLAST alignment results and the processed GFF format files to obtain collinear genes; the collinearity file was processed to obtain the homologous gene pairs of the link. According to the poplar MLP gene family ID selected in the screening and identification of the first poplar MLP gene family members, the MLP genes with homology were selected in the homologous gene pair link file.

[0056] The phylogenetic tree of the two was constructed using the selected poplar MLP gene family members and Arabidopsis MLP gene family members, including 26 Arabidopsis MLPs and 43 poplar MLPs. The results showed that the poplar MLP gene family can be divided into three subfamilies (Class I, Class II and Class III), with 9, 11 and 23 members, respectively. From the evolutionary tree, it can be seen that the poplar MLP members belonging to subfamily II are more closely related to the Arabidopsis MLP family than the other two subfamilies ( Figure 4 ), based on the relationship, we can infer the function of the poplar MLP gene based on the function of the homologous Arabidopsis MLP gene. There are 8 pairs of segmental duplication genes (indicated by red lines) and 13 pairs of tandem duplication genes ( Figure 1 , Figure 5 ). Poplar intraspecific colinearity analysis showed that there were many colinear gene pairs within the poplar genome (shown by gray lines). In the evolution of plant genomes, tandem duplication and segmental duplication promoted the expansion of new gene family members and the generation of new functions. Therefore, segmental duplication and tandem duplication of the PtMLP gene family were analyzed to illustrate the duplication of the family. MCScanX was used to analyze the PtMLP gene family, combined with the results of tandem duplication of previous chromosome analysis, indicating that the generation of PtMLP family members may be related to duplication events, of which tandem duplication and segmental duplication are the main factors.

Claims

1. A method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using a variety of bioinformatics methods, characterized in that The method described is as follows: 1) Obtain the whole genome file, annotation information file, protein sequence file and MLP protein family structure file Bet_v_1.hmm of poplar, conduct preliminary screening, remodeling and re-screening of poplar MLP gene family members to obtain preliminary poplar MLP gene family members; use the protein conserved domain detection website to identify the poplar MLP gene family members obtained by the preliminary screening to obtain the final poplar MLP gene family members; 2) using online physicochemical property analysis software to predict and analyze the physicochemical properties of the screened poplar MLP family member proteins, and using the prediction software to analyze the possible functions of the gene structure, conserved domain, promoter region cis-acting elements and protein subcellular localization of the poplar MLP gene family members in step (1); 3) Use phylogenetic tree software to construct phylogenetic trees of poplar MLP family members and Arabidopsis MLP family members, and analyze their evolutionary analysis and classification.

2. The method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using multiple bioinformatics methods according to claim 1, characterized in that The preliminary screening of the poplar MLP gene family members described in step 1) is as follows: HMMER3.0 was used to screen the MLP protein family structure file Bet_v_1.hmm to obtain the initially screened poplar MLP family members; the line number where the ID of the initially screened poplar MLP family member was located was obtained, the line was intercepted using the sed command, and then the ID number was matched and redirected using the grep command, and the sequence was extracted using the samtools tool to obtain the protein sequence file.

3. The method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using multiple bioinformatics methods according to claim 2, characterized in that The E-value of hmmsearch in HMMER3.0 is set to 10 -20 .

4. The method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using multiple bioinformatics methods according to claim 1, characterized in that The remodeling of the poplar MLP gene family members described in step 1) is as follows: ClustalW software was used to perform multiple sequence alignment of the MLP proteins in the protein sequence files obtained in the preliminary screening. Based on the alignment results, the hidden Markov model was constructed using the hmmbuild algorithm.

5. The method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using multiple bioinformatics methods according to claim 1, characterized in that The poplar MLP gene family members described in step 1) were screened again as follows: The hidden Markov model constructed in the remodeling was used to repeat the initial screening and perform a secondary screening of the poplar MLP family members to obtain the final gene ID and protein sequence.

6. The method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using multiple bioinformatics methods according to claim 1, characterized in that The protein conserved domain detection website was used to identify the poplar MLP gene family members obtained by preliminary screening, and the final poplar MLP gene family members were obtained, as follows: The candidate MLP protein sequences were identified by domain identification using SMART and InterPro online software, and proteins with the Bet_v_1 domain were screened out, which were the final members of the poplar MLP gene family.

7. The method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using multiple bioinformatics methods according to claim 1, characterized in that In step 2), the online physicochemical property analysis software is used to predict and analyze the physicochemical properties of the screened poplar MLP family member proteins, as follows: ExPASy online software was used to analyze the molecular weight, isoelectric point and instability coefficient of poplar MLP family members; MEME online software was used to perform conservative motif analysis on MLP family members.

8. The method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using multiple bioinformatics methods according to claim 1, characterized in that In step 2), prediction software is used to analyze the possible functions of the gene structure, conserved domain, cis-acting elements in the promoter region, and protein subcellular localization of the poplar MLP gene family members in step (1), as follows: The MLP gene structure was analyzed using TBtools software, and the 2000bp sequences of all family member promoters were integrated into one file. The cis-acting elements of the MLP promoter were predicted and analyzed using PlantCARE online software. The results were visualized using Simple BioSequence Viewer, and the subcellular localization of the poplar MLP protein was predicted using the Wolf PSORT tool. The possible functions of the poplar MLP gene family members were analyzed by analyzing the physicochemical properties, conserved motifs, cis-acting elements, and protein localization.

9. The method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using multiple bioinformatics methods according to claim 1, characterized in that The evolutionary tree of the poplar MLP family members and the Arabidopsis MLP family members was constructed using the evolutionary tree software in step 3), and the evolutionary analysis and classification of the two were analyzed as follows: MEGA 7.0 software was used to perform multiple sequence alignment of poplar and Arabidopsis MLP proteins, and the nearest neighbor method was used to construct a phylogenetic tree. The GFF file, FASTA file and protein file of the poplar genome were obtained from JGI. The poplar genome was processed by BLAST and GFF files using Tbtools to obtain BLAST results and processed GFF files. The BLAST alignment results and the processed GFF format files were self-aligned using MCScanX software to obtain collinear genes. The collinearity file was processed to obtain the homologous gene pairs of the link. According to the poplar MLP gene family ID selected in the screening and identification of the first poplar MLP gene family members, MLP genes with homology were selected in the homologous gene pair link file.

10. The method for screening, identifying and functionally analyzing the poplar latex protein family at the whole genome level using multiple bioinformatics methods according to claim 1, characterized in that The phylogenetic tree was constructed using the nearest neighbor method, and the Bootstrap value was set to 1000.

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