Mustard PDR gene family identification and expression analysis method and application thereof

Abstract

The invention belongs to the field of biological information analysis, and particularly relates to a brassica juncea PDR gene family identification and expression analysis method and application thereof. The invention discloses a mustard PDR gene family identification and expression analysis method. A CDS sequence, a DNA sequence and an amino acid sequence of an arabidopsis thaliana PDR gene family are obtained from a database. Loci of the genes in different chromosomes of the mustard are found out through a database containing mustard genomes; the loci are used for finding out a DNA sequence through Oligo software, then a CDS sequence and an amino acid sequence of a leaf mustard PDR gene family are analyzed and predicted through bioinformatics, and the genes are named according to the positions of the genes on chromosomes and the genetic evolution relationship. Furthermore, promoter action element analysis and transcriptome analysis are carried out according to a DNA sequence and a CDS sequence of the leaf mustard PDR gene family and an amino acid sequence of encoded protein, and the effect of the leaf mustard PDR family gene in growth and development of leaf mustard is disclosed.

Description

Technical Field

[0001] The invention belongs to the field of bioinformatics analysis, and in particular relates to a method for identifying and expressing a mustard PDR gene family and an application thereof. Background Art

[0002] Mustard is an important economic crop in the genus Brassica of the cruciferous family and is widely cultivated around the world. Mustard can be divided into various types, such as leaf, stem, stalk and root, according to the different edible organs. In addition to being eaten as a vegetable, it is also an important raw material for edible oil and is the third largest oil crop after soybean and oil palm.

[0003] The pleiotropic drug resistance (PDR) gene subfamily is a member of the ATP-binding cassette transporter (ABC) family. The members of this gene family are mainly distributed on the cell membrane, and the PDR proteins encoded by them can transport substances across the membrane. A large number of studies have reported that PDR proteins play an important role in the stress response of plants to external abiotic stresses. They can not only transport secondary metabolites, but also metal ions and growth regulators. As an important crop, mustard has not yet been reported on the identification and expression patterns of its PDR family members. Summary of the invention

[0004] Based on this, the present invention provides a method for identifying and expressing a PDR gene family in Brassica juncea, comprising the following steps:

[0005] a. Collect the information of the Arabidopsis PDR gene family from the Arabidopsis database;

[0006] b. Find the corresponding site of the mustard chromosome in the database containing mustard gene information based on the PDR gene family information collected in a;

[0007] c. Find the corresponding mustard PDR gene family DNA sequence through Oligo software according to the site found in b, and predict the CDS sequence and amino acid sequence of the encoded protein of the mustard PDR gene family;

[0008] d. Carry out protein gene family identification and evolution analysis based on the DNA sequence, CDS sequence and amino acid sequence of the PDR gene family in c, and name these genes according to their location on the chromosome and their phylogenetic evolutionary relationship;

[0009] e. Based on the DNA sequences, CDS sequences, and amino acid sequences of the mustard PDR gene family in c, perform promoter element analysis and transcriptome analysis to reveal the roles of the mustard PDR family genes in the growth and development of mustard.

[0010] Preferably, in the above method, the information of the Arabidopsis PDR gene family in a includes the DNA sequences, CDS sequences, and amino acid sequences of the Arabidopsis PDR gene family.

[0011] Preferably, in the above method, the protein gene family identification and evolutionary analysis in d include physicochemical property analysis, subcellular localization prediction, chromosome localization analysis, gene structure analysis, protein functional domain analysis prediction, protein higher structure prediction, motif analysis, collinearity analysis, and evolutionary analysis.

[0012] Optionally, the evolutionary analysis is to construct a phylogenetic tree by the ML method using the amino acid sequences of PDR-type proteins of 9 species; the amino acid sequences of PDR-type proteins of the 9 species include: the amino acid sequence of the PDR-type protein of Brassica napus; the amino acid sequence of the PDR-type protein of Brassica rapa; the amino acid sequence of the PDR-type protein of Brassica oleracea; the amino acid sequence of the PDR-type protein of Oryza sativa; the amino acid sequence of the PDR-type protein of Vitis vinifera; the amino acid sequence of the PDR-type protein of Solanum lycopersicum; the amino acid sequence of the PDR-type protein of Cucumis sativus; the amino acid sequence of the PDR-type protein of Arabidopsis thaliana; and, the amino acid sequence of the PDR-type protein of mustard.

[0013] Preferably, in the above method, the promoter element analysis in e is to predict the cis-acting elements in the promoter region for the 1000 - 3000 bp sequence upstream of the mustard PDR gene family.

[0014] Preferably, in the above method, the transcriptome analysis in e is to obtain the transcriptome data of different tissues of the mustard PDR gene family by constructing a cDNA library and Illumina sequencing method, and analyze the expression patterns of the mustard PDR gene family in different tissues.

[0015] Preferably, the different tissues include: the roots, stems, leaves, flower buds, and siliques 7 days or 15 days after flowering of mustard.

[0016] Application of the mustard PDR family genes obtained by the above method in regulating the growth and development of mustard.

[0017] Application of the mustard PDR family genes obtained by the above method in regulating the antibacterial property of mustard.

[0018] Application of the mustard PDR family genes obtained by the above method in regulating the insect resistance of mustard.

[0019] Beneficial effects:

[0020] The present invention performs a genome-wide identification of Brassica juncea PDR family members and analyzes the expression patterns in different tissues by combining transcriptome data, aiming to reveal the role of Brassica juncea PDR family genes in the growth and development of Brassica juncea, and provide theoretical guidance for subsequent gene function verification and gene participation in plant growth regulation.

[0021] Specifically, in the present invention, based on the Arabidopsis thaliana PDR gene information, 43 PDR genes are discovered in Brassica juncea using the method of the present invention, which are divided into 6 subfamilies and have similar conserved motifs and gene structures.

[0022] Specifically, in the present invention, the discovered Brassica juncea PDR promoter region contains cis-acting elements involved in hormone response, environmental stress response, and growth and development.

[0023] Specifically, the expression of 43 Brassica juncea PDR genes of the present invention in Brassica juncea roots, stems, leaves, flower buds, siliques at 7 days and 15 days after flowering, and seeds at 20 days after flowering is detected; and the expression patterns in fertile and sterile second- and fourth-order flower buds are also detected, and the expression under insect and pathogen inoculation conditions is also detected. It is found that the expression patterns of Brassica juncea PDR genes are similar under insect and pathogen inoculation and may be involved in the regulation of insect and pathogen resistance. Brief Description of the Drawings

[0024] Figure 1 : A flow chart for the identification and expression analysis of the Brassica juncea PDR gene family.

[0025] Figure 2 : Chromosome localization of PDR gene family members.

[0026] Figure 3 : Exon-intron structure of PDR gene family members.

[0027] Figure 4 : Domains of PDR gene family members.

[0028] Figure 5 : Tertiary structure of PDR gene family proteins.

[0029] Figure 6 : Analysis of conserved motif motifs of PDR genes.

[0030] Figure 7 : Intraspecific collinearity analysis of PDR gene family members.

[0031] Figure 8 : Phylogenetic tree of PDR gene family members.

[0032] Figure 9 : Map of cis-acting element sites of PDR genes.

[0033] Figure 10 : Tissue-specific expression analysis of PDR gene members.

[0034] Figure 11 : Expression analysis of PDR gene members at the two-fourth flower bud stage.

[0035] Figure 12 : Expression analysis of PDR gene members at 0, 6, 12, 24, and 36 h after inoculation.

[0036] Figure 13 : Expression analysis of PDR gene members at 0, 12, 24, 48, and 72 h after insect infestation. Specific implementation manner

[0037] The present invention provides a method for identifying and analyzing the PDR gene family in mustard, and the process is as Figure 1 shown, including the following steps:

[0038] a. Collect the information of the PDR gene family of Arabidopsis thaliana that has been included in the database of Arabidopsis thaliana;

[0039] Preferably, the information of the PDR gene family of Arabidopsis thaliana in step a includes the CDS sequence, DNA sequence, and amino acid sequence of the encoded protein of the PDR gene family of Arabidopsis thaliana.

[0040] Optionally, the information of the PDR gene family of Arabidopsis thaliana comes from the TAIR (https: / / www.arabidopsis.org / ) database, including AT1G15210, AT1G15520, AT1G59870, AT1G66950, AT2G26910, AT2G29940, AT2G36380, AT2G37280, AT3G16340, AT3G30842, AT3G53480, AT4G15215, AT4G15230, AT4G15233, AT4G15236 。

[0041] b. Find the corresponding sites on the mustard chromosome in the database containing mustard gene information according to the information of the PDR gene family collected in step a;

[0042] Optionally, the database containing mustard gene information is the BRAD (http: / / brassicadb.cn / # / ) database.

[0043] c. Find the corresponding DNA sequence of the mustard PDR gene family through Oligo software according to the sites found in step b, and predict the CDS sequence and amino acid sequence of the encoded protein of the mustard PDR gene family;

[0044] Preferably, the CDS sequences of the predicted mustard PDR gene family and the amino acid sequences of the encoded proteins are obtained using Softberry (http: / / www.softberry.com / ).

[0045] d. Conduct protein gene family identification and evolutionary analysis based on the DNA sequences, CDS sequences, and amino acid sequences of the mustard PDR gene family in c, and name them according to their positions on the chromosome and phylogenetic evolutionary relationships.

[0046] Preferably, the protein gene family identification and evolutionary analysis include physicochemical property analysis, subcellular localization prediction, chromosome localization analysis, gene structure analysis, protein functional domain analysis prediction, protein higher-level structure prediction, motif analysis, collinearity analysis, and evolutionary analysis.

[0047] Optionally, the physicochemical property analysis and the subcellular localization prediction are respectively performed using the online software Expasy (https: / / www.expasy.org / ) and WOLF PSORT (https: / / wolfpsort.hgc.jp / ).

[0048] Optionally, the chromosome localization analysis is to draw the chromosome localization map of the mustard PDR gene family members through the online software MG2C.

[0049] Optionally, the gene structure analysis is to perform gene structure analysis using the online software GSDS (https: / / gsds.gao-lab.org / index.php).

[0050] Optionally, the protein functional domain analysis prediction is to analyze and predict the protein using the online software SMART (http: / / smart.embl-heidelberg.de / smart / set_mode.cgi?NORMAL=1).

[0051] Optionally, the protein higher-level structure prediction is to predict the secondary structure of the protein using the SOPMA online tool (https: / / npsa.lyon.inserm.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html); predict the tertiary structure of the protein using the SWISS-MODEL online tool (https: / / swissmodel.expasy.org / ).

[0052] Optionally, the motif analysis is to perform motif analysis and visualization analysis on the PDR protein sequence of Brassica juncea using the online software MEME (https: / / meme-suite.org / meme / doc / meme.html).

[0053] Optionally, the collinearity analysis is to perform intra-species collinearity analysis on the PDR genes of Brassica juncea based on the Brassica juncea genome annotation information using TBtools.

[0054] Optionally, the evolutionary analysis is to construct a phylogenetic tree using the amino acid sequences of PDR-type proteins of 9 species by the ML (Maximum Likelihood) method; the amino acid sequences of PDR-type proteins of the 9 species include: the amino acid sequence of the PDR-type protein of Brassica napus; the amino acid sequence of the PDR-type protein of Brassica rapa; the amino acid sequence of the PDR-type protein of Brassica oleracea; the amino acid sequence of the PDR-type protein of Oryza sativa; the amino acid sequence of the PDR-type protein of Vitis vinifera; the amino acid sequence of the PDR-type protein of Solanum lycopersicum; the amino acid sequence of the PDR-type protein of Cucumis sativus; the amino acid sequence of the PDR-type protein of Arabidopsis thaliana; and, the amino acid sequence of the PDR-type protein of Brassica juncea.

[0055] e. According to the DNA sequences, CDS sequences and amino acid sequences of the encoded proteins of the Brassica juncea PDR gene family in c, perform promoter cis-element analysis and transcriptome analysis to reveal the roles of the Brassica juncea PDR family genes in the growth and development of Brassica juncea.

[0056] Preferably, the promoter cis-element analysis in e is to predict the cis-acting elements in the promoter region for the sequences 1000 - 3000 bp upstream of the Brassica juncea PDR gene family. More preferably, predict the cis-acting elements in the promoter region for the sequences 2000 bp upstream of the Brassica juncea PDR gene family.

[0057] Optionally, the above prediction is to predict the cis-acting elements in the promoter region through the online software Plant CARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ).

[0058] Preferably, the transcriptome analysis in e is to obtain the transcriptome data of different tissues of the Brassica juncea PDR gene family by constructing a cDNA library and Illumina sequencing method, and analyze the expression patterns of the Brassica juncea PDR gene family in different tissues.

[0059] Preferably, the different tissues include: the roots, stems, leaves, flower buds, and siliques 7 days or 15 days after flowering of Brassica juncea.

[0060] Preferably, the above-mentioned mustard PDR family genes include one or more of: BjuAABCG29-2, BjuAABCG31-2, BjuAABCG32-1, BjuAABCG33-2, BjuAABCG33-3, BjuAABCG34-1, BjuAABCG34-2, BjuAABCG35-2, BjuAABCG35-3, BjuAABCG36-1, BjuAABCG37-1, BjuAABCG42-1, BjuBABCG32-1, BjuBABCG32-2, BjuBABCG33-1, BjuBABCG34-2, BjuBABCG35-1, BjuBABCG35-3, BjuBABCG36-1, BjuBABCG40-1, BjuBABCG41-1, BjuBABCG42-1, BjuBABCG43-1, and BjuBABCG43-2.

[0061] Application of the mustard PDR family gene obtained by the above method in regulating the growth and development of mustard.

[0062] Application of the mustard PDR family gene obtained by the above method in regulating the antibacterial property of mustard.

[0063] Application of the mustard PDR family gene obtained by the above method in regulating the insect resistance of mustard.

[0064] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0065] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0066] Example 1

[0067] Identification and physicochemical properties of the mustard PDR gene family:

[0068] According to the gene IDs of 15 members of the published PDR gene family in Arabidopsis thaliana: AT1G15210, AT1G15520, AT1G59870, AT1G66950, AT2G26910, AT2G29940, AT2G36380, AT2G37280, AT3G16340, AT3G30842, AT3G53480, AT4G15215, AT4G15230, AT4G15233, AT4G15236, the CDS sequences, DNA sequences, and amino acid sequences of the PDR gene family in Arabidopsis thaliana were obtained from the TAIR (https: / / www.arabidopsis.org / ) database. The loci of these genes on different chromosomes of Brassica juncea were found through the BRAD (http: / / brassicadb.cn / # / ) database. Using these loci, the DNA sequences were found through Oligo software, and then the CDS sequences and amino acid sequences of the PDR gene family in Brassica juncea were predicted through Softberry (http: / / www.softberry.com / ). These genes were named according to their positions on the chromosomes and phylogenetic evolutionary relationships.

[0069] The physicochemical properties analysis and subcellular localization prediction of the members of the PDR gene family were carried out using the Expasy (https: / / www.expasy.org / ) online software and WOLF PSORT (https: / / wolfpsort.hgc.jp / ).

[0070] Results: Fifteen AtPDR sequences of Arabidopsis thaliana were aligned with the Brassica juncea genome database, and combined with relevant bioinformatics analysis. Finally, 43 B. juncea PDR genes were obtained. By constructing phylogenetic trees of Arabidopsis thaliana and B. juncea, and comparing and naming their sequence homology through BioEdit software. The physicochemical properties analysis and subcellular localization prediction of 43 B. juncea PDR genes are shown in Table 1. The results show that: The members of the PDR gene family are distributed on 14 chromosomes, with the number of amino acids ranging from 1076 (BjuAABCG33-3) to 1796 (BjuBABCG33-2), and the protein molecular weights ranging from 121.801 KD (BjuAABCG33-3) to 205.167 KD (BjuBABCG33-2); The isoelectric points range from 6.21 (BjuBABCG33-1) to 8.99 (BjuAABCG38-1); There are 28 stable proteins (instability coefficient < 40) and 15 unstable proteins; The fat index ranges from 88.36 (BjuBABCG40-1) to 97.28 (BjuBABCG33-1), indicating that the thermal stability differences among the encoded proteins of the PDR gene family are small; The hydrophobicity index of 3 PDR genes is less than 0, and the rest are greater than 0. The results of subcellular localization show that the PDR genes are located in the plasma membrane, suggesting that the members of the PDR family may play an important role in the plasma membrane.

[0071] Table 1 Basic physicochemical characteristics of the PDR family identified in the B. juncea genome

[0072]

[0073]

[0074] Example 2

[0075] Chromosome localization analysis:

[0076] Based on the B. juncea PDR gene family information obtained in Example 1 and the B. juncea genome annotation information, the chromosome localization map of the B. juncea PDR gene family members was drawn by the online software MG2C. The chromosome localization is as Figure 2 , and the results show that: The 43 PDR genes of B. juncea are distributed on 14 chromosomes, and the most genes, 8 in number, are on the BB-Chr06 chromosome.

[0077] Example 3

[0078] Intron-exon structure of the B. juncea PDR family member genes:

[0079] The DNA sequence and CDS sequence of the mustard PDR gene obtained in Example 1 were input into the online software GSDS (https: / / gsds.gao-lab.org / index.php) for gene structure analysis. The results of the gene exon-intron structure are as Figure 3 , and the analysis results show that there are differences in the number and length of exons of the PDR gene. Most PDR members contain about 20 exons. The most is BjuBABCG33-2 with 28 exons, and the least is BjuAABCG35-3 with 7 exons. In addition, except for BjuBABCG29-2 and BjuBABCG42-1, the gene lengths of the remaining PDR members are all less than 8000 bp.

[0080] Example 4

[0081] Based on the mustard PDR gene family information obtained in Example 1, domain analysis and protein higher-level structure prediction were carried out:

[0082] 1. Use the online software SMART (http: / / smart.embl-heidelberg.de / smart / set_mode.cgi? NORMAL=1) to analyze and predict the functional domains of the PDR protein. The results are as Figure 4 shown: It was found that all 43 predicted protein sequences contain highly conserved domains.

[0083] 2. Use the SOPMA online tool (https: / / npsa.lyon.inserm.fr / cgi-bin / npsa_automat.pl? page= / NPSA / npsa_sopma.html) to predict the secondary structure of the protein, with the parameters being the system default. The results are shown in Table 2: It was found that the secondary structure of PDR is composed of three parts: α-helix, extended strand, and random coil. Among them, the proportion of α-helix of BjuBABCG33-2 is the largest, being 52.12%; the proportion of random coil is the largest in BjuAABCG36-1, being 42.84%.

[0084] Table 2 Basic characteristics of the secondary structure of the proteins of the PDR family identified in the mustard genome

[0085] Protein name Alpha helix (%) Extended strand (%) Random coil (%) Protein name Alpha helix (%) Extended strand (%) Random coil (%) BjuAABCG29-1 49.61 9.84 40.55 BjuBABCG29-2 48.42 10.04 41.54 BjuAABCG31-2 48.13 10.19 41.68 BjuBABCG31-1 48.70 10.78 40.52 BjuAABCG42-1 49.85 10.45 39.70 BjuBABCG32-1 49.50 11.06 39.44 BjuAABCG43-1 50.14 10.60 39.26 BjuBABCG38-1 49.54 9.99 40.47 BjuAABCG34-2 49.19 10.36 40.45 BjuBABCG43-2 48.90 10.91 40.19 BjuAABCG32-1 49.93 9.93 40.14 BjuBABCG42-1 50.53 9.91 39.56 BjuAABCG31-1 49.68 10.29 40.03 BjuBABCG35-3 48.57 10.38 41.05 BjuAABCG34-1 49.20 9.57 41.23 BjuBABCG43-1 49.60 10.40 40.00 BjuAABCG33-1 49.73 10.60 39.67 BjuBABCG35-1 48.57 10.52 40.91 BjuAABCG29-2 49.51 9.54 40.95 BjuBABCG35-2 50.24 9.69 40.07 BjuAABCG33-2 48.70 9.98 41.32 BjuBABCG40-1 48.66 10.16 41.18 BjuAABCG33-3 50.27 11.25 38.48 BjuBABCG33-1 50.53 9.77 39.70 BjuAABCG41-1 50.32 10.47 39.21 BjuBABCG33-2 52.12 10.47 37.42 BjuAABCG38-1 53.32 9.41 37.27 BjuBABCG36-1 50.34 9.88 39.78 BjuAABCG35-1 49.96 9.27 40.77 BjuBABCG37-1 49.62 9.51 40.87 BjuAABCG35-3 51.11 10.49 38.40 BjuBABCG41-1 49.39 10.88 39.73 BjuAABCG40-1 51.07 9.77 39.16 BjuBABCG29-1 50.32 10.33 39.35 BjuAABCG37-1 49.41 9.73 40.86 BjuBABCG34-1 50.27 9.67 40.06 BjuAABCG36-1 47.75 9.41 42.84 BjuBABCG34-3 49.01 10.07 40.92 BjuAABCG35-2 49.34 9.39 41.27 BjuBABCG31-2 49.61 10.12 40.27 BjuBABCG33-3 49.75 9.90 40.35 BjuBABCG32-2 50.43 10.12 39.45 BjuBABCG34-2 45.14 13.27 41.59

[0086] 3. Use the SWEISSMODEL online tool (https: / / swissmodel.expasy.org / ) to predict the tertiary structure of the protein. The results are as Figure 5 shown: The results show that the similarity of the tertiary structure is relatively high, and it is speculated that their functions have a certain similarity.

[0087] Example 5

[0088] Analysis of conserved motifs and collinearity of the PDR gene family in Brassica juncea:

[0089] 1. Use the online software MEME (https: / / meme-suite.org / meme / doc / meme.html) to perform motif analysis on the PDR protein sequences of Brassica juncea, and perform visual analysis on it through Gene StructureView in TBtools software.

[0090] 2. Based on the genome annotation information of Brassica juncea, use TBtools to perform intra-species collinearity analysis on the PDR genes of Brassica juncea.

[0091] The analysis results of 43 PDR gene family protein conserved motifs in Brassica juncea are as Figure 6 shown. A total of 12 motif conserved motifs were identified. It was found that the PDR genes in the same subfamily have similar motifs. The specific distribution of motifs may also be the main reason for the functional differentiation of the PDR gene family. Intra-species collinearity analysis was performed on the PDR genes of Brassica juncea, and the results are as Figure 7 shown. A total of 55 pairs of collinearity relationships were detected among 43 genes on 14 chromosomes, and one gene corresponds to multiple genes, indicating that there may be gene duplication phenomena in the evolution of this gene family.

[0092] Example 6

[0093] Evolutionary analysis of the PDR gene family:

[0094] Download 49 Brassica napus L. PDR-type protein sequences, 21 Brassica rapa L. var. glabra Regel PDR-type protein sequences, and 27 Brassica oleracea L. PDR-type protein sequences from the EnsemblPlants (https: / / plants.ensembl.org / index.html) database (see: Zhu Kang, et al. Identification and phylogenetic analysis of the PDR gene family in Brassica napus [EB / OL]). Download 22 rice PDR-type protein sequences and 31 Vitis vinifera L. PDR-type protein sequences from the Phytozome portal (https: / / phytozome-next.jgi.doe.gov / ) database. Download 23 Solanum lycopersicum L. PDR-type protein sequences from the Solanaceae Genomics Network (https: / / solgenomics.net / ) database (see: ANDOLFO G, et al. Genetic variability and evolutionary diversification of membrane ABC transporters in plants [J / OL]). Download 13 Cucumis sativus L. PDR-type protein sequences from the CuGenDB (http: / / cucurbitgenomics.org / ) database. As well as 15 Arabidopsis thaliana PDR-type protein sequences and 43 Brassica juncea PDR-type protein sequences (see: YUAN Q, et al. Genome-wide characterization, phylogenetic and expression analysis of ABCG gene subfamily in cucumber (Cucumis sativus L.) [J / OL]). Construct a phylogenetic tree using 244 PDR-type protein sequences from 9 species by the ML method (Maximum likelihood).

[0095] The phylogenetic results are as Figure 8 shown, and 244 PDR gene members are divided into 6 subfamilies, namely I, II, III, IV, V, and VI.

[0096] Example 7

[0097] Extract the 2000bp upstream sequence of Brassica juncea PDR family members in TBtools, and predict the cis-acting elements in the promoter region through the online software Plant CARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ).

[0098] The results are as Figure 9 shown. A total of 36 MYB binding sites involved in drought induction, 40 cis-acting elements involved in cold response, 76 cis-acting regulatory elements crucial for anaerobic induction, 26 cis-acting regulatory elements involved in zein metabolism regulation, 117 cis-acting elements involved in abscisic acid responsiveness, 6 MYB binding sites involved in the regulation of flavonoid biosynthesis genes, 21 cis-acting elements involved in defense and stress responses, 13 cis-acting regulatory elements related to meristem expression, 14 cis-acting elements involved in circadian rhythm regulation, 16 MYBHv1 binding sites, 24 auxin response elements, 7 cis-acting elements involved in gibberellin response, 23 cis-acting elements involved in salicylic acid response, 5 enhancer elements involved in hypoxia-specific induction, 24 gibberellin response elements, 115 cis-acting regulatory elements involved in MeJA response, 11 regulatory elements involved in endosperm expression, 2 cis-acting elements involved in cell cycle regulation, 7 cis-acting regulatory elements involved in auxin response, 1 cis-acting regulatory element related to meristem-specific activation, 4 elements involved in palisade mesophyll cell differentiation, 3 cis-acting regulatory elements involved in seed-specific regulation, 3 parts of auxin response elements, and 1 cis-acting element involved in heat stress response. Obviously, most members of the PDR gene family are co-regulated by multiple elements, indicating that different regulatory elements may lead to different functions of the genes.

[0099] Example 8

[0100] Download and analyze the data related to the PDR gene family in different tissues of Brassica juncea through NCBI. Select different tissues of Brassica juncea: roots, stems, leaves, flower buds, siliques at 7 days and 15 days after flowering, and seeds at 20 days after flowering; perform transcriptome analysis on fertile and sterile second- and fourth-level flower buds; perform transcriptome analysis at 0, 12, 24, 48, and 72 h after insect inoculation; perform transcriptome analysis at 0, 6, 12, 24, and 36 h after pathogen inoculation, and visualize the expression levels using TBtools.

[0101] (1) cDNA library construction and Illumina sequencing:

[0102] Using The UltraTM RNA Library Prep Kit was used to construct the library. Oligonucleotide magnetic beads were used to enrich mRNA containing polyA tails, and then the generated mRNA was randomly fragmented in NEB fragmentation buffer. The fragmented mRNA was used as a template, and random oligonucleotides were used as primers to synthesize the first-strand cDNA in the M-MuLV reverse transcriptase system. Then, the RNA strand was degraded by RNaseH in the DNA polymerase I system, and the second-strand cDNA was generated using dNTP. After purification and end repair, the PCR products were purified with AMPure XP beads, and finally a cDNA library was obtained. After the library construction was completed, Qubit 2.0 was first used for preliminary quantitative analysis, and the library concentration was diluted to 1.5 ng / μL. Next, the Agilent 2100 bioanalyzer was used to check the quality of the library. The above library preparation was sequenced on the Illumina HiSeq X-Ten high-throughput sequencing platform, and 150 bp paired-end reads were generated.

[0103] (2) Data processing and analysis:

[0104] To ensure the quality and reliability of data analysis, the raw data was filtered, mainly including removing reads with adapters, reads with undetermined base information, and low-quality reads. Next, all analyses were based on the high-quality clean data. At the same time, the Q20, Q30, and GC content of the clean data were calculated. All clean data were mapped to the mustard reference genome Braju_tum_V2.0 (http: / / www.brassicadb.cn). HISAT2 v2.0.5 was used to build the index of the reference genome and compare the paired-end clean reads with the mustard reference genome. Finally, Fragments Per Kilobase Per Million (FPKM) was used to calculate gene transcriptional expression, and the fold change was calculated through the FPKM values of 09-05A / 09-05B. The DESeq2 R software package was applied to identify DEGs with a p-value < 0.05 and a fold change > 1.5 or < 0.667. To predict and identify transcription factors in DEGs, the Interproscan and Pfam databases were used to annotate each gene.

[0105] 1. Analysis of the relative expression levels of the PDR gene family in different tissues of mustard:

[0106] All values were log10(TPM + 1), and the results showed that ( Figure 10)The PDR genes exhibit different expression patterns in different tissues. BjuAABCG35-3, BjuAABCG36-1, BjuBABCG34-2, and BjuBABCG35-1 have relatively higher expression levels in roots compared to other genes. It is speculated that these genes are involved in the development and growth of mustard roots. By regulating aspects such as cell growth and division, root morphology and structure, stress response, and physiological functions, they promote the normal development and growth of plant roots. BjuAABCG32-1, BjuAABCG36-1, BjuBABCG32-1, and BjuBABCG32-2 are expressed not only at low levels in roots but also in other tissues. It is speculated that these genes play important roles throughout the growth and development of mustard. BjuAABCG36-1, BjuBABCG35-1, and BjuBABCG33-1 are speculated to play important roles in promoting the growth and division of stem cells, thereby driving stem elongation and thickening, as well as aspects such as internode length, diameter, and branching pattern of the stem. BjuAABCG31-2 and BjuAABCG42-1 have relatively higher expression levels in flower buds compared to other tissues, suggesting that these genes may play important roles in the growth and development of mustard, especially in regulating the flowering process. BjuAABCG34-2, BjuAABCG32-1, BjuAABCG33-3, BjuBABCG32-1, BjuBABCG35-1, BjuBABCG36-1, and BjuBABCG32-2 have relatively higher expression levels in siliques and seeds. It is speculated that these genes are an indispensable part of the development and maturation of siliques. By regulating processes such as cell division, growth, differentiation, and the transport and metabolism of substances, they jointly promote the normal development of siliques and the maturation of seeds.

[0107] 2. Analysis of the expression levels of the PDR gene family during the second and fourth stages of mustard flower buds:

[0108] All values were taken as log10(TPM + 1), and the results showed that ( Figure 11)The PDR genes showed different expression patterns in the second- and fourth-stage flower buds of the sterile and fertile lines. BjuAABCG32-1, BjuAABCG36-1, BjuBABCG32-1, and BjuBABCG32-2 were expressed in flower buds at all levels of each line, and the expression levels were relatively high. It is speculated that these genes may be involved in basic metabolic activities during flower bud development, such as energy production, material transport, signal transduction, etc. These genes may be important candidate genes for flower development research; BjuAABCG34-1, BjuAABCG29-2, BjuAABCG33-2, BjuAABCG37-1, BjuBABCG43-2, and BjuBABCG42-1 were higher in the fourth-stage flower buds of the sterile line than in other periods. It is speculated that this gene is involved in the regulation of sterility and may lead to the appearance of the sterile phenotype by affecting processes such as cell division, meiosis, pollen development, and floral organ morphogenesis.

[0109] 3. Analysis of the expression levels of Brassica juncea PDR gene members at 0, 6, 12, 24, and 36 h after inoculation: All values were log10(TPM + 1). The results showed that ( Figure 12 )The PDR gene members BjuAABCG35-3, BjuAABCG36-1, BjuBABCG35-1, and BjuBABCG36-1 were expressed at all time points after inoculation, and the expression levels were relatively high. It is speculated that these genes may be directly involved in pathogen infection, such as encoding toxins, enzymes, or other virulence factors secreted by pathogens. The high expression of these genes helps the pathogen colonize and spread in Brassica juncea, and may also affect the infection process of the pathogen by regulating the defense response of the host plant, such as encoding transcription factors, kinases, and other regulatory proteins to enhance or inhibit the defense ability of Brassica juncea; The expression levels of BjuAABCG31-2, BjuAABCG33-3, BjuAABCG35-2, BjuBABCG34-2, BjuBABCG35-3, BjuBABCG40-1, and BjuBABCG41-1 increased with the increase of inoculation time. It is speculated that as the pathogen spreads and grows in Brassica juncea, the plant will continuously initiate and enhance the defense response, resulting in the continuous increase of the expression levels of related genes with the increase of time after inoculation to maintain effective defense against the pathogen; The expression levels of BjuAABCG32-1, BjuAABCG33-2, BjuBABCG43-1, and BjuBABCG33-1 decreased with the increase of inoculation time. It is speculated that these genes are related to pathogen recognition, signal transduction, and early defense. After pathogen inoculation, Brassica juncea plants will quickly recognize foreign invaders and initiate a series of defense responses to cope with pathogen infection.

[0110] 4. Expression analysis of mustard PDR gene members at 0, 12, 24, 48, and 72 h after insect inoculation: All values were taken as log10(TPM + 1). The results showed that ( Figure 13 ) the PDR gene members were expressed at each time point after insect inoculation in BjuAABCG35-3, BjuAABCG36-1, BjuBABCG35-1, and BjuBABCG36-1, and the expression levels were relatively high. It is speculated that these genes are related to the insect resistance of plants. Their high expression may mean that the plants are actively responding to insect feeding or parasitism by producing toxins, defense proteins, or other chemical substances to resist insect attacks. These genes may encode proteins related to insect perception, attachment, or feeding behavior. The high expression of these genes may enhance the interaction between plants and insects. When plants are attacked by insects, they will activate a complex defense signaling network, including signal pathways such as JA (jasmonic acid) and SA (salicylic acid). The high expression of PDR at each time point after insect inoculation may also indicate that these genes play a key role in these signaling networks, regulating the intensity and duration of plant defense responses, thereby enhancing the resistance of mustard to insects.

[0111] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for identifying and analyzing the PDR gene family in Brassica juncea, characterized in that: The following steps are involved: a. Collect the information of the Arabidopsis PDR gene family from the Arabidopsis database; b. Find the corresponding site of the mustard chromosome in the database containing mustard gene information based on the PDR gene family information collected in a; c. Find the corresponding mustard PDR gene family DNA sequence through Oligo software according to the site found in b, and predict the CDS sequence and amino acid sequence of the encoded protein of the mustard PDR gene family; d. Carry out protein gene family identification and evolution analysis based on the DNA sequence, CDS sequence and amino acid sequence of the PDR gene family in c, and name these genes according to their location on the chromosome and their phylogenetic evolutionary relationship; e. Based on the mustard PDR gene family DNA sequence, CDS sequence and amino acid sequence of the encoded protein in c, promoter action element analysis and transcriptome analysis were performed to reveal the role of mustard PDR family genes in the growth and development of mustard.

2. The method according to claim 1, characterized in that: The information of the Arabidopsis PDR gene family described in a includes the DNA sequence, CDS sequence and amino acid sequence of the encoded protein of the Arabidopsis PDR gene family.

3. The method according to claim 1, characterized in that: The protein gene family identification and evolutionary analysis described in d include physicochemical property analysis, subcellular localization prediction, chromosome localization analysis, gene structure analysis, protein functional domain analysis prediction, protein higher-order structure prediction, motif analysis, colinearity analysis and evolutionary analysis.

4. The method according to claim 3, characterized in that: The evolutionary analysis is to construct a phylogenetic tree using the amino acid sequences of the PDR-type proteins of the nine species through the ML method; The amino acid sequences of the PDR type proteins of the nine species include: the amino acid sequence of the Brassica napus PDR type protein; the amino acid sequence of the Brassica pekinensis PDR type protein; the amino acid sequence of the Cabbage PDR type protein; the amino acid sequence of the Rice PDR type protein; the amino acid sequence of the Grape PDR type protein; the amino acid sequence of the Tomato PDR type protein; the amino acid sequence of the Cucumber PDR type protein; the amino acid sequence of the Arabidopsis thaliana PDR type protein; and the amino acid sequence of the Brassica juncea PDR type protein.

5. The method according to claim 1, characterized in that: The promoter action element analysis described in e is to predict the cis-acting elements in the promoter region of the upstream 1000-3000 bp sequence of the mustard PDR gene family.

6. The method according to claim 1, characterized in that: The transcriptome analysis described in e is to obtain transcriptome data of different tissues of the mustard PDR gene family by constructing a cDNA library and using the Illumina sequencing method, and to analyze the expression patterns of the mustard PDR gene family in different tissues.

7. The method according to claim 6, characterized in that The different tissues include: roots, stems, leaves, flower buds, and siliques 7 or 15 days after flowering of Brassica juncea.

8. Use of the mustard PDR family gene obtained by any one of the methods described in claims 1 to 7 in regulating the growth and development of mustard.

9. Use of the mustard PDR family gene obtained by any one of the methods described in claims 1 to 7 in regulating the antibacterial activity of mustard.

10. Use of the mustard PDR family gene obtained by any one of the methods of claims 1 to 7 in regulating the insect resistance of mustard.

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