Identification, characterization and expression analysis of cardamine violifolia WRKY transcription factor and regulation effect of cardamine violifolia WRKY transcription factor in selenium accumulation

Abstract

The invention discloses identification, characterization and expression analysis of cardamine violifolia WRKY transcription factors and a regulation effect of the cardamine violifolia WRKY transcription factors in selenium accumulation, and belongs to the technical field of biology. WRKY gene family members in cardamine violifolia genes are comprehensively identified, and 120 CvWRKY genes are totally identified. The physical and chemical properties, chromosome localization, phylogeny, gene structure, conserved motif, cis-acting element and colinearity of CvWRKY family gene protein are comprehensively analyzed, and a foundation is laid for understanding the evolutionary relationship and gene function of the CvWRKY gene family. The research finds that the expression level of the CvWRKY34 is in significant positive correlation with the selenium content; meanwhile, both the CvWRKY34 and the CvWRKY021 respond to the induction of Na2SeO3, and the main contribution of the CvWRKY34 and the CvWRKY021 is possibly positive regulation of the selenium absorption process of the root. The research on the cardamine violifolia WRKY family lays a theoretical foundation for analyzing the contribution of the WRKY gene in the high selenium accumulation capacity of the cardamine violifolia, and provides an important clue for functional verification of the cardamine violifolia.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the identification, characterization and expression analysis of a Cardamine violacea WRKY transcription factor and its regulatory role in selenium accumulation. Background Art

[0002] Cardamine violifolia is a cruciferous plant known for its high selenium (Se) accumulation ability and an important source of selenium for humans and animals. The plant is delicious and nutritious, and is widely promoted for cultivation and deep processing. On the one hand, it can enrich people's diet, and on the other hand, it can also bring higher economic income to growers, which is conducive to industrial structure adjustment in areas suitable for cultivating the plant, bringing more social and economic benefits. At the same time, based on the super-selenium accumulation ability of Cardamine violifolia, it is used as a new source of selenium and widely used in the animal husbandry industry. WRKY genes are widely present in plants, and WRKY transcription factors play a vital role in the response of plants to various biotic and abiotic stresses, including cadmium stress, iron deficiency and selenium tolerance. At present, whole genome analysis of the WRKY family has been widely carried out in many sequenced species. However, the WRKY family of Cardamine violifolia has not yet been identified, which has hindered the study of the function of CvWRKY genes to a certain extent. Therefore, this application involves the identification of the WRKY family of Cardamine violata, and systematic bioinformatics analysis and expression analysis were carried out to identify the key WRKY transcription factors involved in regulating the selenium metabolism of Cardamine violata, laying a theoretical foundation for analyzing the molecular mechanism of selenium enrichment in Cardamine violata and strengthening the development and utilization of Cardamine violata. Summary of the invention

[0003] The purpose of the present invention is to provide an identification, characterization and expression analysis of a WRKY transcription factor of Cardamine violata and its regulatory role in selenium accumulation in view of the existing problems.

[0004] The present invention is achieved through the following technical solutions:

[0005] The identification, characterization and expression analysis of a WRKY transcription factor of Cardamine violae comprises the following steps:

[0006] (1) Identification and analysis of the CvWRKY gene family in Cardamine viola

[0007] 1) The genome of Cardamine viola was obtained from the BIG Data Center of the genome repository to analyze the CvWRKY gene family, and the protein conserved regions of CvWRKY candidate genes were identified using the CDD database of NCBI and the online tool SMART;

[0008] 2) multiple sequence alignment was performed to remove sequences with incompletely conserved domains and redundancy to identify members of the CvWRKY gene family;

[0009] 3) Use the online website ExPASy to analyze the physicochemical properties of proteins, and use the Cell-Ploc2.0 online tool to predict subcellular localization information;

[0010] (2) Construction of phylogenetic tree and chromosome location

[0011] 1) ClustalX was used to perform multiple sequence alignment of WRKY proteins from Arabidopsis thaliana and Cardamine viola, and MEGA11 software was used to construct a phylogenetic tree using the neighbor-joining method;

[0012] 2) Use iTOL online software to visualize and beautify the phylogenetic tree;

[0013] 3) Obtain gene density and the distribution of CvWRKY on chromosomes from the annotation files of Cardamine violacea genome data, and use TBtools software to display the localization of CvWRKY protein on chromosomes;

[0014] (3) Motif composition and cis-acting elements of gene structure

[0015] 1) Use the online software MEME to analyze the conserved motifs of CvWRKY family proteins;

[0016] 2) TBtools software was used to extract the upstream 2000 bp sequence of the Cardamine viola genome as the promoter sequence, the online database PlantCARE was used to analyze the promoter cis-acting elements, and the results were visualized using TBtools software;

[0017] (4) Collinearity analysis

[0018] 1) Intraspecific collinearity analysis: TBtools was used to obtain gene density and chromosome skeleton files from the annotation files of the Cardamine violaceae genome data. TBtools was used to perform intraspecific collinearity analysis on Cardamine violaceae and highlight the collinearity of CvWRKY, and the results were visualized;

[0019] 2) Interspecific synteny analysis: The genome data and annotation files of Shepherd's Purse and Arabidopsis thaliana were downloaded from the NCBI database. TBtools was used to analyze the synteny between Shepherd's Purse and Cardamine violaceae and between Arabidopsis thaliana and Cardamine violaceae, and the synteny of CvWRKY was highlighted and the results were visualized.

[0020] (5) Plant materials and treatment

[0021] 1) Sowing Cardamine violacea until 4-5 true leaves grow, and then transplanting into a hydroponic box filled with 1 / 4 concentration Hoagland nutrient solution;

[0022] 2) After 10 days, select and ensure that each hydroponic box contains 11 Cardamine violaceae plants with consistent growth for treatment, and then replace the nutrient solution with 1 / 2 Hoagland nutrient solution; After 10 days, select and ensure that each culture box contains 9 Cardamine violaceae plants with consistent growth for Na2SeO4 treatment, and then replace the nutrient solution with 1 / 2 Hoagland nutrient solution, and add different concentrations of Na2SeO4 for treatment. Each group of treatments contains 3 independent biological replicates, and each replicate contains 9 Cardamine violaceae plants;

[0023] 3) The 1 / 2 Hoagland nutrient solution containing different concentrations of Na2SeO4 was replaced every 10 days. After three consecutive treatments, all plants of each replicate were harvested, rinsed three times with deionized water, and the surface moisture was wiped off with filter paper, frozen with liquid nitrogen, mixed evenly, and stored in a -80°C refrigerator for later use;

[0024] 4) Using the same procedure as above, a batch of Cardamine viola seedlings were re-cultivated, and Cardamine viola plants with consistent growth were selected and cultured continuously for 45 days; then the nutrient solution in the incubator was replaced with 1 / 2 Hoagland nutrient solution containing 100 mg / L Na2SeO3, and 1 / 2 Hoagland nutrient solution without Na2SeO3 was used as a control treatment. Each treatment was repeated three times, and each replication contained 9 Cardamine viola plants. After 15 days of treatment, the aboveground and underground parts were collected, respectively, and after liquid nitrogen quick freezing, they were stored in a -80°C refrigerator for later use;

[0025] (6) Screening and expression analysis of CvWRKY genes involved in metabolism and accumulation of Cardamine viola

[0026] 1) The research team obtained the full-length transcriptome, transcriptome sequencing and selenium content data of Cardamine violata from the plant materials treated for the first time;

[0027] 2) The protein sequences of the CvWRKY gene family were compared with the full-length transcriptome database by using TBtools software, and protein sequences with a similarity greater than 85% were selected from the full-length transcriptome data for further analysis;

[0028] 3) Correlation analysis was performed between the expression levels of the screened genes and the total selenium content, organic selenium content, and inorganic selenium content, and the results were visualized using TBtool;

[0029] (7) Determination of selenium content and qRT-PCR analysis

[0030] 1) Determination of total selenium content in the aboveground and underground parts by hydride atomic fluorescence spectrometry;

[0031] The total selenium detection refers to GB 5009.93-2010, and is measured using a hydride atomic fluorescence spectrometer. Weigh 0.2g of sample into a digestion tank, then add 8mL of nitric acid to each digestion tube, and pre-digest on a fully automatic graphite digestion instrument. The pre-digestion conditions are 180℃, 15 minutes, and after the pre-digestion, cool, screw on the lid, and place in a microwave digestion instrument for further digestion. The program is set as follows: heat to 120℃ within 5 minutes, hold for 3 minutes; heat to 160℃ within 5 minutes, hold for 3 minutes; heat to 180℃ within 5 minutes, hold for 20 minutes. Then, drive the acid at 180℃ on the fully automatic graphite digestion instrument, drive the acid to about 1mL, take it out and cool it to room temperature, and then dilute it to 10mL with the prepared 2% nitric acid solution to obtain the test solution. HG-AFS working conditions: negative high voltage 340V, lamp current 100mA, atomization temperature 800℃, carrier gas flow rate 500mL / min, injection volume 1mL, KBH4 concentration 3.5%, selenium standard purchased from the Chinese Academy of Metrology was used to draw the standard curve, each group of samples had 3 biological replicates, and each sample was measured 3 times;

[0032] 2) qRT-PCR analysis was performed on the total RNA from the aboveground and underground parts of Cardamine violacea treated with Na2SeO3 using the ChamQ Universal SYBR qPCR Master Mix kit.

[0033] Furthermore, when constructing the evolutionary tree described in operation 2) of step (2), the Bootstrap value is set to 1000 times.

[0034] Furthermore, the maximum number of motifs in the motif analysis described in operation 2) of step (3) is set to 20.

[0035] Furthermore, the seeds of Cardamine violacea described in operation 1) of step (5) are provided by Enshi Deyuan Selenium Material Engineering Technology Co., Ltd. and sown into matrix soil for germination.

[0036] Furthermore, the hydroponic environmental conditions in step (5) of operation 1) are: temperature 20±1°C, relative humidity about 75%, light intensity 230 μmol / m 2 / s, lighting time 14h / d.

[0037] Furthermore, the size of the hydroponic box described in step (5) operation 1) is 36 cm×24 cm×12.5 cm.

[0038] Furthermore, the different concentrations of Na2SeO4 are 0, 0.25, 4.0 or 16.0 mg / L respectively.

[0039] Furthermore, in step (7), β-actin 3 was used as the reference gene in qRT-PCR, and 2 -ΔΔCt The relative expression levels of genes were calculated.

[0040] Compared with the prior art, the present invention has the following advantages:

[0041] 1. This application comprehensively identified the WRKY gene family members in Cardamine violaceum genes, and identified a total of 120 CvWRKY genes, which were divided into three groups. In the second group, they were further subdivided into five subgroups. The physicochemical properties, chromosomal localization, phylogeny, gene structure, conserved motifs, cis-acting elements and colinearity of CvWRKY family gene proteins were comprehensively analyzed. Gene structure analysis showed that WRKY variations and mutations were detected in CvWRKYs genes; segmental duplication events were considered to be the main factor driving the expansion of the CvWRKY gene family. The CvWRKY promoter contains a large number of cis-acting elements that respond to stress. This study lays the foundation for understanding the evolutionary relationship and gene function of the CvWRKY gene family.

[0042] 2. This application combines the gene expression level and selenium content data in plant materials under exogenous selenium treatment in previous studies, and refers to the information of AtWRKY47 involved in selenium accumulation in Arabidopsis, and finds that the expression level of CvWRKY34 is significantly positively correlated with the selenium content; at the same time, CvWRKY021 and CvWRKY099 have high homology with the AtWRKY47 gene involved in regulating selenium accumulation in Arabidopsis, and the sequences of CvWRKY021 and AtWRKY47 are highly homologous. Further analysis of the transcriptome data found that CvWRKY021 can respond to the induced expression of sodium selenate, and has a certain positive correlation with the treatment concentration of sodium selenate. At the same time, both CvWRKY34 and CvWRKY021 responded to the induction of Na2SeO3, and their expression was significantly downregulated in the aerial part of Cardamine violaceae, significantly upregulated in the roots, and the selenium content in the roots increased significantly. Therefore, CvWRKY34 and CvWRKY021 are candidate genes involved in the selenium metabolism of Cardamine violaceae, and their main contribution may be to positively regulate the absorption process of selenium in the roots. The study of the WRKY family of Cardamine violaceae in this study laid a theoretical foundation for analyzing the contribution of WRKY genes in the high selenium accumulation ability of Cardamine violaceae, and provided important clues for its functional verification. And the research results of this application illustrate the evolution of CvWRKY genes in the genome of Cardamine violaceae, and provide valuable resources for further analyzing the functional characteristics of WRKY genes related to the super selenium accumulation ability of Cardamine violaceae. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 To locate the chromosome of the CvWRKY gene family in Cardamine viola;

[0044] Figure 2 This is the phylogenetic tree of WRKY protein sequences from Cardamine viola and Arabidopsis thaliana;

[0045] Figure 3 is the multiple sequence alignment of the WRKY domain of CvWRKYs, and gray and yellow indicate conserved amino acid residues;

[0046] Figure 4 The evolutionary tree, motifs, conserved domains and gene structure analysis of the CvWRKY gene family;

[0047] Figure 5 20 conserved motifs of CvWRKYs;

[0048] Figure 6 Analysis of cis-acting elements in CvWRKYs promoter;

[0049] Figure 7 for the colinearity analysis of CvWRKY genes;

[0050] Figure 8 is the expression level of CvWRKYs under treatment with different concentrations of Na2SeO4;

[0051] Fig. 9 For the correlation analysis between gene expression level and selenium content;

[0052] Fig.10 Analysis of candidate genes involved in selenium accumulation in Cardamine viola. DETAILED DESCRIPTION

[0053] In order to further explain the present invention, it is described below in conjunction with the following specific embodiments.

[0054] Example 1

[0055] The identification, characterization and expression analysis of a WRKY transcription factor of Cardamine violae comprises the following steps:

[0056] (1) Identification and analysis of the CvWRKY gene family in Cardamine viola

[0057] The CvWRKY gene family of Cardamine viola genome analysis was obtained from the BIG Data Center of the Genome Repository (https: / / ngdc.cncb.ac.cn / ). The hidden Markov model (HMM) file of the WRKY domain (PF03106) of the Pfam database (http: / / pfam.xfam.org) was used as a seed sequence, and the HMMer software (version 3.3.2) was used to identify genes with an E value < 1e -5 The conserved regions of the proteins of the candidate CvWRKY genes were identified using the NCBI CDD database (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and the online tool SMART (https: / / smart.embl.de / ); multiple sequence alignment was performed to remove sequences with incomplete conserved domains and redundancy, thereby identifying members of the CvWRKY gene family; the online website ExPASy (http: / / web.ExPASy.org / protparam / ) was used to predict the physicochemical properties of the proteins, and the Cell-Ploc2.0 online tool (http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2 / ) was used to predict the subcellular localization information.

[0058] (2) Construction of phylogenetic tree and chromosome location

[0059] The WRKY family gene sequences of Arabidopsis were downloaded from the Arabidopsis database (www.Arabidopsis.org). Multiple sequence alignment of WRKY proteins of Arabidopsis and Cardamine viola was performed using ClustalX, and a phylogenetic tree was constructed using the neighbor-joining method using MEGA 11 software, with the bootstrap value set to 1000 times, and then the phylogenetic tree was visualized and beautified using the online software iTOL (https: / / itol.embl.de / ). The gene density and the distribution of CvWRKY on chromosomes were obtained from the annotation file of the Cardamine viola genome data, and the location of CvWRKY proteins on chromosomes was displayed using TBtools software.

[0060] (3) Motif composition and cis-acting elements of gene structure

[0061] The conserved motifs of CvWRKY family proteins were analyzed using the online software MEME (https: / / meme-suite.org / meme / tools / meme), and the maximum number of motifs was set to 20. The upstream 2000 bp sequence of the Cardamine viola genome was extracted using the TBtools software as the promoter sequence. The online database PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) was used to analyze the promoter cis-acting elements, and the results were visualized using the TBtools software.

[0062] (4) Collinearity analysis

[0063] Intraspecific collinearity analysis: TBtools was used to obtain gene density and chromosome skeleton files from the annotation files of the Cardamine violaceae genome data. TBtools was used to analyze intraspecific collinearity of Cardamine violaceae and highlight the collinearity of CvWRKY, and the results were visualized. Interspecific collinearity analysis: Shepherd's purse and Arabidopsis thaliana genome data and annotation files were downloaded from the NCBI database. TBtools was used to analyze the collinearity between Shepherd's purse and Cardamine violaceae, and between Arabidopsis thaliana and Cardamine violaceae, highlight the collinearity of CvWRKY, and visualize the results.

[0064] (5) Plant materials and treatment

[0065] The seeds of Cardamine violae were provided by Enshi Deyuan Selenium Material Engineering Technology Co., Ltd. After Cardamine violae was sown until 4-5 true leaves grew, it was transplanted into a hydroponic box (36cm×24cm×12.5cm) filled with 1 / 4 concentration Hoagland nutrient solution. The growth environment was: temperature 20±1℃, relative humidity about 75%, light intensity 230μmol / m 2 / s, photoperiod 14h / d; 10 days later, select and ensure that each hydroponic box contains 11 Cardamine violaceae plants with consistent growth for treatment, and then replace the nutrient solution with 1 / 2 Hoagland nutrient solution; 10 days later, select and ensure that each culture box contains 9 Cardamine violaceae plants with consistent growth for Na2SeO4 treatment, and then replace the nutrient solution with 1 / 2 Hoagland nutrient solution, and add different concentrations of Na2SeO4 (0, 0.25, 4.0 or 16.0 mg / L) for treatment, each group of treatments contains 3 independent biological replicates, and each replicate contains 9 Cardamine violaceae plants; the 1 / 2 Hoagland nutrient solution containing different concentrations of Na2SeO4 is replaced every 10 days. After 3 consecutive treatments, all plants of each replicate are harvested, rinsed with deionized water 3 times, and the surface moisture is wiped off with filter paper, frozen with liquid nitrogen, mixed evenly, and stored in a -80℃ refrigerator for later use.

[0066] A batch of Cardamine violaceae seedlings were re-cultivated using the same procedure as above, and Cardamine violaceae plants with consistent growth were selected and cultured continuously for 45 days; then the nutrient solution in the incubator was replaced with 1 / 2 Hoagland nutrient solution containing 100 mg / L Na2SeO3, and 1 / 2 Hoagland nutrient solution without Na2SeO3 was used as the control treatment. Each treatment was repeated three times, and each repeat contained 9 Cardamine violaceae plants. After 15 days of treatment, the aboveground and underground parts were collected respectively, quick-frozen in liquid nitrogen, and stored in a -80°C refrigerator for later use.

[0067] (6) Screening and expression analysis of CvWRKY genes involved in metabolism and accumulation of Cardamine viola

[0068] The research team obtained the full-length transcriptome, transcriptome sequencing and selenium content data of Cardamine violacea from the plant materials treated for the first time; by using TBtools software to compare the CvWRKY gene family protein sequences with the full-length transcriptome database, protein sequences with a similarity greater than 85% were screened out from the full-length transcriptome data for further analysis; the correlation analysis was performed between the expression levels of the screened genes and the total selenium content, organic selenium content and inorganic selenium content, and the results were visualized using TBtools.

[0069] (7) Determination of selenium content and qRT-PCR analysis

[0070] 1) Determination of total selenium content in the aboveground and underground parts by hydride atomic fluorescence spectrometry;

[0071] The total selenium test refers to GB 5009.93-2010, and is measured using a hydride atomic fluorescence spectrometer. Weigh 0.2g of sample into a digestion tank, then add 8mL of nitric acid to each digestion tube, and pre-digest on a fully automatic graphite digestion instrument. The pre-digestion conditions are 180℃, 15 minutes, and after the pre-digestion, cool, screw on the lid, and place in a microwave digestion instrument for further digestion. The program is set as follows: heat to 120℃ within 5 minutes, hold for 3 minutes; heat to 160℃ within 5 minutes, hold for 3 minutes; heat to 180℃ within 5 minutes, hold for 20 minutes. Then, drive the acid at 180℃ on the fully automatic graphite digestion instrument, drive the acid to about 1mL, take it out and cool it to room temperature, and then dilute it to 10mL with the prepared 2% nitric acid solution to obtain the test solution. HG-AFS working conditions: negative high voltage 340V, lamp current 100mA, atomization temperature 800℃, carrier gas flow rate 500mL / min, injection volume 1mL, KBH4 concentration 3.5%, selenium standard purchased from the Chinese Academy of Metrology was used to draw the standard curve, each group of samples had 3 biological replicates, and each sample was measured 3 times;

[0072] 2) qRT-PCR analysis of total RNA from the aboveground and underground parts of Cardamine violae treated with Na2SeO3 was performed using the ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme Biotech), with β-actin 3 as the reference gene and 2 -ΔΔCt The relative expression levels of genes were calculated.

[0073] Specific primers for all genes are listed in Table 1 .

[0074] Table 1 RT-qPCR primer sequences

[0075]

[0076] Example 2

[0077] Experimental Results

[0078] (1) Identification, chromosome distribution and physicochemical properties of CvWRKYs

[0079] A total of 120 CvWRKY gene family members were retrieved from the genome of Cardamine violata and renamed CvWRKY1-CvWRKY120. The lengths of the proteins encoded by these genes ranged from 114 to 1383 amino acids, and the relative molecular masses of the proteins were 13.52-155.75 KDa. The isoelectric points (pI) ranged from 4.76 to 9.86, among which CvWRKY103 had the lowest pI and CvWRKY120 had the highest pI. The total average hydrophilicity values ​​of all CvWRKY proteins were negative, ranging from -1.169 to -0.419, indicating hydrophilicity. The prediction of subcellular location showed that all 120 CvWRKY proteins were located in the nucleus. These results provide a theoretical basis for further studies on the purification, activity and function of CvWRKYs proteins.

[0080] Based on the genome annotation information, 117 CvWRKYs were unevenly located on 16 chromosomes of Cardamine violata, mainly distributed in Chr3 (13), Chr7 (10), Chr8 (10), Chr10 (14) and Chr13 (12) ( Figure 1 ), while there were only three CvWRKYs on chromosome 1, and three CvWRKYs were found not to be mapped to any chromosome.

[0081] (2) Classification and phylogenetic analysis of CvWRKY proteins

[0082] In order to better understand the phylogenetic relationships and classification of WRKY proteins in Cardamine violata, a phylogenetic tree was constructed by the neighbor-joining method based on multiple sequence alignment of the WRKY domains of Arabidopsis thaliana and Cardamine violata, with a bootstrap value of 1000. Phylogenetic analysis showed that members of the CvWRKY gene family proteins clustered well with members of the Arabidopsis subfamily, so referring to the classification of AtWRKY, CvWRKY proteins were divided into three different groups, corresponding to groups I, II, and III ( Figure 2). Group I includes 28 members, whose conserved domain contains two complete WRKY domains, located at the N-terminus and C-terminus of the sequence, and the zinc finger structure type is C2H2 type; Group II accounts for the largest part, with a total of 76 members. In Group II, CvWRKY proteins can be further divided into five subgroups, IIa, IIb, IIc, IId and IIe, with 5, 16, 30, 12 and 13 members, respectively, while proteins from Group II usually contain a WRKY domain, and their zinc finger structure type is C2H2 type; Group III has the least number of CvWRKY proteins, including 16 members, all of which contain a WRKY conserved domain and contain two different zinc finger structure types, namely C2H / C and C2H2. In addition, some exceptions with WRKYGQK mutations were found in CvWRKY proteins. CvWRKY028, CvWRKY048, CvWRKY103, and CvWRKY105 were classified as Group I but contained only one WRKY conserved domain. CvWRKY080, CvWRKY101, and CvWRKY105 proteins lost the zinc finger structure in the WRKY conserved domain near their C-termini. CvWRKY007 (Group IIc) and CvWRKY103 (Group I) had a WRKYGQK mutation to WIKYGQK; CvWRKY101 (Group I) had a WRKYGQK mutation to WQKYGQK; CvWRKY027, CvWRKY085, CvWRKY063, CvWRKY107 (Group IIc), and CvWRKY048 (Group I) had a WRKYGKK substitution for WRKYGQK ( Figure 3 ).

[0083] (3) Analysis of gene structure and conserved motifs of CvWRKYs

[0084] To fully understand the characteristics of the CvWRKY gene family, the sequence features of CvWRKYs, including conserved domains, introns, exons, and motifs, were analyzed. The phylogenetic tree, conserved domains, intron / exon numbers, and motif results together showed that the most closely related CvWRKYs in the same subfamily exhibited the same intron / exon numbers and motifs. The presence of the same motif means that this group has similar biological functions, suggesting that the gene structures are highly correlated ( Figure 4 ).

[0085] This application predicts the motif composition present in CvWRKY protein ( Figure 4 B), 20 different motifs were selected and labeled as motif 1 to motif 20. The length of these conserved motifs ranged from 15 to 50 amino acids ( Figure 5As expected, the same group or subgroup of CvWRKYs have highly consistent conserved motifs, and each CvWRKY protein contains motif1 and motif2, which are annotated as WRKY DNA binding domains ( Figure 4 B) From Figure 4 C shows that all CvWRKYs contain at least one WRKY conserved domain, and combined with the phylogenetic tree classification results, ( Figure 4 A), Group I contains two WRKY conserved domains, Group II and Group III WRKY proteins contain one WRKY domain, and Group IId subfamily also contains Plant-Zn-clust domain. The number of coding exons in CvWRKYs genes varies, ranging from 2 to 15. Among CvWRKY genes, the highest proportion contains 3 coding exons (45.8%), followed by 4 (19.2%), 2 (11.7%), 5 (10.8%), 6 (10%), and 7, 8, and 15 coding exons. The genes have only 1 ( Figure 4 D). Overall, the above results demonstrate the accuracy of the classification of the WRKY gene family and that closely related CvWRKYs in the phylogenetic tree have similar motif compositions and may perform similar functions.

[0086] (4) Cis-acting elements in CvWRKYs promoter

[0087] The promoter of CvWRKY gene contains abundant cis-acting elements, including development-related elements, plant hormone response elements, stress response elements and light response elements, which are related to various biotic and abiotic stresses. Therefore, it is speculated that the cis-acting elements of CvWRKYs gene bind to various stress-related trans-acting factors, thereby regulating the expression and response of stress resistance genes in Cardamine violata. WRKY proteins have been shown to play a role in a variety of abiotic stress conditions, including cadmium stress, salt tolerance and iron deficiency (Han Y, Fan T, Zhu X, Wu X, Ouyang J, Jiang L, Cao S. WRKY12 represses GSH1 expression to negatively regulate cadmium tolerance inArabidopsis. Plant Mol Biol. 2019; 99: 149–159.), (Sheng Y, Yan X, Huang Y, Han Y, Zhang C, Ren Y, Fan T, Xiao F, Liu Y, Cao S. The WRKY transcription factor, WRKY13, activates PDR8 expression to positively regulate cadmium tolerance inArabidopsis. Plant Cell Environ. 2019; 42: 891–903.), (Dai W, Wang M, Gong X, Liu JH. The transcription factor FcWRKY40 of Fortunella crassifolia functions positively in salt tolerance through modulation of ion homeostasis and proline biosynthesis by directly regulating SOS2 and P5CS1 homologs. New Phytol. 2018; 219: 972–989.). However, it is still unclear whether CvWRKY genes play a role in regulating Se tolerance in Cardamine viola.It is generally recognized that there is a close relationship between gene expression and function, so studying the expression pattern of genes in different tissues or under stress is crucial to revealing functional genes (Dutta S, Biswas P, Chakraborty S, Mitra D, Pal A, Das M. Identification, characterization and gene expression analyses of important flowering genes related to photoperiodic pathway in bamboo. BMC Genomics. 2018; 19: 190.). Given the interest in the molecular mechanism of the ability of Cardamine violata to enrich selenium, this study analyzed the correlation between gene expression levels and total selenium, inorganic selenium and organic selenium contents in tissue samples under exogenous selenium treatment (r>0.88, P<0.001), and preliminarily concluded that CvWRKY034 may be involved in the biological process of selenium metabolism and accumulation in Cardamine violata. Studies have reported that AtWRKY47 is involved in regulating the accumulation of selenium (Wu X, Tao M, Meng Y, Zhu X, Cao S. The role of WRKY47 gene in regulating selenium tolerance in Arabidopsis thaliana. Plant Biotechnol Rep. 2020; 14: 121–129.). Genes with similar gene sequences usually have similar gene biological functions (Song H, Cao Y, Zhao L, Zhang J, Li S. Review: WRKY transcription factors: Understanding the functional divergence. Plant Sci. 2023; 334: 111770.). In order to understand the regulatory mechanism of CvWRKYs transcription factors, this application used the PlantCARE online website to predict the cis-acting elements (in the 2000bp sequence upstream of the translation start site (ATG) of the CvWRKYs gene. Figure 6 A). The results showed that 56 cis-acting elements were found in the CvWRKY gene promoter, which can be roughly classified into light-responsive elements, hormone-responsive elements, stress-responsive elements and developmental-responsive elements ( Figure 6B). Among them, there are 28 light-responsive elements, and almost all of its genes contain light-responsive elements. Hormone-related cis-acting elements, including abscisic acid responsiveness (ABRE), auxin responsiveness (AuxRR-core, TGA-element and TGA-box), MeJA responsiveness (TGACG-motif and CGTCA-motif), gibberellin responsiveness elements (GARE-motif, P-box and TATC-box), salicylic acid responsiveness elements (SARE and TCA-element), are widely present in promoter regions. Among them, 303 ABRE cis-acting elements are distributed in the promoter regions of 97 CvWRKYs, and 225 CGTCA-motifs are distributed in the promoter regions of 95 CvWRKYs ( Figure 6 B). Seven stress-related cis-elements were detected, including anaerobic elements (ARE), wound response elements (WUN-motif), low temperature stress response elements (LTR), drought stress response elements (MBS), defense and stress response elements (TC-rich repeats), enhancer-like elements involved in hypoxia-specific induction (GC-motif) and cis-elements involved in dehydration, low temperature and salt stress (DRE). Ten development-related cis-elements were detected, and the number of genes containing such cis-elements was relatively small ( Figure 6 B).

[0088] (5) Collinearity analysis of CvWRKYs within and between species

[0089] In the evolutionary history of species, gene tandem duplication and segmental duplication play a key role in the generation of gene families. In order to explore the genomic amplification mechanism of the CvWRKY gene family, the homologous relationship of CvWRKYs was analyzed. Normally, two or more genes located on the same chromosome are no more than 200kb apart and are considered to be from a gene cluster. This application study detected that 12 tandemly duplicated genes generated 6 gene clusters CvWRKY076 / CvWRKY077 (Chr3), CvWRKY109 / CvWRKY110 (Chr8), CvWRKY113 / CvWRKY114 (Chr8), CvWRKY035 / CvWRKY036 (Chr13), CvWRKY032 / CvWRKY033 (Chr13) and CvWRKY037 / CvWRKY038 (Chr13) ( Figure 7A). A total of 114 of the 120 CvWRKYs (accounting for 95% of the entire gene family members) have homologous relationships, and 127 segment duplication events have occurred, indicating that most WRKY genes in Cardamine violata are derived from the duplication of chromosomal regions or entire chromosomes. In Cardamine violata, the duplicated WRKY genes have similar biological functions, and when treated with exogenous Se, they can form a complex regulatory network and participate in the regulation of gene expression.

[0090] To further explore the molecular evolutionary relationship between species, orthologous WRKY genes between Cardamine violaceae, Shepherd's purse and Arabidopsis were identified using MCScanX. A total of 177 collinear gene pairs were detected between Cardamine violaceae and Shepherd's purse, including 115 CvWRKYs and 68 CvWRKYs, and 53 CvWRKYs shared 2-4 orthologous CvWRKYs; 178 collinear gene pairs were found between Cardamine violaceae and Arabidopsis, including 116 CvWRKYs and 66 AtWRKYs, and 48 CvWRKYs shared 2-4 orthologous AtWRKYs ( Figure 7 B). The present study found that the CvWRKYs gene showed high homology with WRKY in Shepherd's Purse and Arabidopsis thaliana, and Cardamine violacea, Shepherd's Purse and Arabidopsis thaliana are all plants of the Cruciferae family, so the high homology may be attributed to their close relationship.

[0091] (6) Identification of key CvWRKY involved in the regulation of selenium accumulation

[0092] In order to further analyze the WRKY genes involved in regulating selenium metabolism in Cardamine viola, the 120 CvWRKY genes identified from the genome were aligned to the full-length transcriptome database (identity>87.5%), and the duplicate gene IDs were deleted. A total of 67 unique WRKY genes were identified in the transcriptome data. Based on the FPKM values ​​in the transcriptome data, it was found that the 67 WRKY genes under different concentrations of Na2SeO4 treatment were differentially expressed genes, and mainly showed 5 different types of expression patterns ( Figure 8 ), indicating that the above genes can respond to the induction of Na2SeO4 treatment. In order to further explore the key genes that may be involved in the selenium metabolism of Cardamine violaceum, this study further analyzed the FPKM values ​​of 67 genes and the total selenium, organic selenium and inorganic selenium contents in Cardamine violaceum after treatment with different concentrations of Na2SeO4 (Table 2) for correlation analysis. The results showed that the expression level of the CvWRKY034 (F01_transcript / 25855) gene was significantly positively correlated with the selenium content ( Fig. 9 A), and the correlations with total selenium, inorganic selenium, and organic selenium contents were all higher than 0.88 (P<0.001) ( Fig. 9B, C, D), indicating that CvWRKY034 may be involved in the biological process of selenium metabolism and accumulation in Cardamine violata.

[0093] Table 2 Effects of different concentrations of Na2SeO4 on the selenium content of Cardamine violaceum

[0094]

[0095] Studies have reported that AtWRKY47 plays an important role in maintaining selenium homeostasis and tolerance in Arabidopsis thaliana and is involved in selenium accumulation in Arabidopsis thaliana (Wu X, Tao M, Meng Y, Zhu X, Cao S. The role of WRKY47 gene inregulating selenium tolerance in Arabidopsis thaliana. Plant Biotechnol Rep. 2020; 14: 121–129.). In this application, the AtWRKY47 gene sequence is used as the seed sequence. Through sequence alignment, genes with high consistency with the AtWRKY gene sequence are identified in Cardamine viola, namely CvWRKY021 and CvWRKY099, and AtWRKY47, CvWRKY021 and CvWRKY099 are all classified as Group IIb. The sequences of the WRKY conserved domains of the AtWRKY47 and CvWRKY021 genes are completely consistent ( Fig.10 A), and has a close relationship with WRKY genes from other plants, ensuring that this gene belongs to the WRKY gene family ( Fig.10 B). CvWRKY021 and CvWRKY099 were mapped to the transcriptome data, both corresponding to F01_transcript / 22479, and the expression level of the gene increased significantly with the increase of Na2SeO4 treatment concentration, with the highest expression level under 4 mg / L Na2SeO4 treatment; under 16 mg / L Na2SeO4 treatment concentration, the expression level of the gene decreased compared with the expression level under 4 mg / L Na2SeO4 treatment, but there was no significant difference ( Fig.10 C). The above results indicate that the CvWRKY021 gene may be involved in the accumulation of selenium in Cardamine violaceum. The analysis also found that CvWRKY021 and CvWRKY043 could respond to the induced expression of Na2SeO3 treatment; compared with the control group, the above two genes were down-regulated in the aboveground part of Cardamine violaceum and up-regulated in the underground part of Cardamine violaceum ( Fig.10 D), and after being treated with Na2SeO3, the selenium content in both the aboveground and underground parts of Cardamine violacea increased significantly, and the selenium content in the underground part was much higher than that in the aboveground part ( Fig.10E), it is speculated that the main contribution of CvWRKY021 and CvWRKY043 may be involved in the process of selenium absorption by the roots of Cardamine viola.

Claims

1. Identification, characterization and expression analysis of a WRKY transcription factor in Cardamine viola It is characterized in that The steps include: (1) Identification and analysis of the CvWRKY gene family in Cardamine viola 1) The genome of Cardamine viola was obtained from the BIG Data Center of the genome repository to analyze the CvWRKY gene family, and the protein conserved regions of CvWRKY candidate genes were identified using the CDD database of NCBI and the online tool SMART; 2) multiple sequence alignment was performed to remove sequences with incompletely conserved domains and redundancy to identify members of the CvWRKY gene family; 3) Use the online website ExPASy to analyze the physicochemical properties of proteins, and use the Cell-Ploc2.0 online tool to predict subcellular localization information; (2) Construction of phylogenetic tree and chromosome location 1) ClustalX was used to perform multiple sequence alignment of WRKY proteins from Arabidopsis thaliana and Cardamine viola, and MEGA 11 software was used to construct a phylogenetic tree using the neighbor-joining method; 2) Use iTOL online software to visualize and beautify the phylogenetic tree; 3) Obtain gene density and the distribution of CvWRKY on chromosomes from the annotation files of Cardamine violacea genome data, and use TBtools software to display the localization of CvWRKY protein on chromosomes; (3) Motif composition and cis-acting elements of gene structure 1) Use the online software MEME to analyze the conserved motifs of CvWRKY family proteins; 2) TBtools software was used to extract the upstream 2000 bp sequence of the Cardamine viola genome as the promoter sequence, the online database PlantCARE was used to analyze the promoter cis-acting elements, and the results were visualized using TBtools software; (4) Collinearity analysis 1) Intraspecific collinearity analysis: TBtools was used to obtain gene density and chromosome skeleton files from the annotation files of the Cardamine violaceae genome data. TBtools was used to perform intraspecific collinearity analysis on Cardamine violaceae and highlight the collinearity of CvWRKY, and the results were visualized; 2) Interspecific synteny analysis: Download the genome data and annotation files of Shepherd's Purse and Arabidopsis thaliana from the NCBI database, and use TBtools to analyze the synteny between Shepherd's Purse and Cardamine violacea, and between Arabidopsis thaliana and Cardamine violacea, and highlight the synteny of CvWRKY, and visualize the results; (5) Plant materials and treatment 1) Sowing Cardamine violacea until 4-5 true leaves grow, and then transplanting into a hydroponic box filled with 1 / 4 concentration Hoagland nutrient solution; 2) After 10 days, select and ensure that each hydroponic box contains 11 Cardamine violaceae plants with consistent growth for treatment, and then replace the nutrient solution with 1 / 2 Hoagland nutrient solution; After 10 days, select and ensure that each culture box contains 9 Cardamine violaceae plants with consistent growth for Na 2 SeO 4 The nutrient solution was then replaced with 1 / 2 Hoagland nutrient solution and different concentrations of Na 2 SeO 4 Treatments were carried out, each treatment group contained 3 independent biological replicates, and each replicate contained 9 Cardamine violata plants; 3) Contains different concentrations of Na 2 SeO 4 The 1 / 2 Hoagland nutrient solution was replaced every 10 days. After three consecutive treatments, all plants of each replicate were harvested, rinsed with deionized water three times, and the surface moisture was wiped off with filter paper, frozen with liquid nitrogen, mixed evenly, and stored in a -80°C refrigerator for later use; 4) Using the same procedure as above, a batch of Cardamine viola seedlings were re-cultivated, and Cardamine viola plants with consistent growth were selected and cultured for 45 consecutive days; then, 100 mg / L Na 2 SeO 3 1 / 2 of the Hoagland nutrient solution is used to replace the nutrient solution in the incubator to prevent the incubator from using Na 2 SeO 3 1 / 2 Hoagland nutrient solution was used as the control treatment, and each treatment was repeated three times. Each repeat contained 9 Cardamine violae plants. After 15 days of treatment, the aboveground and underground parts were collected, frozen in liquid nitrogen, and stored in a -80°C refrigerator for later use. (6) Screening and expression analysis of CvWRKY genes involved in metabolism and accumulation of Cardamine viola 1) The research team obtained the full-length transcriptome, transcriptome sequencing and selenium content data of Cardamine violata from the plant materials treated for the first time; 2) The protein sequences of the CvWRKY gene family were compared with the full-length transcriptome database by using TBtools software, and protein sequences with a similarity greater than 85% were selected from the full-length transcriptome data for further analysis; 3) Correlation analysis was performed between the expression levels of the screened genes and the total selenium content, organic selenium content, and inorganic selenium content, and the results were visualized using TBtool; (7) Determination of selenium content and qRT-PCR analysis 1) Determination of total selenium content in the aboveground and underground parts by hydride atomic fluorescence spectrometry; Total selenium detection refers to GB 5009.93-2010, using hydride atomic fluorescence spectrometer for determination, weighing 0.2g sample into a digestion tank, then adding 8mL of nitric acid to each digestion tube, pre-digesting on a fully automatic graphite digester, pre-digesting conditions are 180℃, 15 minutes, cooling after pre-digestion, screwing on the lid, placing in a microwave digester for further digestion, the program is set as follows: heating to 120℃ within 5 minutes, holding for 3 minutes; heating to 160℃ within 5 minutes, holding for 3 minutes; heating to 180℃ within 5 minutes, holding for 20 minutes; then, driving acid at 180℃ on a fully automatic graphite digester, driving acid to about 1mL, taking out and cooling to room temperature, then using a prepared 2% nitric acid solution to dilute to 10mL to obtain a test solution; HG-AFS working conditions: negative high voltage 340V, lamp current 100mA, atomization temperature 800℃, carrier gas flow rate 500mL / min, injection volume 1mL, KBH 4 The concentration was 3.5%. The standard curve was drawn using selenium standards purchased from the Chinese Academy of Metrology. Each group of samples had three biological replicates and each sample was measured three times. 2) Use ChamQ Universal SYBR qPCR Master Mix Kit to quantify Na 2 SeO 3 The total RNA from the aboveground and underground parts of Cardamine violacea after treatment was subjected to qRT-PCR analysis.

2. The identification, characterization and expression analysis of a WRKY transcription factor in Cardamine violata according to claim 1, It is characterized in that When constructing the evolutionary tree described in operation 2) of step (2), the Bootstrap value is set to 1000 times.

3. The identification, characterization and expression analysis of a WRKY transcription factor of Cardamine violae according to claim 1, It is characterized in that The maximum number of motifs in the motif analysis described in operation 2) of step (3) is set to 20.

4. The identification, characterization and expression analysis of a WRKY transcription factor in Cardamine violata according to claim 1, It is characterized in that The seeds of Cardamine violacea described in operation 1) of step (5) are provided by Enshi Deyuan Selenium Material Engineering Technology Co., Ltd. and sown into matrix soil for germination.

5. The identification, characterization and expression analysis of a WRKY transcription factor in Cardamine violata according to claim 1, It is characterized in that The hydroponic environmental conditions described in step (5) of operation 1) are: temperature 20±1°C, relative humidity about 75%, light intensity 230 μmol / m 2 / s, lighting time 14h / d.

6. The identification, characterization and expression analysis of a WRKY transcription factor in Cardamine violata according to claim 1, It is characterized in that The size of the hydroponic box described in step (5) operation 1) is 36 cm×24 cm×12.5 cm.

7. The identification, characterization and expression analysis of a WRKY transcription factor in Cardamine violata according to claim 1, It is characterized in that Step (5) The different concentrations of Na 2 SeO 4 0, 0.25, 4.0 or 16.0 mg / L respectively.

8. The identification, characterization and expression analysis of a WRKY transcription factor in Cardamine violata according to claim 1, It is characterized in that In step (7), operation 2) of qRT-PCR, β-actin 3 was used as the reference gene, and 2 -ΔΔCt The relative expression levels of genes were calculated.

Images