CN117447573A - Transcription factor WRKY6 for regulating capsanthin synthase gene and application thereof - Google Patents

Transcription factor WRKY6 for regulating capsanthin synthase gene and application thereof Download PDF

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CN117447573A
CN117447573A CN202311585924.8A CN202311585924A CN117447573A CN 117447573 A CN117447573 A CN 117447573A CN 202311585924 A CN202311585924 A CN 202311585924A CN 117447573 A CN117447573 A CN 117447573A
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wrky6
capsanthin
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欧阳波
唐亚萍
杨生保
甘宇枫
张国儒
沈心彦
石春美
卢永恩
邓萱
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Huazhong Agricultural University
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Abstract

The application relates to the technical field of capsaicin biosynthesis regulation, in particular to a transcription factor WRKY6 for regulating and controlling a capsaicin synthase gene and application thereof. Examples it was found that WRKY6 has the same expression pattern as the capsanthin synthase (CCS) gene in high red material GB 23. The examples demonstrate that WRKY6 is able to bind to the promoter of CCS gene and activate expression of the gene. The embodiment finds that the reduction of the expression quantity of the WRKY6 can obviously reduce the expression of the CCS gene and the content of capsanthin in pepper fruits, and the WRKY6 serving as a regulating factor of the CCS gene can positively regulate the synthesis of capsanthin. The WRKY6 provided in the examples is capable of binding to the promoter of the capsanthin synthase gene CCS and regulating the synthesis of capsanthin in the forward direction.

Description

Transcription factor WRKY6 for regulating capsanthin synthase gene and application thereof
Technical Field
The application relates to the technical field of capsaicin biosynthesis regulation, in particular to a transcription factor WRKY6 for regulating and controlling a capsaicin synthase gene and application thereof.
Background
Capsanthin (Capsanthin) is a natural organic compound with molecular formula of C 40 H 56 O 3 Capsanthin is commonly existing in capsicum fruit tissues, belongs to tetraterpene derivatives, and is a deep carmine needle crystal. Capsanthin as one kind of natural plant pigment has the features of bright color, high color number, high coloring power, high color maintaining effect and high safety, and may be used widely in food, medicine, cosmetics, etc. The economic value is considerable, and the requirements at home and abroad are pretty.
Capsorubin is one of the major carotenoids in peppers, and the color of mature pepper fruits depends primarily on the accumulation of capsorubin and capsorubin. Capsorubin is a metabolic end product of the carotenoid biosynthetic pathway in red pepper fruits, the biosynthesis of which starts with geranylgeranyl pyrophosphate (GGPP), converts 2 molecules of GGPP to phytoene under the catalysis of Phytoene Synthase (PSY), and is further oxidized to lycopene through a series of dehydrogenation and isomerization steps. In capsicum, lycopene is finally converted into capsorubin and capsorubin by the co-participation of a series of enzymes LCYE, LCYB, BCH, ZEP and CCS and oxygen.
At present, although the synthesis route of capsanthin is basically clear, the regulatory mechanism of key enzyme genes (such as CCS) in the synthesis process is still unclear, and a method for breeding varieties with high capsanthin content by intervening in the regulation of the CCS genes is still lacking in the prior art.
Disclosure of Invention
The embodiment of the application discovers a transcription factor WRKY6 which can act on a capsaicin synthase gene CCS promoter, thereby providing technical support for breeding pepper varieties with high capsaicin content.
Therefore, the embodiment of the application at least discloses the following technical scheme:
in a first aspect, the embodiment provides a transcription factor WRKY6 for regulating and controlling a capsanthin synthase gene, wherein the amino acid sequence of the transcription factor WRKY6 is shown as SEQ ID NO. 1.
In a second aspect, the embodiment provides a DNA having a nucleotide sequence shown in SEQ ID NO.2, which encodes the aforementioned transcription factor WRKY6.
In a third aspect, the embodiments provide a recombinant vector comprising the aforementioned DNA.
In a fourth aspect, embodiments provide a method of preparing the aforementioned recombinant vector, comprising: amplifying the DNA serving as a template to obtain a DNA sequence; and recombining the sequence of the DNA into a linearized pGADT7 vector to obtain a pGADT7-CaWRKY6 vector, namely the recombinant vector.
In a fourth aspect, embodiments of the present application provide host microorganisms comprising the aforementioned recombinant vectors.
In a sixth aspect, embodiments of the present application provide a method of increasing capsanthin content comprising the step of encoding the aforementioned DNA into a pepper.
In a seventh aspect, embodiments of the present application provide a method for breeding peppers with high content of capsanthin, comprising the step of breeding peppers with increased content of the transcription factor WRKY6.
In an eighth aspect, embodiments of the present application provide the use of the aforementioned transcription factor WRKY6 or the aforementioned DNA or the aforementioned recombinant vector or the aforementioned method for modulating capsaicin synthesis.
Drawings
FIG. 1 shows phenotypes of GB23 and GB42 materials provided in the examples of the present application in the inversion phase and the red-maturing phase, respectively.
FIG. 2 is a Wen diagram of the differential expression genes of the transition period of the GB23 and GB42 materials provided in the embodiment of the application.
FIG. 3 is a graph of gene enrichment analysis of GB23 material in the color transfer period provided in the examples of the present application.
FIG. 4 is a graph of gene enrichment analysis of GB42 material in the color transfer period provided in the examples of the present application.
FIG. 5 is a Wen diagram of the differential expression genes of GB23/GB42 materials in the color change phase and the red maturation phase provided by the embodiment of the present application.
FIG. 6 is a graph of gene enrichment analysis of GB23/GB42 material in the color transfer phase provided in the examples of the present application.
FIG. 7 is a graph of gene enrichment analysis of GB23/GB42 material in the red ripeness phase provided by the embodiment of the application.
FIG. 8 shows the results of analysis of the expression level of capsanthin synthesis pathway related genes in the transcriptome provided in the examples of the present application.
FIG. 9 shows the results of fluorescence quantitative PCR of key genes of capsanthin synthesis pathway provided in the examples of the present application.
FIG. 10 is a block diagram of the analysis of differential expression genes and carotenoid related materials by the weighted co-expression network provided in the examples of the present application.
FIG. 11 is a graph showing the results of a motif assay for CaCCS promoter binding provided in the examples herein.
FIG. 12 is a graph showing the results of a Y1H study of the interaction of CaWRKY6 with a CaCCS promoter provided in the examples herein.
FIG. 13 is a graph showing the binding of CaWRKY6 to CaCCS promoter as measured by luciferases provided in the examples herein.
FIG. 14 is a graph showing analysis of fruit phenotype and gene expression levels after silencing CaWRKY6 by the VIGS assay provided in the examples of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
Through the research of combining metabonomics with transcriptomics, the application discovers a key transcription factor WRKY6 for regulating and controlling the synthesis of capsanthin in biological materials with high and low capsanthin content. Examples 21 carotenoids were identified in two capsicum materials, wherein the total carotenoids in the GB23 material were 10 times higher than in the GB42 material, with the more diverse carotenoids being β -cryptoxanthin, zeaxanthin, capsanthin and capsanthin. In the carotenoid synthesis pathway, PSY1, BCH1 and CCS genes are remarkably and highly expressed in the red ripe fruits of high pigment material GB 23. Examples WRKY6 and CCS have the same expression pattern in high red material GB23 as determined by weight gene co-expression network analysis. Examples the yeast single hybridization and fluorescence complementation studies demonstrate that WRKY6 is able to bind to the CCS gene promoter and activate gene expression. The embodiment shows that the reduction of the expression quantity of WRKY6 can obviously reduce the expression of CCS genes and the content of capsanthin in pepper fruits in the research of utilizing virus-mediated gene silencing. Therefore, WRKY6 as a regulatory factor of the capsanthin synthase (CCS) gene can positively regulate the synthesis of capsanthin. The WRKY6 provided by the embodiment can be combined with a promoter of a capsanthin synthase gene CCS and positively regulate and control the synthesis of capsanthin, and the discovery of the functional gene provides a research basis for the synthesis of capsanthin.
Based on the above, the embodiment of the application provides a transcription factor WRKY6 for regulating and controlling a capsanthin synthase gene, wherein the amino acid sequence of the transcription factor WRKY6 is shown as SEQ ID NO. 1.
Based on this, the embodiment of the application provides a DNA, the nucleotide sequence of which is shown as SEQ ID NO.2, and the DNA codes for the transcription factor WRKY6.
Based on this, the present examples provide recombinant vectors containing the aforementioned DNA.
In certain embodiments, the heavy vector is a recombinant plasmid.
Based on this, the embodiment of the application also provides a construction method of the recombinant vector containing the DNA, which comprises the following steps: amplifying the DNA serving as a template to obtain a DNA sequence; and recombining the sequence of the DNA into a linearized pGADT7 vector to obtain a pGADT7-CaWRKY6 vector, namely the recombinant vector.
Based on this, embodiments of the present application provide host microorganisms containing the aforementioned recombinant vectors.
Based on this, embodiments of the present application provide a method of increasing capsanthin content comprising encoding the aforementioned DNA into capsicum.
Further, the method comprises the step of expressing the transcription factor WRKY6 in capsicum by using an expression vector which overexpresses the DNA shown as SEQ ID NO. 2.
Based on this, the embodiment of the application provides a method for breeding high-content capsanthin peppers, which comprises the step of breeding peppers with the content of the transcription factor WRKY6 improved.
In certain embodiments, the methods comprise breeding pepper plants that are constructed by genetic engineering means that overexpress the transcription factor WRKY6. For example, an overexpression vector (pHELLSGATE 8-CaWRKY 6) of the WRKY6 gene is constructed, pepper WRKY6 gene overexpression plants are obtained through agrobacterium-mediated genetic transformation and plant tissue culture technology, and the overexpression plants are identified.
The construction process of the WRKY6 gene over-expression vector (pHELLSGATE 8-CaWRKY 6) comprises inserting a target gene shown in SEQ ID NO.2 into pHELLSGATE8 (SnapGene) to obtain a connection product, transferring the connection product into escherichia coli DH5 alpha, and sequencing and verifying the positive bacteria extraction plasmid obtained after transformation.
Wherein, agrobacterium-mediated capsicum genetic transformation is carried out according to GV3101 instruction, and the specific steps comprise: placing 100 full capsicum seeds at the bottom of a small beaker, sterilizing the surfaces of the capsicum seeds with 75% ethanol for 1min, sterilizing the seeds with 8% NaClO solution for 15min, and cleaning with sterile water for multiple times. Sowing the sterilized seeds on a culture medium T0, culturing in dark for 5-7 d, and transferring the seeds to illumination after germination. Cotyledon leaves were cut into small pieces and placed on T1 preculture medium and cultured for 2d under dark conditions. Culturing the constructed agrobacterium in a liquid culture medium until the A600 value is between 0.5 and 0.8, and collecting the thalli by a centrifugal method. Removing the supernatant, adding the MS suspension to resuspend the agrobacterium until the A600 value is 0.1-0.2, and obtaining the infectious microbe liquid. The precultured cotyledons were placed in sterile petri dishes, and the invasive inoculum was poured and shaken well. After 5min of infection, the infected bacterial liquid is sucked up by a pipetting gun, the cotyledons are put back on the preculture medium T1 again, and co-culture is carried out in the dark for 2d. After the co-culture is finished, transferring the explant to a T21 culture medium for selective culture, and transferring the explant to a culture medium T22 for continuous culture when the bud point of the explant grows. And when the bud length is 1-2cm, transferring into a rooting culture medium T3 for rooting, and finally transplanting the rooted pepper regenerated seedlings into soil for planting management after uncovering and hardening.
Wherein, the identification step of the WRKY6 over-expressed pepper plants comprises the following steps: DNA is extracted from wild type and over-expressed pepper plant leaves according to kanamycin resistance sequence design primer in pHELLSGATE8-CaWRKY6, and PCR identification of transgenic pepper plants is carried out by taking the DNA as a template. And detecting the content of capsanthin in fruits of the wild type and the over-expressed pepper plants in the red ripe stage, and finding that the content of capsanthin in fruits of the over-expressed pepper plants in the red ripe stage is 1.2-1.4 times that of the wild type plants.
Based on this, the embodiments of the present application provide the use of the aforementioned transcription factor WRKY6 or the aforementioned DNA or the aforementioned recombinant vector or the aforementioned method for modulating capsaicin synthesis.
The technical solutions of the embodiments of the present application will be clearly and completely described below with reference to more specific embodiments and the accompanying drawings, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments, and therefore, the described embodiments should not be construed as limiting the present application.
1. Screening of capsorubin regulatory genes
In this example, two chilli materials with different red pigment contents are compared, namely GB23 and GB42 respectively, 21 carotenoids are identified, 13 metabolites are derived from the biosynthesis pathway of the carotenoids, the content of the carotenoids is shown in table 1, and reference is made to the detection methods of the red pigment contents in the table, namely "Wang Haihai, huang Yongcai, xiao Qiao, et al, carotenoids modulate kernel texture in maize by influencing amyloplast envelope integery Nature Communications,2020,11:5346". Figure 1 shows the fruit phenotype of both materials GB23, GB42 in the relay colour phase and in the red ripe phase respectively.
TABLE 1
As can be seen from Table 1, the total carotenoid content of fruits in the red ripe stage is higher than that of fruits in the color conversion stage in both GB23 and GB42 materials, and is more obvious in the GB23 materials, while lycopene, epsilon-carotene and canthaxanthin in the capsanthin synthesis pathway are not detected in fruits of both varieties of GB23 and GB42 materials; in addition, in the materials GB23 and GB42, the relative contents of substances such as beta-apocarotin aldehyde, astaxanthin, echinocandine and beta-limonin are low, and the difference is small.
As can be seen from Table 1, the total carotenoid content in GB23 was 168.50 μg/g during the color shift phase, which was significantly lower than that of GB42 (676.24 μg/g); the content of the detected carotenoid is low in GB 23; the accumulation of capsorubin and capsorubin, which are the most important red pigment compounds in capsicum fruits, is low in fruits in the color transfer period of both GB23 and GB42 materials.
As can be seen from Table 1, during the red ripening period, total carotenoids accumulated in large amounts in both capsicum materials, wherein the total carotenoid content in the GB23 fruits reached 28354.75 μg/g, which was 12.8 times the total carotenoid content of GB42; in the red maturation stage, the synthesis of carotenoids in the beta-branch of GB23 is very active, wherein the content of zeaxanthin, beta-cryptoxanthin, capsorubin and capsorubin is higher than GB42; zeaxanthin in GB23 accounts for 34.7% of the total carotenoid content, 24.67 times that in GB 42. Similarly, the content of beta-cryptoxanthin in GB23 is 8049.23 mug/g, which is 26.38 times that of GB42; the content of capsorubin and capsorubin in GB23 fruit is 2885.69 μg/g and 1019.05 μg/g, respectively, which are 7 times and 12 times of GB42 fruit.
From Table 1, it is clear that from the color transition period to the red ripeness period, the total carotenoid content in GB23 was increased by 168.27 times, while the total carotenoid content in GB42 was increased by only 3.26 times. Thus, capsaicin and capsaicin were detected to be more actively biosynthesized in GB23 than in GB 42.
The transcriptomics analysis is carried out on fruits and pericarps of two different capsicum materials in the color conversion stage and the red ripeness stage of GB23 and GB 42. As a result, 5833 and 1361 Differentially Expressed Genes (DEG) were identified from GB23 and GB42, respectively, during the fruit's color change, wherein the total DEG of both materials was 786 (as shown in FIG. 2). In the maturation of the GB23 fruits, 523 genes were up-regulated, 263 genes were down-regulated, and 547 genes were up-regulated and 239 genes were down-regulated in the GB42 fruits in the common DEGs. Through gene function enrichment analysis, the up-regulated differential expression genes mainly participate in the oxidation-reduction process, chloroplast and kinase activity, and the down-regulated differential expression genes mainly participate in DNA template, cytoplasm and protein combination. In the examples herein 5047 differentially expressed genes were specifically detected in GB23 material, most of which are involved in the biosynthesis of metabolic pathways and secondary metabolites; as shown in fig. 3, carotenoid biosynthesis is the most enriched pathway for the fruit development process of GB23 material, which contributes to the accumulation of higher capsanthin red pigment in GB 23; as shown in fig. 4, in GB42 material, most of the differentially expressed genes are also involved in metabolic pathways and biosynthesis of secondary metabolites, the most abundant pathway being fatty acid metabolism, unlike GB23 material.
As shown in fig. 5, the materials GB23 and GB42 have 5934 Differentially Expressed Genes (DEG) in the color transfer phase and 2566 DEG in the red maturation phase, wherein there are 1388 DEG co-existing in the two phases; during the red maturation period, only 1178 DEG's were identified between GB23 and GB42, of which 627 were up-regulated and 551 were down-regulated. FIGS. 6 to 7 show the results of gene function enrichment analysis of GB23 and GB42 materials in the transchronicity stage and the red maturation stage, respectively, as can be seen from the figures, these up-regulated differential genes are involved in the defense reaction, the cell nucleus and the ATP binding, while the down-regulated differential genes are involved in the biological process, the cell nucleus and the protein binding. A large number of differentially expressed genes are involved in the biosynthesis of metabolic pathways and secondary metabolites. As shown in fig. 6, the most gene-rich pathway of difference between these two materials during the color shift phase is carotenoid biosynthesis; as shown in fig. 7, during the red-maturing phase, the most abundant pathway for differential genes is plant-pathogen interaction, followed by carotenoid biosynthesis.
In the examples of the present application, the following conclusions can be drawn from transcriptome data: of the 26 genes involved in the carotenoid synthesis pathway, PSY1, PDS, ZDS, LCYB, BCHs and CCS have higher expression levels; as shown in FIG. 8, in the above high expression genes, the expression differences of PSY1, BCHs and CCS in the two materials GB23 and GB42 are obvious, while the expression amounts of PDS, ZDS and LCYBs in the red ripening period and between the two materials are not obvious; of the 3 PSY genes, PSY1 (Capana 04g 002519) was expressed at the highest level, while the other 2 PSY homologous genes were expressed at very low levels in both varieties of fruits. In GB23 (high pigment system), PSY1 is upregulated 125.14-fold from the relay-to-red ripe stage, whereas in the fruit red ripe stage its expression in GB42 (low pigment system) is even slightly reduced; in the beta-branch of capsanthin synthesis, the transcription level of the gene is far higher than that of the alpha-branch gene, which indicates that the biosynthesis of beta-carotene in capsicum fruits is very active; two BCH genes exist in the beta-branch carotenoid synthesis pathway, wherein the expression quantity of BCH1 (Capana 03g 002170) is far higher than that of BCH2 (Capana 06g 002492) and the difference is at least 20 times; the BCH1 gene was up-regulated 23.37-fold from the transchronicity stage to the red ripening stage in GB23 fruit, while in GB42, stable and high-level expression was maintained in both stages. CCS is the last key enzyme in capsicum synthesis, and CCS genes are highly expressed in fruits of both varieties; meanwhile, it has the highest transcription amount in the red maturation period among all structural genes of the pathway; the CCS gene was only up-regulated in GB23, with a 75.99 fold increase in transcription level from the transfer phase to the red maturation phase. From the above results, it can be seen that PSY1, BCH1 and CCS genes are highly expressed and up-regulated during the maturation of high pigment pepper varieties.
2. Verification of capsorubin regulatory genes
The RNA-seq results of the screened genes are confirmed, namely, the expression of six selected structural genes including PSY1, BCH1, CCS, ZDS, LCYB and LCYB2 genes is verified by adopting qRT-PCR, and the specific qRT-PCR method is as follows:
extracting RNA from capsicum pericarp samples for synthesizing cDNA; 3 biological replicates were performed for each sample using capsicum CaUBI as a control; PCR gene specific primers were designed using Primer 5.0, and specific primers are shown in Table 2 below; the PCR reaction system is as follows: 5. Mu.L of 2X SYBR Green master mix (Thermo Fisher, waltham, mass., U.S.A.), 1. Mu.L of cDNA template, 0.5. Mu.L of each forward and reverse primer, and 3. Mu.L of RNase-free water; qRT-PCR was performed on qTOWER3 (Analytik Jena, jena, germany) with the procedure set to: preheating at 95 ℃ for 2 minutes, then performing 38 cycles, each cycle comprising 95 ℃,15 seconds, 60 ℃,15 seconds and 72 ℃,30 seconds; performing qRT-PCR reaction on each group of three independent RNA samples; the 2-delta CT method was used to calculate gene expression levels.
TABLE 2
Primer name Forward primer (5 'to 3') Reverse primer (5 'to 3')
qCaPSY1 SEQ ID NO.3 SEQ ID NO.4
qCaBCH1 SEQ ID NO.5 SEQ ID NO.6
qCaLCBY1 SEQ ID NO.7 SEQ ID NO.8
qCaLCBY2 SEQ ID NO.9 SEQ ID NO.10
qCaZDS SEQ ID NO.11 SEQ ID NO.12
qCaCCS SEQ ID NO.13 SEQ ID NO.14
qPCR-CaUBI3 SEQ ID NO.15 SEQ ID NO.16
As shown in FIG. 9, the gene shows different expression patterns between two pepper varieties GB23 and GB42, the expression level is relatively high, and the qRT-PCR result is consistent with the RNA-seq result as shown in the graph.
3. Co-expression network analysis (WGCNA) analysis of capsanthin regulatory genes
In order to further dig the regulatory genes in the capsaicin synthesis pathway, the embodiment of the application utilizes the weight gene co-expression network analysis to identify the regulatory factors of capsaicin synthesis; weighted co-expression network analysis (WGCNA) was performed using all DEG identified in the transcriptome and 21 carotenoids identified in the metabolome; cluster analysis of the DEGs expression data resulted in 22 clusters (groups) labeled with different colors.
As shown in fig. 10, 11 modules were obtained by WGCNA analysis between 9319 differentially expressed genes and 21 carotenoid compounds, from which it is known that DEG associated with capsanthin and capsanthin content accumulated in the blue and green modules in higher amounts; most other carotenoid metabolites are also strongly positively correlated with blue and green modules, but negatively correlated with red and black modules; differentially expressed genes in blue and green modules are involved in metabolism, biosynthesis of amino acids and secondary metabolic pathways; differentially expressed genes associated with metabolic pathways, ribosomes, and RNA transport are enriched in the black module, while differentially expressed genes associated with ribosomal, RNA polymerase, and proteasome synthesis are enriched in the red module. From the above modules with high correlation with capsaicin synthesis, examples have mined a transcription factor WRKY6 (Capana 02g 002230) that is highly correlated with CCS gene expression and has a similar expression pattern.
4. Yeast Single hybridization assay (Y1H)
Using GB23 genome DNA as a template, combining corresponding primers, amplifying potential promoter regions of CaCCS, wherein the sequences of the primers for amplification are shown as SEQ ID NO.17 and SEQ ID NO.18, and inserting the amplified promoter fragments into pAbAi vectors linearized by KpnI and XhoI (Thermo Fisher) by a homologous recombination method to generate bait (bait) vectors; to obtain prey vectors, the coding sequence (CDS) of CaWRKY6 was amplified, wherein the primers used for amplification were as shown in SEQ ID No.19, SEQ ID No.20, and recombined into pGADT7 vectors linearized by NdeI and XhoI (Thermo Fisher); the bait vector was transformed into yeast strain Y1H Gold, treated using the PEF/LiAc method, and cultured in SD/-Ura medium; next, pGADT7-CaWRKY6 vector was transferred into Y1H Gold strain containing pAbAi-CCSpro, and plated on SD/-Ura-Leu plates; positive clones were cultured, diluted to different optical densities (OD 600 = 0.1,0.05,0.01,0.001 and 0.0001) with 0.9% NaCl solution, and then spotted on SD/-Ura-Leu plates with or without Aureobasidin A; culturing at 30 deg.c for 2-3 days and photographing the clone; pGADT7 and pAbAi-p53 were used as positive controls, and pGADT7 and pAbAi-empty were used as negative controls.
In order to verify the interaction between WRKY6 and the CaCCS promoter, a yeast single hybridization assay (Y1H) was performed. As shown in FIG. 11, the results of the prediction that the CaCCS promoter contains various cis-elements Y1H of C2H2 and WRKY indicate that CaWRKY6 interacts with the CaCCS promoter as shown in FIG. 12.
5. Bimolecular luciferase assay (LUC)
In the embodiment, double luciferase detection is carried out by using leaves of Nicotiana benthamiana (Nicotiana benthamiana) to test the transcriptional activation activity of CaWRKY6 (TFs) on a target promoter, and the specific steps are as follows:
amplifying the coding sequence of CaWRKY6, wherein the sequences of the primers used for amplification are shown as SEQ ID NO.21 and SEQ ID NO. 22; cloning the coding sequence of CaWRKY6 into pHELLSGATE8 vector (pHG 8); amplifying a promoter sequence of CaCCS, wherein the sequence of a primer for amplification is shown as SEQ ID NO.23 and SEQ ID NO. 24; recombining the promoter sequences of CaCCS into pK7LIC vectors (LUCs), respectively; all recombinant vectors were introduced into agrobacterium Agrobacterium tumefaciens GV3101 and stored at-80 ℃; the agrobacterium culture was diluted to od600=0.5 with an infiltration buffer (10mM MES,10mM MgCl 2 150mM acetosyringone, pH 5.6); the agrobacterium containing transcription factors was mixed with the strain containing promoter plasmid in a volume ratio of 1:1 and then infiltrated into tobacco leaves using a needleless syringe, and the control used an empty pHG8 vector. For each LUC detection of TF-promoter interactions, 6 biological replicates were performed; three days after infiltration, the leaves were treated with 100. Mu.M VivoGlo fluorescein solution (Promega) and kept in the dark for 5 minutes; images and data of the LUC signal were acquired using a low light frozen CCD imaging device (NightSHADE LB985, berthold, germany).
As shown in FIG. 13, luciferase assay results indicate that CaWRKY6 can activate CaCCS expression in vivo.
6. Virus mediated gene silencing studies (VIGS)
The embodiment of the application utilizes virus-mediated gene silencing research to inhibit the expression level of CaWRKY6 gene in capsicum, and specifically comprises the following steps:
cDNA of capsicum was obtained from GB23 to fruit. Designing CaWRKY6 silent fragments by using VIGS-tool (solgenomics. Net), and amplifying target fragments from the cDNA of GB23, wherein the sequences of primers for amplification are shown as SEQ ID NO.25 and SEQ ID NO. 26; inserting the target fragment into a TRV2-C2b vector subjected to SmaI single enzyme digestion by using a homologous recombination method, and sequencing and identifying; the vector was transformed into GV3101, and the monoclonal was cultured overnight at 28℃and resuspended in the invader solution with TRV1 (100mM MES,100mM MgCl) 2 200 mu Macetosporine, pH 5.6) OD600 was 1.0.TRV2-Empty, TRV2-CaPDS was negative and positive control, respectively, and TRV1 (1:1, v/v) was injected into cotyledons of 3 weeks of seedling age of Capsicum GB23, cultured normally after 3d in the dark, positive control leaf phenotype was observed after 4 weeks, and flower morphology changes were observed in the flowering phase.
As shown in fig. 14, the expression level of CCS gene regulating capsanthin in capsicum fruit was significantly reduced, while the content of capsanthin was also significantly reduced compared to the control.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Claims (10)

1. A transcription factor WRKY6 for regulating and controlling capsanthin synthetase gene has an amino acid sequence shown in SEQ ID NO. 1.
2. A DNA having a nucleotide sequence as set forth in SEQ ID No.2, said DNA encoding the transcription factor WRKY6 of claim 1.
3. A recombinant vector comprising the DNA of claim 2.
4. The recombinant vector according to claim 3, which is a recombinant plasmid.
5. The method for constructing a recombinant vector according to claim 3, which comprises:
amplifying the sequence of the DNA of claim 2;
and recombining the sequence of the DNA into a linearized pGADT7 vector to obtain a pGADT7-CaWRKY6 vector, namely the recombinant vector.
6. A host microorganism comprising the recombinant vector of claim 3.
7. A method of increasing capsanthin content comprising the step of encoding the DNA of claim 2 into capsicum.
8. The method of claim 7, further comprising the step of expressing the transcription factor WRKY6 in capsicum using an expression vector over-expressing the DNA shown in SEQ ID No. 2.
9. A method for breeding peppers with high content of capsanthin, comprising the step of breeding peppers with increased content of the transcription factor WRKY6 of claim 1; optionally, the method comprises the step of breeding peppers which are constructed by genetic engineering means and over-express the transcription factor WRKY6 as claimed in claim 1.
10. Use of a transcription factor WRKY6 according to claim 1 or a DNA according to claim 2 or a recombinant vector according to any one of claims 3 to 4 or a method according to any one of claims 7 to 8 for modulating capsaicin synthesis.
CN202311585924.8A 2023-11-22 2023-11-22 Transcription factor WRKY6 for regulating capsanthin synthase gene and application thereof Pending CN117447573A (en)

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