Application of transcription factor PgWRKY2 gene in promotion of punicalagin synthesis

Abstract

The invention provides application of a transcription factor PgWRKY2 gene in promotion of punicalagin synthesis, and belongs to the technical field of gene engineering and molecular biology, experiments verify that the PgWRKY2 gene can positively regulate and control expression of punicalagin synthesis genes PgUGT84A23 and PgPOR9, so that synthesis of punicalagin is promoted; the silent PgWRKY2 in pomegranate peel can inhibit the expression of PgUGT84A23 and PgPOR9, so that the content of punicalagin is reduced. The invention provides an important theoretical basis for improving the pomegranate quality and cultivating excellent pomegranate varieties.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and molecular biology, specifically the application of the transcription factor PgWRKY2 gene in promoting the synthesis of pungent glycosides. Background Technology

[0002] Pomegranate( Punica granatum Pomegranate (Punica granatum) is a distinctive economic forest tree species belonging to the Lythraceae family and the Punica genus. It originated in Central Asia, including Iran and Afghanistan, and has a wide range of adaptability. Pomegranates are rich in ellagic tannins, flavonoids, and alkaloids, possessing potential antioxidant, anti-inflammatory, antibacterial, and anticancer properties. my country is one of the world's major pomegranate producing countries, with a long history of cultivation and abundant germplasm resources.

[0003] Punicantin, a functional component abundant in pomegranates, is responsible for their antioxidant and other benefits. Increasing its content by regulating its biosynthesis is an important way to enhance the economic value of pomegranates. Existing research indicates that... PgUGT84A23 This gene is a key link between shikimic acid and ellagitannin in the punicalin synthesis pathway, encoding uridine diphosphate glucose glycosyltransferase (UGT), which regulates the metabolism of gallic acid to β-glucosidin. Pentagalloylglucoseoxygen oxidoreductase (POR), a type of laccase, plays an important regulatory role in the metabolism of pentagalloylglucose to punicalin, promoting its accumulation. PgPOR9 It is a member of the POR gene family. WRKY is an important family of transcription factors in plants. By binding to specific cis-acting elements on the promoters of target genes, it activates or inhibits the expression of key structural genes, thereby affecting the accumulation of secondary metabolites. The members of the WRKY transcription factor family are closely interconnected in plant growth, development, and responses to biotic and abiotic stresses, forming a highly interactive and complex network that participates in regulating gene expression at different levels.

[0004] Currently, there are few reports on the metabolic regulatory network of pungent glycosides and the mutual regulation between genes. Research on the promotion of pungent glycoside synthesis by transcription factors regulating structural genes is of great theoretical and practical significance for improving pomegranate quality and cultivating superior pomegranate varieties. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of the transcription factor PgWRKY2 gene in promoting the synthesis of pungent glycosides. The PgWRKY2 gene can positively regulate... PgUGT84A23 and PgPOR9 The expression of pungent compounds promotes the synthesis of pungent glycosides.

[0006] The technical solution of this invention is as follows: The application of the transcription factor PgWRKY2 gene in promoting the synthesis of pomegranate glycoside, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Preferably, the amino acid sequence of the PgWRKY2 is shown in SEQ ID NO.2.

[0008] Preferably, the PgWRKY2 gene increases... PgUGT84A23 The expression level of [the substance] promotes the synthesis of pungent glycosides.

[0009] Preferably, the PgWRKY2 gene increases... PgPOR9 The expression level of [the substance] promotes the synthesis of pungent glycosides.

[0010] Preferably, the PgWRKY2 gene increases... PgUGT84A23 and PgPOR9 The expression level of [the substance] promotes the synthesis of pungent glycosides.

[0011] A pomegranate having an exogenous nucleic acid molecule incorporated into the genome, the exogenous nucleic acid molecule containing the PgWRKY2 gene, the nucleotide sequence of the PgWRKY2 gene being shown in SEQ ID NO.1.

[0012] Beneficial effects: This invention provides the application of the transcription factor PgWRKY2 gene in promoting the synthesis of pungent glycosides. Experiments have verified that the PgWRKY2 gene can positively regulate the synthesis of pungent glycosides. PgUGT84A23 and PgPOR9 The expression of [a substance] promotes the synthesis of pungent glycosides; silencing PgWRKY2 in pomegranate peel can inhibit [the synthesis of pungent glycosides]. PgUGT84A23 and PgPOR9 The expression of [the substance] is reduced, thereby lowering the content of pungent glycosides. This invention provides an important theoretical basis for improving pomegranate quality and cultivating superior pomegranate varieties. Attached Figure Description

[0013] Figure 1 The content of pungentin in pomegranate peel at different developmental stages and its relationship with PgWRKY2, PgPOR9, PgUGT84A23 Changes in the level of expression; Figure 2 The distribution of PgWRKY2 in different tissues of pomegranate; Figure 3 Subcellular localization of PgWRKY2 protein; Figure 4 For PgWRKY2 and PgUGT84A23 , PgPOR9 The relationship between expression and pomegranate glycoside metabolism; Figure 5 These are the results of a yeast one-hybrid experiment; Figure 6 These are the results of a dual-luciferase assay; Figure 7 The results are from the VIGS silencing experiment. Detailed Implementation

[0014] The following description is based on specific embodiments: Example 1: Expression level of PgWRKY2 and content of pungent glycosides in pomegranate at different developmental stages Using 'Taishanhong' pomegranate as the research object, qRT-PCR technology was used to detect the content of punicin in the peel of the fruit at different developmental stages (S1, S2, S3, S4, S5, S6). PgWRKY2 , PgPOR9, PgUGT84A23 The dynamic changes in expression levels. Among them, S1 is the young fruit stage, and samples are taken every 10 days from S1, and are respectively recorded as S2, S3, S4, S5, and S6, with S6 being the maturity stage.

[0015] The results are as follows Figure 1 As shown in the figure, Figure A shows the dynamic changes in pungent glycoside content at different developmental stages, while Figures B, C, and D represent... PgWRKY2 , PgPOR9, PgUGT84A23 The dynamic changes in the level of expression. (By...) Figure 1 Analysis shows that the content of pomegranate glycosides is related to... PgWRKY2 , PgPOR9, PgUGT84A23 The trends in expression levels are the same and highly correlated.

[0016] Using qRT-PCR technology, the levels of aril (seeds), flower (flower), leaf (leaf), peel (peel), root (root), and thorn (thorn) in different tissue parts of pomegranate were further detected. PgWRKY2 Level of expression, results as follows Figure 2 As shown. By Figure 2 We can obtain, PgWRKY2 The expression level in pomegranate peel was significantly higher than in other parts.

[0017] Example 2: Subcellular localization of the PgWRKY2 gene Primers WRKY2-F and WRKY2-R were designed and synthesized based on the CDS sequence of PgWRKY2, and the primer sequences are shown in SEQ ID NO. 3~4, respectively. Then, using pGADT7-WRKY2 plasmid (Shandong Nuoding Biotechnology Co., Ltd.) as a template, the CDS sequence of PgWRKY2 was amplified using PrimeSTAR HS DNA polymerase.

[0018] The PC1300S-GFP plasmid (Shandong Nuoding Biotechnology Co., Ltd.) was double-digested with SacI and BamHI. Then, the digested PC1300S-GFP plasmid was ligated with the CDS sequence of PgWRKY2 using a recombinant ligation kit. After incubation at 50℃ for 30 min, the recombinant plasmid PC1300S-GFP-WRKY2 was obtained. PC1300S-GFP-WRKY2 and the empty vector PC1300S-GFP were transformed into DH5α competent cells, respectively, and then plated on LB solid medium containing 50 μg / mL kanamycin. After overnight culture, positive clones were screened and sequenced, confirming the successful construction of the recombinant plasmid vector PC1300S-GFP-WRKY2 (PC1300S-PgWRKY2-GFP).

[0019] Positive clones were selected for bacterial culture propagation, and plasmid PC1300S-GFP-WRKY2 was extracted. Simultaneously, the empty vector PC1300S-GFP was used as a control. PC1300S-GFP-WRKY2 and the empty vector PC1300S-GFP were ligated into plasmid osGhd7-RFP (Shandong Nuoding Biotechnology Co., Ltd.), and co-transformed into Arabidopsis protoplasts (Shandong Nuoding Biotechnology Co., Ltd.). The specific procedures are as follows: The ligated plasmid was dissolved in sterile water to adjust the plasmid concentration to 1.5 μg / μL. 20 μL of the plasmid was transferred to a centrifuge tube, and Arabidopsis protoplasts and 220 μL of PEG4000 solution were added. The mixture was immediately and gently inverted to mix, and incubated at room temperature for 10 min. 800 μL of W5 solution was added to dilute and terminate the transformation reaction. After gentle mixing, the mixture was centrifuged at 100 g for 8 min at room temperature to precipitate the protoplasts. The supernatant was carefully discarded, and the protoplast precipitate was resuspended in 1 mL of W5 solution. The mixture was then incubated at 20–23 °C under low light conditions for 10 h. The results were observed under a laser confocal microscope. Figure 3 As shown in the figure, "GFP, RFP, Chlorophyil fluorescence, Bright field, and Merged" represent green fluorescent protein, red fluorescent protein, chloroplast autofluorescence, bright field, and superimposed field, respectively.

[0020] Depend on Figure 3 Therefore, the PgWRKY2 protein is located in the cell nucleus.

[0021] Example 3: PgWRKY2 and PgUGT84A23 , PgPOR9 Expression of Relationship The biosynthesis of punicalin is directly regulated by UGT (uracil diphosphate-glucosyltransferase) and POR (pentagalloylglucose oxidoreductase). Therefore, using transcriptome sequencing and bioinformatics analysis, the UGT and POR genes in pomegranate were screened, and the results are as follows: Figure 4 As shown, Figure A presents the results of UGT gene screening, and Figure B presents the results of POR gene screening. Figure 4 A total of 25 differentially expressed UGT genes and 15 differentially expressed POR genes were identified. Correlation analysis showed that PgWRKY2 and... PgUGT84A23 , PgPOR9 The expression of punice and the metabolism of punice are significantly positively correlated.

[0022] Example 4: Direct Regulation of PgWRKY2 PgUGT84A23 , PgPOR9 transcription I. Yeast One-Hybrid Experiment: To investigate whether PgWRKY2 regulates PgUGT84A23 and PgPOR9 To assess the effect of PgWRKY2 on regulating the accumulation of pungent glycosides, a yeast one-hybrid (Y1H) experiment was conducted. PgUGT84A23 , PgPOR9 The specific binding status of the promoter is determined. This is done according to the following steps: (1) RNA was extracted from pomegranate peel, and cDNA was obtained by reverse transcription. Using the cDNA as a template, primers pHis-POR9-F and pHis-POR9-R (primer sequences are shown in SEQ ID NO.5~6, respectively) were used to amplify the RNA. PgPOR9 The promoter fragment was obtained and cloned into the pHIS2 plasmid (Shandong Nuoding Biotechnology Co., Ltd.) to obtain the recombinant plasmid PgPOR9pro-pHIS2.

[0023] (2) Using cDNA as a template, primers pHis-UGT84A23-F and pHis-UGT84A23-R (primer sequences are shown in SEQ ID NO. 7~8 respectively) were used to amplify the cDNA. PgUGT84A23 The promoter fragment was obtained and cloned into the pHIS2 plasmid to obtain the recombinant plasmid PgUGT84A23pro-pHIS2.

[0024] (3) Using cDNA as a template, primers AD-WRKY2-F and AD-WRKY2-R (primer sequences are shown in SEQ ID NO. 9~10 respectively) were used to amplify the cDNA. PgWRKY2 and will PgWRKY2 The plasmid AD-PgWRKY2 was cloned into the pGADT7-AD plasmid (Shandong Nuoding Biotechnology Co., Ltd.).

[0025] Set up the following groups respectively: Experimental group 1: Recombinant plasmids AD-PgWRKY2 and PgPOR9pro-pHIS2 were co-transformed into yeast competent cells Y187; Control group 1: Empty vector pGADT7-AD (AD-Empty) and PgPOR9pro-pHIS2 were co-transfected into yeast competent cells Y187; Experimental group 2: Recombinant plasmids AD-PgWRKY2 and PgUGT84A23pro-pHIS2 were co-transformed into yeast competent cells Y187; Control group 2: Empty vector pGADT7-AD (AD-Empty) and PgUGT84A23pro-pHIS2 were co-transfected into yeast competent cells Y187.

[0026] The recombinant yeast cells were cultured on SD / –Leu / –Trp / –His selective plates, and cell growth was observed. The results are as follows: Figure 5 As shown in the figures, Figure A illustrates the growth of yeast cells at a 3-AT concentration of 0 mM, and Figure B illustrates the growth of yeast cells at a 3-AT concentration of 125 mM. 3-AT (3-amino-1,2,4-triazole) is a competitive inhibitor that improves the rigor of the screening system by inhibiting the basal expression of the reporter gene, thereby effectively reducing false-positive clones caused by non-specific binding or reporter gene leakage expression. Figure 5 The recombinant yeast cells in experimental groups 1 and 2 grew vigorously on SD / –Leu / –Trp / –His selective plates, while no significant growth was observed in the recombinant yeast cells in control groups 1 and 2. This result indicates that PgWRKY2 can specifically bind to… PgUGT84A23 , PgPOR9 The promoter region is combined.

[0027] II. Dual-luciferase assay in tobacco: Verification of the effects of PgWRKY2 protein on tobacco using a dual-luciferase system PgUGT84A23 , PgPOR9 The transcriptional activation of the gene was specifically achieved by invading tobacco leaves with the luciferase reporter gene constructs LUC::PgPOR9 (Nos-T1) and LUC::PgUGT84A23 (Nos-T2) along with the 35Spro::PgWRKY2 construct. Simultaneously, the above reporter gene constructs were co-infiltrated with an empty vector (35Spro) as a control group. Here, "LUC" represents the luciferase reporter gene, and "35Spro" represents the cauliflower mosaic virus 35S promoter, a strongly constitutive promoter. A schematic diagram of the construction is shown below. Figure 6 As shown in Figure A.

[0028] Experimental results are as follows Figure 6 Figures B through C in the table show that PgWRKY2 activates the expression of LUC::PgPOR9 and LUC::PgUGT84A23 in tobacco leaves, while Figure C shows the relative luciferase activity. Figures B through C show that, compared to the control group, the co-expression of 35Spro::PgWRKY2 with either LUC::PgUGT84A23 or LUC::PgPOR9 significantly increased luciferase activity, indicating that PgWRKY2 can directly activate... PgUGT84A23 and PgPOR9 Transcription, thereby positively regulating PgUGT84A23 and PgPOR9 The expression.

[0029] Example 5: VIGS Silencing Experiment To verify whether PgWRKY2 can regulate PgUGT84A23 and PgPOR9 The expression of [a specific gene] affects the accumulation of pungent glycosides. This study employed viral gene silencing (VIGS) technology to conduct a transient expression experiment. The specific procedure is as follows: Primers VIGS-WRKY2-F / VIGS-WRKY2-R, VIGS-UGT84A23-F / VIGS-UGT84A23-R, and VIGS-POR9-F / VIGS-POR9-R (primer sequences are shown in SEQ ID NO. 11~16, respectively) were designed. Then, using pGADT7-WRKY2, pGADT7-UGT84A23, and pGADT7-POR9 plasmids (synthesized by Shandong Nuoding Biotechnology Co., Ltd.) as templates, the target fragments were amplified by PCR using PrimeSTAR HS DNA polymerase. PgWRKY2 , PgUGT84A23 and PgPOR9 .

[0030] The pTRV2 plasmid (Shandong Nuoding Biotechnology Co., Ltd.) was double-digested with XbaI and BamHI, incubated at 37℃ for 15 min, detected by agarose gel electrophoresis, and the target fragment was recovered by gel excision; the above target fragment was then ligated using a recombination ligation kit. PgWRKY2 , PgUGT84A23 , PgPOR9The recombinant expression plasmid pTRV2 was ligated with enzyme-digested plasmids and incubated at 50°C for 30 min. The resulting plasmids were then transformed into DH5α competent cells and plated onto LB agar containing 50 μg / mL kanamycin. After overnight incubation, positive clones were screened and sequenced. The correctly sequenced recombinant expression plasmid (silencing vector) and the empty plasmid pTRV2 (control) were transformed into Agrobacterium GV3101 (Shandong Nuoding Biotechnology Co., Ltd.). These were then plated onto LB agar containing 50 μg / mL kanamycin and 20 μg / mL rifampin and incubated. Positive clones were screened after colony growth, thus obtaining the engineered Agrobacterium strains. The obtained engineered Agrobacterium strains contained the empty plasmid pTRV2 (engineering strain 1), the recombinant expression plasmid pTRV2-PgWRKY2 (engineering strain 2), pTRV2-PgUGT84A23 (engineering strain 3), and pTRV2-PgPOR9 (engineering strain 4), respectively.

[0031] During the pomegranate fruit enlargement period, engineered bacteria 1, 2, 3, and 4 were injected into the pomegranate peel using a disposable syringe, and the phenotypic changes of the pomegranate were observed. The results are as follows: Figure 7 As shown in Figure A. (From...) Figure 7 As shown in Figure A, the introduction of engineered bacteria did not cause significant changes in the pomegranate phenotype.

[0032] Five days later, the accumulation of pungent glycosides and the expression levels of related genes in pomegranate peel were measured, and the results are as follows: Figure 7 Figures B through E are shown in the diagram. Figure B shows the silencing vector pTRV2-PgWRKY2 (engineered bacteria 2) in pomegranate peel after its incorporation. pTRV2 , PgWRKY2 , PgUGT84A23 , PgPOR9 The relative expression levels; Figure C shows the expression levels in pomegranate peel after the introduction of the silencing vector pTRV2-PgUGT84A23 (engineered strain 3). pTRV2 , PgWRKY2 , PgUGT84A23 , PgPOR9 The relative expression levels; Figure D shows the expression levels in pomegranate peel after the introduction of the silencing vector pTRV2-PgPOR9 (engineered bacteria 4). pTRV2 , PgWRKY2 , PgUGT84A23 , PgPOR9 The relative expression level; Figure E shows the relative content of punicin in pomegranate peel after the introduction of engineered bacteria 1, 2, 3, and 4.

[0033] As shown in Figure B, the introduction of the silencing vector pTRV2-PgWRKY2 significantly inhibited... PgWRKY2 The expression, and simultaneously caused PgUGT84A23 and PgPOR9The expression level decreased, and the differences between groups were statistically significant.

[0034] From diagrams C and D, we can see that individual silence... PgUGT84A23 or PgPOR9 It only affects the expression of the corresponding gene and does not interfere with... PgWRKY2 or the expression of other target genes.

[0035] As shown in Figure E, at the metabolic level, silencing... PgWRKY2 , PgUGT84A23 or PgPOR9 Any one of the genes can significantly reduce the content of punicin in pomegranate peel.

[0036] The above results indicate that reducing PgWRKY2 expression can significantly inhibit PgUGT84A23 and PgPOR9 The transcription of pungent glycosides is inhibited, thereby hindering the biosynthesis and accumulation of pungent glycosides. This fully demonstrates that PgWRKY2 plays a key upstream regulatory role in the pungent glycoside synthesis pathway.

Claims

1. Application of the transcription factor PgWRKY2 gene in promoting the synthesis of pungent glycosides, wherein the nucleotide sequence of the PgWRKY2 gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The amino acid sequence of PgWRKY2 is shown in SEQ ID NO.

2.

3. The application as described in claim 1, characterized in that, The PgWRKY2 gene increases PgUGT84A23 The expression level of [the substance] promotes the synthesis of pungent glycosides.

4. The application as described in claim 1, characterized in that, The PgWRKY2 gene increases PgPOR9 The expression level of [the substance] promotes the synthesis of pungent glycosides.

5. The application as described in claim 1, characterized in that, The PgWRKY2 gene increases PgUGT84A23 and PgPOR9 The expression level of [the substance] promotes the synthesis of pungent glycosides.

6. A pomegranate, characterized in that, A foreign nucleic acid molecule incorporated into the genome, wherein the foreign nucleic acid molecule comprises the PgWRKY2 gene as described in claim 1.

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