Gene for improving content of resveratrol in grapes and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a gene for increasing the content of resveratrol in grapes and application of the gene. The invention discloses a gene for improving the content of resveratrol in grapes. The DNA sequence of the gene is shown as SEQ ID NO1. The amino acid sequence of the gene synthetic protein disclosed by the invention is as shown in SEQ ID NO2, and the protein is named as VvFHY34D. According to the invention, when four amino acids S124, T128, S156 and T160 of the VvFHY3 are all replaced with aspartic acid to simulate phosphorylation activation, the VvSTS expression quantity in transient over-expression VvFHY34D grapes is higher, and more resveratrol can be synthesized.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a gene for increasing the resveratrol content in grapes and an application thereof. Background Art

[0002] Resveratrol is a class of phenolic secondary metabolites found only in a limited number of plant species. These compounds are called phytoanthins because they are induced upon pathogen infection and play a key role in plant defense against phytopathogens (Adrian et al., 1997; Schnee et al., 2008; Duan et al., 2015). Due to its antioxidant properties, resveratrol also has pharmacological value and is beneficial to human health. The most famous resveratrol is trans-resveratrol (trans-Res), which is believed to help prevent certain types of cancer, cardiovascular disease, diabetes and neurodegenerative diseases (Guerrero et al., 2009; Bavaresco et al., 2012). Plants of the grape family are the main source of resveratrol, and the stilbene synthase VvSTS gene is responsible for the synthesis of resveratrol.

[0003] At the same time, the synthesis of resveratrol is controlled by a complex regulatory system involving multiple transcription factors and multiple conditions, which still needs further study. Summary of the invention

[0004] The present invention aims to provide a gene for increasing the resveratrol content in grapes and its application. The specific scheme is as follows:

[0005] A gene for increasing the resveratrol content in grapes, wherein the DNA sequence of the gene is shown as SEQ ID NO 1.

[0006] A gene for increasing the resveratrol content in grapes, wherein the amino acid sequence of the protein synthesized by the gene is shown as SEQ ID NO2.

[0007] An application of a gene for increasing the resveratrol content in grapes, wherein the gene synthesizes an amino acid sequence as shown in SEQ ID NO 2, and the amino acid is used to increase the resveratrol content in grapes.

[0008] A method for increasing the resveratrol content in grapes, VvBIN2 phosphorylates VvFHY3, so that the Ser-124, Thr-128, Ser-156 and Thr-160 sites of VvFHY3 are phosphorylated (the resulting amino acids are shown in SEQ ID NO2), thereby increasing the resveratrol content in grapes.

[0009] That is, the present invention found that when the four amino acids Ser-124, Thr-128, Ser-156 and Thr-160 of VvFHY3 were replaced with aspartic acid (Asp) to simulate phosphorylation activation, the transient overexpression of VvFHY3 4D Grapes have a higher expression level of VvSTS and can synthesize more resveratrol. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the partial amino acid sequence of VvFHY3 and the amino acids of the present invention.

[0011] Figure 2 Atlas related to VvFHY3 enhancing the resveratrol content in grapes;

[0012] (A) Yeast one-hybrid analysis showed that VvFHY3 directly binds to multiple VvSTSs promoters. The LacZ reporter gene is driven by the VvSTSs promoter containing the FBS element. An empty vector expressing only AD was used as a negative control. (B) EMSA results showed that GST-FHY3N (the first 251 amino acids of VvFHY3) specifically binds to the biotin-labeled proVvSTS29-FBS probe. The arrow indicates GST-VvFHY3N. 50-fold and 100-fold molar excess of unlabeled probe were used in the competition experiment.

[0013] (C) ChIP-qPCR analysis showed that VvFHY3 binds to the promoters of VvSTS29 and VvSTS35. Pro35S:FHY3 transgenic calli cultured for 2 weeks were collected for ChIP and immunoprecipitated using anti-VvFHY3 antibody. The positions of the amplified fragments are shown in the upper figure. Data are mean ± SD; n = 3 biological replicates. Statistical significance was calculated using Student's t test (**P < 0.01).

[0014] (D) Tobacco transient transformation experiments showed that VvFHY3 promoted the expression of VvSTS29 and VvSTS35. The lower panel shows representative images of tobacco (N. benthamiana) leaves 72 hours after Agrobacterium injection. The right panel shows the quantification results of transient expression analysis. Data are mean ± SD; n = 3 biological replicates. Statistical significance was calculated using Student's t test (**P < 0.01).

[0015] (E) Gene expression analysis of VvSTS29 in VvFHY3 overexpression (VvFHY3-OE) and VvFHY3 inhibition (VvFHY3-Ri) transgenic grapes. After 8 minutes of UV-C irradiation, the grapes were placed in the dark for 2 hours, and then the leaves were collected to extract RNA for RT-qPCR analysis. Data are mean ± SD; n = 3 biological replicates. Statistical significance was calculated using Student's t test (*P < 0.05, **P < 0.01). (F) Determination of trans-resveratrol (trans-Res) and trans-resveratrol glycoside (trans-Pd) in VvFHY3-OE and VvFHY3-Ri transgenic grapes. After 8 minutes of UV-C irradiation, the grapes were placed in the dark for 2 hours, and then the leaves were collected to determine the resveratrol content using high-performance liquid chromatography (HPLC). Data are mean ± SD; n = 3 biological replicates. Statistical significance was calculated using Student's t test (*P<0.05, **P<0.01)

[0016] Figure 3 Atlas of VvBIN2 interacting with VvFHY3 and phosphorylating VvFHY3;

[0017] (A) Yeast two-hybrid analysis showed that VvFHY3 interacted with VvBIN2-1 and VvBIN2-2. LexA and AD were used as negative controls.

[0018] (B) Pull-down experiments showed that VvBIN2-1 could directly interact with the N-terminal (amino acids 1-251) and middle region (amino acids 186-576) of VvFHY3, but not its C-terminal fragment (amino acids 541-847).

[0019] (C) Luciferase complementation imaging (LCI) analysis showed that VvFHY3 interacted with VvBIN2-1 and VvBIN2-2 in tobacco leaves. VvFHY3 was fused to the N-terminal fragment of luciferase (nLUC), while VvBIN2-1 and VvBIN2-2 were fused to the C-terminal fragment of luciferase (cLUC). The combination of VvFHY3-nLUC with cLUC or nLUC with cLUC-VvBIN2-1, cLUC-VvBIN2-2 served as negative controls.

[0020] (D) Phosphorylation status of VvFHY3 was detected using in vivo Phos-tag assay. Proteins extracted from two-week-old Pro35S:FHY3 transgenic calli were treated with alkaline phosphatase (CIAP), separated on SDS-PAGE gel containing Phos-tag reagent, and then immunoblotted with anti-VvFHY3 antibody. Arrows indicate phosphorylated or non-phosphorylated VvFHY3, respectively.

[0021] (E) Phos-tag assay was used to analyze the response of VvFHY3 to UV-C treatment and the combined treatment of UV-C and bikinin. Pro35S:FHY3 transgenic callus cultured for 2 weeks was untreated (Mock) or irradiated with UV-C for 8 minutes, or treated with UV-C and bikinin. After UV-C irradiation, callus was placed in the dark for 2 hours and collected for protein extraction. Phosphorylated VvFHY3 was detected using Phos-tag gel (top). Tubulin was used as a loading control (bottom).

[0022] (F) In vitro phosphorylation analysis of VvFHY3 by VvBIN2-1. Upper panel, phosphorylated VvFHY3N and autophosphorylation of VvBIN2-1 were detected using anti-phosphoserine / threonine antibodies. Lower panel, recombinant proteins GST-VvFHY3N, GST-VvFHY3M, GST-VvFHY3C, and His-VvBIN2-1 in CBB-stained gels are indicated by arrows. (G) Phos-tag experiments showed that bikinin and CIAP inhibited the phosphorylation of VvFHY3 by VvBIN2-1. His-VvBIN2-1 and GST-VvFHY3 were separated on SDS-PAGE gels containing Phos-tag reagents after incubation with bikinin or CIAP.

[0023] (H) Schematic diagram of potential phosphorylation sites at the N-terminus of VvFHY3. There are three conserved VvBIN2 phosphorylation sites (S / TXXXS / T) at the N-terminus of VvFHY3, and the Ser (S) and Thr (T) residues in these sites were replaced with Ala (A) residues to generate VvFHY3N S97AS101A 、VvFHY3N S124AT128A 、VvFHY3N S156AT160A and VvFHY3N S124AT128AS156AT160A (VvFHY3N 4A ).

[0024] (I) Determination of phosphorylation sites of VvFHY3 using in vitro phosphorylation assay. S97A S101A 、GST-VvFHY3N S124AT128A 、GST-VvFHY3N S156A T160A and GST-VvFHY3N 4A Incubated with His-VvBIN2-1. Upper panel, immunoblotting using anti-phosphoserine / threonine antibody; lower panel, CBB staining.

[0025] Figure 4 Effect of VvFHY3 phosphorylation on resveratrol synthesis

[0026] (A) In the transient overexpression of VvFHY3-Flag, VvFHY3 4A -Flag and VvFHY3 4D -Flag's 'Crison' grape plant, the gene expression of VvFHY3 was analyzed. WT 、VvFHY3 4A 、VvFHY3 4D ) were transiently transformed into leaves of "Crison" grape. The relative expression level of VvFHY3 in grape leaves after 8 minutes of UV-C irradiation was determined by real-time fluorescence quantitative PCR (RT-qPCR). The right panel of Figure (A) is the immunoblot confirmation of VvFHY3 protein expression. Data are mean ± SD (n = 3). Different letters indicate significant differences (P < 0.05) obtained by two-way analysis of variance (ANOVA). The right panel of Figure (A) is the immunoblot analysis of VvFHY3 protein in transgenic plants. Tubulin was used as a loading control. VvFHY3 protein was detected using anti-Flag antibody.

[0027] (B) Gene expression analysis of VvSTS29 and VvSTS35 in 'Cruzon' plants transiently overexpressing different types of VvFHY3. Data are mean ± SD; n = 3 biological replicates. Statistical significance was calculated using Student's t-test (**P < 0.01).

[0028] (C) and (D) Analysis of UV-C-induced trans-resveratrol (trans-Res) and trans-resveratrol glycoside (trans-Pd) accumulation in response to brassinolide (BL) and bikinin treatment in stably transformed VvFHY3-overexpressing (VvFHY3-OE) plants by high-performance liquid chromatography (HPLC). Empty vector-overexpressing (EV-OE) and VvFHY3-OE plants were pretreated with BL or bikinin and then subjected to UV-C irradiation. The contents of trans-resveratrol and trans-resveratrol glycoside in leaves after UV-C irradiation were determined by HPLC. Data are mean ± SD, n = 3. Different letters indicate significant differences (P < 0.05) by two-way ANOVA. DETAILED DESCRIPTION

[0029] A gene for increasing the resveratrol content in grapes, the DNA sequence of the gene is shown in SEQ ID NO 1. A gene for increasing the resveratrol content in grapes, the amino acid sequence of the protein synthesized by the gene is shown in SEQ ID NO 2 (the amino acid sequence can increase the resveratrol content in grapes).

[0030] A method for increasing the resveratrol content in grapes, wherein VvBIN2 phosphorylates VvFHY3, so that the Ser-124, Thr-128, Ser-156 and Thr-160 sites of VvFHY3 are phosphorylated (reference Figure 1 ), thereby increasing the resveratrol content in grapes.

[0031] The CDS nucleic acid sequence of VvBIN2 is as follows:

[0032] atgtccgacgacaaggaaatctctgcccctgttgttgatgggaatgatcctgtcacaggtcacatcatttccaccacaatt

[0033] ggagggaaaaatggggagcccaaacagaccatcagttacatggcagagcgtgttgtgggtaccgggtcatttggaatt

[0034] gtttttcaggcaaaatgcttggaaactggagagactgtggccataaagaaggttttgcaggacagaagatataagaatc

[0035] gtgagctgcagttaatgcgcacgatggatcatccaaatgtcatttccctaaaacattgtttcttttctactacaagtagag

[0036] atgaacttttcctcaacttggtaatggaatatgtccctgagaccatgtatcgggtcttgaagcactacagcaacgcaaaa

[0037] cagcgaatgcctcttatatatgtgaaactttacacttaccaaattttcaggggcctggcatatatccacagtgttcctgga

[0038] gtttgccatagggatttgaagcctcaaaatcttctagttgatccacttactcaccaagttaagctttgtgactttggaagtg

[0039] caaaagtgctagttaaaggtgaagcaaacatatcatacatatgttctcgtttctaccgagcacctgaactcatatttggg

[0040] gccacagagtatacaacctcaattgatatatggtcagctggttgtgtcctagctgagcttcttctaggccagccattgtttc

[0041] ctggagagaatgcagtggaccagcttgttgagattatcaaggttcttggtacaccaacgcgggaagaaattcgctgtat

[0042] gaatccgagttatactgattttaggtttccacagataaaagcacacccttggcacaaggtctttcacaaacgaatgcctc

[0043] ctgaagcaatcgatcttgcttctcgacttctgcaatactcaccaagtcttcgctgcacagctctagaagcatgtgcacatc

[0044] ctttctttgatgagctccgagaacccaatgcccgcctgccaaatggtcgtccattaccacctctcttcaattttaagcagga

[0045] attatctggggcttctcctgagcttgttaataagttgataccggagcatgtgaggaggcaaataggcctaagttttttcca

[0046] cccagcaggcacataa

[0047] The amino acid sequence of VvBIN2 is as follows:

[0048] MSDDKEISAPVVDGNDPVTGHIISTTIGGKNGEPKQTISYMAERVVGTGSFGIVFQAKCLETGE

[0049] TVAIKKVLQDRRYKNRELQLMRTMDHPNVISLKHCFFSTTSRDELFLNLVMEYVPETMYRVLKH

[0050] YSNAKQRMPLIYVKLYTYQIFRGLAYIHSVPGVCHRDLKPQNLLVDPLTHQVKLCDFGSAKVLVK

[0051] GEANISYICSRFYRAPELIFGATEYTTSIDIWSAGCVLAELLLGQPLFPGENAVDQLVEIIKVLGTPT

[0052] REEIRCMNPSYTDFRFPQIKAHPWHKVFHKRMPPEAIDLASRLLQYSPSLRCTALEACAHPFFDELREPNARLPNGRPLPPLFNFKQELSGASPELVNKLIPEHVRRQIGLSFFHPAGT*

[0053] That is, the present invention found that when the four amino acids S124, T128, S156 and T160 of VvFHY3 were replaced with aspartic acid (Asp) to simulate phosphorylation activation, the transient overexpression of VvFHY3 4D Grapes have a higher expression level of VvSTS and can synthesize more resveratrol.

[0054] Experimental situation

[0055] 1. VvFHY3 regulates the transcription of VvSTS and UV-C-mediated resveratrol synthesis.

[0056] To identify potential regulators involved in UV-C-induced resveratrol biosynthesis, we performed a bioinformatics analysis of cis-elements within all VvSTS promoters. Notably, 21 of the 38 VvSTS promoters contained putative FHY3 / FAR1 binding sites (FBS). We cloned the closest homolog of Arabidopsis FHY3, VvFHY3, and assessed its binding activity using a yeast one-hybrid assay. The results showed that VvFHY3 directly bound to 18 VvSTS promoters ( Figure 2 A). In addition, we selected the VvSTS29 and VvSTS35 promoters and confirmed their binding in vivo and in vitro using electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation analysis (ChIP). Figure 2 B and 2C). To further elucidate the regulatory role of VvFHY3 in VvSTS expression, we performed transient expression analysis in tobacco (N. benthamiana) leaves. The results showed that the expression of VvFHY3 protein significantly activated the firefly luciferase (LUC) reporter gene activity driven by the endogenous promoters of VvSTS29 and VvSTS35 ( Figure 2 D).

[0057] To investigate the role of VvFHY3 in resveratrol biosynthesis, we used Agrobacterium-mediated genetic transformation to generate VvFHY3 overexpression and RNA interference (RNAi) lines in the "seedless white" grape variety. RT-qPCR analysis showed that, compared with the empty vector control, transcripts of VvSTS29 and VvSTS35 were significantly increased in the overexpression lines and significantly decreased in the RNAi lines before and after UV-C irradiation ( Figure 2 E). We then quantified the levels of two major stilbene synthase products, trans-resveratrol (trans-Res) and trans-resveratrol glycoside (trans-Pd). Compared with empty vector plants, the levels of trans-Res and trans-Pd were significantly higher in overexpression plants and lower in RNAi plants ( Figure 2 F).

[0058] 2. VvBIN2 interacts with VvFHY3 and phosphorylates VvFHY3

[0059] The yeast two-hybrid assay ( Figure 3A) The interaction between VvFHY3 and VvBIN2-1 / VvBIN2-2 was verified. Next, we used glutathione S-transferase (GST) pull-down assay to detect their interaction. The results showed that His-VvBIN2-1 could be detected in the pull-down products in GST-VvFHY3N and GST-VvFHY3M experimental treatments, indicating that VvBIN2 can directly interact with the N-terminus (amino acids 1-251) and middle region (amino acids 186-576) of VvFHY3, but not with its C-terminus (amino acids 541-847) ( Figure 3 B). To validate this interaction in vivo, we performed luciferase complementation imaging (LCI) experiments and found that co-expression of the two constructs, VvFHY3-nLuc and VvBIN2-cLuc, restored robust luciferase activity ( Figure 3 C).

[0060] The interaction between VvBIN2 and VvFHY3 prompted us to examine whether VvBIN2 can phosphorylate VvFHY3. We used Phos-tag assay to distinguish the phosphorylated and dephosphorylated states of VvFHY3 immunoprecipitated from 35S:VvFHY3-GFP transgenic calli. The results showed two bands with different protein mobilities, and the upper band was significantly reduced after alkaline phosphatase (CIAP) treatment ( Figure 3 D), indicating that VvFHY3 exists in both phosphorylated and dephosphorylated states in vivo. After UV-C treatment, phosphorylation of VvFHY3 was inhibited in the presence of bikinin, a GSK3-like kinase inhibitor that inactivates BIN2 ( Figure 3 E), indicating that VvBIN2 is involved in regulating the phosphorylation of VvFHY3 under UV-C treatment. In addition, we performed in vitro kinase assays and found that VvBIN2-1 could phosphorylate the N-terminus of VvFHY3, but not its middle region and C-terminus ( Figure 3 F). In addition, Phos-tag experiments showed that the migration of phosphorylated GST-VvFHY3N protein was significantly delayed when incubated with His-VvBIN2-1 in the reaction, while this migration was strongly inhibited by CIAP or bikinin treatment ( Figure 3 G).

[0061] To identify potential phosphorylation sites within the N-terminus of VvFHY3, we found three conserved VvBIN2 phosphorylation motifs (S / TXXXS / T) ( Figure 3 H). To further investigate these sites, we replaced serine (S) and threonine (T) residues with alanine (A) to generate a phosphorylation-inactive mutant: VvFHY3NS97A S101A 、VvFHY3N S124A T128A 、VvFHY3N S156A T160A and VvFHY3N S124A T128A S156A T160A (VvFHY3N 4A ). In vitro phosphorylation and Phos-tag experiments showed that when VvFHY3 contained VvFHY3N S124AT128A and VvFHY3N S156AT160A When two point mutations were detected in VvBIN2-1, the phosphorylation degree of VvFHY3 was reduced to different extents ( Figure 3 I). When all four residues were mutated to alanine, phosphorylation was barely detectable ( Figure 3 I), which indicated that S124, T128, S156, and T160 were the major phosphorylation sites recognized by VvBIN2-1.

[0062] 3. Role of VvFHY3 phosphorylation in resveratrol synthesis

[0063] To investigate the importance of VvFHY3 phosphorylation for resveratrol (Res) synthesis, we transiently overexpressed the non-phosphorylated form of VvFHY3, VvFHY3 4A and the constitutively phosphorylated form VvFHY3 4D , to analyze their effects on VvSTS expression. After examining more than 50 transgenic plants, we obtained three plants with comparable VvFHY3 expression levels ( Figure 4 A). Surprisingly, overexpression of VvFHY3 and VvFHY3 4A Compared with overexpression of VvFHY3 4D UV-C irradiation showed higher VvSTS transcription levels ( Figure 4 B), indicating that the phosphorylation state of VvFHY3 regulated by VvBIN2 plays a role in UV-C-induced resveratrol synthesis. Next, we sought to explore whether the promotion of resveratrol synthesis by VvBIN2 was attributed to the action of VvFHY3. Since it is difficult to transform two genes simultaneously in grapes, we used BL and bikinin to inhibit the activity of VvBIN2 in plants stably transformed with VvFHY3. Figure 4As shown in C and 4D, after treatment with BL, the levels of UV-C-induced trans-resveratrol (trans-Res) and trans-resveratrol glycoside (trans-Pd) in VvFHY3-overexpressing (VvFHY3-OE) plants were still higher than those in the empty vector control. Notably, we found that in VvFHY3-OE plants, despite the overexpression of VvFHY3 transcripts, the inhibitory effects of BL and bikinin on resveratrol synthesis were still very effective ( Figure 4 C and 4D). This finding highlights the importance of VvBIN2 in posttranscriptional regulation of VvFHY3 during resveratrol biosynthesis.

[0064] The above implementation modes are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A gene for increasing the resveratrol content in grapes, characterized in that: The DNA sequence of the gene is shown as SEQ ID NO1.

2. A gene for increasing the resveratrol content in grapes, characterized in that: The amino acid sequence of the gene synthesized protein is shown in SEQ ID NO2.

3. An application of a gene for increasing the resveratrol content in grapes, characterized in that: The gene synthesized sequence is the amino acid shown in SEQ ID NO 2, and the amino acid is used to increase the resveratrol content in grapes.

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