The molecular weight of the ClaF3apos is 100,000; 5 apos; h gene and application thereof

Abstract

The invention relates to a CllaF3 '5' H gene and application thereof. The CllaF3 '5' H gene comprises a nucleotide sequence as shown in SEQ ID NO: 1. The CllaF3 '5' H protein comprises a protein coded by the CllaF3 '5' H gene. According to the invention, a CllaF3 '5' H gene is screened and cloned from clematis lanceolata, and the correlation between the CllaF3 '5' H gene and the content of flavonoid metabolites such as delphinidin is found through research; stable germplasm with blue flower potential can be formed by utilizing the gene.

Description

Technical Field

[0001] This invention belongs to the field of plant variety breeding technology, and in particular relates to the CllaF3′5′H gene and its application. Background Technology

[0002] Flower color is one of the important criteria for judging the ornamental value of ornamental plants. Therefore, research on pigment metabolism is particularly important, and the breeding of blue flowers has become one of the hot topics of international attention.

[0003] Clematis species are mostly woody vines, renowned for their diverse flower shapes and often referred to as the "Queen of Vines." Clematis lanuginosa, distributed in northeastern Zhejiang, is endemic to China and serves as an important original parent for clematis breeding. Currently, most clematis varieties on the domestic market are imported from Europe and Japan, with few varieties possessing independent intellectual property rights in my country. Furthermore, there is a lack of true blue clematis varieties. Therefore, promoting the transformation of flower color to blue-purple and stimulating the accumulation of blue anthocyanins are urgent problems to be solved. The F3'5'H gene, as the core gene for delphinidin synthesis, plays a crucial role in the formation of blue flowers; however, there are currently no reports on the F3'5'H gene in Clematis lanuginosa. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides the CllaF3′5′H gene and its applications.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] The present invention provides the CllaF3′5′H gene, comprising the nucleotide sequence shown in SEQ ID NO:1.

[0007] ATGGTGATCA ATATGTTTGT TTTGAGAGAG CTCAGTTATG CATTTATCTTGTTTTTCATCACCCATATTT TTGGTCGTAG CTTGCTTTCG CACTATGCAC GTAAACTTCCCCCAGGTCCA ACAGGTTTCCCAGTTGTTGG GTCATTGCCA TTTCTCGGAT CCATGCCTCATGTTGCGCTG GCTGAGATGT CAAAGAAGTACGGGCCAGTT ATGTACCTCA AATTAGGTTCGCGAGGGATG GTGGTTGCAT CCACTCCAGA TTCAGCTCGTGCTTTTCTCA AAACCCTAGACCTTAATTTC TCAAATCGCC CAACTGATTC AGGTGCAACC CATATGGCCTATGACTCACAAGACATGGTG TTTGCTGATT ATGGACCACG ATGGAAGTTG CTACGTAGAGTGAGCAACTTACATATGCTT GGTGGGAAGG CTCTAGAGGA TTGGACTAAT GTAAGAAAAAATGAAGTTGGGCACATGCTT CAGGCTATGC ATGAGTCAAG CTGCAGAGGA GAGCCTGTGGTGGTGGCAGACATGTTGGTT TACGCCATGG CTAACATGAT TGGGCAGGTG ATATTGAGTCGCCGTGTGTTTGTCACCAAA GGCACGGAAC TGAACGAGTT CAAAGAAATG GTGGTTGAGCTCATGACGTCAGCTGGCCTT TTTAACGTTG GTGACTACAT CCCTTCTATT GCATGCATGGATTTGCAAGGAATTGTACGA GGCATGAAGA GTTTGCATAA TAAGTTTGAC GTACTTTTGAACAAAATGCTCAAGGAACAT GAGTCAACAA GGCATGAGAG GAAGGAGAAGCCAGACTTGCTGGATGTCTTGCTTGAGAAC AGAGATAACA AGGCTGTAGG GGAGGAAAGGATTACTGACACCAACATCAA GGCTCTTCTT CTGAACTTGT TCACAGCTGGGACTGATACCTCCTCCAGCTCAATTGAATG GGCACTTGCT GAAATGATGA AGAACCCAAG CATCTTTAAACGTGCGCATGCAGAGATGGA TCATGTCATT GGAAACAACC GTAGACTTGA AGAGTCTGACATACCAAACCTCCCATACTT ACAAGCCATA TGCAAAGAAA CATTCAGGAA ACACCCTTCGACACCACTCAATCTTCCAAG AGAAGCTGTG GAATCATGTG AAGTGAATGG ATACTACATCCCCAAAGGAACCAGACTCAG TGTAAATATA TGGGGCATGG GGAGAGACCC GAATGTGTGGGAGAATCCACTGGAATTCAA TCCGGATAGG TTCTTGCGAG TGGAAACTAT GAAAATCGATCCACGGGGAAATCATTTTGA GCTTATACCA TTTGGGGCGG GAAGAAGGAT ATGTGCGGGGAGTAGAATGGCAATTGTTCT TGTTGAGTAC ATGTTGGGGA CACTTGTGCA TTCATTTGATTGGAATTTAGCTGACGGTGT AGAGCTAAAT ATGGATGAAT CATTTGGACT TGCACTCCAAAAGGCTGTCCCACTTGCAGC TATTGTCACC CCTCGCTTGC CACCTAGTGT CTATTATGTT TAA (SEQ ID NO: 1).

[0008] The present invention provides CllaF3′5′H protein, including the protein encoded by the CllaF3′5′H gene described above.

[0009] Furthermore, the nucleotide sequence of the Cl laF3′5′H protein is shown in SEQ ID NO:2.

[0010] MVINMFVLRELSYAFILFFITHIFGRSLLSHYARKLPPGPTGFPVVGSLPFLGSMPHVALAEMSKKYGPVMYLKLGSRGMVVASTPDSARAFLKTLDLNFSNRPTDSGATHMAYDSQDMVFADYGPRW KLLRRVSNLHMLGGKALEDWTNVRKNEVGHMLQAMHESSCRGEPVVVADMLVYAMANMIGQVILSRRVFVTKGTELNEFKEMVVELMTSAGLFNVGDYIPSIACMDLQGIVRGMKSLHNKFDVLLNKML KEHESTRHERKEKPDLLDVLLENRDNKAVGEERITDTNIKALLLNLFTAGTDTSSSSIEWALAEMMKNPSIFKRAHAEMDHVIGNNRRLEESDIPNLPYLQAICKETFRKHPSTPLNLPREAVESCEV NGYYIPKGTRLSVNIWGMGRDPNVWENPLEFNPDRFLRVETMKIDPRGNHFELIPFGAGRRICAGSRMAIVLVEYMLGTLVHSFDWNLADGVELNMDESFGLALQKAVPLAAIVTPRLPPSVYYV(SEQ ID NO:2).

[0011] This invention provides a primer set for amplifying the CllaF3′5′H gene.

[0012] The present invention provides a vector comprising the above-mentioned CllaF3′5′H gene.

[0013] The present invention provides a transformant comprising the above-described carrier.

[0014] This invention provides a method for cultivating blue-purple flowers, comprising the following steps: overexpressing the CllaF3′5′H gene.

[0015] The present invention provides the application of the above-mentioned CllaF3′5′H gene, CllaF3′5′H protein, primer set, vector, and transformant in any one or more of (1) to (7);

[0016] (1) Regulating flower color;

[0017] (2) Synthesize flavonoid metabolites;

[0018] (3) Synthetic delphinidin;

[0019] (4) Synthesize delphinidin derivatives;

[0020] (5) Cultivate blue-flowered plant varieties;

[0021] (6) Promotes the transformation of flower color to blue-purple;

[0022] (7) Promotes the accumulation of blue anthocyanins.

[0023] The CllaF3′5′H gene described above can be used to regulate flower color. Overexpression of the CllaF3′5′H gene causes a change in flower color towards blue. This invention provides that the CllaF3′5′H gene can be used in the synthesis of delphinidin and delphinidin derivatives. Overexpression of CllaF3′5′H significantly increases the accumulation of delphinidin-3-O-arabinoside, delphinidin-3-O-arabinoside, malvidin-3-O-glucoside, petunidin-3-O-(6-O-coumaryl-glucoside), and petunidin-3-O-galactoside, effectively promoting the accumulation of blue anthocyanins.

[0024] This invention cloned the CllaF3′5′H gene from Clematis pubescens, analyzed its expression pattern, tissue and subcellular localization, and correlation with the content of flavonoid metabolites such as delphinidin. Using a tobacco genetic system and the Clematis pubescens VIGS system, it was verified that CllaF3′5′H is a key gene for delphinidin synthesis, and the mechanism by which CllaF3′5′H participates in the formation of blue-purple flowers in Clematis pubescens was preliminarily elucidated. The research results are of great significance for clarifying the flower color formation mechanism of Clematis, and also provide genetic resources and a theoretical basis for the breeding of new Clematis varieties and molecular breeding of blue flowers, which is conducive to the formation of stable germplasm with blue flowers. Attached Figure Description

[0025] Figure 1 Images of flower buds of Clematis pubescens at different developmental stages.

[0026] Figure 2 These are the results of the expression analysis of key enzyme genes.

[0027] Figure 3 The results of RNA extraction and ORF fragment amplification electrophoresis of Clematis samples.

[0028] Figure 4 The structure of CllaF3′5′H protein is shown below, where A represents the protein's hydrophilicity / hydrophobicity; B represents the protein structure; and C represents the protein's secondary structure.

[0029] Figure 5 Subcellular localization of the CllaF3′5′H gene is shown, where A and E are the green fluorescent channels of the target protein; B and F are the red fluorescent channels of the endoplasmic reticulum marker; C and G are the autofluorescence of chloroplasts; D and H are the bright field; and E and I are the superposition of fluorescence and bright field.

[0030] Figure 6 To obtain the CllaF3′5′H tobacco overexpression transgenic line, the process involved: A. tobacco co-culture; B. tobacco induction; C. tobacco first screening; D. tobacco second screening; E. tobacco differentiation; and F. tobacco rooting.

[0031] Figure 7 Positive detection for CllaF3′5′H tobacco overexpression transgenic lines.

[0032] Figure 8 Quantitative detection of CllaF3′5′H tobacco overexpression transgenic lines.

[0033] Figure 9 The anthocyanin content of the CllaF3′5′H tobacco overexpression strain.

[0034] Figure 10 The plot shows the scatter plot of PCA, where QC is the quality control sample, Control is the control group (WT), OE1 is CllaF3′5′H overexpression line 1, OE2 is CllaF3′5′H overexpression line 2, and OE3 is CllaF3′5′H overexpression line 3.

[0035] Figure 11 This is a heatmap of metabolic products.

[0036] Figure 12 This is the transiently silent phenotype of Clematis hairyensis VIGS.

[0037] Figure 13 The anthocyanin content of Clematis armandii after transient VIGS silencing. Detailed Implementation

[0038] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0039] This invention successfully cloned the CllaF3′5′H gene, which is 1533 bp in length, encodes 510 amino acids, and has the molecular formula C1. 4629 H 7727 N 1533 O 1949 S 308With a molecular weight of 125916.85, a theoretical isoelectric point (pI) of 5.01, and an instability index (II) of 41.73, the protein is classified as unstable. Its secondary structure primarily comprises 46.08% α-helices, 12.42% β-sheets, and 37.91% irregular coils. Subcellular localization analysis of Arabidopsis protoplasts and tobacco leaves revealed that the CllaF3′5′H protein is located in the endoplasmic reticulum and cytoplasm. Expression of CllaF3′5′H significantly upregulated during the initial coloring stage of the flower (S4), gradually decreasing with flower opening and senescence.

[0040] Overexpression lines of CllaF3′5′H were constructed using a tobacco genetic transformation system. Overexpression increased the content of delphinidin in the calyx and decreased the content of cyanidin galactoside and paeoniflorin, promoting the biosynthesis of delphinidin and reducing the synthesis of paeoniflorin and cyanidin, thus promoting the color change to blue-purple. Metabolomics analysis revealed that overexpression of CllaF3′5′H significantly increased the accumulation of delphinidin-3-O-arabinoside, delphinidin-3-O-arabinoside, malvidin-3-O-glucoside, petunidin-3-O-(6-O-coumaryl-glucoside), and petunidin-3-O-galactoside, effectively promoting the accumulation of blue anthocyanins.

[0041] After silencing the CllaF3′5′H gene in Clematis pubescens using VIGS technology, the blue-purple color of Clematis pubescens flowers at the initial flowering stage (S6), full bloom stage (S7), and late flowering stage (S8) was significantly lighter, the content of delphinidin and other substances was significantly reduced, and the content of kaempferol and quercetin was significantly increased. Silencing the CllaF3′5′H gene inhibited the synthesis of delphinidin metabolites.

[0042] In the embodiments, Clematis pubescens, Arabidopsis thaliana, and tobacco were independently bred in the inventor's laboratory; pSAT6-EGFP-N, pGADT7, PS1aG-3, and Y1H Gold yeast cells were owned by the Zhejiang Subtropical Crops Research Institute; the above materials are available to the public for non-commercial purposes only to repeat the embodiments described in this invention.

[0043] Agrobacterium (GV3101) was purchased from Beijing Qingke Biotechnology Co., Ltd.; TRV2 vector was purchased from addgene; pTRV1 was purchased from addgene.

[0044] Unless otherwise specified, sequencing and primers in this invention were performed by Beijing Qingke Biotechnology Co., Ltd.

[0045] Unless otherwise specified, the experimental methods described in the examples are conventional methods in the art, or are performed in accordance with the techniques or conditions described in the literature in the art, or in accordance with the product instructions. Unless otherwise specified, the reagents, experimental materials, and instruments used are all conventional products that can be purchased from legitimate channels.

[0046] The following is a detailed description through specific examples.

[0047] Example 1: Screening for CllaF3′5′H gene

[0048] (1) Metabolomics analysis of Clematis pubescens

[0049] Clematis florida is divided into flowering stages based on bud size and developmental stage: S1 (bud height ≤ 1cm), S2 (1cm < bud height ≤ 2cm), S3 (2cm < bud height ≤ 3cm), S4 (3cm < bud height ≤ 4cm), S5 (4cm < bud height ≤ 5cm), S6 (initial flowering stage, corolla half open), S7 (peak flowering stage, corolla fully open), and S8 (late flowering stage). During S5, when the bud height is 4-5cm, anthocyanin accumulation in the sepals can be observed. Figure 1 ).

[0050] Calyx tissue samples were collected from Clematis pubescens during the S3 to S8 stages. Flavonoid metabolomics analysis was performed using broad-targeted metabolomics technology, and a total of 165 metabolites were identified, including 16 anthocyanins and 149 other flavonoid metabolites. Delphinidin and its derivatives were among the main chromogenic pigments.

[0051] Analysis of anthocyanin content showed that the late-stage biosynthesis of anthocyanins in Clematis pubescens mainly focuses on the delphinidin and cyanidin pathways. The detected anthocyanin components were mainly delphinidin, delphinidin 3-O-glucoside, iridin 3-O-glucoside, malvidin-3-acetyl-5-diglucoside, cyanidin 3-O-glucoside, and cyanidin 3-O-rutin, confirming that the delphinidin pathway is one of the key pathways for anthocyanin biosynthesis in Clematis pubescens.

[0052] (2) Transcriptome analysis of Clematis pubescens

[0053] Transcriptome sequencing was performed on the calyx of Clematis pubescens from S1 to S8 stages. The transcriptome sequencing was performed by Shanghai Paisennong Biotechnology Co., Ltd. Based on the combined analysis of KEGG metabolic pathways and the NR database, 87 mRNAs annotated to enzyme genes encoding anthocyanin biosynthesis pathways were found in the Clematis pubescens transcriptome database, as shown in Table 1. These include 3 F3′H genes (named F3′H-1, F3′H-2, and F3′H-3, respectively), 2 F3′5′H genes (named F3′5′H-1 and F3′5′H-2, respectively), 4 DFR genes (named DFR-1, DFR-2, DFR-3, and DFR-4, respectively), 2 ANS genes (named ANS-1 and ANS-2, respectively), and 2 ANR genes (named ANR-1 and ANR-2, respectively).

[0054] Table 1. Identification of genes related to anthocyanin biosynthesis

[0055]

[0056]

[0057] Based on differentially expressed gene information from the transcriptome, 23 key enzyme candidate genes were screened. Pearson correlation analysis was performed on the expression of these key enzyme genes and anthocyanin content, revealing that DFR-1, F3'5'H-1, F3'5'H-2, and ANS-1 had high correlation coefficients with anthocyanin content. Figure 2 Based on differentially expressed gene information from the transcriptome, expression verification by quantitative real-time PCR, and BLAST alignment of amplified sequences, and according to the delphinidin synthesis pathway, F3'5'H is identified as a key node gene in this pathway. Therefore, F3'5'H-2 was determined as the target gene, and its partial gene sequence was obtained. In subsequent examples, the CllaF3′5′H gene is referred to as the F3'5'H-2 gene.

[0058] Example 2

[0059] Cloning the CllaF3′5′H gene: Clematis calyx tissue samples were ground with liquid nitrogen, and total RNA was extracted using the Trizol method. The RNA was then reverse transcribed into cDNA using the Vazyme HiScript II 1st Strand cDNA Synthesis Kit. Primers for amplifying this fragment were designed based on the sequences obtained from transcriptome sequencing and alignment (Table 2). ， cDNA was used as a template and amplified using TOYOBO's KOD FX Neo and its PCR program.

[0060] Table 2 CllaF3′5′H primers

[0061]

[0062]

[0063] The PCR reaction system consisted of: 25 μL of 2×PCR Buffer for KOD FX Neo, 10 μL of 2 mM dNTPs, 1.5 μL of forward primer (10 μM), 1.5 μL of reverse primer (10 μM), 1 μL of template, 1 μL of KOD FX Neo, and distilled water to a final volume of 50 μL.

[0064] PCR reaction conditions included: 94℃, 2 min, 1 cycle; 98℃, 10 sec, 68℃, 1 min 30 sec, 30 cycles; 68℃, 7 min, 1 cycle; 4℃, 30 min, 1 cycle.

[0065] The amplified products all showed a specific single band after electrophoresis. Figure 3 After gel extraction and recovery, sequencing was performed using F-terminal and R-terminal primers. The sequencing results were compared with the reference sequence and confirmed to be the CllaF3′5′H sequence.

[0066] The CllaF3′5′H gene is 1533 bp in length (nucleotide sequence shown in SEQ ID NO:1), encoding 510 amino acids (amino acid sequence shown in SEQ ID NO:2), and belongs to the cytochrome P450 family. The molecular formula of the CllaF3′5′H protein is C1. 4629 H 7727 N 1533 O 1949 S 308 With a molecular weight of 125916.85, a theoretical isoelectric point (pI) of 5.01, and an instability index (II) of 41.73, this protein is classified as unstable. Its secondary structure primarily comprises 46.08% α-helices, 12.42% β-sheets, and 37.91% irregular coils. Figure 4 ).

[0067] CllaF3′5′H gene sequence:

[0068] ATGGTGATCA ATATGTTTGT TTTGAGAGAG CTCAGTTATG CATTTATCTT GTTTTTCATCACCCATATTT TTGGTCGTAG CTTGCTTTCG CACTATGCAC GTAAACTTCC CCCAGGTCCA ACAGGTTTCCCAGTTGTTGG GTCATTGCCA TTTCTCGGAT CCATGCCTCA TGTTGCGCTG GCTGAGATGT CAAAGAAGTACGGGCCAGTT ATGTACCTCA AATTAGGTTC GCGAGGGATG GTGGTTGCAT CCACTCCAGA TTCAGCTCGTGCTTTTCTCA AAACCCTAGA CCTTAATTTC TCAAATCGCC CAACTGATTC AGGTGCAACC CATATGGCCTATGACTCACA AGACATGGTG TTTGCTGATT ATGGACCACG ATGGAAGTTG CTACGTAGAG TGAGCAACTTACATATGCTT GGTGGGAAGG CTCTAGAGGA TTGGACTAAT GTAAGAAAAA ATGAAGTTGG GCACATGCTTCAGGCTATGC ATGAGTCAAG CTGCAGAGGA GAGCCTGTGG TGGTGGCAGA CATGTTGGTT TACGCCATGGCTAACATGAT TGGGCAGGTG ATATTGAGTC GCCGTGTGTT TGTCACCAAA GGCACGGAAC TGAACGAGTTCAAAGAAATG GTGGTTGAGC TCATGACGTCAGCTGGCCTT TTTAACGTTG GTGACTACAT CCCTTCTATTGCATGCATGG ATTTGCAAGGAATTGTACGA GGCATGAAGA GTTTGCATAA TAAGTTTGAC GTACTTTTGAACAAAATGCTCAAGGAACAT GAGTCAACAA GGCATGAGAG GAAGGAGAAGCCAGACTTGCTGGATGTCTTGCTTGAGAAC AGAGATAACA AGGCTGTAGG GGAGGAAAGGATTACTGACACCAACATCAA GGCTCTTCTT CTGAACTTGT TCACAGCTGGGACTGATACCTCCTCCAGCTCAATTGAATG GGCACTTGCT GAAATGATGA AGAACCCAAG CATCTTTAAACGTGCGCATGCAGAGATGGA TCATGTCATT GGAAACAACC GTAGACTTGA AGAGTCTGACATACCAAACCTCCCATACTT ACAAGCCATA TGCAAAGAAA CATTCAGGAA ACACCCTTCGACACCACTCAATCTTCCAAG AGAAGCTGTG GAATCATGTG AAGTGAATGG ATACTACATCCCCAAAGGAACCAGACTCAG TGTAAATATA TGGGGCATGG GGAGAGACCC GAATGTGTGGGAGAATCCACTGGAATTCAA TCCGGATAGG TTCTTGCGAG TGGAAACTAT GAAAATCGATCCACGGGGAAATCATTTTGA GCTTATACCA TTTGGGGCGG GAAGAAGGAT ATGTGCGGGGAGTAGAATGGCAATTGTTCT TGTTGAGTAC ATGTTGGGGA CACTTGTGCA TTCATTTGATTGGAATTTAGCTGACGGTGT AGAGCTAAAT ATGGATGAAT CATTTGGACT TGCACTCCAAAAGGCTGTCCCACTTGCAGC TATTGTCACC CCTCGCTTGC CACCTAGTGT CTATTATGTT TAA(SEQ IDNO:1).

[0069] Nucleotide sequence of CllaF3'5'H protein:

[0070] MVINMFVLRELSYAFILFFITHIFGRSLLSHYARKLPPGPTGFPVVGSLPFLGSMPHVALAEMSKKYGPVMYLKLGSRGMVVASTPDSARAFLKTLDLNFSNRPTDSGATHMAYDSQDMVFADYGPRW KLLRRVSNLHMLGGKALEDWTNVRKNEVGHMLQAMHESSCRGEPVVVADMLVYAMANMIGQVILSRRVFVTKGTELNEFKEMVVELMTSAGLFNVGDYIPSIACMDLQGIVRGMKSLHNKFDVLLNKML KEHESTRHERKEKPDLLDVLLENRDNKAVGEERITDTNIKALLLNLFTAGTDTSSSSIEWALAEMMKNPSIFKRAHAEMDHVIGNNRRLEESDIPNLPYLQAICKETFRKHPSTPLNLPREAVESCEV NGYYIPKGTRLSVNIWGMGRDPNVWENPLEFNPDRFLRVETMKIDPRGNHFELIPFGAGRRICAGSRMAIVLVEYMLGTLVHSFDWNLADGVELNMDESFGLALQKAVPLAAIVTPRLPPSVYYV(SEQ ID NO:2).

[0071] Example 3: Subcellular localization of CllaF3′5′H

[0072] Primers were designed based on the CllaF3′5′H sequence (as shown in Table 3). The CllaF3′5′H clone fragment prepared in Example 2 was used as a template for PCR amplification using the high-fidelity enzyme PrimeSTAR (TaKaRa).

[0073] Table 3 Primers for constructing the CllaF3′5′H subcellular localization vector

[0074]

[0075] The PCR amplification reaction system consisted of: 25 μL PrimeSTAR HS (Premix), 1.5 μL Primer 1 (10 μM), 1.5 μL Primer 2 (10 μM), 1 μL template, and distilled water to a final volume of 50 μL.

[0076] The PCR amplification reaction conditions included: 98℃ for 2 min, cycle number 1; 98℃ for 10 sec, 55℃ for 15 sec, 72℃ for 1 min 30 sec, cycle number 30; 72℃ for 7 min, cycle number 1; and 4℃ for 30 min, cycle number 1.

[0077] The DNA fragments were then recovered via electrophoresis and cloned into the pEASY-Blunt simple P vector (TaKaRa). After confirming the sequence of positive clones, the DNA fragments were recombined with the pAN580 vector. The vector was digested with XbaI / BamHI, and the digested products were purified using a PCR purification kit. The ligation product was then transformed into competent cells using homologous recombination to construct the pAN580::CllaF3′5′H::GFP vector.

[0078] The constructed pAN580::CllaF3′5′H::GFP vector plasmid was transformed into Agrobacterium (GV3101) by electroporation and cultured at 30℃ for 2 days. The cells were then collected by centrifugation at 5000 rpm / min for 4 min at room temperature. The cells were resuspended in 10 mM MgCl2 (containing 120 μM acetylsylcholine, Solarbio) suspension and OD200 was adjusted. 600 The bacterial suspension was obtained by adjusting the concentration to 0.6. Select tobacco plants with good growth conditions and inject 1-2 mL of bacterial suspension into the lower epidermis of tobacco leaves using a 1 mL syringe with the syringe tip removed. Culture the injected tobacco plants in low light for 2 days. Take the tobacco leaves injected with bacterial suspension, make them into glass slides, and observe them under a laser confocal microscope.

[0079] Subcellular localization results from tobacco leaves suggest that the CllaF3′5′H gene may be located in the endoplasmic reticulum.

[0080] To further clarify the subcellular localization of this gene, Arabidopsis protoplasts were used for the study. CllaF3′5′H was fused into the pSAT6-EGFP-N expression vector, and the subcellular localization of CllaF3′5′H was observed in an Arabidopsis protoplast transient expression system. GFP signal was observed on the endoplasmic reticulum under laser confocal microscopy.

[0081] The experimental methods can be found in the following literature: Sang-Dong Yoo, Young-Hee Cho & JenSheen. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols 2, 1565-1572 (2007).

[0082] from Figure 5 It can be seen that the pSAT6-CllaF3′5′H-EGFP fusion protein exists in the endoplasmic reticulum and is partially present in the cytoplasm. CllaF3′5′H is a flavonoid-3′5′-hydroxylase, which is a key enzyme that catalyzes the conversion of dihydroflavones to dihydroflavonols. It catalyzes the hydroxylation process in the anthocyanin biosynthesis pathway on the endoplasmic reticulum.

[0083] Analysis of the expression pattern of CllaF3′5′H revealed that the CllaF3′5′H gene is expressed in leaves, petioles, stems, and floral organs. Specifically, its expression level significantly upregulates during the initial coloring stage of Clematis pubescens flowers (S4), and gradually decreases as the flowers open and senescent.

[0084] Example 4

[0085] Primers were designed based on the CllaF3′5′H sequence (Table 4), and PCR amplification was performed using the CllaF3′5′H clone prepared in Example 2 as a template with the high-fidelity enzyme PrimeSTAR (TaKaRa).

[0086] Table 4 Primers for constructing the CllaF3′5′H overexpression vector

[0087]

[0088] The PCR amplification reaction system included: 25 μL PrimeSTAR HS (Premix), 1.5 μL forward primer (10 μM), 1.5 μL reverse primer (10 μM), 1 μL template, and distilled water to a final volume of 50 μL.

[0089] The PCR amplification reaction conditions included: 98℃ for 2 min, cycle number 1; 98℃ for 10 sec, 55℃ for 15 sec, 72℃ for 1 min 30 sec, cycle number 30; and 72℃ for 7 min, cycle number 1.

[0090] After electrophoresis, the recovered DNA fragments were cloned into the pEASY-Blunt simple P vector (TaKaRa). After confirming the sequence of positive clones, recombination was performed with the pBWA(V)HS vector. The vector was digested with BsaI / Eco31I, and the digested products were purified using a PCR purification kit. The ligation product was then transformed into competent cells using homologous recombination to construct the pBWA(V)HS::CllaF3′5′H::GUS vector (i.e., pBWA(V)HS-CllaF3′5′H-Gus).

[0091] Reference Figure 6The method shown will be used to construct the pBWA(V)HS-CllaF3′5′H-Gus expression vector for heterologous genetic transformation of tobacco.

[0092] It was verified that overexpression of the CllaF3′5′H gene in tobacco resulted in 14 tobacco overexpression lines. Figure 7 ),according to Figure 7 From left to right, they were named L1 to L14. The T1 generation plants were propagated, and the stability of flower color in the progeny was observed. Three overexpression lines were obtained through screening. RT-PCR was used to detect CllaF3′5′H gene expression. It was found that in the L6 (OE1), L7 (OE2), and L9 (OE3) lines, the expression level of CllaF3′5′H was significantly upregulated by 159.23%, 524.89%, and 638.54%, respectively. Figure 8 Functional verification experiments were conducted using T2 generation seeds from these three strains.

[0093] Targeted metabolomics analysis was performed on anthocyanins in tobacco overexpression lines. The anthocyanins included malvidin, pelargonidin, delphinidin, paeoniflorin, cyanidin galactoside, isorhamnetin, cyanidin, cyanidin diglucoside, petunia pigment, quercetin, rutin (rutin) proanthocyanidin B4, luteolin, dimeric cyanidinogen, and kaempferol.

[0094] The experimental method included the following steps: Accurately weigh 100 mg of sample, add 1 mL of extraction buffer (methanol / water / formic acid, 70:30:1, v / v / v), vortex at high speed, then sonicate for 20 min, centrifuge at 12000 rpm for 10 min, and repeat the extraction twice. Combine and mix the supernatants from both extractions, and pass them through an HLB-SPE column: add 1 mL of 100% methanol solution and 1 mL of deionized water respectively, add all the supernatant to the column, and simultaneously add 1 mL of aqueous solution for elution. After drying the liquid, add 1 mL of methanol solution (containing 5% formic acid, volume percentage) for elution, collect the eluent to a new centrifuge tube, slowly dry it under a nitrogen atmosphere, freeze-dry it, and reconstitute it with 0.2 mL of methanol solution (containing 5% formic acid, volume percentage). LC-ESI-MS / MS was used for qualitative and quantitative detection of anthocyanins. Chromatographic conditions: High-performance liquid chromatography (HPLC) using a Vanquish (Thermo, USA) system with a Waters HSS T3 (50 × 2.1 mm, 1.8 μm) column, column temperature 40 °C, injection volume 2 μL. Mobile phase A (0.1% formic acid in water, v / v) and mobile phase B (0.1% formic acid in acetonitrile, v / v). Mass spectrometry conditions: High-resolution mass spectrometer (Q Exactive, Thermo, USA), positive mode detection, sheath gas 40 alb, auxiliary gas 10 alb, ion spray voltage +3000 V, temperature 350 °C, ion transfer tube temperature 320 °C. Scan mode: Single ion detection (SIM), positive ion mode. Primary mass spectrometry scan range (scan m / z range): 200-700.

[0095] Experimental results are as follows Figure 9 As shown, after overexpression of CllaF3′5′H, the delphinidin content increased by 54.88%, the cyanidin galactoside content decreased significantly by 70.85%, the peony pigment content decreased significantly by 99.11%, and the luteolin content decreased significantly by 70.08%. This indicates that overexpression of CllaF3′5′H promoted the delphinidin biosynthesis pathway, increased delphinidin biosynthesis, reduced the synthesis of peony and cyanidin, increased the blue pigment content, and promoted the transformation of flower color to blue-purple.

[0096] Principal component analysis (PCA) showed a significant difference between overexpression and the control group, with good reproducibility within the group. Figure 10Overexpression of CllaF3′5′H significantly increased the accumulation of delphinidin-3-O-arabinoside, dihydrokaempferol, kaempferol-3-O-rutinoside, naringenin-7-O-glucose, delphinidin-3-O-arabinoside, malvidin-3-O-glucose, petunidin-3-O-(6-O-coumaryl-glucose), and petunidin-3-O-galactoside, effectively promoting the accumulation of blue anthocyanins. Figure 11 ).

[0097] Example 5

[0098] Transient silencing (VIGS) of CllaF3′5′H in Clematis pubescens: The CllaF3′5′H gene in Clematis pubescens was silenced using VIGS technology. Based on the specificity of the CllaF3′5′H gene sequence, the silencing region was designed to be 672bp-971bp, and the silencing sequence was designed to be 300bp. Three pairs of primers were designed for amplification. Finally, a TRV2 vector containing the CllaF3′5′H silencing induction sequence fragment was successfully constructed using one pair of primer sequences (Table 5) and transformed into Agrobacterium tumefaciens GV3101.

[0099] Primers were designed based on the CllaF3′5′H sequence, and the CllaF3′5′H clone prepared in Example 2 was used as a template for PCR amplification using the high-fidelity enzyme PrimeSTAR (TaKaRa).

[0100] Table 5 Primers for constructing the CllaF3′5′HVIGS vector

[0101]

[0102] The PCR amplification reaction system included: 25 μL PrimeSTAR HS (Premix), 1.5 μL forward primer (10 μM), 1.5 μL reverse primer (10 μM), 1 μL template, and distilled water to a final volume of 50 μL.

[0103] The PCR amplification reaction conditions included: 98℃ for 2 min, cycle number 1; 98℃ for 10 sec, 55℃ for 15 sec, 72℃ for 30 sec, cycle number 30; 72℃ for 7 min, cycle number 1; and 4℃ for 30 min, cycle number 1.

[0104] After electrophoresis, the recovered DNA fragments were cloned into the pEASY-Blunt simple P vector (TaKaRa). After the positive clones were sequenced and confirmed to be correct, the DNA fragments were digested with EcoRI / XhoI, respectively, along with the pTRV2 vector. The digested DNA fragments were purified using a PCR purification kit and then ligated with T4 ligase. The ligation product was transformed into competent cells to construct the pTRV2::CllaF3′5′H vector (which can also be written as pTRV2-CllaF3′5′H).

[0105] pTRV1+pTRV2 and pTRV1+pTRV2-CllaF3′5′H were injected into the stems of Clematis pubescens to infect the plant and establish a transiently silenced CllaF3′5′H line for Clematis pubescens. Figure 12 The phenotype of Clematis pubescens VIGS transiently silenced lines shows that, based on VIGS technology, the CllaF3′5′H gene of Clematis pubescens was silencing. After transient silencing of the CllaF3′5′H gene, the blue-purple flower color of Clematis pubescens at the initial flowering stage (S6), full bloom stage (S7), and end flowering stage (S8) was significantly lighter.

[0106] qRT-PCR analysis was performed on the calyx tissue of Clematis pubescens. Using the genomic DNA of the calyx tissue as a template, qRT-PCR was performed using the primers listed in Table 6. The qRT-PCR reaction system consisted of: 10 μL SYBR Premix Ex Taq (2×), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 1 μL template, and distilled water to a final volume of 20 μL. The qRT-PCR reaction conditions were: 95℃ for 5 min, cycle number 1; 95℃ for 5 sec, 60℃ for 30 sec, cycle number 40; 95℃ for 1 min, cycle number 1; 55℃ for 1 min, cycle number 1; 55℃ for 10 sec, cycle number 1. The qRT-PCR results showed that the expression level of CllaF3′5′H in the silenced lines was significantly reduced by 88.21%, indicating effective silencing.

[0107] Table 6 CllaF3′5′H qRT-PCR primers

[0108]

[0109] Analysis of transiently silenced Clematis pubescens lines (VIGS) and control groups (WT) at different stages (S4 to S8) revealed that silencing the CllaF3′5′H gene significantly reduced delphinidin content by 81.75% in S6, 55.73% in S7, and 6.11% in S8; simultaneously, cyanidin content significantly decreased by 45.97% in S4, 86.05% in S5, 68.88% in S6, and 41.14% in S7; kaempferol content significantly increased by 72.56% and 77.71% at full bloom and late bloom, respectively. In S8, due to significant decreases in delphinidin and cyanidin content, quercetin content significantly increased by 192.51%, indicating that silencing the CllaF3′5′H gene inhibited the synthesis of delphinidin metabolites. Figure 13 ).

[0110] Analysis revealed that transient silencing of CllaF3′5′H in Clematis pubescens significantly reduced the contents of Mg, Fe, and Cu. Specifically, Mg decreased by 7.43% and 10.80% in S7 and S8, respectively; Fe decreased by 25.86%, 19.62%, and 7.83% in S6, S7, and S8, respectively; and Cu decreased by 45.30%, 10.48%, and 31.55% in S6, S7, and S8, respectively.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The CllaF3′5′H gene, characterized by, Includes the nucleotide sequence shown in SEQ ID NO:

1.

2. Cl laF3′5′H protein, characterized by, Includes the protein encoded by the CllaF3′5′H gene as described in claim 1.

3. A primer set, characterized in that, Used to amplify the CllaF3′5′H gene as described in claim 1.

4. A carrier, characterized in that, Includes the CllaF3′5′H gene as described in claim 1.

5. A transformant, characterized in that, Includes the carrier described in claim 4.

6. A method for cultivating blue-purple flowers, characterized in that, Includes the following steps: Overexpression of the CllaF3′5′H gene as described in claim 1.

7. The use of the CllaF3′5′H gene of claim 1 or the CllaF3′5′H protein of claim 2 in any one or more of (1) to (7), characterized in that, (1) Regulating flower color; (2) Synthesize flavonoid metabolites; (3) Synthetic delphinidin; (4) Synthesize delphinidin derivatives; (5) Cultivate blue-flowered plant varieties; (6) Promotes the transformation of flower color to blue-purple; (7) Promotes the accumulation of blue anthocyanins.

8. The use of the primer set according to claim 3 in any one or more of (1) to (7), characterized in that, (1) Regulating flower color; (2) Synthesize flavonoid metabolites; (3) Synthetic delphinidin; (4) Synthesize delphinidin derivatives; (5) Cultivate blue-flowered plant varieties; (6) Promotes the transformation of flower color to blue-purple; (7) Promotes the accumulation of blue anthocyanins.

9. The use of the carrier according to claim 4 in any one or more of (1) to (7), characterized in that, (1) Regulating flower color; (2) Synthesize flavonoid metabolites; (3) Synthetic delphinidin; (4) Synthesize delphinidin derivatives; (5) Cultivate blue-flowered plant varieties; (6) Promotes the transformation of flower color to blue-purple; (7) Promotes the accumulation of blue anthocyanins.

10. The use of the transformant according to claim 5 in any one or more of (1) to (7), characterized in that, (1) Regulating flower color; (2) Synthesize flavonoid metabolites; (3) Synthetic delphinidin; (4) Synthesize delphinidin derivatives; (5) Cultivate blue-flowered plant varieties; (6) Promotes the transformation of flower color to blue-purple; (7) Promotes the accumulation of blue anthocyanins.

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