The invention relates to an eggplant flavonoid 3apos; , 5apos; gene of hydroxylase and application method thereof

Abstract

The invention discloses an eggplant flavonoid 3 ', 5'-hydroxylase gene and an application method thereof, and relates to the field of biochemistry, the gene sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2; the invention discloses a cloning method of the gene and a gene expression detection method. The gene is applied to creation of new eggplant varieties with different colors, creation of a rare blue system, creation of fruits rich in antioxidant delphinidin anthocyanin, production of natural delphinidin and production of delphinidin through fermentation engineering. According to the invention, on the basis of DNA high-fidelity polymerase, the CDS sequence of the eggplant SmF3 '5' H gene is cloned by using a PCR technology, Sanger sequencing verification is completed, and gene expression quantity detection is carried out. The eggplant SmF3 '5' H gene has huge application potential in the aspects of crop appearance and nutritional quality improvement.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more particularly to a gene for an eggplant flavonoid 3',5'-hydroxylase and its application method. Background Technology

[0002] Anthocyanins are water-soluble natural pigments, a type of flavonoid, found in plants. They impart colors such as red, purple, and blue to plants and are an important pigment pool. Furthermore, as strong reducing agents, anthocyanins possess a powerful ability to scavenge free radicals, playing a crucial role in plant stress resistance. They are also highly beneficial for human anti-aging, anti-inflammation, and prevention of cardiovascular diseases. Eggplant is a horticultural crop rich in anthocyanins, which accumulate mainly in the stems, leaves, petals, sepals, and pericarp. Anthocyanins can serve as morphological markers for identifying varietal purity. Additionally, the content and types of anthocyanins in the pericarp are decisive factors in the appearance and quality indicators of eggplant fruits.

[0003] Of the more than 20 natural anthocyanins identified so far, geranium pigment (Pg), cyanidin pigment (Cy), delphinidin pigment (Dp), peony pigment (Pn), morning glory pigment (Pt), and mallow pigment (Mv) are the most widely distributed and have significant functions. Different types of anthocyanins exhibit functional differentiation due to differences in their chemical structures (different degrees of hydroxylation and methylation): Cy (containing two phenolic hydroxyl groups) has the strongest antioxidant activity and can effectively scavenge superoxide free radicals (O2). - Anthocyanins (Cy3G) and hydrogen peroxide (H2O2) have scavenging efficiencies more than 20 times that of vitamin C, and are enriched in blueberries (wild varieties, 300–700 mg / 100 g), black rice (Cy-3-glucoside, 100–300 mg / 100 g), and red onions (Cy3G, 29.99 mg / 100 g). Dp (containing three phenolic hydroxyl groups) has the best ability to scavenge hydroxyl radicals (·OH) due to its polyphenolic hydroxyl structure, and is a key substance for blue coloration. It needs to co-color with metal ions and auxiliary pigments, and is enriched in eggplant (peel Dp content 110.49 mg / 100 g) and blackcurrant (accounting for 40%–60% of total anthocyanins). Mv and Pn, with high methylation levels, exhibit outstanding stability; acylated Mv (content 90–322 mg / 100 g) is found in purple sweet potatoes. Pn (Pg) has a better ability to scavenge ·OH than ascorbic acid. Grapes and purple corn (such as "Sweet Black Gold 699" with a content of 114.93 mg / 100 g) are rich in Pn (accounting for 30%–50% in wine grapes). In addition, Pg (such as strawberry with a content of 65–100 mg / 100 g) and Pt are known for their antibacterial and anticancer properties and free radical scavenging properties, respectively.

[0004] Anthocyanin biosynthesis begins with phenylalanine (Phe), which is deaminated by phenylalanine ammonia-lyase (PAL) to produce cinnamic acid. Cinnamic acid is then hydroxylated by cinnamic acid 4-hydroxylase (C4H) and activated by 4-coumaroyl-CoA ligase (4CL) to form 4-coumaroyl-CoA. Subsequently, under the action of chalcone synthase (CHS), chalcone is condensed with malonyl-CoA to form naringenin chalcone, which is isomerized by chalcone isomerase (CHI) to form naringenin (flavonoid). Flavonol 3-hydroxylase (F3H) oxidizes naringenin to produce dihydroflavonols (such as dihydrokaempferol). If dihydrokaempferol is directly catalyzed by dihydroflavonol 4-reductase (DFR), it will produce colorless geranyl tannin. If it is catalyzed by flavonol 3'-hydroxylase (F3'H) to undergo hydroxylation at the 3' position of the B ring, it will produce dihydroquercetin, a precursor of cyanidin. If it is catalyzed by flavonol 3',5'-hydroxylase (F3'5'H) to hydroxylate at the 3' and 5' positions of the B ring, it can be converted into a 3',4',5'-trihydroxylated product (such as dihydromyricetin), which is a precursor of delphinidin. These precursors are catalyzed by DFR to produce corresponding colorless anthocyanins, which are then further oxidized by anthocyanin synthase (ANS) to form colored anthocyanin aglycones, and finally glycosylated by UDP-glucosyltransferase (UFGT) to form stable pigments.

[0005] The differences in the distribution and function of anthocyanin types stem from differences in the substituents on their B rings: hydroxylation enhances antioxidant capacity (e.g., Dp > Cy > Pg), while methylation enhances stability (e.g., Mv, Pn). Delphinidin, a key type for blue coloration and high-efficiency antioxidant activity, relies on flavonoid 3',5'-hydroxylase (F3'5'H) for its synthesis. However, most important economic crops lack this enzyme gene and therefore cannot accumulate Dp. Typical examples include: roses, carnations, and chrysanthemums cannot naturally form blue flowers due to the absence of F3'5'H; cereal crops such as rice and wheat can accumulate Cy (e.g., purple rice), but are completely lacking in Dp; and Rosaceae fruits such as apples, cherries, peaches, and strawberries contain only Cy or Pg, with no detectable Dp. In contrast, naturally occurring crops rich in Dp (e.g., eggplant, blackcurrant) and genetically engineered varieties are scarce sources of Dp, highlighting the value of using biotechnology to engineer anthocyanins in important economic crops using the F3'5'H gene.

[0006] In Solanaceae crops, the function of F3'5'H is closely related to anthocyanin diversity. Comparative genomic analysis of *Gynostemma pentaphyllum* showed that copy number variation of the F3'5'H gene is associated with interspecies pigment differences; in the colored potato 'Zhuanxinwu', F3'5'H is highly expressed in tubers, positively correlated with delphinidin accumulation, and its promoter contains a photoresponsive element. In purple peppers, F3'5'H gene expression is much higher than in green peppers. However, in eggplant, a typical delphinidin-rich Solanaceae crop, the gene sequence, expression pattern, and molecular function of F3'5'H remain unclear. Furthermore, other anthocyanin synthesis structural genes in eggplant, such as SmCHS, SmF3H, and SmDFR, have been successfully cloned and published. Therefore, the cloning and functional verification of the eggplant SmF3'5'H gene not only helps to understand the anthocyanin synthesis pathway in eggplant and lays a theoretical foundation for the creation of new eggplant varieties, but also greatly promotes the application of eggplant SmF3'5'H through genetic manipulation to create economically valuable crops with enhanced nutrition and diverse colors.

[0007] Anthocyanins in the plant kingdom are mainly classified into six categories: pelargonidin (Pg), cornflower pigment (Cy), delphinidin (Dp), peony pigment (Pn), morning glory pigment (Pt), and mallow pigment (Mv). Among them, delphinidin exhibits a rare blue-purple color. For example, common and important flowers such as roses, carnations, and chrysanthemums cannot produce blue-purple flowers due to the lack of delphinidin. Furthermore, delphinidin possesses the strongest antioxidant function due to the presence of three hydroxyl groups on its B ring. However, it cannot be normally produced in some common and important fruits and crops, such as apples, cherries, peaches, strawberries, rice (purple rice), and wheat, thus affecting their nutritional value. In plants, flavonoid 3',5'-hydroxylase (F3'5'H) is a key enzyme essential for the catalytic production of delphinidin anthocyanins. Many crops lack the F3'5'H gene in their genome, resulting in the inability of their product organs to synthesize delphinidin normally. Eggplant is a rare economic crop capable of naturally synthesizing large amounts of delphinidin-like anthocyanins, indicating that the SmF3'5'H gene in the eggplant genome can effectively convert anthocyanin precursors into delphinidin-like anthocyanins. Furthermore, with the successful establishment of genetic transformation systems for most economic crops, methods to enrich the color of agricultural products and enhance their nutritional value through genetic manipulation of the genome using biotechnology are readily available. This demonstrates the enormous application potential of the eggplant SmF3'5'H gene in improving crop appearance and nutritional quality.

[0008] Therefore, those skilled in the art are dedicated to developing a gene for a 3',5'-hydroxylase of eggplant flavonoids, as well as a method for cloning and detecting its gene expression, which can be used through genetic manipulation to create and improve new plant varieties and to produce delphinidin. Summary of the Invention

[0009] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is how to develop a gene for eggplant flavonoid 3',5'-hydroxylase, as well as its cloning and gene expression detection method, which can be used for genetic manipulation to create and improve new plant varieties and produce delphinidin.

[0010] To achieve the above objectives, the present invention provides a gene for eggplant flavonoid 3',5'-hydroxylase, the gene sequence of which is shown in SEQ ID NO.1 and its amino acid sequence is shown in SEQ ID NO.2.

[0011] This invention also provides a method for cloning the gene of eggplant flavonoid 3',5'-hydroxylase, comprising the following steps:

[0012] Step 1: Collect the peels of the purplish-black eggplant variety 'Lanshan Hexian';

[0013] Step 2: Extract RNA from the fruit peel sample obtained in Step 1;

[0014] Step 3: Reverse transcribe the RNA obtained in Step 2 into cDNA, use it as a template, and clone it using primers F1 and R1; the sequence of primer F1 is: 5′-ATGGTTATACTTCCTAGTGAGTTG -3′, and the sequence of primer R1 is: 5′-GAAAGGCTCGCTAGACTGATAAAT -3′.

[0015] Furthermore, step 1 also includes collecting the peels under natural growing conditions, once the eggplant plants have grown to market maturity, immediately pre-cooling them with liquid nitrogen, and then storing them in a -80°C freezer.

[0016] This invention also provides a method for detecting the gene expression of 3',5'-hydroxylase in eggplant flavonoids, comprising the following steps:

[0017] Step 1) Collect various tissues of the purplish-black peel eggplant 'Lanshan Hexian' as samples, including roots, stems, leaves, petals, peel, pulp, sepals, and anthers;

[0018] Step 2) Using the gene sequence shown in SEQ ID NO.1 as a template, and primer pairs SmF3'5'HF and SmF3'5'HR, perform RT-qPCR analysis on the sample obtained in Step 1); the sequence of primer SmF3'5'HF is: 5′-TGGACCTCGTTGGAAGTTGCTAAG -3′, and the sequence of primer SmF3'5'HR is: 5′-TGCCATCGCGAACGTCAACATAT -3′.

[0019] Furthermore, step 2) also includes using the actin gene SmACTIN (GU984779.1) as an internal reference gene, with the following RT-qPCR primers:

[0020] SmACTIN-F: 5′-GTCGGAATGGGACAGAAGGATG-3′

[0021] SmACTIN-R: 5′-GTGCCTCAGTCAGGAGAACAGG-3′.

[0022] This invention also provides an application of the gene for eggplant flavonoid 3',5'-hydroxylase in creating new eggplant varieties with different colors.

[0023] This invention also provides an application of the gene for eggplant flavonoid 3',5'-hydroxylase in improving the blue flower color of ornamental flowering plants.

[0024] This invention also provides an application of the gene for eggplant flavonoid 3',5'-hydroxylase in cultivating fruits rich in antioxidant delphinidin anthocyanins.

[0025] This invention also provides an application of the eggplant flavonoid 3',5'-hydroxylase gene in the production of natural delphinidin, specifically: using transgenic technology to overexpress the eggplant flavonoid 3',5'-hydroxylase gene in eggplant or tomato substrate crops, constructing a super crop that synthesizes delphinidin-like anthocyanins for the production of natural delphinidin.

[0026] This invention also provides an application of the eggplant flavonoid 3',5'-hydroxylase gene in the production of delphinidin through fermentation engineering. Specifically, the eggplant flavonoid 3',5'-hydroxylase gene is introduced into an engineered strain, and delphinidin with strong antioxidant function is produced in large quantities using fermentation engineering.

[0027] SEQ ID NO.1:

[0028]

[0029] SEQ ID NO.2:

[0030] MVILPSELIGATIIYIIVYIIIQKLIATGSWRRRRRLPPGPEGWPVIGALPLLGGMPHVALAKMAKKYGPIMYLKVGTCGMVVASTPNAAKAFLKTLDINFSNRPPNAGATHMAYNAQDMVFAPYGPRW KLLRKLSNLHMLGGKALENWANVRANELGHMLKSMFDASHVGERIVVADMLTFAMANMIGQVMLSKRVFVEKGKEVNEFKNMVVELMTVAGYFNIGDFIPQIAWMDLQGIEKGMKKLHKKFDDLLTKM FEEHEATSNERKGKPDFLDFIMANRDNSEGERLSITNIKALLLNLFTAGTDTSSSVIEWALTEMMKNPTIFKKAQQEMDQIIGKNRRFIESDIPNLPYLRAICKEAFRKHPSTPLNLPRVSSDACTID GYYIPKNTRLSVNIWAIGRDPDVWENPLEFIPERFLSEKNAKIEHRGNDFELIPFGAGRRICAGTRMGIVMVEYILGTLIHSFDWKLPNDVVDINMEETFGLALQKAVPLEAIVTPRLSFDIYQSSEPF

[0031] In a preferred embodiment 1 of the present invention, the cloning process of the eggplant SmF3'5'H gene is described in detail.

[0032] In another preferred embodiment 2 of the present invention, the eggplant SmF3'5'H protein sequence information is described in detail;

[0033] In another preferred embodiment 3 of the present invention, the expression detection process of the eggplant SmF3'5'H gene in eggplant roots, stems, leaves, petals, pericarps, pulp, sepals and anther tissues is described in detail.

[0034] In another preferred embodiment 4 of the present invention, the process of creating new eggplant varieties of different colors by regulating the function of the SmF3'5'H gene through genetic manipulation is described in detail.

[0035] In another preferred embodiment 5 of the present invention, the process of creating rare blue roses, carnations and chrysanthemums by introducing the eggplant SmF3'5'H gene through genetic transformation is described in detail.

[0036] In another preferred embodiment 6 of the present invention, the process of creating rare, nutritionally enhanced economic crops such as apples, cherries, peaches, and strawberries by introducing the eggplant SmF3'5'H gene through genetic transformation is described in detail.

[0037] In another preferred embodiment 7 of the present invention, the process of using transgenic technology to overexpress the eggplant SmF3'5'H gene in eggplant base crops to construct a super crop that synthesizes delphinidin-like anthocyanins for the production of natural delphinidin is described in detail.

[0038] In another preferred embodiment 8 of the present invention, the process of introducing the eggplant SmF3'5'H gene into an engineered strain and using fermentation engineering to produce delphinidin with extremely strong antioxidant function in large quantities is described in detail.

[0039] The beneficial technical effects of this invention are as follows:

[0040] This invention clones the CDS sequence of the eggplant SmF3'5'H gene using high-fidelity DNA polymerase PCR technology and verifies it with Sanger sequencing. Based on the CDS sequence of the SmF3'5'H gene, fluorescent quantitative PCR primers were designed, and gene expression levels were detected using Sybr GreenPCR Master Mix. The correlation between the expression level of the eggplant SmF3'5'H gene and the anthocyanin accumulation levels in various tissues and organs was obtained. The disclosed eggplant SmF3'5'H gene has enormous application potential in improving crop appearance and nutritional quality.

[0041] 1. Based on eggplant genetic transformation and CRISPR gene editing system-mediated loss-of-function mutation of the SmF3'5'H gene sequence, a loss-of-function mutant of the SmF3'5'H gene was constructed in eggplant for the first time, obtaining anthocyanin synthesis-deficient eggplant mutants, enriching eggplant fruit colors and meeting diverse market demands. New eggplant varieties with different colors were created by genetically manipulating the expression level of the SmF3'5'H gene;

[0042] 2. By introducing the eggplant SmF3'5'H gene into flowering plants using genetic engineering, the eggplant SmF3'5'H gene can compensate for the lack of the F3'5'H gene in these flowers and convert anthocyanin precursors of other colors in the petals into blue delphinidin-type anthocyanins. This genetic transformation of the eggplant SmF3'5'H gene can create rare blue roses, carnations, and chrysanthemums; it can also create rare and highly sought-after blue roses, greatly enriching the colors of ornamental flowers and avoiding the existing dyeing process.

[0043] 3. By introducing the eggplant SmF3'5'H gene into economic crops such as apples, cherries, peaches, and strawberries through transgenic or gene knock-in technology, the eggplant SmF3'5'H gene can compensate for the lack of the F3'5'H gene in these crops and transform other anthocyanin precursors with low antioxidant capacity in the fruit into delphinidin-like anthocyanins with strong antioxidant capacity. Using genetic transformation to introduce the eggplant SmF3'5'H gene can create rare, nutritionally enhanced varieties of economic crops such as apples, cherries, peaches, strawberries, rice (purple rice), and wheat; and create rare fruits rich in delphinidin-like anthocyanins with strong antioxidant capacity, effectively improving the nutritional value of fruits and enhancing people's dietary health.

[0044] 4. Using transgenic technology, overexpress the eggplant SmF3'5'H gene in eggplant or tomato substrate crops to construct a super crop that synthesizes delphinidin-like anthocyanins for the production of natural delphinidin; it can be used in food processing, cosmetics and healthcare industries.

[0045] 5. The eggplant SmF3'5'H gene was introduced into an engineered strain to produce delphinidin, a compound with strong antioxidant function, in large quantities using fermentation engineering, thus promoting the industrial production of delphinidin. This overcomes the limitations of slow and low-volume industrial anthocyanin production by innovatively constructing an engineered strain that catalyzes the production of delphinidin from phenylalanine or other anthocyanin intermediates.

[0046] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the eggplant SmF3'5'H protein structural domain of a preferred embodiment 2 of the present invention;

[0048] Figure 2 This refers to the anthocyanin content in eggplant roots, stems, leaves, petals, peel, pulp, sepals, and anthers in a preferred embodiment 3 of the present invention.

[0049] Figure 3 This is the relative expression level of the eggplant SmF3'5'H gene in the root, stem, leaf, petal, peel, pulp, sepals and anther of a preferred embodiment of the present invention;

[0050] Figure 4 Correlation analysis of anthocyanin content and relative expression level of SmF3'5'H gene in eggplant roots, stems, leaves, petals, peel, pulp, sepals and anthers in a preferred embodiment 3 of the present invention;

[0051] Figure 5This is the Sanger sequencing analysis result of the target sites of Smf3'5'h-cr#23 and Smf3'5'h-cr#50 CRISPR / Cas9 gene editing lines in a preferred embodiment 4 of the present invention;

[0052] Figure 6 This is the plant phenotype of the Smf3'5'h-cr#23 and Smf3'5'h-cr#50 gene-edited lines of a preferred embodiment 4 of the present invention;

[0053] Figure 7 This is the anthocyanin phenotype of the stems, leaves, petals, and pericarps of the Smf3'5'h-cr#23 and Smf3'5'h-cr#50 gene-edited lines of a preferred embodiment of the present invention (4).

[0054] Figure 8 This refers to the relative expression levels of the main structural genes for anthocyanin synthesis in the pericarps of the Smf3'5'h-cr#23 and Smf3'5'h-cr#50 gene-edited lines in a preferred embodiment 4 of the present invention. Detailed Implementation

[0055] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0056] Example 1: Cloning of the eggplant SmF3'5'H gene

[0057] 1. Collection of plant materials

[0058] The plant material used in this invention is the purplish-black eggplant 'Lanshan Hexian' variety, primarily for constructing an eggplant cDNA library. This material can be obtained from our laboratory and the National Crop Germplasm Resource Platform. Alternatively, purple eggplants can be purchased from the market for mRNA extraction to construct a cDNA library. The experimental materials were cultivated in a plastic greenhouse at the Minhang Campus of Shanghai Jiao Tong University, and routine water and fertilizer management was implemented during the growth period. Under natural growth conditions, once the eggplant plants reached commercial maturity (approximately 20 days after flowering), roots, stems, leaves, petals, pericarps, pulp, sepals, and anther tissues were collected, immediately pre-cooled with liquid nitrogen, and then stored at -80℃ for subsequent RNA extraction and anthocyanin content determination.

[0059] 2. RNA extraction

[0060] Eggplant tissue samples were ground into powder in liquid nitrogen, and 300 mg of the powder was transferred to a 1.5 mL RNase-free centrifuge tube. Total RNA was extracted using the SteadyPure Plant RNA Extraction Kit from Acrylic AgroScience Co., Ltd., following the instructions. The integrity of the RNA was detected by agarose gel electrophoresis, and the purity and concentration of the RNA were determined using a ThermoScientific NANODROP 2000 Spectrophotometer. The RNA was then stored at -80°C for later use.

[0061] 3. Full-length cloning of the SmF3'5'H gene CDS

[0062] 500 ng RNA from various eggplant tissues was reverse transcribed into cDNA using the Takara PrimeScript™ RT reagent Kit with gDNA Eraser, following the manufacturer's instructions. The potato StF3'5'H protein sequence (NP_001274807.2) was compared with the eggplant genome database to find the homologous sequence of the eggplant SmF3'5'H gene. Based on this, specific primers for cloning the full-length CDS were designed using Primer 5 software.

[0063] F1: 5′-ATGGTTATACTTCCTAGTGAGTTG-3′

[0064] R1: 5′-GAAAGGCTCGCTAGACTGATAAAT-3′

[0065] Using an eggplant pericarp cDNA library as a template, the full-length CDS of the gene was cloned by PCR using the high-fidelity DNA polymerase premixed solution-FS from Acrel Biotech. The PCR products were purified by agarose gel electrophoresis, and the target DNA fragment was recovered using the FastPureGel DNA Extraction Mini Kit from Novizuma. The recovered product was ligated into the intermediate vector Blunt Simple Vector (Beijing TransGen Biotech Co., Ltd.), transformed into *E. coli* DH5α, and positive clones were screened by colony PCR using primers M13-F and M13-R. These clones were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, yielding the CDS sequence of the SmF3'5'H gene, SEQ ID NO. 1.

[0066] Example 2: Eggplant SmF3'5'H protein sequence information

[0067] The full-length CDS sequence of the eggplant SmF3'5'H gene in this invention is 1542 bp, and the detailed sequence is shown in SEQ ID NO.1. The amino acid sequence of eggplant SmF3'5'H, translated from the CDS coding sequence, consists of 513 amino acid residues, and the detailed sequence is shown in SEQ ID NO.2. Using the online analysis website ProtParam, the molecular weight of the protein was predicted to be 57.8 kDa, and the theoretical isoelectric point (pI) was 8.8, therefore the SmF3'5'H protein is basic. The protein domains of the SmF3'5'H amino acid sequence were analyzed using the online website SMART, as shown... Figure 1 As shown, SmF3'5'H was found to possess the P450 (37-496aa) conserved domain of the P450 family, indicating that SmF3'5'H belongs to the cytochrome P450-dependent monooxygenase family. Using the amino acid sequence of SmF3'5'H protein as a template, homologous proteins from Solanaceae (P37120.1, Solanum virginianum), potato (NP_001274807.2, Solanum tuberosum), tomato (NP_001234840.2, Solanum lycopersicum), pepper (NP_001361341.1, Solanum pennellii), and petunia (AUI38394.1, Petunia x hybrida) were selected from the Blast homologous protein sequence on the NCBI website. Multiple sequence alignment analysis was performed using DNAMAN software, and it was found that the homology of SmF3'5'H protein sequence in Solanaceae crops was greater than 80%, indicating that the function of SmF3'5'H is relatively conserved.

[0068] Example 3: Expression of the eggplant SmF3'5'H gene in eggplant roots, stems, leaves, petals, pericarps, pulp, sepals, and anther tissues.

[0069] 1. Determination of anthocyanin content in various tissues of 'Lanshan Hexian' eggplant

[0070] Samples of eggplant roots, stems, leaves, petals, pericarps, pulp, sepals, and anther tissues were collected according to Example 1. Three independent plants were collected for each tissue, with 0.3 g of sample per sample. For the pericarp, a thin layer was removed using a sharp scalpel, minimizing the amount of pulp tissue included. Each tissue was placed in 5 mL of methanol extraction buffer containing 0.1% hydrochloric acid and extracted at 4 °C for 12 h. Subsequently, the extract was centrifuged at 4 °C for 5 min, and the extract was collected in a new 5 mL centrifuge tube. Anthocyanin content was determined using a pH differential assay. 4 mL of buffer A and buffer B were transferred to new 5 mL centrifuge tubes. 1 mL of the anthocyanin solution to be tested was added to each of the 4 mL buffers A and B, and mixed thoroughly. 200 µL of each buffer solution was transferred to a 96-well microplate, and the absorbance values ​​at 510 nm and 700 nm were read using a microplate reader. Each biological replicate was performed in triplicate. The formula for calculating total anthocyanin content is: TA = 100 × (ΔA × F × M) / (ε × m × d), ΔA = [A510 nm (pH 1.0) - A700 nm (pH 1.0)] - [A510 nm (pH 4.5) - A700 nm (pH 4.5)], where M is the standard molecular weight, with a value of 449.2; m is the sample mass; ε is the molar absorptivity, with a value of 26,900; F is the dilution factor (5 here); and d is the optical path length, with a value of 0.6. 0.05% hydrochloric acid methanol solution: 0.5 mL concentrated hydrochloric acid was diluted to 1 L with methanol; pH 1.0 buffer A: 0.2 M HCl: 0.2 M KCl: H2O = 50:97:53, where 0.2 M hydrochloric acid was taken from 1 volume of 36... % concentrated hydrochloric acid was diluted with water to 60 mL; 1.491 g of 0.2 M potassium chloride solid was dissolved in 100 mL of water; pH 4.5 buffer B: 19.29 g of sodium acetate was added to 24 mL of glacial acetic acid, and the volume was adjusted to 500 mL with water. The anthocyanin content in eggplant roots, stems, leaves, petals, peel, pulp, sepals, and anthers is as follows: Figure 2 As shown. After extracting and determining the anthocyanin content, SPSS 19.0 software was used for comparison. One-way ANOVA test: post-hoc multiple comparisons (Duncan, significance level P<0.05), and the significance analysis results are expressed in lowercase letters (af). It was found that anthocyanins could accumulate in eggplant stems, leaves, petals, pericarps and sepals. The pericarp had the highest anthocyanin content, reaching 400 (mg / 100 g, FW), followed by the sepals, with an anthocyanin content of about 160 (mg / 100 g, FW). Anthocyanins were not detected in roots, pulp and anthers.

[0071] 2. Obtaining the cDNA library

[0072] For the collection of samples from eggplant roots, stems, leaves, petals, pericarps, pulp, sepals, and anther tissues, and the procedures for RNA extraction and reverse transcription to obtain cDNA libraries, please refer to Example 1.

[0073] 3. Primer design for real-time PCR

[0074] Using the full-length CDS sequence (SEQ ID NO.1) of the SmF3'5'H gene obtained in Example 1 as a template, specific primer pairs with PCR product sizes of 150-250 bp were searched in the primer design software Primer 5. The primer pair with the highest score was selected for real-time quantitative PCR (RT-qPCR) to detect the relative expression level of the SmF3'5'H gene in different tissues. The primer pair sequences are as follows:

[0075] SmF3'5'HF: 5′- TGGACCTCGTTGGAAGTTGCTAAG -3′

[0076] SmF3'5'HR:5′-TGCCATCGCGAACGTCAACATAT-3′

[0077] In addition, the actin gene SmACTIN (GU984779.1) was used as an internal reference gene, and its RT-qPCR primers were as follows:

[0078] SmACTIN-F: 5′-GTCGGAATGGGACAGAAGGATG-3′

[0079] SmACTIN-R: 5′-GTGCTCAGTCAGGAGAACAGG-3′

[0080] 4. Construct standard curves for the SmF3'5'H and SmACTIN genes.

[0081] The cDNA library obtained in Example 1 was serially diluted 2, 4, 8, 12, and 24-fold using ddH2O. Then, using the diluted cDNA solutions as templates, RT-qPCR analysis was performed using Novizan's ChamQ Universal SYBR qPCR Master Mix. The reaction mixture consisted of: 7.2 µL RNase-free ddH2O + 0.4 µL each of forward and reverse quantitative primers + 2 µL cDNA template + 10 µL ChamQ Universal SYBR qPCR Master Mix (total 20 µL). The reaction program used a two-step method: one cycle at 95 °C for 30 s, followed by 39 cycles at 95 °C for 10 s and 60 °C for 30 s. Signal acquisition was set at 60 °C, and melting curves were obtained using the qTOWER instrument.3G Default parameters. After the reaction, plot the melting curve of the target gene and the standard curve. Analyze the melting curve to determine whether the target gene and internal reference gene have been specifically amplified by PCR based on whether the peaks are singular. Determine the appropriate dilution factor of the template cDNA using the standard curve.

[0082] 5. Analysis of the relative expression levels of the SmF3'5'H gene in various tissues

[0083] Referring to the standard curve, this invention uses a cDNA library diluted 8-fold for RT-qPCR analysis, with three biological replicates and three operational replicates for each tissue. After the reaction, the PCR amplification cycle number (Ct value) of SmF3'5'H and SmACTIN genes in different tissues was obtained. ∆Ct was calculated using the Ct value of SmACTIN as a control, and then ∆∆Ct was calculated using the lowest ∆Ct value (in this invention, pericarp tissue) as a control. The relative expression level of the SmF3'5'H gene in different tissues and organs was calculated using the 2-∆∆Ct analysis method. The relative expression levels of the eggplant SmF3'5'H gene in roots, stems, leaves, petals, pericarp, pulp, sepals, and anthers are shown below. Figure 3 As shown. Finally, the mean-one-way ANOVA test was performed in SPSS 19.0 software: post-hoc multiple comparisons (Duncan, significance level P<0.05), and the significance analysis results are expressed in lowercase letters (ad) to obtain the differential expression of the SmF3'5'H gene in eggplant root, stem, leaf, petal, pericarp, pulp, sepal, and anther tissues. The results showed that the SmF3'5'H gene was expressed only in anthocyanin-rich stems, leaves, petals, pericarp, and sepals, and the expression level in pericarp was significantly higher than in other tissues, showing dominant expression.

[0084] 6. Based on the anthocyanin content and SmF3'5'H gene expression levels in various eggplant tissues, a linear relationship between SmF3'5'H gene expression levels and anthocyanin accumulation levels was fitted in EXCEL, yielding a determination coefficient R² = 0.72. Therefore, the Pearson correlation coefficient r = 0.85 was calculated between SmF3'5'H gene expression levels and anthocyanin accumulation in eggplant. Correlation analysis of anthocyanin content and relative SmF3'5'H gene expression levels in eggplant roots, stems, leaves, petals, pericarps, pulp, sepals, and anthers is as follows: Figure 4 As shown, the expression level of the eggplant SmF3'5'H gene is highly correlated with the amount of anthocyanin accumulation. The expression pattern of the SmF3'5'H gene in various tissues is highly consistent with the anthocyanin accumulation pattern, which means that the SmF3'5'H gene may be involved in the anthocyanin synthesis pathway in eggplant.

[0085] Example 4: Creating new eggplant varieties with different colors by genetically manipulating the function of the SmF3'5'H gene.

[0086] Using the full-length CDS sequence of the SmF3'5'H gene cloned in Example 1 as a template, editing targets were screened on the online gene editing target design website CRISPRdirect. The reference genome was Eggplant (Solanum melongena)genome, r2.5.1. After analysis, a highly specific target located on the first exon of the SmF3'5'H gene was selected, with the sequence shown in SEQ ID NO.9. This target sequence is 20 bp long, has a GC content of 60%, and a Tm value of 79.50℃. Following the method described in the previously published article "Cloning of the Eggplant U6 Promoter and Establishment of a CRISPR / Cas9-Mediated Gene Editing System," the target sequence was integrated into the SmU6-1P::sgRNA-Cas9 gene editing vector. The recombinant vector was transformed into E. coli DH5α, and after propagation, plasmids with correct sequencing results were selected for transformation into Agrobacterium GV3101 for transgenic operations. The background material used for transgenics was 'Lanshan Hexian' eggplant, and the transgenic operation procedure followed the method described in the previously published article "Establishment of an Agrobacterium-Mediated Transgenic System for Eggplant Leaves." Gene editing target + PAM sequence:

[0087] 5′-TAATAGCCACCGGCAGCTGGCGG-3′

[0088] After eggplant tissue culture, regenerated lines were obtained. Leaf DNA was extracted using the CTAB method, and a target PCR cloning primer pair was designed based on the SmF3'5'H genome sequence, approximately 200 bp upstream and downstream of the gene editing target site. The primer sequences are shown in SEQ ID NO.10 and SEQ ID NO.11, and the PCR product of this primer pair is 435 bp long. Using the regenerated plant DNA as a template, the target sequence of the regenerated plant was cloned using the primers shown in SEQ ID NO.10 and SEQ ID NO.11 with the 2×Easy Taq PCR SuperMix(+dye) Taq enzyme from Beijing TransGen Biotech Co., Ltd. The obtained PCR product was then sent to Beijing Qingke Biotechnology Co., Ltd. for forward Sanger sequencing using the sequencing primer SEQ ID NO.10. Finally, the sequencing results of the regenerated plants were compared with the wild-type (WT) sequencing results using SnapGene software. The selected CRISPR / Cas9 gene-editing positive lines were then planted in a plastic greenhouse for routine management.

[0089] The PCR primer pair sequences are as follows:

[0090] 5′-ATGGTTATACTTCCTAGTGAGT-3′ and

[0091] 5′-ATTGTACGCAGACCTTACC-3′

[0092] Sanger sequencing analysis results of the target sites of Smf3'5'h-cr#23 and Smf3'5'h-cr#50 CRISPR / Cas9 gene editing lines are as follows: Figure 5 As shown. Based on the sequencing results, the Smf3'5'h-cr#23 and Smf3'5'h-cr#50 lines were selected for further analysis. Smf3'5'h-cr#23 is heterozygous, and the sequencing results show a double peak; Smf3'5'h-cr#50 is homozygous, and the sequencing results show an insertion of a single "C" base at the target site. The plant phenotypes of the Smf3'5'h-cr#23 and Smf3'5'h-cr#50 gene-edited lines are shown below. Figure 6 As shown. Observation of the whole plant phenotype revealed no significant variation except for color; other aspects (such as plant shape and leaf shape) showed no obvious differences. This indicates that knocking out the SmF3'5'H gene may specifically affect anthocyanin synthesis in eggplant.

[0093] Visual observation revealed that the Smf3'5'h-cr#23 heterozygote exhibited a lighter purple color in its stems, leaves, and petals, and striped purple pericarp; the Smf3'5'h-cr#50 homozygous mutant showed green stems, leaves, and pericarp, and white petals. Anthocyanins were extracted from the stems, leaves, petals, and pericarp of wild-type, Smf3'5'h-cr#23, and Smf3'5'h-cr#50 plants using the method described in Example 3. Prism9 software was used to plot the anthocyanin content, and a tailless t-test assuming homogeneity of variance was performed using the anthocyanin content in the wild-type as a control (****P<0.001). The results showed that no anthocyanins were detected in the stems and leaves of the Smf3'5'h-cr#23 heterozygote, while the anthocyanin content in the petals and pericarp was significantly lower than that in the WT petals and pericarp. The anthocyanin phenotypes of stems, leaves, petals, and pericarps of the Smf3'5'h-cr#23 and Smf3'5'h-cr#50 gene-edited lines are as follows: Figure 7 As shown, anthocyanins were not detected in the stems, leaves, petals, and peels of the Smf3'5'h-cr#50 homozygous mutant. This indicates that the SmF3'5'H gene is essential for anthocyanin synthesis in eggplant plants and peels, and that it does not contain any functionally redundant genes.

[0094] cDNA libraries from the pericarp tissue of WT, Smf3'5'h-cr#23, and Smf3'5'h-cr#50 plants were obtained according to Example 1. RT-qPCR experiments were performed according to Example 3 to analyze the gene expression changes of the major anthocyanin synthesis structural genes SmCHS, SmF3H, SmF3'5'H, SmDFR, and SmANS in the pericarp tissue. Using the gene expression level in WT pericarp as a control, a tailless t-test with assumed homogeneity of variance was performed in Prism9 software (ns. indicates no difference, **P<0.01, ***P<0.005, ****P<0.001). The relative expression levels of the major anthocyanin synthesis structural genes in the pericarp of the Smf3'5'h-cr#23 and Smf3'5'h-cr#50 gene-edited lines are shown below. Figure 8 As shown in the figure, the results indicated that, except for the SmCHS gene, the expression levels of other genes SmF3H, SmF3'5'H, SmDFR, and SmANS in the pericarp of Smf3'5'h-cr#23 and Smf3'5'h-cr#50 plants were significantly lower than their expression levels in the WT pericarp. This suggests that the gene expression trends of most anthocyanin synthesis structural genes are consistent.

[0095] Example 5: Creating rare blue-colored roses, carnations, and chrysanthemums by introducing the eggplant SmF3'5'H gene through genetic transformation.

[0096] 1. Cloning and expression vector construction of eggplant SmF3'5'H gene

[0097] Using eggplant pericarp as material, the full-length CDS of the eggplant SmF3'5'H gene was cloned following the method described in Case Study 1. The gene amplification-specific primers were: F1: 5′-ATGGTTATACTTCCTAGTGAGTTG -3′, R1: 5′-GAAAGGCTCGCTAGACTGATAAAT -3′. The full-length SmF3'5'H CDS sequence (1542 bp) was obtained by high-fidelity PCR amplification. The purified gene fragment was cloned into the multiple cloning site of the plant expression vector pCAMBIA2300, positioned between an anthocyanin synthesis tissue-specific promoter (such as ANSpro) or a petal tissue-specific expression promoter and the Nos terminator. The vector carried kanamycin (NptII), hygromycin (HYG), or other resistance selection marker genes.

[0098] 2. Genetic transformation of flowers such as roses, carnations, and chrysanthemums.

[0099] (1) Prepare recipient materials: leaves of sterile tissue culture seedlings of roses (variety: 'Peace'), stem segments of carnations (variety: 'Scania'), leaves or stem segments of chrysanthemums (variety: 'Shenma').

[0100] (2) Agrobacterium-mediated transformation: The recombinant vector was transformed into Agrobacterium LBA4404 by heat shock, following the instructions. The recipient material was pre-cultured in pre-medium for 2 days. Transformed Agrobacterium single clones were picked, and PCR-positive single clones were detected using universal primers for the vector and SmF3'5'H gene-specific primers. Agrobacterium was cultured overnight in LB medium (50 mg / L kanamycin, 25 mg / L rifampin) (28℃, 220 rpm, 18-20 h), and the transformation was performed using OD200. 600 Infect with Agrobacterium suspension at 0.5–0.6 for 10–15 min.

[0101] (3) Cultured for 3 days (22℃, dark culture).

[0102] (4) Screening culture: Transfer to differentiation medium (MS + TDZ / 6-BA + NAA) containing 500 mg / L cephalosporin (antibacterial) and appropriate concentrations of kanamycin (rose: 100 mg / L; chrysanthemum: 50 mg / L).

[0103] (5) Subculture every 2 weeks, and resistant callus and regenerated shoots are obtained after 4–8 weeks.

[0104] 3. Molecular identification of transgenic plants

[0105] PCR verification: Genomic DNA was extracted from regenerated plants using the CTAB method and amplified using eggplant SmF3'5'H gene-specific primers. The PCR product of transgenic positive plants showed a clear target band at a size of 1500 bp.

[0106] qRT-PCR analysis: mRNA was extracted from petals during full bloom according to the method in Example 1; the expression level of gene SmF3'5'H was detected by qRT-PCR according to the method in Example 3. The quantitative primers were: SmF3'5'HF (SEQ ID NO.5): 5′-TGGACCTCGTTGGAAGTTGCTAAG -3′; SmF3'5'HR (SEQ ID NO.6): 5′-TGCCATCGCGAACGTCAACATAT -3′. The expression level in transformed plants was significantly higher than that in wild-type plants, indicating successful gene transcription.

[0107] 4. Flower color phenotype and biochemical analysis

[0108] (1) Detection of anthocyanin components: Petals from full bloom were ground with liquid nitrogen and the pigments were extracted with methanol containing 1% HCl. HPLC analysis (C18 reversed-phase column, acetonitrile-0.1% formic acid gradient elution) was performed. The results should be as follows: Eggplant SmF3'5'H gene-positive plants: a characteristic peak (λ) appeared at a retention time of 12.8 min. ax =550 nm), identified by mass spectrometry as delphinidin-3-O-glucoside. Wild type: only other types were detected, such as pelargonidin and cyanidin derivatives (retention time <10 min).

[0109] (2) Quantitative analysis of flower color: Blue flowers were compared with other flowers using a colorimeter. The results showed that the color parameters of blue flowers differed significantly in terms of brightness (L*), red-green saturation (a*), and yellow-blue saturation (b*). Specifically, blue flowers typically had higher blue saturation (b value close to -1), while the b values ​​of red and yellow flowers were significantly positive. The L and a values ​​of blue flowers also showed significant differences. These color differences effectively distinguished blue flowers from other flower colors, indicating the success of transgenic plants in improving flower color.

[0110] 5. Verification of genetic stability

[0111] In-depth phenotypic analysis was conducted on the obtained transgenic flower plants to observe the stability of their flower color and phenotypic characteristics. The genetic stability of the flower color characteristics was verified through multiple generations of propagation. Simultaneously, their growth performance under different environmental conditions was evaluated to ensure that the transgenic flowers can maintain good performance and flower color stability in actual cultivation.

[0112] Example 6: Creating rare, nutritionally enhanced economic crops such as apples, cherries, peaches, and strawberries by introducing the SmF3'5'H gene into eggplant through genetic transformation.

[0113] 1. Cloning and expression vector construction of eggplant SmF3'5'H gene

[0114] Using eggplant pericarp as material, the full-length CDS of the eggplant SmF3'5'H gene was cloned following the method described in Case Study 1. The gene amplification-specific primers were: F1: 5′-ATGGTTATACTTCCTAGTGAGTTG -3′, R1: 5′-GAAAGGCTCGCTAGACTGATAAAT -3′. The full-length SmF3'5'H CDS sequence (1542 bp) was obtained by high-fidelity PCR amplification. The purified gene fragment was cloned into the multiple cloning site of the marker-free plant expression vector pMF-FFT, which contained: ① a fruit-specific promoter (MdMYB10 promoter for apples, FaPG1 promoter for strawberries); ② a Cre / loxP marker knockout system (hygromycin phosphotransferase HPT was used as the selection gene); and ③ a 35S-driven Cre recombinase expression cassette.

[0115] 2. Genetic transformation of cash crops such as apples, strawberries, cherries, and peaches.

[0116] (1) Preparation of recipient materials: sterile seedling stem segments of apple (Malus domestica 'Gala'); 8 mm leaf discs were prepared from unfolded leaves of strawberry (Fragaria × ananassa 'Zhangji'); mature hypocotyls of cherry (Prunus avium 'Meizao') were extracted; and cotyledon callus tissue of peach (Prunus persica 'Chunxue') was selected for transformation. All materials underwent a 2-day pre-culture period before transformation.

[0117] (2) Agrobacterium-mediated transformation: The pMF-FFT-SmF3'5'H vector, which integrates the eggplant SmF3'5'H gene, was transformed into Agrobacterium EHA105 via heat shock. The pre-cultured explants were then immersed in Agrobacterium (OD) containing the recombinant plasmid. 600 The sample was treated with a shaking solution (MS + 100 μM acetylsalicylic acid) containing 0.4-0.5 μM acetylsalicylic acid for 15 min.

[0118] (3) After co-culturing for 3 days (22 ℃, in the dark), the culture was transferred to the Stage I screening medium (MS + 500 mg / L cephalosporin + 25 mg / L hygromycin + corresponding hormones) and subcultured every 14 days.

[0119] (4) After the formation of resistant callus, it was transferred to stage II differentiation medium (apple: MS + 1.0 mg / L TDZ + 0.1 mg / L IBA; strawberry: MS + 2.0 mg / L Zeatin + 0.05 mg / L NAA).

[0120] (5) When the differentiated shoots grow to 2 cm, cut them off and transfer them to stage III rooting medium (1 / 2 MS + 0.5 mg / L IBA + 20 mg / L hygromycin).

[0121] (6) After obtaining complete plants, the selection marker was removed by expressing Cre gene driven by the 35S promoter.

[0122] 3. Molecular identification of transgenic plants

[0123] PCR verification: Genomic DNA was extracted from regenerated plants of apple, strawberry, cherry and peach using the CTAB method and amplified using eggplant SmF3'5'H gene-specific primers. The PCR product of transgenic positive plants showed a clear target band at a size of 1500 bp.

[0124] qRT-PCR analysis: mRNA was extracted from the pericarp during the peak fruiting period according to the method in Example 1; the expression level of gene SmF3'5'H was detected by qRT-PCR according to the method in Example 3. The quantitative primers were: SmF3'5'HF: 5′-TGGACCTCGTTGGAAGTTGCTAAG -3′; SmF3'5'HR: 5′-TGCCATCGCGAACGTCAACATAT -3′. The expression level in the transformed plants was significantly higher than that in the wild type, indicating that the gene was successfully transcribed.

[0125] 4. Phenotypic validation of nutritional fortification

[0126] Collect mature fruits for biochemical analysis:

[0127] (1) Quantification of anthocyanin components: UPLC-ESI-MS / MS (Waters ACQUITY UPLC® HSS T3 column, 0.1% formic acid-acetonitrile gradient elution) was used. Delphinidin-3-O-galactoside (m / z 465→303, retention time 6.8 min) was detected in the peel of apple, peach and cherry transgenic eggplant SmF3'5'H gene, but not in wild type; delphinidin-3-O-glucoside was detected in the pulp of strawberry transgenic eggplant SmF3'5'H gene, but not in wild type.

[0128] (2) Antioxidant capacity: The ORAC method was used to detect the free radical scavenging capacity of transgenic apples, cherries, peaches and strawberries. Compared with wild type, transgenic fruits have significantly stronger antioxidant capacity.

[0129] (3) Fruit nutritional composition detection: Non-targeted metabolomics (LC-QTOF-MS) was used to detect various nutrients in transgenic and wild-type fruits to confirm that the total flavonoid content was significantly increased, while the core nutrients such as sugar and organic acids did not change significantly (p>0.05).

[0130] 5. Genetic stability

[0131] Asexual propagation was carried out using micrografting (apple) and stolon division (strawberry), and it was determined that the phenotypic stability reached 100% within three generations, with no significant changes in the anthocyanin content and antioxidant capacity of the fruit.

[0132] 6. Field trials and evaluation

[0133] Select suitable experimental fields for field trials, planting transgenic economic crops (such as apples, cherries, peaches, and strawberries) and comparing them with control groups (wild-type plants). Long-term monitoring of the growth of transgenic plants in the natural environment will be conducted, including evaluations of growth rate, fruit yield, disease resistance, and adaptability. This aims to obtain reliable data on the application of this transgenic technology in actual agricultural production.

[0134] Example 7: Using transgenic technology, the eggplant SmF3'5'H gene was overexpressed in eggplant basal crops to construct a super crop that synthesizes delphinidin-like anthocyanins for the production of natural delphinidin.

[0135] 1. Gene cloning

[0136] First, using eggplant pericarp as material, the full-length CDS of the eggplant SmF3'5'H gene was cloned following the method described in Case Study 1. The gene amplification-specific primers were: F1: 5′-ATGGTTATACTTCCTAGTGAGTTG -3′, R1: 5′-GAAAGGCTCGCTAGACTGATAAAT -3′. The full-length CDS sequence of SmF3'5'H (1542 bp) was obtained by high-fidelity PCR amplification.

[0137] 2. Construction of plant expression vectors

[0138] The cloned SmF3'5'H gene was inserted into a plant expression vector (such as pHB, pBI121, or pCAMBIA2300), and a strong promoter sequence (such as the 2×CaMV 35S promoter or the eggplant ubiquitin expression strong promoter) was added upstream to ensure high-level gene expression in transgenic plants. In addition, selection marker genes (such as the antibiotic kanamycin selection gene and the hygromycin gene) were added to facilitate the screening of transgenic plants. The completed transgenic plant expression vector was constructed using restriction enzyme digestion and ligation reactions, and the correctness of the insertion was confirmed by restriction enzyme analysis and sequencing.

[0139] 3. Transformation of recipient plants

[0140] A suitable eggplant variety (facilitating transgenic development, such as 'February Eggplant' or 'LSHX' in this invention) was selected as the substrate crop. Agrobacterium-mediated transformation was used to transfer the constructed plant expression vector into eggplant cells. Specific methods are detailed in "Establishment of Agrobacterium-Mediated Transgenic Eggplant Leaf System". In short, firstly, Agrobacterium and the transformation vector were co-cultured under suitable conditions, allowing Agrobacterium to transfer the SmF3'5'H gene into plant cells. Subsequently, the co-cultured cells were transferred to a selective medium, and successfully transformed cells were screened. Transgenic integration was confirmed by PCR and Southern blot analysis.

[0141] 4. Regeneration and screening of transgenic plants

[0142] The successfully transformed cells were regenerated and cultured, and callus formation was induced using growth regulators (such as zeatin and 6-BA). After a series of cultures and selections, transgenic eggplant plants containing the SmF3'5'H gene were obtained. PCR verification was performed on the regenerated plants to confirm whether the target gene was successfully integrated into their genome, and its expression level was detected. The specific method is described in Example 3, and the overexpression capacity of the transgenic plants for the SmF3'5'H gene was evaluated.

[0143] 5. Analysis of the delphinidin synthesis pathway

[0144] Metabolic analysis was performed on validated transgenic eggplant, focusing on the activities of key enzymes and related metabolites in the delphinidin synthesis pathway. Anthocyanin content, particularly delphinidin accumulation, in transgenic plants was analyzed using HPLC and mass spectrometry. Metabolic differences between transgenic and non-transgenic plants were investigated to determine the role of the SmF3'5'H gene in anthocyanin synthesis and to optimize the synthesis pathway to increase delphinidin yield.

[0145] 6. Extraction and purification of delphinidin

[0146] After the transgenic plants reached a suitable growth point, eggplant fruits, leaves, stems, and other organs rich in anthocyanins were collected for delphinidin extraction. Extraction was performed using a suitable solvent system (such as methanol or ethanol) to remove cell wall residues. The extract was then obtained by centrifugation and filtration. Next, column chromatography (such as silica gel column chromatography or reversed-phase high-performance liquid chromatography) was used to separate and purify the extract, obtaining high-purity delphinidin. Quantitative analysis was performed using HPLC to ensure extraction efficiency and purity.

[0147] 7. Functional verification and application development

[0148] A series of bioactivity assessments were conducted on the extracted delphinidin, particularly verifying its antioxidant, anti-inflammatory, and anti-tumor functions. The biological effects of delphinidin were tested using in vitro experiments, cell experiments, and animal models. Simultaneously, market research was carried out to explore the application potential of delphinidin in health supplements, food additives, and cosmetics, promoting its industrialization. Collaboration with relevant enterprises was undertaken to develop products containing delphinidin for commercial application.

[0149] Example 8: The eggplant SmF3'5'H gene was introduced into an engineered strain, and delphinidin, which has a strong antioxidant function, was produced in large quantities using fermentation engineering, thus promoting the industrial production of delphinidin.

[0150] 1. Gene cloning and expression vector construction

[0151] First, using eggplant pericarp as material, the full-length CDS of the eggplant SmF3'5'H gene was cloned following the method described in Case Study 1. The gene amplification-specific primers were: F1: 5′-ATGGTTATACTTCCTAGTGAGTTG -3′, R1: 5′-GAAAGGCTCGCTAGACTGATAAAT -3′. The full-length SmF3'5'H CDS sequence (1542 bp) was obtained by high-fidelity PCR amplification. Next, the purified gene fragment was cloned into a suitable expression vector (e.g., pET or pGEX series), and the insertion was confirmed by restriction enzyme digestion and ligation reactions. Simultaneously, the anthocyanin synthesis pathway was reconstructed, and the constructed expression vector was transformed into *E. coli* for subsequent expression and screening.

[0152] 2. Construction and screening of engineered strains

[0153] Select suitable fermentation strains (e.g., modified E. coli or Saccharomyces cerevisiae) and transform the constructed expression vector into the selected strains. Successfully transformed strains are screened using antibiotics in the culture medium, and preliminary screening is performed to detect the expression of the eggplant SmF3'5'H gene. The expression level of the target gene in the engineered strains is confirmed using qRT-PCR, gene sequencing, or Western blot techniques.

[0154] 3. Optimize fermentation conditions

[0155] Shake-flask fermentation experiments were conducted on engineered strains to optimize their growth and metabolic activity by varying culture medium composition, temperature, pH, and shaking speed. The study focused on the expression stability of the eggplant SmF3'5'H gene during fermentation and its impact on delphinidin synthesis. High-performance liquid chromatography (HPLC) was used to analyze the delphinidin content in the fermentation broth to determine the optimal fermentation conditions.

[0156] 4. Extraction and purification of delphinidin

[0157] Large-scale fermentation was conducted under optimized conditions, and the fermentation broth was collected. Delphinidin in the fermentation broth was extracted using an organic solvent (such as ethanol or ethyl acetate), and cell debris was removed by centrifugation and filtration. Subsequently, the extract was purified using column chromatography (such as silica gel column or reversed-phase column) to obtain high-purity delphinidin. HPLC analysis was performed at each extraction and purification step to ensure the purity and yield of delphinidin.

[0158] 5. Functional verification of delphinidin

[0159] The antioxidant activity of the extracted delphinidin was tested to evaluate its function in scavenging free radicals and delaying oxidation. Various in vitro experiments, including the DPPH free radical scavenging assay and the ABTS assay, were used to compare the antioxidant capacity of delphinidin with control samples (such as common antioxidants like vitamin C or E). Simultaneously, cell model experiments were conducted to verify the antioxidant effects and potential biological activities of delphinidin at the cellular level.

[0160] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A gene for an eggplant flavonoid 3',5'-hydroxylase, characterized in that, The gene sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.

2.

2. A method for cloning the gene of eggplant flavonoid 3',5'-hydroxylase as described in claim 1, characterized in that, The method includes the following steps: Step 1: Collect the peels of the purplish-black eggplant variety 'Lanshan Hexian'; Step 2: Extract RNA from the fruit peel sample obtained in Step 1; Step 3: Reverse transcribe the RNA obtained in Step 2 into cDNA, use it as a template, and clone it using primers F1 and R1; the sequence of primer F1 is: 5′-ATGGTTATACTTCCTAGTGAGTTG-3′, and the sequence of primer R1 is: 5′-GAAAGGCTCGCTAGACTGATAAAT-3′.

3. The gene cloning method as described in claim 2, characterized in that, Step 1 also includes, under natural growing conditions, waiting for the eggplant plants to grow to market maturity, collecting the peels, immediately pre-cooling them with liquid nitrogen, and then storing them in a -80°C freezer.

4. A method for detecting the gene expression of 3',5'-hydroxylase in eggplant flavonoids, characterized in that, The method includes the following steps: Step 1) Collect various tissues of the purplish-black peel eggplant 'Lanshan Hexian' as samples, including roots, stems, leaves, petals, peel, pulp, sepals, and anthers; Step 2) Using the gene sequence shown in SEQ ID NO.1 as a template, and primer pairs SmF3'5'HF and SmF3'5'HR, perform RT-qPCR analysis on the sample obtained in Step 1); the sequence of primer SmF3'5'HF is: 5′-TGGACCTCGTTGGAAGTTGCTAAG-3′, and the sequence of primer SmF3'5'HR is: 5′-TGCCATCGCGAACGTCAACATAT-3′.

5. The detection method as described in claim 4, characterized in that, Step 2) further includes using the actin gene SmACTIN as an internal reference gene; the actin gene SmACTIN has the NCBI nucleotide accession number GU984779.1; its RT-qPCR primers are: SmACTIN-F: 5′-GTCGGAATGGGACAGAAGGATG-3′ SmACTIN-R: 5′-GTGCCTCAGTCAGGAGAACAGG-3′.

6. The application of the eggplant flavonoid 3',5'-hydroxylase gene as described in claim 1 in the creation of new eggplant varieties with different colors.

7. The application of the eggplant flavonoid 3',5'-hydroxylase gene as described in claim 1 in the improvement of blue flower color in ornamental flowering plants.

8. The application of the eggplant flavonoid 3',5'-hydroxylase gene as described in claim 1 in cultivating fruits rich in antioxidant delphinidin anthocyanins.

9. The application of the eggplant flavonoid 3',5'-hydroxylase gene as described in claim 1 in the production of natural delphinidin, characterized in that, The specific application method is as follows: using transgenic technology to overexpress the gene of eggplant flavonoid 3',5'-hydroxylase in eggplant or tomato substrate crops, constructing a super crop that synthesizes delphinidin anthocyanins, and using it to produce the natural delphinidin.

10. The application of the eggplant flavonoid 3',5'-hydroxylase gene as described in claim 1 in the production of delphinidin via fermentation engineering, characterized in that... The specific application method is as follows: the gene of eggplant flavonoid 3',5'-hydroxylase is introduced into an engineered strain, and the delphinidin with strong antioxidant function is produced in large quantities by fermentation engineering.

Images