Method for screening key hormones and genes for lycium ruthenicum anthocyanin synthesis

Abstract

The invention provides a method for screening key hormones and genes for lycium ruthenicum anthocyanin synthesis. The key hormones comprise 1-aminocyclopropane carboxylic acid, salicylic acid, abscisic acid and dihydrozeatin. The key genes comprise an MYB80 gene, an MYB102 gene, a K21383 enzyme family gene, an ARR2 gene and a BHLH14 gene; the screening method comprises the following steps: collecting lycium ruthenicum and lycium barbarum samples, washing with ultrapure water, removing surface moisture, freezing and fixing with liquid nitrogen, performing freeze drying, grinding, sieving, and respectively performing transcriptome analysis and hormone group analysis. The invention further provides application of the key hormones and the genes screened by the method, and the key hormones and the genes can be used as molecular markers or genetic manipulation targets to research and regulate signal pathways for synthesizing anthocyanin of lycium ruthenicum. Key hormones and key genes participating in synthesis of lycium ruthenicum anthocyanin are analyzed and screened by using a hormone group and a transcriptome, and the synthesis of the lycium ruthenicum anthocyanin jointly regulated by dynamic transcriptional regulation and hormone interaction is disclosed.

Description

Technical Field

[0001] This invention belongs to the field of anthocyanin synthesis technology, specifically relating to a method for screening key hormones and genes involved in the synthesis of anthocyanins in black goji berries. Background Technology

[0002] The polymorphism of fruit color in wolfberry (Lycium ruthenicum) is an important adaptive trait that has evolved in extreme habitats (such as the saline-alkali land of the Qinghai-Tibet Plateau). Black-fruited varieties are rich in anthocyanins, whose accumulation not only gives the fruit a deep purple color but also enhances stress resistance through antioxidant activity; while white-fruited varieties are milky white due to defects in anthocyanin synthesis, suggesting a unique regulatory mechanism. Anthocyanin biosynthesis is controlled by a three-tiered regulatory network: 1) transcriptional activation of structural genes (such as CHS, DFR, ANS, and UFGT); 2) the core driver of the MYB-bHLH-WD40 (MBW) transcriptional complex; and 3) the integrated response of upstream hormonal signals and environmental stress. Among these, plant hormones (such as jasmonic acid JA, abscisic acid ABA, and auxin) modify the activity of the MBW complex, becoming a key hub connecting developmental programs and stress responses.

[0003] Recent studies have shown that JA signaling dynamically regulates anthocyanin synthesis through the JAZ-MYC2 module: in Arabidopsis, JAZ proteins inhibit MYC2 transcription factors, while stress-induced JA accumulation promotes SCF / COI1 ubiquitination and degradation of JAZ, releasing MYC2 to activate genes such as DFR and ANS. ABA, through the PYL-PP2C-SnRK2 signaling cascade, phosphorylates MYB transcription factors (such as MdMYB1 / MdMYB75), directly binding to anthocyanin gene promoters. Auxin exhibits a dual role: low concentrations of ARF (auxin response factor) activate UFGT expression, while high concentrations inhibit ARF activity through IAA / AUX proteins. Notably, the spatiotemporal regulation of anthocyanin synthesis by hormone interaction networks (such as SA-JA antagonism and ET-ABA synergy) and cytokinin activity forms (zeatin vs. inactivated glycosides) remains a gap in non-model fruits.

[0004] The explosive accumulation of anthocyanins during fruit ripening is closely related to the temporal expression of core transcription factors. In grapes, VvMYBA1 / VvMYBA2 are specifically activated during veraison, while in apples, MdMYB10 expression increases a hundredfold under light induction. bHLH factors (such as TT8 and GL3) act as scaffolds for the MBW complex, and their stability is regulated by the COP1-SPA ubiquitination system, mediating the coupling of light signals with anthocyanin synthesis. However, most studies focus on single developmental nodes, and in-depth research is needed on the dynamic transcriptional regulation during continuous development and its interaction with hormone signaling.

[0005] Previous genomic studies have revealed that black goji berries are rich in anthocyanin pathway genes (such as LrANS and LrUFGT), but no inactivation mutations were found in homologous genes in ginkgo varieties, suggesting that differences in transcriptional regulation or hormonal microenvironment imbalance may be the main cause of color differentiation. MAPK cascade pathways (such as MKK4-MPK3) have been shown to integrate cold stress with anthocyanin synthesis in tomatoes, but their role in the stress response-color association in goji berries remains unexplored. Furthermore, the contradictory phenomenon of high ABA but low anthocyanin content in ginkgo challenges the understanding of ABA as a "universal inducer," suggesting the existence of species-specific signal decoding mechanisms. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for screening key hormones and genes involved in the synthesis of anthocyanins in black goji berries, which addresses the shortcomings of the prior art. This method uses a combination of hormonomics and transcriptomics analysis to screen key hormones and genes involved in the synthesis of anthocyanins in black goji berries, revealing that dynamic transcriptional regulation and hormone interaction jointly regulate the synthesis of anthocyanins in black goji berries.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for screening key hormones and genes for anthocyanin synthesis in black goji berries, wherein the key hormones for anthocyanin synthesis in black goji berries include 1-aminocyclopropanecarboxylic acid, salicylic acid, abscisic acid, and dihydrozeatin. The key genes for screening anthocyanin synthesis in black wolfberry include the MYB80 gene, MYB102 gene, K21383 enzyme family gene, ARR2 gene, and BHLH14 gene. The filtering method is as follows: S1. Selection and pretreatment of plant samples: Black wolfberry and white wolfberry samples were collected respectively. The samples were rinsed with ultrapure water and the surface moisture was removed with sterile filter paper to obtain the rinsed black wolfberry samples and the rinsed white wolfberry samples respectively. S2. The washed black goji berry samples and washed white goji berry samples obtained in S1 were frozen and fixed in liquid nitrogen, then freeze-dried, ground, sieved, and stored in the dark at -20℃ to obtain ground black goji berry samples and ground white goji berry samples respectively. S3. Transcriptome and hormoneome analyses were performed on the ground black goji berry samples and ground white goji berry samples obtained in S2, respectively.

[0008] Preferably, the freeze-fixation time in S2 is 20 min; the freeze-drying conditions are: drying for 48 h under a vacuum of 10 Pa and a temperature of -50 °C; and the sieve mesh size is 100 mesh.

[0009] Preferably, the hormone analysis in S3 is performed using high performance liquid chromatography-mass spectrometry.

[0010] This invention also provides the application of the key hormones and genes for anthocyanin synthesis in black goji berries selected above. These key hormones and genes can be used as molecular markers or genetic manipulation targets to study the signaling pathways regulating anthocyanin synthesis in black goji berries.

[0011] Preferably, when the MYB80 gene, MYB102 gene and MYB58 are upregulated in black goji berries, and the K21383 enzyme family gene is upregulated in the anthocyanin biosynthesis pathway map00942, it is used to catalyze anthocyanin modification. When the ARR2 gene is expressed in black goji berries and not expressed in white goji berries, and the BHLH14 gene is upregulated in BS4, the cytokinin signaling pathway is activated in black goji berries. Compared with the prior art, the present invention has the following advantages: This invention utilizes a combined hormonal and transcriptomic analysis to examine the plant hormone content in *Lycium barbarum* (WS4) and *Lycium chinense* (BS4) fruits. It also screens key metabolic pathways related to anthocyanin biosynthesis and plant hormone signal transduction, identifying differentially expressed genes and elucidating the regulatory network by which endogenous hormones precisely regulate anthocyanin biosynthesis. This pioneering combined hormonal and transcriptomic analysis strategy fills a gap in existing research on *Lycium barbarum*, which relies on single-dimensional analysis. High-performance liquid chromatography-mass spectrometry (HPLC-MS) precisely quantifies the hormonal differences between *Lycium barbarum* and *Lycium barbarum* (e.g., dihydrozein content in *Lycium barbarum* is 11.9 times higher than in *Lycium barbarum*, and salicylic acid content in *Lycium barbarum* is 65.2% higher in *Lycium barbarum*). Simultaneously, transcriptomic sequencing identifies key genes (e.g., MYB102 is specifically expressed in *Lycium barbarum*, and MYB80 expression is 6.01 times higher in *Lycium barbarum*). This clearly reveals the molecular mechanism by which precise regulation by endogenous hormones and dynamic gene transcription jointly drive anthocyanin synthesis, and also answers the species-specific regulatory contradiction of high abscisic acid but low anthocyanin content in *Lycium barbarum*. Furthermore, the sample processing procedure of this invention is rigorous and controllable, and the data is reliable. The four key hormones (1-aminocyclopropanecarboxylic acid, salicylic acid, abscisic acid, and dihydrozeatin) and five key genes (MYB80, MYB102, etc.) can be directly used as molecular markers or genetic manipulation targets, which not only serve the screening of superior varieties of black goji berries, but also provide precise molecular markers for the targeted regulation of anthocyanin synthesis and the promotion of the industrialization of functional products.

[0012] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0013] Figure 1 This is a volcano analysis diagram of differentially expressed genes WS4_vs_BS4 in Example 1 of the present invention.

[0014] Figure 2This is a KEGG enrichment analysis diagram of the differentially expressed genes WS4_vs_BS4 in Example 1 of the present invention. Detailed Implementation

[0015] Example 1 This embodiment uses combined hormonal and transcriptomic analysis to screen key hormones and genes involved in the synthesis of anthocyanins in black goji berries, revealing that dynamic transcriptional regulation and hormone interactions jointly regulate the synthesis of anthocyanins in black goji berries.

[0016] (I) Identification and content determination of key anthocyanin compounds in black wolfberry: (1) Sample selection: Goji berry fruits with consistent growing environment (same plot, same water and fertilizer conditions, same growth cycle) and uniform maturity (fully colored fruit, black or white) were selected as experimental samples and divided into two groups: black fruit group (denoted as BS4): 3 biological replicates, sample numbers BS4_1, BS4_2, and BS4_3; white fruit group (denoted as WS4): 3 biological replicates, sample numbers WS4_1, WS4_2, and WS4_3. The sample size for each replicate in each group was no less than 50g to ensure that the sample size met the requirements for pretreatment and repeated testing.

[0017] (2) Sample preprocessing: Freshly collected fruit samples were rinsed three times with ultrapure water (MilliQ grade, Millipore) to remove surface mud and impurities, and the surface moisture was blotted dry with sterile filter paper. The dried fruit was then quickly placed in liquid nitrogen for freeze fixation (20 min) to prevent anthocyanin degradation. The frozen samples were then transferred to a freeze dryer (vacuum of 10 Pa, temperature of -50℃) and freeze-dried for 48 h until the samples were completely dry (no moisture residue, crisp texture). The freeze-dried samples were ground into a fine powder using an agate mortar and pestle, passed through a 100-mesh nylon sieve, and the sieved powder was collected, sealed in centrifuge tubes, and stored in the dark at -20℃ for later use.

[0018] (3) Reagents and instruments: All reagents meet the requirements for chromatography-mass spectrometry detection grade. Specific reagents and their uses are shown in Table 1, and instruments and their uses are shown in Table 2.

[0019] Table 1. Reagents and Uses Table 2. Instruments and Applications (4) Sample pretreatment (anthocyanin extraction and purification): Preparation of extract: Prepare a 50% methanol aqueous solution (containing 0.1% hydrochloric acid). Take 500 mL of methanol (chromatographic grade) and mix with 500 mL of ultrapure water. Add 1 mL of hydrochloric acid (analytical grade), vortex to mix, and store at 4°C in the dark for later use. Sample weighing and extraction: Under liquid nitrogen protection, accurately weigh 50 mg (accuracy 0.1 mg) of lyophilized sample powder and place it in a 2 mL sterile centrifuge tube. Add 500 μL of the above extraction solution and tighten the tube cap. Mixing and centrifugation: Place the centrifuge tube on a vortex mixer and vortex at room temperature for 10 min (2000 r / min) to ensure complete dispersion of the sample powder; then place the centrifuge tube in a high-speed refrigerated centrifuge and centrifuge at 12000 r / min for 3 min at 4°C. Use a pipette to transfer the supernatant to a new 2 mL centrifuge tube. Secondary extraction: Add 500 μL of extraction solution to the remaining sample residue, vortex for 10 min, centrifuge at 12000 r / min for 3 min at 4℃, collect the supernatant and combine it with the first supernatant; Filtration and purification: The combined supernatant was filtered through a 0.22 μm organic phase filter membrane, and the filtrate was collected into a 2 mL sample vial and stored at 4 °C in the dark for detection by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) (the sample must be detected within 24 h to avoid anthocyanin degradation).

[0020] (5) UPLC-MS / MS detection conditions: Ultra-high performance liquid chromatography (UPLC) conditions: Column: Waters ACQUITY BEHC18 reversed-phase column (particle size 1.7 μm, column length × column inner diameter = 100 mm × 2.1 mm); Mobile phase: Phase A: ultrapure water (containing 0.5% formic acid, v / v), i.e., 0.5 mL formic acid (chromatographic grade) is added to every 100 mL of ultrapure water, and ultrasonically degassed for 15 min; Phase B: methanol (containing 0.5% formic acid, v / v), i.e., 0.5 mL formic acid (chromatographic grade) is added to every 100 mL of methanol, and ultrasonically degassed for 15 min; Gradient elution program is shown in Table 3, column temperature is 40℃ (column oven temperature fluctuation ±0.5℃), injection volume is 2 μL (injector cleaning program: clean with methanol 3 times and ultrapure water 3 times before injection); Detection mode: UV-Vis assisted detection, detection wavelength 520 nm (characteristic absorption wavelength of anthocyanins).

[0021] Table 3 Gradient elution program for UPLC-MS / MS Note: Maintain phase B at 95% from 12.00 min to 14.00 min, and equilibrate the column from 14.00 min to 16.00 min to ensure stable sample detection baseline.

[0022] Tandem mass spectrometry (MS / MS) conditions: Ion source was electrospray ionization (ESI), positive ion mode (ESI+); ion source parameters were: ion source temperature: 550℃, ion spray voltage (IS): 5500V, curtain gas (CUR): 35psi, nebulizer gas (GS1): 50psi, auxiliary gas (GS2): 50psi; detection mode was multiple reaction monitoring (MRM), with the declustering voltage (DP) and collision energy (CE) optimized for each anthocyanin standard to ensure maximum response intensity of the target ion; data acquisition: Analyst 1.6.3 software (Sciex) was used to control the instrument and acquire data, recording the parent ion (Q1, Da), characteristic daughter ion (Q3, Da), and corresponding retention time (RT, min) for each anthocyanin.

[0023] (6) Qualitative and quantitative analysis methods for anthocyanins: Qualitative analysis is achieved by matching the standard database with sample detection data. The specific steps are as follows: ① Standard database construction: All target anthocyanin standards (such as cyanidin-3-O-sophoroside, delphinidin-3-O-glucoside, petunidin-3-O-(p-coumaryl)rhamnoside-5-O-glucoside, etc.) are detected, and the retention time (RT, min), precursor ion mass number (Q1, Da), and characteristic daughter ion mass number (Q3, Da) of each standard are recorded. ① **Anthocyanin Standard Information Database:** This involves several steps: 1) **Anthocyanin Matching in Samples:** The chromatographic peaks detected in the sample are compared with the standard database. If the following three conditions are met, the sample is identified as containing that anthocyanin: ① The retention time of the sample peak deviates from that of the standard by ≤ ±0.05 min; ② The mass numbers of the precursor ion (Q1) and daughter ion (Q3) detected in the sample under MRM mode are completely consistent with the standard (mass deviation ≤ ±0.1 Da); ③ The peak shape of the sample peak is consistent with that of the standard peak (symmetry factor deviation ≤ ±0.1). 2) **Qualitative Result Confirmation:** The main anthocyanin species in black goji berries can be identified using the above methods. Anthocyanin quantification is achieved using the external standard method combined with a standard curve. Each sample undergoes three technical replicate tests, and the average of the three test results is taken as the final content of the corresponding anthocyanin in the sample, ensuring the repeatability of the quantitative results (relative standard deviation RSD ≤ 10%).

[0024] (7) Types and contents of anthocyanins in black goji berries: HPLC-MS / MS analysis identified six key anthocyanin compounds in black goji berries, including: ① Cyanin derivatives: cyanin-3-O-rutin glycoside, cyanin-3-O-rutin glycoside, and cyanin-rutin-rhamnoside are the main pigment sources for the purplish-red color of BS4 goji berries; ② Malvaceae pigments: malvaceae-3-O-glucoside, malvaceae-3-O-galactoside, and malvaceae-3-O-(feruloyl)xyloside-5-O-(p-coumaroyl)glucoside, with the first two being important contributors to the antioxidant activity of black goji berries; ③ Delphinidin derivatives: delphinidin-3-O-glucoside, malvaceae-3-O-galactoside, and malvaceae-3-O-(feruloyl)xyloside-5-O-(p-coumaroyl)glucoside. -O-rutin-5-O-glucoside, delphinidin-3-O-glucoside, and delphinidin-3-O-galactoside are the most abundant anthocyanin categories in black goji berries; ④ petunia compounds: petuniadin-3-O-feruloyl-xyloside-rutin, petuniadin-3-(6-Op-p-coumaryl)glucoside, etc., whose acylation modification structure enhances the stability of the compounds; ⑤ peonidin compounds: peonidin-3-O-(caffeoyl)rhamnoside and other derivatives are distributed in trace amounts in black goji berries; ⑥ pelargonidin compounds: pelargonidin-3-O-galactoside is a characteristic minor anthocyanin component of black goji berries.

[0025] Based on three parallel quantitative analyses of black goji berry samples using HPLC-MS / MS, the average content (unit: mg / g dry weight) of each anthocyanin compound was calculated and sorted in descending order to identify the top 5 high-content anthocyanin compounds. The high-content anthocyanins in black goji berries include: petunidin-3-O-(p-coumaryl)rhamnoside-5-O-glucoside (average content 218.0931 mg / g, more than 2240 times that of ginkgo goji berries), petunidin-3-O-rutinoside-5-O-rhamnoside (average content 208.5191 mg / g, 2070 times that of ginkgo goji berries), and petunidin-3-O-feruloyl-xyloside-rutinoside (average content 44.498 mg / g). The five most abundant anthocyanins in black goji berries are petunidin (0.0234 mg / g in ginkgo biloba), petunidin-3-O-caffeoyl-rhamnoside-rutin (average content 4.1920 mg / g, only 0.0129 mg / g in ginkgo biloba), and delphinidin-3-O-glucoside (average content 0.9087 mg / g, 13.2% higher than in ginkgo biloba). Petunidins are the top four most abundant anthocyanins in black goji berries, and all contain acylation and polysaccharide chain modifications. These structural features are key to their high stability and activity. Although delphinidin-3-O-glucoside, the fifth most abundant anthocyanin, is less abundant than petunidins, it is a "representative component" of delphinidins and has indicative significance for variety selection. These five anthocyanins together constitute the core antioxidant system of black goji berry BS4, providing clear target components for its functional product development and quality control.

[0026] (II) Determination of the types and contents of key anthocyanin synthesis hormones in black wolfberry: In this experiment, the selection and processing of black and white wolfberry samples were the same as in steps (1) and (2). The hormone extraction and purification methods were as follows: ① Extraction: Weigh 100 mg of freeze-dried fruit sample powder into a 2 mL centrifuge tube, add 1 mL of extraction solution (methanol:water:formic acid = 80:19:1, v / v / v), vortex for 30 s, and stand at 4℃ in the dark for 12 h; ② Centrifugation: Centrifuge at 12000 rpm for 15 min at 4℃, and collect the supernatant into a new centrifuge tube; ③ Re-extraction: Add 0.8 mL of the above extraction solution to the residue, repeat the vortexing, standing and centrifugation steps, and combine the two supernatants; ④ Purification: Filter the supernatant through a 0.22 μm organic phase filter membrane, transfer the filtrate to a sample bottle, store at 4℃ in the dark, and wait for HPLC-MS / MS detection.

[0027] The HPLC-MS / MS detection conditions were as follows: ① Chromatographic conditions: Column: C18 reversed-phase column (2.1 mm × 150 mm, 1.8 μm); Mobile phase: Phase A (0.1% formic acid aqueous solution), Phase B (acetonitrile); Gradient elution program: 0 min–2 min, 5% B; 2 min–10 min, 5%–95% B; 10 min–12 min, 95% B; 12 min–12.1 min, 95%–5% B; 12.1 min–15 min, 5% B; Column temperature: 30℃; Injection volume: 5 μL; Flow rate: 0.3 mL / min. ② Mass spectrometry conditions: Ion source: Electrospray ionization (ESI), negative ion mode; Detection mode: Multiple reaction monitoring (MRM); Spray voltage: -4500 V; Ion source temperature: 500℃; Curtain gas pressure: 35 psi; Nebulizer gas pressure: 50 psi; Auxiliary gas pressure: 50 psi.

[0028] Standard curve plotting: Prepare concentration gradients of various hormone standards (0.25, 0.5, 1, 5, 10, 50, 100, 500, 1000 ng / ml), and detect them under the above HPLC-MS / MS conditions. Plot a standard curve with standard concentration as the x-axis and peak area as the y-axis and obtain the linear equation. Content calculation: Based on the peak area of ​​the hormone in the sample, substitute it into the corresponding linear equation to calculate the absolute content (ng) of the hormone in the sample, and then convert it to the content per unit mass of fruit (ng / g, based on the weight of the freeze-dried sample). Data validation: Add QC samples (a mixture of various hormone standards) to each batch of samples for testing, calculate the RSD of each hormone in the QC sample, and judge if RSD < 0.3. The quantitative and qualitative results are reliable; the regression coefficient R of the linear equation is verified to be ≥0.998, ensuring a good linear relationship; data statistics: the average content of each hormone in each sample group (BS4, WS4) is calculated using Excel; independent samples t-test is performed using SPSS software to calculate the p-value; difference judgment: FoldChange = WS4 average content / BS4 average content, FoldChange > 1 indicates hormone upregulation in WS4, < 1 indicates downregulation; p < 0.05 is considered significant, p < 0.01 is considered highly significant; high-content hormone identification: hormones with an average content ≥10ng / g are screened, and the type of high-content hormone is determined by combining the content fold difference (compared with other hormones).

[0029] The hormone content determination results of black and white wolfberries are shown in Table 4. Among them, the hormones with high content in black wolfberry are: 1-aminocyclopropanecarboxylic acid (ACC) (the ACC content in black wolfberry was 22.6% higher than that in white wolfberry, a highly significant difference, with a QCRSD of 0.016497, indicating reliable quantitative results). ACC is a direct precursor of ethylene, and its content directly reflects the ethylene synthesis capacity. The high ACC content in black wolfberry suggests that ethylene may promote anthocyanin accumulation in black wolfberry; and salicylic acid (SA) (the SA content in black wolfberry was 65.2% higher than that in white wolfberry, a highly significant difference, with a QCRSD of 0.048825, indicating reliable quantitative results). SA usually participates in plant stress response and secondary metabolism regulation. The association between high SA content and low anthocyanin synthesis in WS4 is also relevant. Further investigation is needed; abscisic acid (ABA) (the ABA content in ginkgo biloba was 64.8% higher than that in black goji berries, a highly significant difference, with a QCRSD of 0.053378, indicating reliable quantitative results), ABA has been shown to induce anthocyanin synthesis in various plants, but the high ABA content in ginkgo biloba is accompanied by low anthocyanin synthesis, suggesting that there may be a unique mechanism for the regulation of anthocyanins by ABA in black goji berries; dihydrozein (DHZ) (the DHZ content in black goji berries was 11.9 times higher than that in ginkgo biloba, a highly significant difference, with a QCRSD of 0.014007, indicating reliable quantitative results), DHZ, as an active cytokinin, may participate in anthocyanin synthesis by regulating cell metabolism and gene expression, and the high DHZ content in black goji berries is significantly associated with high anthocyanin accumulation.

[0030] Table 4 Hormone content in black goji berries and white goji berries Note: p < 0.05 indicates a significant difference between the two groups, and p < 0.01 indicates a highly significant difference; Fold Change > 1 indicates that the hormone content in ginkgo goji berries is upregulated compared to that in black goji berries, and < 1 indicates downregulation.

[0031] (III) Transcriptome sequencing of black goji berries and white goji berries: Total RNA was extracted from the fruits of black goji berry (BS4) and white goji berry (WS4) and sent to Wuhan Maiwei Metabolic Biotechnology Co., Ltd. for transcriptome sequencing. Volcano plot analysis of differentially expressed genes was performed using the DESeq2 method, and KEGG analysis of differentially expressed genes was performed using the clusterProfiler method.

[0032] The results of the volcano plot analysis of differentially expressed genes WS4_vs_BS4 are as follows: Figure 1As shown, red spheres represent genes upregulated in BS4, blue spheres represent genes upregulated in WS4, and gray spheres represent genes with no difference in expression between WS4 and BS4. The top ten upregulated genes (significantly increased expression) sorted by absolute log2 Fold Change value are concentrated in functional types such as ribosomal proteins (e.g., RPL16), cell wall modifying enzymes (e.g., pectin esterase, xyloglucan endoglucosidase XTH26), and transport proteins (e.g., ABCG22). No genes related to hormone regulation or flavonoid (e.g., anthocyanin) metabolism were detected. Among the top ten downregulated genes (significantly decreased expression), the detection of Lr08g000448 (PIN2) is of key significance. This gene encodes an auxin export carrier and is a core regulator of plant auxin polar transport. Its significant downregulation indicates that the auxin transport process may play an irreplaceable regulatory role in the formation of differential phenotypes between the two groups of samples. Auxin, as a core hormone regulating plant growth and development, directly affects key physiological processes such as lateral root development, apical dominance maintenance, and growth posture adjustment under stress through PIN2-mediated directional transport of auxin between root tips and organs. Changes in auxin expression can profoundly alter plant growth patterns and organogenesis, highlighting the fundamental role of hormone regulation in shaping plant phenotypic differences. Furthermore, although the key gene Lr03g005135 (CHS1B, chalcone synthase, log2Fold Change=7.013), involved in flavonoid (anthocyanin) synthesis, was not among the top ten upregulated genes, its significant upregulation as the rate-limiting initiation enzyme of the anthocyanin synthesis pathway suggests that the flavonoid metabolic pathway was activated in both groups of samples. Flavonoids are not only important secondary metabolites in plants (involved in flower color formation, oxidative damage resistance, and UV protection), but they can also participate in environmental adaptation and growth coordination through interactions with hormone signaling pathways (such as regulating auxin transport efficiency and enhancing ABA-mediated stress responses), further confirming the auxiliary regulatory value of flavonoids in plant regulatory networks. In summary, although this analysis only found hormone-related genes (PIN2) among the top ten downregulated genes, and key flavonoid genes did not rank highly, the differential expression characteristics of the two are clearly evident: hormones directly dominate the regulation of plant growth and development through signal transduction and substance transport, while flavonoids assist in optimizing the regulatory effect through the interaction of metabolite synthesis and signaling pathways. Together, they constitute an important regulatory mechanism for plants to respond to changes in the internal and external environment and form differential phenotypes. The specific activation or inhibition of their regulatory effects is one of the core clues for understanding the phenotypic differences between WS4 and BS4 samples.

[0033] like Figure 2As shown, KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of differentially expressed genes in WS4_vs_BS4 revealed that hormone-related pathways and flavonoid-related pathways were the core pathways regulating the phenotypic differences between the two groups, and both showed significant enrichment characteristics. Within the hormone-related pathways, the plant hormone signal transduction pathway (map04075) was enriched with 298 differentially expressed genes (106 upregulated and 192 downregulated), covering signal transduction genes of various hormones such as auxin, cytokinin, brassinolide, and abscisic acid, including PIN (purine indole) family auxin export carriers and ARR family cytokinin response factors. The proportion of downregulated genes was higher (64.4%), suggesting that WS4 exhibits overall inhibition of hormone signal response relative to BS4. The zeatin biosynthesis pathway (map00908) was enriched with 53 differentially regulated genes (20 upregulated and 33 downregulated). Downregulation of key zeatin synthesis enzymes such as IPT (isopentenyltransferase) may reduce the content of active zeatin in WS4 cells, inhibiting cell division and growth. In the flavonoid-related pathways, the core flavonoid biosynthesis pathway (map00941) was enriched with 75 differentially regulated genes (29 upregulated and 46 downregulated). Upregulation of rate-limiting enzyme genes such as CHS (chalcone synthase) and CHI (chalcone isomerase) promoted precursor synthesis, while some downstream genes such as DFR (dihydroflavonol 4-reductase) and ANS (anthocyanin synthase) were downregulated, leading to a metabolic bias towards flavonol accumulation. Downstream branches such as flavonoid and flavonol biosynthesis (map00944) and isoflavone biosynthesis (map00943) also exhibited differential regulation, further refining the direction of flavonoid metabolism.

[0034] Based on KEGG pathway analysis, key metabolic pathways and differentially expressed genes related to anthocyanin biosynthesis and plant hormone signal transduction were screened. In the anthocyanin biosynthesis pathway (map00942), four upregulated genes were identified, all members of the K21383 enzyme family (Lr05g000447, Lr05g000453, Lr05g000464, and Lr05g000468). These genes may encode key enzymes in the anthocyanin synthesis pathway, participating in the modification and accumulation of anthocyanins. Simultaneously, six downregulated genes were also found in this pathway, indicating that anthocyanin synthesis is subject to multi-level fine-tuning. In the plant hormone signal transduction pathway (map04075), a wider range of gene expression changes were discovered, with a total of 261 differentially expressed genes, of which 73 were upregulated and 188 were downregulated. These genes are involved in multiple plant hormone signaling pathways, including auxin, cytokinin, gibberellin, abscisic acid, ethylene, and brassinosteroids, encompassing key components such as hormone receptors, signal transduction components, and transcriptional regulators. Such large-scale alterations in the expression of hormone-related genes indicate a significant reprogramming of the plant's hormone signaling network, which may be an important regulatory mechanism for plant responses to changes in the internal and external environment. Furthermore, significant changes in gene expression were also observed in hormone-specific pathways such as brassinosteroid biosynthesis (map00905) and zeatin biosynthesis (map00908), further confirming the central role of hormone metabolism and signal transduction in plant physiological processes.

[0035] Based on expression level data (FPKM values), key genes related to the regulation of anthocyanin and zeatin accumulation were screened, and their expression differences under BS4 and WS4 conditions were described in detail. As shown in Table 5, in terms of anthocyanin regulation, several MYB family transcription factors were upregulated in BS4, which may positively regulate the anthocyanin biosynthesis pathway. The average expression level of MYB58 (gene Lru01G00008) was 0.6196 in BS4, while it was 0.2588 in WS4, with a ratio (BS4 / WS4) of 2.39 and a Log2FC of 1.26, indicating that its expression was enhanced in BS4. The expression level of MYB80 (Lru01G00029) was 0.2999 in BS4 and 0.0499 in WS4, with a high ratio of 6.01 and a log2FC of 2.59, showing significant upregulation. MYB102 (Lru01G00409) was expressed at 0.1877 in BS4 but not detected in WS4, suggesting that it is specifically expressed in BS4 and may be a key driver of anthocyanin synthesis. Conversely, WRKY34 (Lru01G00022) was highly expressed in WS4 and may negatively regulate anthocyanin accumulation.

[0036] Table 5 Differentially expressed genes related to anthocyanin accumulation As shown in Table 6, regarding zeatin regulation, the expression level of the cytokinin response regulator ARR2 (Lru01G00360) was 0.1874 in BS4 but not expressed in WS4, indicating that the cytokinin signaling pathway is active in BS4, potentially promoting zeatin accumulation or signal transduction. TPK1 (Lru01G00055) was highly expressed in WS4, but only 1.7262 in BS4, with a log2FC of -0.47, possibly inhibiting cytokinin signaling. Furthermore, BHLH14 (Lru01G00559) was significantly upregulated in BS4 (log2FC of 4.54), possibly indirectly affecting zeatin metabolism through transcriptional regulation. Overall, these expression differences suggest that under BS4 conditions, anthocyanin accumulation may be promoted through the upregulation of MYB transcription factors, while zeatin accumulation may be achieved through ARR2 upregulation and enhanced cytokinin signaling, providing crucial insights into the molecular mechanisms of secondary metabolite accumulation in BS4.

[0037] Table 6 Differentially expressed genes related to zeatin accumulation. Comparative transcriptomic analysis of black goji berry BS4 and white goji berry WS4 revealed clear differences in the molecular mechanisms underlying anthocyanin and zeatin accumulation between the two species. In BS4, MYB80 (expression increased 6-fold, log2FC=2.59) and MYB102 (specific expression from absent to present) act as core transcriptional activators, synergistically driving upstream regulation of the anthocyanin synthesis pathway. At the hormonal regulation level, the specific expression of ARR2 in BS4 (complete absence in WS4) clearly indicates strong activation of the cytokinin signaling pathway, while the highly significant upregulation of the co-regulatory factor BHLH14 (log2FC=4.54) further amplifies the physiological effects of zeatin. In contrast, the high expression of WRKY34 and the upregulation of TPK1 in WS4 inhibited anthocyanin synthesis and zeatin signaling, respectively. The synergistic regulation of these key genes ultimately leads to the large accumulation of anthocyanins (resulting in black fruit) and enhanced cytokinin signaling in BS4, while WS4, lacking this activation network, maintains a white phenotype and basal hormone levels.

[0038] In summary, the regulatory network for anthocyanin synthesis in black goji berries involves a synergistic regulatory mechanism between the hormonome and transcriptome. At the hormonome level, 1-aminocyclopropanecarboxylic acid (ACC, a precursor to ethylene), salicylic acid (SA), and dihydrozein (DHZ, an active cytokinin) accumulate significantly in black goji berries (BS4) (DHZ is 11.9 times higher than in ginkgo, and SA is 65.2% higher), forming the core hormonal signaling environment for anthocyanin synthesis. At the transcriptome level, this hormonal environment induces the upregulation of MYB family transcription factors such as MYB80 (6.01 times the expression level in ginkgo) and MYB102 (BS4-specific expression), while simultaneously activating K21383 enzyme family genes (upregulated in the anthocyanin pathway map00942), which may be involved in catalyzing anthocyanin modification. ARR2 (BS4-specific expression) is closely related to DHZ signaling activation of the cytokinin pathway. In contrast, the ginkgo biloba (WS4) has low DHZ content, no ARR2 expression, and low transcription of key genes such as MYB102. Even with a high ABA content (64.8% higher than BS4), it cannot activate the anthocyanin synthesis pathway. Ultimately, the differences in hormone signaling and gene expression differentiation determine the regulatory network of anthocyanin accumulation in goji berry fruit.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for screening key hormones and genes involved in anthocyanin synthesis in black wolfberry, characterized in that, The key hormones for screening anthocyanin synthesis in black goji berries include 1-aminocyclopropanecarboxylic acid, salicylic acid, abscisic acid, and dihydrozeatin. The key genes for screening anthocyanin synthesis in black wolfberry include the MYB80 gene, MYB102 gene, K21383 enzyme family gene, ARR2 gene, and BHLH14 gene. The filtering method is as follows: S1. Selection and pretreatment of plant samples: Black wolfberry and white wolfberry samples were collected respectively. The samples were rinsed with ultrapure water and the surface moisture was removed with sterile filter paper to obtain the rinsed black wolfberry samples and the rinsed white wolfberry samples respectively. S2. The washed black goji berry samples and washed white goji berry samples obtained in S1 were frozen and fixed in liquid nitrogen, then freeze-dried, ground, sieved, and stored in the dark at -20℃ to obtain ground black goji berry samples and ground white goji berry samples respectively. S3. Transcriptome and hormoneome analyses were performed on the ground black goji berry samples and ground white goji berry samples obtained in S2, respectively.

2. The method for screening key hormones and genes involved in anthocyanin synthesis in black wolfberry according to claim 1, characterized in that, The freeze-fixation time in S2 is 20 min; the freeze-drying conditions are: drying at a vacuum of 10 Pa and a temperature of -50 °C for 48 h; and the sieve mesh size is 100 mesh.

3. The method for screening key hormones and genes involved in anthocyanin synthesis in black wolfberry according to claim 1, characterized in that, The hormone analysis described in S3 was performed using high performance liquid chromatography-mass spectrometry.

4. The application of the key hormones and genes for anthocyanin synthesis in black wolfberry selected by the method described in any one of claims 1-3, characterized in that, The key hormones and genes mentioned can be used as molecular markers or genetic manipulation targets to study the signaling pathways that regulate anthocyanin synthesis in black goji berries.

5. The application according to claim 5, characterized in that, When the MYB80, MYB102 and MYB58 genes are upregulated in black goji berries, and the K21383 enzyme family genes are upregulated in the anthocyanin biosynthesis pathway map00942, they are used to catalyze anthocyanin modification. When the ARR2 gene is expressed in black goji berries, not in white goji berries, and the BHLH14 gene is upregulated in BS4, the cytokinin signaling pathway is activated in black goji berries.

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