Licomt gene for regulating synthesis of lycorine glycosides in lily and application thereof

Abstract

The application provides a LiCOMT gene for regulating synthesis of lycoris kingianus glycosides and application thereof, belongs to the technical field of plant genetic engineering, and the nucleotide sequence of the LiCOMT gene is shown as SEQ ID NO. 7, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 8; the application screens out a candidate gene LiCOMT significantly related to lycoris kingianus glycoside accumulation from lily bulb development period transcriptome data through weighted gene co-expression network analysis (WGCNA), and confirms that silencing the LiCOMT gene can reduce the content of lycoris kingianus glycoside A, lycoris kingianus glycoside B and lycoris kingianus glycoside F in lily bulbs by about 60%, 42% and 43% respectively by using a virus-induced gene silencing (VIGS) technique; and the application provides important gene resources and theoretical basis for lily secondary metabolism regulation and molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the LiCOMT gene that regulates the synthesis of lily glycosides in Lilium argenteum and its applications. Background Technology

[0002] Lily (Lilium spp.) is native to my country. Its underground bulbs are rich in nutrients and functional components, and it was one of the first plants announced as having both medicinal and edible uses. Currently, research on the chemical components and functional activities of lily is quite in-depth, and the public has a high level of awareness of its health benefits, demonstrating broad research prospects and development and application value. Lilium glycosides, as phenolic secondary metabolites unique to the Lilium genus, show great development potential in functional foods, natural cosmetics, and pharmaceuticals due to their unique chemical structure and potential pharmacological activities such as hypoglycemic and immunomodulatory effects. With a deeper understanding of the functional activities of lily glycosides, their status as core indicator components for lily quality evaluation is becoming increasingly prominent. Existing studies have suggested using lily glycoside A, lily glycoside B, and lily glycoside C as quality markers for lily slices, providing a scientific basis for lily quality control. Simultaneously, in comparing the impact of different field management models on lily quality, the content of lily glycosides A, B, C, F, H, and total polysaccharides is also used as quality indicators.

[0003] As a phenolic glycerol ester compound, the biosynthesis of lily glycoside mainly originates from the phenylpropane metabolic pathway, involving a series of modification reactions such as hydroxylation, acylation, and methylation. Among them, caffeic acid-O-methyltransferase (COMT) is one of the key enzymes in the phenylpropane metabolic pathway, mainly responsible for catalyzing the methylation reaction of caffeoyl derivatives, and plays an important role in the structural modification and diversity formation of phenolic substances (Li Yuanyu et al., 2022, Research progress of plant caffeic acid-O-methyltransferase. Chinese Journal of Biotechnology, 38(06): 2187-2200.). Studies have shown a positive correlation between COMT expression levels and the accumulation of phenolic acids. In transgenic tobacco plants with downregulated MsCOMT in Miscanthus sinensis, the total lignin content decreased while the total flavonol concentration significantly decreased, indicating that COMT regulates phenolic acid methylation and thus affects the accumulation of downstream phenolic metabolites (Seong E Set al., 2013, Antisense-overexpression of the MsCOMT gene induces changes in lignin and total phenol contents in transgenic tobacco plants. Molecularbiology reports, 40(2): 1979-1986.). However, the molecular regulatory mechanism of lily glycoside biosynthesis is still unclear, and the discovery of key synthase genes is seriously lagging behind, which restricts the targeted breeding and industrial application of lily germplasm with high lily glycoside content. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to address the shortcomings of existing technologies by providing the LiCOMT gene that regulates the synthesis of lily glycosides and its applications. This invention clones the key gene regulating the biosynthesis of lily glycosides and analyzes its expression regulatory network. This not only provides core gene resources and theoretical support for the molecular improvement of the edible and medicinal quality of lilies, but also lays a solid foundation for the heterologous and efficient production of lily glycosides based on synthetic biology strategies, and promotes the in-depth development and high-value utilization of lily's unique resources.

[0005] Technical solution: The present invention describes a LiCOMT gene, the nucleotide sequence of which is shown in SEQ ID NO. 7.

[0006] A LiCOMT protein, the amino acid sequence of which is shown in SEQ ID NO.8.

[0007] Recombinant expression vectors containing the aforementioned LiCOMT gene.

[0008] Recombinant microorganisms containing the LiCOMT gene or the recombinant expression vector described above.

[0009] The application of the aforementioned LiCOMT gene, the aforementioned protein, the aforementioned recombinant expression vector, or the aforementioned recombinant microorganism in regulating the synthesis of lily glycosides in Lilium argenteum.

[0010] Furthermore, the lily is the 'Orange Sunshine' lily.

[0011] Furthermore, silencing the LiCOMT gene inhibits the synthesis of lily glycoside A, lily glycoside B, and lily glycoside F in lily bulbs.

[0012] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows:

[0013] Licoside, a unique phenolic secondary metabolite of lily plants, has been a hot topic in phytochemistry and pharmacology due to its structural diversity and functional specificity. Caffeic acid-O-methyltransferase (COMT) is a key enzyme in the phenylpropanoid metabolic pathway, participating not only in lignin biosynthesis but also extensively in the methylation modification of various phenolic compounds in plants, providing a structural basis for many plant secondary metabolites. However, its role in regulating the synthesis of lily glycosides in lily bulbs has not yet been reported. This invention, based on lily transcriptome and metabolome data, used WGCNA analysis to identify and screen the LiCOMT gene in the phenylpropanoid synthesis pathway, and cloned the full-length gene. Spatiotemporal expression analysis showed that the spatiotemporal expression trend of the LiCOMT gene during lily bulb development was initially flat, then increased, and then decreased, consistent with the overall trend of licoside synthesis. Furthermore, this invention used VIGS to clarify that silencing LiCOMT significantly inhibits the synthesis of licoside A, licoside B, and licoside F. The results showed that LiCOMT plays a positive regulatory role in the synthesis of lily glycosides in the bulb of lily 'Orange Sunshine'. Attached Figure Description

[0014] Figure 1 Weighted gene co-expression network analysis and correlation analysis of lily glycoside metabolites were performed, including: (A) a heatmap of lily glycoside metabolite content at different developmental stages. Color intensity indicates content level (red indicates high content, blue indicates low content). (B) a gene co-expression network clustering tree diagram. This shows the clustering of gene modules, with color labels at the bottom indicating different co-expression modules (module color). (C) a module-feature relationship heatmap. This shows the correlation coefficients and significant P-values ​​between different gene modules (rows) and lily glycoside metabolites (columns). Color intensity indicates the strength of the correlation (red indicates positive correlation, blue indicates negative correlation). (D) expression patterns and functional annotations of key gene clusters. This shows a bubble chart of the expression status of specific gene clusters in different samples, with corresponding NR descriptions or KEGG pathway information labeled on the right.

[0015] Figure 2 Phylogenetic and expression pattern analyses of the LiCOMT gene were performed, with (A) showing the phylogenetic tree of LiHMGR. The evolutionary relationships between LiCOMT and COMT proteins from other species are illustrated. Numbers on nodes represent confidence levels. (B) and (C) show the expression analysis of LiCOMT at different flower developmental stages and in different tissues. S1, 0d refrigeration; S2, 30d refrigeration; S3, 60d refrigeration; S4, seedling stage; S5, budding stage; S6, full bloom stage; S7, decline stage. Bulb, bulb; Ovary, ovary; Petal, petal; Filament, filament; Anther, anther; Style, style; Stem, stem; Leaf, leaf; Root, root. Bar charts represent mean ± standard deviation; different letters indicate significant differences (P < 0.05).

[0016] Figure 3 Transient silencing of the LiCOMT gene in lily bulbs. (A) Sampling status of silent plants and control plants, scale bar 1 cm. (B) Detection of relative expression level of LiCOMT gene. Comparison of LiCOMT enzyme expression levels in TRV2 control group and TRV2-LiCOMT silencing group. ** indicates extremely significant difference (P < 0.01). (C) Detection of the content of limonene A, limonene B, and limonene F in silent plants and control plants. Values ​​represent mean ± standard deviation. Each circle represents a biological replicate. ** indicates extremely significant difference (P < 0.01). Detailed Implementation

[0017] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the embodiments described.

[0018] The materials used in the following embodiments include:

[0019] 1. Plant materials and treatment

[0020] Bulbs of the lily 'Chengse Yangguang' (Lilium hybrid cultivar 'Chengse Yangguang') were collected from the Flower Germplasm Innovation Experimental Base of the Fengpu Branch of the Shanghai Academy of Agricultural Sciences. Bulbs of uniform maturity and free from mechanical damage and pests / diseases were selected as experimental materials. Each group had three biological replicates. During sampling, the outer scales were peeled off, and the healthy scale tissue from the middle was taken, flash-frozen in liquid nitrogen, and stored at -80℃ for later use.

[0021] 2. The test strains and vectors are shown in Table 1.

[0022] Table 1

[0023]

[0024] All the main kits used in the experiment were purchased from Beijing TransGen Biotech Co., Ltd.: RNA extraction: TransZolUp Plus RNA Kit; Reverse transcription: TransScript® Uni All-in-One First-Strand cDNASynthesis SuperMix for qPCR; DNA gel extraction: EasyPure® Quick Gel Extraction Kit.

[0025] The experimental methods used in the following embodiments include:

[0026] 1. Carrier Construction

[0027] Based on existing sequence information in the lily transcriptome database, primers for cloning were designed, and PCR was performed using lily 'Orange Sunshine' cDNA as a template. The reaction system and reaction procedure are shown in Table 2.

[0028] Table 2

[0029]

[0030] The obtained PCR products were validated by running on agarose gels using the EasyPure® Quick Gel Extraction Kit. Then, the desired vector was double-digested with 1 μg of enzymes. The system is shown in Table 3.

[0031] Table 3

[0032]

[0033] The fragment enzymatic digestion reaction system was denatured at 37℃ for 30 min and 80℃ for 20 min, and the liquid was recovered.

[0034] Next, the enzyme digestion products recovered in the previous step were ligated with T4 ligase, and the ligation product was transformed into competent E. coli (DH5α) cells for transformation. Selected bacterial cultures that had been successfully transformed and verified by sequencing were cultured overnight in 50 mL centrifuge tubes. Afterwards, plasmids were extracted from E. coli using a TransGold plasmid miniprep kit and stored for later use.

[0035] The primers used in the experiment, the LiCOMT gene sequence, and the corresponding protein sequence they encode are shown in Table 4.

[0036] Table 4

[0037]

[0038] 2. LiCOMT gene cloning

[0039] The LiCOMT gene sequence fragment was obtained from transcriptome data of 'Orange Sunshine' bulbs during their developmental stage, and the full-length sequence was amplified using 2×ApexHF FS PCR Master Mix. Phylogenetic analysis was performed using MEGA 7 and neighbor-joining with 1,000 bootstrap replicates.

[0040] 3. Quantitative qRT-PCR analysis

[0041] Total RNA was extracted from 'Orange Sunshine' bulbs using the TransZol Up Plus RNA Kit. First-strand cDNA was synthesized using the TransScript® Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) reverse transcription kit, with 1 μg of total RNA as a template. PerfectStart was used... ® qRT-PCR reactions were performed using Green qPCR SuperMix (20 μL volume containing 1 μL cDNA template). The lily EF1 gene was used as an internal control. PCR primers are listed in Table 4. Primers ending in -F are forward primers, and primers ending in -R are reverse primers.

[0042] 4. Virus-induced transient silencing system

[0043] To construct the VIGS silencing vector, a 300bp specific fragment of the LiCOMT gene, as shown in SEQ ID NO. 9, was constructed into the pTRV2 vector and named TRV2-LiCOMT. The vectors (TRV2-LiCOMT, TRV2, and TRV1) were transformed into 50 µL of semi-thawed Agrobacterium tumefaciens competent cells, mixed thoroughly, incubated on ice for 10 min, at 37 °C for 5 min, in liquid nitrogen for 5 min, and then incubated on ice for 5 min. Subsequently, 700 µL of antibiotic-free LB medium was added, and the cells were incubated at 28 °C at 200 rpm for approximately 3 h. Then, 100 µL of the culture was plated and incubated upside down at 28 °C for 2-3 days. Eight single colonies were selected and transferred to 500 μL of LB medium containing Kan+Rif, and incubated with shaking at 28 °C at 200 rpm for 14 h. Following this, PCR detection was performed. The bacterial culture of successfully detected positive clones was transferred to 5 mL of LB liquid containing Kan+Rif (using a 50 mL centrifuge tube) and incubated at 28 °C with shaking at 200 rpm for 10 h. Then, 5 mL of the cultured medium-shaken bacterial culture was transferred to 500 mL of LB liquid containing Kan+Rif and incubated at 28 °C with shaking at 200 rpm for 12 h (in a 1000 mL culture flask). The cultured large-shaken bacterial culture was collected and centrifuged at 5,000 rpm for 10 min, and the supernatant was discarded. The bacterial cells were resuspended in infection solution (containing 200 mM acetylsalicylic acid, 10 mM magnesium chloride, and 10 mM MES), and the OD was adjusted. 600 The concentration was increased to 0.8. TRV2 and its recombinant vector, resuspended in the infection solution, were mixed with TRV1 in equal proportions and incubated in the dark for 3 hours. Healthy, uniform lily bulbs that had broken dormancy were selected for infection using a vacuum method. The vacuum was reduced to 0.7 atm, maintained for 15 minutes, and then slowly released for 10 minutes, repeated twice. The infected bulbs were washed three times and then planted in the substrate. After culturing under normal light for 30 days, samples were collected for silencing efficiency testing and scale sampling. The primers used were TRV2-LiCOMT-F and TRV2-LiCOMT-R.

[0044] 5. Detection of the content of lily glycoside A, lily glycoside B and lily glycoside F

[0045] A method for determining the content of lily glycosides in lily bulbs using high performance liquid chromatography (HPLC) was developed. 0.2 g of lily bulb powder was placed in a container, 5 mL of 70% ethanol was added, and the mixture was weighed. After soaking for 1 hour, ultrasonic extraction was performed in a water bath at 70℃-95℃ for 30-60 minutes. The mixture was then removed, cooled, and weighed again. The weight was made up with 70% ethanol, the mixture was shaken well, filtered, and the filtrate was used for analysis. Chromatographic conditions: Thermo-C18 column (250*4.6mm 5µm); mobile phase A:B = (chromatographic acetonitrile): (0.2% glacial acetic acid). Elution program: gradient elution (0-12 min, 8%-15% A; 12-20 min, 15%-30% A; 20-30 min, 8% A); detection wavelength: 300 nm; flow rate: 1 mL / min; column temperature: 35℃.

[0046] Example 1

[0047] Screening of key regulatory factors in the biosynthesis of lily glycosides

[0048] Wangliu glycosides, unique secondary metabolites found in lily bulbs, were analyzed in detail for their distribution across seven developmental stages. Previous K-means clustering analysis identified a subclass of metabolites whose accumulation pattern aligned with the bulb's developmental process, detecting eight different Wangliu glycosides within this subclass. Figure 1 As shown in Figure A, the content of lily glycoside A was the highest, but remained relatively stable across different stages. In contrast, lily glycosides C, B, I, and F exhibited significant dynamic changes, with lily glycoside F showing the most pronounced variation. During cold storage treatment (S1-S3), lily glycosides C and F increased by 2.04-fold and 1.72-fold, respectively, from S1 to S2, but decreased significantly in S3 (lily glycosides B, I, and F decreased by 53%, 54%, and 50%, respectively). After transplanting (S4), lily glycosides B, I, and F rebounded, increasing by 2.45-fold, 2.24-fold, and 1.77-fold, respectively. Lily glycosides C and F reached their peak values ​​during the flowering stage (S6) (increasing by 1.66-fold and 1.56-fold, respectively), but decreased by 48% and 39% during the senescence stage (S7). To elucidate the molecular mechanisms underlying these metabolic changes, we integrated transcriptomic and metabolomic data using WGCNA technology, identifying 18 co-expression modules, each containing 43 to 6451 genes. Figure 1 B). Module-feature relationship analysis showed that the MEbrown module (5202 genes) exhibited the highest positive correlation with all eight lily glycosides. Figure 1 C)

[0049] The top 10 hub genes are primarily highly expressed on S6. Figure 1D). Functional annotation of MEbrown genes revealed significant enrichment in phenylpropanoid biosynthesis (ko00940), starch and sucrose metabolism (ko00500), and plant hormone signal transduction (ko04075). In the Hub genes, Cluster-114994.3, annotated as caffeoyl-CoA O-methyltransferase (COMT), was selected as a key candidate regulator due to its high connectivity and consistent expression pattern with lipofusin accumulation. These results constructed a complete regulatory network for lipofusin biosynthesis and laid the foundation for functional validation.

[0050] Example 2

[0051] Phylogenetic analysis and expression pattern analysis of the LiCOMT gene in lilies

[0052] The coding region sequence of the gene was obtained from the transcriptome database of lily bulb development. Using 'Orange Sunshine' cDNA as a template, the CDS region of COMT was successfully cloned and named LiCOMT according to the NCBI alignment results. Its ORF is 741 bp, encoding 246 amino acids, with a theoretical molecular mass of 27.6 kDa. The amino acid sequence of LiCOMT was compared using the BLAST function of the NCBI website. The amino acid sequences of COMT from 10 other plants with high similarity were downloaded, and homology comparisons were performed on these proteins. The LiCOMT protein was found to be closely related to *Lilium regale*. Figure 2 A) indicates a high degree of sequence conservation within the genus *Lili*. Multiple sequence alignment revealed two highly conserved domains: a SAM-dependent methyltransferase domain and an AdoMet-MTase domain, both crucial for its catalytic activity. Key catalytic residues, including the SAM-binding motif, are retained in *LiCOMT*, suggesting enzymatic functions similar to *COMT* in other species. Quantitative PCR analysis at seven developmental stages revealed distinct expression patterns of *LiCOMT*. Figure 2 (B) The expression level was relatively low from S1 to S5, increased significantly during the full flowering stage (S6), and then decreased during the senescence stage (S7). Specifically, the expression level at S6 was approximately 45 times that at S1. This expression pattern showed a strong positive correlation with the accumulation trends of limonene C and F, further demonstrating that LiCOMT is a key regulator of limonene biosynthesis.

[0053] Tissue-specific expression analysis of nine organs showed that LiCOMT is universally expressed, but its abundance varies. Figure 2C). LiCOMT expression was highest in the filaments, followed by the petals, consistent with the tissue distribution pattern of LiCOMT. Notably, LiCOMT expression was moderate in the bulbs and lowest in the leaves. The different letters above the bars indicate significant differences as determined by Duncan's test following one-way ANOVA. These results suggest that LiCOMT primarily functions in the reproductive tissues during the flowering stage, coinciding with the peak accumulation period of the target metabolite.

[0054] Example 3

[0055] Obtaining LiCOMT transiently silenced plants and identifying the content of lily glycosides.

[0056] To elucidate the role of LiCOMT in the synthesis of lily glycosides in 'Orange Sunshine' lily king, a TRV2-LiCOMT recombinant vector was constructed. Dormant lily bulbs were infected with Agrobacterium and samples were collected 30 days after infection. No significant phenotypic differences were observed between TRV2-LiCOMT-silenced plants and TRV2 control plants in terms of overall growth vigor, plant height, or root development. Figure 3 A) qRT-PCR was used to detect effectively silenced LiCOMT lines, and the content of these silenced lines was analyzed. For example... Figure 3 As shown in Figure B, the abundance of LiCOMT transcripts in the silenced lines was significantly reduced by 87% compared to the control plants, indicating that gene silencing is highly efficient. Targeted metabolite analysis was performed to determine the effect of LiCOMT silencing on the accumulation of limonene. Figure 3 As shown in Figure C, in the silent samples, the levels of all three detected lily glycosides were significantly reduced. Lily glycoside A showed the most significant change, decreasing by approximately 60%, while lily glycosides B and F decreased significantly by 42% and 43%, respectively. This provides strong evidence for LiCOMT directly regulating the biosynthesis of lily glycosides.

[0057] These functional verification results, combined with relevant WGCNA data ( Figure 1 ) and tissue-specific expression patterns ( Figure 2 This study established LiCOMT as a key positive regulator of regaloside biosynthesis in lily bulbs. The consistent reduction in the content of various regalosides after LiCOMT silencing suggests that this enzyme may play a role at a branch point or upstream of the regaloside biosynthetic pathway.

[0058] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A LiCOMT gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

7.

2. A LiCOMT protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

8.

3. A recombinant expression vector containing the LiCOMT gene as described in claim 1.

4. Recombinant microorganisms containing the LiCOMT gene of claim 1 or the recombinant expression vector of claim 3.

5. The application of the LiCOMT gene of claim 1, the protein of claim 2, the recombinant expression vector of claim 3, or the recombinant microorganism of claim 4 in regulating the synthesis of lily glycosides in Lilium argenteum.

6. The application according to claim 5, characterized in that, The lily in question is the 'Orange Sunshine' lily.

7. The application according to claim 5, characterized in that, Silencing the LiCOMT gene inhibits the synthesis of lily glycoside A, lily glycoside B and lily glycoside F in lily bulbs.

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