LiHMGR gene for regulating and controlling biosynthesis of lilium steroid saponin substances and application of LiHMGR gene
By cloning the LiHMGR gene of the lily 'Orange Sunshine' and regulating the HMGR enzyme activity, the problem of gene deficiency in the synthesis of lily steroidal saponins was solved, thus improving the medicinal components of lily.
Patent Information
- Application Number
- CN202511179905.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-22
- Publication Date
- 2025-11-21
AI Technical Summary
The lack of existing technologies for gene regulation of the biosynthesis of lily steroidal saponins has led to a shortage of medicinal plant resources in the lily industry.
The LiHMGR gene of the lily 'Orange Sunshine' was cloned and expressed. By silencing or overexpressing HMGR enzyme activity, the synthesis of mevalonic acid was affected, thereby regulating the production of steroidal saponins.
It significantly inhibits or increases the production of steroidal saponins in lily bulbs, thereby increasing the content of medicinal components in lilies and meeting the needs of medicinal plant resources.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology, specifically relating to a LiHMGR gene isolated and cloned from the lily 'Orange Sunshine' and its application in the regulation of steroidal saponin synthesis. Background Technology
[0002] Lily (Lilium spp.) is native to my country. Its underground bulbs are rich in nutrients and functional components, and it is one of the first plants listed as both food and medicine by the National Health Commission of China. There is considerable research on the chemical components and functional activities of lily, and the public has a good understanding of its functionalities, indicating significant potential for research and development. Currently, various active substances have been extracted from lily bulbs, revealing anti-tumor, antidepressant, antioxidant, sedative, and hypnotic effects. It also has certain therapeutic effects on lung inflammation and emphysema, with saponins being the pharmacological basis for its functions. There are 39 health food products containing lily raw materials (data from the State Administration for Market Regulation's Special Food Information Query Platform). The main active ingredients are saponins, polysaccharides, and flavonoids, with 30 products primarily containing saponins. However, only 8 of these health food products have lily as the primary ingredient. The medicinal lily *Lilium lancifolium* Thunb., rich in functional components, has a planting area of 62,000 mu (approximately 4,800 hectares), accounting for only about 20% of the national edible lily planting area. Therefore, the lily industry should seize the current new opportunities and make improving the quality of its edible and medicinal products its urgent task in breeding.
[0003] Steroidal saponins are the basic substances for the medicinal effects of lilies. Their biosynthesis mainly occurs through the mevalonate (MVA) pathway in the cytoplasm. Among them, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the first key synthase in the process, which is responsible for catalyzing the conversion of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) into the isoprene precursor compound mevalonate (MVA). This is an irreversible reaction (Zheng Ting et al., 2022, Research progress of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in plants. Acta Physiologica Sinica, 58(6):1037-1044.). Studies have shown that the content, activity, and yield of subsequent products of HMGR are positively correlated (Kim et al., 2014, Functional analysis of 3-Hydroxy-3-methylglutaryl coenzyme Areductase encoding genes in triterpenesaponin-producing ginseng. Plant Physiology, 165(1):373-387). Currently, using biotechnologies such as genetic engineering and fermentation engineering to regulate key genes in the biosynthetic pathways of secondary metabolites at the molecular level and promote their expression, thereby achieving the large-scale production of effective components from medicinal plants, has become a major trend in the modernization of traditional Chinese medicine and an important way to solve the increasingly scarce medicinal plant resources. Compared with other plants, research on the regulatory mechanism of lily steroidal saponin synthesis is relatively lagging. There are currently no reports on the genes that regulate the biosynthesis of lily steroidal saponins. Summary of the Invention
[0004] One of the objectives of this invention is to provide a LiHMGR gene that regulates the biosynthesis of lily steroidal saponins, the nucleotide sequence of which is shown in SEQ ID NO.9.
[0005] A second objective of this invention is to provide a protein encoded by the LiHMGR gene, the amino acid sequence of which is shown in SEQ ID NO. 10.
[0006] A third objective of this invention is to provide a recombinant expression vector containing the aforementioned LiHMGR gene.
[0007] The fourth objective of this invention is to provide recombinant microorganisms containing the above-mentioned LiHMGR gene or the above-mentioned recombinant expression vector.
[0008] The fifth objective of this invention is to provide the application of the above-mentioned LiHMGR gene, the above-mentioned protein, the above-mentioned recombinant expression vector, or the above-mentioned recombinant microorganism in regulating the synthesis of lily steroidal saponins.
[0009] Furthermore, the lily is the 'Orange Sunshine' lily.
[0010] Furthermore, silencing the LiHMGR gene inhibits the synthesis of steroidal saponins in lily bulbs; overexpression of the LiHMGR gene increases the synthesis of steroidal saponins in lily bulbs.
[0011] Furthermore, silencing the LiHMGR gene inhibits the activity of HMGR enzyme in lily bulbs, thereby inhibiting the synthesis of mevalonic acid.
[0012] Furthermore, overexpression of the LiHMGR gene increases the activity of HMGR enzyme in lily bulbs and enhances the synthesis of mevalonic acid.
[0013] Steroidal saponins, as hallmark secondary metabolites of lilies, have been a hot topic in phytochemistry and pharmacology due to their structural diversity, identification techniques, and functional correlations. HHMGR is the first rate-limiting enzyme in the mevalonic acid (MVA) pathway, a biosynthetic pathway of steroidal saponins, catalyzing the conversion of HMG-CoA to mevalonic acid and providing precursors for secondary metabolites such as phytosterols, terpenes, and carotenoids. However, its role in regulating the synthesis of steroidal saponins in lily bulbs has not yet been reported. This invention found that the spatiotemporal expression trend of the LiHMGR gene during lily bulb development was first decreased, then increased, and then decreased again, consistent with the overall trend of steroidal saponin synthesis. Furthermore, this invention clarified through VIGS that silencing LiHMGR significantly inhibited mevalonic acid synthesis, while overexpression of LiHMGR significantly increased mevalonic acid content. The results indicate that the LiHMGR protein plays a positive regulatory role in the synthesis of steroidal saponins in the bulbs of lily 'Orange Sunshine'. Attached Figure Description
[0014] Figure 1 Predicted conservative structural domains for LiHMGR.
[0015] Figure 2 This is the phylogenetic tree of LiHMGR.
[0016] Figure 3 Expression analysis of LiHMGR in different flower development stages (A) and different tissues (B) for this invention. S1, 0d of cold storage; S2, 30d of cold storage; S3, 60d of cold storage; 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.
[0017] Figure 4Transient silencing of the LiHMGR gene in lily bulbs was performed, with (A) representing the silencing efficiency of LiHMGR, (B) representing the enzyme activity of HMGR in silenced lily bulbs, and (C) representing the content of mevalonate (MVA) in silenced lily bulbs.
[0018] Figure 5 The transient overexpression of the LiHMGR gene in lily bulbs was shown in (A), where (B) was the detection of LiHMGR overexpression efficiency, (C) was the detection of HMGR enzyme activity in overexpressing lily bulbs, and (D) was the detection of mevalonate (MVA) content in overexpressing lily bulbs. Detailed Implementation
[0019] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.
[0020] The materials used in the following embodiments include:
[0021] 1. Plant materials and treatment
[0022] Bulbs of the lily 'Chengse Yangguang' (Lilium hybridcultivar '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, free from mechanical damage and pests / diseases, were selected as experimental materials. Three biological replicates were set up for each group. During sampling, the outer scales were peeled off, and healthy scale tissue from the middle was collected, flash-frozen in liquid nitrogen, and stored at -80℃ for later use.
[0023] 2. The test strains and vectors are shown in Table 1.
[0024] Table 1
[0025] Common strains or plasmids Model or name E. coli DH5α Agrobacterium EHA105 Cloning vector pClone-007 Overexpression vector Super1300-GFP(C) VIGS carrier pTRV1, pTRV2
[0026] All main reagent kits used in the experiment were purchased from Beijing TransGen Biotech Co., Ltd.: RNA extraction: TransZolUp Plus RNA Kit; Reverse transcription: Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR; DNA gel recovery: Quick Gel Extraction Kit.
[0027] The experimental methods used in the following embodiments include:
[0028] 1. Carrier Construction
[0029] 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.
[0030] Table 2
[0031]
[0032] The obtained PCR products were validated by running on agarose gels, and the gel extraction kit from the All-Gold assay was used. The Quick Gel Extraction Kit was used. Then, the desired vector was double-digested, with 1 μg digested. The system is shown in Table 3.
[0033] Table 3
[0034]
[0035] The fragment enzymatic digestion reaction system was denatured at 37℃ for 30 min and 80℃ for 20 min, and the liquid was recovered.
[0036] 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.
[0037] The primers used in the experiment, the LiHMGR gene sequence, and the corresponding protein sequences they encode are shown in Table 4.
[0038] Table 4
[0039]
[0040]
[0041] 2. Cloning of the LiHMGR gene
[0042] The LiHMGR 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×ApexHFFS PCRMaster Mix. Phylogenetic analysis was performed using MEGA7 and neighbor-joining with 1,000 bootstrap replicates.
[0043] 3. Quantitative qRT-PCR analysis
[0044] Total RNA was extracted from 'Orange Sunshine' bulbs using the TransZol Up Plus RNA Kit. The Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) reverse transcription kit was used to synthesize first-strand cDNA using 1 μg of total RNA as a template. 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.
[0045] 4. Virus-induced transient silencing system
[0046] To construct the VIGS silencing vector, a 302 bp specific fragment of the LiHMGR gene, as shown in SEQ ID NO.11, was constructed into the pTRV2 vector and named TRV2-LiHMGR. The vectors (TRV2-LiHMGR, 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 on ice for 5 min. Then, 700 μL of antibiotic-free LB medium was added, and the cells were incubated at 28°C and 200 rpm for approximately 3 h. Subsequently, 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 at 28°C and 200 rpm with shaking for 14 h. Colony PCR was then performed. Transfer the bacterial culture of successfully detected positive clones to 5 mL of LB liquid containing Kan+Rif (using a 50 mL centrifuge tube) and incubate at 28°C with shaking at 200 rpm for 10 h. Then, transfer the 5 mL culture of the partially cultured bacterial culture to 500 mL of LB liquid containing Kan+Rif and incubate at 28°C with shaking at 200 rpm for 12 h (in a 1000 mL culture flask). Collect the cultured partially cultured bacterial culture by centrifugation at 5,000 rpm for 10 min and discard the supernatant. Resuspend the bacterial cells in infection buffer (containing 200 mM acetylsalicylic acid, 10 mM magnesium chloride, and 10 mM MES) and adjust the OD. 600The 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 a substrate. After culturing under normal light for 30 days, samples were collected for silencing efficiency testing and scale sampling. The primers used were TRV2-LiHMGR-F and TRV2-LiHMGR-R.
[0047] 5. Agrobacterium-based overexpression system
[0048] Transient overexpression vector construction for lily: The full-length LiHMGR was constructed into the pSuper1300-GFP vector and named pSuper1300-LiHMGR. The successfully constructed recombinant plasmid and the empty pSuper-GFP vector were transformed into Agrobacterium tumefaciens EHA105, and the culture conditions were the same as above. OD200 of the pSuper-GFP recombinant plasmid and the empty vector were adjusted. 600 The concentration was increased to 0.8. Infection and subsequent culturing were then performed using the same method. Sampling was conducted approximately 4 days after culture for overexpression efficiency detection and for enzyme activity and mevalonic acid content determination.
[0049] 6. Detection of HMGR enzyme activity
[0050] Tissue homogenization: The tissue was broken up and mixed with pre-cooled PBS (0.01M, pH=7.4) (1g of tissue sample corresponds to 9mL of PBS). The homogenate was thoroughly ground on ice, and then centrifuged at 5000×g for 10 minutes. The supernatant was collected for analysis. The enzyme activity of HMGR in lily bulbs was detected using a plant hydroxymethylglutaryl-CoA reductase (HMGR) ELISA kit.
[0051] 7. Detection of Mevaleric Acid (MVA) Content
[0052] Sample processing and test solutions were performed using the same enzyme activity assay kit, with the mevalonic acid content detected by the plant mevalonic acid (MVA) ELISA kit.
[0053] Example 1
[0054] LiHMGR gene screening
[0055] The inventors conducted a combined metabolome and transcriptome analysis of the 'Orange Sunshine' lily bulbs throughout their entire growth cycle, and screened for enzymes related to steroidal saponin synthesis using WGCNA analysis. Analysis of their conserved domains revealed the presence of an HMGL-like domain. Figure 1The gene coding region sequence was obtained from the lily bulb development transcriptome database. Using 'Orange Sunshine' cDNA as a template, the full-length 1365bp coding region (see SEQ ID NO.9) was successfully cloned, encoding 454 amino acids (see SEQ ID NO.10), with a theoretical molecular mass of 48.7 kDa. Using the BLAST function of the NCBI website, proteins from 10 other plants with high similarity were downloaded for homology comparison. The results showed high homology with HMGR protein from Phoenix dactylifera. Based on the NCBI comparison results, it was named LiHMGR. Figure 2 ).
[0056] Example 2
[0057] Tissue-specific expression analysis of LiHMGR in lily
[0058] The expression characteristics of LiHMGR in different flower development stages and tissues of lilies were analyzed. LiHMGR showed an overall trend of first decreasing, then increasing, and then decreasing again throughout the entire growth period. The highest expression level was observed in stage S4, which was 3.5 times higher than the lowest expression levels in stages S2 and S7. Figure 3 (A). In different tissues, LiHMGR was highly expressed in the anthers, followed by the petals, filaments, and styles, at 4.7, 3.7, 3.4, and 2.3 times higher than in the bulbs, respectively. No significant differences were observed in other tissues. Figure 3 (B)
[0059] Example 3
[0060] Obtaining transiently silenced lily plants (LiHMGR) and identifying their steroidal saponin content.
[0061] To clarify whether LiHMGR plays a role in the synthesis of steroidal saponins in 'Orange Sunshine' lily bulbs, transient silencing function was verified using VIGS technology. First, the TRV2-LiHMGR recombinant vector was constructed and infected with dormant lily bulbs via Agrobacterium-mediated infection. Silent plants were selected using qRT-PCR. The expression of LiHMGR transcripts was significantly downregulated, at 35% of the control. Figure 4 In the control group (A), the activity of HMGR enzyme was also significantly inhibited, decreasing by 35% compared to the control. Figure 4 (B). Meanwhile, the content of its downstream product, mevalonic acid (MVA), decreased significantly by 7%. Figure 4 (C). The above results indicate that LiHMGR positively regulates the synthesis of steroidal saponins.
[0062] Example 4
[0063] Obtaining LiHMGR transient overexpression plants in lily and identifying their steroidal saponin content.
[0064] The pSuper1300-LiHMGR recombinant vector was constructed, and Agrobacterium-mediated infection of lily bulbs was used to clarify the role of LiHMGR in the synthesis of lily steroidal saponins. qRT-PCR was used to detect endogenous LiHMGR expression to select plants with effective overexpression for further analysis. Results showed that LiHMGR was successfully overexpressed, increasing expression by 5.2-fold (…). Figure 5 (A). The enzyme activity of HMGR was then measured, and the results showed a significant increase of 35% ( ). Figure 5 In the control group (B), the content of mevalonic acid increased by 24% compared to the control group. Figure 5 (C). The above results indicate that overexpression of LiHMGR can significantly enhance the activity of HMGR enzyme, thereby increasing the synthesis of steroidal saponins.
Claims
1. A LiHMGR gene, the nucleotide sequence of which is shown in SEQ ID NO.
9.
2. A LiHMGR protein, the amino acid sequence of which is shown in SEQ ID NO.
10.
3. A recombinant expression vector containing the LiHMGR gene as described in claim 1.
4. Recombinant microorganisms containing the LiHMGR gene of claim 1 or the recombinant expression vector of claim 3.
5. The application of the LiHMGR 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 steroidal saponins from lily bulbs.
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 LiHMGR gene inhibits the synthesis of steroidal saponins in lily bulbs; overexpression of the LiHMGR gene increases the synthesis of steroidal saponins in lily bulbs.