UGT gene cluster for regulating and controlling synthesis of steroid saponin of lily and application of UGT gene cluster

Abstract

The invention discloses a glycosyl transferase gene cluster for regulating and controlling lily steroid saponin biosynthesis and application of the glycosyl transferase gene cluster, and belongs to the field of plant genetic engineering, the gene cluster comprises three genes LiUGT82A1, LiUGT87A1 and LiUGT90A1, the nucleotide sequences of the genes LiUGT82A1, LiUGT87A1 and LiUGT90A1 are respectively shown as SEQ ID NO: 7, SEQ ID NO: 16 and SEQ ID NO: 25, and the amino acid sequences of coded proteins are respectively shown as SEQ ID NO: 8, SEQ ID NO: 17 and SEQ ID NO: 26; the three genes present an expression trend which is consistent with steroidal saponin accumulation in a lily'orange sunlight 'bulb development process, and expression of any gene is specifically inhibited through a virus-induced gene silencing technology, so that the contents of total saponins and timosaponin AIII in lily bulbs are remarkably reduced; the positive regulation effect on the synthesis of the steroid saponin is proved; the key UGT gene participating in synthesis of steroid saponin is cloned from lilium brownii and functionally verified, and core gene resources and theoretical basis are provided for lilium brownii saponin synthesis regulation mechanism analysis, molecular breeding and high-added-value product development.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the UGT gene cluster that regulates the synthesis of lily steroidal saponins and its applications. Background Technology

[0002] Lily (Lilium spp.) originated in my country. Its underground bulbs are rich in various nutrients and active substances, and it was one of the first plants included in the National Health Commission's list of plants with both medicinal and edible uses. With the improvement of living standards and the promotion of the "broad food concept," people are increasingly focusing on the diversified utilization of flower resources. As a multifunctional plant with ornamental, edible, and medicinal value, lily has broad development prospects. Currently, various active ingredients have been isolated and identified from lily bulbs, and studies have confirmed that they have anti-tumor, antidepressant, antioxidant, sedative, and hypnotic effects, and also have a certain therapeutic effect on lung inflammation and emphysema. The material basis of these pharmacological activities is mainly steroidal saponins. According to statistics, there are already 39 health food products containing lily raw materials (data from the State Administration for Market Regulation's Special Food Information Query Platform), of which 30 products have saponins as the main active ingredient, highlighting the core position of saponins in the edible and medicinal value of lily. Therefore, in-depth research on the biosynthesis and regulation mechanism of steroidal saponins in lily bulbs is of great significance for improving the quality of lilies and the added value of the industry.

[0003] The biosynthesis of steroidal saponins is mainly accomplished through the mevalonate (MVA) pathway in the cytoplasm. In this pathway, UDP-glycosyltransferases (UGTs), as key post-modification enzymes, are responsible for catalyzing the glycosylation of the sapogenin backbone, specifically transferring glycosyl groups from activated UDP-glycoside donors (such as UDP-glucose and UDP-galactose) to specific hydroxyl groups or other functional groups of the sapogenin, thereby significantly increasing the structural diversity and bioactivity of steroidal saponins. Previous studies have shown that in medicinal plants such as *Paris polyphylla*, multiple UGT genes have been identified as participating in the sequence glycosylation process of steroidal saponins, and a series of new compounds have been successfully obtained by utilizing their substrate heterogeneity (Yun Taowen et al., 2013; Chen et al., 2023). Targeted regulation of key genes in secondary metabolic pathways using modern biotechnologies such as genetic engineering and fermentation engineering has become an important strategy for increasing the yield of effective components in medicinal plants and alleviating resource shortages, and it is also a direction for the modernization of traditional Chinese medicine.

[0004] However, compared with other medicinal plants, research on the biosynthetic regulatory mechanisms of lily steroidal saponins is still relatively lagging. Currently, there are no publicly available reports on the cloning and functional identification of the key UGT gene regulating lily steroidal saponin synthesis. In particular, for lily varieties with important edible and medicinal value, such as "Orange Sunshine," the molecular regulatory network of steroidal saponin synthesis in their bulbs remains unclear, hindering the improvement of lily quality and industrial upgrading.

[0005] Therefore, cloning and identifying the key UGT gene involved in steroidal saponin synthesis in lily, and elucidating its expression pattern and function during bulb development, is of great theoretical and practical significance for understanding the biosynthetic pathway of lily steroidal saponins, realizing metabolic engineering regulation, cultivating new lily varieties with high saponin content, and promoting the transformation of the traditional lily industry into a high-value-added health food industry. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to address the shortcomings of existing technologies by providing a UGT gene cluster that regulates the synthesis of steroidal saponins in lilies and its applications. This invention clones three genes, LiUGT82A1, LiUGT87A1, and LiUGT90A1, from lilies and confirms that their expression trends during lily bulb development are consistent with the accumulation trends of steroidal saponins.

[0007] Technical solution: The present invention discloses an isolated glycosyltransferase gene that regulates the biosynthesis of lily steroidal saponins, wherein the gene is LiUGT82A1, LiUGT87A1, or LiUGT90A1; the nucleotide sequence of the LiUGT82A1 gene is shown in SEQ ID NO:7; the nucleotide sequence of the LiUGT87A1 gene is shown in SEQ ID NO:16; and the nucleotide sequence of the LiUGT90A1 gene is shown in SEQ ID NO:25.

[0008] A protein encoded by the aforementioned gene.

[0009] Furthermore, the amino acid sequence of the protein encoded by the LiUGT82A1 gene is shown in SEQ ID NO:8;

[0010] Alternatively, the protein encoded by the LiUGT87A1 gene has the amino acid sequence shown in SEQ ID NO:17;

[0011] Alternatively, the protein encoded by the LiUGT90A1 gene has the amino acid sequence shown in SEQ ID NO: 26.

[0012] A recombinant expression vector containing the above-mentioned genes.

[0013] Furthermore, the carrier is a VIGS carrier.

[0014] A recombinant microorganism containing the above-mentioned genes or the above-mentioned recombinant expression vector.

[0015] Furthermore, the microorganism is Agrobacterium or Escherichia coli.

[0016] The application of the aforementioned genes, proteins, recombinant expression vectors, or recombinant microorganisms in regulating the synthesis of lily steroidal saponins.

[0017] Furthermore, the lily is the 'Orange Sunshine' lily.

[0018] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows:

[0019] This invention cloned three genes, LiUGT82A1, LiUGT87A1, and LiUGT90A1, from lily and confirmed that their expression trends during lily bulb development were consistent with the accumulation trends of steroidal saponins. Transient silencing of these genes using VIGS technology significantly reduced the contents of total saponins and anemarrhena saponin AⅢ in lily bulbs, indicating that these three genes play a positive regulatory role in lily steroidal saponin synthesis. This invention provides key gene resources and theoretical basis for elucidating the regulatory mechanism of lily saponin synthesis and for high-quality lily molecular breeding. Attached Figure Description

[0020] Figure 1 Predicting the conservative structural domains of LiUGT82A1;

[0021] Figure 2 The phylogenetic tree of LiUGT82A1;

[0022] Figure 3 Expression analysis of LiUGT82A1 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; Petal; Filament; Anther; Style; Stem; Leaf; Root;

[0023] Figure 4 Transient silencing of the LiUGT82A1 gene in lily bulbs, where (A) is the detection of the silencing efficiency of LiUGT82A1, (B) is the detection of the total saponin content in the silenced lily bulbs, and (C) is the detection of the content of Anemarrhena saponin AⅢ in the silenced lily bulbs.

[0024] Figure 5Predicting the conservative structural domains of LiUGT87A1;

[0025] Figure 6 The phylogenetic tree of LiUGT87A1;

[0026] Figure 7 Expression analysis of LiUGT87A1 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; Petal; Filament; Anther; Style; Stem; Leaf; Root;

[0027] Figure 8 Transient silencing of the LiUGT87A1 gene in lily bulbs, where (A) is the detection of silencing efficiency of LiUGT87A1, (B) is the detection of total saponin content in silenced lily bulbs, and (C) is the detection of content of Anemarrhena saponin AⅢ in silenced lily bulbs.

[0028] Figure 9 Predicting the conservative structural domains of LiUGT90A1;

[0029] Figure 10 The phylogenetic tree of LiUGT90A1;

[0030] Figure 11 Expression analysis of LiUGT90A1 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; Petal; Filament; Anther; Style; Stem; Leaf; Root;

[0031] Figure 12 Transient silencing of the LiUGT90A1 gene in lily bulbs, where (A) is the detection of the silencing efficiency of LiUGT90A1, (B) is the detection of the total saponin content in the silenced lily bulbs, and (C) is the detection of the content of Anemarrhena saponin AⅢ in the silenced lily bulbs. Detailed Implementation

[0032] 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.

[0033] (I) About the LiUGT82A1 gene

[0034] 1. Plant materials and treatment

[0035] Bulbs of the lily 'Chengse Yangguang' (Lilium hybrid cultivar 'Chengse Yangguang') were collected from the Flower Germplasm Innovation Experimental Base of the Fengpu Campus of the Shanghai Academy of Agricultural Sciences. Bulbs of uniform maturity, 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.

[0036] 2. The test strains and vectors are shown in Table 1.

[0037] Table 1

[0038]

[0039] 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.

[0040] The experimental methods used in the following embodiments include:

[0041] 1. Carrier Construction

[0042] 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.

[0043] Table 2

[0044]

[0045] 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.

[0046] Table 3

[0047]

[0048] The fragment enzymatic digestion reaction system was denatured at 37℃ for 30 min and 80℃ for 20 min, and the liquid was recovered.

[0049] 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.

[0050] The primers used in the experiment, the LiUGT82A1 gene sequence, and the corresponding protein sequence it encodes are shown in Table 4.

[0051] Table 4

[0052]

[0053]

[0054] 2. Cloning of the LiUGT82A1 gene

[0055] The LiUGT82A1 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.

[0056] 3. Quantitative qRT-PCR analysis

[0057] 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.

[0058] 4. Virus-induced transient silencing system

[0059] To construct the VIGS silencing vector, a 250 bp specific fragment of the LiUGT82A1 gene, as shown in SEQ ID NO. 9, was constructed into the pTRV2 vector and named TRV2-LiUGT82A1. The vectors (TRV2-LiUGT82A1, TRV2, and TRV1) were transformed into 50 µL of semi-thawed Agrobacterium tumefaciens competent cells, aspirated and mixed, 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 at 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 with shaking at 28 °C at 200 rpm for 14 h. Colony PCR was then performed for detection. 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-LiUGT82A1-F and TRV2-LiUGT82A1-R.

[0060] 5. Total saponin content detection

[0061] The total saponin content of lily was determined using the vanillin method. Approximately 0.1 g of sample was weighed, and 2 mL of 80% ethanol extract was added and ground into a homogenate. The sample was then extracted using ultrasonic extraction at 55°C (300 W power) for 30 min. After centrifugation at 12,000 rpm for 10 min at room temperature, 30 μL of the supernatant was taken and 20 μL of vanillin solution was added. Then, 80 μL of sulfuric acid was added and mixed. The mixture was reacted at 60°C for 15 min and then placed in an ice box for 5 min. Subsequently, 500 μL of the solution was added and mixed. 200 μL of the solution was transferred to a 96 empty plate and measured at 540 nm.

[0062] 6. Detection of Anemarrhena saponin AⅢ content

[0063] A method for determining the content of anemarrhena saponin AⅢ in lily bulbs using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was employed. Lily bulb powder was passed through a No. 4 sieve, and approximately 0.5 g was accurately weighed into a test tube. The extract was ultrasonically extracted at 4 ℃ for 30 min, followed by centrifugation at 5,000 rpm for 5 min at 4 ℃. The supernatant was collected, and 1 mL of chromatographic acetonitrile was added to the precipitate. The extraction steps were repeated. The supernatants were then combined, dried under nitrogen, reconstituted with methanol, filtered through a 0.22 μm organic phase filter membrane, and stored at -20 ℃ for analysis. Chromatographic conditions: Agilent Poroshell 120 EC-C18 column (2.7µm, 3.0 x 100 mm); mobile phase: A:B = (0.1% formic acid water): (chromatographic acetonitrile) solution gradient elution (0 min, 90% A; 0–6 min, 1% A; 6–9 min, 1% A; 9–9.1 min, 90% A; 9.1–13 min, 90% A); column temperature: 30℃; flow rate: 0.4 mL / min. Mass spectrometry conditions: ionization mode: electrospray ionization (ESI), multi-stage reaction monitoring (MRM), positive and negative ion switching scan.

[0064] Example 1

[0065] Screening of LiUGT82A1 gene

[0066] The inventors conducted a combined analysis of the metabolome and transcriptome of the 'Orange Sunshine' lily bulbs throughout their entire growth period, and screened for enzymes related to steroidal saponin synthesis through correlation analysis. Analysis of their conserved domains revealed the presence of a Glycosyltransferase_GTB-type 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 1338 bp coding region (see SEQ ID NO. 7) was successfully cloned, encoding 445 amino acids (see SEQ ID NO. 8), with a theoretical molecular mass of 49.4 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 UGT90A1 of Acorus gramineus. Based on the NCBI comparison results, it was named LiUGT82A1. Figure 2 ).

[0067] Example 2

[0068] Tissue-specific expression analysis of LiUGT82A1 in lily

[0069] The expression characteristics of LiUGT82A1 in different flower development stages and tissues of lilies were analyzed. The expression of LiUGT82A1 throughout the entire growth period showed an overall trend of first decreasing, then increasing, and then decreasing again. The highest expression level was observed in stage S6, while the relative expression level approached 0 in stages S2-S5. Figure 3 (A). In different tissues, LiUGT82A1 was highly expressed in roots, with no significant difference in other tissues, followed by bulbs and anthers, and lowest expression in petals and ovaries. Figure 3 (B)

[0070] Example 3

[0071] Obtaining transiently silenced lily plants (LiUGT82A1) and identifying their steroidal saponin content.

[0072] To clarify whether LiUGT82A1 plays a role in the synthesis of steroidal saponins in 'Orange Sunshine' lily bulbs, transient silencing function was verified using VIGS technology. First, a TRV2-LiUGT82A1 recombinant vector was constructed and infected with Agrobacterium-mediated transformation of dormant lily bulbs. Silent plants were selected using qRT-PCR. The expression of the LiUGT82A1 transcript was significantly downregulated, at 20.6% of the control. Figure 4 In the control group (A), the total saponin content was also significantly inhibited, decreasing by 20.9% compared to the control. Figure 4 In the middle B), the content of Anemarrhena saponin AⅢ also decreased by 31.9% ( Figure 4 (C). The above results indicate that LiUGT82A1 positively regulates the synthesis of steroidal saponins.

[0073] (II) Regarding the LiUGT87A1 gene

[0074] The materials used in the following embodiments include:

[0075] 1. Plant materials and treatment

[0076] Bulbs of the lily 'Chengse Yangguang' (Lilium hybrid cultivar 'Chengse Yangguang') were collected from the Flower Germplasm Innovation Experimental Base of the Fengpu Campus of the Shanghai Academy of Agricultural Sciences. Bulbs of uniform maturity, 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.

[0077] 2. The test strains and vectors are shown in Table 5.

[0078] Table 5

[0079]

[0080] 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.

[0081] The experimental methods used in the following embodiments include:

[0082] 1. Carrier Construction

[0083] 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 6.

[0084] Table 6

[0085]

[0086] 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 7.

[0087] Table 7

[0088]

[0089] The fragment enzymatic digestion reaction system was denatured at 37℃ for 30 min and 80℃ for 20 min, and the liquid was recovered.

[0090] 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.

[0091] The primers used in the experiment, the LiUGT87A1 gene sequence, and the corresponding protein sequence it encodes are shown in Table 8.

[0092] Table 8

[0093]

[0094]

[0095] 2. Cloning of the LiUGT87A1 gene

[0096] The LiUGT87A1 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.

[0097] 3. Quantitative qRT-PCR analysis

[0098] 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.

[0099] 4. Virus-induced transient silencing system

[0100] To construct the VIGS silencing vector, a 250 bp specific fragment of the LiUGT87A1 gene, as shown in SEQ ID NO. 9, was constructed into the pTRV2 vector and named TRV2-LiUGT87A1. The vectors (TRV2-LiUGT87A1, TRV2, and TRV1) were transformed into 50 µL of semi-thawed Agrobacterium tumefaciens competent cells, aspirated and mixed, 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 at 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 with shaking at 28 °C at 200 rpm for 14 h. Colony PCR was then performed for detection. 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-LiUGT87A1-F and TRV2-LiUGT87A1-R.

[0101] 5. Total saponin content detection

[0102] The total saponin content of lily was determined using the vanillin method. Approximately 0.1 g of sample was weighed, and 2 mL of 80% ethanol extract was added and ground into a homogenate. The sample was then extracted using ultrasonic extraction at 55°C (300 W power) for 30 min. After centrifugation at 12,000 rpm for 10 min at room temperature, 30 μL of the supernatant was taken and 20 μL of vanillin solution was added. Then, 80 μL of sulfuric acid was added and mixed. The mixture was reacted at 60°C for 15 min and then placed in an ice box for 5 min. Subsequently, 500 μL of the solution was added and mixed. 200 μL of the solution was transferred to a 96 empty plate and measured at 540 nm.

[0103] 6. Detection of Anemarrhena saponin AⅢ content

[0104] A method for determining the content of anemarrhena saponin AⅢ in lily bulbs using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was employed. Lily bulb powder was passed through a No. 4 sieve, and approximately 0.5 g was accurately weighed into a test tube. The extract was ultrasonically extracted at 4 ℃ for 30 min, followed by centrifugation at 5,000 rpm for 5 min at 4 ℃. The supernatant was collected, and 1 mL of chromatographic acetonitrile was added to the precipitate. The extraction steps were repeated. The supernatants were then combined, dried under nitrogen, reconstituted with methanol, filtered through a 0.22 μm organic phase filter membrane, and stored at -20 ℃ for analysis. Chromatographic conditions: Agilent Poroshell 120 EC-C18 column (2.7µm, 3.0 x 100 mm); mobile phase: A:B = (0.1% formic acid water): (chromatographic acetonitrile) solution gradient elution (0 min, 90% A; 0–6 min, 1% A; 6–9 min, 1% A; 9–9.1 min, 90% A; 9.1–13 min, 90% A); column temperature: 30℃; flow rate: 0.4 mL / min. Mass spectrometry conditions: ionization mode: electrospray ionization (ESI), multi-stage reaction monitoring (MRM), positive and negative ion switching scan.

[0105] Example 4

[0106] Screening of LiUGT87A1 gene

[0107] The inventors conducted a combined analysis of the metabolome and transcriptome of the 'Orange Sunshine' lily bulbs throughout their entire growth period, and screened for enzymes related to steroidal saponin synthesis through correlation analysis. Analysis of their conserved domains revealed the presence of a Glycosyltransferase_GTB-type domain. Figure 5The gene coding region sequence was obtained from the lily bulb development transcriptome database. Using 'Orange Sunshine' cDNA as a template, the full-length 1428 bp coding region (see SEQ ID NO. 16) was successfully cloned, encoding 476 amino acids (see SEQ ID NO. 17), with a theoretical molecular mass of 48.9 kDa. Using the BLAST function on the NCBI website, proteins from 10 other plants with high similarity were downloaded for homology comparison. The results showed high homology with UGT87A1 of Cinnamomum micranthum. Based on the NCBI comparison results, it was named LiUGT87A1. Figure 6 ).

[0108] Example 5

[0109] Tissue-specific expression analysis of LiUGT87A1 in lily

[0110] The expression characteristics of LiUGT87A1 in different flower development stages and tissues of lilies were analyzed. The expression of LiUGT87A1 throughout the entire growth period showed an overall trend of first decreasing, then increasing, and then decreasing again. The highest expression level was observed in stage S6, while the relative expression level approached 0 in stages S2-S4. Figure 7 (A). In different tissues, LiUGT87A1 was highly expressed in roots, bulbs, and ovaries, with the highest expression level in bulbs and the lowest expression level in stems and filaments. Figure 7 (B)

[0111] Example 6

[0112] Obtaining transiently silenced lily plants (LiUGT87A1) and identifying their steroidal saponin content.

[0113] To clarify whether LiUGT87A1 plays a role in the synthesis of steroidal saponins in the bulbs of 'Orange Sunshine' lily, transient silencing function was verified using VIGS technology. First, a TRV2-LiUGT87A1 recombinant vector was constructed and infected with dormant lily bulbs via Agrobacterium-mediated infection. Silent plants were selected using qRT-PCR. The expression of the LiUGT87A1 transcript was significantly downregulated, at 7.4% of the control. Figure 8 In the control group (A), the total saponin content was also significantly inhibited, decreasing by 23.4% compared to the control. Figure 8 In the middle B), the content of Anemarrhena saponin AⅢ also decreased significantly by 45.2% ( Figure 8 (C). The above results indicate that LiUGT87A1 positively regulates the synthesis of steroidal saponins.

[0114] (III) Regarding the LiUGT90A1 gene

[0115] The materials used in the following embodiments include:

[0116] 1. Plant materials and treatment

[0117] Bulbs of the lily 'Chengse Yangguang' (Lilium hybrid cultivar 'Chengse Yangguang') were collected from the Flower Germplasm Innovation Experimental Base of the Fengpu Campus of the Shanghai Academy of Agricultural Sciences. Bulbs of uniform maturity, 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.

[0118] 2. The test strains and vectors are shown in Table 9.

[0119] Table 9

[0120]

[0121] 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.

[0122] The experimental methods used in the following embodiments include:

[0123] 1. Carrier Construction

[0124] 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 10.

[0125] Table 10

[0126]

[0127] 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 11.

[0128] Table 11

[0129]

[0130] The fragment enzymatic digestion reaction system was denatured at 37℃ for 30 min and 80℃ for 20 min, and the liquid was recovered.

[0131] 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.

[0132] The primers used in the experiment, the LiUGT90A1 gene sequence, and the corresponding protein sequence it encodes are shown in Table 12.

[0133] Table 12

[0134]

[0135]

[0136] 2. Cloning of the LiUGT90A1 gene

[0137] The LiUGT90A1 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.

[0138] 3. Quantitative qRT-PCR analysis

[0139] 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 12. Primers ending in -F are forward primers, and primers ending in -R are reverse primers.

[0140] 4. Virus-induced transient silencing system

[0141] To construct the VIGS silencing vector, a 250 bp specific fragment of the LiUGT90A1 gene, as shown in SEQ ID NO. 27, was constructed into the pTRV2 vector and named TRV2-LiUGT90A1. The vectors (TRV2-LiUGT90A1, TRV2, and TRV1) were transformed into 50 µL of semi-thawed Agrobacterium tumefaciens competent cells, aspirated and mixed, 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 at 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 with shaking at 28 °C at 200 rpm for 14 h. Colony PCR was then performed for detection. 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-LiUGT90A1-F and TRV2-LiUGT90A1-R.

[0142] 5. Total saponin content detection

[0143] The total saponin content of lily was determined using the vanillin method. Approximately 0.1 g of sample was weighed, and 2 mL of 80% ethanol extract was added and ground into a homogenate. The sample was then extracted using ultrasonic extraction at 55°C (300 W power) for 30 min. After centrifugation at 12,000 rpm for 10 min at room temperature, 30 μL of the supernatant was taken and 20 μL of vanillin solution was added. Then, 80 μL of sulfuric acid was added and mixed. The mixture was reacted at 60°C for 15 min and then placed in an ice box for 5 min. Subsequently, 500 μL of the solution was added and mixed. 200 μL of the solution was transferred to a 96 empty plate and measured at 540 nm.

[0144] 6. Detection of Anemarrhena saponin AⅢ content

[0145] A method for determining the content of anemarrhena saponin AⅢ in lily bulbs using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was employed. Lily bulb powder was passed through a No. 4 sieve, and approximately 0.5 g was accurately weighed into a test tube. The extract was ultrasonically extracted at 4 ℃ for 30 min, followed by centrifugation at 5,000 rpm for 5 min at 4 ℃. The supernatant was collected, and 1 mL of chromatographic acetonitrile was added to the precipitate. The extraction steps were repeated. The supernatants were then combined, dried under nitrogen, reconstituted with methanol, filtered through a 0.22 μm organic phase filter membrane, and stored at -20 ℃ for analysis. Chromatographic conditions: Agilent Poroshell 120 EC-C18 column (2.7µm, 3.0 x 100 mm); mobile phase: A:B = (0.1% formic acid water): (chromatographic acetonitrile) solution gradient elution (0 min, 90% A; 0–6 min, 1% A; 6–9 min, 1% A; 9–9.1 min, 90% A; 9.1–13 min, 90% A); column temperature: 30℃; flow rate: 0.4 mL / min. Mass spectrometry conditions: ionization mode: electrospray ionization (ESI), multi-stage reaction monitoring (MRM), positive and negative ion switching scan.

[0146] Example 7

[0147] Screening of LiUGT90A1 gene

[0148] The inventors conducted a combined analysis of the metabolome and transcriptome of the 'Orange Sunshine' lily bulbs throughout their entire growth period, and screened for enzymes related to steroidal saponin synthesis through correlation analysis. Analysis of their conserved domains revealed the presence of a Glycosyltransferase_GTB-type domain. Figure 9The gene coding region sequence was obtained from the lily bulb development transcriptome database. Using 'Orange Sunshine' cDNA as a template, the full-length 1410 bp coding region (see SEQ ID NO. 25) was successfully cloned, encoding 470 amino acids (see SEQ ID NO. 26), with a theoretical molecular mass of 52.0 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 UGT90A1 of coconut palm protein (Cocos nucifera). Based on the NCBI comparison results, it was named LiUGT90A1. Figure 10 ).

[0149] Example 8

[0150] Tissue-specific expression analysis of LiUGT90A1 in lily

[0151] The expression characteristics of LiUGT90A1 in different flower development stages and tissues of lilies were analyzed. The expression of LiUGT90A1 showed an overall trend of first decreasing and then increasing throughout the entire growth period, with the highest expression level observed in stage S7, which was 5.2 times higher than that in stage S3, the lowest expression level. Figure 11 (A). In different tissues, LiUGT90A1 was highly expressed in roots and anthers, with the highest expression level in anthers, while there were no significant differences in other tissues, and the lowest expression level was found in petals and bulbs. Figure 11 (B)

[0152] Example 9

[0153] Obtaining transiently silenced lily plants (LiUGT90A1) and identifying their steroidal saponin content.

[0154] To clarify whether LiUGT90A1 plays a role in the synthesis of steroidal saponins in the bulbs of 'Orange Sunshine' lily, transient silencing function was verified using VIGS technology. First, a TRV2-LiUGT90A1 recombinant vector was constructed and infected with dormant lily bulbs via Agrobacterium-mediated infection. Silent plants were selected using qRT-PCR. The expression of the LiUGT90A1 transcript was significantly downregulated, at 40.6% of the control. Figure 12 In the control group (A), the total saponin content was also significantly inhibited, decreasing by 24.5% compared to the control. Figure 12 In the middle B), the content of Anemarrhena saponin AⅢ also decreased significantly by 48.0%. Figure 12 (C). The above results indicate that LiUGT90A1 positively regulates the synthesis of steroidal saponins.

[0155] In the above embodiments, the sequence list corresponding to SEQ ID NO: 1-27 has been submitted separately, wherein: SEQ ID NO: 1-9 correspond to LiUGT82A1 related sequences; SEQ ID NO: 10-18 correspond to LiUGT87A1 related sequences; and SEQ ID NO: 19-27 correspond to LiUGT90A1 related sequences. The accuracy and completeness of all sequences have been verified by sequencing.

[0156] 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. An isolated glycosyltransferase gene regulating the biosynthesis of lily steroidal saponins, characterized in that: The gene is LiUGT82A1, LiUGT87A1, or LiUGT90A1; The nucleotide sequence of the LiUGT82A1 gene is shown in SEQ ID NO: 7; The nucleotide sequence of the LiUGT87A1 gene is shown in SEQ ID NO: 16; The nucleotide sequence of the LiUGT90A1 gene is shown in SEQ ID NO:

25.

2. A protein encoded by the gene of claim 1.

3. The protein according to claim 2, characterized in that: The protein encoded by the LiUGT82A1 gene has the amino acid sequence shown in SEQ ID NO: 8; The protein encoded by the LiUGT87A1 gene has the amino acid sequence shown in SEQ ID NO:17; The protein encoded by the LiUGT90A1 gene has the amino acid sequence shown in SEQ ID NO:

26.

4. A recombinant expression vector containing the gene of claim 1.

5. The recombinant expression vector according to claim 4, characterized in that, The carrier is a VIGS carrier.

6. A recombinant microorganism containing the gene of claim 1, or the recombinant expression vector of claim 4 or 5.

7. The recombinant microorganism according to claim 6, characterized in that, The microorganisms mentioned are Agrobacterium or Escherichia coli.

8. The use of the gene of claim 1, the protein of claim 2 or 3, the recombinant expression vector of claim 4 or 5, or the recombinant microorganism of claim 6 or 7 in regulating the synthesis of lily steroidal saponins.

9. The application according to claim 8, characterized in that, The lily in question is the 'Orange Sunshine' lily.

Images