Application of Lycium ruthenicum LrAOMT gene and its encoded protein in regulating anthocyanin biosynthesis in Lycium barbarum
By cloning and overexpressing the LrAOMT gene of the black fruit wolfberry and its encoding protein, recombinant expression vectors were constructed and Agrobacterium was transformed, the problem of insufficient anthocyanins content of wolfberry wolfberry was solved, and the content of anthocyanins such as delphinium chloride-3-O-galactoside was significantly improved, and its medicinal value and application potential were expanded.
Patent Information
- Application Number
- CN202411304814.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-19
- Publication Date
- 2025-07-08
- Estimated Expiration
- 2044-09-19
AI Technical Summary
The lack of effective regulatory measures in the prior art to improve the biosynthesis of anthocyanins of black fruit wolfberry, resulting in insufficient content in plants, limiting the medicinal value and application potential of anthocyanins.
By cloning and overexpressing the LrAOMT gene of black fruit wolfberry and its encoding protein, recombinant expression vectors were constructed and Agrobacterium was transformed. The recombinant bacteria were used to improve the synthesis of anthocyanins in plants, especially the content of delphinin-3-O-galactosin chloride, paeonin chloride-3-O-glucoside, cornflower-O-sylic acid, cornflower-3-glucoside and proanthocyanin B1.
The above-mentioned anthocyanin compounds in overexpressed plants have been significantly increased, and new regulatory measures have been provided, new ways to biosynthesis and genetic improvement of anthocyanins, which have enhanced the medicinal value and application potential of plants.
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Figure CN119351413B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to Lycium ruthenicum LrAOMT genes and their encoded proteins in regulating the biosynthesis of wolfberry anthocyanins. Background Art
[0002] Lycium ruthenicum Lycium ruthenicum Murr.) is rich in anthocyanins, and the content of anthocyanins is higher than that of blackcurrant or blueberry. Anthocyanins are a class of plant-specific water-soluble natural pigments. Different anthocyanins have different biological activities, such as antioxidant, anti-inflammatory, anti-tumor, etc., and have high medicinal value; they can also be used as natural food pigments in food. Moreover, some anthocyanins also play an important role in promoting plant reproduction and protecting plants from biotic and abiotic stresses.
[0003] The anthocyanin biosynthesis pathway has been relatively well studied in plants, mainly including structural genes such as PAL, C4H, 4CL, CHS, CHI, F3H, F3'H, F3'5'H, DFR, ANS, and UFGT. Different anthocyanins have different biological activities or uses. It is necessary to continue to study the genes regulating anthocyanin synthesis to provide more options and references for the biosynthesis and genetic improvement of wolfberry anthocyanins. Summary of the Invention
[0004] Aiming at the above technical problems, the present invention provides the application of Lycium ruthenicum LrAOMT genes and their encoded proteins in regulating the biosynthesis of wolfberry anthocyanins. The present invention discovers for the first time LrAOMT genes can participate in the biosynthesis of anthocyanins and regulate the accumulation of anthocyanin compounds, thereby providing a new way to obtain anthocyanin compounds.
[0005] To achieve the above invention object, the present invention adopts the following technical scheme:
[0006] The first aspect of the present invention provides the application of Lycium ruthenicum LrAOMT genes in increasing the content of plant anthocyanins, and the nucleotide sequence of the Lycium ruthenicum LrAOMT gene is as shown in SEQ ID NO.1, or a protein encoded by an amino acid sequence as shown in SEQ ID NO.2.
[0007] The present invention discovers through research that LrAOMT genes have a significant regulatory effect on the biosynthesis of anthocyanins. Overexpression of LrAOMTIn plants with the gene, delphinidin 3-O-galactoside chloride (CAS No.: 28500-00-7), peonidin-3-O-glucoside chlorid (CAS No.: 6906-39-4), cyanidin O-syringic (the structural formula is as shown in Formula I), cyanidin 3-glucoside (the structural formula is as shown in Formula II), and procyanidin B1 (CAS No.: 20315-25-7) were significantly up-regulated.
[0008]
[0009] Formula I
[0010]
[0011] Formula II
[0012] The antioxidant capacities of different types of anthocyanins vary. It is reported that delphinidin-type anthocyanins have the highest antioxidant activity, followed by cyanidin-type anthocyanins. Delphinidin 3-O-galactoside not only has antioxidant effects but also has anti-inflammatory, anti-excitatory, and antibacterial effects. Cyanidin O-syringic has significant antioxidant capacity, can inhibit the inflammatory response, and also shows certain effects in cancer research. Cyanidin 3-glucoside has various healthcare effects such as significant antioxidant, anti-tumor, protecting the nervous system, restoring transient visual impairment, and reducing glomerulosclerosis and renal interstitial fibrosis in patients with diabetic nephropathy. It can also be used as a plant protectant to protect cotton leaves from the damage of cotton bollworms. Although the antioxidant effect of peonidin-3-O-glucoside is relatively low, it has the effect of increasing the glucose uptake of HepG2 cells and can be used as an active substance for insulin secretion promotion in type 2 diabetes complications. By overexpressing the LrAOMT gene, the biosynthesis of the above-mentioned anthocyanin compounds can be selectively regulated, providing a new technical means for obtaining the above-mentioned anthocyanin compounds.
[0013] Preferably, the plant is Lycium barbarum.
[0014] Preferably, the anthocyanins include delphinidin 3-O-galactoside chloride, peonidin-3-O-glucoside chlorid, cyanidin O-syringic, cyanidin 3-glucoside, and procyanidin B1.
[0015] The second aspect of the present invention provides the use of a protein with an amino acid sequence shown in SEQ ID NO. 2 in increasing the anthocyanin content of plants.
[0016] Preferably, the plant is Lycium barbarum.
[0017] Preferably, the anthocyanins include delphinidin-3-O-galactoside chloride, peonidin-3-O-glucoside chloride, cyanidin-O-syringate, cyanidin-3-glucoside, and procyanidin B1.
[0018] The third aspect of the present invention provides a recombinant expression vector overexpressing the above-mentioned Lycium ruthenicum LrAOMT gene.
[0019] Preferably, the recombinant expression vector includes a recombinant plasmid and a recombinant bacterium.
[0020] More preferably, the recombinant bacterium is Agrobacterium.
[0021] The fourth aspect of the present invention provides a method for constructing a recombinant plasmid overexpressing the above-mentioned Lycium ruthenicum LrAOMT gene: Using primers OE- LrAOMT -F with a sequence shown in SEQ ID NO. 5 and primers OE- LrAOMT -R with a sequence shown in SEQ ID NO. 6, transform the Lycium ruthenicum LrAOMT gene into the plasmid, and the recombinant plasmid overexpressing the Lycium ruthenicum LrAOMT gene is obtained.
[0022] Preferably, the plasmid is pCambia 1300-35s.
[0023] The fifth aspect of the present invention provides a method for constructing a recombinant bacterium overexpressing the above-mentioned Lycium ruthenicum LrAOMT gene: Using primers OE- LrAOMT -F with a sequence shown in SEQ ID NO. 5 and primers OE- LrAOMT -R with a sequence shown in SEQ ID NO. 6 to construct a recombinant plasmid, and transform the recombinant plasmid into Agrobacterium, and the recombinant bacterium overexpressing the Lycium ruthenicum LrAOMT gene is obtained.
[0024] Preferably, the plasmid is pCambia 1300-35s.
[0025] Preferably, the Agrobacterium is Agrobacterium GS115, Agrobacterium LBA4404, Agrobacterium GV3101, or Agrobacterium EHA105.
[0026] The sixth aspect of the present invention provides the above-mentioned recombinant Lycium ruthenicum LrAOMTApplication of Recombinant Bacteria of Gene in Improving Anthocyanin Content in Plants.
[0027] Preferably, the recombinant bacteria is Agrobacterium tumefaciens GV3101.
[0028] Preferably, the plant is Lycium barbarum.
[0029] Preferably, the anthocyanins include delphinidin-3-O-galactoside chloride, peonidin-3-O-glucoside chloride, cyanidin-O-syringate, cyanidin-3-glucoside and procyanidin B1.
[0030] Preferably, the bacterial suspension of the above recombinant bacteria is injected into the plant.
[0031] The beneficial effect of the present invention is that: the gene is first identified from Lycium barbarum in the present invention, and it is first discovered through techniques such as homologous cloning, transient transformation of Lycium barbarum, and fluorescence quantitative detection that LrAOMT the gene can regulate the biosynthesis of anthocyanin compounds. LrAOMT In the plants with overexpression of the gene, the contents of delphinidin-3-O-galactoside chloride, peonidin-3-O-glucoside chloride, cyanidin-O-syringate, cyanidin-3-glucoside and procyanidin B1 increase extremely significantly, while the content of rhamnoglucoside chloride of cyanidin decreases significantly. Therefore, LrAOMT the gene can be used to regulate the quality of Lycium barbarum and provides a new target for the biosynthesis of the above anthocyanin compounds. The construction method of the recombinant plasmid and recombinant bacteria with overexpression of LrAOMT the gene of Lycium ruthenicum in the present invention can obtain a recombinant expression vector with overexpression of LrAOMT the gene, and the obtained recombinant expression vector can be applied to the biosynthesis of the above anthocyanin compounds. LrAOMT Description of the Drawings
[0032] Figure 1 is the expression level of LrAOMT gene in the overexpression plants and the control in Example 1 of the present invention; LrAOMT
[0033] Figure 2 is the content of anthocyanin compounds in the overexpression plants and the control of OE- LrAOMT in Example 1 of the present invention. Detailed Embodiments
[0034] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.
[0035] Anthocyanin is one of the most important nutritional components of Lycium ruthenicum Murr. Through the comprehensive application of techniques such as homologous cloning, transcriptome differential expression, and transient transformation detection of Lycium barbarum, the present invention has found important structural genes that regulate the biosynthesis of anthocyanin in Lycium ruthenicum. LrAOMT Through experimental research, it has been found that this gene can be effectively applied to regulate the biosynthesis of anthocyanin in Lycium ruthenicum: positively mediating the biosynthesis of delphinidin 3-O-galactoside chloride, peonidin-3-O-glucoside chlorid, cyanidin O-syringic, cyanidin 3-glucoside, and procyanidin B1.
[0036] Based on the above research results, the embodiments of the present invention provide the application of the Lycium ruthenicum LrAOMT gene in increasing the anthocyanin content of plants. The nucleotide sequence of the Lycium ruthenicum LrAOMT gene is shown in SEQ ID NO.1, or the protein encoded by the amino acid sequence shown in SEQ ID NO.2. Without affecting the protein structure and activity, the amino acid sequences obtained by substituting, deleting, or adding one or more amino acids or terminal modifications to the amino acid sequence shown in SEQ ID NO.2, as well as the nucleotide sequences encoding them, also fall within the protection scope of the present invention.
[0037] The embodiments of the present invention also provide a recombinant expression vector overexpressing the above-mentioned Lycium ruthenicum LrAOMT gene and its construction method.
[0038] The following will be further described in conjunction with specific embodiments.
[0039] Unless otherwise specified, the raw materials, reagents, drugs, or instruments used in the following examples are all conventional commercially available products obtained through commercial channels. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art.
[0040] Example 1
[0041] This example provides the cloning and functional verification of the genes of Lycium ruthenicum Murr. LrAOMT
[0042] 1. Cloning of the genes of Lycium ruthenicum Murr. LrAOMT
[0043] The total RNA of Lycium ruthenicum Murr. fruits was isolated using the RNA extraction kit from Takara (Takara, Dalian, China). The single-stranded cDNA of the LrAOMT gene was prepared using the Reverse Aid First-strand cDNA Synthesis Kit from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). The open reading frame (CDS) of the LrAOMT gene was amplified using primers LrAOMT -F and LrAOMT -R. The components of the amplification system were as follows: 25 μL of 2×PCR buffer, 10 μL of dNTP (2 mM), 2 μL of the upstream primer (10 μM), 2 μL of the downstream primer (10 μM), 5 μL of single-stranded cDNA, 1 μL of KOD FX Neo (Toyobo Life Science, Osaka, Japan), and 10 μL of Millipore H2O. The PCR reaction program was set as follows: pre-denaturation at 98°C for 3 minutes, followed by 30 cycles, each cycle including denaturation for 10 seconds, annealing at 58°C for 30 seconds, and extension at 68°C for 2 minutes; finally, extension at 68°C for 5 minutes. The amplification products were purified and independently cloned into the pMD18-T vector (Toyobo LifeScience, Osaka, Japan) for Sanger sequencing.
[0044] LrAOMT -F (shown in SEQ ID NO. 3): ATGGCTTCGCTTTCCAACTC;
[0045] LrAOMT -R (shown in SEQ ID NO. 4): TCACATGAGTCGCCTGCAAAC.
[0046] LrAOMT The full length of the open reading frame (CDS) is 720 bp, and the nucleotide sequence is shown in SEQ ID NO. 1:
[0047] ATGGCTTCGCTTTCCAACTCTAATTCCGACTCCAACTCCAAGGGACTGTTGCAGAGTCGAGAATTGCATGAGTATGTACTGGAGACTGCTGTGTACCCACGGGAGCCAGAGCTTCTCAAAGAGATCAGAGTTATCACTACAAATCATCCAGAGTGTATAATGACAACTGCACCAGAAGCAGGCCAACTGATGGCGTTGCTCTTGAAACTGACAAACGCTAAAAAGACAATTGAAATTGGAGTGTTCACTGGATACTCTTTGCTCCTCACAGCCCTTACAATTCCTGAAGATGGAAAGATTACGGCTATAGATCTGAATCGGGATACATATGAGATGGGATTGCCAGTTATCAAAAAGGCTGGAGTTGAGCACAAAATCAATTTCATTCAGTCGCCAGCATTATTAGCCCTTGATGTACTCTTGAAAGATAATGACAATAAAGGAAGTTTCGATTTTGCTTTTGTTGATGCGGACAAAGTTAGCTATCAAAAGTACCATGAGAGACTGGTGGAGTTGGTGAAGGTGGGTGGTATAATAGTGTATGATAATACACTCTGGTTTGGAACAGTTGCTATGCCAGAGGAGTTCGTAAGGGAAGGACTGAAACCAAACAGGCATCACATCATTGAATTTAATAAATTTGTAGCTGCTGATACTCGCATTCAAATTTCTCAAGTCCCTTTAGGTGATGGAATCACCGTTTGCAGGCGACTCATGTGA。
[0048] LrAOMT The gene encodes 239 amino acids, and the sequence is shown in SEQ ID NO. 2:
[0049] MASLSNSNSDSNSKGLLQSRELHEYVLETAVYPREPELLKEIRVITTNHPECIMTTAPEAGQLMALLLKLTNAKKTIEIGVFTGYSLLLTALTIPEDGKITAIDLNRDTYEMGLPVIKKA GVEHKINFIQSPALLALDVLLKDNDNKGSFDFAFVDADKVSYQKYHERLVELVKVGGIIVYDNTLWFGTVAMPEEFVREGLKPNRHHIIEFNKFVAADTRIQISQVPLGDGITVCRRLM.
[0050] 2. Black wolfberry LrAOMT Functional verification of genes
[0051] 2.1 Transient overexpression transformation
[0052] Using primer OE- LrAOMT -F and OE- LrAOMT -R, clone the obtained LrAOMT The sequence was constructed into the pCambia 1300-35s vector and OE- LrAOMT Overexpression vector. LrAOMT The overexpression vector was transformed into Agrobacterium GV3101 to obtain OE- LrAOMT Overexpression engineered bacteria. The engineered bacteria were adjusted to an OD of 0.04 in infection buffer (5 g / L D-glucose, 50 mM MES, 2 mM Na3PO4·12 H2O and 0.1 mM acetobutanone). 600 The concentration of 0.5-0.6 was injected into the leaves of Ningxia wolfberry seedlings that had been cultured for 30 days in advance, and 5-10 mL was injected per seedling; pCambia1300-35 s-GFP was used as a negative control. The leaves injected with the engineered bacterial suspension were collected 72 hours after injection for subsequent real-time fluorescence quantitative PCR (qRT-PCR) and anthocyanin content detection.
[0053] OE- LrAOMT -F (as shown in SEQ ID NO. 5): TTCCGACTCCAACTCCAAGG;
[0054] OE- LrAOMT -R (as shown in SEQ ID NO. 6): TCAGTTTCAAGAGCAACGCC.
[0055] 2.2 qRT-PCR detection
[0056] Using Lycium barbarum Ef1a as the internal reference gene, the SYBR Green dye method was adopted, and the BIO-RAD fluorescence quantitative PCR instrument was used to detect and analyze the overexpression of the LrAOMT gene in Lycium barbarum leaves (primers: CCATACCAGCATCACCATTCTTC, as shown in SEQ ID NO. 7; GTCACACTTCCCACATTGCC, as shown in SEQ ID NO. 8).
[0057] The qRT-PCR reaction system was: 12 μL Power SYBR® Green PCR Master Mix, 1 μL qRTF (10 μM), 5 μL cDNA template, and 20 μL ddH2O; the reaction conditions were: pre-denaturation at 95.0 °C for 5 min; denaturation at 95.0 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 20 s, (40 cycles); the melting curve rose from 65 °C to 95 °C, with an increase of 0.5 °C for each reading. The 2 −ΔΔCT method was used to determine the relative expression level of the target gene.
[0058] By detecting the OE- LrAOMT overexpression plants and the control, the results showed that in the OE- LrAOMT overexpression plants, the LrAOMT gene expression was significantly higher than that of GFP, as Figure 1 shown, and this construction method achieved the LrAOMT overexpression of the gene.
[0059] 2.3 Detection of anthocyanin content
[0060] The liquid chromatography tandem mass spectrometry (LC-MS / MS) method was used to determine the anthocyanin content of the OE- LrAOMT overexpression plants.
[0061] 2.3.1 Sample pretreatment
[0062] Accurately weigh 0.2 g of the sample into a 2 ml centrifuge tube, add 1 ml of 5% formic acid, extract on ice for 30 min, centrifuge at 12000 rpm for 10 min, and take the supernatant for LC-MS / MS analysis.
[0063] 2.3.2 Chromatography and mass spectrometry acquisition conditions
[0064] The chromatographic system used a Waters ultra-high performance liquid system (AcQuity UPLC, Waters, USA), a Waters HSS T3 (100 * 2.1 mm, 1.8 μm) liquid chromatography column, the injection volume was 2 μL, and the column temperature was 40 °C; mobile phase A (0.1% formic acid aqueous solution), mobile phase B (0.1% formic acid - acetonitrile).
[0065] The mass spectrometry system uses the Q exactive high-resolution mass spectrometry detection system of Thermo Company in the United States, equipped with an electrospray ionization (ESI) source and an Xcalibur workstation. The mass spectrometry conditions mainly include: using the electrospray ionization source (ESI), the analyte is analyzed in the single ion detection (SIM) mode under positive ion simultaneous scanning, and this mode can greatly improve the sensitivity; the sheath gas pressure is 40 arb; the auxiliary gas pressure is 10 arb; the ion spray voltage is +3000 V; the temperature is 350 °C; the temperature of the ion transfer tube is 320 °C.
[0066] By detecting the OE- LrAOMT overexpressing plants and the control, the results (as Figure 2 shown) indicate that in the OE- LrAOMT overexpressing plants, delphinidin 3-O-galactoside chloride, peonidin-3-O-glucoside chloride, cyanidin O-syringic, cyanidin 3-glucoside, and procyanidin B1 in the anthocyanin biosynthesis pathway are significantly up-regulated. It shows that LrAOMT positively mediates the biosynthesis of delphinidin 3-O-galactoside chloride, peonidin-3-O-glucoside chloride, cyanidin O-syringic, cyanidin 3-glucoside, and procyanidin B1.
[0067] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. Application of overexpressed Lycium ruthenicum Murr. LrAOMT gene in increasing anthocyanin content in plants, characterized in that The Lycium ruthenicum LrAOMT has a nucleotide sequence of a gene as shown in SEQ ID NO. 1, or the Lycium ruthenicum LrAOMT gene encodes a protein with an amino acid sequence as shown in SEQ ID NO. 2; the plant is Lycium barbarum; the anthocyanins are delphinidin-3-O-galactoside, peonidin-3-O-glucoside, cyanidin-O-syringate, cyanidin-3-glucoside, and procyanidin B1.
2. Use of a protein with an overexpressed amino acid sequence as shown in SEQ ID NO. 2 in increasing the anthocyanin content of plants, characterized in that, The plant is Lycium barbarum; the anthocyanins are delphinidin-3-O-galactoside chloride, peonidin-3-O-glucoside, cyanidin-O-syringic acid, cyanidin-3-glucoside and procyanidin B1.
3. A recombinant expression vector overexpressing the Lycium ruthenicum Murr. described in claim 1 LrAOMT gene.
4. Application of the recombinant bacterium overexpressing the Lycium ruthenicum gene described in claim 1 in increasing the anthocyanin content of plants, characterized in that, LrAOMT The plant is Lycium barbarum; the anthocyanins include delphinidin-3-O-galactoside chloride, peonidin-3-O-glucoside, cyanidin-O-syringic acid, cyanidin-3-glucoside and procyanidin B1. 5. The application according to claim 4, characterized in that, The recombinant bacterium is Agrobacterium tumefaciens GV3101; and / or Inject the bacterial suspension of the recombinant bacterium into the plant.