DpCYP707A2 gene regulating dioscin synthesis and its application
By providing the DpCYP707A2 gene and its recombinant vector, regulating diosacin synthesis has solved the problem of insufficient diosacin production capacity, achieving a significant increase in diosacin content, and promoting the development of high-quality and high-yield yam cultivation and molecular breeding.
Patent Information
- Application Number
- CN202510706703.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-29
- Publication Date
- 2025-09-02
- Estimated Expiration
- 2045-05-29
AI Technical Summary
In the prior art, the synthesis of diosinosaponins lacks effective gene regulation methods in plants, resulting in insufficient production capacity of diosinosaponins and difficult to meet market demand.
It provides a DpCYP707A2 gene that regulates dysfunction synthesis and its applications, and transiently transforms yam by constructing a recombinant vector, overexpressing or silencing the gene to regulate dysfunction synthesis.
By regulating the DpCYP707A2 gene synthesised by diosinosaponin, the content of diosinosaponin in yam has been significantly improved, providing theoretical support for the biosynthesis of plant-specific metabolites, high-quality and high-yield yam cultivation and molecular breeding, and has important economic and social benefits.
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Abstract
Description
Technical Field
[0001] The present application belongs to the field of plant genetic engineering technology, and specifically relates to a DpCYP707A2 gene for regulating dioscin synthesis and its application. Background Art
[0002] Yam belongs to the Dioscoreaceae family, Dioscorea genus ( Dioscoreae ) vine. Yams can be traced back to Jiexiu and Pingyao in Shanxi Province, China, and are currently distributed primarily in Northwest China, North China, and the Yangtze River Basin, as well as in North Korea and Japan. They are highly adaptable, preferring warmth and being cold-resistant, and possess deep roots. As a food ingredient with both medicinal and nutritional value, yam is abundant, enjoys high market demand, and boasts stable exports. In agriculture, yam, due to its high yield and high efficiency, is considered a key crop for adjusting cropping structures and promoting sustainable agricultural development, and holds significant promotional value.
[0003] Yam contains many bioactive substances, and dioscin is one of them with important biological activities and medicinal value, exhibiting anti-inflammatory, anti-tumor, and cardiovascular protective effects. The synthesis of dioscin in plants is completed through the steroidal metabolic pathway, starting with acetyl-CoA. The mevalonate pathway (MVA pathway) generates isopentenyl pyrophosphate (IPP) and its isomers, which are then synthesized into squalene. Squalene is then epoxidized to 2,3-oxidosqualene, which is then converted to a steroidal skeleton (such as cycloartenol) by cyclases (such as cycloartenol synthase). Cytochrome P450 oxidase catalyzes the steroidal skeleton through hydroxylation and oxidation reactions to produce diosgenin. Finally, glycosyltransferases attach sugars such as glucose and rhamnose to the diosgenin to form dioscin. Cytochrome P450 oxidase is the key enzyme for hydroxylation, but there are few reports on the regulation of dioscin synthesis by CYP450 genes in yam.
[0004] Industrially, diosgenin is primarily extracted from Dioscorea plants (such as Dioscorea serrata and Dioscorea zingiberensis), and then synthesized into dioscin through chemical modifications (such as acid hydrolysis and glycosylation). Recent attempts have been made to synthesize steroidal saponin precursors using metabolic engineering of microorganisms (such as yeast), but these efforts are still at the research stage. Increasing dioscin content in plants through genetic engineering has important theoretical and practical implications for improving dioscin production in the production of secondary metabolites. Summary of the Invention
[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide a DpCYP707A2 gene for regulating dioscin synthesis and its application, specifically adopting the following technical solutions:
[0006] In a first aspect, the present invention provides a DpCYP707A2 gene for regulating the synthesis of dioscin. The nucleotide sequence of the DpCYP707A2 gene is shown in SEQ ID NO.1.
[0007] SEQ ID NO.1:
[0008]
[0009] The DpCYP707A2 gene provided by the present invention is obtained by PCR amplification using a forward primer (as shown in SEQ ID NO.2) and a reverse primer (as shown in SEQ ID NO.3) and Ruichang yam cDNA as a template.
[0010] SEQ ID NO.2: 5'-ATGGTTTCCATTGTTAGCATCAT-3';
[0011] SEQ ID NO. 3: 5'-TCATCCATTGTTCCTTGATGTCT-3'.
[0012] In a second aspect, the present invention provides a DpCYP707A2 protein that regulates the synthesis of dioscin. The DpCYP707A2 protein that regulates the synthesis of dioscin is encoded by the above-mentioned DpCYP707A2 gene. The amino acid sequence of the DpCYP707A2 protein is shown in SEQ ID NO.4.
[0013] SEQ ID NO.4:
[0014] MVSIVSIMVNTISLLALPSFVYLLWCLVSKAWAWAWSTSRTLPTEAQRKLSLPPGSMGYPYIGETFQLYSKNPNIFFALKQKRYGAIFKTHILGCPCVMLSSPEAAKFVLVTKAQLFKPTFPA SKERMLGRQAIFFQQGDYHARLRRLVLRAFMPDAIRSTVADIETVALTTLSSWDGRMVNTFKEMKTYAFNVALLSIFGKDEIGHIEELKQCYYTLEKGYNSMPINLPGTLFHKAMKARKQLGE IVAKILASRRESKVQANDLLASFMEDREALTDAQIADNIIGVIFAARDTTASVLTWIVKYLGEYPGVLQAVREEQEEIMRIKEMGQDKNDVDKCLTWADTKKMPMTSRVIQETMRVASILSFT FREAVEDVEFEGYLIPKGWKVLPLFRNIHHSPDNFPDPEKFDPSRFEKSPKPNTYMPFGNGTHSCPGNELAKLEMLVLLHHLTTRYRWSLSGTESGIQFGPFALPLNGLPIRFFRKTSRNNG.
[0015] In a third aspect, the present invention provides a recombinant vector for expressing the DpCYP707A2 gene, wherein the recombinant vector comprises the above-mentioned DpCYP707A2 gene.
[0016] The present invention cloned the DpCYP707A2 gene in yam, constructed a recombinant expression vector, and transiently transformed the gene into yam radix schizonepetae. It was found that the content of dioscin in the radix schizonepetae changed accordingly.
[0017] As a further preferred embodiment, the original vector of the recombinant vector comprises a plant expression vector. In the embodiment of the present invention, the original vector is preferably pCAMBIA1300. The recombinant vector expressing the DpCYP707A2 gene of the present invention inserts the nucleotide sequence shown in SEQ ID NO.1 between the Pst I and Kpn I restriction sites of pCAMBIA1300.
[0018] In a fourth aspect, the present invention also provides a target sequence for silencing the above-mentioned DpCYP707A2 gene, the sequence of which is shown in SEQ ID NO. 5. The target sequence is a target sequence for silencing the DpCYP707A2 gene based on VIGS technology. According to the results of the embodiments of the present invention, the target sequence can be used to specifically silence the DpCYP707A2 gene and reduce the expression level of the DpCYP707A2 gene.
[0019] SEQ ID NO.5:
[0020] 5'-GAAGACTTATGCATTTAATGTGGCACTTCTATCTATATTCGGGAAAGATGAGATTGGTCACATAGAAGAGCTGAAGCAGTGCTACTACACGCTGGAGAAGGGGTATAACTCGATGCCCATCAATCTCCCTGGGACCCTCTTCCACAAGGC CATGAAAGCCAGGAAGCAATTGGGTGAGATTGTGGCCAAGATACTGGCATCCCGAAGGGAAAGCAAGGTCCAGGCCAATGACTTGCTGGCTTCCTTCATGGAGGACAGAGAAGCTCTCACCGACGCCCAGATTGCTGACAACATCATCGG-3'.
[0021] In a fifth aspect, the present invention provides a recombinant bacterium expressing the DpCYP707A2 gene, wherein the recombinant bacterium comprises the above-mentioned recombinant vector.
[0022] In a sixth aspect, the present invention provides the use of the aforementioned DpCYP707A2 gene, the aforementioned DpCYP707A2 protein, the aforementioned recombinant vector, the aforementioned recombinant bacteria, or the aforementioned target sequence in increasing the dioscin content in plants or cultivating plants with high dioscin content.
[0023] As a further preferred embodiment, the plant includes at least one of a dicotyledonous plant, a monocotyledonous plant, a cruciferous plant and Arabidopsis thaliana.
[0024] In a seventh aspect, the present invention provides a method for cultivating plants with a high content of dioscin, by overexpressing the above-mentioned DpCYP707A2 gene in the plants.
[0025] As a further preferred embodiment, the method for overexpressing the DpCYP707A2 gene in plants comprises infecting plants with the recombinant bacteria; the recombinant bacteria are obtained by transferring the recombinant vector into Agrobacterium.
[0026] As a further preferred embodiment, the plant includes at least one of a dicotyledonous plant, a monocotyledonous plant, a cruciferous plant and Arabidopsis thaliana.
[0027] The beneficial effects of the present invention are:
[0028] The present invention provides a DpCYP707A2 gene sequence for regulating dioscin synthesis, as shown in SEQ ID NO: 1. Results from the present invention's examples demonstrate that transient overexpression of the DpCYP707A2 gene obtained from yam reduced the dioscin content in the yam, while silencing the gene increased the dioscin content. This indicates that the yam DpCYP707A2 gene described herein can regulate dioscin synthesis and has broad application prospects in fields such as metabolic regulation. It can provide theoretical support for further research into the biosynthesis of plant-specific metabolites, high-quality and high-yield cultivation of yam, and molecular breeding, and has significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0030] Figure 1 Shown are the cloning results of the DpCYP707A2 gene from Dioscorea opposita;
[0031] Figure 2The figure shows the relative expression of the DpCYP707A2 gene and the dioscin content analysis at different developmental stages of yam in Example 2; wherein, a in the figure represents the relative expression of the DpCYP707A2 gene at different developmental stages of yam, and b in the figure represents the dioscin content at different developmental stages of yam;
[0032] Figure 3 The figure shows the expression level identification result of DpCYP707A2 gene in Example 3;
[0033] Figure 4 Shown is the dioscin content analysis of DpCYP707A2 in Example 3;
[0034] Figure 5 Shown are the results of the identification of DpCYP707A2 gene expression in Example 4;
[0035] Figure 6 Shown is the dioscin content analysis of DpCYP707A2 in Example 4. DETAILED DESCRIPTION
[0036] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.
[0037] Example 1
[0038] Acquisition of DpCYP707A2 gene
[0039] The present invention discovered a cytochrome P450 oxidase from Ruichang yam, named DpCYP707A2, and the gene sequence is shown in Sequence 1 (SEQ ID NO. 1) in the sequence listing. The specific process is as follows:
[0040] (1) The roots, stems, leaves, tubers and seeds of Ruichang yam were ground in liquid nitrogen and evenly mixed. RNA was extracted using the polysaccharide and polyphenol plant RNA extraction kit provided by Nanjing Novozyme Biotechnology Co., Ltd.
[0041] (2) cDNA synthesis method: Using the RNA from step (1) as a template, the reverse transcription kit Hifair Ⅲ 1 of Shanghai Yisheng Biotechnology Co., Ltd. st Strand cDNA Synthesis Kit (gDNA digester plus) instructions: Reverse transcription to synthesize first-strand cDNA.
[0042] (3) Using the cDNA obtained in step (2) as a template, PCR amplification was performed using forward primers and reverse primers, wherein the forward primer was 5'-ATGGTTTCCATTGTTAGCATCAT-3' (SEQ ID NO. 2); the reverse primer was 5'-TCATCCATTGTTCCTTGATGTCT-3' (SEQ ID NO. 3); the amplification system was: 2 μL forward primer (primer concentration 10 μM), 2 μL reverse primer (primer concentration 10 μM), 5 μL template (Ruichang yam cDNA, template concentration 500 ng / μL), 25 μL 2×Hieff canace Plus PCR Master Mix (With Dye) (Shanghai Yisheng Biological Co., Ltd.), and 16 μL sterile ddH2O. The PCR amplification program was as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 58°C for 20 s, extension at 72°C for 1 min, for a total of 32 cycles, and final extension at 72°C for 5 min.
[0043] (4) The PCR product from step (3) is 1476 bp in total. The specific results are as follows: Figure 1 The PCR product was sent for sequencing, and the nucleotide sequence of the PCR amplified product was shown as SEQ ID NO. 1. The gene was named DpCYP707A2 according to its classification in the yam CYP450 gene family.
[0044] Example 2
[0045] Analysis of DpCYP707A2 gene expression at different stages
[0046] The yam tubers were cut into pieces and sown and cultivated normally. The underground tubers were sampled 150 days (T1), 170 days (T2), 190 days (T3), 210 days (T4), and 240 days (T5) after sowing. The total RNA of the tubers was extracted and the first-strand cDNA of the tubers was synthesized by reverse transcription.
[0047] The first-strand cDNA of tubers was used as template for fluorescence quantitative PCR detection. Actin The gene was used as the internal reference gene, and the gene expression was detected by fluorescence quantitative PCR.
[0048] The fluorescence quantitative PCR amplification system is preferably performed in 20 µL using the fluorescence quantitative reagent (Hieff UNICON Universal Blue qPCR SYBR Green Master Mix) provided by Shanghai Yisheng Biotechnology Co., Ltd., China, including: Hieff UNICON Universal Blue qPCR SYBR Green Master Mix 10 µL, forward primer (10 µM) 0.4 µL, reverse primer (10 µM) 0.4 µL, enzyme-free sterile water (RNase-free ddH2O) 7.2 µL and cDNA template 2 µL.
[0049] The fluorescence quantitative PCR reaction procedure included: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, and annealing at 60°C for 30 s, for a total of 39 cycles. The melting curve was between 65°C and 95°C, with an increase of 0.5°C every 5 s.
[0050] The primer pair consisting of qpDpCYP707A2-F and qpDpCYP707A2-R was used to detect the expression of DpCYP707A2 gene, and the primer pair consisting of ACTIN-F and ACTIN-R was used to detect the expression of ACTIN gene. Actin Gene expression.
[0051] qpDpCYP707A2-F (SEQ ID NO.6): 5'-TTCATGCCCGACGCCATT-3';
[0052] qpDpCYP707A2-R (SEQ ID NO.7): 5'-CATGCGGCCATCCCAAGA-3';
[0053] ACTIN-F (SEQ ID NO.8): 5'-GAGCAAGGAAATCACAGCAC-3';
[0054] ACTIN-R (SEQ ID NO. 9): 5'-TCAGGGAAGCCAAGATAGAG-3'.
[0055] The results are as follows Figure 2 As shown in a: The results showed that the relative expression level of DpCYP707A2 gene was higher in the fourth stage of yam development and lower in other stages.
[0056] Example 3
[0057] Transient overexpression analysis of DpCYP707A2 gene
[0058] 1. Recombinant vector
[0059] The specific construction process of the recombinant vector pCAMBIA1300-DpCYP707A2 that overexpresses the DpCYP707A2 gene is as follows:
[0060] Total RNA was extracted from tubers at different stages and reverse transcribed into cDNA. The cDNAs from different stages were mixed and then PCR amplified using the mixed cDNA as a template with a primer pair consisting of pCAMBIA1300-DpCYP707A2_F and pCAMBIA1300-DpCYP707A2_R. The amplification system was as follows: 2 μL forward primer (primer concentration 10 μM), 2 μL reverse primer (primer concentration 10 μM), 5 μL template (mixed cDNA, template concentration 500 ng / μL), 25 μL 2×Hieff canace Plus PCR Master Mix (With Dye) (Shanghai Yisheng Biological Company), and 16 μL sterile ddH2O. The PCR amplification program was as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 58°C for 20 s, extension at 72°C for 1 min, for a total of 32 cycles, and a final extension at 72°C for 5 min to obtain the amplified product; the primer sequences are as follows:
[0061] pCAMBIA1300-DpCYP707A2_F (SEQ ID NO.10):
[0062] 5'-GACTCTAGAAAGCTTCTGCAGATGGTTTCCATTGTTAGCATCATGG-3';
[0063] pCAMBIA1300-DpCYP707A2_R (SEQ ID NO.11):
[0064] 5'-CGCCCTTGCTCACCATGGTACCTCCATTGTTCCTTGATGTCTTCC-3';
[0065] The pCAMBIA1300 vector was double-digested with restriction endonucleases Pst I and Kpn I. The double-digestion reaction system consisted of 5 µL of rCutSmart buffer, 1 µL of Pst I, 1 µL of Kpn I, 3.5 µL of pCAMBIA1300 vector plasmid (plasmid concentration 400 ng / µL), and 40 µL of ddH2O. The double-digestion reaction conditions were: digestion at 37°C for 3 h, followed by enzyme inactivation at 65°C for 20 min to linearize the vector. The amplified product and the linearized vector were then homologously recombined to generate the recombinant vector pCAMBIA1300-DpCYP707A2.
[0066] 2. Recombinant Agrobacterium
[0067] The recombinant vector pCAMBIA1300-DpCYP707A2 obtained in the above step 1 was introduced into Agrobacterium GV3101 by freeze-thaw method to obtain recombinant Agrobacterium GV3101 / pCAMBIA1300-DpCYP707A2.
[0068] 3. Agrobacterium-mediated transient overexpression of yam
[0069] (1) Activation of Agrobacterium: streak the pCAMBIA1300-DpCYP707A2 and pCAMBIA1300 empty-loaded Agrobacterium stored in the ultra-low temperature freezer for 48 h, select a single well-growing plaque, add it to 5 mL of LB liquid culture medium containing 50 µg / mL Kana and 25 µg / mL Rif antibiotics, and shake the culture overnight at 28°C and 200 rpm.
[0070] (2) Agrobacterium: Pipette 500 μL of Agrobacterium from step (1) and add it to 100 mL LB liquid medium (containing 50 μg / mL Kana and 25 μg / mL Rif antibiotics). Cultivate at 28°C and 200 rpm until the OD 600 =0.8-1.
[0071] (3) Add the Agrobacterium that reaches OD 0.05 to a 50 ml centrifuge tube, centrifuge at 5000 rpm for 8 min, remove the supernatant, add invasion dye solution (prepared as follows: add 10 mM MES, 200 μM acetosyringone and 10 mM MgCl2 to MS culture medium) and resuspend the bacterial solution to OD 0.05. 600 The infection solution is MS liquid culture medium containing only 10 mM magnesium chloride, 10 mM 2-morpholineethanesulfonic acid and 200 µM acetosyringone.
[0072] (4) Select healthy and plump radix schizonepetae, clean them and dry the surface moisture. Use the sterile needle of a 5 mL syringe to evenly pierce the radix schizonepetae. Immerse the pierced radix schizonepetae completely in the infection solution and vacuum for 15 min. After completion, wipe off the excess bacterial solution on the surface and place it in the dark at 25°C for 3 days. After 3 days, extract RNA from the pierced part to measure the expression level. At the same time, freeze-dry some samples to measure the dioscin content.
[0073] 4. Analysis of relative expression of DpCYP707A2 gene
[0074] The relative expression levels of the DpCYP707A2 gene in the nulliparous plants of the pCAMBIA1300 empty-infected group (OE-control) and the pCAMBIA1300-DpCYP707A2-infected group (OE-DpCYP707A2) were analyzed using the fluorescence quantitative method in Example 2. Figure 3 As shown, it can be found that the relative expression level of DpCYP707A2 gene in the complement sample of transiently overexpressing DpCYP707A2 gene is significantly increased compared with that in the OE-control.
[0075] 5. Determination of dioscin content in transiently overexpressed Dioscorea zingiberensis
[0076] Dioscin was extracted from the lyophilized sample of the genus Eupatorium using isopropanol ultrasonic extraction. The method was as follows: 200 mg of lyophilized Eupatorium genus was weighed, 1.2 mL of isopropanol was added, and the mixture was sonicated at low temperature for 10 minutes. The extracted solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. The supernatant was filtered through a 0.22 μm organic filter membrane and transferred to a 2 mL chromatography vial. 0.81 mL of the supernatant was concentrated to dryness at 4°C and reconstituted in 135 μL of isopropanol. The sample was filtered through a 0.22 μm microporous organic filter membrane and stored in a vial for UPLC-MS / MS analysis. The 6-fold concentrated sample was used for dioscin detection.
[0077] A Waters ACQUITY Premier UPLC BEH C18 1.8 µm, 2.1 mm × 100 mm column was used. The mobile phases A and B consisted of ultrapure water (0.04% HAC) and acetonitrile (0.04% HAC). The flow rate was 0.3 mL / min, the column temperature was 45°C, and the injection volume was 5 µL. Mass spectrometry conditions included an electrospray ionization (ESI) temperature of 500°C, ion source voltages of 5500 V (positive), -4500 V (negative), ion source gas I (GS I) at 50 psi, gas II (GS II) at 60 psi, and curtain gas (CUR) at 30 psi. Collision-activated dissociation (CAD) parameters were set to Medium. In a triple quadrupole (Qtrap), each ion transition was scanned and detected based on optimized declustering potential (DP) and collision energy (CE). Mass spectrometric peak intensity data for the corresponding quantitative signals were acquired for each standard concentration. A standard curve was plotted with standard concentration as the abscissa and peak area as the ordinate. The standard curve was calculated as y = 4181.31654 x + 478.30600, with an r = 0.99892.
[0078] The results are as follows Figure 4 As shown, the dioscin content in the DpCYP707A2 gene-overexpressing DpCYP707A2 samples was significantly reduced compared with the control, with the DpCYP707A2 gene-overexpressing DpCYP707A2 gene-overexpressing DpCYP707A2 sample as the control.
[0079] Example 4
[0080] VIGS silencing of the DpCYP707A2 gene
[0081] 1. Construction of pTRV2-DpCYP707A2 silencing expression vector
[0082] (1) Using the mixed cDNA from step 1 in Example 3 as a template, PCR amplification was performed using a primer pair consisting of pTRV2-DpCYP707A2_F and pTRV2-DpCYP707A2_R. The amplification system was as follows: 2 μL forward primer (primer concentration 10 μM), 2 μL reverse primer (primer concentration 10 μM), 5 μL template (mixed cDNA, template concentration 500 ng / μL), 25 μL 2×Hieff canace Plus PCR Master Mix (With Dye) (Shanghai Yisheng Biological Co., Ltd.), and 16 μL sterile ddH2O. The PCR amplification procedure was as follows: initial denaturation at 98°C for 3 min; 32 cycles of denaturation at 98°C for 10 s, annealing at 58°C for 20 s, and extension at 72°C for 30 s, followed by a final extension at 72°C for 5 min. A PCR amplification product containing the DpCYP707A2 gene-specific fragment (shown in SEQ ID NO. 5) was obtained. The primer sequences are as follows:
[0083] pTRV2-DpCYP707A2_F (SEQ ID NO.12):
[0084] 5'-GTGAGTAAGGTTACCGAATTCGAAGACTTATGCATTTAATGTGGCAC-3';
[0085] pTRV2-DpCYP707A2_R (SEQ ID NO.13):
[0086] 5'-CGTGAGCTCGGTACCGGATCCCCGATGATGTTGTCAGCAATCT-3';
[0087] The pTRV2 vector was double-digested with restriction endonucleases EcoRI-HF and BamHI-HF. The double-digestion reaction system consisted of 5 µL of rCutSmart buffer, 1 µL of EcoRI-HF, 1 µL of BamHI-HF, 3 µL of pTRV2 vector plasmid (plasmid concentration 500 ng / µL), and 40 µL of ddH2O. The double-digestion reaction conditions were: digestion at 37°C for 3 h, followed by enzyme inactivation at 65°C for 20 min to linearize the vector. The amplified product and the linearized vector were then homologously recombined to generate the recombinant vector pTRV2-DpCYP707A2.
[0088] 2. Recombinant Agrobacterium
[0089] The recombinant vector pTRV2-DpCYP707A2 obtained in the above step 1 was introduced into Agrobacterium GV3101 by freeze-thaw method to obtain recombinant Agrobacterium GV3101 / pTRV2-DpCYP707A2.
[0090] 3. Agrobacterium-mediated VIGS silencing of Yam
[0091] (1) Activation of Agrobacterium: streak the pTRV2-DpCYP707A2, pTRV2 empty vector, pTRV1 empty vector, and P19 Agrobacterium stored in an ultra-low temperature freezer for 48 h. Select a single well-growing plaque and add it to 5 mL of LB liquid culture medium containing 50 µg / mL Kana and 25 µg / mL Rif antibiotics. Shake the culture overnight at 28°C and 200 rpm.
[0092] (2) Agrobacterium: 200 μL of Agrobacterium from step (1) were taken and added to 50 mL of LB liquid culture medium (containing 50 μg / mL Kana and 25 μg / mL Rif antibiotics), and cultured at 28°C and 200 rpm until OD 600 =0.8-1.
[0093] (3) Add the Agrobacterium that reaches OD 0.05 to a 50 ml centrifuge tube, centrifuge at 5000 rpm for 8 min, remove the supernatant, add invasion dye solution (prepared as follows: add 10 mM MES, 200 μM acetosyringone and 10 mM MgCl2 to MS culture medium) and resuspend the bacterial solution to OD 0.05. 600 The concentration of the culture medium was 0.8-1. Equal volumes of the infection medium after resuspension of the pTRV1 empty load, the infection medium after resuspension of the pTRV2 empty load, and the infection medium after resuspension of the P19 were mixed to obtain the infection medium of the control group. Equal volumes of the infection medium after resuspension of the pTRV1 empty load, the infection medium after resuspension of the pTRV2-DpCYP707A2 empty load, and the infection medium after resuspension of the P19 empty load were mixed to obtain the infection medium of the test group. The mixed bacterial solution was allowed to stand in the dark at 25°C for 4-6 hours. The infection medium was MS liquid medium containing only 10 mM magnesium chloride, 10 mM 2-morpholineethanesulfonic acid, and 200 µM acetosyringone.
[0094] (4) Select healthy and plump radix schizonepetae, clean them and dry the surface moisture. Use the sterile needle of a 5 mL syringe to evenly pierce the radix schizonepetae. Immerse the pierced radix schizonepetae completely in the infection solution and vacuum for 15 min. After completion, wipe off the excess bacterial solution on the surface and place it in the dark at 25°C for 3 days. After 3 days, extract RNA from the pierced part to measure the expression level. At the same time, freeze-dry some samples to measure the dioscin content.
[0095] 4. Analysis of relative expression of DpCYP707A2 gene
[0096] The relative expression levels of the DpCYP707A2 gene in the nulliparous plants of the pTRV2 empty-infected group (VIGS-control) and the pTRV2-DpCYP707A2-infected group (VIGS-DpCYP707A2) were analyzed using the same fluorescence quantitative method as in Example 2. Figure 5 As shown in the Figure 3, the relative expression level of DpCYP707A2 gene in the DpCYP707A2 gene silenced by VIGS was significantly decreased compared with that in the VIGS-control.
[0097] 5. Determination of dioscin content in transiently overexpressed Dioscorea zingiberensis
[0098] The determination method is the same as that of step 5 in Example 3, and the results are as follows: Figure 6 As shown: Taking the cotyledons of the pTRV2 empty infection group (VIGS-control) as the control, the dioscin content in the cotyledons samples in which the DpCYP707A2 gene was silenced by VIGS was significantly increased compared with the control.
[0099] In summary, VIGS silencing of the DpCYP707A2 gene in yam increased dioscin content in yams, demonstrating that the cytochrome P450 oxidase gene DpCYP707A2 plays a key role in regulating dioscin biosynthesis in plants. This invention not only has applications in the biosynthesis of specific metabolites but also provides theoretical support for high-quality, high-yield cultivation and molecular breeding of yam, with significant economic and social benefits.
[0100] The embodiments of the present application are described above in conjunction with the accompanying drawings. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of the present application, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present application and the claims, all of which are within the protection of the present application.
Claims
1. Use of the DpCYP707A2 gene in increasing the dioscin content in plants or cultivating plants with high dioscin content; The plant is Chinese yam; The nucleotide sequence of the DpCYP707A2 gene is shown in SEQ ID NO. 1; the application increases the dioscin content in plants or cultivates plants with high dioscin content by silencing the DpCYP707A2 gene.
2. A method for cultivating yam with high dioscin content, characterized in that: The DpCYP707A2 gene is silenced in yam; the nucleotide sequence of the DpCYP707A2 gene is shown in SEQ ID NO.
1.
3. The method according to claim 2, characterized in that The method for silencing the DpCYP707A2 gene in yam comprises infecting yam with recombinant bacteria; the recombinant bacteria are obtained by transferring a recombinant vector containing the silenced DpCYP707A2 gene into Agrobacterium.