Use of pgNAC72 in regulating ginsenoside biosynthesis

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to the application of PgNAC72 protein or gene in regulating ginsenoside biosynthesis. The sequence of the PgNAC72 protein is shown in SEQ ID NO.1, and the sequence of the PgNAC72 gene is shown in SEQ ID NO.2. This invention verified in vitro and in vivo, using EMSA and dual-luciferase reporter gene assays, that PgNAC72 can bind to the cis-acting elements ABRE and CACG on the PgDDS promoter, thereby promoting PgDDS transcription. These results indicate that PgDDS is a target gene regulated by PgNAC72.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology. Specifically, PgNAC72 regulates the biosynthesis of ginsenosides by promoting the expression of the dammarene diol synthase (DDS) gene. Background Technology

[0002] Ginseng (Panax ginseng CAMeyer) is a perennial herbaceous plant belonging to the Araliaceae family with a long history of medicinal use. Ginsenosides are the main active components of ginseng, possessing anti-cancer, antioxidant, and anti-inflammatory functions. However, due to low ginsenoside production, it is difficult to meet the growing medicinal demand. In recent years, key enzyme genes in the ginsenoside biosynthesis pathway have been gradually identified; however, regulatory factors in this pathway have been rarely reported. Transcription factors, as proteins that can specifically bind to cis-acting elements, play an important role in the complex regulatory network of plants. Exploring the biological functions of transcription factors related to ginsenoside synthesis will help reveal the accumulation patterns of ginsenosides in ginseng and also provide a foundation for the production of ginsenosides using plant synthetic biology techniques.

[0003] Currently, a total of 4439 transcription factors belonging to 94 gene families have been discovered in the ginseng genome. Transcription factors of the WRKY, bHLH, and MYB families that have been reported to play a key role in ginsenoside biosynthesis have been reported. Wang found that low temperature stimulation can promote the accumulation of ginsenosides. Five ginsenoside biosynthesis genes, GPS, SS, CYP716A53v2, UGT74AE2 and UGT94Q2, and the expression levels of three PgWRKYs (PgWRKY1, PgWRKY3 and PgWRKY8) are strongly positively correlated with the yield of ginsenosides. These PgWRKYs may participate in the biosynthesis of ginsenosides by regulating related pathway genes [1]. Chu et al. conducted a whole-genome study on bHLH transcription factors and identified a total of 169 PgbHLH genes and divided them into 24 subfamilies. Combining gene expression patterns and saponin chemical content, Chu found that six PgbHLH genes from four subfamilies may participate in the regulation of ginsenoside biosynthesis [2]. Liu screened out a MeJA-induced R2R3 type MYB gene, PgMYB2. Through yeast one-hybrid and dual-luciferase reporter gene experiments, it was found that PgMYB2 can bind to the DDS promoter and promote the expression of PgDDS, which suggests that PgMYB2 may be involved in the biosynthesis of ginsenosides [3]. Due to the difficulty of genetic transformation of ginseng, there are few studies on the role of transcription factors in regulating ginsenoside synthesis. Exploring the role of other transcription factor families in saponin synthesis is still of pioneering significance.

[0004] NACs are a class of transcription factors unique to plants and are also the most widely found. According to the Plant Transcription Factor Database, 19,997 NAC transcription factors from over 150 species have been included, with 101, 328, and 138 transcription factors found in tomato, rice, and Arabidopsis thaliana, respectively. http: / / planttfdb.gao-lab.or g). Liu conducted a preliminary identification and analysis of the NAC gene family of ginseng. Liu screened 251 PgNACs in the ginseng genome database and divided them into 11 subgroups through evolutionary analysis. At the same time, he also discovered 5 PgNACs that responded to cold stress (PgNAC05-2, PgNAC41-2, PgNAC48, PgNAC56-1, and PgNAC59)[4].

[0005] References:

[0006] [1]WANG S, LIANG W, YAO L, et al. Effect of temperature on morphology, ginsenosides biosynthesis, functional genes, and transcriptional factors expression in Panax ginseng adventitious roots [J]. Journal of FoodBiochemistry, 2019, 43.

[0007] [2]CHU Y,XIAO S,SU H,et al.Genome-wide characterization and analysis of bHLH transcription factors in Panax ginseng[J].Acta Pharmaceutica SinicaB, 2018,8:666-677.

[0008] [3]LIU T,LUO T,GUO

[0009] [4] LIU Q, SUN C, HAN J, et al. Identification, characterization and functional differentiation of the NAC gene family and its roles in response to cold stress in ginseng, Panax ginseng CAMeyer[J]. Plos One, 2020,15.

[0010] [5]LIN T,DU J,ZHENG X,et al.Comparative transcriptome analysis ofMeJA-responsive AP2 / ERF transcription factors involved in notoginsenosidesbiosynthesis[J].3Biotech,2020,10.

[0011] [6] Hu Honghong. Isolation and functional identification of stress-related transcription factors in rice [D]. Huazhong Agricultural University, 2006. Summary of the Invention

[0012] This invention aims to provide the application of PgNAC72 in regulating the biosynthesis of ginsenosides.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] The application of PgNAC72 protein in regulating ginsenoside biosynthesis, the sequence of which is shown in SEQ ID NO.1.

[0015] SEQ ID NO.1 is:

[0016] MGVPETDPLSQLSLPPGFRFYPTDEELLVQYLCRKVAGQHFSLQIIGEIDLYKFDPWVLPSKAIFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKVITTEGRKVGIKKALVFYVGKAPKGTKTNWIMHEYRLSDPQRKNGSARLDDWVLCRIYKKNSSAQKPVLGDID SKEHSHSHSHSHSHGSSSSSSQFEDVLESLPEIEDRFFTLPRMNSSLNDKLNFQNLGSGNFDWAILAGLNSMPEHVPGTQAPMQTQTQGLMNNNNQNYMCVPSTSPLGHVDTRFGKSMEEEVESGLRNNQRVDNSGFLNSNSNSSCSVDPFAIRYPIQSGNMGFTLGCENCT.

[0017] The application of the PgNAC72 gene in regulating ginsenoside biosynthesis, the sequence of which is shown in SEQ ID NO.2.

[0018] SEQ ID NO.2 is:

[0019]

[0020] In one preferred embodiment, the application is that the PgNAC72 gene and PgNAC72 protein regulate the synthesis of ginsenosides by regulating the expression of the ginseng PgDDS gene.

[0021] In one preferred embodiment, the application is to regulate the synthesis of ginsenosides by overexpressing the PgNAC72 gene or PgNAC72 protein.

[0022] In one preferred embodiment, the reagent for overexpressing the PgNAC72 gene or PgNAC72 protein includes methyl jasmonate.

[0023] In one preferred embodiment, the application is performed by transfecting ginseng suspension cells with an overexpression plasmid containing the PgNAC72 gene.

[0024] In one preferred embodiment, the ginsenoside is a dammarane-type ginsenoside.

[0025] This invention also claims the use of a recombinant vector in regulating ginsenoside biosynthesis, wherein the recombinant vector overexpresses the PgNAC72 gene.

[0026] The present invention also claims a kit comprising a reagent for overexpressing the PgNAC72 gene or the PgNAC72 protein, or an overexpression vector having the PgNAC72 gene.

[0027] This invention also claims protection for the use of the kit in regulating ginsenoside biosynthesis.

[0028] In fact, there are many transcription factors that are upregulated in ginseng root hairs after MeJA (methyl jasmonate) induction, but most of them cannot regulate the synthesis of ginsenosides. For example, Lin analyzed the differential transcriptome of Panax notoginseng induced by MeJA and found 16 significantly differentially expressed AP2 / ERF transcription factors. Real-time quantitative PCR (RT-qPCR) and co-expression network analysis of these 16 AP2 / ERF transcription factors revealed that only PnERF2 and PnERF3 were significantly correlated with the key genes for Panax notoginseng saponin synthesis, dammarene diol II synthase gene (DS) and squalene epoxidase gene (SE) [5]. After a large number of experimental screenings, we selected PgNAC72 (Pg_S5466.10), which was significantly upregulated after MeJA induction, as the research object. The amino acid sequence, structure and physicochemical properties of the protein encoded by the PgNAC72 gene were analyzed using bioinformatics methods. The expression pattern of the PgNAC72 gene was investigated using qRT-PCR, and the subcellular localization of the PgNAC72 protein in tobacco was determined. To explore the function of PgNAC72, we overexpressed it in ginseng callus tissue, finding a significant increase in dammarane-type ginsenoside content in the transgenic callus. We then used qRT-PCR to detect the expression of key enzyme genes in the ginsenoside synthesis pathway, thus screening for the candidate target gene PgDDS. Based on these studies, we subsequently conducted DNA-protein interaction experiments, gradually elucidating the role of the PgNAC72 transcription factor in the ginsenoside synthesis pathway. This study is the first to provide a relatively complete functional analysis of the hormone-responsive NAC gene in ginseng, and its results can provide a new avenue for the study of ginseng secondary metabolites.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] This invention clones the PgNAC72 gene, constructs a plant expression vector, and transforms ginseng callus tissue using Agrobacterium-mediated transformation. Positive callus tissue overexpressing PgNAC72 was obtained by PCR, Western blotting (WB), and qRT-PCR. The total saponin content in the positive callus tissue was significantly increased. Terpenoid metabolomics analysis revealed that the increased content was mainly of dammarane-type ginsenosides, indicating that the PgNAC72 gene promotes the biosynthesis of dammarane-type ginsenosides.

[0031] Overexpression of PgNAC72 in this invention significantly increased the expression of key enzyme genes PgDDS and PgSS3 in the ginsenoside biosynthesis pathway, but the fold change in PgDDS was much higher than that in PgSS3; while the expression trends of other key enzyme genes (PgHMGR, PgFPS, PgSE1, PgPDDS) were inconsistent with those of PgNAC72. We verified in vitro and in vivo, using EMSA and dual-luciferase reporter gene assays, that PgNAC72 can bind to the cis-acting elements ABRE and CACG on the PgDDS promoter, thereby promoting PgDDS transcription. These results indicate that PgDDS is a target gene regulated by PgNAC72. Attached Figure Description

[0032] Figure 1 Subculture of ginseng callus;

[0033] Figure 2 Detection of total RNA in ginseng callus;

[0034] Figure 3 PCR amplification of the PgNAC72 fragment gel electrophoresis results, where M is the gene marker;

[0035] Figure 4 The flowchart shows the genetic transformation process of ginseng callus; Figure A: Callus tissue 1 month after Agrobacterium infection; Figure B: Callus tissue 2 months after Agrobacterium infection; Figure C: Newly formed callus tissue transferred to Hgy-free medium; Figure D: General flowchart of genetic transformation of ginseng callus.

[0036] Figure 5 GUS staining analysis of ginseng callus tissue;

[0037] Figure 6 Detection of gDNA in ginseng callus;

[0038] Figure 7 PCR identification of transgenic callus;

[0039] Figure 8 qRT-PCR was used to verify the expression level of PgNAC72 in transgenic callus tissue;

[0040] Figure 9 Detection of total saponin content in ginseng callus; Figure A: Indication of saponin extract using vanillin-perchloric acid colorimetric system, the higher the saponin content, the darker the color of the extract; Figure B: Total saponin content in PgNAC72 transgenic callus, with the total saponin content of the WT group as a reference.

[0041] Figure 10 Terpenoid metabolomics analysis of differentially expressed metabolites;

[0042] Figure 11 Expression of key enzyme genes in PgNAC72 transgenic ginseng callus;

[0043] Figure 12 EMSA investigated the binding of the PgNAC72 protein to DNA. Probe A has an ABRE site, and probe B has a CACG site.

[0044] Figure 13 The dual-luciferase reporter gene assay was used to verify the regulatory role of PgNAC72 on PgDDS. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0046] The experimental materials used in the examples are as follows:

[0047] 1 plant material

[0048] The ginseng callus tissue was obtained from previous subculture in the laboratory; the wild-type tobacco seeds were obtained from previous harvesting and preservation in the laboratory.

[0049] 2. Main biochemical reagents

[0050] Table 1 Biochemical reagents used in this invention

[0051]

[0052] 3. Main experimental instruments

[0053] Table 2 Experimental instruments used in this invention

[0054]

[0055]

[0056] Example 1

[0057] Cloning of the PgNAC72 gene

[0058] 1. Establishment of a Ginseng Callus Subculture System

[0059] 1.1 Preparation of 6,7-V culture medium

[0060] Table 3 6,7-V culture medium

[0061]

[0062] Prepare 6,7-V culture medium according to the above formula, adjust the pH to 5.8. If preparing solid culture medium, add 8g of agar powder. Autoclave at 121℃ for 15min. After sterilization, wait for the culture medium to cool to about 60℃, add 1mL of sterilized 2,4-D (stock solution concentration of 1mg / mL) to the culture medium in a clean bench, and dispense and seal it.

[0063] 1.2 Subculture of Ginseng Callus

[0064] Using sterile forceps in a clean bench, healthy, light yellow ginseng callus tissue (derived from ginseng callus tissue previously subcultured and preserved by Professor Luo Zhiyong's research group at Central South University) was picked up and transferred to freshly prepared 6,7-V solid culture medium for subculture, resulting in ginseng callus tissue with good viability. Figure 1 As shown. During the culture process, try to keep the callus tissue clustered together to avoid excessive dispersion. After sealing with plastic film, place the culture bottle in a 25℃ incubator and incubate statically in the dark. Observe the growth and bacterial contamination of the callus tissue every 2-3 days, and replace with fresh 6,7-V medium approximately every 20-30 days depending on its growth status.

[0065] Total RNA extraction from ginseng callus

[0066] (1) Add 1 ml of Trizol to an RNase-free centrifuge tube and place it on ice. Grind the sample into powder using a mortar and pestle. Liquid nitrogen needs to be added to the mortar continuously during this process. Add 50 mg of powder to the centrifuge tube and mix using a vortex mixer. Let stand at room temperature for 5 min.

[0067] (2) Centrifuge at 12,000×g at 4℃ for 5 min, carefully aspirate 950uL of the supernatant into a new RNase-free centrifuge tube, add 1 / 5 of the volume of Trizol in chloroform, mix thoroughly, and let stand at room temperature for 5 minutes;

[0068] (3) Centrifuge at 12,000×g at 4℃ for 10 min. The homogenate will be divided into three layers: the supernatant containing RNA, the intermediate protein layer, and the lower organic phase.

[0069] (4) Transfer 450 μL of supernatant to another new RNase-free centrifuge tube (do not aspirate the intermediate protein layer); add 450 μL of isopropanol equal to the volume of Trizol, mix thoroughly, and let stand at room temperature for 10 min.

[0070] (5) Transfer the above mixture to the adsorption column RA, centrifuge at 13,000 rpm for 2 min, and discard the filtrate;

[0071] (6) Add 500 μL of protein removal solution RW1, place at room temperature for 3 min, centrifuge at 13,000 rpm for 30 s, and discard the filtrate;

[0072] (7) Add 500 μL of washing buffer RW pre-added with anhydrous ethanol, centrifuge at 13,000 rpm for 30 seconds, discard the filtrate; repeat the operation once.

[0073] (8) Place the adsorption column back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes;

[0074] (9) Place the adsorption column in a new RNase-free centrifuge tube, open the cap and let it sit at room temperature for 2 min to evaporate the residual ethanol; add 30 μL of RNase-free water preheated to 70-90℃ to the adsorption membrane, let it sit at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

[0075] After centrifugation, 2 μL was taken for agarose gel electrophoresis to verify RNA quality. The electrophoresis results are as follows: Figure 2 As shown in the image: there are three distinct rRNA bands, and the 28S band is about twice as bright as the 18S band. The bands do not show obvious tailing, indicating that the extracted RNA has not been degraded and can be used for subsequent experiments.

[0076] 3. Reverse transcription

[0077] (1) Removal of gDNA

[0078] Add the following reagents to the RNase-free PCR tube in sequence:

[0079] Table 4 PCR System

[0080]

[0081] After mixing the above reagents by pipetting, incubate them in a PCR instrument at 42°C for 2 minutes, then remove and place on ice.

[0082] (2) cDNA synthesis

[0083] Add 4 μL of 5×HiScriptⅡ Enzyme Mix to the PCR tube containing the above reaction solution, and perform the following reaction in a PCR instrument: 50℃, 15 min; 85℃, 5 s; 4℃, ∞. After the reaction is complete, store the cDNA at -20℃.

[0084] 4. Construction of PgNAC72 gene overexpression vector

[0085] 4.1 PCR amplification of the PgNAC72 gene fragment

[0086] PCR amplification was performed using the high-fidelity enzyme PrimeSTAR Max DNA Polymerase from Takara. Primers were designed using Primer Premier 5.0 software and synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are as follows:

[0087] Primer Sequence (5′→3′)

[0088] PgNAC-OF CGGGGTACCATGGGTGTGCCGGAGACTG(SEQ ID NO.3)

[0089] PgNAC-OR

[0090] CGCGGATCCTTACTTATCATCATCATCCTTATAATCAGTACAATTTTCACATCCTAAAGTAAAC(SEQID NO.4)

[0091] The reaction system is as follows:

[0092] Table 5. PCR amplification system for the PgNAC72 gene fragment.

[0093]

[0094] The PCR reaction program settings are as follows:

[0095]

[0096] After the reaction is complete, quickly centrifuge the liquid on the tube wall and place it on ice for subsequent agarose gel purification or temporarily store it in a -20°C freezer.

[0097] A PgNAC72 fragment with restriction enzyme sites at both ends was amplified. The PCR product was then detected by agarose gel electrophoresis. The results are as follows: Figure 3 As shown, its product fragment is 1050bp.

[0098] 4.2 Purification of PCR Products

[0099] PCR products were purified by gel electrophoresis to remove impurities. The gel containing the target fragment was purified using a gel extraction kit from Novizan. All steps were performed at room temperature, as detailed below:

[0100] (1) Mix the PCR product obtained in the previous step with 6× Loading Buffer, separate the target fragment from the impurities by agarose gel electrophoresis for 30 min, quickly cut the agarose gel containing the DNA fragment of the target size under UV light, and then place it in a clean 1.5 ml centrifuge tube. Note that the blade needs to be cleaned in advance.

[0101] (2) Weigh the gel and add Buffer GDP according to the ratio of 100 μL per 0.1 g gel. Then, incubate the centrifuge tube in a 50°C water bath for 10 min, gently inverting and mixing 3 times during the process. If the gel is not completely dissolved, the water bath time can be extended appropriately.

[0102] (3) Transfer the completely dissolved solution to the adsorption column, centrifuge at 12,000×g for 30s. To improve the recovery rate, the liquid in the collection tube can be poured back into the adsorption column, centrifuged at 12,000×g for 30s, and the filtrate can be discarded. If the total volume of the solution is greater than 700μL, it can be passed through the column multiple times.

[0103] (4) Add 500 μL Buffer GDP to the adsorption column, let stand for 1 min, centrifuge at 12,000 × g for 30 s, discard the filtrate, and put the adsorption column back into the collection tube.

[0104] (5) Confirm that anhydrous ethanol has been added to Buffer DW in advance, add 700 μL of Buffer DW along the tube wall to the adsorption column, centrifuge at 12,000×g for 30s, discard the filtrate, put the adsorption column back into the collection tube, and repeat the step once.

[0105] (6) Place the adsorption column back into the empty collection tube and centrifuge the empty tube at 12,000×g for 2 min.

[0106] (7) Discard the collection tube, put the adsorption column into a new dry 1.5ml centrifuge tube, open the adsorption column cap and dry at room temperature for 2-5 minutes to evaporate the residual ethanol;

[0107] (8) Preheat the Elution Buffer to 60℃, add 30 μL to the adsorption membrane, let stand for 2 min, centrifuge at 12,000×g for 2 min, and store the final DNA solution at -20℃. The purified product was verified by agarose gel electrophoresis, as shown below. Figure 3 Consistent.

[0108] 4.3 Purification of fragments, enzyme digestion, and linearization of pCambia1301s vector

[0109] The purified fragment obtained in the previous step and the pCambia1301s vector (provided by the Institute of Subtropical Agriculture Ecology, Chinese Academy of Sciences, and modified by the laboratory of Huazhong Agricultural University, i.e., the double CaMV 35S promoter was introduced into the pCambia1301 vector [6]) were digested with restriction endonucleases BamHⅠ and KpnⅠ to produce sticky ends, which are beneficial for subsequent ligation reactions. The enzyme digestion system was prepared in 200 μL PCR tubes according to the following table:

[0110] Table 6 Enzyme digestion system

[0111]

[0112] 4.4 The purified PgNAC72 fragment was ligated into the linearized pc1301s vector.

[0113] The enzyme digestion product obtained in the previous step was purified. The purified PgNAC72 fragment was ligated to the vector using T4 DNA ligase. The amount of PgNAC72 fragment used was calculated based on a vector to insert molar ratio of 3:1. The ligation system was prepared in a 200 μL PCR tube according to the table below:

[0114] Table 7 Connection System

[0115]

[0116] 4.5 Transformation of ligation products into Escherichia coli and screening

[0117] (1) Take out a 100 μL LDH5α competent cell from the -80℃ freezer, quickly insert it into ice to thaw for 10 min, add 10 μL of ligation product to the bacterial culture and gently pipette to mix, then let it stand on ice for 30 min.

[0118] (2) Place the above bacterial solution in a 42°C water bath for 1 minute for heat shock, and then quickly insert it into ice;

[0119] (3) In a clean bench, add 700 μL of antibiotic-free LB liquid culture medium to the tube, gently invert to mix, and incubate at 180 rpm for 60 min.

[0120] (4) Centrifuge at 4,000×g for 3 min to collect the bacterial cells, discard 700 μL of supernatant, and use about 100 μL of culture medium to resuspend the bacterial cells. After thoroughly mixing by pipetting and aspiration, transfer the bacterial solution to LB solid medium containing Amp (100 μg / mL) antibiotic, spread it evenly with a spreader, and invert the plate in a 37℃ incubator overnight after the bacterial solution has been completely absorbed.

[0121] (5) Pick 5 single colonies of suitable size and inoculate them into 300 μL of LB liquid medium containing Amp (100 μg / mL) antibiotic. Incubate at 37℃ with shaking at 200 rpm for 2 h. Take the corresponding bacterial solutions for PCR identification. The reaction system is as follows:

[0122] Table 8 Conversion System

[0123]

[0124]

[0125] The PCR reaction procedure is as follows:

[0126]

[0127] All PCR products were subjected to agarose gel electrophoresis to determine whether the bands were single and whether their size matched the prediction. The correctly identified bacterial cultures were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. After correct sequence alignment, the remaining bacterial cultures were expanded, and a portion of the culture was mixed with an equal volume of 50% glycerol and stored at -80°C.

[0128] 4.6 Large-scale culture of Escherichia coli

[0129] Remove the stored bacterial culture from the -80℃ freezer and place it on ice. Using an inoculation loop, streak an appropriate amount of the culture onto LB solid medium containing Amp (100 μg / mL) for revival. Incubate overnight at 37℃. Then, using a 10 μL sterile tip, pick a single colony and transfer it to 4 mL of LB liquid medium containing the corresponding antibiotic. Incubate overnight at 37℃ with shaking at 200 rpm. The LB medium formulation is as follows (volume 1 L):

[0130] Table 9. Large-scale culture system of Escherichia coli

[0131]

[0132] If preparing LB solid medium, add 15 g / L of agar powder to the above formula, autoclave at 121°C for 15 min, and add the required antibiotics after the medium cools to about 60°C. It can be stored at room temperature for one week.

[0133] 4.7 Small-scale extraction of E. coli plasmid DNA

[0134] Using Beijing Qingke Biotechnology Co., Ltd. Plasmid Mini Kit is used for plasmid DNA mini-preparation, and all steps are performed at room temperature:

[0135] (1) Take 4 mL of overnight cultured bacterial solution, centrifuge at 12,000×g for 1 min, collect the bacterial cells, and remove as much supernatant as possible;

[0136] (2) Add 250 μL of Buffer PA containing RNase A to the bacterial cells and mix by blowing and aspirating until there are no obvious bacterial clumps;

[0137] (3) After adding 250 μL of Buffer PB, gently invert and mix 6-8 times to fully lyse the cells;

[0138] (4) After adding 350 μL of Buffer PC, gently invert and mix 6-8 times. After mixing thoroughly, centrifuge at 12,000 rpm for 10 min.

[0139] (5) Transfer the supernatant to the adsorption column, being careful not to remove the precipitate. Centrifuge at 12,000 rpm for 1 min, discard the filtrate, and return the adsorption column to the collection tube.

[0140] (6) Add 600 μL of Buffer PW containing anhydrous ethanol along the wall of the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and repeat this step once.

[0141] (7) Place the adsorption column back into the collection tube and centrifuge the empty tube at 12,000 rpm for 2 min.

[0142] (8) Place the adsorption column into a new, clean 1.5 mL centrifuge tube, open the cap, and let it sit at room temperature for 2 minutes to evaporate the residual ethanol.

[0143] (9) Add 35-50 μL of preheated Elution Buffer (60°C) to the center of the adsorption membrane, let stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 2 min. Store the final plasmid solution at -20°C.

[0144] Example 2

[0145] Construction of ginseng callus overexpressing PgNAC72

[0146] Preparation of Agrobacterium EHA105 chemocompetent cells

[0147] (1) Remove the preserved strain EHA105 from the -80℃ freezer and place it on ice. Use an inoculation loop to take an appropriate amount of bacterial solution and streak it onto YEB solid medium containing Rif (50μg / mL) antibiotic for revival. Incubate upside down in a 28℃ incubator for 1-2 days. The YEB medium formula is as follows:

[0148] Table 9 YEB Culture Medium Formulation

[0149]

[0150] If preparing YEB solid medium, add 15 g / L of agar powder to the above formula, autoclave at 121°C for 15 min, and add the required antibiotics after the medium has cooled to about 60°C. It can be stored at room temperature for one week.

[0151] (2) Pick a single colony of suitable size and inoculate it into 5 mL of YEB liquid medium containing Rif (50 μg / mL), and incubate overnight at 28°C with shaking at 200 rpm;

[0152] (3) Inoculate the overnight cultured bacterial suspension into 50 mL of fresh YEB liquid medium at a ratio of 1:100, and culture at 28°C with shaking at 200 rpm until OD. 600 The concentration was approximately 0.5. The bacterial culture was then transferred to a 50 mL centrifuge tube and incubated on ice for 30 minutes.

[0153] (4) Place the bacterial culture in a pre-cooled centrifuge at 4°C and centrifuge at 4,000 rpm for 10 min;

[0154] (5) Discard the supernatant, add 10 mL of pre-cooled 0.15 M NaCl solution, gently blow and aspirate on ice to mix and resuspend the bacterial solution, and centrifuge at 4 °C and 4,000 rpm for 10 min.

[0155] (6) Discard the supernatant, add 1 mL of pre-cooled 20 mM CaCl2 solution, gently blow and aspirate on ice to mix and resuspend the bacterial solution, dispense into pre-cooled 1.5 mL sterile centrifuge tubes, freeze in liquid nitrogen and store at -80℃ for later use.

[0156] Transformation of Agrobacterium EHA105 competent cells

[0157] (1) Take out a 100μL EHA105 competent cell from the -80℃ freezer and thaw it on ice for 10min; in the clean bench, add 2μL of plasmid to the bacterial culture, gently pipette and mix, and then let it stand on ice for 30min.

[0158] (2) Quick freeze in liquid nitrogen for 2 min, heat shock in water bath at 37℃ for 5 min, add 900 μL of YEB liquid culture medium in a clean bench, and revive culture at 28℃ and 100 rpm for 4-6 h.

[0159] (3) Centrifuge at 4,000 rpm for 3 min, discard 850 μL of supernatant, use the remaining culture medium to resuspend the cells, spread evenly on YEB solid medium containing Rif (50 μg / mL) and Kan (50 μg / mL), and incubate upside down in an incubator at 28℃ for 2 days;

[0160] (4) Select single colonies for PCR identification, using the same method as in Example 1. Select positive bacteria with single and correct band size for amplification culture, and add 50% glycerol to store at -80℃.

[0161] Genetic transformation of ginseng callus

[0162] 3.1 Preparation of Agrobacterium EH105

[0163] (1) The successfully verified recombinant bacteria EHA105 / pCambia1300s-PgNAC72 was inoculated into 50 mL of YEB liquid medium and cultured at 28 °C with shaking at 200 rpm until OD. 600 When the concentration reaches 0.4, add 50 μL of 100 mM AS to the bacterial culture and continue shaking incubation until the OD value reaches 0.4. 600 Reaching 0.6;

[0164] (2) Transfer the bacterial culture to a sterile 50 mL centrifuge tube, centrifuge at 4,500 rpm for 10 min, discard the supernatant, and resuspend the bacterial cells in 50 mL of liquid MS medium containing 100 μM AS for later use.

[0165] 3.2 Transformation of ginseng callus

[0166] (1) Select ginseng callus in good growth condition as infection material, and cut it into pieces with a diameter of about 1 cm using a sterile scalpel; spread the cut ginseng callus pieces on MS medium (containing 0.5 mg / L 6-BA, 2.0 mg / L NAA) and pre-culture for 2 days;

[0167] (2) Place the pre-cultured ginseng callus in the bacterial solution in a clean bench and infect it for 15 min at 28℃ and 200 rpm.

[0168] (3) Filter the bacterial solution through a sterile funnel, wipe the ginseng callus dry with sterile filter paper, and place the treated callus on MS solid medium (containing 100 μM AS) for 1 day.

[0169] (4) In a clean bench, ginseng callus was immersed in MS liquid medium (containing 400 mg / L Cef) for 10 min with intermittent shaking; the medium was filtered out with a sterile funnel, and the ginseng callus was wiped dry with sterile filter paper. The treated callus was placed on 6,7-V solid medium (containing 100 μM AS, 1.5 mg / L 2,4-D, and 400 mg / L Cef) and cultured at 24°C in the dark for about 2 months, during which the medium was changed every 20 days.

[0170] (5) Transfer the callus to 6,7-V solid medium containing 50 mg / L hygromycin (Hgy) (other hormones and antibiotics are the same as above); new callus will grow after about 2 months of screening. Transfer the new callus to 6,7-V solid medium without Hgy for further culture.

[0171] Figure 4 The image shows ginseng callus tissue infected with Agrobacterium tumefaciens one month later. Four weeks later, the infected callus tissue was transferred to 6,7-V medium containing 50 mg / L hygromycin for selection until new callus tissue grew. This new callus tissue was then separated and cultured further for identification. Figures B and C show callus tissue grown under hygromycin selection pressure and newly formed callus tissue, respectively. The general process of genetic transformation of ginseng callus tissue is shown in Figure D. Obtaining transgenic callus tissue requires at least 6 months.

[0172] Example 3

[0173] Identification of transgenic ginseng callus

[0174] 1. GUS staining for identification of transgenic callus

[0175] Since the pCambia1301s vector contains the β-D-glucuronidase (GUS) gene and ginseng cells do not contain an endogenous GUS gene, the GUS staining kit from Coolaber was used to identify transgenic ginseng callus. The staining principle is that GUS catalyzes the decomposition of the substrate 5-bromo-4-chloro-3-indole-β-glucuronide (X-Gluc) to produce a blue compound. For substrate preparation details, please refer to the product instructions. 0.1 g of the ginseng callus to be tested was immersed in the substrate solution and reacted at 37°C in the dark for 2 hours. The staining of the callus was then observed.

[0176] The results are as follows Figure 5 As shown: Two groups of callus tissues showed blue color after GUS staining, and were preliminarily identified as transgenic positive callus tissues, named OE-1 and OE-2 respectively.

[0177] Extraction of genomic DNA from ginseng callus tissue

[0178] The gDNA extraction from ginseng callus tissue utilized the SteadyPure Plant Genomic DNA Extraction Kit from Akerui, and the specific steps are as follows:

[0179] (1) Weigh 100mg of ginseng callus tissue and grind it into powder using liquid nitrogen. Transfer the powder to a 1.5mL EP tube containing 500μL Buffer LS-4, add 10μL RNase A, shake to mix, and place the centrifuge tube in a 56℃ water bath for 10min, inverting and mixing 3 times during the process.

[0180] (2) Add 62.5 μL of Buffer PA and mix thoroughly. Place on ice for 5 min, centrifuge at 12,000 rpm for 5 min, take the supernatant and add an equal volume of Buffer BS-2, and mix thoroughly.

[0181] (3) Transfer the above solution to the adsorption column, let it stand at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0182] (4) Add 500 μL of Buffer WA to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0183] (5) Add 500 μL of Buffer WB pre-added with anhydrous ethanol to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; repeat this step once.

[0184] (6) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 min;

[0185] (7) Place the adsorption column into a new 1.5 mL centrifuge tube, open the cap and let it stand at room temperature for 2 min to evaporate residual ethanol. Add 50 μL of preheated 60 °C Elution Buffer to the center of the membrane, let it stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 2 min. Take 5 μL of gDNA solution for agarose gel electrophoresis to ensure extraction quality. The results are as follows: Figure 6 As shown, the extracted gDNA band was single and without tailing, indicating high extraction quality. The remaining solution was stored at -20°C.

[0186] Primers were designed to specifically amplify the insert fragment, and PCR verification was performed on the extracted positive callus tissue gDNA. The PCR system and experimental steps were as described in Example 1, and the primer sequences are as follows:

[0187] gDNA-NAC-F CTACTCTATTTGGGCGTGAC(SEQ ID NO.5)

[0188] gDNA-NAC-R CATCATTGCGATAAAGGAAA(SEQ ID NO.6)

[0189] The results are as follows Figure 7As shown: transgenic positive callus tissue can amplify the target fragment with a length of 550bp, while WT has no corresponding band.

[0190] 3. qRT-PCR was used to identify the expression of the PgNAC72 gene in positive callus tissue.

[0191] RNA extraction and reverse transcription from positive callus tissue were performed as described in Example 1. The differential expression of PgNAC72 in positive callus tissue was detected by qRT-PCR. The reaction system was prepared using Vazyme's ChamQ SYBR qPCR Master Mix, and the qPCR primer sequences for the relevant genes are as follows:

[0192] Primer Sequence (5′→3′)

[0193] q-PgNAC72-F AGAGTAGCAGGGTCGGGTTA (SEQ ID NO.7)

[0194] q-PgNAC72-R TGGTTTTGGTTCCTTTTGG (SEQ ID NO.8)

[0195] q-PgActin-F TGCCCCAGAAGAGCACCCTGT (SEQ ID NO.9)

[0196] q-PgActin-R AGCATACAGGGAAAGATCGGCTTGA (SEQ ID NO.10)

[0197] Prepare the following reaction system on ice:

[0198] Table 10 qRT-PCR system

[0199]

[0200] Avoid strong light exposure during setup. Each experiment should have four replicates. Use the Bio-Rad CFXConnect Real-Time PCR Detection System for qRT-PCR reactions. The reaction program settings are as follows:

[0201]

[0202] Fluorescence signals were acquired during the 72℃ extension step, and the melting curve was set as follows:

[0203] 95℃ 15s

[0204] 60℃ 20s

[0205] 95℃ 15s

[0206] Three independent biological replicate experiments were conducted, and the relative expression levels of genes were calculated using the 2-ΔΔCt method.

[0207] The results are as follows Figure 8 As shown, compared with the wild-type group, the expression of the PgNAC72 gene was increased by 12.0 times in the OE-1 group and by 15.8 times in the OE-2 group.

[0208] Example 4

[0209] Detection of changes in ginsenoside content in callus tissue overexpressing PgNAC72

[0210] 1. Determination of total ginsenoside content

[0211] The total saponin content of ginseng was determined using the total saponin content kit from Shanghai Zhuocai Biotechnology Co., Ltd.

[0212] (1) Take an appropriate amount of the sample to be tested into a 15mL centrifuge tube, place it in a -80℃ freezer overnight, and freeze-dry the completely frozen sample under vacuum for 24h.

[0213] (2) Grind the thoroughly dried sample into powder, weigh 0.05g, add 1mL of extraction solution, extract by sonication for 1h, and centrifuge at 8,000×g for 10min;

[0214] (3) Take 0.5 mL of supernatant into a 1.5 mL centrifuge tube. For the blank control group, take 0.5 mL of extract directly and place it in a 70 °C oven to evaporate to dryness. Add 0.2 mL of reagent one and 0.8 mL of perchloric acid and incubate in a 55 °C water bath for 20 min.

[0215] (4) Pipette 40 μL of reaction solution into a 96-well plate, set 3 replicates for each group, add 200 μL of glacial acetic acid, mix thoroughly, and measure the absorbance at 589 nm using an ELISA reader. The experimental group is recorded as A1 and the blank group is recorded as A2. Calculate ΔA = A1 - A2.

[0216] (5) Calculation of total saponin content: using oleanolic acid as a reference standard.

[0217] Total saponin content (μg / g dry weight) = 5555.6 × (ΔA + 0.012).

[0218] The results are as follows Figure 9As shown: the staining degree of the extracts from OE-1 and OE-2 callus tissues was darker than that of the control group; the total saponin content was calculated using oleanolic acid as a standard, and the total saponin content per gram of wild-type callus tissue was 633.9 μg. The total saponin content of the two groups of transgenic ginseng callus tissues were 1584.9 μg / g dry weight (DW) and 1953.1 μg / g DW, respectively. Compared with the wild type, the total saponin content of OE-1 and OE-2 lines increased by 2.5 times and 3.1 times, respectively.

[0219] 2. Terpenoid metabolomics analysis

[0220] Ginseng callus samples from the OE-1 and WT groups were prepared according to the method described in 2.2.12. Three replicate samples were prepared for each group. The frozen samples were sent to Wuhan Metawell Biotechnology Co., Ltd. for terpene metabolite detection.

[0221] After freeze-drying, metabolites were accurately identified and quantified using high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and the company's proprietary database MWDB (metware database). Sample quality control analysis, orthogonal partial least squares discriminant analysis (OPLS-DA), and screening of differentially expressed metabolites were all performed by Metawell Biotech. The screening criterion for differentially expressed metabolites was based on the variable importance projection (VIP) obtained from the OPLS-DA model; metabolites with a VIP > 1 were generally considered significantly different.

[0222] The screening of differentially expressed metabolites was based on the following principles: VIP > 1, fold change ≥ 2 or fold change ≤ 0.5. The screening results are as follows: Figure 10 As shown, compared to the WT group ginseng callus, the OE group ginseng callus showed a decrease in the content of 5 terpenoids and an increase in the content of 48 terpenoids. Among these, the content of dammarane-type ginsenosides (including PPD-type ginsenosides Rd, Rb1, F2 and PPT-type ginsenosides Re, Rg2, Rg1, Rh1) was significantly increased, with Rd showing a highly significant increase of 836.6 times. The increases in other saponin contents ranged from 2.0 to 6.2 times. There was no significant difference in the content of oleanane-type ginsenosides (ginsenoside R0). The DDS gene is a key node in the biosynthetic pathway of dammarane-type ginsenosides; therefore, we can infer that PgNAC72 enhances the synthesis of dammarane-type ginsenosides by promoting the expression of the PgDDS gene.

[0223] Example 5

[0224] Mechanism of PgNAC72 in regulating ginsenoside biosynthesis

[0225] 1. Analysis of gene expression of key enzymes in the ginsenoside synthesis pathway

[0226] Using the transgenic and wild-type callus cDNA obtained previously as templates, qRT-PCR was used to identify the expression differences of key enzyme genes in the ginsenoside synthesis pathway in WT, OE-1, and OE-2 cells. The method was as described above, and the primer sequences used are as follows:

[0227] Primer Sequence (5′→3′)

[0228] qRT-PgACT-F TGCCCCAGAAGAGCACCCTGT(SEQ ID NO.11)

[0229] qRT-PgACT-RAGCATACAGGGAAAGATCGGCTTGA(SEQ ID NO.12)

[0230] qRT-PgDDS-F TGAGATTAGATGAAACGAAC(SEQ ID NO.13)

[0231] qRT-PgDDS-RGGCAATGATAAGGGGAGGTGT(SEQ ID NO.14)

[0232] qRT-PgFPS-F CAAGTGCTCCTGGTTGGTAGT(SEQ ID NO.15)

[0233] qRT-PgFPS-R TCATACTCGGCAAATACATCC(SEQ ID NO.16)

[0234] qRT-PgHMGR-F GGTTCCCCAAAAGCATAAA(SEQ ID NO.17)

[0235] qRT-PgHMGR-R CCGCCACTACTGCGTTAA(SEQ ID NO.18)

[0236] qRT-PgPPDS-F CGGTTAAGAAATACACGGTCA(SEQ ID NO.19)

[0237] qRT-PgPPDS-R TGGCACGATTCATAGCAGTC(SEQ ID NO.20)

[0238] qRT-PgSE1-FTCTTTGCCGTGGCTATCTAT(SEQ ID NO.21)

[0239] qRT-PgSE1-R CATTTGTCGAAGTCCTTCTGA (SEQ ID NO.22)

[0240] qRT-PgSS3-FTTCAACAGCTCGGACCTCA(SEQ ID NO.23)

[0241] qRT-PgSS3-RGAAAAGTGCCAGTCGTTATCAT(SEQ ID NO.24)

[0242] The results are as follows Figure 11 As shown, compared to WT, the expression of the PgDDS gene increased by 10.3 and 6.0 times in OE-1 and OE-2, respectively. While the expression of other key enzyme genes in the pathway, such as PgSS3, was significantly increased in both OE-1 and OE-2, the increase was much smaller than that of the PgDDS gene. The expression of PgHMGR and PgFPS was downregulated in OE-1 but upregulated in OE-2. PgSE1 was significantly upregulated in OE-1 but showed no significant difference in expression in OE-2. PgPDDS, on the other hand, showed a significant decrease in expression in both OE-1 and OE-2. This suggests that the PgDDS gene is likely a target gene regulated by the PgNAC72 transcription factor.

[0243] 2. EMSA investigation into the binding of PgNAC72 protein to DNA

[0244] 2.1 Marking and Annealing of Probes

[0245] (1) Based on the ABRE and CACG site sequences in PgDDSpro, the following probes were designed:

[0246] Primer Sequence (5′→3′)

[0247] EMSA-ABRE-F GAAT ACGTG ACGGGAAT ACGTG ACGGGAAT ACGTG ACGG (SEQ ID NO.25)

[0248] EMSA-ABRE-R CCGTCACGTATTCCCGTCACGTATTCCCGTCACGTATTC(SEQ ID NO.26)

[0249] EMSA-CACG-F TTTA CACG ACTGTTTA CACG ACTGTTTA CACG ACTG (SEQ ID NO.27)

[0250] EMSA-CACG-R CAGTCGGTAAACAGTCGGTAAACAGTCGTGTAAA(SEQ ID NO.28)

[0251] EMSA-mABRE-F TTT ATTTG ACTG TTT ATTTG ACTG TTT ATTTG ACTG (SEQ ID NO.29)

[0252] EMSA-mABRE-R CAGTCAAATAAACAGTCAAATAAACAGTCAAATAAA(SEQ ID NO.30)

[0253] EMSA-mCACG-F GAAT ATTTC ACGGGAAT ATTTC ACGGGAAT ATTTC ACGG (SEQ ID NO.31)

[0254] EMSA-mCACG-R CCGTGAAATATTCCCGTGAAATATTCCCGTGAAATATTC (SEQ ID NO. 32)

[0255] The underlined portion represents the predicted NAC binding site and its mutation site. The biotin tag was added to the 5′ end of the F chain and purified by HPLC. The synthesis, labeling and purification of the probe were all completed by Beijing Qingke Biotechnology Co., Ltd.

[0256] (2) Take an appropriate amount of equimolar ratio of the forward and reverse strands and mix them in a clean centrifuge tube. Dilute the probe to a final concentration of 1 pmol / μL with Tris buffer (containing 10 mM Tris, 1 mM EDTA, and 50 mM NaCl adjusted to pH 8.0). Take 50 μL of the dilution buffer into a 0.2 mL PCR tube and perform an annealing reaction in a PCR instrument. The reaction program is: 95℃, 5 min, -1℃ / cycle, for a total of 70 cycles, with each cycle lasting 1 min. After the reaction is complete, remove the PCR tube, aliquot the probe, and store it in a -20℃ freezer.

[0257] 2.2 Gel migration retardation experiment

[0258] (1) Prepare 6% TBE gel according to the formula. Note that air bubbles should be prevented during the gel pouring process. After the gel solidifies, rinse the sample well with 0.5×TBE. Perform pre-electrophoresis at 100V for 30 minutes. The electrophoresis buffer is 0.5×TBE.

[0259] (2) Prepare the protein-probe binding reaction system on ice according to the following formula (all volumes are in μL):

[0260] Table 11 Protein-probe binding reaction system

[0261]

[0262] Where X represents the TF chaperone protein, Y represents the PgNAC72:TF fusion protein, Poly(dI·dC) can inhibit the specific binding of the protein to DNA. Before adding the labeled probe, the total system was reacted at room temperature for 5 min, and after adding the labeled probe, it was allowed to stand at room temperature for 20 min.

[0263] (3) Electrophoresis: After the binding reaction is complete, add 5 μL of 5× Loading Buffer, mix thoroughly, and then load the sample. The electrophoresis buffer is 0.5×TBE, the voltage is 100V, and electrophoresis is stopped when the indicator reaches 3 / 4 of the gel.

[0264] (4) Transfer: Take out the nylon membrane and immerse it in 0.5×TBE for at least 10 minutes. Place it in the order of cathode plate-sponge-filter paper-glue-membrane-filter paper-sponge-positive plate and remove air bubbles with a glass rod. Place the transfer tank in an ice bath and transfer the membrane at a constant voltage of 100V for 1 hour.

[0265] (5) UV crosslinking: Place the membrane on clean filter paper (with the bromophenol blue side facing up), and then quickly place it under a UV lamp at a distance of 10 cm for 15 min.

[0266] (6) Blocking: Place the UV-crosslinked nylon membrane in a clean petri dish, add 20 mL of Blocking Buffer, and shake gently for 15 min;

[0267] (7) Hybridization: Gently pour out the Blocking Buffer, add 66.7 μL of Stabilized Streptavidin-Horseradish Peroxidase Conjugate and 20 mL of Blocking Buffer mixture, and shake gently for 15 min;

[0268] (8) Rinse: Gently pour off the binding blocking solution, add 20 mL of 1×Wash Buffer, gently shake and rinse for 5 min, repeat rinsing 3 times;

[0269] (9) Equilibration: Transfer the nylon membrane to a new petri dish, add 30 mL of Substrate Equilibration Buffer, and gently shake for 5 min;

[0270] (10) Luminescence test: Remove the nylon membrane, absorb the excess liquid, place it in a new petri dish, gently pour the substrate working solution onto the membrane so that it can completely cover the membrane surface, and let it stand in the dark for 5 minutes to react; remove the nylon membrane, absorb the excess chemiluminescent substrate from the side, being careful not to let the membrane dry completely, and place it under a chemiluminescence imaging instrument for development.

[0271] The results are as follows Figure 12 As shown, the TF protein did not bind to either the ABRE or CACG labeled probes, while the PgNAC72:TF fusion protein bound to both labeled probes, forming a DNA-protein complex, which resulted in a delayed electrophoretic migration band. In the competitive reaction, we further verified this binding using 50-fold and 200-fold unlabeled competitive probes and a 200-fold mutated competitive probe, respectively. Figure 12 It is known that the PgNAC72:TF fusion protein cannot bind to the mutated ABRE and CACG probes. In summary, we can confirm that the PgNAC72 protein can bind to the ABRE and CACG sites in the PgDDS promoter.

[0272] 3. Dual-luciferase reporter gene assay

[0273] To verify the binding of the PgNAC72 transcription factor to the PgDDS gene promoter in vivo, we cloned the PgDDS promoter sequence, constructed a reporter vector, and conducted a dual-luciferase reporter gene experiment in tobacco leaf cells.

[0274] 3.1 Construction of reporter gene vector

[0275] The PgDDS promoter sequence was retrieved from the NCBI website. BamHI and KpnHI were selected as the restriction enzyme sites for the pGreenⅡ0800-LUC vector and fragment. Specific primers were designed to amplify the full-length PgDDSpro. The primer sequences are as follows:

[0276] Primer Sequence (5′→3′)

[0277] DDSpro-F CGGGGTACCTCCGATGTAGTTAAACTTGA(SEQ ID NO.33)

[0278] DDSpro-R CGCGGATCCGTGCTTTAGGTGCCTATAGA(SEQ ID NO.34)

[0279] The PCR amplification, enzyme digestion, purification, ligation, transformation and identification steps are as described in Example 1;

[0280] 3.2 Transient expression of Agrobacterium-mediated transdermal expression in tobacco leaf lower epidermal cells via Agrobacterium injection permeation method

[0281] The overexpression vector pCambia1301s-PgNAC72 and the reporter gene vector were transformed into Agrobacterium GV3101, respectively, using the transformation method described above. The successfully verified Agrobacterium strains were mixed at a 1:1 volume ratio and co-infected the lower epidermal cells of tobacco leaves using an injection permeation method. Three tobacco leaves were infected from each sample group, using the infection method described in section 2.2.7 above. The co-transfection combinations for each group are as follows:

[0282] Table 12 Cotransfection System

[0283]

[0284] 3.3 Dual-fluorescence quantitative detection

[0285] The quantitative detection of firefly luciferase and Renilla luciferase was performed using the TransGen dual-luciferase assay kit. The specific steps are as follows:

[0286] (1) Sampling begins 2 days after injection. Use a punch with an inner diameter of 2 cm to take samples, being careful to avoid large leaf veins. Take one sample from each leaf and quickly put it into a 1.5 mL centrifuge tube. Then freeze it with liquid nitrogen.

[0287] (2) Use an electric drill to drive the grinding rod to quickly crush the sample, add 100 μL of protein extraction solution (containing 1 mM DTT and pH 7.8 of PBS buffer) in two portions, homogenize, and centrifuge at 4°C and 12,000 rpm for 10 min.

[0288] (3) Take the supernatant to detect the activities of firefly luciferase and kidney luciferase. The detection steps are performed according to the kit instructions. Calculate the ratio of firefly luciferase activity to kidney luciferase activity.

[0289] The results are as follows Figure 13 As shown: The PgNAC72 expression vector (Effector, full name pCambia1300s-PgNAC72) and the reporter gene vector (Reporter, full name pGreenⅡ0800-LUC-DDSpro) were successfully constructed. The effector and reporter vectors were co-transformed into tobacco leaf cells using Agrobacterium tumefaciens GV3101-mediated transformation. The control group was transfected with the empty vector plasmid pCambia1301s+Reporter. After 2 days of culture (16 h light / 8 h dark), the activities of firefly luciferase (LUC) and Renilla luciferase (REN) in both groups were detected using a multi-functional microplate reader. The relative activity of the experimental group was calculated based on the relative luciferase activity of the control group (LUC / REN). The experimental results are shown below. Figure 13As shown in (B): Compared with the control group, the relative activity in the experimental group increased by 2.4 times. This result indicates that PgNAC72 can bind to the PgDDS promoter and promote the expression of PgDDS.

[0290] The above embodiments should be understood as being used only to illustrate the present invention more clearly, and not to limit the scope of the present invention. After reading the present invention, any modifications of the present embodiments by those skilled in the art will fall within the scope defined by the appended claims.

Claims

1. The application of PgNAC72 protein in upregulating ginsenoside biosynthesis, characterized in that, The sequence of the PgNAC72 protein is shown in SEQ ID NO.1; the ginsenoside is a dammarane-type ginsenoside.

2. Use of a vector overexpressing the PgNAC72 gene in up-regulating ginsenoside biosynthesis, characterized in that, The sequence of the protein encoded by the PgNAC72 gene is shown in SEQ ID NO.1; the ginsenoside is a dammarane-type ginsenoside.

3. Use according to claim 2, characterized in that, The application involves transfecting ginseng suspension cells with a plasmid that overexpresses the PgNAC72 gene.

4. Use of a recombinant vector in up-regulating ginsenoside biosynthesis, characterized in that, The recombinant vector overexpresses the PgNAC72 gene; the sequence of the protein encoded by the PgNAC72 gene is shown in SEQ ID NO.1; the ginsenoside is a dammarane-type ginsenoside.

5. Use of a kit in upregulating ginsenoside biosynthesis, characterized in that, The kit includes a vector overexpressing the PgNAC72 gene; the sequence of the protein encoded by the PgNAC72 gene is shown in SEQ ID NO.1; the ginsenoside is a dammarane-type ginsenoside.

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