CN116375837A - Application of PgNAC72 in regulation and control of ginsenoside biosynthesis - Google Patents

Abstract

The invention belongs to the technical field of biological genetic engineering, and particularly relates to application of PgNAC72 protein or gene in regulating ginsenoside biosynthesis, wherein the sequence of the PgNAC72 protein is shown as SEQ ID NO.1, and the sequence of the PgNAC72 gene is shown as SEQ ID NO. 2. According to the invention, in vitro and in vivo experiments of EMSA and dual-luciferase reporter genes prove that PgNAC72 can be combined with cis-acting elements ABRE and CACG on a PgDDS promoter so as to promote transcription of PgDDS. These results indicate that PgDDS is a target gene regulated by PgNAC 72.

Description

Application of PgNAC72 in regulation and control of ginsenoside biosynthesis

Technical Field

The invention belongs to the technical field of biological genetic engineering, and particularly relates to a method for realizing regulation and control of ginsenoside biosynthesis by promoting expression of dammarenediol synthase (DDS) genes by PgNAC 72.

Background

Ginseng (Panax ginseng c.a. meyer) is a perennial herb of the family araliaceae and has a long history of medicinal use. Ginsenoside is the main active ingredient of ginseng and has the functions of resisting cancer, resisting oxidation, resisting inflammation and the like, however, the increasing medicinal demands are difficult to meet due to low yield of the ginsenoside. In recent years, key enzyme genes on the biosynthesis pathway of ginsenoside are gradually identified, but regulatory factors on the pathway are rarely reported. The transcription factor is used as a protein capable of being specifically combined with cis-acting elements, plays an important role in a complex regulation network of plants, explores the biological functions of transcription factors related to ginsenoside synthesis, is beneficial to revealing the accumulation rule of ginsenoside in ginseng, and simultaneously provides a foundation for producing ginsenoside by utilizing plant synthesis biological means.

Currently, 4439 transcription factors belonging to 94 gene families are found in total in the ginseng genome. Transcription factors of the WRKY, bHLH, MYB family have been reported to play a key role in ginsenoside biosynthesis. Wang found that low temperature stimulation can promote accumulation of ginsenoside, 5 ginsenoside biosynthesis genes GPS, SS, CYP716a53v2, UGT74AE2 and UGT94Q2, and expression levels of 3 PgWRKY (PgWRKY 1, pgWRKY3 and PgWRKY 8) are strongly positively correlated with ginsenoside yield, and these PgWRKY might be involved in ginsenoside biosynthesis by regulating related pathway genes [1]. Chu et al performed a whole genome study on bHLH transcription factors, identified 169 PgbHLH genes altogether and divided them into 24 subfamilies, and Chu found that 6 PgbHLH genes from 4 subfamilies could be involved in the regulation of ginsenoside biosynthesis in combination with gene expression patterns and saponin chemistry [2]. Liu screened out a MeJA-induced R2R3 type MYB gene PgMYB2, and found through yeast single hybridization and double luciferase reporter gene experiments that PgMYB2 can be combined with a DDS promoter and promote the expression of PgDDS, which suggests that PgMYB2 may be involved in the biosynthesis of ginsenoside [3]. Because of the great difficulty of genetic transformation of ginseng, less research is needed for regulating and controlling the synthesis of ginsenoside by transcription factors, and the research on the effects of other transcription factor families in the synthesis of the ginsenoside still has an original meaning.

NAC is a plant-specific class of transcription factors, and is also the most widely occurring class. 19997 NAC transcription factors from 150 or more species have been included based on plant transcription factor database statistics, with 101, 328 and 138 transcription factors found in tomato, rice and Arabidopsis, respectivelyhttp://planttfdb.gao-lab.org) A. The invention relates to a method for producing a fibre-reinforced plastic composite Liu preliminary identification and analysis of the NAC gene family of Ginseng radix, liu co-screened 251 PgNACs in the genomic database of Ginseng radix, and evolutionarily analyzed to separate them into 11 subgroups, while also mining 5 cold-responsive PgNACs (PgNAC 05-2, pgNAC41-2, pgNAC48, pgNAC56-1, and PgNAC 59) [4 ]]。

Reference is made to:

[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 Food Biochemistry,2019,43.

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

[3]LIU T,LUO T,GUO X,et al.PgMYB2,a MeJA-Responsive Transcription Factor,Positively Regulates the Dammarenediol Synthase Gene Expression in Panax Ginseng[J].International journal of molecular sciences,2019,20.

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

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

[6] hu Gonggong isolation and functional identification of transcription factors related to rice stress [ D ]. University of agriculture in China, 2006.

Disclosure of Invention

The invention aims to provide an application of PgNAC72 in regulating and controlling ginsenoside biosynthesis.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the application of PgNAC72 protein in regulating ginsenoside biosynthesis is provided, and the sequence of the PgNAC72 protein is shown in SEQ ID NO. 1.

SEQ ID NO.1 is:

MGVPETDPLSQLSLPPGFRFYPTDEELLVQYLCRKVAGQHFSLQIIGEIDLYKFDPWVLPSKAIFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKVITTEGRKVGIKKALVFYVGKAPKGTKTNWIMHEYRLSDPQRKNGSARLDDWVLCRIYKKNSSAQKPVLGDIDSKEHSHSHSHSHSHSHGSSSSSSSQFEDVLESLPEIEDRFFTLPRMNSLNDKLNFQNLGSGNFDWAILAGLNSMPEHVPGTQAPMQTQTQGLMNNNNQNYMCVPSTSPLGHVDTRFGKSMEEEVESGLRNNQRVDNSGFLNSNSNSSCSVDPFAIRYPIQSGNMGFTLGCENCT。

the application of the PgNAC72 gene in regulating ginsenoside biosynthesis is provided, and the sequence of the PgNAC72 gene is shown in SEQ ID NO. 2.

SEQ ID NO.2 is:

ATGGGTGTGCCGGAGACTGACCCTCTTTCACAGCTAAGCTTGCCTCCGGGTTTCCGGTTTTATCCGACGGACGAGGAGCTTCTGGTTCAGTACCTTTGCCGGAAAGTCGCCGGACAACATTTCTCTCTGCAAATTATTGGCGAAATTGATTTGTACAAATTCGATCCCTGGGTTTTGCCTAGTAAAGCAATAATTGGGGAGAAAGAATGGTACTTTTTCAGTCCAAGAGATAGAAAATACCCAAATGGGTCACGCCCAAATAGAGTAGCAGGGTCGGGTTACTGGAAGGCAACCGGAACCGATAAGGTCATAACTACAGAGGGAAGAAAAGTTGGAATCAAGAAAGCTCTGGTTTTTTACGTCGGTAAAGCTCCAAAAGGAACCAAAACCAACTGGATCATGCACGAGTACAGGCTCTCAGATCCCCAAAGGAAAAATGGAAGCGCAAGGTTGGATGATTGGGTACTTTGTCGGATTTACAAGAAGAACTCGAGTGCACAAAAACCCGTGTTGGGTGATATTGATAGTAAAGAACATAGTCATAGTCATAGTCATAGTCATAGTCATAGTCATGGTTCTTCTTCTTCATCTTCCTCTCAATTCGAGGATGTGCTGGAATCCTTGCCGGAGATTGAGGACCGGTTCTTTACCCTGCCTAGAATGAACTCGCTCAATGACAAGCTCAATTTTCAGAATTTGGGTTCTGGTAATTTTGACTGGGCAATTCTCGCAGGACTTAATTCGATGCCGGAGCATGTCCCCGGAACTCAGGCTCCGATGCAAACTCAAACTCAAGGATTAATGAACAACAACAATCAGAATTACATGTGTGTCCCCTCAACCTCACCGCTTGGCCACGTGGACACCAGATTTGGAAAAAGTATGGAGGAAGAGGTGGAGAGTGGACTCAGAAATAATCAGCGGGTTGACAATTCAGGCTTCCTCAACTCCAACTCCAACTCTTCTTGTTCGGTTGATCCGTTTGCTATTCGATACCCGATTCAATCCGGAAACATGGGGTTTACTTTAGGATGTGAAAATTGTACTTAA。

in one preferred embodiment, the use is that the PgNAC72 gene and PgNAC72 protein regulate ginsenoside synthesis by regulating expression of the PgDDS gene of ginseng.

In one preferred embodiment, the use is to regulate ginsenoside synthesis by over-expression of the PgNAC72 gene or PgNAC72 protein.

In one preferred embodiment, the agent that overexpresses the PgNAC72 gene or PgNAC72 protein comprises methyl jasmonate.

In one preferred embodiment, the application is transfection into ginseng suspension cells by an over-expression plasmid with the PgNAC72 gene.

In one preferred embodiment, the ginsenoside is dammarane type ginsenoside.

The invention also claims the application of a recombinant vector in regulating and controlling the biosynthesis of ginsenoside, and the recombinant vector overexpresses PgNAC72 gene.

The invention also claims a kit comprising an agent that overexpresses the PgNAC72 gene or PgNAC72 protein or an overexpression vector with the PgNAC72 gene.

The invention also claims the application of the kit in regulating and controlling the biosynthesis of ginsenoside.

In fact, there are many transcription factors that are upregulated by ginseng roots after MeJA (methyl jasmonate) induction, but most of them do not regulate ginsenoside synthesis, for example, lin finds 16 AP2/ERF transcription factors that are significantly differentially expressed by analysis of MeJA-induced differential transcriptome of notoginseng, and real-time quantitative PCR (RT-qPCR) and co-expression network analysis of these 16 AP2/ERF transcription factors found that only key genes of the synthesis of PnERF2 and PnERF3 with notoginseng saponins, damascene diol II synthase gene (DS) and squalene epoxidase gene (SE), have significant correlation [5]. Whereas we selected, after screening by a number of experiments, pgNAC72 (Pg_S 5466.10) that was significantly up-regulated after MeJA induction as the subject. The amino acid sequence, structure and physicochemical properties of the protein encoded by the PgNAC72 gene are analyzed by using a bioinformatics method. The subcellular localization of the PgNAC72 protein was determined in tobacco by qRT-PCR exploring the expression pattern of the PgNAC72 gene. In order to explore the function of PgNAC72, we realize the overexpression of PgNAC72 in ginseng callus, find that the content of dammarane type ginsenoside in transgenic callus is obviously improved, and detect the expression of key enzyme genes on the ginsenoside synthesis path by qRT-PCR, thereby screening candidate target genes PgDDS thereof. Based on the above studies, we have subsequently performed experiments on DNA-protein interactions, and the role of PgNAC72 transcription factor in the ginsenoside synthesis pathway has been gradually elucidated. The research carries out relatively perfect functional analysis on the NAC gene of hormone response in ginseng for the first time, and the result can provide a new approach for researching secondary metabolites of ginseng.

Compared with the prior art, the invention has the beneficial effects that:

the invention constructs a plant expression vector by cloning PgNAC72 genes, transforms ginseng callus by using an agrobacterium transformation method, and obtains positive callus which over-expresses PgNAC72 through PCR, WB and qRT-PCR identification. The total saponin content in the positive callus is obviously improved, and the terpenoid metabonomics detection shows that the dammarane type ginsenoside content is mainly improved, which indicates that the PgNAC72 gene promotes the biosynthesis of the dammarane type ginsenoside.

The invention over-expresses PgNAC72 to find that the key enzyme genes PgDDS and PgSS3 on the ginsenoside biosynthesis pathway are obviously improved in expression, but the change multiple of PgDDS is far higher than that of PgSS3; while the trend of the other key enzyme genes (PgHMGR, pgFPS, pgSE, pgPDDS) was not consistent with PgNAC 72. We verified from in vitro and in vivo experiments with EMSA and dual luciferase reporter genes, respectively, that PgNAC72 was able to bind to the cis-acting elements ABRE and CACG on the PgDDS promoter to promote transcription of PgDDS. These results indicate that PgDDS is a target gene regulated by PgNAC 72.

Drawings

FIG. 1 subculture of ginseng callus;

FIG. 2 detection of total RNA of ginseng callus;

FIG. 3 shows the result of PCR amplification of PgNAC72 fragment gel electrophoresis, M being the gene Marker;

FIG. 4 is a flow chart of genetic transformation of ginseng callus; graph a: callus 1 month after infection with agrobacterium; graph B: callus 2 months after infection with agrobacterium; graph C: neonatal callus transferred to media without Hgy; graph D: general procedure of genetic transformation of ginseng callus;

FIG. 5 GUS staining to detect ginseng callus;

FIG. 6 detection of ginseng callus gDNA;

FIG. 7 PCR identification of transgenic calli;

FIG. 8 qRT-PCR verifies the expression level of PgNAC72 in transgenic calli;

FIG. 9 detection of total saponins content of ginseng callus; graph a: using the indication of vanillin-perchloric acid color system to extract saponin, the higher the saponin content, the darker the extract color; graph B: the content of total saponins in the transgenic callus of the over-expressed PgNAC72 is taken as a reference by taking the content of total saponins in the WT group;

FIG. 10 differential metabolite analysis by terpenoid metabonomics;

FIG. 11 overexpression of the key enzyme gene in PgNAC72 transgenic ginseng calli;

FIG. 12 EMSA explores the binding of PgNAC72 protein to DNA, probe A with ABRE site and probe B with CACG site;

FIG. 13 shows the regulation of PgDDS by PgNAC72 in a dual luciferase reporter assay.

Detailed Description

The invention will be described in detail below with reference to the drawings in connection with embodiments. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.

The experimental materials used in the examples are as follows:

1 plant material

The ginseng callus is obtained from the laboratory early-stage subculture; wild type tobacco seeds were harvested and stored from a laboratory at a earlier stage.

2 main biochemical reagent

TABLE 1 Biochemical reagents used in the present invention

3 main experimental instrument

TABLE 2 Experimental apparatus used in the present invention

Example 1

Cloning of PgNAC72 Gene

1 establishment of ginseng callus subculture system

1.1 Configuration of 6,7-V Medium

TABLE 36, 7-V Medium

Preparing a 6,7-V culture medium according to the formula, regulating the pH to 5.8, adding 8g of agar powder if preparing a solid culture medium, sterilizing at 121 ℃ for 15min under high pressure, cooling the culture medium to about 60 ℃ after sterilization, adding 1mL of sterilized 2,4-D (the concentration of mother liquor is 1 mg/mL) into the culture medium in an ultra-clean workbench, packaging and sealing.

1.2 subculture of Ginseng radix callus

The ginseng callus with good growth vigor and light yellow color (the ginseng callus which is obtained from the early-stage subculture and preservation of the subject group of the teacher of the university of middle and south Luo Zhiyong) is picked up by using sterile forceps in an ultra-clean workbench, and transferred to a freshly prepared 6,7-V solid medium for subculture, so that the ginseng callus with better activity is obtained, as shown in figure 1. The callus is gathered together as much as possible in the culture process, so that excessive dispersion is avoided. After being sealed by a plastic film, the culture flask is placed into a culture box at 25 ℃ for standing and light-shielding culture. The growth and the infection of the callus were observed every 2-3d, and the new 6,7-V medium was changed about 20-30d according to the growth state.

2 extraction of total RNA from ginseng callus

(1) 1ml Trizol was added to a RNase-Free centrifuge tube in advance and placed on ice. Grinding the sample into powder by using a mortar pestle, wherein the process needs to continuously supplement liquid nitrogen into the mortar, taking 50mg of powder, adding into a centrifuge tube, and uniformly mixing by a vortex instrument; standing at room temperature for 5min;

(2) Centrifuging at 12,000Xg at 4deg.C for 5min, carefully sucking 950uL supernatant into a new RNase free centrifuge tube, adding 1/5 of the volume of Trizol chloroform, and mixing thoroughly; standing at room temperature for 5 minutes;

(3) Centrifugation is carried out for 10min at 12,000Xg and 4 ℃, and the homogenate is divided into three layers, namely: a supernatant containing RNA, an intermediate protein layer and a lower organic phase;

(4) Transfer 450ul of supernatant to another new RNase free centrifuge tube (no aspiration of the intermediate protein layer); adding 450uL of isopropanol with the volume equal to that of Trizol, fully and uniformly mixing, and standing at room temperature for 10min;

(5) Transferring the mixture into an adsorption column RA, centrifuging at 13,000rpm for 2min, and discarding the filtrate;

(6) Adding 500 μl deproteinized solution RW1, standing at room temperature for 3min, centrifuging at 13,000rpm for 30s, and discarding the filtrate;

(7) Adding 500 μl of rinsing solution RW with absolute ethanol added in advance, centrifuging at 13,000rpm for 30 seconds, and discarding the filtrate; repeating the operation one time;

(8) The adsorption column was put back into the empty collection tube and centrifuged at 13,000rpm for 2 minutes;

(9) Placing the adsorption column in a new RNase free centrifuge tube, opening the cover, and standing at room temperature for 2min to volatilize residual ethanol; to the adsorption film was added dropwise 30. Mu.L of RNase free water preheated to 70-90℃and left at room temperature for 2min at 12,000rpm for 1min.

After centrifugation, 2. Mu.L was taken and the quality of RNA was verified by agarose gel electrophoresis. The electrophoresis results are shown in FIG. 2: the three rRNA bands are obvious in the picture, the brightness of the 28S band is about twice that of 18S, no obvious tailing phenomenon exists in the bands, and the extracted RNA is not degraded and can be used for subsequent experiments.

3 reverse transcription

(1) gDNA removal

The following reagents were added to the RNase-free PCR tube in this order:

TABLE 4 PCR System

After blowing and sucking the reagent, incubating the reagent in a PCR instrument at 42 ℃ for 2min, taking out the reagent and placing the reagent on ice;

(2) cDNA Synthesis

To the PCR tube containing the reaction solution, 4. Mu.L of 5 XHiScript II Enzyme Mix was added, and the following reaction was performed in a PCR apparatus: 50 ℃ for 15min;85 ℃,5s;4 ℃ and infinity. After completion of the reaction, the cDNA was stored at-20 ℃.

Construction of 4 PgNAC72 gene over-expression vector

4.1 PCR amplification of PgNAC72 Gene fragment

The PCR amplification was performed using Takara high fidelity enzyme PrimeSTAR Max DNA Polymerase, and primers were designed by Primer Premier 5.0 software and synthesized by Beijing Optimago Co., ltd, and the Primer sequences were as follows:

Primer Sequence(5′→3′)

PgNAC-O-F CGGGGTACCATGGGTGTGCCGGAGACTG(SEQ ID NO.3)

PgNAC-O-R

CGCGGATCCTTACTTATCATCATCATCCTTATAATCAGTACAATTTTCACATCCTAAAGTAAAC(SEQ ID NO.4)

the reaction system is as follows:

TABLE 5 PCR amplification System of PgNAC72 Gene fragment

The PCR reaction procedure was set as follows:

after the reaction, the liquid on the tube wall is quickly centrifuged down and placed on ice for subsequent agarose gel purification or temporarily stored in a-20 ℃ refrigerator.

The PgNAC72 fragment with enzyme cutting sites at two ends is amplified, the PCR product is detected by agarose gel electrophoresis, the detection result is shown in figure 3, and the product fragment is 1050bp.

4.2 PCR product purification

Removing the impurity band of the PCR product by gel electrophoresis, purifying the gel containing the target fragment, and adopting a gel recovery kit of Norwezan company, wherein all the steps are carried out at room temperature, and the method comprises the following steps:

(1) Mixing the PCR product obtained in the last step with a 6×loading Buffer, separating a target fragment and a impurity fragment by agarose gel electrophoresis for 30min, rapidly cutting agarose gel containing the DNA fragment with the target size under the projection of an ultraviolet lamp, and then placing the agarose gel into a clean 1.5ml centrifuge tube, wherein the blade needs to be cleaned in advance;

(2) Weighing the gel, adding Buffer GDP according to the proportion of adding 100 mu L of gel into each 0.1g of gel, then carrying out water bath on a centrifuge tube at 50 ℃ for 10min, slightly and reversely mixing for 3 times during the water bath, and properly prolonging the water bath time if the gel is not completely dissolved;

(3) Transferring the completely dissolved solution to an adsorption column, centrifuging for 30s at 12,000Xg, pouring the liquid in the collection tube back to the adsorption column again for improving recovery rate, centrifuging for 30s at 12,000Xg, and discarding the filtrate; if the total volume of the solution is more than 700 mu L, the solution can be separated into a plurality of times of column passing;

(4) Adding 500 μl Buffer GDP into the adsorption column, standing for 1min, centrifuging at 12,000Xg for 30s, discarding the filtrate, and placing the adsorption column back into the collection tube;

(5) Confirming that absolute ethyl alcohol is added into the Buffer DW in advance, adding 700 mu L of Buffer DW into an adsorption column along the pipe wall, centrifuging for 30s at 12,000Xg, discarding the filtrate, placing the adsorption column back into a collecting pipe, and repeating the steps once;

(6) Placing the adsorption column into an empty collecting pipe, and centrifuging the empty pipe for 2min at a speed of 12,000Xg;

(7) Discarding the collecting tube, placing the adsorption column into a new dry 1.5ml centrifuge tube, opening the cover of the adsorption column, and drying at room temperature for 2-5min to volatilize residual ethanol;

(8) The solution Buffer was preheated to 60℃and 30. Mu.L of the solution was added to the adsorption film, the solution was allowed to stand for 2min and centrifuged at 12,000Xg for 2min, and the finally obtained DNA solution was stored in a refrigerator at-20 ℃. The purified product was verified by agarose gel electrophoresis, as shown in FIG. 3.

4.3 purification of fragment restriction enzyme and pCambia1301s vector linearization

The purified fragment obtained in the previous step and pCambia1301S vector (the vector is provided by subtropical agricultural ecology research in the department of China, modified by the university of agriculture laboratory in China, namely, double CaMV 35S promoter [6 ]) is introduced on the pCambia1301 vector, and is digested with restriction enzymes BamHI and KpnI to generate cohesive ends which facilitate subsequent ligation reactions, and a digestion system is configured in a 200 mu L PCR tube according to the following table:

TABLE 6 enzyme digestion system

4.4 ligation of PgNAC72 purified fragment to linearized pc1301s vector

Purifying the enzyme-cleaved product obtained in the last step by adopting T ₄ The purified enzyme-cut PgNAC72 fragment is connected with a carrier by DNA ligase, the consumption of the PgNAC72 fragment is calculated according to the molar concentration ratio of the carrier to the insert of 3:1, and a connection system is configured in a 200 mu L PCR tube according to the following table:

table 7 connection system

4.5 transformation of ligation products into E.coli and screening thereof

(1) Taking out a 100 mu LDH5 alpha competent cell from a refrigerator at the temperature of minus 80 ℃, rapidly inserting the competent cell into ice to melt for 10min, adding 10 mu L of a connection product into bacterial liquid, slightly blowing and sucking the connection product to mix the connection product, and then standing the connection product on the ice for 30min;

(2) Placing the bacterial liquid into a water bath at 42 ℃ for heat shock for 1min, and rapidly inserting into ice;

(3) In an ultra-clean workbench, 700 mu L of LB liquid medium without antibiotics is added into the tube, and the mixture is gently inverted and mixed uniformly, and shake-cultured for 60min at 180 rpm;

(4) Centrifuging at 4,000Xg for 3min, collecting thallus, discarding 700 μl of supernatant, leaving about 100 μl of culture medium for re-suspending thallus, fully blowing and sucking, mixing, transferring bacterial liquid onto LB solid culture medium containing Amp (100 μg/mL) antibiotics, coating with coater uniformly, and placing the plate in 37 deg.C incubator for culturing overnight after bacterial liquid is completely absorbed;

(5) 5 single colonies with moderate sizes are selected and inoculated into 300 mu L of LB liquid medium containing Amp (100 mu g/mL) antibiotics, the culture is carried out for 2 hours at 37 ℃ under shaking at 200rpm, and corresponding bacterial solutions are taken for PCR identification, and a reaction system is prepared as follows:

TABLE 8 conversion System

The PCR reaction procedure was as follows:

all PCR products were used for agarose gel electrophoresis to identify if the bands were single and consistent in size with the predicted. And (3) delivering the identified correct bacterial liquid to Beijing qing biological technology Co-Ltd for sequencing, after the sequence comparison is correct, performing expansion culture on the residual bacterial liquid, uniformly mixing part of bacterial liquid with 50% glycerol with the same volume, and storing in a refrigerator at the temperature of minus 80 ℃.

4.6 expanded culture of E.coli

The preserved bacterial liquid is taken out from a refrigerator at the temperature of minus 80 ℃ and placed on ice, a proper amount of bacterial liquid is dipped in an inoculating loop to be streaked and revived on LB solid medium containing Amp (100 mug/mL) antibiotics, after overnight culture in a 37 ℃ incubator, single colony is picked up to 4mL of LB liquid medium containing the corresponding antibiotics by a10 mug sterile Tip, and the culture is carried out at the temperature of 37 ℃ and shaking at 200rpm for overnight. The formula of LB medium is as follows (volume 1L):

TABLE 9 extended culture System for E.coli

If LB solid culture medium is prepared, 15g/L agar powder is added on the basis of the above formula, and the culture medium is autoclaved at 121 ℃ for 15min, and the required antibiotics are added when the culture medium is cooled to about 60 ℃ and can be stored for one week at room temperature.

4.7 Small extraction of E.coli plasmid DNA

By Beijing Optimu Corp

Plasmid Mini Kit for Plasmid DNA miniprep, all steps were performed at room temperature:

(1) Taking 4mL of bacterial liquid cultured overnight, centrifuging for 1min at 12,000Xg, collecting bacterial cells, and removing the supernatant as much as possible;

(2) 250 mu L Buffer PA containing RNase A is added into the thalli, and the mixture is blown and sucked until no obvious bacteria blocks exist;

(3) After 250 mu LBuffer PB is added, the mixture is gently inverted and uniformly mixed for 6 to 8 times, so that the thalli are fully cracked;

(4) Adding 350 μL Buffer PC, gently mixing for 6-8 times, and centrifuging at 12,000rpm for 10min;

(5) Transferring the supernatant to an adsorption column without sucking out precipitate, centrifuging at 12,000rpm for 1min, discarding the filtrate, and placing the adsorption column back into the collection tube;

(6) Adding 600 μl Buffer PW containing absolute ethanol along the wall of the adsorption column, centrifuging at 12,000rpm for 1min, discarding the waste liquid, and repeating the steps once;

(7) Placing the adsorption column back into the collecting tube, and centrifuging at 12,000rpm for 2min;

(8) Placing the adsorption column into a new clean 1.5mL centrifuge tube, opening a tube cover, and standing at room temperature for 2min to volatilize residual ethanol;

(9) 35-50. Mu.L of an adsorption Buffer preheated to 60℃was added to the center of the adsorption film, and the mixture was allowed to stand at room temperature for 2min and centrifuged at 12,000rpm for 2min. The resulting plasmid solution was stored in a-20deg.C refrigerator.

Example 2

Construction of PgNAC 72-overexpressed ginseng callus

Preparation of 1 Agrobacterium EHA105 chemocompetent cells

(1) The stored strain EHA105 was taken out from the-80℃refrigerator and placed on ice, and a proper amount of bacterial liquid was dipped in an inoculating loop and streaked on a YEB solid medium containing Rif (50. Mu.g/mL) antibiotics for resuscitation, and the strain EHA105 was cultured in an inversion manner in a 28℃incubator for 1-2d, with the following formula:

table 9 YEB Medium formulation

If yes solid culture medium is prepared, adding 15g/L agar powder based on the above formula, sterilizing at 121deg.C for 15min, cooling to 60deg.C, adding required antibiotic, and standing at room temperature for one week;

(2) Picking single colony with moderate size, inoculating into 5mL of YEB liquid culture medium containing Rif (50 mug/mL), and shaking culturing at 28 ℃ and 200rpm for overnight;

(3) The overnight cultured bacterial liquid is inoculated into 50mL of fresh YEB liquid culture medium according to the proportion of 1:100, and the bacterial liquid is cultured at 28 ℃ under shaking at 200rpm until the bacterial liquid reaches OD ₆₀₀ About 0.5, transferring the bacterial liquid into a 50mL centrifuge tube, and carrying out ice bath for 30min;

(4) Placing the bacterial liquid in a pre-cooling centrifuge at 4 ℃, and centrifuging at 4,000rpm for 10min;

(5) Removing the supernatant, adding 10mL of precooled 0.15M NaCl solution, slightly blowing and sucking on ice to mix uniformly, and centrifuging at 4 ℃ and 4,000rpm for 10min;

(6) The supernatant was discarded and 1mL of pre-chilled 20mM CaCl was added ₂ The solution is lightly blown and sucked on ice to mix the bacteria suspension again, the bacteria suspension is subpackaged into a precooled 1.5mL sterile centrifuge tube, and the bacteria suspension is placed at the temperature of minus 80 ℃ for standby after quick freezing by liquid nitrogen.

Transformation of competent cells of Agrobacterium EHA105

(1) Taking out 100 μl of EHA105 competent cells from-80deg.C refrigerator, and thawing on ice for 10min; sucking 2 mu L of plasmid in an ultra-clean workbench, adding the plasmid into bacterial liquid, slightly blowing and sucking the plasmid, uniformly mixing the plasmid and the bacterial liquid, and standing the mixture on ice for 30min;

(2) Quick-freezing with liquid nitrogen for 2min, performing heat shock in 37 ℃ water bath for 5min, adding 900 mu L of YEB liquid culture medium into an ultra-clean workbench, and performing slow shaking recovery culture at 28 ℃ and 100rpm for 4-6h;

(3) Centrifuging at 4,000rpm for 3min, discarding 850 μL supernatant, allowing the rest culture medium to be used for re-suspending thallus, uniformly coating on YEB solid culture medium containing Rif (50 μg/mL), kan (50 μg/mL), and culturing in an incubator at 28deg.C for 2d;

(4) Single colonies were picked for PCR identification in the same manner as in example 1, positive bacteria with single bands and correct sizes were selected for amplification culture, and 50% glycerol was added and stored at-80 ℃.

Genetic transformation of Ginseng callus

3.1 preparation of Agrobacterium EH105

(1) Inoculating recombinant strain EHA105/pCambia1300s-PgNAC72 to 50mL YEB liquid culture medium, and shake culturing at 28deg.C and 200rpm to OD ₆₀₀ When the concentration was 0.4, 50. Mu.L of 100mM AS was added to the bacterial liquid, and the shaking culture was continued until the OD was reached ₆₀₀ Up to 0.6;

(2) The bacterial solution was transferred to a sterile 50mL centrifuge tube, centrifuged at 4,500rpm for 10min, the supernatant was discarded, and the bacterial cells were resuspended in 50mL of liquid MS medium containing 100. Mu.M AS for further use.

3.2 transformation of Ginseng callus

(1) Selecting ginseng callus with good growth state as an infection material, and cutting the ginseng callus into blocks with the diameter of about 1cm by using a sterilized scalpel; spreading the cut massive ginseng callus on MS culture medium (containing 0.5 mg/L6-BA and 2.0mg/L NAA), and pre-culturing for 2d;

(2) Placing the pre-cultured ginseng callus in a bacterial liquid in an ultra-clean workbench, and infecting for 15min at 28 ℃ and 200 rpm;

(3) Filtering the bacterial liquid by using a sterile funnel, wiping the ginseng callus with sterile filter paper, placing the treated callus on an MS solid culture medium (containing 100 mu M AS), and co-culturing for 1d;

(4) Soaking ginseng callus in MS liquid culture medium (containing 400mg/L Cef) in an ultra-clean workbench for 10min, and intermittently shaking; filtering the culture medium with a sterile funnel, wiping the ginseng callus with sterile filter paper, placing the treated callus on a 6,7-V solid culture medium (containing 100 mu M AS, 1.5 mg/L2, 4-D and 400mg/L Cef), and culturing at 24deg.C in dark for about 2 months, wherein the culture medium is replaced every 20D;

(5) Calli were transferred to 6,7-V solid medium (other hormone and antibiotic content consistent with the above) containing 50mg/L Hygromycin (Hgy); about 2 months of screening will give rise to new calli, and the new calli will be transferred to 6,7-V solid medium without Hgy for expansion.

FIG. 4 shows ginseng calli after 1 month of Agrobacterium infection. After 4 weeks, the infected calli were transferred to 6,7-V medium containing 50mg/L hygromycin for selection until new calli developed, which were then divided for expansion and subsequently identified, and figure B, C shows calli grown under hygromycin selection pressure and new calli, respectively. General procedure for genetic transformation of ginseng callus as shown in panel D, the acquisition of transgenic callus required at least 6 months.

Example 3

Identification of transgenic ginseng callus

Identification of transgenic callus by 1 GUS staining

Since the vector pCambia1301s contains the beta-D-Glucuronidase (GUS) gene and no endogenous GUS gene is present in the ginseng cells, the transgenic ginseng callus was identified using the GUS staining kit from Coolaber company. The principle of staining is that GUS is able to catalyze the decomposition of the substrate 5-bromo-4-chloro-3-indole- β -glucuronide (abbreviated as X-Gluc) to give a blue compound. The preparation of the substrate is shown in the specification of the product, 0.1g of ginseng callus to be detected is soaked in the substrate solution, and the substrate is placed in a dark condition at 37 ℃ for reaction for 2 hours, and the callus is taken out for observation of the dyeing condition of the callus.

The results are shown in FIG. 5: two groups of calli showed blue color after GUS staining, and were initially identified as transgenic positive calli, designated OE-1 and OE-2, respectively.

2 extraction of genomic DNA from callus of Ginseng radix

The extraction of ginseng callus gDNA uses SteadyPure Plant Genomic DNA Extraction Kit of Ai Kerui company, and the specific steps are as follows:

(1) Weighing 100mg of ginseng callus, grinding the ginseng callus into powder by using liquid nitrogen, transferring the powder into a 1.5mL EP tube containing 500 mu LBuffer LS-4, adding 10 mu L RNaseA, shaking and mixing uniformly, placing a centrifuge tube in a 56 ℃ water bath, heating for 10min, and reversing and mixing uniformly for 3 times;

(2) Adding 62.5 μl Buffer PA, mixing, centrifuging at 12,000rpm for 5min on ice for 5min, collecting supernatant, adding equal volume Buffer BS-2, and mixing;

(3) Transferring the solution to an adsorption column, standing at room temperature for 1min, centrifuging at 12,000rpm for 1min, and discarding the filtrate;

(4) Adding 500 μl Buffer WA into the adsorption column, centrifuging at 12,000rpm for 1min, and discarding the filtrate;

(5) Adding 500 μl of Buffer WB with absolute ethanol added in advance into the adsorption column, centrifuging at 12,000rpm for 1min, and discarding the filtrate; repeating the step once;

(6) Placing the adsorption column back into the collecting tube, and centrifuging at 12,000rpm for 2min;

(7) Placing the adsorption column in a new 1.5mL centrifuge tube, uncovering the centrifuge tube, standing at room temperature for 2min to volatilize residual ethanol, adding 50 mu L of an absorption Buffer preheated to 60 ℃ into the center of the membrane, standing at room temperature for 2min, and centrifuging at 12,000rpm for 2min; the extraction quality was ensured by agarose gel detection of 5. Mu.L gDNA solution, and the result is shown in FIG. 6, the extracted gDNA has single band and no tailing phenomenon, which indicates that the extraction quality is high, and the residual solution is stored in a refrigerator at-20 ℃.

Primers were designed to specifically amplify inserts and PCR verification was performed on gDNA from positive calli extracted, wherein the PCR system and experimental procedure were as described in example 1, and the primer sequences were as follows:

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

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

the results are shown in FIG. 7: transgenic positive calli can amplify a fragment of interest 550bp in length, whereas WT has no corresponding band.

Identification of positive callus PgNAC72 Gene expression Using 3 qRT-PCR

RNA extraction and reverse transcription procedure of positive calli the difference in PgNAC72 expression in positive calli was detected by qRT-PCR experiments as described in example 1, using the Vazyme company ChamQ SYBR qPCR Master Mix formulation reaction system, wherein the qPCR primer sequences of the relevant genes were as follows:

Primer Sequence(5′→3′)

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

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

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

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

the following reaction system was prepared on ice:

TABLE 10 qRT-PCR System

Note that strong light exposure was avoided during the configuration, 4 duplicate wells were set up for each set of experiments, and qRT-PCR reactions were performed using CFX Connect Real-Time PCR Detection System from Bio-Rad corporation, with the following reaction procedure set up:

fluorescence signals were collected at 72 ℃ extension step, with the dissolution profile set as follows:

95℃ 15s

60℃ 20s

95℃ 15s

three independent biological replicates were performed and the relative expression levels of the genes were calculated using the 2- ΔΔct method.

As a result, as shown in FIG. 8, the expression of PgNAC72 gene was increased 12.0 times in the OE-1 group and 15.8 times in the OE-2 group, compared to the wild-type group.

Example 4

Detection of changes in ginsenoside content in PgNAC72 overexpressing callus

1 determination of total ginsenoside content

The total ginsenoside content was determined using a total saponin content kit from Shanghai pullup biotechnology Co.

(1) Taking a proper amount of sample to be tested in a 15mL centrifuge tube, placing the sample in a refrigerator at the temperature of minus 80 ℃ for freezing overnight, and carrying out vacuum freeze drying on the completely frozen sample for 24 hours;

(2) Grinding thoroughly dried sample into powder, weighing 0.05g, adding 1mL of extract, ultrasonic extracting for 1h, centrifuging for 10min at 8,000Xg;

(3) Taking 0.5mL of supernatant to a 1.5mL centrifuge tube, wherein the blank control group directly takes 0.5mL of extracting solution, and placing the extracting solution in a baking oven at 70 ℃ for volatilizing until the extracting solution is dry; adding 0.2mL of reagent I and 0.8mL of perchloric acid, and carrying out water bath at 55 ℃ for 20min;

(4) Sucking 40 mu L of reaction liquid to a 96-well plate, arranging 3 compound wells in each group, adding 200 mu L of glacial acetic acid, fully and uniformly mixing, measuring absorbance at 589nm by using an enzyme-labeling instrument, wherein an experimental group is marked as A1, a blank group is marked as A2, and calculating delta A=A1-A2;

(5) Calculation of total saponins content: oleanolic acid as reference substance

Total saponin content (μg/g dry weight) = 5555.6 × (Δa+0.012).

The results are shown in FIG. 9: the extract of OE-1 and OE-2 calli stained to a greater extent than the control; the total saponin content is calculated by taking oleanolic acid as a reference substance, wherein each gram of wild type callus contains 633.9 mug of total saponins, and the total saponin content of two groups of transgenic ginseng callus is 1584.9 mug/g Dry Weight (DW) and 1953.1 mug/g DW respectively, and compared with the wild type, the total saponin content of OE-1 and OE-2 systems is respectively improved by 2.5 times and 3.1 times.

2 terpenoid metabonomics analysis

OE-1 and WT ginseng callus samples were prepared according to the method described by 2.2.12, 3 replicates were prepared for each group, and frozen samples were sent to the Whan Meier Biotechnology Co., ltd for terpene metabolite detection.

After sample freeze-drying, metabolites were accurately characterized and quantified using high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and corporate self-established database MWDB (metware database). Sample quality control analysis, orthogonal partial least squares discriminant analysis (OPLS-DA) and screening of differential metabolites were all performed by michaelsholtzian. The differential metabolite screening criteria were: the metabolite of VIP >1 is generally considered to be significantly different based on the Variable Importance Projection (VIP) obtained by the OPLS-DA model.

Screening for differential metabolites is based on the following principle: VIP (VIP)>1, fold change is greater than or equal to 2 or fold change is less than or equal to 0.5, and the screening result is shown in FIG. 10: compared with WT ginseng callus, OE ginseng callus has 5 terpenes reduced and 48 terpenes increased, wherein detected dammarane type ginsenoside (including PPD type ginsenoside Rd, rb1, F2 and PPT type ginsenoside Re, rg2, rg1, rh 1) have increased content, rd content change is very remarkable, 836.6 times is increased, and other saponin content increases are between 2.0 and 6.2. While oleanane-type ginsenoside (ginsenoside R) ₀ ) The content is not significantly different. The DDS gene is a key node in the dammarane type ginsenoside biosynthesis pathway, so we can infer that PgNAC72 improves the synthesis of dammarane type ginsenoside by promoting the expression of PgDDS gene.

Example 5

Mechanism research of PgNAC72 for regulating ginsenoside biosynthesis

1 analysis of Gene expression of Key enzymes in the ginsenoside Synthesis pathway

The expression difference of key enzyme genes on a ginsenoside synthesis path in WT, OE-1 and OE-2 is identified by qRT-PCR by using the obtained transgene and wild callus cDNA as templates, and the method is as described above, and the used primer sequences are as follows:

Primer Sequence(5′→3′)

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

qRT-PgACT-R AGCATACAGGGAAAGATCGGCTTGA(SEQ ID NO.12)

qRT-PgDDS-F TGAGATTAGATGAAAACGAAC(SEQ ID NO.13)

qRT-PgDDS-R GGCAATGATAAGGGGAGGTGT(SEQ ID NO.14)

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

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

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

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

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

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

qRT-PgSE1-F TCTTTGCCGTGGCTATCTAT(SEQ ID NO.21)

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

qRT-PgSS3-F TTCAACAGCTCGGACCTCA(SEQ ID NO.23)

qRT-PgSS3-R GAAAAGTGCCAGTCGTTATCAT(SEQ ID NO.24)

the results are shown in FIG. 11: compared with WT, the expression of PgDDS genes in OE-1 and OE-2 is improved by 10.3 times and 6.0 times respectively, while the expression of other key enzyme genes on the pathway in OE-1 and OE-2 groups is obviously improved, but the growth factor is far lower than that of PgDDS genes, the expression of PgHMGR and PgFPS in OE-1 is down-regulated, the expression of PgSE1 in OE-2 is up-regulated, but the expression of PgSE1 in OE-1 is obviously up-regulated, but the expression of PgPDDS in OE-2 groups is obviously reduced, which suggests that the PgDDS genes are likely target genes regulated by PgNAC72 transcription factors.

2 EMSA investigation of the binding of PgNAC72 protein to DNA

2.1 labelling and annealing of probes

(1) The following probes were designed based on the ABRE and CACG site sequences in PgDDSpro:

Primer Sequence(5′→3′)

EMSA-ABRE-F GAATACGTGACGGGAATACGTGACGGGAATACGTGACGG(SEQ ID NO.25)

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

EMSA-CACG-F TTTACACGACTGTTTACACGACTGTTTACACGACTG(SEQ ID NO.27)

EMSA-CACG-R CAGTCGTGTAAACAGTCGTGTAAACAGTCGTGTAAA(SEQ ID NO.28)

EMSA-mABRE-F TTTATTTGACTG TTTATTTGACTG TTTATTTGACTG(SEQ ID NO.29)

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

EMSA-mCACG-F GAATATTTCACGGGAATATTTCACGGGAATATTTCACGG(SEQ ID NO.31)

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

the underlined part indicates the predicted NAC binding site and its mutation site, biotin tag was added at the 5' end of F chain and purified by HPLC, and synthesis, labeling and purification of the probe were all completed by Beijing qingke organism company;

(2) Mixing proper amount of forward chain and reverse chain with equal molar ratio in a clean centrifuge tube, and diluting the probe to a final concentration of 1 pmol/. Mu.L with Tris buffer (containing 10mM Tris, 1mM EDTA, 50mM NaCl and adjusting pH to 8.0); taking 50 mu L of diluent into a 0.2mL PCR tube, and carrying out annealing reaction in a PCR instrument, wherein the reaction program is as follows: and (3) carrying out 70 cycles at 95 ℃ for 5min, -1 ℃/cycle, taking out the PCR tube after the reaction is completed for 1min, sub-packaging the probe, and storing the probe in a refrigerator at-20 ℃.

2.2 gel migration blocking experiments

(1) Preparing 6% TBE gel according to the formula, taking care of preventing bubbles in the gel filling process, flushing a sample loading hole with 0.5 XTBE after gel fixation, and pre-electrophoresis for 30min at 100V, wherein the electrophoresis buffer is 0.5 XTBE;

(2) The protein and probe binding reaction system (volume units are all μl) was formulated on ice according to the following formulation:

table 11 protein and probe binding reaction System

Wherein X represents TF chaperone protein, Y represents PgNAC72, and Poly (dI.dC) can inhibit the specific binding of protein and DNA, the total system is reacted for 5min at room temperature before adding a labeled probe, and then the reaction is carried out for 20min at room temperature after adding the labeled probe;

(3) Electrophoresis: after the completion of the binding reaction, 5. Mu.L of 5×loading Buffer was added, and the mixture was thoroughly mixed and then loaded. The electrophoresis buffer solution is 0.5 XTBE, the voltage is 100V, and the electrophoresis is stopped when the indicator reaches 3/4 of the gel;

(4) Transferring: taking out nylon membrane, soaking in 0.5 XTBE for at least 10min, placing in the order of cathode plate-sponge-filter paper-glue-membrane-filter paper-sponge-positive plate, and removing bubbles with glass rod; placing the film transfer groove in an ice bath, and transferring the film for 1h at a constant voltage of 100V;

(5) Ultraviolet crosslinking: placing the membrane on clean filter paper (with the side with bromophenol blue facing upwards), and rapidly placing under an ultraviolet lamp at 10cm for irradiation for 15min;

(6) Closing: placing the nylon membrane after ultraviolet crosslinking in a clean plate, adding 20mL Blocking Buffer, and gently shaking for 15min;

(7) Hybridization: gently pouring out the Blocking Buffer, adding 66.7 mu L Stabilized Streptavidin-Horseradish Peroxidase Conjugate and 20mL Blocking Buffer mixed solution, and gently shaking for 15min;

(8) Rinsing: gently pouring out the binding blocking solution, adding 20mL of 1 xWash Buffer, gently shaking and rinsing for 5min, and repeatedly rinsing for 3 times;

(9) Balance: transferring the nylon membrane to a new plate, adding 30mL Substrate Equilibration Buffer, and shaking for 5min;

(10) Luminescence inspection: taking out the nylon membrane to suck redundant liquid, placing the nylon membrane in a new plate, pouring substrate working solution onto the membrane lightly to enable the substrate working solution to cover the surface of the membrane completely, and standing the nylon membrane in a dark place for reaction for 5min; the nylon film was removed and the excess chemiluminescent substrate was blotted off the side, taking care not to allow the film to dry completely, and developed under a chemiluminescent imager.

The results are shown in FIG. 12: the TF protein and the labeled probes containing ABRE and CACG are not combined, and the PgNAC72, the TF fusion protein and the two labeled probes can be combined to form a DNA-protein complex, so that the complex appears as a hysteretic electrophoresis migration band. In the competition reaction, we further validated the binding using 50-fold, 200-fold unlabeled and 200-fold mutated competitive probes, respectively, as seen in FIG. 12, pgNAC72: TF fusion protein was unable to bind to mutated ABRE, CACG probes. In summary, we can confirm that PgNAC72 protein is able to bind to ABRE and CACG sites in PgDDS promoter.

3 double luciferase reporter experiments

In order to verify the combination of PgNAC72 transcription factor and PgDDS gene promoter in vivo, we cloned the promoter sequence of PgDDS, constructed a report vector, and performed a double luciferase reporter gene experiment in tobacco leaf cells.

3.1 reporter vector construction

Inquiring PgDDS promoter sequence on NCBI website, selecting BamHI and KpnI as enzyme cutting sites of pGreenII 0800-LUC vector and fragment, designing specific primer to amplify PgDDSpro full length, the primer sequence is as follows:

Primer Sequence(5′→3′)

DDSpro-F CGGGGTACCTCCGATGTAGTTAAACTTGA(SEQ ID NO.33)

DDSpro-R CGCGGATCCGTGCTTTAGGTGCCTATAGA(SEQ ID NO.34)

PCR amplification, cleavage, purification, ligation, transformation and identification steps are described in example 1;

3.2 Agrobacterium injection infiltration method for mediating transient expression of epidermal cells under tobacco leaf

The overexpression vector pCambia1301s-PgNAC72 and the reporter gene vector were transformed into Agrobacterium GV3101, respectively, as described above. Uniformly mixing the successfully verified agrobacterium with a volume ratio of 1:1, and infecting the lower epidermal cells of the tobacco leaves together by an injection infiltration method, wherein each group of samples infects 3 tobacco leaves respectively, the invasion method is as described in the foregoing 2.2.7, and the co-transfection of the groups is carried out according to the following steps:

TABLE 12 cotransfection System

3.3 double fluorescence quantitative detection

The quantitative detection of firefly luciferase and Renilla luciferase is carried out by using a double-luciferase detection kit of TransGen company, and the specific steps are as follows:

(1) Sampling after 2d injection, sampling by a puncher with the inner diameter of 2cm, taking each sample once while avoiding the large veins, rapidly filling the sample into a 1.5mL centrifuge tube, and quick freezing by liquid nitrogen;

(2) The sample was rapidly crushed using an electric drill driven grinding rod, 100. Mu.L of protein extract (PBS buffer containing 1mM DTT and having a pH of 7.8) was added in two portions, homogenized, and centrifuged at 12,000rpm at 4℃for 10min;

(3) Collecting supernatant to detect firefly luciferase and Renilla luciferase activities, wherein the detection steps are carried out according to the instruction of the kit; the ratio of firefly luciferase activity to Renilla luciferase activity was calculated.

The results are shown in FIG. 13: the PgNAC72 expression vector (effect, vector is called pCambia1300s-PgNAC 72) and the Reporter gene vector (Reporter, vector is called pGreenII 0800-LUC-DDSpro) are successfully constructed, the effect vector and the Reporter vector are co-transformed into tobacco leaf cells by an Agrobacterium tumefaciens GV3101 mediated method, the transfected vector of the control group is empty plasmid pCambia1301s+reporter, after 16h light/8 h dark culture is carried out for 2 days, a multifunctional enzyme-labeling instrument is used for respectively detecting the firefly luciferase activity (LUC) and the Renilla luciferase activity (REN) of the two groups, and the relative activity (Relative activity) of the experimental group is calculated according to the relative luciferase activity (=LUC/REN) of the control group. The experimental results are shown in fig. 13 (B): compared with the control group, the relative activity of the experimental group is improved by 2.4 times. This result demonstrates that PgNAC72 can bind to and promote expression of PgDDS promoter.

The foregoing examples are set forth in order to provide a more thorough description of the present invention, and are not intended to limit the scope of the invention, since modifications of the present invention, in which equivalents thereof will occur to persons skilled in the art upon reading the present invention, are intended to fall within the scope of the invention as defined by the appended claims.

Claims (9)

1. The application of PgNAC72 protein or gene in regulating ginsenoside biosynthesis is characterized in that the sequence of the PgNAC72 protein is shown as SEQ ID NO.1, and the sequence of the PgNAC72 gene is shown as SEQ ID NO. 2.
2. 2. The use according to claim 1, wherein the use is of PgNAC72 gene and PgNAC72 protein to regulate ginsenoside synthesis by regulating expression of the PgDDS gene of ginseng.
3. 3. The application of the over-expressed PgNAC72 gene or the over-expressed PgNAC72 protein in regulating ginsenoside biosynthesis is characterized in that the sequence of the PgNAC72 protein is shown as SEQ ID NO.1, and the sequence of the PgNAC72 gene is shown as SEQ ID NO. 2.
4. 4. Use according to claim 3, wherein the agent that overexpresses the PgNAC72 gene or PgNAC72 protein comprises methyl jasmonate.
5. 5. The use according to any one of claims 1 to 4, characterized in that it is transfected into ginseng suspension cells by means of an overexpression plasmid with the PgNAC72 gene.
6. 6. The use according to claim 5, wherein the ginsenoside is dammarane type ginsenoside.
7. 7. An application of a recombinant vector in regulating ginsenoside biosynthesis, wherein the recombinant vector overexpresses PgNAC72 gene.
8. 8. A kit comprising an agent that overexpresses the PgNAC72 gene or PgNAC72 protein or an overexpression vector having the PgNAC72 gene.
9. 9. Use of the kit according to claim 8 for modulating ginsenoside biosynthesis.

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