CN116694672A - Method for heterologously synthesizing ginsenoside Rg3 in plant by utilizing polygene coexpression - Google Patents

Abstract

The invention discloses a method for heterologously synthesizing ginsenoside Rg3 in plants by utilizing polygene coexpression, belonging to the technical field of biology. The genes involved in the polygene coexpression include: isopentenyl pyrophosphate isomerase-encoding gene, farnesyl pyrophosphate synthase-encoding gene, squalene epoxidase-encoding gene, dammarenediol synthase-encoding gene, protopanaxadiol synthase-encoding gene, uridine diphosphate glycosyltransferase Pn 1-31-encoding gene, and uridine diphosphate glycosyltransferase Pn 3-31-encoding gene. The invention discloses that the gene is co-expressed in plants for the first time, and rare ginsenoside Rg3 can be synthesized in transgenic plants. The invention provides a feasible technical scheme for synthesizing rare ginsenoside Rg3 by utilizing a plant chassis.

Description

Method for heterologously synthesizing ginsenoside Rg3 in plant by utilizing polygene coexpression

Technical Field

The invention relates to the technical field of biology, in particular to a method for heterologously synthesizing ginsenoside Rg3 in plants by utilizing polygene coexpression.

Background

Ginseng is recorded as a medicinal plant and can be seen in Shennong Ben Cao Jing in Qin Han period at the earliest, and is considered in books to have the effects of tonifying qi, soothing nerves, prolonging life and the like, so that the ginseng is a health food with homology of medicine and food. Ginsenoside (ginsenosides) is one of the main bioactive substances in ginseng. In recent years, many studies on pharmacological activities of ginsenoside have shown that ginsenoside can act on central nervous system, vascular system and immune system, and has anti-inflammatory, antioxidant, immunoregulatory, anti-arteriosclerosis, antihypertensive, anticancer, neuroprotection and other effects (Ratan Z A, haidere M F, hong Y H, et al, pharmaceutical potential of ginseng and its major component ginsenosides [ J ],2020 ]), and thus, have received much attention.

Ginsenoside can be classified into dammarane type, oleanolic acid type and October type according to the different skeleton structures. In nature, ginsenoside is mainly tetracyclic triterpene dammarane type. According to the difference between the attached glycoside and the hydroxy ligand, dammarane Type ginsenosides can be classified into protopanaxadiol Type (PPD-Type) ginsenosides and protopanaxatriol Type (PPT-Type) ginsenosides, wherein the PPD-Type mainly comprises ginsenosides Ra1, ra2, ra3, rb1, rb2, rb3, rc, rd, rg3, rh2 and the like, and the PPT-Type comprises ginsenosides Re, rf, rg1, rg2, rh1, F1, R2 and the like. Ginsenoside has low content in Panax plants, especially rare saponins, and usually less than one ten thousandth of dry weight.

The traditional preparation method of the saponin is mostly directly extracted from the ginseng plant, is highly dependent on cultivation and planting of the plant, and has long period, complex extraction and purification process and high cost. With the development of biotechnology, the biosynthesis of rare ginsenosides by a biological method has become a research hotspot, for example, the team of Zhou Zhihua research has successively completed the biosynthetic pathway analysis of ginsenoside CK (Cell research, 2014), rh2 and Rg3 (Metabolic engineering, 2015) and F1 and Rh1 (Molecular plant, 2015) series rare ginsenosides by re-splicing the transcriptome data of ginseng plants and mining biological elements, and created a yeast Cell factory, realizing the de novo synthesis of the above rare ginsenosides. The team cloned and identified two UDP glycosyltransferases UGTPg45 and UGTPg29 from ginseng in 2015, established a yeast cell factory based on two UGTs and a yeast chassis Producing Protopanaxadiol (PPD), specifically UGTPg45 selectively transferred the glucose moiety to the hydroxyl group at the C3 position of PPD to produce ginsenoside Rh2, UGTPg29 selectively transferred the glucose moiety to the C3 glucose of Rh2 to form 1-2-glycosidic linkages to synthesize Rg3 (Pw A, yw A, yun F, et al production of bioactive ginsenosides Rh and Rg3 by metabolically engineered yeasts [ J ], metabolic engineering,2015,29:97-105 ].

Compared with microorganisms, plants are used as natural products of plants to produce chassis, and have many natural superior conditions, such as photosynthesis systems, extremely rich enzyme libraries, such as cytochrome P450 and the like, so that the plants have great production and application potential as the chassis.

Disclosure of Invention

The invention aims to provide a method for heterologously synthesizing ginsenoside Rg3 by utilizing a plant chassis, which realizes the scale production of rare ginsenoside and promotes the wide application of medicinal ginsenoside.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides application of polygene coexpression in heterologous synthesis of ginsenoside Rg3 in plants, wherein the polygene coexpression participated genes comprise: isopentenyl pyrophosphate isomerase (isopentenyl diphosphate isomerase, IDI) encoding gene, farnesyl pyrophosphate synthase (farnesyl pyrophosphate synthase, FPS) encoding gene, squalene synthetase (squalene synthase, SS) encoding gene, squalene epoxidase (squalene epoxidase, SE) encoding gene, dammarenediol synthetase (dammarenediol synthase, DDS) encoding gene, protopanaxadiol synthetase (Protopanaxadiol synthase, PPDS) encoding gene, uridine diphosphate glycosyltransferase Pn1-31 encoding gene and uridine diphosphate glycosyltransferase Pn3-31 encoding gene.

The coding genes of the enzymes participating in the ginsenoside Rg3 synthesis pathway are integrated into the genome of plant chassis cells, so that the genes are expressed in plants, a ginsenoside Rg3 synthesis pathway is constructed, and meanwhile, the ginsenoside Rg3 synthesis precursor substances existing in the plants are utilized to realize the heterologous synthesis of the ginsenoside Rg3.

Specifically, the sequence information of isopentenyl pyrophosphate isomerase is NCBI Reference Sequence:NP-197148.3, the sequence information of farnesyl pyrophosphate synthase is GenBank:AAD17204.1, the sequence information of squalene synthase is GenBank:BAD08242.1, the sequence information of squalene epoxidase is GenBank:BAD15330.1, the sequence information of damascene glycol synthase is GenBank:ACZ71036.1, the sequence information of protopanaxadiol synthase is GenBank: AEY75213.1, the sequence information of uridine diphosphate glycosyltransferase Pn1-31 is GenBank: QOJ43864.1, and the sequence information of uridine diphosphate glycosyltransferase Pn3-31 is GenBank: QOJ43866.1.

Specifically, the action modes of the proteases participating in the synthesis of rare ginsenoside Rg3 are as follows: ginsenoside is a triterpene compound among terpenoids, and in higher plants, biosynthesis of terpenoids starts from isopentenyl pyrophosphate (IPP) or its isomer methallyl pyrophosphate (DMAPP), which is a major precursor generated by the Mevalonate (MVA) synthesis pathway located in the cytoplasm and the methylerythritol phosphate (MEP) synthesis pathway located in the plastid. IPP and DMAPP can be mutually converted under the action of isopentenyl pyrophosphate isomerase (IDI). Thereafter, two IPPs are condensed with one DMAPP end to end under the action of Farnesyl Pyrophosphate Synthase (FPS) to produce farnesyl pyrophosphate (FPP) having 15 carbon atoms, and then two FPP molecules are subjected to a coupling reaction under the action of Squalene Synthase (SS) to produce squalene having 30 carbon atoms. Squalene is an important precursor of triterpenes and their related derivatives. Squalene is further catalyzed by Squalene Epoxidase (SE) to form 2, 3-oxidized squalene, which is a common precursor of all ginsenosides, and increasing its synthesis plays an important role in increasing the yield of ginsenosides. Then, the dammarenediol-II, namely the skeleton of dammarane type ginsenoside, is generated under the action of dammarenediol synthetase (DDS). dammarenediol-II can synthesize protopanoxadiol (PPD) under the action of protopanoxadiol synthase (PPDS). PPD selectively transfers the glycosyl moiety to the hydroxyl group at the C3 position of protopanoxadiol under the catalysis of uridine diphosphate glycosyltransferase Pn1-31 to produce ginsenoside Rh2, after which ginsenoside Rh2 is catalytically synthesized to Rg3 by uridine diphosphate glycosyltransferase Pn3-31.

Further, the application includes: the coding genes are integrated into the genome of plant chassis cells by using a biological technology means to obtain a transgenic plant with polygene over-expression, and ginsenoside Rg3 is extracted from the transgenic plant after cultivation.

Preferably, the TransGene Stacking II system is used for polygenic assembly. The TransGene Stacking II system is a polygene assembly vector system, see Chinese patent application No. 2017103841977.

Further, the plants include, but are not limited to, tobacco.

According to the codon preference of the chassis cells, the coding genes are subjected to codon optimization to synthesize corresponding gene fragments for multi-gene assembly. Preferably, when the acceptor plant is tobacco, the nucleotide sequence of the isopentenyl pyrophosphate isomerase encoding gene is shown as SEQ ID NO. 1; the nucleotide sequence of the farnesyl pyrophosphate synthase encoding gene is shown as SEQ ID NO. 2; the nucleotide sequence of the squalene synthetase encoding gene is shown as SEQ ID NO. 3; the nucleotide sequence of the squalene epoxidase encoding gene is shown as SEQ ID NO. 4; the nucleotide sequence of the dammarenediol synthetase encoding gene is shown as SEQ ID NO. 5; the nucleotide sequence of the protopanoxadiol synthetase coding gene is shown as SEQ ID NO. 6; the nucleotide sequence of the coding gene of the uridine diphosphate glycosyltransferase Pn1-31 is shown as SEQ ID NO. 7; the nucleotide sequence of the coding gene of the uridine diphosphate glycosyltransferase Pn3-31 is shown as SEQ ID NO. 8.

Preferably, each coding gene contains an over-expressed promoter upstream. Selection of appropriate promoters for driving overexpression of the coding genes is based on the chassis cells, and overexpression of each gene is achieved by integrating the promoters upstream of each coding gene.

Preferably, the isopentenyl pyrophosphate isomerase-encoding gene is upstream of the RbcS3B promoter; the upstream of farnesyl pyrophosphate synthase encoding gene, squalene synthase encoding gene and dammarenediol synthase encoding gene are provided with CaMV35S promoter; upstream of the squalene epoxidase encoding gene there is an RbcS1A promoter; an RbcS3A promoter is arranged at the upstream of the protopanaxadiol synthase coding gene; the gene encoding uridine diphosphate glycosyltransferase Pn3-31 has an RbcST1 promoter at the upstream; the MALD1 promoter is located upstream of the gene encoding uridine diphosphate glycosyltransferase Pn3-31. Wherein the RbcS promoter and the MALD1 promoter can drive the target gene to specifically express in leaves, so that the target gene is selected for part of genes.

Specifically, the nucleotide sequence of the RbcS3B promoter is shown as SEQ ID NO. 9; the nucleotide sequence of the CaMV35S promoter is shown as SEQ ID NO. 10; the nucleotide sequence of the RbcS1A promoter is shown as SEQ ID NO. 11; the nucleotide sequence of the RbcS3A promoter is shown as SEQ ID NO. 12; the nucleotide sequence of the RbcST1 promoter is shown as SEQ ID NO. 13; the nucleotide sequence of the MALD1 promoter is shown in SEQ ID NO. 14.

The invention also provides a method for heterologously synthesizing ginsenoside Rg3 in tobacco, which comprises the following steps:

(1) Integrating an IDI gene fragment, an FPS gene fragment, an SS gene fragment, an SE gene fragment, a DDS gene fragment, a PPDS gene fragment, a Pn1-31 gene fragment and a Pn3-31 gene fragment into a receptor vector by utilizing a polygene assembly technology, and constructing and obtaining a polygene vector;

(2) The target gene segment in the polygene vector is led into tobacco receptor by using transgenic technology, the polygene overexpressed transgenic plant is obtained by cultivating, and then ginsenoside Rg3 is extracted from the transgenic plant.

Further, in step (1), the polygene assembly was performed using a TransGene Stacking II system. pYL322, pYL and 322d2 are taken as donor carriers in the system, and pYLTAC380GW is taken as an acceptor carrier.

The upstream of each gene fragment contains an over-expressed promoter, and the corresponding gene fragments containing the over-expressed promoters are respectively constructed on donor vectors pYL d1 and pYL322d2 from which the original 35S promoter is knocked out by using a Gibson Assembly method.

In the step (2), the constructed polygene fragments are introduced into a receptor plant to enable the polygene fragments to be expressed in a tobacco plant body, and the expressed proteases are involved in the synthesis of rare ginsenoside Rg3, so that the tobacco plant can synthesize the rare ginsenoside Rg3 in the body.

Further, agrobacterium-mediated techniques are used to introduce the multiple gene segments into recipient plants. The agrobacterium employs EHA105.

Further, the tobacco acceptor uses medium tobacco 100.

The invention has the beneficial effects that:

(1) The invention discloses IDI gene, FPS gene, SS gene, SE gene, DDS gene, PPDS gene, pn1-31 gene and Pn3-31 gene for the first time to co-express in plants, and rare ginsenoside Rg3 can be synthesized in transgenic plants. The invention provides a feasible technical scheme for synthesizing rare ginsenoside Rg3 by utilizing a plant chassis.

(2) Compared with a large-scale fermentation tank and a harsh growth environment required by microorganism culture, the invention utilizes the plant chassis to produce the ginsenoside, and plants can be planted in a field with only water and CO ₂ Can thrive with chemical fertilizer. The cost can be greatly reduced by the plant cultivation expression extraction, the process almost does not need to be adjusted and changed in a large scale along with the mass production amplification, and the comprehensive cost can be reduced by 5-10 times.

Drawings

FIG. 1 is a schematic diagram of 380MF-10G vector construction.

FIG. 2 is a diagram showing NotI cleavage of 380MF-10G vector.

FIG. 3 is an electrophoretogram of the positive detection of transgenic plants 4529B, wherein the first behavior is positive detection with IPPSCX-F/FPSCX-R primer, fragment size 1770bp; the second action uses DDSQ-F/CYP716CX-R2 primer to carry out positive detection, and the fragment size is 1347bp; the third behavior is that SSQ-F/SECX-R2 primer is used for positive detection, and the fragment size is 2626bp; the fourth row of UGT29 CX-F/RBST 1CX-R primer is used for positive detection, and the fragment size is 1458bp.

FIG. 4 shows the expression levels of foreign genes in transgenic tobacco 4529B, wherein A-H respectively represent the expression levels of AtIDI gene, aaFPS gene, pgDDS gene, pgPPDS gene, pgSE gene, pgSS gene, pn1-31 gene and Pn3-31 gene.

FIG. 5 is a liquid chromatogram of a transgenic tobacco 4529B extract, wherein A-E represents 4529B-25, 4529B-26, 4529B-30, wild-type, standard Rg3, respectively.

Fig. 6 is a mass spectrum of the transgenic tobacco 4529B extract.

Detailed Description

The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

pYL322d1, pYL d2 and pYLTAC380GW (TransGene Stacking II system) give the teaching laboratory of the university of agricultural China Liu Yaoguang for construction methods see China patent application No. 2017103841977.

Example 1 construction of large fragment rare ginsenoside Rg3 Metabolic pathway Module DNA vector

By analyzing transcriptome data of Arabidopsis, rice and ginseng plants, 8 genes involved in the synthesis route of rare ginsenoside Rg3 are respectively AtIDI gene, aaFPS gene, pgSS gene, pgSE gene, pgDDS gene, pgPPDS gene, pn1-31 gene and Pn3-31 gene, and cloned into Zhongyan 100 for heterologous expression.

Wherein, the sequence information of AtIDI is shown in gene accession number: 831505; sequence information of AaFPS is shown in gene accession number: AF112881.1; the sequence information of PgSS is shown in gene accession number: AB115496.1; the sequence information of PgSE is shown in gene accession number: AB122078.1; the sequence information of PgDDS is shown in the gene accession number: GU183405.1; the sequence information of PgPPDS is shown in the gene accession number: JN604537.1; the sequence information of Pn1-31 is shown in GenBank: MT551198.1, and the sequence information of Pn3-31 is shown in GenBank: MT551200.1.

Based on the results of the https:// www.ncbi.nlm.nih.gov/related gene transcripts query, codon optimisation was performed based on the codon preference of the chassis cells. Specifically, the coding sequence of the AtIDI gene is shown as SEQ ID NO.1, the coding sequence of the AaFPS gene is shown as SEQ ID NO.2, the coding sequence of the PgSS gene is shown as SEQ ID NO.3, the coding sequence of the PgSE gene is shown as SEQ ID NO.4, the coding sequence of the PgDDS gene is shown as SEQ ID NO.5, the coding sequence of the PgPPDS gene is shown as SEQ ID NO.6, the coding sequence of the Pn1-31 gene is shown as SEQ ID NO.7, and the coding sequence of the Pn3-31 gene is shown as SEQ ID NO. 8.

Furthermore, the AtIDI gene is designed to be driven to express by an RbcS3B promoter (the nucleotide sequence is shown as SEQ ID NO. 9), the AaFPS gene, the PgSS gene and the PgDDS gene are designed to be driven to express by a CaMV35S promoter (the nucleotide sequence is shown as SEQ ID NO. 10), the PgSE gene is designed to be driven to express by an RbcS1A promoter (the nucleotide sequence is shown as SEQ ID NO. 11), the PgPPDS gene is designed to be driven to express by an RbcS3A promoter (the nucleotide sequence is shown as SEQ ID NO. 12), the Pn1-31 gene is designed to be driven to express by an RbcST1 promoter (the nucleotide sequence is shown as SEQ ID NO. 13), and the Pn3-31 gene is designed to express by a MALD1 promoter (the nucleotide sequence is shown as SEQ ID NO. 14). The commercial company was entrusted with synthesizing a gene fragment carrying the above promoter.

Constructing corresponding gene fragments on donor vectors pYL d1 and pYL d2 by using a Gibson Assembly method, sequentially assembling the genes on a pYLT A C380GW vector by using a multi-gene polymerization vector system TransGene Stacking II system to obtain a plant expression vector of pYLT A C380GW-AtIDI-AaFPS-PgSE-PgSS-PgPPDS-PgDDS-Pn1-31-Pn3-31, and carrying out Bp recombination reaction to obtain pYLMFH-P _Bnm1 Herbicide-resistant screening marker gene on carrier and rape pollen specific expression promoter P _Bnm1 The driven Cre gene is recombined to obtain a corresponding expression vector 380GW-AtIDI-AaFPS-PgSE-PgSS-PgPPDS-PgDDS-Pn1-31-Pn3-31-Bar-Cre (380 MF-10G) (figure 1).

The specific construction process of the 380MF-10G vector is as follows:

(1) The pYL d1 and pYL d2 vectors are digested with BamHI and EcoRI, the original 35S promoter on the vector backbone is removed, and the digested vector backbone is subjected to homologous recombination connection with a target gene fragment containing the promoter, so that a donor vector pYL d1-AtIDI, pYL322d2-AaFPS, pYL322d1-PgSE, pYL322d2-PgSS pYL322d1-PgPPDS, pYL322d2-PgDDS, pYL322d1-Pn1-31 and pYL322d2-Pn3-31 is constructed.

(2) The donor vector pYL d1-AtIDI and the acceptor vector pYLTAC380GW (1:1 to 2:1) were mixed in NS3529 competent for cotransformation by heat shock method, ice bath for 30min, heat shock for 90s, ice bath for 2-3min, in LB without antibiotic, 37 ℃ and at 200rpm for 2h resuscitated, smeared on LA plate containing kanamycin (Km, 25 mg/L) and chloramphenicol (Chl, 15 mg/L), after about 18h monoclonal was grown, and ddH was used ₂ O all the monoclonal were washed into the tube and the mixed plasmid was extracted.

(3) About 50-100ng of the mixed plasmid was digested with homing endonuclease 0.5 mu L I-SceI (NEB) in a 10. Mu.L system for 4-5 hours, transformed E.coli strain XL10 (Vazyme) or NEB 10-beta (Bomeid biosciences Co., ltd.) was spread on LA plates containing kanamycin (Km, 25 mg/L), after 37℃for 15 hours, the monoclonal was picked up, cultured in LB (containing 25mg/L Km and 0.5mM IPTG) and subjected to bacterial liquid PCR identification, and further extracted plasmids capable of amplifying the bright band were each obtained by digestion verification in a 20. Mu.L reaction system using Green Taq Mix, four bands were generated, and further gene sequencing was confirmed to be the desired positive clone pYLTAC380 GW-IDI by 2.03K bp band.

(4) The donor vector pYL d2-AaFPS and (3) the acceptor vector pYLTAC380GW-AtIDI (1:1 to 2:1) were mixed in NS3529 competence for cotransformation, transformed according to (2) method, plated on LA plates containing kanamycin (Km, 25 mg/L) and ampicillin (Amp, 70 mg/L) and after about 18h a single clone was grown, and ddH was used ₂ O all the monoclonal were washed into the tube and the mixed plasmid was extracted.

(5) About 40-90ng of the mixed plasmid was digested with 0.5. Mu.L of PI-SceI (NEB), added with 0.5. Mu.L of BSA, and digested in a 10. Mu.L system for 4-5h, followed by transformation and verification according to the method in (3) to appear four bands, and the positive clone pYLTAC380GW-AtIDI-AaFPS containing the target genes AtIDI and AaFPS, which contained a band size of 4.28K bp (2.25K bp+2.03K bP), was obtained.

(6) More rounds of recombination, cross-using donor vectors containing different genes to co-rotate with the acceptor vector constructed in the previous round, constructing 380GW-AtIDI-AaFPS-PgSE-PgSS-PgPPDS-PgDDS-Pn1-31-Pn3-31, finally carrying out BP reaction at 25 ℃, and combining 380GW-AtIDI-AaFPS-PgSE-PgSS-PgPPDS-PgDDS-Pn1-31-Pn3-31 with PYLMFH-P _Bnml (100 ng) was reacted with 1. Mu.L of a 5 XBP enzyme mixture in 5. Mu.L of the reaction system for 5 hours. Then 1. Mu.L of protease K solution was added to terminate the reaction at 37℃for 10 minutes. Transferring into NEB 10-beta (Bomeid biotechnology Co., ltd.) for competence, and selecting monoclonal identification. And (3) carrying out enzyme digestion detection by using Not I, and finally obtaining a correct positive final vector 380GW-AtIDI-AaFPS-PgSE-PgSS-PgPPDS-PgDDS-Pn1-31-Pn3-31-Bar-Cre (380 MF-10G) with 11 DNA bands (figure 2), selecting positive clones for full plasmid sequencing analysis, selecting a correct 380MF-10G plasmid to be transformed into agrobacterium EHA105, and placing the transformed agrobacterium strain at the temperature of-80 ℃ for later use.

Example 2 acquisition of 380MF-10G transgenic tobacco

1. The leaf disc method is adopted to obtain the transgenic tobacco, and the specific steps are as follows:

1. the EHA105 bacterial liquid containing the verified correct carrier plasmid 380MF-10G is streaked on a LA+Rif+Kana plate, 28 ℃ for 36 hours, selected and monoclonal cultured in 3-5ml LB culture medium at 200rpm for 28 ℃ for 36 hours, 50ml is expanded and cultured for 3-5 hours to OD=0.6 according to the proportion of 1:100-1:50, then the bacterial liquid is centrifuged, and the bacterial liquid is suspended to OD=0.4 by MS0 liquid culture medium (MS+3%subculture, PH=5.8, 50 ml) for infection;

2. a first fully expanded healthy leaf (4-5 weeks) was selected, cut with a scalpel to a square size of 0.5cm (cut out the margin to avoid the main vein, 10 pieces/dish), and dark-cultured for 2-3d at 25℃on MS1 solid medium (MS+0.5 mg/L IAA+2.0mg/L BA+3% sucrosia+0.6-0.8% Phytagel, pH=5.8) with the upper surface of the leaf facing downward.

3. Adding the pre-cultured tobacco leaves into the bacterial liquid for infection, carrying out vortex oscillation to ensure that leaf cuts are immersed by the bacterial liquid, standing for 10min, and sucking the attached bacterial liquid by using sterile filter paper; placing the upper surface of the infected leaf on an MS1 solid culture medium downwards for dark culture at 28 ℃ for 2-3d; leaf upper surface was transferred upwards to MS1 solid medium containing antibiotic (timentin+hygromycin) and incubated at 25 ℃ for 2 weeks under light (L: d=16:8); when She Yuanchang buds and can be separated (more than 1 cm), the buds are excised and transferred to MS2 (MS+0.5 mg/L IAA+3% sucrose+0.6-0.8% Phytagel, PH=5.8) solid medium containing antibiotics (Tintin+hygromycin), roots grow out after two weeks, the cover of the seedling box is opened, and seedlings are planted for one week and transferred to greenhouse culture.

2. PCR detection of transgenic plants

The CTAB method is adopted to extract the total DNA of the transgenic tobacco leaves. T pair by PCR ₀ The positive detection is carried out on the transgenic tobacco, the sizes of the used primers and the detection fragments are shown in the table 1 and the table 2, and the obtained transgenic tobacco is referred to as 4529B for short:

TABLE 1 Gene transferred to transgenic tobacco 4529B

TABLE 2 primers for positive detection

The PCR reaction system is as follows: 50ng of synthesized template DNA, 0.2. Mu.L of F primer, 0.2. Mu.L of R primer, 10. Mu.L of GreenTaqmix, ddH ₂ O was made up to 20. Mu.L.

The PCR reaction procedure was as follows: 94 ℃ for 5min;94℃for 30s, 58℃for 30s, 72℃for 90s,32 cycles; and at 72℃for 5min.

For all T ₀ And carrying out PCR detection on the generation plants, and analyzing the integration conditions of the AtIDI gene, the AaFPS gene, the PgDDS gene, the PgPPDS gene, the PgSE gene, the PgSS gene, the Pn1-31 gene and the Pn3-31 gene in the transgenic plants. A total of 33 tobacco transformed plants were examined, in which 17 positive tobacco were present in the presence of 8 genes of AtIDI gene, aaFPS gene, pgDDS gene, pgPPDS gene, pgSE gene, pgSS gene, pn1-31 gene and Pn3-31 gene (FIG. 3).

Example 3: expression quantity detection of exogenous gene of transgenic positive plant

(1) Extraction of total RNA from tobacco leaves

The RNA extraction of tobacco leaves and rice ears adopts RNA isolater Total RNA Extraction Reagent (Trizol) reagent of Novozan company, and the specific operation process is as follows: the transgenic tobacco leaves are taken to be placed into liquid nitrogen for quick freezing, leaf tissues are rapidly ground into powder in the liquid nitrogen, 0.1g of sample is added into a precooled 1.5mL centrifuge tube, and 1mL of Trizol is added and then is rapidly and evenly mixed. Standing at room temperature for 5-10min. 200. Mu.L of chloroform/isoamyl alcohol (volume ratio of 24:1) was added to each tube, vigorously shaken for 30s, and allowed to stand at room temperature for 5min. Centrifuge 12000r/min at 4deg.C for 10min, at which time the sample phase separated into three layers. The supernatant, 600 μl, was carefully pipetted into a new centrifuge tube. Adding 500 μl of isopropanol, gently inverting and mixing, standing at room temperature for 5-10min. Centrifuge 12000r/min at 4deg.C for 10min, precipitating white RNA at the bottom of the centrifuge tube, and discarding supernatant. 1mL of 75% ethanol is added to each tube, RNA is washed by flicking, and centrifuged at 12000r/min for 5min at 4 ℃, and the supernatant is discarded. After a slight centrifugation, the residual 75% ethanol was carefully aspirated with a 20. Mu.L pipette. The RNA pellet was blown down onto a super clean bench to a semitransparent gel and then 50-100. Mu.L of sterilized DEPC water was added to the pellet in a 65℃water bath until the RNA was completely dissolved. And (3) measuring the concentration of the completely dissolved RNA sample, detecting the quality by electrophoresis, and storing at-80 ℃ after the RNA sample is qualified for mRNA reverse transcription.

(2) Reverse transcription of RNA

The extracted RNA was reverse transcribed with HiScript III 1st Strand cDNASynthesis Kit from Norwegian corporation. Mu.g RNA was pipetted into a 0.5mL centrifuge tube of new RNase free and DEPC water was added to 8. Mu.L. Heating at 65deg.C for 5min, and rapidly standing on ice for 2min. The residual DNA in the digested sample was treated with 2. Mu.L of 5 XgDNA wind Mix and, after completion of the water bath at 42℃for 2min, 10 XRT Mix 2. Mu.L, hiScript III Enzyme Mix. Mu.L, oligo (dT) was added to each tube ₂₀ VN 1μL，Rnase-free ddH ₂ O5. Mu.L, was mixed and placed in a 37℃water bath for 45min. And (3) supplementing water to 200 mu L at 85 ℃ for 5 seconds, and preserving at-20 ℃ for later use.

(3) Real-time fluorescent quantitative PCR (qRT-PCR) detection

qRT-PCR was performed using an ABI 7500 instrument,for reactions from Northenan CorpUniversal SYBR qPCR Master Mix reagent.

The real-time fluorescent quantitative PCR reaction system (11. Mu.L) used was as follows: cDNA 5. Mu.L, upstream primer (10. Mu.M) 0.2. Mu.L, downstream primer (10. Mu.M) 0.2. Mu.L, high ROX Dye (100X) 0.1. Mu.L, chemoHS qPCR Mix 5.5. Mu.L.

The qRT-PCR reaction parameters were as follows: pre-denaturation: 1 cycle, 95 ℃ for 5min;95 ℃ for 5s, 60 ℃ for 25s,40 cycles; the dissolution profile was set using the instrument default program.

qPCR detection is carried out on transgenic tobacco 4529B, wherein the plant numbers of which AtIDI, aaFPS, pgSS, pgSE, pgDDS, pgPPDS, pn1-31 and Pn3-31 are higher are 4529B-25, 4529B-26 and 4529B-30, and the relative expression detection diagrams of the three plants, which are equivalent to the internal reference gene activation, are shown in figure 4. The plants with high exogenous gene expression can be used for subsequent detection and analysis of the content of rare ginsenoside Rg3.

Example 4: detection of rare ginsenoside Rg3 content

(1) Extracting ginsenoside from transgenic tobacco leaves: the ground slurry sample was weighed 1g using an analytical balance. The sample and 5mL of pure methanol are added into a10 mL centrifuge tube, and a horn of an ultrasonic cell disruption instrument is inserted into the sample, wherein the distance from the tail end of the horn to the bottom is not less than 5mm. The ultrasonic condition is 50 ℃,20kHz, ultrasonic treatment for 2s and intermittent treatment for 2s. The ultrasonic time was 30min.

Taking out the ultrasonic sample, naturally cooling, centrifuging for 5min at 13000r/min at normal temperature, collecting supernatant, filtering the supernatant with 0.45 μm microporous membrane, and measuring on a machine.

(2) LC-MS detects the content of rare ginsenoside Rg 3: the chromatographic conditions used are as shown in Table 3.

TABLE 3 gradient elution of formic acid (A)/acetonitrile (B) mobile phase (v/v)

The LC-MS selects an MRM ion mode, a chromatographic column adopts ACQUITY UPLC BEH C (2.1 mm is 100mm,1.7 mu m), the column temperature is 40 ℃, the flow rate is 0.3mL/min, the acquisition mode adopts an ESI-mode, and the sample injection amount is 5 mu L.

The study utilizes LC-MS to detect and identify rare ginsenoside Rg3 in tobacco leaves of transgenic tobacco 4529B. The linear regression equation for the standard sample solution is shown in Table 4.

TABLE 4 Linear regression equation for Standard samples

Standard name of product	Regression equation	Linear correlation coefficient R
			Ginsenoside Rg3	y＝3.126064*X+11.211565	0.99981711

The formula used in this study to calculate the ginsenoside content is:

W＝[(C-C ₀ )×V×N]/m

wherein W: the content of the target in the sample is mg/kg; c: measuring the concentration of a target in the liquid in mg/L; c (C) ₀ : concentration of target in mg/L in blank; v: volume is fixed, unit mL; n: dilution factor; m: the amount of sample taken in g.

And taking 4529B-25, 4529B-26 and 4529B-30 strain leaves of transgenic tobacco 4529B, grinding the leaves, extracting ginsenoside therein by using an ultrasonic instrument, detecting ginsenoside Rg3 in the extract by using LC-MS, and obtaining LC chromatograms of all samples by taking the retention time as an abscissa and the response value as an ordinate, as shown in figure 5. The retention time of the standard ginsenoside Rg3 standard is found to be 4.117min in the chromatogram. Compared with wild type tobacco, transgenic tobacco lines 4529B-25, 4529B-26 and 4529B-30 all showed a new peak at 4.117min, indicating that a new compound was present in the transgenic tobacco lines, the retention time in the chromatogram of this new compound corresponded to that of the standard ginsenoside Rg3, and the mass spectrum cleavage pattern of this compound was also identical to that of the standard ginsenoside Rg3 (FIG. 6), indicating that rare ginsenoside Rg3 could be synthesized in the transgenic tobacco lines, and the results were consistent with experimental expectations.

The ginsenoside Rg3 contents of the three transgenic lines 4529B-25, 4529B-26 and 4529B-30 used for detection were 0.745. Mu.g/g, 0.552. Mu.g/g and 0.766. Mu.g/g respectively.

Claims (10)

1. The application of polygene coexpression in the heterologous synthesis of ginsenoside Rg3 in plants is characterized in that the polygene coexpression-involved genes comprise: isopentenyl pyrophosphate isomerase-encoding gene, farnesyl pyrophosphate synthase-encoding gene, squalene epoxidase-encoding gene, dammarenediol synthase-encoding gene, protopanaxadiol synthase-encoding gene, uridine diphosphate glycosyltransferase Pn 1-31-encoding gene, and uridine diphosphate glycosyltransferase Pn 3-31-encoding gene.

2. The application of claim 1, wherein the application comprises: the coding genes are integrated into the genome of plant chassis cells by using a biological technology means to obtain a transgenic plant with polygene over-expression, and ginsenoside Rg3 is extracted from the transgenic plant after cultivation.

3. The use of claim 2, wherein the polygenic assembly is performed using a TransGene Stacking II system.

4. The use according to claim 1 or 2, wherein the plant is tobacco.

5. The use according to claim 4, wherein the nucleotide sequence of the gene encoding isopentenyl pyrophosphate isomerase is shown in SEQ ID NO. 1; the nucleotide sequence of the farnesyl pyrophosphate synthase encoding gene is shown as SEQ ID NO. 2; the nucleotide sequence of the squalene synthetase encoding gene is shown as SEQ ID NO. 3; the nucleotide sequence of the squalene epoxidase encoding gene is shown as SEQ ID NO. 4; the nucleotide sequence of the dammarenediol synthetase encoding gene is shown as SEQ ID NO. 5; the nucleotide sequence of the protopanoxadiol synthetase coding gene is shown as SEQ ID NO. 6; the nucleotide sequence of the coding gene of the uridine diphosphate glycosyltransferase Pn1-31 is shown as SEQ ID NO. 7; the nucleotide sequence of the coding gene of the uridine diphosphate glycosyltransferase Pn3-31 is shown as SEQ ID NO. 8.

6. The use according to claim 1 or 5, wherein each coding gene comprises an over-expressed promoter upstream thereof.

7. The use according to claim 6, wherein the gene encoding isopentenyl pyrophosphate isomerase has an RbcS3B promoter upstream of the gene; the upstream of farnesyl pyrophosphate synthase encoding gene, squalene synthase encoding gene and dammarenediol synthase encoding gene are provided with CaMV35S promoter; upstream of the squalene epoxidase encoding gene there is an RbcS1A promoter; an RbcS3A promoter is arranged at the upstream of the protopanaxadiol synthase coding gene; the gene encoding uridine diphosphate glycosyltransferase Pn3-31 has an RbcST1 promoter at the upstream; the MALD1 promoter is located upstream of the gene encoding uridine diphosphate glycosyltransferase Pn3-31.

8. A method for heterologously synthesizing ginsenoside Rg3 in tobacco, which is characterized by comprising the following steps:

(1) Integrating an IDI gene fragment with a nucleotide sequence shown as SEQ ID NO.1, an FPS gene fragment with a nucleotide sequence shown as SEQ ID NO.2, an SS gene fragment with a nucleotide sequence shown as SEQ ID NO.3, an SE gene fragment with a nucleotide sequence shown as SEQ ID NO.4, a DDS gene fragment with a nucleotide sequence shown as SEQ ID NO.5, a PPDS gene fragment with a nucleotide sequence shown as SEQ ID NO.6, a Pn1-31 gene fragment with a nucleotide sequence shown as SEQ ID NO.7 and a Pn3-31 gene fragment with a nucleotide sequence shown as SEQ ID NO.8 into a receptor vector by utilizing a multi-gene assembly technology to construct a multi-gene vector;

(2) The target gene segment in the polygene vector is led into tobacco receptor by using transgenic technology, the polygene overexpressed transgenic plant is obtained by cultivating, and then ginsenoside Rg3 is extracted from the transgenic plant.

9. The method of claim 8, wherein in step (2) the polygenic fragment is introduced into the recipient plant using agrobacterium-mediated techniques.

10. The method of claim 8, wherein the tobacco is medium smoke 100.