CN109295080B - Application of rhizoma panacis majoris beta-balsamol synthetase gene Pj beta-AS - Google Patents
Application of rhizoma panacis majoris beta-balsamol synthetase gene Pj beta-AS Download PDFInfo
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Abstract
The invention discloses a rhizoma panacis majoris beta-amyrin synthetase genePjβ‑ASThe application of the beta-amyrin synthase gene in promoting the synthesis of oleanane type saponin by panax notoginseng cells, namely the application of the beta-amyrin synthase genePjβ‑ASThe nucleotide sequence of (A) is shown as SEQ ID NO. 1; obtaining rhizoma Panacis Majoris by gene cloning (Panax japonicus) Beta-amyrin alcohol synthase gene (a)Pjβ‑AS) (ii) a Construction of a plasmid containingPjβ‑ASA plant expression vector for the gene; transforming the vector into agrobacterium tumefaciens EHA105 to obtain an agrobacterium tumefaciens strain containing the gene; transforming Notoginseng radix cell with Agrobacterium tumefaciens strain to obtain positive transformantPjβ‑ASDetecting the types and the contents of main oleanane type saponin in the transgenic panax notoginseng cells by using the panax notoginseng cell plants of the genes; in the transgenic panax notoginseng cell obtained by the invention, the contents of panax japonicus saponin IV and panax japonicus saponin IVa belonging to oleanane type saponin are 0.20 mg/g and 0.42 mg/g respectively.
Description
Technical Field
The invention belongs to the technical field of medicinal plant genetic engineering, and particularly relates to a rhizoma panacis majoris beta-resinol synthetase genePjβ-ASThe application of the beta-amyrin synthetase gene and a method for synthesizing oleanane type saponin in pseudo-ginseng cells by transferring the beta-amyrin synthetase gene into the pseudo-ginseng cells.
Background
Notoginseng (radix Notoginseng)Panax notoginseng) Is used as a medicine for roots and rhizomes of plants in the genus of Panax in the family of Araliaceae, is an important component of Yunnan province genuine medicinal materials and also is a Yunnan white drug powder. The pseudo-ginseng isTraditional famous and precious Chinese herbal medicines have double effects of promoting blood circulation and stopping bleeding, and the Qing Dynasty pharmaceutical works recorded in Ben Cao gang mu Shi Yi (supplement of Qi with Ginseng) and Qi with Panax Notoginseng have the same flavor and action, so the traditional Chinese medicine is called Panax Notoginseng, which is the most precious of the traditional Chinese medicines. At present, pseudo-ginseng medicinal materials are all cultivated products, 98% of pseudo-ginseng raw medicinal materials are produced in Yunnan, and pseudo-ginseng becomes the most important medicinal material resource in Yunnan province. The notoginsenoside is the main medicinal component of Notoginseng radix, is composed of dammarane type tetracyclic triterpene saponin, and has no oleanane type saponin, which is similar to rhizoma Panacis Majoris (rhizoma Panacis Majoris) (Panax ginseng C.A. Mey) of PanaxPanax japonicus) The saponin components contained in the composition have obvious difference.
Root of Redbud Stichopus japonicusPanax japonicusAs the root and stem are used as the medicine, they are recorded in Dian nan Ben Cao, a kind recorded in the Chinese pharmacopoeia of the calendar edition. The panax japonicus medicinal material is mainly produced in Yunnan, is a traditional medicine for minority nationalities such as Yi nationality, Bai nationality, Naxi nationality, Lisu nationality and the like, has the effects of tonifying lung and yin, removing blood stasis and relieving pain and stopping bleeding, and is clinically applied to deficiency of both qi and yin, dysphoria with smothery sensation and thirst, consumptive disease and cough, traumatic injury, arthralgia, hemoptysis, hematemesis, traumatic bleeding and the like. The rhizoma Panacis Majoris saponin is the main active ingredient of rhizoma Panacis Majoris, and comprises dammarane type and oleanane type triterpene saponin. At present, more than 30 saponin components are separated from rhizome and leaf of panax japonicus, mainly comprising panax japonicus saponin IVa, panax japonicus saponin IV, panax japonicus saponin V and ginsenoside R0Ginsenoside Re, ginsenoside Rd, ginsenoside Rb1, etc. Compared with ginseng and pseudo-ginseng of the same genus, the saponin components contained in the rhizoma panacis majoris have obvious difference in component types and content of each component, and the rhizoma panacis majoris has special clinical application due to containing a large amount of oleanane type saponin.
Pseudo-ginseng and rhizoma panacis majoris are perennial medicinal materials, the root of pseudo-ginseng and rhizoma panacis majoris are used as medicines, rotation is needed, the cultivation time cost is high, the land utilization rate is low, in addition, the medicinal material demand is increased year by year in recent years, so that the supply and demand contradictions of different degrees appear in the pseudo-ginseng and the rhizoma panacis majoris, and the price of the medicinal materials is high. In view of the disadvantages of long artificial cultivation time of medicinal materials and unclear chemical synthesis mechanism and route of medicinal saponin, the production of medicinal saponin by using a biological engineering technology and a synthetic biological method gradually becomes a research hotspot.
At present, homologous expression of plant terpenoid drugs has been applied industrially, including large-scale culture of ginsenoside; the research work on heterologous expression systems has also made important progress, such as the use of metabolic engineering means to achieve accumulation of target products or precursor substances in E.coli or yeast, including the expression of artemisinin and its precursors in yeast, the expression of paclitaxel precursors in yeast and E.coli and the synthesis of ginsenosides in Saccharomyces cerevisiae. The genetic characteristic of the secondary metabolic pathway of the medicinal plant is modified by utilizing the genetic engineering technology, the content of effective components of the medicinal plant is improved, and a cell line capable of accumulating a large amount of target secondary metabolites is cultivated, so that the medicinal plant is more and more concerned by people. However, no report is available on the current method for synthesizing oleanane type saponin in pseudo-ginseng cells by utilizing a biological technology.
Our previous work has studied in detail the biosynthetic pathway of notoginsenoside and the regulatory techniques for this pathway, but oleanane-type saponins have never been found in notoginseng cells. Oleanane-type saponins are triterpene saponins, which are mainly synthesized by mevalonic acid (MVA) pathway, the steps for forming saponin skeleton are mostly the same as those for synthesizing dammarane-type saponins, and the synthesis of oleanane-type and dammarane-type saponins share precursor 2, 3-oxidosqualene. Because no oleanane-type saponin is found in panax notoginseng at present, but the panax japonicus contains a large amount of oleanane-type saponin, wherein panax japonicus saponin IVa and panax japonicus saponin IV are the marked components of the panax notoginseng, the oleanane-type saponin synthesis way is constructed by transferring the oleanane-type saponin beta-coumarol synthetase gene in the panax notoginseng into panax notoginseng cells, so that the panax notoginseng can be synthesized into the oleanane-type saponin after artificial modification. The invention synthesizes oleanane type saponin, namely panax japonicus saponin IVa and panax japonicus saponin IV, in panax notoginseng for the first time through redesigning and reforming the synthesis way of the panax notoginseng saponin, and has stronger innovation.
Disclosure of Invention
The invention provides a rhizoma panacis majoris beta-amyrin synthase genePjβ-ASThe new application of the compound is in promoting the cells of the pseudo-ginsengApplication of oleanane-type saponin synthesis, namely, beta-amyrin synthetase gene of panax japonicasPjβ-ASThe nucleotide sequence of (A) is shown as SEQ ID NO. 1.
The technical scheme adopted for solving the technical problems comprises the following steps:
(1) obtaining of genes: extracting total RNA of rhizoma Panacis Majoris, reverse transcribing to synthesize first strand cDNA, and amplifying by RT-PCRPj β-ASThen connecting the full-length coding region to a pGEM-T easy vector, and obtaining a clone with a target gene through sequencing verification;
(2) construction and genetic transformation of plant expression vectors: using restriction endonucleasesSacI andXbai enzyme digestion pGEM-T-Pj β-ASPlasmid, and obtaining a target gene segment through glue recovery; digesting the plant expression vector pCAMBIA1300S with the same endonuclease, and recovering the gel to obtain a large vector fragment; connecting the target gene fragment with pCAMBIA1300S vector fragment to construct plant over-expression vector pCAMBIA1300S-Pjβ-AS(ii) a pCAMBIA1300S-Pjβ-ASIntroducing the plasmid into an agrobacterium strain EHA 105; by utilizing agrobacterium-mediated genetic transformation methodPjβ-ASIntroducing pseudo-ginseng cells for expression; screening positive transgenic cell lines by antibiotic screening and qRT-PCR;
(3) and (3) detecting the content of the saponin in the transgenic cell line: extracting saponin from transgenic and non-transgenic cell lines of Notoginseng radix, and analyzing the difference of saponin types and contents between transgenic and non-transgenic cell lines.
The invention provides a new method for producing oleanane type saponin, which can synthesize oleanane type saponin in notoginseng cells by utilizing a biological engineering technology and a gene regulation method, and overcomes the defects of long artificial cultivation period, unclear chemical synthesis mechanism and route and the like; key enzymesPjβ-ASThe gene is introduced into the notoginseng cell for expression, so that the notoginseng cell can produce oleanane type saponin, and theoretical reference and scientific basis are provided for large-scale industrialized production of oleanane type saponin.
Drawings
FIG. 1 is an electrophoretogram of transgenic Panax notoginseng cells detected by PCR; marker in the figure: DL2000 DNA Marker, which consists of six DNA segments of 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp and 100 bp;
FIG. 2 shows the real-time fluorescent quantitative PCR detection of transgenic notoginseng cellsPjβ-ASThe relative expression of the genes was detected by using T1 as a wild type cell line (control) and T4, T5, T9 and T12 as transitionsPjβ-ASA gene cell line;
FIG. 3 is a chromatogram of high performance liquid chromatography for determining oleanane-type saponin (panax japonicus saponin IVa, panax japonicus saponin IV) content in transgenic panax notoginseng cells; wherein SMS is a liquid phase detection map of oleanane type saponin standard product, TCL is a liquid phase detection map of oleanane type saponin in transgenic notoginseng cells, and WT is a liquid phase detection map of oleanane type saponin in unmodified notoginseng cells.
Detailed Description
The invention is further illustrated by the following figures and examples, without however restricting its scope to these.
Example 1: cloning of full-length ORF of beta-amyrin synthetase gene of rhizoma Panacis Majoris
(1) Extraction of total RNA of Panax japonicum
Selecting 35-day-old Stichopus japonicus cells, extracting total RNA by guanidinium isothiocyanate method, cleaning all the cells, soaking in 0.1% diethyl pyrocarbonate (DEPC) water solution with shaking overnight for more than 24 hr, taking out, and standing at 121 deg.C and 1.034 × 105 Sterilizing with Pa steam for 40 min, and oven drying at 50 deg.C. A suitable amount of alcohol was added to the cleaned mortar to burn the mortar and the spatula, and after the mortar was cooled, 1.0 g of the Panax japonicum cells were taken, put in liquid nitrogen and ground into powder, and 5.0 mL of guanidinium isothiocyanate extraction buffer and 50.0. mu.L of beta-mercaptoethanol were added. The triturate was transferred to a 2.0 mL EP tube, 1/10 volumes of 2M sodium acetate (pH 4.0), 500. mu.L water-saturated phenol and 100. mu.L chloroform were added, shaken well and incubated on ice for 5 min. The supernatant was centrifuged at 12000 rpm for 15 minutes at 4 ℃ to collect a supernatant. The supernatant layer was transferred to a new 2.0 mL EP tube, and an equal volume of water-saturated phenol: chloroform (1: 1) was shaken and incubated on ice for 5 min. Centrifuging at 4 deg.C and 12000 rpm for 15 min, collecting supernatantExtracted once with chloroform. The supernatant was taken and added with 1/10 volumes of 3M sodium acetate (pH 5.2) and mixed well, after adding equal volume of isopropanol and shaking slowly, the mixture was precipitated for 60 min at-20 ℃. Centrifuge at 12500 rpm for 25 minutes at 4 ℃. Washing RNA precipitate with 1.0 mL of 75% ethanol aqueous solution in ice bath, washing the tube wall of the centrifuge tube by inversion, vortexing the sample, suspending the precipitate as much as possible, repeating the process once, and drying the precipitate in the shade appropriately. The pellet was dissolved in 50.0. mu.L of RNase-free water and the integrity was checked by electrophoresis on a 120V, 1% agarose gel.
(2) First Strand cDNA Synthesis
Selecting RNA with better quality and synthesizing a first cDNA chain by using a GoScript reverse transcription system of Promega company; add total RNA of Panax japonicus (. ltoreq.5. mu.g), 1.0. mu.L Random Primers (0.5. mu.g/. mu.L), 1.0. mu.L Oligo (dT)15Primer (0.5 mu g/mu L) and RNase-free aqueous solution to 10.0 mu L, mixing the reaction solution, incubating at 70 ℃ for 5min, and rapidly placing on ice; sequentially adding into the reaction solution
Mixing the reaction mixture, annealing at 25 deg.C for 5min, incubating at 42 deg.C for 60 min, incubating at 70 deg.C for 15 min to terminate the reaction, and storing the cDNA obtained by reverse transcription at-20 deg.C for use.
(3) PCR amplification of ORF full length of P-amyrin synthetase gene
Designing and amplifying an upstream Primer and a downstream Primer of a complete coding frame by using Premier Primer 5.0 software according to the cDNA sequence (shown as a sequence table SEQ ID NO: 1) of the rhizoma panacis majoris beta-balsamol synthetase gene, and respectively introducing the upstream Primer and the downstream PrimerSacI andXbarestriction sites to construct expression vectors. Constructing an upstream primer PjAS-F of 5'-GAGCTCATGTGGAGGCTAATGACAGGCCAAGGG-3'; the downstream primer PjAS-R is as follows: 5'-TCTAGATCAGACGCTTTTAGGTGGTAATCGAACA-3' are provided. Using the cDNA of the panax japonicus as a template, amplifying by polymerase chain reaction, separating the obtained amplification product by electrophoresis on 1 percent agarose gel, and obtaining the targetThe fragments were recovered and ligated with pGEM T-easy vector. Transferring into Escherichia coli DH5 alpha, randomly selecting the obtained positive clone, detecting by colony polymerase chain reaction, and sequencing by bio-engineering (Shanghai) corporation. The sequencing result shows that the cloned sequence is consistent with the ORF full-length sequence (GenBank Acc. No.: KP 658156) of the rhizoma panacis majoris beta-resinol synthetase gene reported in GenBank, and is shown as a sequence table SEQ ID NO. 1.
The method adopts a gene cloning method to obtain the rhizoma panacis majoris beta-balsamic alcohol synthetase gene with correct sequence from the rhizoma panacis majoris, and provides an important key enzyme gene for producing oleanane type saponin (panax japonicus saponin IV and panax japonicus saponin IVa) by transgenic panax notoginseng cells.
Example 2: construction of plant expression vector containing rhizoma Panacis Majoris beta-amyrin synthetase gene
Using pCAMBIA1300S as plant expression vectorSacI andXbathe method comprises the following steps of carrying out double enzyme digestion on a pGEM T-easy vector containing a rhizoma panacis majoris beta-resinol synthetase gene and a pCAMBIA1300S plant expression vector, recovering the rhizoma panacis majoris beta-resinol synthetase gene and a pCAMBIA1300S large fragment, carrying out connection transformation, selecting a single clone, extracting a plasmid, and carrying out polymerase chain reaction and enzyme digestion verification to obtain the plant expression vector containing the genes.
The rhizoma panacis majoris beta-resinol synthetase gene is connected with the regulation and control sequence of the plant expression vector to form the plant expression vector containing the gene, and the expression vector can be used for producing oleanane type saponins (panax japonicus saponin IV and panax japonicus saponin IVa) through a gene engineering strategy.
Example 3: obtaining of Agrobacterium tumefaciens strain containing panax japonicus beta-amyrin synthetase gene plant expression vector
Transferring the plant expression vector containing the rhizoma panacis majoris beta-resinol synthetase gene into agrobacterium tumefaciens EHA105, and carrying out polymerase chain reaction verification, wherein the verification result shows that the plant expression vector containing the rhizoma panacis majoris beta-resinol synthetase gene is successfully constructed into an agrobacterium tumefaciens strain. The specific construction method is as follows:
preparing competent agrobacterium EHA105 by a calcium chloride method, adding 5mL of LB liquid culture medium (containing 25mg/L of rifamycin) into a 50mL sterile centrifuge tube, picking a well-grown EHA105 single colony from a plate by using a sterilized toothpick, inoculating the well-grown EHA105 single colony into the centrifuge tube, culturing the well-grown EHA105 single colony in a constant temperature shaking table at 28 ℃ for 16-18 hours at 180 r/min, inoculating 2mL of culture in the next day into a conical flask containing 100 mL of LB liquid culture medium (containing 25mg/L of rifamycin), and quickly culturing the well-grown EHA105 single colony in the constant temperature shaking table at 28 ℃ for 180 r/min to OD6000.4-0.6, transferring the bacterial liquid to two autoclaved 50mL centrifuge tubes under aseptic condition, centrifuging at 4 ℃ for 5 minutes at 5000 rpm, collecting cells, discarding supernatant, adding 10.0 mL precooled 0.1 moL CaCl2The solution was gently resuspended in bacteria, ice-bathed for 25 minutes, centrifuged at 5000 rpm at 4 ℃ for 5 minutes to collect cells, the supernatant was discarded, and 4.0 mL of pre-cooled 0.1 moL CaCl was added2The solution (containing 15% of glycerol) is lightly mixed, then is subpackaged in a 1.5 mL centrifuge tube, is frozen quickly by liquid nitrogen and is stored at minus 80 ℃. The agrobacterium tumefaciens strain containing the plant expression vector of the rhizoma panacis majoris beta-amyrin synthase gene is obtained by the following method:
dissolving EHA105 competent cells on ice, adding 3.0 μ g of plant expression vector plasmid containing the beta-amyrin synthase gene of panax japonicus into 100 μ L of the dissolved competent cells, carrying out ice bath for 30 minutes, transferring the centrifugal tube into liquid nitrogen for quick freezing for 5 minutes after the ice bath is finished, carrying out heat shock in a water bath kettle rapidly at 37 ℃ for 5 minutes, transferring the liquid culture medium to the ice for 1-2 minutes, adding 600 μ L of common LB liquid culture medium into a test tube, carrying out recovery culture at 28 ℃ for 100 rpm for 4 hours, uniformly coating the bacterial liquid on an LB solid culture medium containing rifamycin (25mg/L) and kanamycin sulfate (50 mg/L), carrying out inverted culture in a constant-temperature incubator at 28 ℃ for 36-48 hours, selecting resistant single bacterial colonies, inoculating the bacterial colonies in 600 μ L of LB liquid culture medium (containing kanamycin sulfate 50.0mg/L and rifamycin 25mg/L), after shaking culture at 28 deg.c and 200 rpm for 24 hr, the beta-amyrin synthetase gene of Panax schinseng was amplified by colony polymerase chain reaction. The results show that the plant expression vector containing the rhizoma panacis majoris beta-amyrin synthase gene has been successfully transferred into the agrobacterium tumefaciens strain.
Example 4: obtaining transgenic notoginseng cell
(1) Agrobacterium tumefaciens mediated panax japonicas beta-amyrin synthase gene transformed pseudo-ginseng cell
And sucking 600 mu L of the monoclonal bacterial liquid positive to PCR, uniformly spreading the monoclonal bacterial liquid in an LB solid culture medium containing Kan (50.0 mg/L) and Rif (25mg/L), and performing inverted culture at 28 ℃ until thick lawn grows on the plate (2-3 days). Scraping appropriate amount of pellet with inoculating loop, placing into MGL liquid culture medium (containing 40mg/L AS), and performing shake culture at 28 deg.C and 200 rpm to OD600About 0.6. The same day as the Agrobacterium solution was spread on the plate, pre-culture of the Notoginseng cells was performed. Appropriate amount of Notoginseng radix cells with good growth state are transferred into pre-culture medium (MS culture medium with 2.0 mg/L2, 4-D, 1.0mg/L KT and 40.0mg/L AS), and pre-cultured at 25 deg.C in dark for 3 days. After the pre-culture is finished, soaking the pseudo-ginseng cells in the bacterial liquid obtained in the step 1 for dip dyeing, and carrying out shaking culture on a shaking table at the temperature of 28 ℃ and at the speed of 105 r/min for 20 min. After the impregnation is finished, filtering out bacterial liquid, then flatly paving the pseudo-ginseng cells on sterilized filter paper to suck residual bacterial liquid, fully sucking to be dry, transferring the pseudo-ginseng cells to a co-culture medium (2.0 mg/L2, 4-D, 1.0mg/L KT and 40mg/L AS are added to an MS culture medium), and placing the culture medium under the dark condition for co-culture at 25 ℃ for 3 days. After the co-culture is finished, the notoginseng cells are transferred to a sterilized can bottle and washed 3-5 times with sterile water containing 400mg/L Cef. After the washing, the notoginseng cells are paved on sterilized filter paper to absorb residual water, then the notoginseng cells are transferred to a sterilization culture medium (MS culture medium is added with 2.0 mg/L2, 4-D, 1.0mg/L KT and 400.0 mg/L Cef), and placed under dark conditions for sterilization culture at 25 ℃ for 15 days. During the period, whether the agrobacterium is over-propagated on the surface of the panax notoginseng cell is carefully observed, and the content of Cef in the culture medium and the sterile water can be properly adjusted for control. After the sterilization is finished, the notoginseng cells are transferred to a screening medium (the MS medium is added with 2.0 mg/L2, 4-D, 1.0mg/L KT and 25mg/L Hyg), and are subcultured once a month on average (the subculture interval time can be properly adjusted according to the cell growth condition). Through 4-5 times of screening, the Hyg resistant transgenic strain can be obtainedPjASGene notoginseng cell.
(2) Polymerase chain reaction detection of transgenic panax notoginseng cells
Extracting DNA of the transgenic and control pseudo-ginseng cells by using an improved Cetyl Trimethyl Ammonium Bromide (CTAB) method, and performing polymerase chain reaction detection on a target gene by using upstream and downstream primers of a Hyg resistance gene on T-DNA, wherein an electrophoresis chart is shown in figure 1, and M in figure 1 is a DNA molecular weight standard which is 2000, 1000, 750, 500, 250 and 100 bp from top to bottom in sequence. Lane 13 is the non-transformed notoginseng cell, lane 14 is the positive control, i.e. the plant expression vector containing the rhizoma panacis majoris beta-resinol synthetase gene, lanes 1-12 are different transgenic lines, as can be seen from figure 1, the specific primer of polymerase chain reaction can amplify the specific DNA fragment of 450 bp, while when the non-transformed notoginseng genome DNA is used as the template, no fragment is amplified, which indicates that the vector construction and transformation strategy is correct, the target gene has been inserted into the notoginseng cell genome, and the acquisition of the transgenic notoginseng cell provides the basic material for producing oleanane-type saponin.
Example 5: method for detecting expression of rhizoma panacis majoris beta-resinol synthetase gene in transgenic pseudo-ginseng cell by fluorescence quantification-polymerase chain reaction
(1) Design and Synthesis of primers
Designing rhizoma Panacis Majoris beta-resinol synthetase gene and Notoginseng radix housekeeping gene by Premier Primer 5.0 software18S rRNAThe primer of (3) is used for carrying out semi-quantitative reverse transcription-polymerase chain reaction amplification, and the amplification of a target segment is compared with that of a non-transformed control pseudo-ginseng cell line after the polymerase chain reaction product is subjected to gel electrophoresis.
(2) Extraction of panax notoginseng total RNA
Selecting transgenic Notoginseng radix cell growing for 35 days, extracting total RNA with guanidinium isothiocyanate method, cleaning all the used glassware, gun head, centrifuge tube, soaking in 0.1% diethyl pyrocarbonate (DEPC) water solution shaken overnight for more than 24 hr, taking out, and standing at 121 deg.C for 1.034 × 105 Sterilizing with Pa steam for 40 min, and oven drying at 50 deg.C. Adding appropriate amount of alcohol into the cleaned mortar, heating the mortar and spoon, cooling the mortar, collecting 1g of rhizoma Panacis Majoris cells,the mixture was ground to a powder in liquid nitrogen, and 5mL of guanidinium isothiocyanate extraction buffer and 50. mu.L of beta-mercaptoethanol were added.
The reagents used above were purchased from Biotechnology (Shanghai) Inc. The triturate was transferred to a 2mL EP tube, 1/10 volumes of 2M sodium acetate (pH 4.0), 500. mu.L of water-saturated phenol and 100. mu.L of chloroform were added, shaken well and incubated on ice for 5 min. The supernatant was centrifuged at 12000 rpm for 15 minutes at 4 ℃ to collect a supernatant. The supernatant layer was transferred to a new 2mL EP tube, and an equal volume of water-saturated phenol: chloroform (1: 1) was shaken and incubated on ice for 5 min. The mixture was centrifuged at 12000 rpm at 4 ℃ for 15 minutes, and the supernatant was extracted once with chloroform. The supernatant was taken and added with 1/10 volumes of 3M sodium acetate (pH 5.2) and mixed well, after adding equal volume of isopropanol and shaking slowly, the mixture was precipitated for 60 min at-20 ℃. Centrifuge at 12500 rpm for 25 minutes at 4 ℃. Washing RNA precipitate with 1mL of 75% ethanol aqueous solution in ice bath, washing the tube wall of the centrifuge tube by inversion, vortexing the sample, suspending the precipitate as much as possible, repeating the process once, and drying the precipitate in the shade appropriately. The pellet was dissolved in 50. mu.L of RNase-free water and the integrity was checked by electrophoresis on a 1% agarose gel at 120 volts.
(3) First Strand cDNA Synthesis
Selecting RNA with better quality and synthesizing a first cDNA chain by using a GoScript reverse transcription system of Promega company; transgenic Panax notoginseng cell total RNA (less than or equal to 5 μ g), 1.0 μ L Random Primers (0.5 μ g/. mu.L), 1.0 μ L Oligo (dT) were added to the reaction tube15Primer (0.5. mu.g/. mu.L), and RNase-free aqueous solution to 10.0. mu.L, the reaction solution was mixed well, incubated at 70 ℃ for 5min, and then rapidly placed on ice. Sequentially adding into the reaction solution
Mixing the reaction mixture, annealing at 25 deg.C for 5min, incubating at 42 deg.C for 60 min, and incubating at 70 deg.C for 15 min to terminate the reaction. The cDNA obtained by reverse transcription was stored at-20 ℃ for future use.
(4) Method for detecting expression of rhizoma panacis majoris beta-resinol synthetase gene in transgenic pseudo-ginseng cell by fluorescence quantification-polymerase chain reaction
The expression level of the beta-amyrin synthetase gene of the panax japonicus is determined by fluorescence quantitative-polymerase chain reaction. The reaction parameters were as follows: hot start at 95 ℃ for 2 min; denaturation 95 ℃ for 15 s, annealing/extension 60 ℃ for 1 min, for 45 cycles. Each gene corresponding to each sample was tested in duplicate 3 times. The results show that the transgenic notoginseng cellsPjβ-ASThe expression level of the gene was significantly increased (FIG. 2), indicating thatPjβ-ASThe gene successfully realizes the overexpression in the pseudo-ginseng cells; in the figure, T-1 is a wild-type cell line; t-4, T-5, T-9 and T-12 represent different experimental groups of transgenic cell lines, respectively.
Example 6: high performance liquid chromatography is utilized to determine the oleanane type saponin content in the transgenic panax notoginseng cells
(1) High performance liquid chromatography conditions and system applicability and preparation of standard solution
Adopting a 1260 high performance liquid chromatograph (including an autosampler, an ultraviolet detector and the like) of Agilent Technologies company in America, wherein the chromatographic column is a Waters silica gel matrix column (5 mu m C18 reverse column, 4.6 mm multiplied by 250 mm), and performing gradient elution by using phosphoric acid water solution with volume fraction of 0.05% and acetonitrile as a mobile phase, and the mobile phase is subjected to gradient elution for 0-20 minutes, the volume of the acetonitrile is 20%, and the volume of the phosphoric acid water with volume fraction of 0.05% is 80%; 20-30 minutes, the volume of acetonitrile is increased from 20% to 35%, and the volume of phosphoric acid water with the volume fraction of 0.05% is decreased from 80% to 65%; 30-40 minutes, the volume of acetonitrile is 35%, and the volume of phosphoric acid water with the volume fraction of 0.05% is 65%; 40-50 min, the volume of acetonitrile is increased from 35% to 40%, and the volume of phosphoric acid water with the volume fraction of 0.05% is decreased from 65% to 60%; the volume of acetonitrile is increased from 40 percent to 100 percent and the volume of phosphoric acid water with the volume fraction of 0.05 percent is reduced from 60 percent to 0 percent in 50-60 min; the column temperature is 30 ℃, the flow rate is 1.0 mL/min, the detection wavelength is 203 nm, and the sample injection amount is 30 mu L.
Preparing a standard solution: weighing 10.0mg of each of the panax japonicus saponin IV and the panax japonicus saponin IVa standard substance, placing the panax japonicus saponin IV and the panax japonicus saponin IVa standard substance in a10 mL volumetric flask, adding methanol to a constant volume to obtain a standard substance mixed solution, and storing the standard substance mixed solution in a refrigerator at 4 ℃.
According to the mobile phase gradient elution program adopted by the invention, the retention time of the panax japonicus saponin IV and the panax japonicus saponin IVa is respectively 37.1 minutes and 38.9 minutes, the peak types are good, and the separation of the panax japonicus saponin IV and the panax japonicus saponin IVa from other saponin components can be ensured.
(2) Drawing a standard curve
Performing chromatographic detection under the conditions of different sample volumes (10.0 muL, 8.0 muL, 6.0 muL, 4.0 muL, 2.0 muL, 1.0 muL and 0.5 muL), recording the spectrum and chromatographic parameters, and performing regression analysis on the quality of each standard product by respectively using the peak area of each standard product to obtain a linear regression equation of each standard product, wherein the linear regression equation is as follows:
y=595.19x+75.464（r2=0.9978）、y=535.06x-36.982（r2=0.9985）
wherein x represents the mass of each standard in the sample, the unit is μ g, y represents the peak area of each standard, and r represents the correlation coefficient.
(3) Preparation of sample solution
Collecting a quantity of the rotorPjβ-ASGene notoginseng cell (different strains) and untransformedPjβ-ASAnd (3) drying the gene pseudo-ginseng cells in an oven at 50-55 ℃ (about 12 hours), and fully grinding the dried gene pseudo-ginseng cells into powder. 0.5g of each sample was weighed out without rotationPjASGenes and transgenesPjASThe gene pseudo-ginseng cell powder is respectively placed in 50mL centrifuge tubes, 50mL 70% methanol solution is respectively added and soaked overnight, and then ultrasonic wave is utilized to carry out crushing treatment (60W, ultrasonic 4s, intermittent 2 s) for 1.5-2.0 h until the pseudo-ginseng cell powder is completely crushed. And after the ultrasonic treatment is finished, transferring the mixture to a centrifugal machine, centrifuging the mixture for 30min at 4000 rpm, collecting supernatant, and drying the supernatant in an oven at 50-55 ℃. After drying, it was dissolved in 10mL of distilled water and extracted 3 times with equal volume of water saturated with n-butanol. Collecting the extract liquor, and drying the extract liquor in an oven at 50-55 ℃. Drying, dissolving with appropriate amount of methanol solution, diluting to 25mL, and filtering with 0.45 μ M microporous membrane to obtain saponin solution.
(4) High performance liquid chromatography
And (3) sequentially carrying out chromatographic analysis on each sample, recording the peak area of the oleanane type saponin in each sample, respectively substituting into a linear regression equation, and calculating to obtain the content of each oleanane type saponin. As can be seen from FIG. 3, the average contents of oleanane-type saponin IV and panax japonicus saponin IVa in the panax notoginseng cells grown for 35 days were 0.20 mg/g and 0.42 mg/g, respectively; as can be seen from fig. 3, the notoginseng cells after transgenic operation contain oleanane-type saponins (panax japonicus saponin IVa, panax japonicus saponin IV), while the wild type notoginseng cells that are not modified do not contain oleanane-type saponins (panax japonicus saponin IVa, panax japonicus saponin IV).
In the embodiment, the transgenic panax notoginseng cells for producing oleanane type saponin are obtained by using a genetic engineering strategy of transforming the beta-amyrin synthase gene of the panax japonicus, the content of the oleanane type saponin in the transgenic panax notoginseng cells is measured by using a high performance liquid chromatography, and an ideal method is provided for large-scale production of the oleanane type saponin.
Sequence listing
<110> university of Kunming science
<120> application of rhizoma panacis majoris beta-balsalaol synthetase gene Pj beta-AS
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2286
<212> DNA
<213> Panax japonicus
<400> 1
atgtggaggc taatgacagc caagggaggc aacgatctgt atttgtacag tacaaataac 60
ttcatagggc ggcagacatg ggagtttgat cctgactatg gaaccccggc ggagagggct 120
gaggttgagg aggctcgtct ccatttctgg aataaccggt atcaggttaa gcccagcggc 180
gatgtcctct ggcgaatgca gtttctaaaa gagaagaatt tcaaacaaat tattccccaa 240
gtgaaggtgg aggatggtga agaaatttcc tatgaagcag ccacaaccac attgaggaga 300
gctgtccact acttttcagc attgcaggct gatgatgggc actggcctgc cgaaaatgct 360
ggcccgttat ttttccttcc acccttggtc atgtgtctgt atattacagg gcatcttaat 420
actgtattcc ctgcagagta tcgcatagaa attctacgct acatttactg tcatcagaac 480
gaggatggtg gctggggatt acatatcgag ggccacagca ccatgttttg tacagctctc 540
agctacatct gcatgcgtat acttggagaa ggacgcgatg gtggtgaaaa caatgcctgt 600
gccagagcaa gaaaatggat ccttgatcat ggtagtgtga cagcaatacc ctcctgggga 660
aagacatggc tttcgatact tggcttattc gattggtcag gaagcaaccc aatgccccca 720
gagttttgga tccttcctcc tttccttcct atgcatccag caaaaatgtg gtgctattgc 780
cggatggttt acatgcctat gtcatatttg tatgggaaga ggtttgtggg gccgatcact 840
cctctcattt tacaactgag agaagaactt tacgcccaag catatgatga aattaactgg 900
aggaaagtgc gacataattg tgcaaaggaa gacctctact atccccatcc tttgatacaa 960
gatttgatgt gggatagcct ctacatattt acggagcctt tcttgactcg ttggcctttt 1020
aacaagttga gagagaaagc tcttcaaacc actatgaaac atatccatta tgaagatgag 1080
aacagtagat acatcactat aggatgtgtg gaaaaggttt tgtgtatgct tgcttgttgg 1140
gttgaggatc caaatggtga ttacttcaag aagcacctcg ctaggatccc agattatata 1200
tgggttgctg aagatggaat gaaaatgcag agttttggca gtcaagagtg ggatactggt 1260
tttgccatac aagcattgtt ggcgagtgat ctcactgatg aaattcgtcc tacactgatg 1320
aaagggcatg acttcataaa aaagtcccag gtcaaggaga acccttctgg cgacttcaaa 1380
agcatgcatc gccacatttc taaaggatcc tggacctttt cagatcaaga tcatggatgg 1440
caagtttcgg attgtactgc agaagctttg aagtgttgcc tactcttttc aaggatgcca 1500
acagaaatag ttggtgataa aatggaagac agccaattgt ttgatgctgt caatatactg 1560
ctatccctac agagcaaaaa tggcggccta gctgcatggg agcctgcagg atcctcagaa 1620
tggttggagc tgctcaatcc tacagaattc tttgaagaca ttgtcattga acatgagtat 1680
gtcgaatgca cttcatcagc aattcaggct atggttatgt ttaagaagtt ataccctggg 1740
cataggaaga aagagattga agtttcaatc acaaatgctg tacagtacct tgaagacata 1800
caaatgcctg atggttcatg gtacggaaac tggggtgtgt gcttcacata tggtacttgg 1860
tttgctatgg gaggtctaac cgcggctgga aagacataca acaacagcca aactcttcat 1920
aaagcagtgg attttctaat aaaatggcaa cgcagtgatg gtggttgggg agaaagctat 1980
ctttcttgcc caaacaagga atatacacct ttagaaggaa ataggtcaaa tttggtacac 2040
acttcatggg ccatgatggg tctgattcat tctgggcagg ctgaaagaga cccaacacct 2100
cttcatcgtg cagccaagtt gttgatcaat tcccaaatgg aaagtggtga ttttccccaa 2160
caggaaatca ctggagtttt catgaagaac tgcatgttac actatgcagc gtatagaaac 2220
atatatccgt tgtgggcttt agcagaatat cgaaaaaatg ttcgattacc acctaaaagc 2280
gtctga 2286
<210> 2
<211> 33
<212> DNA
<213> Artificial sequence (Artificial)
<400> 2
gagctcatgt ggaggctaat gacaggccaa ggg 33
<210> 3
<211> 34
<212> DNA
<213> Artificial sequence (Artificial)
<400> 3
tctagatcag acgcttttag gtggtaatcg aaca 34
Claims (1)
1. Rhizoma panacis majoris beta-amyrin synthase genePjβ-ASApplication of panax japonicus beta-amyrin synthase gene in promoting synthesis of oleanane type saponin by panax notoginseng cellsPjβ-ASNucleotide sequence ofShown as SEQ ID NO. 1;
the synthesized oleanane type saponin is chikusetsusaponin IVa and chikusetsusaponin IV.
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| CN109266626B (en) * | 2018-09-19 | 2021-11-09 | 云南农业大学 | Oleanolic acid glucuronyl transferase and coding gene and application thereof |
| CN110343678B (en) * | 2019-06-12 | 2022-09-27 | 云南农业大学 | Panax japonicus glycosyltransferase UGTPjm1 gene and application thereof in preparation of ginsenoside Ro |
| CN110938640B (en) * | 2019-12-06 | 2022-04-08 | 华南农业大学 | Alpha-amyrin synthetase gene EjAAS1 and application thereof |
| CN111647589A (en) * | 2020-06-08 | 2020-09-11 | 上海大学 | Euphorbia dienol synthase and coding gene and application thereof |
| CN115927280B (en) * | 2022-07-29 | 2023-08-15 | 中国中医科学院中药研究所 | Horse chestnut 2, 3-oxidation squalene cyclase and encoding gene and application thereof |
| CN115725620B (en) * | 2022-09-12 | 2023-09-15 | 昆明理工大学 | Method for synthesizing panax japonicus saponins in pseudo-ginseng cells |
| CN116515872A (en) * | 2022-09-30 | 2023-08-01 | 云南农业大学 | Cyclocarya paliurus Liu San terpene synthase CpalOSC2 gene and application thereof in preparation of beta-amyrin |
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