CN109295069B

CN109295069B - Application of rhizoma panacis majoris transcription factor gene PjMYB1

Info

Publication number: CN109295069B
Application number: CN201811092438.1A
Authority: CN
Inventors: 葛锋; 于怡琳; 姜森; 姜利; 向盈盈; 刘迪秋
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2018-09-19
Filing date: 2018-09-19
Publication date: 2021-08-20
Anticipated expiration: 2038-09-19
Also published as: CN109295069A

Abstract

The invention discloses a rhizoma panacis majoris transcription factor genePjMYB1The application of the beta-cyclodextrin in improving the biosynthesis of key enzyme genes of the panaxsaponinPjASApplication of expression quantity and increasing saponin content in panax japonicus callus, and panax japonicus transcription factor genePjMYB1The nucleotide sequence of (1) is shown in SEQ ID NO. 1, and the MYB transcription factor is coded; the invention adopts the related technologies of functional genomics and metabolic engineering to prove that the rhizoma panacis majoris PjMYB1 transcription factor has the function of positively regulating and controlling the biosynthesis of the rhizoma panacis majoris saponin; the ginseng beads of the invention are addedPjMYB1The transcription factor gene is constructed on a plant expression vector and is transferred into the callus of the rhizoma panacis majoris to cause the overexpression of the gene,not only enhances the expression quantity of key enzyme genes in the synthesis path of the panaxsaponin, but also improves the yield of the panaxsaponin.

Description

Application of rhizoma panacis majoris transcription factor gene PjMYB1

Technical Field

The invention relates to the field of molecular biology and genetic engineering, in particular to a transcription factor gene for regulating and controlling biosynthesis of panaxsaponinPjMYB1The use of (1).

Background

Root of Redbud Stichopus japonicusPanax japonicus C. A. Mey.var. major(Burk.) C.Y.Wu et K.M. Feng is Araliaceae PanaxPanaxThe plants are named because the rhizomes are slender in internodes and the nodes are expanded into globose marbles. The rhizoma panacis majoris is used as a traditional Chinese herbal medicine, is recorded in Yunnan materia medica, and is a recorded variety in Chinese pharmacopoeia of the calendar edition. The medicinal rhizoma panacis majoris is produced mainly in Yunnan and is a traditional medicine for the minority nationalities such as the Yi nationality, the Naxi nationality, the Bai nationality, the Tibetan nationality, the Lisu nationality and the like. At present, wild rhizoma panacis majoris is mainly distributed in northwest Yunnan and northeast Yunnan, and is commonly found in subalpine coniferous forests and broad-leaved forests with the elevation of 2500-. The rhizoma panacis majoris is bitter, sweet and slightly cold in nature, belongs to liver, lung and stomach channels, has the effects of tonifying lung and yin, removing blood stasis and relieving pain and stopping bleeding, and is clinically applied to qi and yin deficiency, dysphoria with smothery sensation and thirst, consumptive disease and cough, traumatic injury, arthralgia, hemoptysis, hematemesis, traumatic hemorrhage and the like.

Rhizoma Panacis Majoris SaponinPanax japonicussaponins, PJS) is the major active ingredient of panax japonicus, including dammarane-type and oleanane-type triterpene saponins. At present, more than 30 saponin components are separated from rhizome and leaves of panax japonicus, and the representative components are panax japonicus saponin IVa, panax japonicus saponin IV, panax japonicus saponin V and ginsenoside R₀And the like oleanane-type saponins. And a representative species of the genus Panax, GinsengPanax ginseng) Mainly contains dammarane type saponin, and only ginsenoside R with very small content is found in oleanane type saponin₀(ii) a 'Sanchi' (Notoginseng)Panax notoginseng) Only contains dammarane type saponin, and does not contain oleanane type saponin. Compared with ginseng and pseudo-ginseng belonging to the same genus, the saponin components contained in the rhizoma panacis majoris have obvious differences in component types and content of each component, and the rhizoma panacis majoris has special clinical application due to the fact that the rhizoma panacis majoris contains a large amount of oleanane type saponin. Modern pharmacological research shows that the panaxsaponin has anti-inflammatory and analgesic effectsImproving myocardial ischemia, increasing cerebral blood flow, resisting tumor, regulating immunity, and treating leukopenia.

Rhizoma panacis majoris is a herbaceous plant used as a perennial drug, and can grow for many years. The long-term over-mining of wild resources leads to the gradual exhaustion of the resources of the rhizoma panacis majoris. In recent years, with the continuous and deep pharmacological research of the panax japonicus, the medicinal value of the panax japonicus is gradually accepted and valued, the social cognition degree on the medicinal effect of the panax japonicus is gradually improved, the market of medicines taking the panax japonicus as a component is rapidly expanded, the demand of the panax japonicus medicinal material is rapidly increased, and the contradiction between supply and demand is gradually highlighted. In view of the disadvantages of long artificial cultivation time, unclear chemical synthesis mechanism and route of medicinal components and the like, the production of panaxsaponin by using a biological engineering technology and a gene regulation method gradually becomes a research hotspot.

The transcriptional activation of the transcription factor on the gene is an important regulation link in the secondary metabolic process of the plant, and the condition that a single key enzyme gene in the metabolic engineering operation is insufficient in function and a plurality of key enzyme genes are likely to generate constitutive lethal expression is compensated by the unique advantage of 'multi-point regulation'. The transcription factor starts or closes the secondary metabolic synthesis pathway by activating the expression of enzyme genes with function related in the plant secondary metabolic synthesis pathway, thereby regulating and controlling the synthesis of secondary metabolites.

MYB transcription factors are important members of plant transcription factor families, and since the discovery of MYB, more and more researches show that the MYB transcription factors have close relationship with plant growth and development and secondary metabolites, and in recent years, the interaction relationship between the MYB transcription factors and other transcription factors for regulating the secondary metabolites is discovered. Such as gentian (Gentiana scabra) The over-expression of two R2R3-MYB type transcription factors GtMYBP3 and GtMYBP4 is beneficial to the biosynthesis and accumulation of flavone, and the transient expression finds that the transcription factors GtMYBP3 and GtMYBP4 are promoter activities of genes FNSII and F3H for improving early synthesis of flavone, so that the biosynthesis of endogenous flavone and the accumulation of flavone are promoted; AtMYBl23/TT2, TTl and R2R3-MYB can be used for Arabidopsis thaliana (A)Arabidopsis thaliana) The biosynthesis of Procyanidins (PAs) from the seed coat acts as a synergistic control.

With the elucidation of the deep analysis and regulation mechanism of the plant secondary metabolic network, especially the separation and identification of the transcription factor regulating the synthesis of specific secondary metabolites, the genetic engineering based on the transcription factor provides a more effective means for developing and utilizing the plant secondary metabolites.

Disclosure of Invention

The invention aims to clarify a transcription factor gene which is obtained by cloning from rhizoma panacis majoris and can regulate biosynthesis of rhizoma panacis majoris saponinPjMYB1The application of the beta-cyclodextrin in improving the biosynthesis of key enzyme genes of the panaxsaponinFPS、DS、ASExpression level and increase of saponin content in the callus of the Panax japonicus.

The invention is based on the principle of homologous cloning, and clones cDNA of MYB transcription factor genes from phyllanthus niruri and carries out functional identification on coding proteins of the cDNA. The inventors named this genePjMYB1Wherein the cDNA is shown as a sequence table SEQ ID NO. 1, and codes an amino acid sequence protein shown as a sequence table SEQ ID NO. 2; the sequence analysis of the gene shows thatPjMYB1The cDNA has a size of 735 bp, is just an Open Reading Frame (ORF) and encodes a protein containing 244 amino acids. The method adopts an agrobacterium-mediated method and uses a plant overexpression vector to carry out the methodPjMYB1The transcription factor gene is introduced into the rhizoma panacis majoris cell, so that the expression level of the key enzyme gene in the rhizoma panacis majoris saponin synthesis pathway can be improved, and the yield of the rhizoma panacis majoris saponin is increased.

The transcription factor gene can be applied to the positive regulation of the biosynthesis of the panaxsaponin, and the specific operation is as follows:

(1) obtaining a target gene: extracting total RNA of the panax japonicus, and performing reverse transcription to synthesize a first cDNA chain; amplified by RT-PCRPjMYB1The full-length coding region is connected with a pMD18-T easy vector, and the clone with the target gene is obtained through sequencing verification;

(2) construction and genetic transformation of plant expression vectors: using restriction endonucleasesXbaI andSmai enzyme digestion pMD18-T-PjMYB1Plasmid and plant expression vector pCAMBIA2300S, and recovering target gene fragment and vector large fragment. Connecting target gene fragment with pCAMBIA2300S vector sheetSegment, construction of plant overexpression vector pCAMBIA2300S-PjMYB1(ii) a pCAMBIA2300S-PjMYB1After the plasmid is introduced into an agrobacterium strain EHA105, an overexpression vector containing a target gene is introduced into a panax japonicus cell to be expressed by utilizing an agrobacterium-mediated genetic transformation method, and a positive transgenic cell line is screened by antibiotics and qRT-PCR;

(3) and (3) detecting the content of the saponin in the transgenic cell line: extracting saponin from the transgenic cell line and the non-transgenic cell line of the panax japonicus, and analyzing the difference of the saponin content between the transgenic cell line and the non-transgenic cell line.

The invention provides a new method for improving the content of saponin in the panax japonicus, the method for synthesizing the panax japonicus saponin with higher efficiency by utilizing the biological engineering technology and the gene regulation and control method, and the defects of long artificial cultivation period, unclear chemical synthesis mechanism and route and the like are overcome; transcription factorPjMYB1The gene is introduced into the panax japonicus cell for expression, so that the expression quantity of key enzyme genes related to the biosynthetic pathway of the panax japonicus saponin is improved, the yield of the panax japonicus saponin is increased, and theoretical reference and scientific basis are provided for large-scale industrialized production of the panax japonicus saponin.

Drawings

FIG. 1 is an electrophoretogram of total RNA of Panax japonicum;

FIG. 2 isPjMYB1 Detecting the result of RT-PCR; wherein marker (M): DL2000 DNA Marker, which consists of six DNA segments of 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp and 100 bp; 1 is a positive cell line;

FIG. 3 is a three-dimensional structure prediction for PjMYB 1;

FIG. 4 is a qRT-PCR result analysis diagram showing that the synthesis pathway of panaxsaponin is controlled by PjMYB1PjFPS、 PjDS、PjASThe level of gene expression in wild type and transgenic cell lines; c represents wild type (control), 1, 2, 3, 4 represent different over-expressionsPjMYB1A cell line of the gene;

FIG. 5 shows the result of measuring the content of panaxsaponin.

Detailed Description

The invention is further illustrated by the following figures and examples, without however restricting its scope to these.

Example 1:PjMYB1cloning of genes and bioinformatic analysis

Extracting total RNA of rhizoma Panacis Majoris (figure 1) by modified guanidinium isothiocyanate method, and referring to GoScript with the extracted total RNA of rhizoma Panacis Majoris callus as template^TMThe first strand cDNA is synthesized by the Reverse Transcriptase System kit instruction, and the reaction System and the operation process are as follows: 5. mu.g of Total RNA was added to 50 ng of oligo (dT)₁₅2 μ L dNTP (2.5 mM each), DEPC water to a reaction volume of 13.5 μ L; mixing gently, performing pre-denaturation at 70 deg.C for 5min, rapidly cooling on ice for 5min, sequentially adding 4 μ L of 5 XFirst-stand buffer, 0.5 μ L of RNase (200U), and 1 μ L M-MLV (200U), mixing well and centrifuging instantly, annealing at 25 deg.C for 5min, warm-bathing at 42 deg.C for 1.5 h, heating in 70 deg.C water bath for 10min to inactivate transcriptase to terminate reaction, and storing the synthesized First chain of cDNA at-20 deg.C for use.

And (3) designing specific primers by taking the synthesized first-strand cDNA as a template according to the MYB transcription factor gene cDNA sequence related to the regulation and control of notoginsenoside biosynthesis in the panax notoginseng, and performing high-fidelity PCR amplification, wherein the sequences of the primers are 5'-ATGGGGAGGAGCCCTTGCTGTGC-3' and 5'-TCAAGACAGCCAATCTCCTCCGGAC-3' respectively. And (3) PCR reaction conditions: 5min at 94 ℃; 30 s at 94 ℃, 30 s at 55 ℃, 1min at 72 ℃ and 32 cycles; 10min at 72 ℃. The reaction system (50. mu.L) was 4. mu.L of first strand cDNA, 5. mu.L of Ex Taq PCR Buffer (10X), 4. mu.L of dNTP mix (2.5 mM), 0.5. mu.L of forward primer (10. mu.M), 0.5. mu.L of reverse primer (10. mu.M), 0.5. mu.L of Ex Taq DNA polymerase (5U/. mu.L), 35.5. mu.L of ddH₂And O. After the PCR was completed, 5. mu.L of the PCR product was subjected to agarose gel electrophoresis, and the specificity and size of the amplified product were measured, and the remaining PCR product was gel recovered (FIG. 2). The target fragment glue recovery product is subjected to T-A cloning, and the reaction system and the operation process are as follows: collecting 3 μ L of gel, recovering the product, sequentially adding 1 μ L of pMD18-T vector, 5 μ L of Solution I and 1 μ L of ddH₂Mixing O, centrifuging instantaneously, and reacting at 16 deg.C for 30 min. The ligation products were transformed into E.coli Trans1-T1 using heat shock transformation. By using a composition containing ampicillinLB solid medium of essence (Ampicillin, Amp) for screening positive clones. Selecting several colonies, shaking, and amplifyingPjMYB1Identifying the multiple cloning site insertionPjMYB1The identified clones are sequenced.

Finally obtainedPjMYB1The cDNA size was 735 bp, which was found to be exactly an open reading frame by NCBI ORF finder (http:// www.ncbi.nlm.nih.gov/gorf. html) analysis (see sequence listing).PjMYB1The molecular weight of the encoded protein is about 27.7KD, the isoelectric point is 8.85, the instability coefficient is 68.98, and the prediction is thatPjMYB1The encoded protein is unstable. Bioinformatics predicts that PjMYB1 contains no transmembrane region, no signal peptide, and a conserved domain characteristic of MYB transcription factors. The possibility of PjMYB1 localization to the nucleus was predicted by the online tool iPSORT. Three-dimensional structural modeling was performed with the SWISS-MODEL tool, PjMYB1 using the 1a5j.1.a protooncogene protein as a template (fig. 3), and the results showed that 1a5j.1.a has a spatial structure with sequence similarity of 43.93%.

Example 2: plant expression vector construction

Based on the multiple cloning site of the plant expression vector pCAMBIA2300S, Primer Premier 5.0 pair was usedPjMYB1The gene sequence is used for enzyme cutting site selection and primer design. The insertion is extracted by adopting a SanPrep column type plasmid DNA small extraction kit (Shanghai worker)PjMYB1The E.coli plasmid pMD18-T-PjMYB1And the plasmid of the plant expression vector pCAMBIA2300S, and taking 1 mu L of the plasmid for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. By usingXbaI (TaKaRa) andSmai (TaKaRa) plasmid pMD18-T-PjMYB1And pCAMBIA2300S (100 mu L system), wherein the reaction system and the operation process are as follows: take 20. mu.L of pMD18-T-PjMYB1Or pCAMBIA2300S plasmid, sequentially adding 10 μ L10 XM buffer and 5 μ L Xba I、5 μL SmaI、60 μL ddH₂And O, mixing uniformly, and placing in a water bath kettle at 37 ℃ for enzyme digestion for 6 hours. Use of SanPrep column type DNA gel recovery kit (Shanghai Producer)PjMYB1The fragment and the large fragment of pCAMBIA2300S were separately recovered by gel. Taking 1 μ L of the recovered product, detecting the size and concentration of the recovered fragment by agarose gel electrophoresis, and placing inStoring at 20 deg.C for use.

The recovered DNA fragments were ligated with T4 DNA Ligase to construct a recombinant vector pCAMBIA2300S-PjMYB1. The reaction system (20. mu.L) and the procedure were as follows: taking 10 μ LPjMYB1The DNA fragment was sequentially added with 2. mu.L of pCAMBIA2300S vector DNA, 2. mu.L of 10 XT 4 DNA Ligase Buffer, 1. mu. L T4 DNA Ligase, and 5. mu.L of ddH₂O, mixed well and subjected to ligation reaction overnight at 16 ℃. Then transferring the ligation product into Escherichia coli Trans1-T1 by heat shock transformation, coating the transformed bacterial liquid on LB solid culture medium containing 50 mg/L kanamycin, culturing at 37 deg.C under inversion overnight, picking out monoclonal antibody, shaking, using bacterial liquid as template, and culturing with Bacillus coli Trans1-T1PjMYB1The upstream and downstream primers are used for carrying out PCR detection on bacteria liquid, agarose gel electrophoresis is used for detecting PCR products, and recombinant plasmid pCAMBIA2300S- PjMYB1Adding 20% glycerol into the Escherichia coli monoclonal bacterial liquid, mixing, and storing at-80 deg.C.

Extracting and purifying pCAMBIA2300S from the above Escherichia coli with plasmid extraction kit-PjMYB1A plasmid. Competent cells of the Agrobacterium EHA105 strain were prepared and distributed in 1.5 mL centrifuge tubes, 150. mu.L per tube, frozen with liquid nitrogen and stored at-80 ℃ for future use. The plant expression vector pCAMBIA2300S constructed above is frozen and thawed by adopting liquid nitrogenPjMYB1Transferring into the prepared agrobacterium EHA105 competent cell. The operation steps are as follows: taking 3 μ g of pCAMBIA2300S-PjMYB1The plasmid was added to a centrifuge tube containing 150. mu.L of competent cells, gently mixed and ice-cooled for 30min, then transferred to liquid nitrogen for quick freezing for 5min, and then quickly placed in a 37 ℃ water bath for 5min, followed by immediate ice-cooling for 2 min. The transformed Agrobacterium-infected competent cells were transferred to LB liquid medium (without any antibiotics), cultured with shaking at 200 rpm at 28 ℃ for 4 hours to resuscitate the cells, spread on LB solid medium containing 50 mg/L Km and 25 mg/L rifampicin, and cultured in 28 ℃ incubator upside down for about 48 hours. Selecting single clone, shaking, detecting PCR of bacterial liquid, screening recombinant vector pCAMBIA2300S-PjMYB1The Agrobacterium EHA105 infects the Phymatopsis japonica callus cells, and the strain glycerol is stored at-80 ℃ for later use.

Example 3: agrobacterium mediated genetic transformation of Panax japonicum

And (3) selecting the panax japonicus cells with good growth state (the cells have no particles and are in light yellow), transferring the panax japonicus cells to a panax japonicus cell pre-culture medium (containing 35 mg/L acetosyringone), paving the surface of the culture medium, and performing dark culture at 25 ℃ for 3 days. The preserved product containing pCAMBIA2300S was taken out from a-80 deg.C refrigerator- PjMYB1The plasmid Agrobacterium EHA105 strain was inoculated at 0.1% inoculum size in LB liquid medium containing 50 mg/L kanamycin and 25 mg/L rifampicin, and shake-cultured at 28 ℃ and 200 rpm to OD₆₀₀The value is 0.6-0.8, then the mixture is subpackaged in a 50mL centrifuge tube, and the thalli are collected by centrifugation at 5000 rpm for 5min at room temperature. Transferring the thallus into MGL liquid culture medium containing 40 mg/L acetosyringone, performing shake culture at 28 deg.C and 200 rpm to OD₆₀₀The value is 0.6 to 0.8; completely immersing the callus cells of the panax japonicus which are pre-cultured for 3 days into a bacterial solution, shaking and culturing for 20 min at a shaker of 25 ℃ and 110 rpm, and then removing the bacterial solution on the surface of the panax japonicus callus by using a Buchner funnel and filter paper. Transferring the panax japonicus cells to a co-culture medium with sterile filter paper paved on the surface, and co-culturing for 3 days at 25 ℃ in the dark;

after the co-culture is finished, the panax japonicus cells are transferred to a sterilized beaker, and are washed for 5-6 times by using sterile water containing 400mg/L of cefamycin so as to sufficiently remove agrobacterium. After the washing, the liquid on the surfaces of the stichopus japonicus callus cells was removed again using a buchner funnel and filter paper. The Stichopus japonicus cells are transferred to a sterilization culture medium containing 400mg/L of cefamycin for dark culture for 15 days, so that the overgrowth of agrobacterium is prevented. Finally, the callus was transferred to a selection medium, subcultured every 45 days. And finally separating out a transgenic cell line with Km resistance for subsequent detection after 4-5 times of screening.

Example 4:PjMYB1gene overexpression key enzyme gene for phyllanthus niruri saponin synthesis pathwayPjFPS、PjDS、PjASInfluence of expression amount

Selecting about 25 days old and good growing status rhizoma Panacis Majoris positive transgenic cell line and wild type cell line, respectively extracting RNA, and performing GoScript^TMThe Reverse Transcriptase System kit reversely transcribes the extracted RNA into cDNA, and the reaction SystemAnd the operation process is as follows: add 5. mu.g total RNA, 1. mu.L Random Primer and 1. mu.L Oligo (dT) to the centrifuge tube₁₅Adding into 10 μ L with nucleic-free Water, mixing, pre-denaturing at 70 deg.C for 5min, and immediately placing on ice bath for 5 min. Then, the centrifugal tube is subjected to short-time centrifugation in a centrifuge, so that the reaction liquid is collected at the bottom of the tube, and 4 mu L of GoScript is added into the reaction liquid^TM 5×Reaction Buffer、2µL MgCl₂(25 mM), 1 μ L PCR Nucleotide Mix (10 mM), 0.5 μ L Recombinant RNase ribonuclear Inhibitor and 1 μ L GoScript^TMReverse Transcriptase, mixing, instantaneously centrifuging, annealing at 25 deg.C for 5min, extending at 42 deg.C for 1.5 h, finally placing in 70 deg.C water bath for 15 min to inactivate Reverse Transcriptase, finishing reaction, centrifuging for a short time, and storing at-20 deg.C for use.

And (3) diluting the cDNA synthesized by reverse transcription by 5 times, namely diluting 20.0 muL cDNA to 100.0 muL. Diluted cDNA as template according to GoTaq^®2-Step RT-qPCR System kit instruction and Panax japonicus18S rRNAA gene (accession number: AB 088018.1), a Farnesyl diphosphate synthase (FPS) gene (accession number: KP 684141),βPrimers were designed for the beta-amyrin synthase (beta AS) gene (accession number: KP 658156) and dammarane synthase gene (dammarane synthase) and subjected to fluorescent quantitative PCR.

The primer sequence isFPS _F：5’-AGAATGAGCGATCTGAAGACGAG-3’， FPS _R：5’-ACAGACAACAAC TTCCCCTCCAT-3’；βAS _F：5’-GTATTCCCTGTAGAGCATCGCAT-3’，βAS _R：5’-GGCACAGGCGTTGTTTTCAC-3’；DS _F：5’-TGGGAGTTTCAGCCCGATG-3’，DS _R：5’-GGGGAGGTGTATAAAGTAAAGAGCC-3’； 18S _F：5’-GTTGTTGCAG TTAAAAAGCTCGTAG-3’，18S _R：5’-ACCTCTGACTATGAAATACGAATGC-3’。

The specific reaction system and the operation process are as follows: 20 ng of cDNA and 25 muL of GoTaq were added to the PCR tube^®qPCR Master Mix (2X) and 0.2 μ L qPCR Primers (2X)18S _F /18S _R，FPS _F / FPS _R，βAS _F/ βAS _R，DS _F / DS _R10 mM), supplemented to 50 μ L with Nuclear-Free Water. And (2) after the reaction system is uniformly mixed in a vortex mode, centrifuging and collecting the reaction system to the bottom of the tube, then placing the tube into a fluorescence quantitative PCR instrument for reaction, and performing fluorescence quantitative PCR by adopting a two-step method, wherein the reaction parameters are as follows: hot start at 95 ℃ for 2 min; denaturation 95 ℃ for 15s, annealing/extension 60 ℃ for 1min, for 45 cycles. Each gene corresponding to each sample was tested in duplicate 2 times.

Results of qRT-PCR showed, transitionPjMYB1In the cells of the genetic rhizoma panacis majorisPjFPS、PjDS、PjASThe expression level of the gene is higher than that of the wild type (figure 4), which shows that PjMYB1 is used as a transcription factor and can promote the key enzyme gene in the anabolic pathway of the phyllanthus niruri saponinPjFPS、PjDS、PjAS(ii) expression of (a); in the figure, C represents a wild-type cell line in a control group, and 1, 2, 3 and 4 represent different experimental groups of transgenic cell lines, respectively.

Example 5:PjMYB1influence of Gene overexpression on the amount of Polystichopus japonicus Saponin synthesized

Collecting the transgenic panax japonicus cells and wild type cells which grow for about 35 days, respectively placing the transgenic panax japonicus cells and wild type cells in an oven at 55 ℃ to be dried to constant weight, fully grinding the cells into powder, and sieving the powder through a 100-mesh sieve. Transgenic and wild type Panax japonicum cell powders, 0.5g each, were weighed and placed in a clean flask containing 50mL of methanol solution for overnight immersion. And carrying out ultrasonic treatment for 1.5-2.0 h under the ultrasonic condition of 60W, ultrasonic treatment for 4s and intermittent treatment for 2 s. Centrifuging at 4,000 rpm for 30min at room temperature, collecting supernatant, i.e. total saponin solution of rhizoma Panacis Majoris, and storing in refrigerator at 4 deg.C.

Precisely sucking 150 μ L of the total saponin solution corresponding to each prepared panax japonicus cell strain into a test tube with a plug (each sample is provided with 3 parallel), volatilizing the solvent at 55 ℃, adding 200 μ L of a newly prepared 5% vanillin-glacial acetic acid solution and 800 μ L of perchloric acid, plugging the test tube plug, carrying out vortex mixing, heating in a water bath at 60 ℃ for 15 min, and fully shaking once every 5min to promote full reaction. Immediately after the reaction, the test tube was cooled in ice, and 5 mL of glacial acetic acid was added to the reaction solutionMixing, and standing at room temperature for 10 min. And measuring the light absorption value of each reaction solution at the wavelength of 550nm by using an ultraviolet spectrophotometer, and calculating the content of the total saponin in each transgenic and wild type panax japonicus cell strain according to a standard curve. Result display, turnPjMYB1The saponin content in the gene rhizoma panacis majoris cells is obviously higher than that in wild cells (figure 5), which shows that the PjMYB1 transcription factor participates in anabolic regulation of rhizoma panacis majoris saponin, and is beneficial to the improvement of the saponin yield. C represents a wild-type cell line of a control group, and 1, 2, 3 and 4 represent different experimental groups of transgenic cell lines.

Sequence listing

<110> university of Kunming science

Application of <120> phyllanthus niruri transcription factor gene PjMYB1

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 735

<212> DNA

<213> Panax japonicus

<400> 1

atggggagga gcccttgctg tgcaaaggta gggctaaaca aaggtgcatg gacttctgtt 60

gaggacaaga ttctcagaga ctacattaag ctccatggtg aaggcagatg gagaagcctc 120

cccaaaagag caggtttaaa gagatgtggg aagagttgca ggcttagatg gttgaattat 180

ctgagaccag acatcaagag aggcaacatc tcccctgatg aagaagaact catcatccgc 240

cttcacaagc tcttgggaaa tagatggtct ctaatagctg ggaggcttcc ggggcgaaca 300

gacaatgaaa taaagaacta ctggaataca accttgagaa aaaaggttca tgacaatcac 360

tcctcctctg cttcagcttc agcttcagct ccagctccag ctcctcccaa gaaagactgg 420

cagcagcagc agcatttggt tcggagcaga tcggcgcatg agtgggataa taaaattgat 480

gattccagtt caaaggcaga agaaaaatat gtagcaaaag aatgtattag tccagctgca 540

ttggatttgg tgcatccgga tgtggaattt tgcttaacgg agcttttaaa ctcggatttc 600

tggggtttat ttgagagccc tctgcagccg gaggctaatt ggaatgtcgg tgattttgtt 660

caaccaaatg tggcttccac tcatcattat tttgctaata atatttctga gtccggagga 720

gattggctgt cttga 735

<210> 2

<211> 244

<212> PRT

<213> Panax japonicus

<400> 2

Met Gly Arg Ser Pro Cys Cys Ala Lys Val Gly Leu Asn Lys Gly Ala

1 5 10 15

Trp Thr Ser Val Glu Asp Lys Ile Leu Arg Asp Tyr Ile Lys Leu His

20 25 30

Gly Glu Gly Arg Trp Arg Ser Leu Pro Lys Arg Ala Gly Leu Lys Arg

35 40 45

Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp

50 55 60

Ile Lys Arg Gly Asn Ile Ser Pro Asp Glu Glu Glu Leu Ile Ile Arg

65 70 75 80

Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu

85 90 95

Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Thr Leu

100 105 110

Arg Lys Lys Val His Asp Asn His Ser Ser Ser Ala Ser Ala Ser Ala

115 120 125

Ser Ala Pro Ala Pro Ala Pro Pro Lys Lys Asp Trp Gln Gln Gln Gln

130 135 140

His Leu Val Arg Ser Arg Ser Ala His Glu Trp Asp Asn Lys Ile Asp

145 150 155 160

Asp Ser Ser Ser Lys Ala Glu Glu Lys Tyr Val Ala Lys Glu Cys Ile

165 170 175

Ser Pro Ala Ala Leu Asp Leu Val His Pro Asp Val Glu Phe Cys Leu

180 185 190

Thr Glu Leu Leu Asn Ser Asp Phe Trp Gly Leu Phe Glu Ser Pro Leu

195 200 205

Gln Pro Glu Ala Asn Trp Asn Val Gly Asp Phe Val Gln Pro Asn Val

210 215 220

Ala Ser Thr His His Tyr Phe Ala Asn Asn Ile Ser Glu Ser Gly Gly

225 230 235 240

Asp Trp Leu Ser

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

atggggagga gcccttgctg tgc 23

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 4

tcaagacagc caatctcctc cggac 25

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence (Artificial)

<400> 5

agaatgagcg atctgaagac gag 23

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence (Artificial)

<400> 6

acagacaaca acttcccctc cat 23

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence (Artificial)

<400> 7

gtattccctg tagagcatcg cat 23

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 8

ggcacaggcg ttgttttcac 20

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence (Artificial)

<400> 9

tgggagtttc agcccgatg 19

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 10

ggggaggtgt ataaagtaaa gagcc 25

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 11

gttgttgcag ttaaaaagct cgtag 25

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 12

acctctgact atgaaatacg aatgc 25

Claims

1. Polygala tenuifolia transcription factor genePjMYB1Key enzyme gene for improving biosynthesis of panaxsaponinPjASThe application of the expression quantity and the increase of the total saponin content in the panax japonicus callus is characterized in that: the rhizoma panacis majoris transcription factor genePjMYB1The nucleotide sequence of (A) is shown as SEQ ID NO. 1.