CN100494387C - Method for increasing catharanthus roseus hairy root terpenes indole alkaloid content - Google Patents
Method for increasing catharanthus roseus hairy root terpenes indole alkaloid content Download PDFInfo
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Abstract
The present invention belongs to the field of biology technology, and is method of increasing the TIAs content in hairy root of catharantus roseus. The present invention includes cloning str and g10h genes from catharantus roseus, constructing plant expression vector of str and g10h genes, genetically transforming to catharantus roseus to obtain transgenic catharantus roseus hairy root, determining the TIAs content in hairy root of catharantus roseus in high efficiency liquid chromatography process, and determining the expression of biological synthesized gene of TIAs in hairy root of catharantus roseus via fluorometric quantitative PCR. The transgenic catharantus roseus hairy root has obviously raised TIAs content, especially 234 times raisedVinblastine content. The present invention provides one new method of raising TIAs content in hairy root of catharantus roseus and producing Vinblastine and Vincristine as important clinical anticancer medicine.
Description
Technical field
What the present invention relates to is a kind of method of biological technical field, particularly the method for a kind of pair of key gene cotransformation metabolic engineering strategy raising catharanthus roseus hairy root terpenes indole alkaloid (TIAs) content.
Background technology
Vinca (Catharanthus roseus) is Apocynaceae (Apocynaceae) Vinca plant, people have separated more than 100 kind of terpene indole alkaloid (Terpenoid indolealkaloids from the Vinca herb at present, TIAs), as vinealeucoblastine(VLB) (Vinblastine), vincristine(VCR) (Vincristine), raubasine (Ajmalicine), Serpophite (Serpentine), vindoline alkali (Vindoline), Catharanthine (Catharanthine), Luo Keding alkali (Lochneridine), Pai Wenli alkali (Perivine) etc.Most of TIAs are widely used in the modern medicine owing to having biologic activity, can be used for treating Hokdkin disease, malignant lymphoma, acute lymphoblastic type leukemia, chorioepithelium cell carcinoma and other cancer as famous antitumor drug vinealeucoblastine(VLB) and vincristine(VCR), its vitriol has been widely used in clinical, is present most widely used natural phant antitumor drug.Other TIAs in the Vinca is efficient depressor as raubasine and Serpophite, and vindoline, Catharanthine and leurosine (Leurosine) tool hypoglycemic activity can be treated diabetes, and vindoline and Catharanthine are hypolipidemics.Vinealeucoblastine(VLB) and vincristine(VCR) are maximum, the most widely used anticancer TIAs of medicinal value in the present Vinca.They mainly are to extract from natural Vinca plant, but natural phant in-vivo content pettiness extremely, make Vinca material source difficulty.The content of vinealeucoblastine(VLB) and vincristine(VCR) also is subjected to the influence of the stage of development of the plant, cell subregion, tissue differentiation and environment in the Vinca, only extracts TIAs and can not meet the need of market far away from natural phant.Vinealeucoblastine(VLB) and vincristine(VCR) are because complex structure, and chemosynthesis and semi-synthetic cost are too high, do not have a commercial promise.Hairly root and cell culture system have brought hope for producing these valuable alkaloids.Although can produce monoterpenes indole alkaloid raubasine by hairly root and cell culture system, its content is too low does not have a commercial promise, and cell culture system can not produce the diterpenoids indole alkaloid vinealeucoblastine(VLB) and the vincristine(VCR) of tool using value.Therefore, improving the plant biological alkali content is very important with change alkaloid composition.Metabolic engineering for a change vegetable cell or hairly root composition and increase vegetable cell or the hairly root alkaloid yield provides a tempting method.In order more effectively to produce vinca alkaloids, carry out TIAs biosynthetic pathway and katalaze enzyme thereof and even gene expression and regulation research, and carry out TIAs metabolic engineering research will raising TIAs content and the final rare effective ways of antitumor TIAs medicine that solve.
Find by retrieval, Geraniol-10-desaturase (Geraniol 10-hydroxylase in the existing document, G10H) and different lima bean glycosides synthetic enzyme (Strictosidine synthase STR) is key enzyme in the Vinca TIAs biosynthetic pathway, is the important target spot of TIAs metabolic engineering.Adopt genetic engineering means; with described two key gene str and g10h cotransformation Vinca; to break TIAs biosynthesizing speed limit bottleneck; obtain the Vinca rosea hairly root or the plant of Vinca TIAs high yield; for large-scale production Vinca TIAs provides a new way, but find relevant report with the mentioned two key gene cotransformation strategies raising Vinca rosea hairly root TIAs content of theme of the present invention as yet.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of method that improves catharanthus roseus hairy root terpenes indole alkaloid content is provided.The key gene clone who the present invention relates to, vector construction, genetic transformation, Molecular Detection, TIAs extraction and assay, quantitative fluorescent PCR analysis are used for the present invention, set up the method that improves Vinca rosea hairly root TIAs content, establish solid basis for the large-scale industrialized production of Vinca rosea hairly root, promoted the development of China's Vinca technical field of pharmaceuticals.
The present invention is achieved by the following technical solutions: the present invention clones str and g10h gene from Vinca, structure contains the plant expression vector of described dna molecular, with the Agrobacterium rhizogenes mediation, str and g10h gene are imported the Vinca cell and the hairly root of regenerating simultaneously; Integration and the expression of PCR and fluorescence quantitative PCR detection external source goal gene str and g10h, TIAs content in the high-performance liquid chromatogram determination Vinca rosea hairly root.
The present invention includes following concrete steps:
(1) adopt gene clone method to obtain Vinca key gene str and g10h;
(2) str and g10h gene operability ground are connected in expression regulation sequence, form the plant expression vector that contains str and g10h gene;
(3) will contain the plant expression vector transforming agrobacterium rhizogenes of str and g10h gene, obtain to be used to transform the agrobacterium rhizogene strain that contains str and g10h gene plant expression vector of Vinca;
(4) utilize constructed agrobacterium rhizogene strain to transform the Vinca cell, obtain the transgenosis hairly root that detects through PCR;
(5) quantitative fluorescent PCR is measured the expression of TIAs biosynthesis related genes in the Vinca rosea hairly root;
(6) TIAs content in the transgenosis hairly root that obtains is carried out high effective liquid chromatography for measuring, obtain the transgenosis Vinca rosea hairly root that TIAs content significantly improves.
In the described PCR detection, the primer of synthetic rolB, rolC, hpt, str and g10h gene of design carries out DNA cloning respectively, and the positive strain that the purpose band is observed in ultraviolet ray down is to be transgenosis Changchun hairly root.
The method of described high-performance liquid chromatogram determination raubasine, vinealeucoblastine(VLB) and vincristine(VCR) content is as follows: chromatographic column C-18 reverse phase silica gel post, moving phase acetonitrile: phosphoric acid buffer, volume ratio is 25:75, detect wavelength 220nm, 30 ℃ of column temperatures, flow velocity 1.0mL/min, sample size 10 μ L, sensitivity is AUFS=1.0, and theoretical plate number is pressed vinealeucoblastine(VLB), vincristine(VCR) and raubasine peak and calculated 〉=2000.
The method that described quantitative fluorescent PCR is measured the expression of TIAs biosynthesis related genes in the Vinca rosea hairly root is: the primer of 11 genes in the synthetic Vinca TIAs biosynthetic pathway of design and house-keeping gene 18SrRNA respectively, carry out fluorescent quantitative PCR, with relative quantification method analyzing gene relative expression quantity.
Of the present invention pair of key gene cotransformation strategy improves the method for Vinca rosea hairly root TIAs content, adopts gene engineering method, and two key genes are imported in the Vinca rosea hairly root, obtained the Vinca rosea hairly root clone of TIAs high yield.The acquisition of TIAs high yield Vinca rosea hairly root, can significantly increase the content of important anticancer TIAs in the Vinca rosea hairly root, the content of vinealeucoblastine(VLB), vincristine(VCR) and raubasine is respectively 234.8 times, 3.3 times and 40.6 times of non-conversion contrast root in the hairly root, to solve anticancer TIAs medicine source scarcity, to satisfy the large-scale industrialized production needs of medicine industry significant.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Be interpreted as: these embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The clone of Vinca str and g10h gene
1. separate tissue (Isolation)
With the Vinca seed with 75% alcohol immersion 1min, soak 10min with 2%NaClO again, aseptic water washing 3-4 time, blot surface-moisture with aseptic thieving paper, be inoculated in MS (Murashige and Skoog, the 1962) solid medium of no hormone, 25 ℃, 12h/12h illumination cultivation, can obtain the Vinca aseptic seedling, treat that seedling grows to about 5cm after, cut blade and stem section and be used for RNA and extract.
2.RNA separation (RNA isolation)
Take by weighing the described Vinca sterile test tube of 0.5g seedling, behind liquid nitrogen flash freezer, grind with mortar rapidly, add and fill 1mL TRIzol (TRIzol Reagents, GIBCO BRL is in 1.5mL Eppendorf pipe USA), fully after the vibration, under room temperature, place 5min, add 200 μ L chloroforms, use forced oscillation 15sec, after room temperature is placed 2-3min, in 4 ℃, 12, the centrifugal 15min of 000g; Supernatant liquor (about 600 μ L) is sucked in the clean 1.5mL Eppendorf pipe, adds isopyknic Virahol, put upside down mixing, place 10min under the room temperature after, in 4 ℃, 12, the centrifugal 10min of 000g; Abandon supernatant, add 1mL75% ethanol and clean, after the vibration, in 4 ℃, 7, the centrifugal 5min of 500g; Be dissolved in behind the drying at room temperature 15-20min in an amount of (30-50 μ L) RNAase-free water; Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
3. gene clone (Cloning of the genes)
3.1. the first chain cDNA's is synthetic
3.2. the pcr amplification of Vinca str and g10h gene coding region
According to the encoding sequence (SEQ ID NO.1) of described str gene and the encoding sequence (SEQIDNO.2) of g10h gene, design amplifies the upstream and downstream primer of complete encoder block respectively, and on the upstream and downstream primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively, so that construction of expression vector.With the described first chain cDNA is template, checks order behind pcr amplification.Rich inferior or brilliant safe biotechnology Services Co., Ltd adopts 3730 automatic sequencers to finish to determined dna sequence by Shanghai.Sequencing result shows, Vinca str gene (SEQ ID NO.1) and g10h gene (the SEQ ID NO.1) encoding sequence reported among the sequence of being cloned and the GenBank are consistent.
Present embodiment adopts gene clone method to obtain sequence correct TIAs biosynthesizing key gene str and g10h from Vinca, provides two important key genes for improve Vinca TIAs content by two key enzyme gene transformation strategies.
Embodiment 2
The structure that contains the plant binary expression vector of str and g10h gene
1. intermediate carrier pCAMBIA1304
+Structure
Selecting pBI121 and pCAMBIA1304 for use is primary element, makes up double base plant expression vector pCAMBIA1304
+Particularly, HindIII and EcoRI double digestion pBI121 and pCAMBIA1304; Reclaim GUS expression cassette and the big fragment of pCAMBIA1304 of pBI121; Connect and reclaim product, transformation and selection is taken out the plasmid enzyme restriction checking.
2. plant expression vector pCAMBIA1304
+The structure of+str
With described pCAMBIA1304
+Be expression vector, replace gus gene on it with str among the embodiment 1.Particularly, BamHI/SacI double digestion pGEM T-easy+str and pCAMBIA1304
+, reclaim str and pCAMBIA1304
+Big fragment connects conversion, the picking mono-clonal, and the extraction plasmid is done the PCR detection and enzyme is cut checking.
3. plant expression vector pCAMBIA1304
+The structure of+str+g10h
With described pCAMBIA1304
++ str is the basis, replaces pCAMBIA1304 with the g10h among the embodiment 1
+Gfp+gus gene on the+str.Particularly, BglII/BstEII double digestion pCAMBIA1304
++ str and pGEM T-easy+g10h reclaim pCAMBIA1304
+Big fragment of+str and g10h gene small segment connect conversion, the picking mono-clonal, and the extraction plasmid is done the PCR detection and enzyme is cut checking.The result shows that the g10h gene successfully is building up to plant expression vector pCAMBIA1304
+Among+the str, thereby acquisition contains the plant expression vector pCAMBIA1304 of str and g10h
++ str+g10h.
Present embodiment is connected in expression regulation sequence with TIAs biosynthetic pathway key gene str and g10h operability ground, formation contains the plant expression vector of str and g10h gene, and this expression vector can be used for improving Vinca TIAs content by the metabolic engineering strategy.
Embodiment 3
Agrobacterium rhizogenes mediation str and g10h gene genetic transform Vinca and obtain the transgenosis hairly root
1. contain the acquisition of str and g10h gene double base plant expression vector Agrobacterium rhizogenes engineering bacteria
Change the plant binary expression vector that contains str and g10h gene among the embodiment 2 over to Agrobacterium rhizogenes (as C58C1), the performing PCR of going forward side by side checking.The result shows, contains str and g10h gene plant binary expression vector successfully is building up to agrobacterium rhizogene strain.
2. Agrobacterium rhizogenes mediates str and g10h gene transformation Vinca
2.1. the pre-cultivation of explant
Vinca aseptic seedling blade among the clip embodiment 1 (0.5cm x 0.5cm) and stem section (0.5cm) explant are inoculated on the pre-culture medium (MS+AS 100 μ mol/L), 25 ℃ of dark 2d that cultivate.
2.2. the common cultivation of Agrobacterium and explant
With described pre-cultivation Vinca blade and stem explants, after putting into the 1/2MS suspension that contains the good described Agrobacterium rhizogenes engineering bacteria of activation and soaking 5min (shake gently explant is fully contacted with bacterium liquid), pour out suspension, blot surperficial surplus bacterium with aseptic thieving paper, forward in the common culture medium (1/2MS+AS100 μ mol/L), secretly cultivate 2d.With the blade and the stem explants that are immersed in the 1/2MS liquid nutrient medium that does not have Agrobacterium rhizogenes is contrast.
2.3. inducing and succeeding transfer culture of hairly root
The described Vinca explant of cultivating 2d altogether is transferred to takes off bacterium solid medium (1/2MS+Cef250mg/L) and go up in 25 ℃, 16h/8h illumination cultivation and about 20 days, can go out hairly root from explant edge notches director.Clip hairly root (approximately 2-3cm), to originate from single celled every root as a clone, be inoculated in and take off dark the cultivation for two weeks in the bacterium culture medium (1/2MS+Cef 250mg/L), the hairly root fast, that branch is good of selecting to grow changes over to and takes off subculture screening in the bacterium culture medium (1/2MS+Cef 250mg/L), per two all succeeding transfer culture once can take off bacterium fully through behind 5-6 subculture.Again well-grown Vinca rosea hairly root is changed over to and continue on the 1/2MS substratum of antibiotic-free about the dark 20d of cultivation, the transgenosis growth of hair root rapidly, Gen Mao and branch increases gradually, geotropism forfeiture and can growing fast on the substratum of no plant hormone, have typical hairly root feature.
3. the PCR of transgenosis Vinca rosea hairly root detects
Design Ri plasmid T-DNA district rol gene-specific primer carries out Molecular Detection with PCR method to described hairly root.Since goal gene str and g10h on the plant expression vector with hygromycin gene (hpt) in same border, detect the hpt gene to confirm the transgenosis hairly root.According to goal gene str sequence (SEQ ID NO.1) and g10h sequence (SEQ ID NO.2) design primer goal gene is detected.The result shows, utilizes the PCR special primer of rolB, rolC and hpt gene, can amplify the specific DNA fragment of 423bp, 622bp and 812bp respectively.And when being template, do not amplify any fragment with the common kan gene group of non-conversion Vinca DNA.PCR primer amplification Vinca rosea hairly root DNA with goal gene str and g10h can amplify the special purpose fragment consistent with expected results.This explanation induces hpt, str and the g10h gene in rolB, rolC gene and the T-DNA district of hairly root formation to be incorporated in the Vinca genome.
Present embodiment is with described plant expression vector transforming agrobacterium rhizogenes, acquisition is used to transform the agrobacterium rhizogene strain that contains str and g10h gene plant expression vector of Vinca, utilize constructed agrobacterium rhizogene strain to transform the Vinca cell, obtain the transgenosis hairly root that detects through PCR.The acquisition of transgenosis Vinca rosea hairly root provides direct material for the hairly root that screening obtains TIAs content high yield.
Embodiment 4
The expression of TIAs biosynthesis gene in the fluorescence quantitative PCR detection transgenosis Vinca rosea hairly root
1. primer design is with synthetic
According to Vinca TIAs biosynthetic metabolism pathway gene (orca3, g10h, dxs, ggpps, as α, cpr, str, tdc, sgd, d4h and dat, totally 11) and house-keeping gene 18S rRNA complete sequence design primer.Primer amplification gene fragment length is between 150-400bp.The primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.
2.RNA extraction, the preparation and the standard working curve of standard substance
With reference to method described in the embodiment 1, from Vinca rosea hairly root, extract RNA, remove genomic dna among the RNA with DNaseI, carry out the synthetic of the first chain cDNA with ThermoScript II XL (AMV), be template with the first chain cDNA again, carry out the amplified band that pcr amplification obtains corresponding 11 genes with 11 pairs of primers respectively.
PCR product electrophoresis reclaims, and measures the concentration and the purity of all goal gene stostes with ultraviolet spectrophotometer.Standard substance obtain the goal gene standard substance of series concentration with 10 times of gradient dilution methods.Each standard substance is measured with fluorescence quantifying PCR method, obtains the Ct value.Logarithm with each normal concentration is mapped to their Ct value, obtains 11 satisfied gene standard working curves.11 the gene-correlation coefficients (about slope-3.3, relation conefficient is greater than 0.95) that obtain show between the logarithm of original template concentration and the cycle threshold Ct and exist extremely strong linear relationship that this provides the foundation for quantitative accuracy.Typical curve shows that simultaneously quantitative fluorescent PCR (FQ-PCR) reaction linearity range is extremely wide, can reach the scope (10-9-10-5 μ g/ μ L) of 5 log values, the susceptibility height, and initial concentration also can obtain good quantitative effect at 10-9 μ g/ μ L.The reference standard working curve can obtain the concentration of starting template in the sample exactly.
3. fluorescence quantitative PCR detection house-keeping gene 18S rRNA expression of gene
For adjusting the difference of each sample room reaction efficiency in RNA extracting and reverse transcription process, need detect house-keeping gene 18S rRNA expression of gene in the time of the testing goal gene expression amount.The reaction system of the quantitative fluorescent PCR of detection 18S rRNA and goal gene is as follows:
PCR reaction conditions: 94 ℃ of 15min of warm start and sex change, 50 circulations (72 ℃ are extended 30sec and detect fluorescence for 94 ℃ of sex change 15sec, 55 ℃ of annealing 30sec, and 80 ℃ of 20sec also detect fluorescence).
4. the detection of melting curve analysis and quantitative fluorescent PCR product
All PCR products make temperature slowly rise to 95 ℃ from 72 ℃ with the speed of 0.2 ℃/sec after 50 circulations are finished.The amplified production double-stranded DNA with the rising of temperature gradually sex change unwind and be strand, SYBR Green I dyestuff discharges gradually, fluorescent signal weakens gradually, 0.2 ℃ of variation that detects primary first-order equation inner fluorescent tube intensity of the every rising of instrument, obtain the melting curve (Meltingcurve) of amplified production fluorescence signal intensity to temperature variation at last, the quantitative PCR instrument is mapped to temperature T with dF/dT automatically.Shape and peak value according to melting curve are identified the amplified production in the reaction tubes.For eliminating the influence of primer dimer to Ct, the plate temperature of reading in each circulation is heightened 80 ℃, the primer dimer sex change of two strands discharges bonded SYBR-Green dyestuff under this temperature, thereby has eliminated the fluorescent signal that primer dimer produces.
According to typical curve, obtain the original concentration of each goal gene among each testing sample cDNA, the concentration of goal gene in each sample is proofreaied and correct divided by the concentration of the house-keeping gene 18S rRNA among every part of cDNA with goal gene concentration among every part of cDNA then.The corrected concentrations of every duplicate samples goal gene is then represented the relative expression quantity of sample to non-conversion contrast root divided by the corrected concentrations of this goal gene in the non-conversion contrast root, and the expression amount of non-conversion contrast root is 1.
By research, provide a kind of real-time fluorescence quantitative PCR to measure the method for Vinca TIAs biosynthesis gene expression amount among the present invention.Use method of the present invention, have advantages such as quick, accurate, highly sensitive, that the amplification pollution is little, simultaneously, same sample can be analyzed the expression of a plurality of goal gene in the TIAs biosynthetic pathway simultaneously, has saved cost greatly.
5. 12 expression of gene in the fluorescence quantitative PCR detection transgenosis Vinca rosea hairly root
Measure the expression of TIAs biosynthesis gene in the embodiment 3 transgenosis Vinca rosea hairly roots with fluorescent quantitative PCR technique, the result shows, str and g10h gene import the expression that Vinca rosea hairly root can be induced as α and ggpps in the elementary metabolism, the expression that also can induce g10h in the secondary metabolism, str, tdc, cpr, sgd, dat and d4h.
Present embodiment adopts fluorescent quantitative PCR technique to measure the expression of TIAs biosynthesis related genes in the transgenosis Vinca rosea hairly root, and the height by gene expression amount can preliminary screening goes out the Vinca rosea hairly root of TIAs high yield.
Embodiment 5
Utilize HPLC to measure TIAs content in the transgenosis Vinca rosea hairly root
1. hairly root liquid culture
Choose the Vinca rosea hairly root clone rapidly of growing among the embodiment 3, on Bechtop, take out the hairly root culture, after aseptic water washing falls agar, blot surface-moisture with aseptic filter paper, hairly root is cut into the long root segment of about 5cm, put into the 150mL triangular flask that 30mL 1/2MS liquid nutrient medium is housed, initial inoculum size is 0.5-1.0g fresh weight/bottle.It is on the rotary shaking table of 110rpm that culturing bottle is placed rotating speed, shaking culture under 25 ℃ of dark.Randomly draw 3 bottles of hairly root cultures every 3d, blot the hairly root surface-moisture with filter paper after, take by weighing fresh weight, and calculate the fresh weight increment multiple of hairly root culture.By research, the postvaccinal 1-9d of Vinca rosea hairly root in the present invention, growth of hair root is slower, is in the lag phase of growth of hair root.The 9-21d growth of hair root is rapid, is the logarithmic phase of growing fast, and the hairly root breeding coefficient increases to 47.87 from 3.89.Behind the 21d, growth velocity begins to descend, and enters the growth platform phase.After cultivating 40d, because the consumption of nutritive ingredient in the substratum, culture begins brownization aging, and becomes light yellow gradually.
2. the preparation of chromatographic condition and system suitability and standardized solution
Chromatographic column: C-18 reverse phase silica gel post (SymmetryShieldTM C18,5 μ m, 250 * 4.6mm, Waters).Moving phase is acetonitrile: phosphoric acid buffer (volume ratio 25:75).Detect wavelength 220nm, 30 ℃ of column temperatures, flow velocity 1.0mL/min, sample size 10 μ L, sensitivity (AUFS=1.0), theoretical plate number is calculated by vinealeucoblastine(VLB), vincristine(VCR) and raubasine peak and is not less than 2000.
Precision takes by weighing vinealeucoblastine(VLB), vincristine(VCR) and raubasine standard substance 10.0mg, 5.0mg and 10.0mg, dissolve fully with 1mL methyl alcohol, obtain 10mg/mL vinealeucoblastine(VLB), 5mg/mL vincristine(VCR) and 10mg/mL raubasine reference substance solution respectively, as the standard substance stock solution, be stored in-20 ℃ standby.
The mixed solution that HPLC moving phase is made up of acetonitrile and 5mM Na2HPO4 (regulating pH value to 6.0 with H3PO4).Moving phase acetonitrile-phosphoric acid buffer is when volume ratio 25:75 among the present invention, and the retention time of vincristine(VCR), raubasine and vinealeucoblastine(VLB) is respectively 3.195min, 4.727min and 6.232min, and the peak type is good, and can guarantee three kinds of terpene indole alkaloid good separation.Theoretical plate number is calculated by vinealeucoblastine(VLB), vincristine(VCR) and raubasine and is not less than 2000.
3. the making of typical curve
Described reference substance solution is diluted 1,2,5,10 times respectively, sample introduction under corresponding chromatographic condition, record collection of illustrative plates and chromatographic parameter, (X mg/L) carries out regression analysis to standard substance concentration with peak area (Y) respectively.By research, vinealeucoblastine(VLB) presents good linear relationship at 2.5-25mg/L, raubasine at 2.5-25mg/L, vincristine(VCR) among the present invention in the 2.5-25mg/L scope.The equation of linear regression of vinealeucoblastine(VLB), vincristine(VCR) and raubasine reference substance is respectively:
| Yvinblastine＝586912.3X-1287.4 | r＝0.99993 |
| Yvincristine＝308665.5X-1526.5 | r＝0.99994 |
| Yajmalicine＝136029.4X-1553.2 | r＝0.99996 |
4. the preparation of sample and TIAs Determination on content
Collect the catharanthus roseus transgenic hairly root of described liquid culture 30d, blot surface medium, take by weighing fresh weight, to constant weight, claim its dry weight in 60 ℃ of oven dry 48h with filter paper.Hairly root is put into mortar, add a small amount of quartz sand, grind, add 95% ethanol (1:5, W/V), 50 ℃ of supersound extraction 30min, be cooled to room temperature, centrifugal (12,000rpm) 10min draws supernatant liquor to extracting solution, in 12, the centrifugal again 10min of 000rpm draws supernatant liquor, measures TIAs content with the HPLC method.
Draw described reference substance solution respectively and for each 10 μ L of test agent solution, inject high performance liquid chromatograph, write down each component peaks area, the substitution equation of linear regression calculates and promptly gets sample TIAs content.
Corotation str/g10h gene significantly improves Vinca rosea hairly root TIAs content in the present invention.The average content of vinealeucoblastine(VLB), vincristine(VCR) and raubasine is respectively 61.07mg/g DW, 5.91mg/g DW and 2.11mg/g DW in the corotation str/g10h gene Vinca rosea hairly root, is 234.8 times, 3.3 times of common of non-conversion and 40.6 times (0.26mg/g DW vinealeucoblastine(VLB), 1.81mg/g DW vincristine(VCR) and 0.052mg/g DW raubasine).The total TIAs content of corotation str/g10h gene hairly root is 69.09mg/g DW, is 32.6 times (2.12mg/g DW) of common of non-conversion.In the corotation str/g10h Vinca rosea hairly root clone of being tested, TIAs content the highest (78.13mg/g DW) among the SG11.
Present embodiment adopts the HPLC method to measure transgenosis Vinca rosea hairly root TIAs content.The metabolic engineering strategy of employing cotransformation str and g10h gene has obtained the Vinca rosea hairly root of TIAs high yield, solves the TIAs scarcity for large-scale production Vinca TIAs is also final a kind of Perfected process is provided.
Sequence that the present invention relates to and mark apportion are as follows:
The information of SEQIDNO.1
＜110〉Shanghai Communications University
＜120〉two key enzyme gene transformation strategies improve Vinca rosea hairly root TIAs content
<140>
<141>2005-12-05
<160>1
<170>PatentIn version3.1
<210>1
<211>1044
<212>DNA
＜213〉Vinca (Catharanthus roseus)
<400>1
atgatggcag ttttcttcat gtttttcctt cttcttcttt cttcttcttc ttcttcttct 60
tcttcttcac caattttgaa aaagattttt attgaaagcc cttcctatgc tccgaatgcc 120
ttcaccttcg attcaactga taaagggttc tacacttccg tccaagatgg ccgagttatc 180
aaatatgaag ggccaaattc aggcttcact gacttcgcct acgcatctcc cttctggaac 240
aaagcttttt gtgagaacag caccgatcca gagaaaagac cattgtgtgg gaggacatat 300
gatatttcct atgactataa gaacagccaa atgtacattg ttgatggcca ttaccatctt 360
tgtgtggttg gaaaagaagg tgggtatgcc acacaactag ccacaagtgt gcaaggagtg 420
ccattcaaat ggctctatgc agtaactgtt gatcagagaa cagggattgt ttatttcact 480
gatgttagct ccatacatga tgacagtccc gaaggtgtgg aagaaatcat gaatacaagt 540
gatagaacag ggagattaat gaagtatgat ccttcaacaa aagaaaccac cttattattg 600
aaagagctac atgttcccgg cggtgcagaa atcagcgcag atggttcctt tgttgtagta 660
gcagaatttt taagcaatcg gatagtgaag tattggctag aagggccaaa gaaaggcagt 720
gcagagttct tagttacaat cccaaatcca ggaaatataa agaggaattc tgatggccat 780
ttttgggtgt cttcaagtga agaattagat ggaggtcaac atggaagtgt tgtttcaaga 840
ggaattaagt ttgatggatt tgggaatatt cttcaagtta taccacttcc accaccatat 900
gaaggtgaac attttgaaca gattcaagag cacgatggtt tgttatacat tggaagtctc 960
tcccatagct ctgtgggtat attagtgtat gatgatcatg ataacaaggg aaattcttat 1020
gtttctcagc tagtcattaa ttga 1044
The information of SEQ ID NO.2
<210>2
<211>1482
<212>DNA
＜213〉Vinca (Catharanthus roseus)
<400>2
atggattacc ttaccataat attaacttta ctatttgcct tgactctcta tgaagccttc 60
agctacctat ccagaagaac caaaaacctt cctccaggac catcgccatt gccgttcatc 120
ggaagcctcc atttattagg cgaccaacca cacaaatcct tagcaaaact ttccaaaaaa 180
cacggtccaa ttatgagtct caaattaggc cagatcacta caatcgtcat atcttcatca 240
acaatggcga aagaagttct tcaaaaacag gatttagcat tttcaagcag atcagttcca 300
aacgcactcc acgctcacaa tcaattcaaa ttctccgttg tatggcttcc ggtagcctca 360
cgatggagaa gtcttcgaaa agttttgaat tctaatatat tttccggcaa tcggctcgac 420
gctaatcaac atttgagaac tagaaaagta caggaactaa ttgcgtattg ccggaaaaat 480
agccagagcg gagaagcggt tgacgtcggc cgagctgctt ttagaacttc gttgaatttg 540
ttgtcgaatt tgattttttc aaaggatttg acggatcctt attcggattc tgccaaggaa 600
ttcaaggatt tggtttggaa tataatggtt gaggcgggga aacctaattt ggtcgatttt 660
tttcccctgc ttgaaaaagt tgatcctcaa ggtatacgac atcgtatgac gattcacttt 720
ggggaagttc ttaagctttt tggtggactt gttaatgaaa gattggagca aagaagatca 780
aaaggggaaa aaaatgatgt gttggatgta cttctaacta ccagccaaga aagccctgag 840
gaaatcgata gaactcacat tgagcgaatg tgcttggacc tgtttgtagc agggacggac 900
acaacatcaa gcacattaga atgggcaatg tcagaaatgc ttaaaaaccc agacaaaatg 960
aagaaaaccc aagatgaact tgcacaagta atcggcagag gaaaaacaat agaagaatcc 1020
gatattaacc gcttacctta cttaagatgc gttatgaaag aaaccttaag gatacatcca 1080
ccagttccct tcttaattcc tcgcaaagtg gaacaaagtg ttgaggtttg tggatacaat 1140
gtccctaaag gatcacaagt tcttgtgaat gcttgggcaa ttggacgtga tgaaactgtt 1200
tgggatgatg ctttggcatt caaacccgag agatttatgg aatctgaatt ggatatccgt 1260
ggaagagatt tcgagctgat tccgttcggt gctggccgaa gaatttgccc agggttgcca 1320
ttggcactaa ggactgtgcc tttgatgctt ggttctttgt tgaactcttt taattggaag 1380
cttgaaggtg ggatggctcc aaaagatttg gatatggagg agaagtttgg tattacactg 1440
cagaaggctc atcctttgcg tgctgtacca agcacccttt aa 1482
Claims (4)
1. method that improves catharanthus roseus hairy root terpenes indole alkaloid content, it is characterized in that, the different lima bean glycosides synthase gene str of clone and Geraniol-10-dehydrogenase gene g10h from Vinca, structure contains the plant expression vector of described dna molecular, mediate with Agrobacterium rhizogenes, str and g10h gene are imported the Vinca cell and the hairly root of regenerating simultaneously, integration and the expression of PCR and fluorescence quantitative PCR detection external source goal gene str and g10h, terpene indole alkaloid TIAs content in the high-performance liquid chromatogram determination Vinca rosea hairly root.
2. the method for raising catharanthus roseus hairy root terpenes indole alkaloid content according to claim 1 is characterized in that, comprises following concrete steps:
(1) adopt gene clone method to obtain Vinca key gene str and g10h;
(2) str and g10h gene operability ground are connected in expression regulation sequence, form the plant expression vector that contains str and g10h gene;
(3) will contain the plant expression vector transforming agrobacterium rhizogenes of str and g10h gene, obtain to be used to transform the agrobacterium rhizogene strain that contains str and g10h gene plant expression vector of Vinca;
(4) utilize constructed agrobacterium rhizogene strain to transform the Vinca cell, obtain the transgenosis hairly root that detects through PCR;
(5) quantitative fluorescent PCR is measured the expression of TIAs biosynthesis related genes in the Vinca rosea hairly root;
(6) TIAs content in the transgenosis hairly root that obtains is carried out high effective liquid chromatography for measuring, obtain the transgenosis Vinca rosea hairly root that TIAs content significantly improves.
3. the method for raising catharanthus roseus hairy root terpenes indole alkaloid content according to claim 2, it is characterized in that, the described transgenosis hairly root that detects through PCR, its detection method is: the primer of root of hair locus gene B rolB, root of hair locus gene C rolC, hygromycin phosphotransferase gene hpt, str gene and 10h gene is synthesized in design respectively, carry out DNA cloning, the positive strain that the purpose band is observed in ultraviolet ray down is to be the transgenosis Vinca rosea hairly root.
4. the method for raising catharanthus roseus hairy root terpenes indole alkaloid content according to claim 2, it is characterized in that, described TIAs content in the transgenosis hairly root that obtains is carried out high effective liquid chromatography for measuring, concrete measuring method is as follows: chromatographic column C-18 reverse phase silica gel post, moving phase is acetonitrile: phosphoric acid buffer, the volume ratio of acetonitrile and phosphoric acid buffer is 25:75, detect wavelength 220nm, 30 ℃ of column temperatures, flow velocity 1.0mL/min, sample size 10 μ L, sensitivity is AUFS=1.0, theoretical plate number is pressed vinealeucoblastine(VLB), vincristine(VCR) and raubasine peak calculate 〉=2000.
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Non-Patent Citations (4)
| Title |
|---|
| ENGINEERING TERPENOID INDOLE ALKALOIDSBIOSYNTHETIC PATHWAY IN CATHARANTHUS ROSEUSHAIRY ROOT CULTURES BY OVEREXPRESSING THEGERANIOL 10-HYDROXYLASE GENE. GONG YI-FU, ET AL.JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY(SCIENCE),Vol.E-10 No.S1. 2005 |
| ENGINEERING TERPENOID INDOLE ALKALOIDSBIOSYNTHETIC PATHWAY IN CATHARANTHUS ROSEUSHAIRY ROOT CULTURES BY OVEREXPRESSING THEGERANIOL 10-HYDROXYLASE GENE. GONG YI-FU, ET AL.JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY(SCIENCE),Vol.E-10 No.S1. 2005 * |
| 植物次级代谢产物研究. 安俊龙等.河北化工，第6期. 2002 |
| 植物次级代谢产物研究. 安俊龙等.河北化工，第6期. 2002 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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