CN102191333A - Method for identifying gynostemma pentaphylla and making distinction between gynostemma pentaphylla and cayratia japonica at deoxyribonucleic acid (DNA) level - Google Patents
Method for identifying gynostemma pentaphylla and making distinction between gynostemma pentaphylla and cayratia japonica at deoxyribonucleic acid (DNA) level Download PDFInfo
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Abstract
The invention discloses a method for identifying gynostemma pentaphylla and making a distinction between gynostemma pentaphylla and cayratia japonica at deoxyribonucleic acid (DNA) level. The method is characterized by designing gynostemma pentaphylla SCAR marked specific primer pair WJS-T1 and WJS-T2 by using the result of medicinal plant cucurbitaceae gynostemma pentaphylla DNA characteristic segment SCAR marked clone sequencing, designing internal reference primer pair WHL-18S-C1 and WHL-18S-C2 according to a cucurbitaceae gynostemma pentaphylla 18S rRNA conserved region, forming a polymerase chain reaction (PCR) composite system 1, and amplifying the cucurbitaceae gynostemma pentaphylla DNA by using a touchdown PCR program to obtain two zones, namely a 359bp zone and a 293bp zone; and designing the internal reference primer pair WPT-18S-C1 and WPT-18S-C2 according to a vitaceae 18S rRNA conserved region, combining the internal reference primer pair WPT-18S-C1 and WPT-18S-C2 and the gynostemma pentaphylla SCAR marked specific primer pair WJS-T1 and WJS-T2 to form a PCR composite system 2, and amplifying a vitaceae cayratia japonica DNA by using the touchdown PCR program to obtain an 18S rRNA 177bp zone. Therefore, the DNA level of medicinal plant cucurbitaceae gynostemma pentaphylla is identified, and the distinction between the gynostemma pentaphylla and the vitaceae cayratia japonica which is easy to confound with the cucurbitaceae gynostemma pentaphylla in shape is made.
Description
Technical field
The present invention relates to a kind of dna level identify gynostemma pentaphylla and with the method for Japanese Cayratia Herb difference.
Background technology
There is abundant resources of medicinal plant in China, and is of a great variety because of it, various places homonym or synonym phenomenon ubiquity; Same kind famous-region drug and non-famous-region drug, not fairly obvious in the difference of features such as form, histological structure, but quality is widely different sometimes, is difficult to identification.Even if same kind, the place of production not simultaneously its active constituent content also may there are differences.The situation that drives adulteration because of interests in sales of Chinese herbal medicine happens occasionally, so the kind of herbal medicine and quality evalution seem particularly important.
The traditional authentication method of Chinese medicinal herbs mainly is proterties and/or organizes microscopical identification that some has developed thin layer look (light) spectrum, electrophoretic method, X-ray diffraction method etc. according to the medium-height grass the effective elements of the medicine.These methods respectively have relative merits and limitation thereof.Along with infiltration and the application of molecular genetic marker technique in the STUDIES ON PHARMACEUTICAL BOTANY field, increasing plant medicine material begins to attempt the genetic analysis evaluation of molecular level.Because the genetic composition of plant species can not change with its physical form, physiology and external conditions, is more objective, more reliable identification marking, and the plant base source feature of herbal medicine can be provided better.Based on the dna molecular marker technology of PCR, as low-Cot DNA fingerprinting, RAPD, AP-PCR, PCR-RFLLP, Amplified DNA RFLP etc. have brought into play important effect in the Chinese medicinal herbs real and fake discrimination.This can not only carry out the hereditary feature analysis to medicinal plant Ji Yuan, and can progressively accumulate the genetic molecule information of these medicinal plants, helps protecting the advantage species resource.Differentiated with available AFLP of Radix Panacis Quinquefolii (Panax quinquefolius) (Amplified Fragment Length Polymorphism) and DAMD (Directed Amplification of Minisatellite Region DNA) as famous Chinese medicine genseng (Panax ginseng); RAPD also is used to distinguish the Radix Astragali in source, the different place of production, as the Korea S's Radix Astragali or the Chinese Radix Astragali; The cluster and the signature analysis that are used for medicinal plant Ji Yuan such as Semen Plantaginis, Herba Centellae.The progress of external this respect is very quick, existing multinomial patent.At present, Shang Weijian is relevant for the method for gynostemma pentaphylla dna molecular evaluation.
RDNA is the gene of coding rrna rRNA, exists to repeat continuous arrangement mode in plant, comprises coding region, non-encoding transcription district that evolutionary rate does not wait.18S, 5S rRNA conservative in the evolution have been used to various Eukaryotic sibship researchs.18S rRNA gene is a highly repetitive sequence, and about 800～10000 copies are present in the cell with continuous arrangement mode.Simultaneously 18S rRNA gene order structure, function are very conservative, and evolutionary rate is slower, if their sequence exists variation just can become a kind of good dna marker.18S rRNA gene conservative is that the gene identification and the authenticity that carry out Chinese medicinal herbs have been laid good molecular basis.At present, 18S rRNA gene sequencing technology has been used to the Ji Yuan discriminating of genseng, pseudo-ginseng, Chinese yam, rheum officinale, turmeric etc.According to some plants of in GenBank, having logined of our inquiry or the cluster analysis that moss 18S rRNA sequence is done, the result shows that 18S rRNA evolutionary rate in equal platymiscium is slower, but in not equal platymiscium monoid, have, or can be used as the fine mark that the medicinal plant classification is identified than big difference.
Gynostemma pentaphylla (Gynostemma pentaphyllum (Thrunb.) Mak) is a cucurbitaceae genus gynostemma.China's gynostemma pentaphylla resource is very abundant.The main effective constituent of gynostemma pentaphylla is saponin(e, and isolating at present gypenoside has kind more than 80, is the tetracyclic triterpene dammarane type, has 6 kinds of saponin(es identical with the ginsenoside structure, also has some can obtain ginsenoside through hydrolysis.Gynostemma pentaphylla is the plant that contains ginsenoside few in number beyond the Araliaceae.Pharmacological mechanism to gypenoside has also had more research report at present.Some discover that gypenoside can weaken the formation of aminofluorene-dna adduct in the cervical cancer cell, suppress the expression of human cervical carcinoma's kharophen transferring enzyme; Gypenoside can also suppress nitric oxide production synthetic by suppressing iNOS (inducible nitric oxide synthase) iNOS protein expression active and that weaken NF-κ B mediation, as one of mechanism of its performance therapeutic action; With gypenoside handler liver cancer cell, can come cell death inducing by rise Bax and Bak, downward modulation Bcl-2, release wire plastochondria cytochrome C, activation cascade reaction; Gypenoside can also be protected the neurone of rat brain ischemia-reperfusion district damage; Can come lowering blood glucose concentration or the like by changing the hepatic glucose metabolic enzyme activity.The demand to gypenoside is also more and more higher on the medical market at present.
Gynostemma pentaphylla has formed numerous kinds and cultivated form gradually under secular physical environment and tame condition.According to its bird foot shape compound leaf vanelets number branch, seven leaves or five leaf gynostemma pentaphyllums are arranged; Divide pleasantly sweet or the bitter taste gynostemma pentaphylla according to its blade taste.Gynostemma pentaphylla genetic diversity complexity, plant phenotype and saponin(e kind, content all have certain difference.Our result of study shows that the triterpenoid saponin content of seven leaf gynostemma pentaphyllum blades is that the triterpenoid saponin content of 6.78%, five leaf gynostemma pentaphyllum blade has only 3.69%; The triterpenoid saponin content of gynostemma pentaphylla blade is 4.72%, and the content of its stem has only 1.85%, and the triterpenoid saponin content that south Guangxi is produced five leaf gynostemma pentaphyllum blades is 4.1%, and the northern triterpenoid saponin content that produces five leaf gynostemma pentaphyllums in its Guangxi is 3.6%.In addition, also there is the adulterant of some other plants in the gynostemma pentaphylla medicinal material, as Japanese Cayratia Herb (Cayratia japonica (Thunb.) Gagnep.) of Vitaceae etc.Therefore, gynostemma pentaphylla being carried out analysis of genetic diversity and molecular biology identification, is highly significant to the high gynostemma pentaphylla kind of preferred saponin content, the problem that solves the pseudo-product of gynostemma pentaphylla.
Summary of the invention
Purpose of the present invention: for the tradition that overcomes medicinal plant Curcurbitaceae gynostemma pentaphylla identifies mainly be with proterties or/and organize the limitation of methods such as microscopical identification and the technical deficiency of complicacy, at Curcurbitaceae gynostemma pentaphylla dna sequence dna specificity, set up a more objective and easy row dna level identify the Curcurbitaceae gynostemma pentaphylla and with its form on the method for the Vitaceae Japanese Cayratia Herb difference easily obscured.
The present invention is achieved in that
A kind of dna level identify gynostemma pentaphylla and with the method for Japanese Cayratia Herb difference, it is characterized in that: the result who utilizes the order-checking of medicinal plant Curcurbitaceae gynostemma pentaphylla dna characteristics fragment SCAR marker clone, the special primer of design Curcurbitaceae gynostemma pentaphylla SCAR mark is right, in conjunction with Curcurbitaceae gynostemma pentaphylla and Vitaceae Japanese Cayratia Herb plant 18S rRNA gene conserved regions design primer separately to as confidential reference items, be combined into PCR compound system 1 respectively and PCR compound system 2 increases, amplified production is carried out electrophoretic analysis, identify thereby medicinal plant Curcurbitaceae gynostemma pentaphylla is carried out dna level, and with form on easily and the Vitaceae Japanese Cayratia Herb obscured of gynostemma pentaphylla distinguish mutually.
Above-described PCR compound system 1 comprises the segmental primer of amplification Curcurbitaceae gynostemma pentaphylla SCAR mark 359bp to WJS-T
1And WJS-T
2, the segmental confidential reference items primer of amplification Curcurbitaceae gynostemma pentaphylla 18S rRNA 293bp is to WHL-18S-C
1And WHL-18S-C
2, use touchdown PCR program amplification Curcurbitaceae gynostemma pentaphylla DNA, obtain 359bp and the 293bp district band of two Curcurbitaceae gynostemma pentaphylla, the process of foundation is as follows:
(1) adopts the analysis of biological information method, filter out 19 10mer RAPD random primers, set up the RAPD analysis system, find out the total SCAR molecule marker J-750 of Curcurbitaceae gynostemma pentaphylla; 19 random primers that employing screens see attached list 1, carry out pcr amplification, analyze the dna polymorphism difference of bright sample of Curcurbitaceae gynostemma pentaphylla (bright sample source is used in experiment, sees attached list 2) and dry sample (experiment sees attached list 3 with the dry sample source).Wherein, adopt the Curcurbitaceae gynostemma pentaphylla DNA in source, the domestic different place of production, 8 parts of Guangxi of No. 10 primer amplifications total amplified band all to occur, length is about 750bp, sees accompanying drawing 4.To the DNA sample of 17 parts of dry Curcurbitaceae gynostemma pentaphylla medicinal materials, this feature band that also increases can be designated as J-750 as the molecule marker of gynostemma pentaphylla, sees accompanying drawing 5 and accompanying drawing 6.Adopt the method for molecular cloning, carry out cloning and sequencing respectively, obtain the dna sequence dna of 8 length at 766～770bp.To obtaining the Curcurbitaceae gynostemma pentaphylla Partial Feature dna sequence dna of 8 parts of different sourcess, length is in 766～770bp scope, through adopting the Blast software analysis of NCBI, the Curcurbitaceae gynostemma pentaphylla J-750 that obtains 8 parts of different sourcess is the DNA of plants sequence of not logining at NCBI as yet of one section new clone; Cloned sequence to the Curcurbitaceae gynostemma pentaphylla molecule marker J-750 of 8 parts of different sourcess being obtained uses Vector NTI software analysis sequencing result.The result shows that the consistence of above-mentioned 8 sequences has the height consistence between 97.4%～99.9%, see attached list 4.Through the restriction enzyme site analysis, article 8, sequence all contains the restriction enzyme site of 11 kinds of restriction endonucleases, wherein sequence J1-750, J6-750 have a SspI restriction enzyme site, J3-750, J4-750, J5-750, J7-750, J8-750 have two SspI restriction enzyme sites, J2-750 has three SspI restriction enzyme sites, other 10 kinds of restriction endonucleases are single restriction enzyme site at the restriction enzyme site of 8 sequences, see attached list 5.
The gynostemma pentaphylla molecule marker J-750DNA fragment that 8 parts of different sources gynostemma pentaphylla come out through No. 10 primer amplifications is cloning and sequencing, logins in GenBank, and obtains accession number, sees attached list 6 and the nucleotides sequence tabulation, but still unexposed.
Blast program in the utilization NCBI gene database is carried out the homology compare of analysis to Curcurbitaceae gynostemma pentaphylla feature band, the result shows that 99 gene orders from other species have the part similarity among 8 sequences and the GenBank, but similar site fragment is only between 40-90bp, whole homology is little, sees accompanying drawing 7～14.Illustrate that 8 Curcurbitaceae gynostemma pentaphylla dna sequence dnas among the present invention are new sequence.
(3) the gynostemma pentaphylla SCAR molecule marker J-750 that utilizes Oligo 6 softwares to obtain at the present invention designs special primer to WJS-T
1And WJS-T
2, set up PCR system amplification Curcurbitaceae gynostemma pentaphylla DNA, amplification obtains the special district band that length is 359bp;
Subordinate list 1:RAPD analyzes 19 10mer random primer sequence table look-ups of usefulness
Subordinate list 2: test with bright sample source table look-up
(4) according to Curcurbitaceae gynostemma pentaphylla 18S rRNA gene conserved regions design primer to WHL-18S-C
1And WHL-18S-C
2, set up PCR system amplification Curcurbitaceae gynostemma pentaphylla DNA, amplification obtains the total band of Curcurbitaceae gynostemma pentaphylla 18S rRNA gene conserved regions that a segment length is 293bp;
(5) with Curcurbitaceae gynostemma pentaphylla SCAR molecule marker primer to WJS-T
1And WJS-T
2With 18S rRNA gene conserved regions primer to WHL-18S-C
1And WHL-18S-C
2Combination, set up PCR complex reaction system 1, adopt touchdown PCR program amplification Curcurbitaceae gynostemma pentaphylla dna profiling, the PCR product carries out the agarose gel electrophoresis analysis and obtains Liang Tiao district band, wherein 1 is the special band of Curcurbitaceae gynostemma pentaphylla SCAR of 359bp, and 1 is the conservative band of Curcurbitaceae gynostemma pentaphylla 18S rRNA gene regions of 293bp in addition.
Subordinate list 3: experiment is with the dry sample table look-up of originating
Subordinate list 4:8 bar different sources gynostemma pentaphylla molecule marker J-750DNA sequence identity is table look-up relatively
Subordinate list 5:8 bar different sources gynostemma pentaphylla molecule marker J-750DNA sequence restriction enzyme digestion sites is analyzed table look-up
Subordinate list 6:8 part different sources gynostemma pentaphylla molecule marker J-750 is cloning and sequencing dna sequence dna table look-up
Above-described PCR compound system 2 comprises the segmental primer of amplification Curcurbitaceae gynostemma pentaphylla SCAR mark 359bp to WJS-T
1And WJS-T
2, the segmental confidential reference items primer of 18S rRNA 177bp of amplification Vitaceae Japanese Cayratia Herb is to WPT-18S-C
1And WPT-18S-C
2, use touchdown PCR program amplification Vitaceae Japanese Cayratia Herb DNA, obtain the 18S rRNA 177bp district band of 1 Vitaceae Japanese Cayratia Herb, the process of foundation is as follows:
(1) according to Vitaceae Japanese Cayratia Herb 18S rRNA gene conserved regions design primer to WPT-18S-C
1And WPT-18S-C
2, set up PCR system amplification Vitaceae Japanese Cayratia Herb DNA, obtaining a segment length is the total band of Vitaceae Japanese Cayratia Herb 18S rRNA gene conserved regions of 177bp;
(2) with Vitaceae Japanese Cayratia Herb 18S rRNA gene conserved regions primer to WPT-18S-C
1And WPT-18S-C
2, with Curcurbitaceae gynostemma pentaphylla SCAR molecule marker special primer to WJS-T
1And WJS-T
2Be combined into PCR complex reaction system 2, adopt the touchdown PCR program that Vitaceae Japanese Cayratia Herb dna profiling is increased, the PCR product carries out the agarose gel electrophoresis analysis, obtains the Vitaceae Japanese Cayratia Herb 18S rRNA gene conserved regions band of 1 177bp, no Curcurbitaceae gynostemma pentaphylla SCAR fragment 359bp district band.
Among the present invention, adopt PCR compound system 1 or 2, the Curcurbitaceae gynostemma pentaphylla is identified in amplification, and sees attached list 7 with the information list of Vitaceae Japanese Cayratia Herb difference:
Subordinate list 7: PCR compound system 1 or 2 amplification gynostemma pentaphylla or Japanese Cayratia Herb dna profiling information lists
Among the present invention, the special primer of amplification gynostemma pentaphylla SCAR mark is to WJS-T
1And WJS-T
2Sequence, gynostemma pentaphylla 18S rRNA gene confidential reference items primer is to WHL-18S-C
1And WHL-18S-C
2Sequence, Vitaceae 18S rRNA gene confidential reference items primer is to WPT-18S-C
1And WPT-18S-C
2Sequence sees attached list 8.
The primer sequence table look-up that subordinate list 8: PCR compound system 1 and 2 is used
The present invention set up dna level identify gynostemma pentaphylla and with the method for Japanese Cayratia Herb difference, identify the Curcurbitaceae gynostemma pentaphylla and with phytomorph on application in the Vitaceae Japanese Cayratia Herb difference easily obscured mutually with the Curcurbitaceae gynostemma pentaphylla, the results are shown in accompanying drawing 1～3.
Advantage of the present invention and effect thereof:
1, the inventive method is utilized Curcurbitaceae gynostemma pentaphylla dna sequence dna specificity, with the special primer of amplification Curcurbitaceae gynostemma pentaphylla SCAR mark to WJS-T
1And WJS-T
2, Curcurbitaceae gynostemma pentaphylla 18S rRNA gene confidential reference items primer is to WHL-18S-C
1And WHL-18S-C
2Form PCR complex reaction system 1, adopt the touchdown PCR amplification program, can amplify 359bp, 293bp two bands to Curcurbitaceae gynostemma pentaphylla DNA, and the Vitaceae Japanese Cayratia Herb does not have this two band; With the special primer of amplification Curcurbitaceae gynostemma pentaphylla SCAR mark to WJS-T
1And WJS-T
2, Vitaceae Japanese Cayratia Herb 18S rRNA gene confidential reference items primer is to WPT-18S-C
1And WPT-18S-C
2Be combined into PCR complex reaction system 2, adopt the touchdown PCR amplification program, can amplify a 177bp band to Vitaceae Japanese Cayratia Herb DNA, and the Curcurbitaceae gynostemma pentaphylla does not have this 177bp band, thus to medicinal plant Curcurbitaceae gynostemma pentaphylla carry out that dna level is identified and with form on easily and the Vitaceae Japanese Cayratia Herb obscured of gynostemma pentaphylla distinguish mutually.
2, the inventive method tradition of having overcome medicinal plant Curcurbitaceae gynostemma pentaphylla identifies mainly to be with proterties or/and organize the limitation of methods such as microscopical identification and the technical deficiency of complicacy, at Curcurbitaceae gynostemma pentaphylla dna sequence dna specificity, set up a more objective and easy row evaluation Curcurbitaceae gynostemma pentaphylla and with its form on the method for the Vitaceae Japanese Cayratia Herb difference easily obscured.
Description of drawings
Fig. 1 is Curcurbitaceae gynostemma pentaphylla fresh sample (J1～J8) and the fresh product DNA of Vitaceae Japanese Cayratia Herb, PCR product agarose gel electrophoresis figure;
Fig. 2 is Curcurbitaceae gynostemma pentaphylla dry sample (JG1～JG8) and Vitaceae Japanese Cayratia Herb dry sample DNA, PCR product agarose gel electrophoresis figure;
Fig. 3 is Curcurbitaceae gynostemma pentaphylla dry sample (JG10～JG17) and Vitaceae Japanese Cayratia Herb dry sample DNA, PCR product agarose gel electrophoresis figure;
Fig. 4 is after to be No. 10 random primers to the DNA of the bright product J1～J8 of 8 portions of gynostemma pentaphylla carry out the RAPD amplification, and the electrophoresis swimming of product is schemed;
Fig. 5 is after No. 10 random primers carry out the RAPD amplification to gynostemma pentaphylla dry product JG1～JG8DNA, the electrophorogram of its product;
Fig. 6 is after No. 10 random primers carry out the RAPD amplification to gynostemma pentaphylla dry product JG9～JG17DNA, the electrophorogram of its product;
Fig. 7 compares the similar fragment distribution plan that obtains for known dna sequence among sequence J1-750 and the GenBank;
Fig. 8 compares the similar fragment distribution plan that obtains for known dna sequence among sequence J2-750 and the GenBank;
Fig. 9 compares the similar fragment distribution plan that obtains for known dna sequence among sequence J3-750 and the GenBank;
Figure 11 compares the similar fragment distribution plan that obtains for known dna sequence among sequence J4-750 and the GenBank;
Figure 10 compares the similar fragment distribution plan that obtains for known dna sequence among sequence J5-750 and the GenBank;
Figure 12 compares the similar fragment distribution plan that obtains for known dna sequence among sequence J6-750 and the GenBank;
Figure 13 compares the similar fragment distribution plan that obtains for known dna sequence among sequence J7-750 and the GenBank;
Figure 14 compares the similar fragment distribution plan that obtains for known dna sequence among sequence J8-750 and the GenBank.
Specific embodiment
Embodiment
1. extract DNA:
Adopt inventor's improved method of CTAB to extract Curcurbitaceae gynostemma pentaphylla and Vitaceae Japanese Cayratia Herb DNA respectively.
(1) Curcurbitaceae gynostemma pentaphylla fresh sample DNA extraction
1. take by weighing the 0.5g blade and place the mortar of precooling, add liquid nitrogen grinding and become fine powder, be transferred to rapidly in the 7ml centrifuge tube, the 1.5 * CTAB that adds the 3ml preheating extracts damping fluid, and mixing is incubated 20min in 65 ℃ of water-baths.
2. will 1. go on foot extracting solution and be cooled to room temperature, the centrifugal 10min of 7500rpm.Get supernatant, add isopyknic chloroform/primary isoamyl alcohol (24/1) and carry out extracting, mixing, the centrifugal 10min of 7500rpm.
3. get and 2. go on foot centrifugal supernatant, the 1 * CTAB that adds 1/10 volume extracts damping fluid and isopyknic chloroform/primary isoamyl alcohol (24/1), mixing, the centrifugal 10min of 7500rpm.
4. get and 3. go on foot centrifugal supernatant, add isopyknic 1 * CTAB precipitation buffering liquid, light and slow putting upside down to forming flocks.If no obvious sediment is separated out, then in 65 ℃ of water-baths, be incubated 30min, the centrifugal 10min of 7500rpm.
5. will 4. go on foot centrifugal sediment and dissolve the centrifugal insolubles of removing with 0.5mlNaCl (1mol/L).Add the dehydrated alcohol of the precooling of 2.5 times of volumes in the dna solution, in-20 ℃ of refrigerators, leave standstill 15min, the centrifugal 10min of 8000rpm.
6. will 5. go on foot centrifugal sediment with 1ml70% washing with alcohol twice, and in ventilating kitchen, volatilize the back naturally and fully dissolve the back as DNA sample stock solution, preserve standby down in-20 ℃ with 100～200 μ l sterilization tri-distilled water.
(2) Vitaceae Japanese Cayratia Herb fresh sample DNA extraction
On the basis of above extracting method, improved.Improvements mainly are that sample is carried out pre-treatment: sample adds 10%PVP powder (W/W) and grinds jointly in liquid nitrogen; Powder after grinding is divided into two parts and is transferred to rapidly in the 7ml centrifuge tube, and the cleaning buffer solution that does not contain CTAB that every pipe adds the precooling of 3ml ice cleans, and adds 2% beta-mercaptoethanol, abundant mixing, the centrifugal 10min of 7500rpm discards supernatant liquid, and this step repeats 1～2 time.1.5 * the CTAB that adds the 1.5ml preheating in the powder of handling with cleaning buffer solution extracts damping fluid, and 30 μ l beta-mercaptoethanols (final concentration is 2%) are incubated 20min behind the mixing that turns upside down again in 65 ℃ of water-baths.Upper strata extracting solution with two pipes after centrifugal is merged into a pipe, carries out extracting with isopyknic chloroform isoamyl alcohol again.Following step is with Curcurbitaceae gynostemma pentaphylla fresh sample DNA extraction.
2.RAPD amplification system:
The PCR reaction system contains 10 * Buffer2.5 μ l, 25mmol/L Mg
2+2 μ l, each 1 μ l of 10 μ M, 10 base random primers, ExTaqDNA polysaccharase 1U is about template DNA 60ng.The RAPD-PCR amplification program is: 94 ℃ of pre-sex change 3min, and then by 94 ℃ of sex change 50s, 37 ℃ of annealing 50s, 72 ℃ are extended 120s, carry out 40 circulations, and last 72 ℃ are extended 10min, and product detects with 1.5% agarose gel electrophoresis, the results are shown in accompanying drawing 4～6.
3. the clone and the order-checking of Curcurbitaceae gynostemma pentaphylla J-750SCAR marker DNA band:
The DNA that adopts the Curcurbitaceae gynostemma pentaphylla of 8 parts of fresh different sourcess of subordinate list 1 to extract respectively is a template, obtains the dna fragmentation of about 750bp with No. 10 primer amplifications.Reclaim test kit with glue and reclaim purifying, dna fragmentation behind the purifying is connected with the pMD18-TVector carrier, transforms the JM109 competent cell, obtain positive recombinant chou by the white screening of indigo plant, extract test kit in a small amount with plasmid DNA and extract plasmid, BamH I, Hind III double digestion are identified.Send the order-checking of order-checking company with positive colony, the results are shown in subordinate list 6.
4.DNA sequential analysis:
Use VectorNTI software analysis sequencing result.The gynostemma pentaphylla sequence identity of 8 different sourcess the results are shown in subordinate list 4; Restriction enzyme site in the sequence sees attached list 5.Blast program in the utilization NCBI gene database is carried out the homology comparison to Curcurbitaceae gynostemma pentaphylla feature band, the results are shown in accompanying drawing 7～14.
5. Curcurbitaceae gynostemma pentaphylla dna level is identified, distinguishes with Japanese Cayratia Herb:
(1) presses the composition of subordinate list 9, prepare PCR compound system 1 and PCR compound system 2 respectively.
The composition table of subordinate list 9:PCR compound system 1 and PCR compound system 2
(2) condition by subordinate list 10 adopts the touchdown PCR response procedures to carry out pcr amplification.
Subordinate list 10:PCR amplification condition table
The PCR product carries out 1.5% agarose gel electrophoresis, the results are shown in accompanying drawing 1, accompanying drawing 2 and accompanying drawing 3.
Annotate: 8 parts of different sources gynostemma pentaphylla molecule marker J-750 cloning and sequencing dna sequence dna see sequence table
Claims (5)
- One kind dna level identify gynostemma pentaphylla and with the method for Japanese Cayratia Herb difference, it is characterized in that: the result who utilizes the order-checking of medicinal plant Curcurbitaceae gynostemma pentaphylla dna characteristics fragment SCAR marker clone, the special primer of design Curcurbitaceae gynostemma pentaphylla SCAR mark is right, in conjunction with Curcurbitaceae gynostemma pentaphylla and Vitaceae Japanese Cayratia Herb plant 18S rRNA gene conserved regions design primer separately to as confidential reference items, be combined into PCR compound system 1 respectively and PCR compound system 2 increases, amplified production is carried out electrophoretic analysis, identify thereby medicinal plant Curcurbitaceae gynostemma pentaphylla is carried out dna level, and with form on easily and the Vitaceae Japanese Cayratia Herb obscured of gynostemma pentaphylla distinguish mutually.
- 2. according to claim 1 dna level identify gynostemma pentaphylla and with the method for Japanese Cayratia Herb difference, it is characterized in that: described PCR compound system 1 comprises the segmental primer of amplification Curcurbitaceae gynostemma pentaphylla SCAR mark 359bp to WJS-T 1And WJS-T 2, the segmental confidential reference items primer of amplification Curcurbitaceae gynostemma pentaphylla 18S rRNA 293bp is to WHL-18S-C 1And WHL-18S-C 2, use touchdown PCR program amplification Curcurbitaceae gynostemma pentaphylla DNA, obtain 359bp and the 293bp district band of two Curcurbitaceae gynostemma pentaphylla, the process of foundation is as follows:(1) adopts the analysis of biological information method, filter out 19 10mer RAPD random primers, set up the RAPD analysis system, find out the total SCAR molecule marker J-750 of Curcurbitaceae gynostemma pentaphylla;(2) the total SCAR molecule marker J-750 of acquired Curcurbitaceae gynostemma pentaphylla is carried out cloning and sequencing, obtain the Curcurbitaceae gynostemma pentaphylla Partial Feature dna sequence dna of 8 parts of different sourcess, length is in 766～770bp scope, through adopting the Blast software analysis of NCBI, the Curcurbitaceae gynostemma pentaphylla J-750 that obtains 8 parts of different sourcess is the DNA of plants sequence of not logining at NCBI as yet of one section new clone;(3) utilize Oligo 6 softwares to design special primer to WJS-T at gynostemma pentaphylla SCAR molecule marker J-750 1And WJS-T 2, set up PCR system amplification Curcurbitaceae gynostemma pentaphylla DNA, amplification obtains the special district band that length is 359bp;(4) according to Curcurbitaceae gynostemma pentaphylla 18S rRNA gene conserved regions design primer to WHL-18S-C 1And WHL-18S-C 2, set up PCR system amplification Curcurbitaceae gynostemma pentaphylla DNA, amplification obtains the total band of Curcurbitaceae gynostemma pentaphylla 18S rRNA gene conserved regions that a segment length is 293bp;(5) with Curcurbitaceae gynostemma pentaphylla SCAR molecule marker primer to WJS-T 1And WJS-T 2With 18S rRNA gene conserved regions primer to WHL-18S-C 1And WHL-18S-C 2Combination, set up PCR complex reaction system 1, adopt touchdown PCR program amplification Curcurbitaceae gynostemma pentaphylla dna profiling, the PCR product carries out the agarose gel electrophoresis analysis and obtains Liang Tiao district band, wherein 1 is the special band of Curcurbitaceae gynostemma pentaphylla SCAR of 359bp, and 1 is the conservative band of Curcurbitaceae gynostemma pentaphylla 18S rRNA gene regions of 293bp in addition.
- 3. according to claim 1 dna level identify gynostemma pentaphylla and with the method for Japanese Cayratia Herb difference, it is characterized in that: described PCR compound system 2 comprises the segmental primer of amplification Curcurbitaceae gynostemma pentaphylla SCAR mark 359bp to WJS-T 1And WJS-T 2, the segmental confidential reference items primer of 18S rRNA 177bp of amplification Vitaceae Japanese Cayratia Herb is to WPT-18S-C 1And WPT-18S-C 2, use touchdown PCR program amplification Vitaceae Japanese Cayratia Herb DNA, obtain the 18S rRNA 177bp district band of 1 Vitaceae Japanese Cayratia Herb, the process of foundation is as follows:(1) according to Vitaceae 18S rRNA gene conserved regions design primer to WPT-18S-C 1And WPT-18S-C 2, set up PCR system amplification Vitaceae Japanese Cayratia Herb DNA, obtaining a segment length is the total band of Vitaceae Japanese Cayratia Herb 18S rRNA gene conserved regions of 177bp;(2) with Vitaceae Japanese Cayratia Herb 18S rRNA gene conserved regions primer to WPT-18S-C 1And WPT-18S-C 2, with Curcurbitaceae gynostemma pentaphylla SCAR molecule marker special primer to WJS-T 1And WJS-T 2Be combined into PCR complex reaction system 2, adopt the touchdown PCR program that Vitaceae Japanese Cayratia Herb dna profiling is increased, the PCR product carries out the agarose gel electrophoresis analysis, obtains the Vitaceae Japanese Cayratia Herb 18S rRNA gene conserved regions band of 1 177bp, no Curcurbitaceae gynostemma pentaphylla SCAR fragment 359bp district band.
- Claim 1 described dna level identify gynostemma pentaphylla and with the method for Japanese Cayratia Herb difference, identify the Curcurbitaceae gynostemma pentaphylla and with phytomorph on application in the Vitaceae Japanese Cayratia Herb difference easily obscured mutually with the Curcurbitaceae gynostemma pentaphylla.
- 5. according to claim 1 dna level identify gynostemma pentaphylla and with the method for Japanese Cayratia Herb difference, it is characterized in that: the gynostemma pentaphylla molecule marker J-750DNA fragment sequence that 8 parts of different sources gynostemma pentaphylla come out through No. 10 primer amplifications:The J1-750 sequence(1) sequence signature:A. length: 769basesB. type: DNAC. chain: two strands(2) molecule type: nucleic acid(3) sequence description:ctcaccgtcc?atatagctat?cggtaccggg?acaatataaa?tcagaaggca?tttcgtaaac?60gggccaaaca?tctttgttct?gctttcaatt?taaccaaaat?tttccatgat?cagtataagt?120taaaaaaact?acaactacaa?gtaaagaaga?gaaaaaaaaa?ataggaaaaa?ccttattata?180caaactatac?tccagataag?atgaacaggc?cataaggcaa?atataatacc?aatatcaccc?240aaagcaatca?caagaaacac?aaagggactg?aatagagttg?ctgcacataa?tatttaaaaa?300tcaataaaca?ctataattaa?tcaccattaa?agtaaaatga?aaatgaagga?aaatagagaa?360agaaatgtac?ctttgataaa?tccaagaata?aagagaaacc?aaaaaacagg?aaggaagaag?420atgaagtaga?agagataaca?gcaacatcct?cttggaattt?tcatttttca?aattctatca?480actttctcag?ccacaaagct?tcatctaata?aactattatt?tttataacat?ttgtgagtgt?540tcgggtcagt?ttacacgcat?ttcaactcaa?cttcaataca?caatttaagt?aaaccccaaa?600tttgaatgaa?atcatacaaa?cttcaaggct?agaaatcaag?tttagaatct?gggtcttcaa?660aatcttgttc?caattcgacc?acaatctaat?taaaaatgta?aaatttaacg?attgaagtgg?720gagaaagttg?ggtttttctc?aggacccaga?tagagcagag?gacggtgag 769The J2-750 sequence(1) sequence signature:A. length: 766basesB. type: DNAC. chain: two strands(2) molecule type: nucleic acid(3) sequence description:ctcaccgtcc?atatagccat?cggtaccggg?acaattaaaa?tcagaaggca?tttcgtaaac?60cgacgaaaca?tctttgttct?gctttcaatt?taaccaaaat?attccatgat?cagtattagt?120taaaaaacta?caactacaag?taaagaagag?aaaaaaaaaa?ggaaaaacct?tattatacaa?180actatactcc?agataagatg?aacaggccat?aaggcaaata?taataccaat?atcacccaaa?240gcaatcacaa?gaaacacaaa?gggactgaat?agagttgctg?cacataatat?ttaaaaatca?300ataaacacta?taattaatca?ccattaaagt?aaaatgaaaa?tgaaggaaaa?tagagaaaga?360aatgtacctt?tgataaatcc?aagaataaag?agaaaccaaa?aaacaggaag?gaagaagatg?420aagtagaaga?gataacagca?acatcctctt?ggaattttca?tttttcaaat?tctatcaact?480ttctcagcca?caaagcttca?tctaataaac?tattattttt?ataatatttg?tgagtgttcg?540ggtcaattta?cacgcacttc?aactcaactt?caatacacaa?tttaagtaaa?ccccaaattt?600gaatgaaatc?atacaaattt?caaggctgga?aatcaagttt?agaatctggg?tcttcaaaat?660cttgttctaa?ttcgaccaca?atctaattaa?aaatgtaaaa?tataacgatt?gaagtgggag?720aaagttgggt?ttttctcagg?acccagatag?agcagaggac?ggtgag 766The J3-750 sequence:(1) sequence signature:A. length: 766basesB. type: DNAC. chain: two strands(2) molecule type: nucleic acid(3) sequence description:ctcaccgtcc?atatagctat?cggtaccggg?acaatataaa?tcagaaggca?tttcgtaaac?60gggccaaaca?tctttgttct?gctttcaatt?taaccaaaat?tttccatgat?cagtataagt?120caaaaaaact?acaactacaa?gtaaagaaga?gaaaaaaaaa?taggaaaaac?cttattatac?180aaactatact?ccagataaga?tgaacaggcc?ataaggcaaa?tataatacca?atatcaccca?240aagcaatcac?aagaaacaca?aagggactga?atagagttgc?tgcacataat?atttaaaaat?300caataaacac?tataattaat?caccattaaa?gtaaaatgaa?aatgaaggaa?aatagagaaa?360gaaatgtacc?tttgataaat?ccaagaataa?agagaaacca?aaaaacagga?aggaagaaga?420tgaagtagaa?gagataacag?caacatcctc?ttggaatttt?catttttcaa?attctatcaa?480ctttctcagc?cacaaagctt?catctaataa?actattattt?ttataatatt?tgtgagtgtt?540cgggtcagtt?tacacgcact?tcaactcaac?ttcaatacac?aatttaagta?aaccccaaat?600ttgaatgaaa?tcatacaaac?ttcaaggcta?gaaatcaagt?ttagaatctg?ggtcttcaaa?660atcttgttct?aattcgacca?caatctaatt?aaaaatgtaa?aatttaacga?ttgaagtggg?720agaaagttgg?gtttttctca?ggacccagat?agagcagagg?acggtgag 768The J4-750 sequence:(1) sequence signature:A. length: 767basesB. type: DNAC. chain: two strands(2) molecule type: nucleic acid(3) sequence description:ctcaccgtcc?atatagctat?cggtaccggg?acaattaaaa?tcagaaggca?tttcgtaaac?60gggccaaaca?tctttgttct?gctttcaatt?taaccaaaat?tttccatgat?cagtataagt?120taaaaaaact?acaactacat?gtaaagaaga?gaaaaaaaaa?aggaaaaacc?ttattataca?180aactatactc?cagataagat?gaacaggcca?taaggcaaat?ataataccaa?tatcacccaa?240agcaatcaca?agaaacacaa?agggactgaa?tagagttgct?gcacataata?tttaaaaatc?300aataaacact?ataattaatc?accattaaag?taaaatgaaa?atgaaggaaa?atagagaaag?360aaatgtacct?ttgataaatc?caagaataaa?gagaaaccaa?aaaacaggaa?ggaagaagat?420gaagtagaag?agataacagc?aacatcctct?tggaattttc?atttttcaaa?ttctatcaac?480tttctcagcc?acaaagcttc?atctaataaa?ctattatttt?tataatattt?gtgagtgttc?540gggtcagttt?acacgcactt?caactcaact?tcaatacaca?atttaagtaa?accccagatt?600tgaatgaaat?catacaaact?tcaaggctag?aaatcaagtt?tagaatctgg?gtcttcaaaa?660tcttgttcta?attcgaccac?aatctaatta?aaaatgtaaa?atctaaggat?tgaagtggga?720gaaagttggg?tttttctcag?gacccagata?gagcagagga?cggtgag 767The J5-750 sequence:(1) sequence signature:A. length: 770basesB. type: DNAC. chain: two strands(2) molecule type: nucleic acid(3) sequence description:ctcaccgtcc?atatagctat?cggtaccggg?acaattaaaa?tcagaaggca?tttcgtaaac?60gggccaaaca?tctttgttct?gctttcaatt?taaccaaaat?tttccatgat?cagtataagt?120taaaaaaact?acaactacat?gtaaagaaga?gaaaaaaaaa?aaaaggaaaa?accttattat?180acaaactata?ctccagataa?gatgaacagg?ccataaggca?aatataatac?caatatcacc?240caaagcaatc?acaagaaaca?caaagggact?gaatagagtt?gctgcacata?atatttaaaa?300atcaataagc?actataatta?atcaccatta?aagtaaaatg?aaaatgaagg?aaaatagaga?360aagaaatgta?cctttgataa?atccaagaat?aaagagaaac?caaaaaacag?gaaggaagaa?420gatgaagtag?aagagataac?agcaacatcc?tcttggaatt?ttcatttttc?aaattctatc?480aactttctca?gccacaaagc?ttcatctaat?aaactattat?ttttataata?tttgtgagtg?540ttcgggtcag?tttacacgca?cttcaactca?acttcaatac?acaatttaag?taaaccccag?600atttgaatga?aatcatacaa?acttcaaggc?tagaaatcaa?gtttagaatc?tgggtcttca?660aaatctcgtt?ctaatacgac?cacaatctaa?ttaaaaatgt?aaaatttaac?gattgaagtg?720ggagaaagtt?gggtttttct?caggacccag?ttagagcaga?ggacggtgag 770The J6-750 sequence:(1) sequence signature:A. length: 769basesB. type: DNAC. chain: two strands(2) molecule type: nucleic acid(3) sequence description:ctcaccgtcc?atatagctat?cggtaccggg?acaatataaa?tcagaaggca?tttcgtaaac?60gggccaaaca?tctttgttct?gctttcaatt?taaccaaaat?tttccatgat?cagtataagt?120taaaaaaact?acaactacaa?gtaaagaaga?gaaaaaaaaa?ataggaaaaa?ccttattata?180caaactatac?tccagataag?atgaacaggc?cataaggcaa?atataatacc?aatatcaccc?240aaagcaatca?caagaaacac?aaagggactg?aatagagttg?ctgcacataa?tatttaaaaa?300tcaataaaca?ctataattaa?tcaccattaa?agtaaaatga?aaatgaagga?aaatagagaa?360agaaatgtac?ctttgataaa?tccaagaata?aagagaaacc?aaaaaacagg?aaggaagaag?420atgaagtaga?agagataaca?gcaacatcct?cttggaattt?tcatttttca?aattctatca?480actttctcag?ccacaaagct?tcatctaata?aactattatt?tttataacat?ttgtgagtgt?540tcgggtcagt?ttacacgcat?ttcaactcaa?cttcaataca?caatttaagt?aaaccccaaa?600tttgaatgaa?atcatacaaa?cttcaaggct?agaaatcaag?tttagaatct?gggtcttcaa?660aatcttgttc?taattcgacc?acaatctaat?taaaaatgta?aaatttaacg?attgaagtgg?720gagaaagttg?ggtttttctc?aggacccaga?tagagcagag?gacggtgag 769The J7-750 sequence:(1) sequence signature:A. length: 766basesB. type: DNAC. chain: two strands(2) molecule type: nucleic acid(3) sequence description:ctcaccgtcc?atatagctat?cggtaccggg?acaattaaaa?tcagaaggca?tttcgtaaac?60gggccaaaca?tctttgttct?gctttcaatt?taaccaaaat?tttccatgat?caatataagt?120taaaaaaact?acaactacat?gtaaagaaga?gaaaaaaaaa?ggaaaaacct?tattatacaa?180actatactcc?agataagatg?aacaggccat?aaggcaaata?taataccaat?atcaaccaaa?240gcaatcacaa?gaaacacaaa?gggactgaat?agagttgctg?cacataatat?ttaaaaatca?300ataaacacta?taattaatca?ccattaaagt?aaaatgaaaa?tgaaggaaaa?tagagaaaga?360aatgtacctt?tgataaatcc?aagaataaag?agaaaccaaa?aaacaggaag?gaagaagatg?420aagtagaaga?gataacagca?acatcctctt?ggaattttca?tttttcaaat?tctatcaact?480ttctcagcca?caaagcttca?tctaataaac?tattattttt?ataatatttg?tgagtgttcg?540ggtcagttta?cacggacttc?aactcaactt?caatacacaa?tttaagtaaa?ccccagattt?600gaatgaaatc?atacaaactt?caaggctaga?aatcaagttt?agaatctggg?tcttcaaaat?660cttgttctaa?ttcgaccaca?atctaattaa?aaatgtaaaa?tttaacgatt?gaagtgggag?720aaagttgggt?ttttctcagg?acccagatag?agcagaggac?ggtgag 766The J8-750 sequence:(1) sequence signature:A. length: 768basesB. type: DNAC. chain: two strands(2) molecule type: nucleic acid(3) sequence description:ctcaccgtcc?atatagctat?cggtaccggg?acaattaaaa?tcagaaggca?tttcgtaaac?60gggccaaaca?tctttgttct?gctttcaatt?taaccaaaat?tttccatgat?cagtataagt?120taaaaaaaac?tacaactaca?tgtaaagaag?agaaaaaaaa?aaggaaaaac?cttattatac?180aaactatact?ccagataaga?tgaacaggcc?ataaggcaaa?tataatacca?atatcaccca?240aagcaatcac?aagaaacaca?aagggactga?atagagttgc?tgcacataat?atttaaaaat?300caataaacac?tataattaat?caccattaaa?gtaaaatgaa?aatgaaggga?aatagagaaa?360gaaatgtacc?tttgataaat?ccaagaataa?agagaaacca?aaaaacagga?aggaagaaga?420tgaagtagaa?gagataacag?caacatcctc?ttggaatttt?catttttcaa?attctatcaa?480ctttctcagc?cacaaagctt?catctaataa?actattattt?ttataatatt?tgtgagtgtt?540tgggtcagtt?tacacgtact?tcaactcaac?ttcaatacac?aatttaagta?aaccccagat?600ttgtatgaaa?tcatacaaac?ttcaaggcta?gaaatcaagt?ttagaatctg?ggtcttcaaa?660atcttgttct?aattcgacca?caatctaatt?aaaaatgtaa?aatttaacga?ttgaagtggg?720agaaagttgg?gtttttctca?ggacccagat?agagcagagg?acggtgag。768
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| CN103131781A (en) * | 2013-02-28 | 2013-06-05 | 中国科学院成都生物研究所 | PCR detection method of DNA of biological counterfeit drug in Chinese patent drug |
| CN114990246A (en) * | 2021-11-19 | 2022-09-02 | 中国药科大学 | Rapid identification method of cayratia japonica and counterfeit product thereof |
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| 周娟: "广西产药用植物绞股蓝遗传多样性分析及分子水平鉴定方法的建立", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 周娟等: "8份广西产绞股蓝种质的RAPD引物筛选及其遗传多样性分析", 《基因组学与应用生物学》 * |
| 陈慧浓: "绞股蓝与乌蔹莓的鉴别研究", 《浙江中药杂志》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103131781A (en) * | 2013-02-28 | 2013-06-05 | 中国科学院成都生物研究所 | PCR detection method of DNA of biological counterfeit drug in Chinese patent drug |
| CN103131781B (en) * | 2013-02-28 | 2015-10-28 | 中国科学院成都生物研究所 | The PCR detection method of biological species adulterant DNA in Chinese patent medicine |
| CN114990246A (en) * | 2021-11-19 | 2022-09-02 | 中国药科大学 | Rapid identification method of cayratia japonica and counterfeit product thereof |
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