CN101182572A - Molecule identification method of different producing area changium root plants - Google Patents
Molecule identification method of different producing area changium root plants Download PDFInfo
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- CN101182572A CN101182572A CNA2007101352900A CN200710135290A CN101182572A CN 101182572 A CN101182572 A CN 101182572A CN A2007101352900 A CNA2007101352900 A CN A2007101352900A CN 200710135290 A CN200710135290 A CN 200710135290A CN 101182572 A CN101182572 A CN 101182572A
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- 241000530402 Changium Species 0.000 title claims abstract description 96
- 238000000034 method Methods 0.000 title claims abstract description 18
- 241000196324 Embryophyta Species 0.000 title claims description 8
- 238000004519 manufacturing process Methods 0.000 claims abstract description 38
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 12
- 238000007400 DNA extraction Methods 0.000 claims abstract description 7
- 108020004414 DNA Proteins 0.000 claims description 50
- 101100213970 Schizosaccharomyces pombe (strain 972 / ATCC 24843) ypt3 gene Proteins 0.000 claims description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 238000013461 design Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 10
- 238000012163 sequencing technique Methods 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 238000011144 upstream manufacturing Methods 0.000 claims description 7
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 6
- 238000012850 discrimination method Methods 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 238000011160 research Methods 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 5
- 108091023242 Internal transcribed spacer Proteins 0.000 claims description 3
- 238000012937 correction Methods 0.000 claims description 3
- 238000007405 data analysis Methods 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract 1
- 241000530419 Changium smyrnioides Species 0.000 description 8
- 239000003814 drug Substances 0.000 description 6
- 229940079593 drug Drugs 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000208173 Apiaceae Species 0.000 description 1
- 241000282461 Canis lupus Species 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- CEJLBZWIKQJOAT-UHFFFAOYSA-N dichloroisocyanuric acid Chemical compound ClN1C(=O)NC(=O)N(Cl)C1=O CEJLBZWIKQJOAT-UHFFFAOYSA-N 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 235000015961 tonic Nutrition 0.000 description 1
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Abstract
The invention relates to a molecular identification method of medicinal changium root from different production areas. The method, on the basis of the processes of general DNA extraction, PCR magnification, rDNA sequence detection and analysis, finds the basic group points for identifying the medicinal changium root from different production areas. When the r DNAITS sequence of the detected medicinal changium root accords with the DNA sequence of medicinal changium root from different production areas or has the special point of the medicinal changium root, the medicinal changium root is from the production area. Based on the r DNAITS sequences of the medicinal changium root from different production areas, a high-special identification primer is designed for identifying the production area of the medicinal changium root (Jurong, Jiangsu) with which the high-special PCR identification of the production areas of the medicinal changium root can be implemented successfully.
Description
Technical field:
The present invention relates to the molecular identification method of different places of production Root of Medicinal Changium plant, belong to the pharmacognosy field.
Background technology:
The Root of Medicinal Changium Changium smyrnioides Wolf beginning sees supplementary Amplifications of the Compendium of Materia Medica, be that the umbelliferae Root of Medicinal Changium belongs to single kind of plant, be distributed in the narrow and small scope such as Jiangsu, Zhejiang, Anhui, Hubei, Jiangxi of China East China, dry root is because of having in strengthening by means of tonics, moistening the lung and resolving the phlegm, the detoxifcation of flat liver, the peace and effect such as stomach is recorded " in the Chinese pharmacopoeia, being the distinctive rare traditional Chinese medicine of China, also is one of Jiangsu famous-region drug simultaneously.
Famous-region drug are the former species of this medicinal material in the generation evolution of the kind system in its place of production and fauna, the special outcome that is being subjected to breed the history environment condition of these species and mankind's activity effect for a long time and is forming, be to have specific output zone (geography and ecology), long production and marketing history (commodity distribution centre), its product has good social prestige and can produce the medicinal material of considerable economic benefit, its product goods is true, of fine quality, and stay in grade is reliable, is the common people person of speaking approvingly of.Between the place of production of Chinese medicine and quality thereof, curative effect substantial connection is arranged,, always whether gives very big attention from the country of origin to medicinal material traditionally, often with real estate, authentic medicinal herbs as sign of fine quality.Because the profile of medicinal plant of the same race is basic no difference in its range of distribution, is difficult to their source is differentiated from form.The Root of Medicinal Changium in genuine producing region and non-genuine producing region does not have difference on form, only gap is being arranged qualitatively, and utilization morphology means can not be to its discriminating of originating.
Summary of the invention:
The purpose of this invention is to provide a kind of molecular identification method of differentiating different places of production Root of Medicinal Changium plant, its special card is with specific dna sequence dna the Root of Medicinal Changium discriminating of originating, make the source discriminating of Root of Medicinal Changium more reliable, accurate, the discriminating for famous-region drug simultaneously provides the high specific discrimination method.
The present invention at first checks order to the rDNAITS district of different places of production Root of Medicinal Changium, obtain part ITS1 sequence and complete 5.8S and the sequence of ITS2, find the distinctive site of different places of production Root of Medicinal Changium again by sequence alignment, and on this basis the Root of Medicinal Changium of Jurong, Jiangsu, genuine producing region has been carried out high specific PCR and identified.
Utilize rDNA ITS district dna sequence dna that Root of Medicinal Changium is originated among the present invention and differentiate that the step that is adopted is as follows:
(1) total DNA extraction
Get the fresh blade of Root of Medicinal Changium plant to be checked, after handling with sterilized water, grind to form fine powder in liquid nitrogen, extract total DNA with the CTAB method, the DNA purifying of producing with the rich Bioexperiment Materials Research Laboratories of Beijing Lay reclaims the test kit purifying then, is dissolved in the sterilization distilled water.
(2) pcr amplification
The primer of Root of Medicinal Changium ITS district amplification is ITS district universal primer ITS4 and ITS5, and primer sequence is:
ITS4:5`-TCCTCCGCTTATTGATATGC-3` is positioned on the 18S;
ITS5:5`-GGAAGTAAAAGTCGTAACAAGG-3` is positioned on the 26S.
Utilize this that primer is carried out amplified reaction to total DNA, and with ddH
2O replaces masterplate DNA to make blank.
(3) determined dna sequence
Adopt PCR product direct sequencing, unidirectional order-checking, sequencing primer is PCR primer I TS5, sequencing reaction carries out on ABI 3730 full-automatic sequenators.
(4) discriminating of dna sequence data analysis and plant origin
The sequence in gained rDNA ITS district is arranged software (CLUSTAL software) with contraposition carry out the contraposition arrangement, and be aided with artificial check and correction, analyze the difference of each sequence in sample DNA sequence to be checked and the database with genetic analysis software (MEGA software), by analyzing relatively the source of final decision inspection Root of Medicinal Changium plant.
The present invention also utilizes high specific PCR diagnostic primers that the Root of Medicinal Changium of Jurong, Jiangsu, genuine producing region has been carried out the source discriminating.At first with reference to the rDNAITS region sequence of different places of production Root of Medicinal Changium, design the high specific diagnostic primers of Jurong, a pair of Jiangsu Root of Medicinal Changium, the design reference of upstream diagnostic primers JR:5`-TGAGTGTCATGTATCGTGTC-3` the 5.8S and the ITS2 part in Root of Medicinal Changium rDNAITS district, Jurong, Jiangsu, the base in the 8th site of Jurong, Jiangsu 3 Root of Medicinal Changium samples ITS2 sequence all is C, and the base of 18 Root of Medicinal Changium samples in other places of production in this site all is T, so this site is decided to be first base of upstream diagnostic primers 3` end, in view of the GC content of one section sequence before this specificity site higher, so when swimming diagnostic primers in design the base in 3 sites is changed, so rDNA ITS region sequence of upstream diagnostic primers and Jurong, Jiangsu Root of Medicinal Changium and not exclusively complementary.The downstream diagnostic primers is the universal primer ITS4 of ITS district amplification.
Before carrying out high specific PCR discriminating, at first the Root of Medicinal Changium of different sources is increased with primer I TS4 and ITS5, detect all samples whether and can both amplify the fragment about about 700bp and the brightness of amplified fragments, with the quality of checking masterplate DNA, the concentration of adjustment masterplate DNA, with diagnostic primers JR and ITS4 masterplate DNA is increased then, when annealing temperature is controlled at 68 ℃, have only the Root of Medicinal Changium of Jurong, Jiangsu to be amplified out, and the Root of Medicinal Changium in other places of production is all negative, can detect the result of amplified reaction with agarose gel electrophoresis.
Can design the diagnostic primers of other place of production Root of Medicinal Changium according to similar approach.
Beneficial effect of the present invention:
The present invention utilizes specific repetition multiple copied rDNA ITS region sequence, analyzes comparison by analysis softwares such as CLUSTAL, MEGA, has realized that finally the source of Root of Medicinal Changium is differentiated.The sequential analysis comparative result shows, have than big-difference between the rDNAITS region sequence of different places of production Root of Medicinal Changium, rDNA ITS region sequence difference between the Root of Medicinal Changium individuality of the same place of production (population) is between 0-3 base, variation is little, and the sequence subordinate list is only listed the rDNA ITS region sequence of a sample of each population Root of Medicinal Changium.Can differentiate its source of coming in well by analyzing the variant sites that relatively obtains, therefore, utilize present method that Root of Medicinal Changium is originated and differentiate accuracy with height.In recent years, the dna sequencing cost constantly reduces, and utilizing the method that checks order to differentiate becomes a kind of trend.
Because the order-checking expense is higher relatively and need certain plant and instrument, is very restricted in the actually operating of differentiating that medicinal material in enormous quantities is originated.The present invention designs the diagnostic primers of producing region, discriminator track ground (Jurong, Jiangsu) Root of Medicinal Changium also according to the rDNA ITS region sequence of different places of production Root of Medicinal Changium, differentiates thereby carry out high specific PCR, for the discriminating of famous-region drug provides quick, practical method.
Embodiment
Embodiment:
Utilize high specific PCR diagnostic primers to identify Jurong, Jiangsu Root of Medicinal Changium plant
Present embodiment is got 7 places of production fresh blade of totally 32 Root of Medicinal Changium individualities or the blade of silica dehydrator, number consecutively is JR1-JR5 (Jurong, Jiangsu), NJ1-NJ5 (Nanjing), AQ1-AQ5 (Anqing), QY1-QY5 (Qingyang, Anhui), HZ1-HZ5 (Zhejiang Hangzhou), NB1-NB5 (Ningbo of Zhejiang), PZ1-PZ2 (Pengze, Jiangxi), all differentiate by following steps:
(1) plant total DNA extraction to be checked
Get the fresh blade of Root of Medicinal Changium plant sample to be checked, after the sterilized water processing, in liquid nitrogen, grind to form fine powder, extract total DNA with the CTAB method, the DNA purifying of producing with the rich Bioexperiment Materials Research Laboratories of Beijing Lay reclaims the test kit purifying then, is dissolved in the sterilization distilled water.
(2) quality examination of masterplate DNA
With ITS district universal primer ITS4 and ITS5 Root of Medicinal Changium sample total DNA to be checked is carried out amplified reaction, detect all samples whether and can both amplify the fragment about about 700bp and the brightness of amplified fragments, with the quality of checking masterplate DNA, adjust the concentration of masterplate DNA.
Pcr amplification reaction carries out on PE-9600 PCR instrument, the component and the final concentration of 20 μ L reaction systems are: 1 * TaqDNA enzyme buffer liquid (10mmol/LTris-HCl, 50mmol/LKCl, 0.1%Trion x-100, pH8.4), 2.5mmol/LMgCl2,1U Taq enzyme, about 50ng template, 0.3 μ mol/L primer, 0.15mmol/L dNTP.Amplification program is: 94 ℃, and pre-sex change 3.0min; 94 ℃ of sex change 45s, 68 ℃ of annealing 30s, 72 ℃ are extended 1min, and 35 circulations replace masterplate DNA to make blank with ddH2O.
(3) utilization diagnostic primers JR and ITS4 increase to masterplate DNA
Operation in the quality examination of pcr amplification reaction and masterplate DNA is the same, only amplimer ITS5 wherein is replaced by JR.When annealing temperature is controlled at 68 ℃, have only 5 Root of Medicinal Changium samples of Jurong, Jiangsu to be amplified out, and the Root of Medicinal Changium sample in other places of production is all negative, can detect the result of amplified reaction with agarose gel electrophoresis.
In sum, the accurate Root of Medicinal Changium of Jurong, Jiangsu, producing region, discriminator track ground of the high specific PCR diagnostic primers of the present invention design and PCR method.Set up the rDNAITS district of different places of production Root of Medicinal Changium plant, comprise part ITS1 sequence and complete 5.8S and the sequence library of ITS2 and the specificity discriminating site of each place of production Root of Medicinal Changium plant, it is characterized in that carrying out as follows:
(a) total DNA extraction
Get the fresh blade of Root of Medicinal Changium plant to be checked, after handling with sterilized water, grind to form fine powder in liquid nitrogen, extract total DNA with the CTAB method, the DNA purifying of producing with the rich Bioexperiment Materials Research Laboratories of Beijing Lay reclaims the test kit purifying then, is dissolved in the sterilization distilled water:
(b) pcr amplification
The primer of Root of Medicinal Changium ITS district amplification is ITS district universal primer ITS4 and ITS5, and primer sequence is:
ITS4:5`-TCCTCCGCTTATTGATATGC-3` is positioned on the 18S;
ITS5:5`-GGAAGTAAAAGTCGTAACAAGG-3` is positioned on the 26S.
Utilize this that primer is carried out amplified reaction to total DNA, and with ddH
2O replaces masterplate DNA to make blank.
(c) determined dna sequence
PCR product direct sequencing is adopted in order-checking, unidirectional order-checking, and sequencing primer is pcr amplification primer ITS5, sequencing reaction carries out on ABI 3730 full-automatic sequenators.
(d) discriminating of dna sequence data analysis and plant origin
The sequence one in the rDNAITS district of Root of Medicinal Changium plant to be checked after measured, arrange software (CLUSTAL software) with contraposition and carry out the contraposition arrangement, and be aided with artificial check and correction, analyze the difference of each sequence in sample DNA sequence to be checked and the database with genetic analysis software (MEGA software), by analyzing relatively the source of final decision Root of Medicinal Changium plant to be checked.
2. the high specific PCR discrimination method of genuine producing region (Jurong, Jiangsu) Root of Medicinal Changium plant is characterized in that the rDNAITS sequence by more different places of production Root of Medicinal Changium plant, designs the high specific primer of differentiating Jurong, Jiangsu Root of Medicinal Changium plant.The diagnostic primers of Jurong, Jiangsu Root of Medicinal Changium plant is: the upstream diagnostic primers is JR:5`-TGAGTGTCATGTATCGTGTC-3`, and the downstream diagnostic primers is the universal primer ITS4 of ITS district amplification.Concrete steps are:
(1) total DNA extraction
Get the fresh blade of Root of Medicinal Changium plant to be checked, after handling with sterilized water, grind to form fine powder in liquid nitrogen, extract total DNA with the CTAB method, the DNA purifying of producing with the rich Bioexperiment Materials Research Laboratories of Beijing Lay reclaims the test kit purifying then, is dissolved in the sterilization distilled water.
(2) quality examination of masterplate DNA
With ITS district universal primer ITS4 and ITS5 the total DNA of product to be checked is increased, with the concentration of validating DNA masterplate quality, adjustment masterplate DNA.ITS4 and ITS5 sequence are:
ITS4:5`-TCCTCCGCTTATTGATATGC-3` is positioned on the 18S;
ITS5:5`-GGAAGTAAAAGTCGTAACAAGG-3` is positioned on the 26S.
(3) utilization diagnostic primers JR, ITS4 increase to masterplate DNA, and annealing temperature is risen to 68 ℃, and what can be amplified out promptly derives from the Jurong, Jiangsu, and the Root of Medicinal Changium in other sources is all negative.
(4) utilize high specific PCR diagnostic primers that the Root of Medicinal Changium of Jurong, Jiangsu, genuine producing region has been carried out the source discriminating, rDNA ITS region sequence with reference to different places of production Root of Medicinal Changium, design the high specific diagnostic primers of Jurong, a pair of Jiangsu Root of Medicinal Changium, the design reference of upstream diagnostic primers JR:5`-TGAGTGTCATGTATCGTGTC-3` the 5.8S and the ITS2 part in Root of Medicinal Changium rDNA ITS district, Jurong, Jiangsu, the base in the 8th site of Jurong, Jiangsu 3 Root of Medicinal Changium samples ITS2 sequence is C all, and the base of 18 Root of Medicinal Changium samples in other places of production in this site all is T.
Sequence table
＜110〉Institute of Botany
＜120〉molecular identification method of different place of production Root of Medicinal Changium plant
<160>9
<210>1
<211>555
<212>DNA
＜213〉derive from Root of Medicinal Changium (Changium smyrnioides)-Jurong, Jiangsu population
<400>1
ggcaagcgtc?ggtgggcttg?attcccccgt?tgcgaacccc?aggaaggtgg?gcgctcccgg 60
gcgctcgtcg?gccgacgaaa?tcaaccgggc?gcggaatgcg?ccaaggaaat?cagaatagaa 120
ctgtacgttt?gcttcccgtt?cgtgggaggc?ggcgtctttc?ctaaacacaa?acgactctcg 180
gcaacggata?tcccggctct?tgcatcgatg?aagaacgtag?cgaaatgcga?tacttggtgt 240
gaattgcaga?atcccgtgaa?ccatcgagtc?tttgaacgca?agttgcgccc?gaagccacta 300
ggctgagggc?acgcctgcct?gggtgtcacg?catcgtgtcg?ccccctgacc?actcactcct 360
tggggagctg?cattggttac?agggcggata?ttggcctccc?gtgccttgcg?gtgcggttgg 420
cctaaaagcg?agtctctggt?gacagatgtt?gcgatatttg?tggttgaaat?aaataccctc 480
ttctcttgtc?gtgcgaatgc?ctgtcacctc?ggtgagctca?aggaccctcg?agtgccgcaa 540
acggcgcgcg?cttta 555
<210>2
<211>555
<212>DNA
＜213〉derive from Root of Medicinal Changium (Changium smyrnioides)-Nanjing population
<400>2
ggcaagcgtc?ggtgggcttg?attcccccgt?tgcgaacccc?aggaaggtgg?gcgctcccgg 60
gcgcccatcg?gccgacgaaa?tcaaccgggc?gcggaatgcg?ccaaggaaat?cagaatagaa 120
ctgtacgttt?gcttcccgtt?cgtgggaggc?ggcgtctttc?ctaaacacaa?acgactctcg 180
gcaacggata?tcccggctct?tgcatcgatg?aagaacgtag?cgaaatgcga?tacttggtgt 240
gaattgcaga?atcccgtgaa?ccatcgagtc?tttgaacgca?agttgcgccc?gaagccacta 300
ggctgagggc?acgcctgcct?gggtgtcacg?catcgtgttg?ccccctgacc?actcactcct 360
tgaggagctg?cattggttac?ggggcggata?ttggcctccc?gtgccttgcg?gtgcggttgg 420
cctaaaagcg?agtctctggt?gacagatgtt?gcgatatttg?tggttgaaat?aaataccctc 480
ttctcttgtc?gtgcgaatgc?ctgtcacctc?ggtgagctca?aggaccctcg?agtgccgcaa 540
acggcgcgcg?cttta 555
<210>3
<211>555
<212>DNA
＜213〉derive from Root of Medicinal Changium (Changium smyrnioides)-Anqing population
<400>3
ggcaagcgtc?ggtgggcttg?agtcccccgt?tgcgaacccc?aggaaggtgg?gcgctcccgg 60
gcgcccatcg?gccgacgaaa?tcaaccgggc?gcggaatgcg?ccaaggaaat?cagaatagaa?120
ctgtacgttt?gcttcccgtt?cgtgggaggc?ggcgtctttc?ctaaacacaa?acgactctcg 180
gcaacggata?tcccggctct?tgcatcgatg?aagaacgtag?cgaaatgcga?tacttggtgt 240
gaattgcaga?atcccgtgaa?ccatcgagtc?tttgaacgca?agttgcgccc?gaagccacta 300
ggctgagggc?acgcctgcct?gggtgtcacg?catcgtgttg?ccccctgacc?actcactcct 360
tgaggagctg?cattggttac?agggcagata?ttggcctccc?gtgccttgcc?gtgcggttgg 420
cctaaaagcg?agtctctggt?gacagatgtt?gcgatatttg?tggttgaaat?aaataccctc 480
ttctcttatc?gtgcgaatgc?ctgtcacctt?ggtgagctca?aggaccctcg?agtgccgcaa 540
acggcgcgcg?cttta 555
<210>4
<211>555
<212>DNA
＜213〉derive from Root of Medicinal Changium (Changium smyrnioides)-Qingyang, Anhui population
<400>4
ggcaagcgtc?ggtgggcttg?gttcccccgt?tgcgaacccc?aggaaggtgg?gcgctcccgg 60
gcgctcatcg?gccgacgaaa?tcaaccgggc?gcggaatgcg?ccaaggaaat?caaaatagaa?120
ttgtacgttt?gcttcccgtt?cgtgggaggc?ggcgtctttc?ctaaacacaa?acgactctcg?180
gcaacggata?tcccggctct?tgcatcgatg?aagaacgtag?cgaaatgcga?tacttggtgt?240
gaattgcaga?atcccgtgaa?ccatcgagtc?tttgaacgca?agttgcgccc?gaagccgcta?300
ggctgagggc?acgcctgcct?gggtgtcacg?catcgtgctg?ccccctgacc?actcgctcct?360
tgaggagctg?cattggttac?agggcggata?ttggcctccc?gtgccttgcg?gtgcggttgg?420
cctaaaagtg?agtctctggt?gacagatgtt?gcgatatttg?tggttgaaat?aaataccctc?480
ttctcttgtc?gtgcgaatgc?ctgtcacctt?ggtgagctca?aggaccctca?agcgccgcaa?540
acggcgcgcg?cttta 555
<210>5
<211>555
<212>DNA
＜213〉derive from Root of Medicinal Changium (Changium smyrnioides)-Zhejiang Hangzhou population
<400>5
ggcgagcgtc?ggtgggcttg?gttcccccgt?tgcgaacccc?aggaaggtgg?gcgctcccgg 60
gcgctcatcg?gccgacgaaa?tcaaccgggc?gcggaatgcg?ccaaggaaat?caaaatagaa?120
ttgtacgttt?gcttcccgtt?cgtgggaggc?ggcgtctttc?ctaaacacaa?acgactctcg?180
gcaacggata?tcccggctct?tgcatcgatg?aagaacgtag?cgaaatgcga?tacttggtgt?240
gaattgcaga?atcccgtgaa?ccatcgagtc?tttgaacgca?agttgcgccc?gaagccgcta?300
ggctgagggc?acgcctgcct?gggtgtcacg?catcgtgctg?ccccctgacc?actcgctcct?360
tgaggagctg?cattggttac?agggcggata?ttggcctccc?gtgccttgcg?gtgcggttgg?420
cctaaaagtg?agtctctggt?gacagatgtt?gcgatatttg?tggttgaaat?aaataccctc?480
ttctcttgtc?gtgcgaatgc?ctgtcacctt?ggtgagctca?aggaccctca?agcgccgcaa?540
acggcgcgcg cttta 555
<210>6
<211>555
<212>DNA
＜213〉derive from Root of Medicinal Changium (Changium smyrnioides)-Ningbo of Zhejiang population
<400>6
ggcaagcgtc?ggtgggcttg?attcccccgt?tgcgaacccc?aggcaggtgg?gcgctcccgg 60
gcgctcatcg?gccgaccaaa?tcaaccgggc?gcggaatgcg?ccaaggaaaa?cagaatagaa?120
ctgtacgttt?gcttcccgtt?cgtgggaggc?ggcgtctttc?ctaaacacaa?acgactctcg?180
gcaacggata?tcccggctct?tgcatcgatg?aagaacgtag?cgaaatgcga?tacttggtgt?240
gaattgcaga?atcccgtgaa?ccatcgagtc?tttgaacgca?agttgcgccc?gaagccgcta?300
ggctgagggc?acgcctgcct?gggtgtcacg?catcgtgttg?ccccctgacc?actcgctcct?360
tgaggagctg?cattggttac?agggcggata?ttggcctccc?gtgccttgcg?gtgcggttgg?420
cctaaaagcg?agtctctggt?gacagatgtt?gcgatatttg?tggttgaaat?aaataccctc?480
ttctcttgtc?gtgcgaatgc?ctgtcacctc?ggtgagctca?aggaccctcg?agtgccgcaa?540
acggcgcgcg?cttta 555
<210>7
<211>555
<212>DNA
＜213〉derive from Root of Medicinal Changium (Changium smyrnioides)-Pengze, Jiangxi population
<400>7
ggcaagcgtc?ggtgggcttg?gttcccccgt?tgcgaacccc?aggaaggtgg?gcgctcccgg 60
gcgctcgtcg?gccgacgaaa?tcaaccgggc?gcggaatgcg?ccaaggaaat?caaaatagaa 120
ttgtacgttt?gcttcccgtt?cgtgggaggc?ggcgtctttc?ctaaacacaa?acgactctcg 180
gcaacggata?tcccggctct?tgcatcgatg?aagaacgtag?cgaaatgcga?tacttggtgt 240
gaattgcaga?atcccgtgaa?ccatcgagtc?tttgaacgca?agttgcgccc?gaagccgcta 300
ggctgagggc?acgcctgcct?gggtgtcacg?catcgtgctg?ccccctgacc?actcgctcct 360
tgaggagctg?cattggttac?agggcggata?ttggcctccc?gtgccttgcg?gtgcggttgg 420
cctaaaagtg?agtctctggt?gacagatgtt?gcgatatttg?tggttgaaat?aaataccctc 480
ttctcttgtc?gtgcgaatgc?ctgtcacctt?ggtgagctca?aggaccctca?agcgccgcaa 540
acggcgcgcg?cttta 555
<210>8
<211>20
<212>DNA
＜213〉artificial primer
<400>8
tcctccgctt?attgatatgc 20
<210>9
<211>22
<212>DNA
＜213〉artificial primer
<400>9
ggaagtaaaa?gtcgtaacaa gg?22
Claims (3)
1. the dna molecular discrimination method of a different places of production Root of Medicinal Changium plant, set up the rDNAITS district of different places of production Root of Medicinal Changium plant, comprise part ITS1 sequence and complete 5.8S and the sequence library of ITS2 and the specificity discriminating site of each place of production Root of Medicinal Changium plant, it is characterized in that carrying out as follows:
(1) total DNA extraction
Get the fresh blade of Root of Medicinal Changium plant to be checked, after handling with sterilized water, grind to form fine powder in liquid nitrogen, extract total DNA with the CTAB method, the DNA purifying of producing with the rich Bioexperiment Materials Research Laboratories of Beijing Lay reclaims the test kit purifying then, is dissolved in the sterilization distilled water:
(2) pcr amplification
The primer of Root of Medicinal Changium ITS district amplification is ITS district universal primer ITS4 and ITS5, and primer sequence is:
ITS4:5`-TCCTCCGCTTATTGATATGC-3` is positioned on the 18S;
ITS5:5`-GGAAGTAAAAGTCGTAACAAGG-3` is positioned on the 26S;
Utilize this that primer is carried out amplified reaction to total DNA, and with ddH
2O replaces masterplate DNA to make blank;
(3) determined dna sequence
PCR product direct sequencing is adopted in order-checking, unidirectional order-checking, and sequencing primer is pcr amplification primer ITS5, sequencing reaction carries out on ABI 3730 full-automatic sequenators;
(4) discriminating of dna sequence data analysis and plant origin
The sequence one in the rDNAITS district of Root of Medicinal Changium plant to be checked after measured, arrange software (CLUSTAL software) with contraposition and carry out the contraposition arrangement, and be aided with artificial check and correction, analyze the difference of each sequence in sample DNA sequence to be checked and the database with genetic analysis software (MEGA software), by analyzing relatively the source of final decision Root of Medicinal Changium plant to be checked.
2. according to the dna molecular discrimination method of the different place of production of claim 1 Root of Medicinal Changium plant, the high specific PCR discrimination method of genuine producing region (Jurong, Jiangsu) Root of Medicinal Changium plant, it is characterized in that rDNA ITS sequence by more different places of production Root of Medicinal Changium plant, design the high specific primer of differentiating Jurong, Jiangsu Root of Medicinal Changium plant, the diagnostic primers of Jurong, Jiangsu Root of Medicinal Changium plant is: the upstream diagnostic primers is JR:5`-TGAGTGTCATGTATCGTGTC-3`, and the downstream diagnostic primers is the universal primer ITS4 of ITS district amplification; Concrete steps are:
(1) total DNA extraction
Get the fresh blade of Root of Medicinal Changium plant to be checked, after handling with sterilized water, grind to form fine powder in liquid nitrogen, extract total DNA with the CTAB method, the DNA purifying of producing with the rich Bioexperiment Materials Research Laboratories of Beijing Lay reclaims the test kit purifying then, is dissolved in the sterilization distilled water;
(2) quality examination of masterplate DNA
With ITS district universal primer ITS4 and ITS5 the total DNA of product to be checked is increased, with the concentration of validating DNA masterplate quality, adjustment masterplate DNA; ITS4 and ITS5 sequence are:
ITS4:5`-TCCTCCGCTTATTGATATGC-3` is positioned on the 18S;
ITS5:5`-GGAAGTAAAAGTCGTAACAAGG-3` is positioned on the 26S;
(3) utilization diagnostic primers JR, ITS4 increase to masterplate DNA, and annealing temperature is risen to 68 ℃, and what can be amplified out promptly derives from the Jurong, Jiangsu, and the Root of Medicinal Changium in other sources is all negative.
3. according to the dna molecular discrimination method of the different place of production of claim 1 Root of Medicinal Changium plant, special card of the present invention is to utilize high specific PCR diagnostic primers that the Root of Medicinal Changium of Jurong, Jiangsu, genuine producing region has been carried out the source discriminating, rDNA ITS region sequence with reference to different places of production Root of Medicinal Changium, design the high specific diagnostic primers of Jurong, a pair of Jiangsu Root of Medicinal Changium, the design reference of upstream diagnostic primers JR:5`-TGAGTGTCATGTATCGTGTC-3` the 5.8S and the ITS2 part in Root of Medicinal Changium rDNA ITS district, Jurong, Jiangsu, the base in the 8th site of Jurong, Jiangsu 3 Root of Medicinal Changium samples ITS2 sequence is C all, and the base of 18 Root of Medicinal Changium samples in other places of production in this site all is T.
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| CNA2007101352900A CN101182572A (en) | 2007-11-15 | 2007-11-15 | Molecule identification method of different producing area changium root plants |
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| CN104762370A (en) * | 2014-11-28 | 2015-07-08 | 江苏省中国科学院植物研究所 | Nucleotide sequence and method used for identifying and differentiating species and varieties of Taxus chinensis |
| CN104762370B (en) * | 2014-11-28 | 2018-08-17 | 江苏省中国科学院植物研究所 | The nucleotide sequence and method of Chinese Taxus kind and kind are distinguished in identification |
| JP2018201501A (en) * | 2017-05-31 | 2018-12-27 | 株式会社ツムラ | Primer set for discriminating herbal medicine and method for discriminating herbal medicine using the same |
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| CN107236797A (en) * | 2017-06-07 | 2017-10-10 | 苏州市李良济健康产业有限公司 | A kind of primer pair and its application for Identification chinese herbs medicine Radix Codonopsis |
| JP2020103279A (en) * | 2018-12-27 | 2020-07-09 | 株式会社ツムラ | Primer set for discriminating herbal medicine and method for discriminating herbal medicine using the same |
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