CN103173532B - Method for identifying honeysuckle and lonicera confuse and application of same - Google Patents
Method for identifying honeysuckle and lonicera confuse and application of same Download PDFInfo
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- CN103173532B CN103173532B CN201210372344.6A CN201210372344A CN103173532B CN 103173532 B CN103173532 B CN 103173532B CN 201210372344 A CN201210372344 A CN 201210372344A CN 103173532 B CN103173532 B CN 103173532B
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- 241001570521 Lonicera periclymenum Species 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 22
- 241000245240 Lonicera Species 0.000 title abstract description 12
- 239000000463 material Substances 0.000 claims abstract description 20
- 238000012408 PCR amplification Methods 0.000 claims abstract description 16
- 108020004414 DNA Proteins 0.000 claims abstract description 14
- 239000000843 powder Substances 0.000 claims abstract description 7
- 108091029795 Intergenic region Proteins 0.000 claims abstract description 3
- 241000100289 Lonicera confusa Species 0.000 claims description 37
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 32
- 244000167230 Lonicera japonica Species 0.000 claims description 30
- 235000017617 Lonicera japonica Nutrition 0.000 claims description 29
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 2
- 230000003321 amplification Effects 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 102000053602 DNA Human genes 0.000 abstract 3
- 238000003752 polymerase chain reaction Methods 0.000 abstract 2
- 239000002773 nucleotide Substances 0.000 abstract 1
- 125000003729 nucleotide group Chemical group 0.000 abstract 1
- 241001170080 Lonicera hypoglauca Species 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 238000007400 DNA extraction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
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- 238000012163 sequencing technique Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 241000628997 Flos Species 0.000 description 2
- 235000009388 Parthenocissus quinquefolia Nutrition 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 238000000498 ball milling Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 102000054766 genetic haplotypes Human genes 0.000 description 2
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- 230000004048 modification Effects 0.000 description 2
- 241000208828 Caprifoliaceae Species 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241001500894 Lonicera fulvotomentosa Species 0.000 description 1
- 241001170076 Lonicera macranthoides Species 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- WYROLENTHWJFLR-ACLDMZEESA-N queuine Chemical compound C1=2C(=O)NC(N)=NC=2NC=C1CN[C@H]1C=C[C@H](O)[C@@H]1O WYROLENTHWJFLR-ACLDMZEESA-N 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
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Abstract
The invention relates to a method for identifying honeysuckle and lonicera confuse and an application of the method. The method disclosed by the invention comprises the step of performing PCR (polymerase chain reaction) amplification on ITS2 nucleotide sequence, and particularly comprises the steps of 1) extracting the DNA (deoxyribonucleic acid) of a sample; 2) performing PCR amplification on the segments of the ITS2 sequence containing ribosome DNA; 3) splicing the amplification products to obtain a complete ITS2 intergenic region; and 4) establishing the NJ (neighbour-joining) tree of the ITS2 sequence of the sample. The method disclosed by the invention can be used for effectively solving the problems of variety identification, variety improvement and breeding of honeysuckle medicinal materials, development and utilization of germplasm resources, and the like; and the method disclosed by the invention is wide in applicability, simple to operate, easy to grasp, high in accuracy, and capable of successfully realizing rapid and accurate identification on honeysuckle medicinal materials or powder and lonicera confuse medicinal materials or powder.
Description
Technical field
The present invention relates to Chinese medicinal materials source cultivar identification technical field, be specifically related to utilize the ITS2 fragment of rDNA to differentiate method and the application thereof of Japanese Honeysuckle and Lonicera confusa DC. medicinal material.
Background technology
The dry flower that Japanese Honeysuckle (Flos Lonicerae Japonica) is caprifoliaceae plant honeysuckle Lonicerajaponica Thunb. or the flower that band is just opened.The flower that Lonicera confusa DC. (Flos Lonicerae) is caprifoliaceae plant largeflower-like honeysuckle flower Lonicera macranthoides Hand.-Mazz., lonicera hypoglauca miq Lonicera hypoglauca Miq., Lonicera confusa Lonicera confusa DC. becomes the dry flower of Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms Lonicera fulvotomentosa Hsu et S.C.Cheng or band just to open.Japanese Honeysuckle and Lonicera confusa DC. are all conventional rare traditional Chinese medicines, all be widely used clinically, have clearing heat and detoxicating, effect of dispelling wind and heat pathogens, but both are different on content of drug effect components, effect also emphasizes particularly on different fields, and their proterties is very similar, with traditional method is extremely difficult, the two is distinguished, the method of the character identification adopted in pharmacopeia has certain subjectivity, and necessarily requires medicinal material profile complete, not damaged, operability is not strong, and identification person needs abundant expertise and identification of experience simultaneously.There is the situation that Japanese Honeysuckle is mixed with Lonicera confusa DC. in the market, have impact on quality and the safety of medication to a certain extent.For this situation, be badly in need of finding a kind of stable, reliably, simply method be used for differentiating fast Japanese Honeysuckle and Lonicera confusa DC. medicinal material or powder.
Summary of the invention
The object of the invention is to provide the method utilizing the ITS2 fragment of rDNA to differentiate Japanese Honeysuckle and Lonicera confusa DC. medicinal material.
Due to the flower that Japanese Honeysuckle and Lonicera confusa DC. medicinal material are all dry bud and just open, their DNA may there occurs certain degraded, and Japanese Honeysuckle and Lonicera confusa DC. all contain more polysaccharide, Japanese Honeysuckle provided by the invention and Lonicera confusa DC. medicinal material DNA extraction method, can efficiently, the DNA of rapid extraction Japanese Honeysuckle and Lonicera confusa DC. medicinal material.
Specifically comprise the following steps: extracting honeysuckle, Lonicera confusa DC. medicinal material, before extracting genome DNA, add the PVP40 powder of 5%.
The ITS2 rapid PCR amplification method of the rDNA of Japanese Honeysuckle provided by the invention and Lonicera confusa DC. medicinal material mainly for the high feature of its sequence GC content, by improving annealing temperature and adding the ITS2 sequence of the dimethyl sulfoxide (DMSO) of 5% and the Trimethyl glycine Successful amplification Japanese Honeysuckle of 2% and Lonicera confusa DC..
The authentication method of Japanese Honeysuckle provided by the invention and Lonicera confusa DC., comprises pcr amplification ITS2 gene.Specifically comprise the steps:
(1) testing sample DNA is extracted,
(2) pcr amplification contains the fragment of the ITS2 sequence of rDNA;
(3) two-way order-checking is carried out to amplified production, splicing, remove 5.8S and the 28S constant gene segment C at sequence two ends, obtain complete ITS2 intergenic region.
The present invention's amplimer sequence used is:
ITS2-HF 5’-ATGCGATACTTGGTGTGAAT-3’
ITS2-HR 5’-GACGCTTCTCCAGACTACAAT-3’
The system of described pcr amplification is every 25 μ L:
10 × PCR Buffer2.5 μ L, 25mmol/L Mg
2+2 μ L, 2.5mmol/LdNTPs2 μ L, 2.5 μm of each 1.0 μ L of ol/L primer, DNA profiling 30ng, Taq archaeal dna polymerase 1.0U, the dimethyl sulfoxide (DMSO) 1.0 μ L of 5%, the Trimethyl glycine 0.5 μ L of 2%, mend sterilizing distilled water to 25 μ L.
The condition of described pcr amplification is 94 DEG C of sex change 30s, and 58 DEG C of annealing 30s, 72 DEG C extend 45s, 40 circulations.
(4) based on K2P model, build the NJ tree of the ITS2 sequence of testing sample, Japanese Honeysuckle and Lonicera confusa DC. can be differentiated intuitively by NJ tree.
The method of the invention can be applicable to identify woodbine.Described woodbine is preferably Japanese Honeysuckle, Lonicera confusa DC..
Beneficial effect of the present invention:
(1) the ITS2 fragment preparation method of traditional Chinese medicine honeysuckle rDNA provided by the invention effectively can solve the cultivar identification of traditional Chinese medicine honeysuckle, breed improvement, breeding, and the problem such as the exploitation of germ plasm resource.
(2) the inventive method suitability is wide, simple to operate, is easy to grasp, and accuracy is high.Successfully can realize quick, the precise Identification to Japanese Honeysuckle and Lonicera confusa DC. medicinal material or powder.
Accompanying drawing explanation
Fig. 1 is in the PCR reaction system of Trimethyl glycine 0.5 μ L of the dimethyl sulfoxide (DMSO) 1.0 μ L and 2% adding 5%, the pcr amplification electrophorogram of Japanese Honeysuckle and Lonicera confusa DC. ITS2 sequence; M is molecular weight standard; 1,26 is negative control; 2-25 is Japanese Honeysuckle; 27-31 is largeflower-like honeysuckle flower; 32-36 is Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms; 37-38 is lonicera hypoglauca miq; 39-40 is Lonicera confusa;
Fig. 2 is in the PCR reaction system of Trimethyl glycine 0.5 μ L of the dimethyl sulfoxide (DMSO) 1.0 μ L and 2% not adding 5%, the pcr amplification electrophorogram of Japanese Honeysuckle and Lonicera confusa DC. ITS2 sequence; M is molecular weight standard; 1,26 is negative control; 2-25 is Japanese Honeysuckle; 27-31 is largeflower-like honeysuckle flower; 32-36 is Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms; 37-38 is lonicera hypoglauca miq; 39-40 is Lonicera confusa;
Fig. 3 is that the NJ of Japanese Honeysuckle and Lonicera confusa DC. sets qualification result figure.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.Instrument of the present invention is table model high speed centrifuge, PTC-100 gene-amplificative instrament, electrophoresis apparatus, ultraviolet Labworks image acquisition and analysis software, MM400 ball milling instrument (German Retsch).
The percentage sign " % " related in the present invention, if not specified, refers to mass percent; But the per-cent of solution, unless otherwise specified, refers in solution 100ml containing solute some grams; Per-cent between liquid, the ratio of capacity when referring to 20 DEG C.
Embodiment 1
1, experiment material, as shown in table 1.
Table 1 Japanese Honeysuckle and Lonicera confusa DC. sample
| Sequence number | Sample ID | Sample number into spectrum | Sampling position |
| 1 | Honeysuckle | RD-1-1 | Pingyi, shandong Province |
| 2 | Honeysuckle | RD-1-2 | Pingyi, shandong Province |
| 3 | Honeysuckle | RD-1-3 | Pingyi, shandong Province |
| 4 | Honeysuckle | RD-1-4 | Pingyi, shandong Province |
| 5 | Honeysuckle | RD-1-5 | Pingyi, shandong Province |
| 6 | Honeysuckle | RD-2-1 | Henan Xixia Mine |
| 7 | Honeysuckle | RD-2-2 | Henan Xixia Mine |
| 8 | Honeysuckle | RD-2-3 | Henan Xixia Mine |
| 9 | Honeysuckle | RD-2-4 | Henan Xixia Mine |
| 10 | Honeysuckle | RD-3-1 | Fengqiu |
| 11 | Honeysuckle | RD-3-2 | Fengqiu |
| 12 | Honeysuckle | RD-4 | Henan Zhoukou City |
| 13 | Honeysuckle | RD-5-1 | Donghai Area, Jiangsu county |
| 14 | Honeysuckle | RD-5-2 | Donghai Area, Jiangsu county |
| 15 | Honeysuckle | RD-5-3 | Donghai Area, Jiangsu county |
| 16 | Honeysuckle | RD-5-4 | Donghai Area, Jiangsu county |
| 17 | Honeysuckle | RD-5-5 | Donghai Area, Jiangsu county |
| 18 | Honeysuckle | RD-6 | Nanning |
| 19 | Honeysuckle | RD-7 | Guizhou Liu Panshui |
| 20 | Honeysuckle | RD-8 | Chongqing Kaixian |
| 21 | Honeysuckle | RD-9 | Wanzhou prefecture of Chongqing |
| 22 | Honeysuckle | RD-10 | Anhui Shucheng County |
| 23 | Honeysuckle | RD-11 | Huanggang |
| 24 | Honeysuckle | RD-12 | Hebei Julu |
| 25 | Largeflower-like honeysuckle flower | HZMRD-1 | Liuzhi guizhou |
| 26 | Largeflower-like honeysuckle flower | HZMRD-2 | Chongqing Ba Nan |
| 27 | Largeflower-like honeysuckle flower | HZMRD-3 | Hunan Longhui |
| 28 | Largeflower-like honeysuckle flower | HZMRD-4 | Sichuan Nan Jiang |
| 29 | Largeflower-like honeysuckle flower | HZMRD-5 | Pu'er, Yunnan |
| 30 | Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms | HHMRD-1 | Anlong, Guizhou |
| 31 | Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms | HHMRD-2 | Xingren County, Guizhou Province |
| 32 | Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms | HHMRD-3 | Zhenfeng, Guizhou |
| 33 | Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms | HHMRD-4 | Guizhou is then flourish |
| 34 | Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms | HHMRD-5 | Xingyi of Guizhou |
| 35 | Lonicera hypoglauca miq | HXRD-1 | Nanning |
| 36 | Lonicera hypoglauca miq | HXRD-2 | South china |
| 37 | Lonicera confusa | HNRD-1 | Sichuan Stone dish |
| 38 | Lonicera confusa | HNRD-2 | Jianyang of Sichuan |
2, DNA extraction
Get 20mg medicinal material respectively, in the upper grinding of MM400 ball milling instrument (German Retsch), the PVP40(polyvinylpyrrolidone PVP-40 of sample size 5% is added before grinding) powder, extracts test kit with plant genome DNA and extracts STb gene (TIANGEN Biotech (Beijing) Co., Ltd.).
3, pcr amplification
Primer sequence is ITS2F 5 '-ATGCGATACTTGGTGTGAAT-3 '
ITS3R 5 '-GACGCTTCTCCAGACTACAAT-3 ', is synthesized by company limited of Sheng Gong bio-engineering corporation (Beijing).Primer dissolves with aseptic deionization and is diluted to 2.5 μm of ol/L.
25 μ L reaction systems: PCR Buffer (10 ×) 2.5 μ L, Mg
2+2 μ L (25mmol/L), dNTPs mixture 2 μ L(2.5mmol/L), the each 1.0 μ L of primer (2.5 μm of ol/L), DNA profiling is about 30ng, Taq archaeal dna polymerase 1.0U, add the Trimethyl glycine 0.5 μ L of the dimethyl sulfoxide (DMSO) 1.0 μ L and 2% of 5%, add sterilizing distilled water to 25 μ L, in the enterprising performing PCR amplification of PTC-100 gene-amplificative instrament.
PCR reaction parameter: 95 DEG C of denaturation 5min, 94 DEG C of sex change 30s; 58 DEG C of annealing 30s, 72 DEG C extend 45s; 40 circulations, 72 DEG C extend 10min.
Arrange the negative control not adding template DNA in PCR experiment, PCR terminates the rear pcr amplification product getting 5 μ L respectively, and the sepharose with 1% carries out electrophoresis, detects electrophoresis result (Fig. 1) with gel imaging instrument.
4, DNA sequencing
PCR primer direct Song Meiji company carries out two-way order-checking, the same PCR primer of sequencing primer (ITS2F and ITS3R).
5, sequence assembly
The present embodiment adopts application software CodonCode Aligner 2.06(CodonCode Co., Germany) carry out sequence assembly and check and correction.First, utilize this software to remove the guiding region at sequence two ends, then carry out sequencing quality assessment and pre-treatment, namely remove the inferior quality part at sequencing result two ends, and quality evaluation is carried out to remainder.
Concrete steps are: slide from sequence 5 ' end and 3 ' end respectively with the window of 20bp, if there is the Q value more than 2 bases to be less than 20 in window, then delete a base, window continues to slide, if the number that in window, base Q value is less than 20 is less than or equal to 2, window stops sliding, and namely eliminates low-quality region.After carrying out sequence assembly, then according to Hidden Markov Model (HMM) model, remove sequence two ends 5.8S and 28S gene regions, obtain the ITS2 intergenic sequence of complete Japanese Honeysuckle and Lonicera confusa DC..
6, sequence alignment
With MEGA5.0 software to order-checking and the ITS2 intergenic sequence of each sample spliced compare, result shows, the ITS2 sequence of 24 traditional Chinese medicine honeysuckles only has a variant sites, can be divided into two haplotypes, as shown in SEQ ID NO.1-2.There is variation between the ITS2 sequence of four kinds of Original plants of Lonicera confusa DC., but respective sequence does not make a variation yet and obtains two haplotypes of traditional Chinese medicine honeysuckle listed by present method, the ITS2 sequence of Lonicera confusa DC. is as shown in SEQ ID NO.3-6.
7, the ITS2 sequence construct NJ of K2P distance model to the Japanese Honeysuckle obtained and Lonicera confusa DC. medicinal material is utilized to set (Fig. 3), for the identification of Japanese Honeysuckle and Lonicera confusa DC..
Test example 1
1, experiment material: with embodiment 1;
2, DNA extraction: with embodiment 1;
3, pcr amplification: with embodiment 1, difference is, does not add the Trimethyl glycine 0.5 μ L of the dimethyl sulfoxide (DMSO) 1.0 μ L and 2% of 5% in reaction system.
Pcr amplification product, the sepharose with 1% carries out electrophoresis, detects electrophoresis result (Fig. 2) with gel imaging instrument.
Test example 1 is compared with embodiment 1: the Trimethyl glycine 0.5 μ L not adding the dimethyl sulfoxide (DMSO) 1.0 μ L and 2% of 5% in test example 1, electrophoresis result is found out: goal gene band is not obvious, can not be used for differentiating Japanese Honeysuckle and Lonicera confusa DC..
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (2)
1. differentiate a method for Japanese Honeysuckle and Lonicera confusa DC., comprise the following steps:
(1) extract traditional Chinese medicine honeysuckle or Lonicera confusa DC. medicinal material DNA, before extracting DNA, add the polyvinylpyrrolidone PVP-40 powder of sample size 3%-5%;
(2) pcr amplification contains the fragment of the ITS2 sequence of rDNA;
(3) two-way order-checking is carried out to amplified production, splicing, remove 5.8S and the 28S constant gene segment C at sequence two ends, obtain complete ITS2 intergenic region;
Amplimer sequence used is:
ITS2-HF 5’-ATGCGATACTTGGTGTGAAT-3’；
ITS2-HR 5’-GACGCTTCTCCAGACTACAAT-3’；
The system of described pcr amplification is every 25 μ L:
10 × PCR Buffer2.5 μ L, 25mmol/L Mg
2+2 μ L, 2.5mmol/LdNTPs2 μ L, 2.5 μm of each 1.0 μ L of ol/L primer, DNA profiling 30ng, Taq archaeal dna polymerase 1.0U, the dimethyl sulfoxide (DMSO) 1.0 μ L of 5%, the Trimethyl glycine 0.5 μ L of 2%, mend sterilizing distilled water to 25 μ L;
The condition of described pcr amplification is 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, and 72 DEG C extend 45s, 40 circulations;
(4) based on K2P model, build the NJ tree of the ITS2 sequence of testing sample, set by NJ and differentiate Japanese Honeysuckle and Lonicera confusa DC. intuitively.
2. method described in claim 1 is differentiating the application in traditional Chinese medicine honeysuckle and Lonicera confusa DC. medicinal material.
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| CN113265482A (en) * | 2021-05-10 | 2021-08-17 | 长沙学院 | SNP (single nucleotide polymorphism) sites for identifying honeysuckle and lonicera confusa and amplification primers |
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