CN107190082B - For differentiating the molecular specificity labeled primers and its discrimination method of national improved tea variety ' meeting frost ' - Google Patents
For differentiating the molecular specificity labeled primers and its discrimination method of national improved tea variety ' meeting frost ' Download PDFInfo
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- CN107190082B CN107190082B CN201710573478.7A CN201710573478A CN107190082B CN 107190082 B CN107190082 B CN 107190082B CN 201710573478 A CN201710573478 A CN 201710573478A CN 107190082 B CN107190082 B CN 107190082B
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Abstract
The present invention discloses a kind of molecular specificity labeled primers and its discrimination method for being used to differentiate national improved tea variety ' meeting frost '.The primer sequence is：Sense primer S4YSF：5’‑TGAGCTGAGTGAGTTGAA‑3’；Anti-sense primer S4YSR：5’‑GTCGGTACTGACTATGAA‑3’.The molecular specificity labeled primers S4YSF/S4YSR designed using the present invention, is expanded, it is possible to achieve the quick and precisely discriminating of tea tree breed ' meeting frost ' by PCR.S4YSF/S4YSR is the specificity amplification primer of tea tree breed ' meeting frost ', and testing sample can then amplify the DNA fragment specific of 1346bp sizes, then be negative reaction if other tea tree breeds if tea tree breed ' meeting frost '；The inventive method is easy to operate, as a result accurately, and it is time-consuming shorter.
Description
Technical field
The present invention relates to one kind to differentiate national improved tea variety ' meeting frost ' Camellia sinensis cv.
The molecular specificity labeled primers of ' Yingshuang ', and quick discriminating is carried out to ' meeting frost ' using the specificity labeled primers
Method.
Background technology
' meeting frost ' Camellia sinensis cv. ' Yingshuang ' are ground by Hangzhou City Agricultural Science Research Inst.'s tealeaves
Study carefully the tea tree breed that institute's seed selection forms, have that bud-leaf fertility is strong, holds that tender property is strong, yield is high, the strong, quality better of cutting propagation power etc.
Advantage.By Zhejiang provincial-level appraisal, national tea tree was regarded as by the national Crop breed audit committee in 1987 within 1980
Breeding, numbering GS13041-1987.The kind is suitable to make black tea by spreading the leaves on withering racks to dry, green tea, its tea quality is excellent, it is fragrant it is high, taste is dense fresh.Zhejiang Province
Department of Agriculture ' will meet frost ', and tea tree breed is classified as one of Zhejiang Province's planting industry Variety comprehensive.The most of Chan Cha areas in the whole nation introduce a fine variety.Mesh
Before, there is larger area cultivation the provinces and regions such as Zhejiang, Jiangsu, Jiangxi, Anhui, Henan, Hunan, Hubei and Guangxi.
' meeting frost ' bud-leaf is closely similar (see attached for the tea tree breed of green with other bud-leafs in green, its morphological feature
Fig. 1).Therefore, it is difficult to distinguish them using traditional form discrimination method, particularly in seedling process of exchange, tea grower
Fake and forged seedling usually is bought because of being difficult to recognize, causes economic loss serious.This also for the improved tea variety protection and
Using bringing certain difficulty.
DNA molecular marker technology can be analyzed by gene order comparison in difference and provided directly for plant discriminating, genealogical classification
The evidence connect.As the focus industrial crops in China, at present, common molecular labelling technique is ground in the genetic diversity of tea tree breed
Studying carefully aspect has more application.Such as：RAPD (Chen Zhidan etc., the south of Fujian Province oolong tea Tea Germplasm RAPD finger-print structures
Build and analysis of genetic diversity, Molecular Plant Breeding, 2012,10 (6)：731-739), SSR (Zhang Mingze, 60 parts of the south of Guizhou Province tea tree kind
The ssr analysis of matter resource genetic diversity, northwest Botany Gazette, 2016,36 (6)：1117-1124), SCoT (Chen Xi etc., SCoT
The genetic diversity of labeled analysis Shaanxi Resources of Tea Plant, tea science, 2016,36 (2)：131-138), ISSR and SRAP (are utilized
The genetic diversity of SRAP and ISSR labeled analysis Tea plant Germplasms in Guangdong Province, nuclear agricultural science report 2010,24 (5)：948-955).This
A little researchs have established important foundation for the Genetic diversity evaluation of tea tree breed and protection.However, conventional molecular marking technique
Performing PCR amplification is entered using general random primer mostly, its PCR primer finger-print is more complicated, repeatability is poor.Therefore, this
A little applications of the method in plant germplasm particularly cultivar identification are not strong.The primer of specific molecular marker technology has fine
Stability and selectivity, specific amplification can be carried out, therefore the technology has application well in plant germplasm identification
Prospect.There has been no the research report that specific molecular marker technology application tea tree breed ' meeting frost ' differentiates at present.This patent passes through
A kind of molecular specificity labeled primers for meeting frost are developed, and its discrimination method are established, to realize the true and false of the famous-brand and high-quality tea tree breed
Differentiate and protection provides technical support.
The content of the invention
First purpose of the present invention is to provide for differentiating ' meeting frost ' Camellia sinensis cv.
The molecular specificity labeled primers of ' Yingshuang ', the primer sequence are as follows：
Sense primer S4YSF：5 '-TGAGCTGAGTGAGTTGAA-3 ', as shown in SEQ ID NO.1；
Anti-sense primer S4YSR：5 '-GTCGGTACTGACTATGAA-3 ', as shown in SEQ ID NO.2.
Specificity labeled primers combination is to use Standard PCR technology, by a large amount of DNA fingerprinting comparative analysis, sieve
Acquisition is selected to meet white DNA fragment specific.Then, clone and be sequenced by gel extraction, TA, obtain the DNA sequence dna, it is basic herein
White molecular specificity labeled primers are met in upper design, and using the primer as PCR primer, ' meeting frost ' and its close tea tree breed are carried out
PCR expand, the primer only reacted with meeting white sample DNA, obtain 1346bp sizes specific fragment, and with other tea trees
Kind sample does not react.In order to further verify specific primer S4YSF/S4YSR stability, with primer combination pair
The sample DNA that white individual is met from 10 differences enters performing PCR amplification, as a result all to meet white sample and amplify 1346bp sizes
Specific band, illustrate that the primer has higher stability and application.
Second object of the present invention is to provide above-mentioned molecular specificity labeled primers and carries out mirror method for distinguishing to meeting frost.Institute
The method of stating is：Specificity amplification primer is used as using above-mentioned primer combination (S4YSF/S4YSR), with tea tree plant sample to be measured
DNA is template, enters performing PCR amplification, is detected through agarose gel electrophoresis, if electrophoresis result molecular weight occurs for 1346bp sizes
Specific fragment, then tea tree plant sample to be measured is ' meeting frost ', on the contrary then be not.
Specifically, methods described is as follows：
(1) extracting genome DNA：The fresh blade of tea tree plant sample to be measured is taken, liquid feeding nitrogen is ground to powder, then, profit
Extracted with UNIQ-10 pillar plant genome DNA extraction agents box (ordering in Shanghai Sheng Gong bioengineering limited company)
The genomic DNA of testing sample.
(2) PCR is expanded：Using the testing sample DNA of step (1) extraction as template, with the molecular specificity labeled primers
(S4YSF/S4YSR) amplimer is used as, enters performing PCR amplification.
PCR reaction systems (μ l of cumulative volume 20)：1 μ l sense primers S4YSF (10 μM), 1 μ l anti-sense primers S4YSR (10 μ
M), 10 μ l 2 ×PCR SuperMix (are ordered in Beijing Quanshijin Biotechnology Co., Ltd), 1 μ l templates
DNA (50ng/ μ l), 7 μ l ddH2O。
PCR response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 50s, 57 DEG C of annealing 50s, 72 DEG C of extension 1.5min, altogether
32 circulations；Finally, 72 DEG C of extension 10min.
(3) electrophoresis detection：10 μ l steps (2) PCR primers are taken, electrophoresis detection, Ran Houli are carried out with 1.5 ﹪ Ago-Gels
With gel imaging system (BIO-RAD MolecularGel DocTMXR+System with Image
LabTMSoftware) take pictures detection.If there is the DNA bands that molecular weight is 1346bp sizes, testing sample in electrophoretogram passage
It is on the contrary then be not to meet frost.
Main beneficial effect of the invention is shown as：The primer combination (S4YSF/S4YSR) of design is tea tree breed ' meeting frost '
Specificity amplification primer, if the sample DNA of other tea tree breeds, then PCR reactions are feminine gender, and then realize that tea tree breed ' is met
Frost ' quick discriminating, testing result is accurate, and application method is easy, and operation is time-consuming short.
Brief description of the drawings
Fig. 1 be the present invention relates to 18 tea tree breeds two leaves and a bud phenotype photo.1st, Dragon Well tea 43；2nd, Dragon Well tea is grown
Leaf；3rd, middle tea 102；4th, middle tea 108；5th, black ox is early；6th, Fuding white tea, 7, cyclopentadienyl it is green；8th, frost is met；9th, spring rain 1,10, spring rain 2
Number；11st, Zhejiang agriculture 113；12nd, Zhenong 117；13rd, strength peak；14th, Cui Feng；15th, middle tea 125；16th, middle tea 126；17th, middle tea 127；18、
Middle tea 128.Fig. 1 shows that ' meeting frost ' and the leaf morphology of other 17 tea tree breeds are closely similar.
Fig. 2 is the molecular specificity labeled primers (S4YSF/S4YSR) designed using the present invention to 18 tea tree sample DNAs
Enter the electrophoretogram of performing PCR amplification, wherein M is that DNA molecular amount standard Trans2K DNA Marker (are ordered in the full Shi Jinsheng in Beijing
Thing Technology Co., Ltd.)；Passage 1：Dragon Well tea 43；2：Longjing Changye；3：Middle tea 102；4：Middle tea 108；5：Black ox is early；6：Fuding is big
White tea, 7：Cyclopentadienyl is green；8：Meet frost；9：Spring rain 1；10：Spring rain 2；11：Zhejiang agriculture 113；12：Zhenong 117；13：Strength peak；14：It is emerald green
Peak；15：Middle tea 125；16：Middle tea 126；17：Middle tea 127；18：Middle tea 128.Electrophoretogram shows that only ' meeting frost ' sample amplification goes out
Molecular weight is the specific DNA band of 1346bp sizes.
Fig. 3 is to use to meet the DNA progress that white molecular specificity labeled primers S4YSF/S4YSR meets 10 parts of differences white individual
The PCR amplification electrophoretograms of detection.Wherein, M is that DNA molecular amount standard Trans2K DNA Marker (are ordered in the full formula gold in Beijing
Bioisystech Co., Ltd)；Passage 1-10：10 difference ' meeting frost ' samples are corresponded to, numbering is respectively YS1-YS10.
Embodiment
The present invention more can fast and accurately differentiate the associated sample of tea tree breed ' meeting frost ' on a molecular scale.Below
The present invention is described further in conjunction with specific embodiments, but protection scope of the present invention is not limited to that：
Embodiment 1：The exploitation design of tea tree breed ' meeting frost ' molecular specificity labeled primers
1. sample gene group DNA is extracted
The similar tea tree sample of clip 18 (specifying information is shown in that accompanying drawing 1 illustrates) fresh blade 100mg is put into mortar, adds immediately
Enter liquid nitrogen thoroughly to grind, then (order using UNIQ-10 pillar plant genome DNA extraction agents box and give birth to work biology in Shanghai
Engineering stock Co., Ltd) extraction genomic DNA, gained DNA carries out electrophoresis detection with 1% agarose gel, and uses ultraviolet spectrometry
Photometer detectable concentration, it is diluted to 50ng/ μ l, 4 DEG C of preservations, for subsequent PCR amplification.
2. molecular specificity labeled primers S4YSF/S4YSR is designed
Using standard PCR amplification technology, by a large amount of DNA fingerprinting comparative analysis, the specific core of frost is met in screening acquisition
Nucleotide sequence, by gel extraction, clone and sequencing, obtain the DNA sequence dna and (serve Hai Shenggong bioengineering limited company
Sequencing), it is then based on DNA sequence dna design acquisition and meets white specific primer S4YSF/S4YSR (S4YSF：
TGAGCTGAGTGAGTTGAA；S4YSR：GTCGGTACTGACTATGAA) (primer sequence is by the deep fast limited public affairs of biotechnology in Suzhou
Department's synthesis).
Embodiment 2：Specificity labeled primers S4YSF/S4YSR PCR amplifications and electrophoresis detection
The specificity labeled primers combination S 4YSF/S4YSR developed using the present invention is amplimer, to Different Tea Varieties
Sample DNA (being specifically shown in accompanying drawing 1 to illustrate) enters performing PCR detection.
PCR reaction systems (μ l of cumulative volume 20)：Sense primer S4YSF (10 μM) 1 μ l, anti-sense primer S4YSR (10 μM) 1 μ
L, 2 ×The μ l of PCR SuperMix (ordering in Beijing Quanshijin Biotechnology Co., Ltd) 10, template DNA
(50ng/ μ l) 1 μ l, ddH2O 7μl。
PCR response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 50s, 57 DEG C of annealing 50s, 72 DEG C of extension 1.5min, altogether
32 circulations；Finally, 72 DEG C of extension 10min.
Electrophoresis detection is carried out to PCR primer on 1.5 ﹪ Ago-Gels, utilizes gel imaging system (BIO-RAD
MolecularGel DocTMXR+System with Image LabTMSoftware detection of taking pictures) is carried out.Electrophoresis
(in figure, passage M is DNA molecular amount standard Trans2K DNA Marker to figure as shown in Figure 2；Passage 1~18 is 18 differences
The sample of tea tree breed, it is specifically shown in brief description of the drawings).From accompanying drawing 2 it can be seen that only meeting white sample (passage 8) can amplify
The DNA fragment specific of 1346bp sizes.And other tea tree breeds do not amplify any band, this shows that the present invention's is special
Property labeled primer selectivity is good, high sensitivity, can be used for the quick discriminating of tea tree breed ' meeting frost '.
Embodiment 3：Molecular specificity labeled primers S4YSF/S4YSR is further verified
In order to further verify the primer combination S4YSF/S4YSR of this patent exploitation stability and application, utilize
Primer S4YSF/S4YSR enters performing PCR augmentation detection, the electrophoretogram such as institute of accompanying drawing 3 to 10 parts from the different sample DNAs for meeting white individual plant
Show that (in figure, M is DNA molecular amount standard Trans2K DNA Marker；Passage 1-10：Correspond to meet white sample number into spectrum YS1-
YS10).Accompanying drawing 3 show it is all meet white sample and can amplify the specific band of 1346bp sizes, therefore, of the invention is special
Property labeled primer S4YSF/S4YSR has good stability and application.
SEQUENCE LISTING
<110>Tea Inst., Chinese Academy of Agricultural Sciences
<120>For differentiating the molecular specificity labeled primers and its discrimination method of national improved tea variety ' meet frost '
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 1
tgagctgagt gagttgaa 18
<210> 2
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 2
gtcggtactg actatgaa 18
Claims (4)
1. the molecular specificity labeled primers of tea tree breed ' meeting frost ', it is characterised in that the specific primer sequence is as follows：
Sense primer S4YSF：5 '-TGAGCTGAGTGAGTTGAA-3 ', as shown in SEQ ID NO.1；
Anti-sense primer S4YSR：5 '-GTCGGTACTGACTATGAA-3 ', as shown in SEQ ID NO.2.
2. a kind of molecular specificity labeled primers S4YSF/S4YSR using described in claim 1 enters to tea tree breed ' meeting frost '
The method of row quick discriminating, it is characterised in that methods described is：
(1) genomic DNA of tea tree breed to be measured is extracted；
(2) using the testing sample DNA of step (1) extraction as amplification template, amplification is used as using the molecular specificity labeled primers
Primer, enter performing PCR amplification；
(3) row agarose gel electrophoresis detection is entered to the PCR primer of step (2), if the spy of 1346bp sizes occurs in electrophoresis result
Specific fragment, then testing sample is meets frost, on the contrary then be not.
3. method as claimed in claim 2, it is characterised in that described PCR amplification system：1 μ l concentration is that 10 μM of upstream is drawn
Thing S4YSF, 1 μ l concentration are 10 μM of anti-sense primer S4YSR, 10 μ lPCR SuperMix, 1 μ l concentration are
50ng/ μ l template DNA, 7 μ l ddH2O；Cumulative volume is 20 μ l.
4. method as claimed in claim 2, it is characterised in that described PCR amplification programs are：94 DEG C of pre-degeneration 5min；94℃
50s is denatured, 57 DEG C of annealing 50s, 72 DEG C of extension 1.5min, totally 32 circulate；Finally, 72 DEG C of extension 10min.
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| CN108220471B (en) * | 2018-01-25 | 2020-02-18 | 中国农业科学院茶叶研究所 | Molecular specific marker primer for identifying national-grade tea tree improved variety Fuyun No. 6 and identification method thereof |
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| WO2000017341A1 (en) * | 1998-09-23 | 2000-03-30 | Business And Research Management Pty. Ltd. | Myrtaceae microsatellites |
| CN1955312A (en) * | 2006-09-12 | 2007-05-02 | 浙江大学 | Albino tea tree breed 'sajin' specific DNA molecular mark and its identification method |
| KR101114312B1 (en) * | 2009-03-19 | 2012-03-02 | 재단법인 하동녹차연구소 | Identification of Hadong Choigo tea tree base sequence and Method for detection of Hadong Choigo tea using the markers |
| CN104651515B (en) * | 2015-02-27 | 2016-08-10 | 福建农林大学 | A kind of method building Camellia sinensis DNA fingerprinting |
| CN105624321B (en) * | 2016-03-28 | 2018-11-02 | 安徽农业大学 | Differentiate the method for yellow stalwart tea tree breed using SSR finger-prints |
| CN106591460B (en) * | 2016-12-27 | 2020-04-28 | 中国农业科学院茶叶研究所 | Method for identifying variety of 'Zhongcha 302' tea tree by SSR molecular marker and application |
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