CN106119388A - A kind of PCR primer differentiating lily cultivar Huang Tempo is to, test kit and method - Google Patents
A kind of PCR primer differentiating lily cultivar Huang Tempo is to, test kit and method Download PDFInfo
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Abstract
The invention belongs to genetic engineering field, be specifically related to a kind of PCR primer differentiating lily cultivar Huang Tempo to, test kit and method.This PCR primer pair, a primer therein has the nucleotide sequence of the SEQ ID NO:1 in sequence table, and another primer has the nucleotide sequence of the SEQ ID NO:2 in sequence table；Or a primer therein has the nucleotide sequence of the SEQ ID NO:3 in sequence table, another primer has the nucleotide sequence of the SEQ ID NO:4 in sequence table.This test kit includes this PCR primer pair.The method includes: pcr amplification reaction and detecting step.The present invention can identify from molecular level whether this product to be measured is lily cultivar Huang Tempo, and it is more accurate to compare from differentiating in appearance.
Description
Technical field
The invention belongs to genetic engineering field, be specifically related to a kind of PCR primer differentiating lily cultivar Huang Tempo to, reagent
Box and method.
Background technology
OT type Bulbus Lilii (Lilium spp.) is to be that parent hybridizes with oriental hybrid lily (O type) and loudspeaker (T-shaped) Bulbus Lilii
Selection-breeding forms, and the east series lily cultivar that compares easily is degenerated, and cultivation technique is required more high unfavorable factor, OT type hundred
Conjunction has obvious advantage, such as: flower is big, abnormal smells from the patient delicate fragrance, and the cultivation phase is short and easy, strong stress resistance, is difficult to degeneration etc..Cause
This, in recent years, the share that OT type Bulbus Lilii commercially occupies is increasing, and is gradually liked by consumers in general.
Yellow Tempo (Manissa) is one of the most popular OT type lily cultivar, and flower is very large, pattern
Golden yellow, stem stalk is tall and straight sturdy, has good market prospect.Timber (Conca d ' or) is also OT type lily cultivar, pattern
Very much like with yellow Tempo with profile, the two kind is all from Holland.Owing to yellow Tempo has higher ornamental value and excellent
Breediness, therefore plant ball wholesale price and exceed 12.5% than timber, single flower price 1-2 higher than timber unit, is timber
The 120-150% of price.So many Bulbus Lilii growers utilize the spy that both of which outward appearance is similar with a lot of florist's shop retail dealers
Point, with the yellow Tempo of timber personation, cheats vast consumer.And, only poor from distinguishing the two accuracy in appearance, it is not enough to
For fields higher to identification result precise requirements such as customs's cultivar identification, technically offer legal basis.
So, need one can distinguish lily cultivar Huang sky from molecular level effectively, easily in the market
Despot and timber and the method for other common OT type lily cultivar, and this type of method at home and abroad there is no report.
Summary of the invention
It is an object of the invention to provide a kind of PCR primer differentiating lily cultivar Huang Tempo to, test kit and method.
It is an object of the invention to be achieved through the following technical solutions:
A kind of PCR primer pair differentiating lily cultivar Huang Tempo, a primer therein has the SEQ ID in sequence table
The nucleotide sequence of NO:1, another primer has the nucleotide sequence of the SEQ ID NO:2 in sequence table；Or one therein
Primer has the nucleotide sequence of the SEQ ID NO:3 in sequence table, and another primer has the SEQ ID NO:4 in sequence table
Nucleotide sequence.
A kind of specific band differentiating lily cultivar Huang Tempo, is obtained carrying out PCR reaction by this primer；This specificity
Band, has the nucleotide sequence of SEQ ID NO:5 in sequence table；Or there is the nucleoside of SEQ ID NO:6 in sequence table
Acid sequence.
A kind of PCR kit differentiating lily cultivar Huang Tempo, including: above-mentioned PCR primer pair；
In a preferred embodiment, this test kit also includes dNTP, magnesium chloride, archaeal dna polymerase and PCR buffer；
In a preferred embodiment, final concentration of in PCR reaction system of the component that this test kit includes: described in draw
Two each 0.2-0.8mmol/L of primer of thing centering, described dNTP 5-20 μm ol/L, described magnesium chloride 0.1-2mmol/L, described
Archaeal dna polymerase 0.2-0.5U/ μ L, Tris-HCL (pH7.5-9.0) 1-5 mmol/L and KCL0.6-5 mmol/L is as described
PCR buffer.
In a preferred embodiment, the component that this test kit includes in PCR reaction system, the volume of PCR premixed liquid
Percentage ratio is 85-95%, two primer each 0.1-1.5 μm ol/L of described primer centering.
A kind of method differentiating lily cultivar Huang Tempo, comprises the following steps:
PCR reactions steps: utilize above-mentioned PCR primer to or above-mentioned PCR kit, with Plant Genome to be measured as template,
Carry out PCR reaction, obtain pcr amplification product；
Detecting step: described pcr amplification product is detected, as described pcr amplification product contains specific band,
Treat described in then that measuring plants is described lily cultivar Huang Tempo.
In a preferred embodiment, the response procedures of this pcr amplification reaction is:
Degeneration: 94-95 DEG C 2-5 minute；Amplification: 94-95 DEG C of 10-50 second, 54-60 DEG C of 15-50 second, 72 DEG C of 25-50 seconds, altogether
Count 30-35 circulation；Extend: 72 DEG C 4-10 minute.
In a preferred embodiment, in the method:
When described PCR primer to for: a primer therein has the nucleotide base of the SEQ ID NO:1 in sequence table
Sequence, when another primer has the nucleotide base sequence of the SEQ ID NO:2 in sequence table, described specific band has
The nucleotide sequence of the SEQ ID NO:5 in sequence table；
When described PCR primer to for: a primer therein has the nucleotide base of the SEQ ID NO:3 in sequence table
Sequence, when another primer has the nucleotide base sequence of the SEQ ID NO:4 in sequence table, described specific band has
The nucleotide sequence of the SEQ ID NO:6 in sequence table.
The invention have the benefit that
1, the present invention utilizes specific PCR primer pair, and the genome treating measuring plants carries out PCR reaction, and detection amplification is produced
Whether thing contains specific band；So can identify from molecular level whether this product to be measured is lily cultivar Huang sky
Despot, it is more accurate to compare from differentiating in appearance.
2, present invention only requires and treat the genome of measuring plants and carry out PCR reaction and detect, thus easy, efficiently.
3, the present invention has wide range of applications, and is particularly well-suited to the field higher to identification result accuracy requirement, such as: (1)
Can be used for assisting customs to carry out cultivar identification；(2) can be used for providing foundation and method for country's lily cultivar Markers for Detection；
(3) can be used for as producing dispute offer detection foundation.
Accompanying drawing explanation
Fig. 1 is the two kinds of Bulbus Liliies of embodiment 1 PCR product electrophoresis patterns in different primers.
Fig. 2 is the PCR product electrophoresis pattern of lower 3 the RAPD primers of different annealing temperature of embodiment 1.
Fig. 3 is the PCR product electrophoresis pattern of the bacterium colony PCR of embodiment 1.
Fig. 4 is the PCR product electrophoresis pattern of the rear 6 pairs of primers that check order of embodiment 1.
Fig. 5 is 6 kinds of common lily cultivar PCR product electrophoresis patterns of embodiment 2.
Fig. 6 is two kinds of common lily cultivar PCR product electrophoresis patterns of embodiment 3.
Detailed description of the invention
First aspect, the present invention provides a kind of PCR primer pair differentiating lily cultivar Huang Tempo.Primer tool therein
The nucleotide sequence of the SEQ ID NO:1 in ordered list, another primer has the nucleoside of the SEQ ID NO:2 in sequence table
Acid sequence；Or a primer therein has the nucleotide sequence of the SEQ ID NO:3 in sequence table, another primer has sequence
The nucleotide sequence of the SEQ ID NO:4 in list.
Second aspect, the present invention provides a kind of specific band differentiating lily cultivar Huang Tempo.This specific band by
Above-mentioned primer carries out pcr amplification reaction to SEQ ID NO:1 and SEQ ID NO:2 and obtains, and has the SEQ ID in sequence table
The nucleotide sequence of NO:5；Or by above-mentioned primer, SEQ ID NO:3 and SEQ ID NO:4 is carried out pcr amplification reaction and obtain, tool
The nucleotide sequence of the SEQ ID NO:6 in ordered list.
The third aspect, the present invention provides a kind of PCR kit differentiating lily cultivar Huang Tempo.Included by this test kit
Final concentration of in PCR reaction system of component: above-mentioned PCR primer is to (SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID
NO:3 and SEQ ID NO:4) in two each 0.2-0.8mM of primer, also include dNTP 5-20 μm ol/L, magnesium chloride 0.1-
2mmol/L, archaeal dna polymerase 0.2-0.5U/ μ L；Possibly together with PCR buffer, (this PCR buffer contains at reactant this test kit
In system final concentration of 1-5 mM Tris-HCL (pH7.5-9.0) and in reaction system the KCL of final concentration of 0.6-5mM)；Should
PCR buffer is formed by the dilution of former PCR premixed liquid, and the concentration of this former PCR premixed liquid is typically at the 1.1-10 for this PCR buffer
Times.
In above-mentioned PCR reaction system, also include the template DNA of final concentration of 1-3.75ng/ μ L.
Preferably, final concentration of in PCR reaction system of the component included by above-mentioned dose of box: above-mentioned PCR primer pair
Two each 0.6mM of primer in (SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:3 and SEQ IDNO:4), also include
DNTP 10 μm ol/L, magnesium chloride 0.5mmol/L, archaeal dna polymerase 0.25U/ μ L, PCR buffer are (containing 2 mM Tris-HCL
(pH8.0) and 1.5mM KCL)；Preferably, in above-mentioned PCR reaction system, also include the template of final concentration of 1.5ng/ μ L
DNA。
Exemplarily, the final concentration of each component in above-mentioned reaction system can be: template DNA 1ng/ μ L, 1.5 ng/
Model between any number and any number in μ L, 2 ng/ μ L, 2.5 ng/ μ L, 3 ng/ μ L, 3.5 ng/ μ L, 3.75 ng/ μ L
Enclose, in each 0.2 mM of two primers of primer centering, 0.4 mM, 0.5 mM, 0.6 mM, 0.8 mM any number and any number it
Between scope, in magnesium chloride 0.1mmol/L, 0.5 mmol/L, 1 mmol/L, 1.5 mmol/L, 2mmol/L any number and appoint
Scope between meaning numerical value, any number and any number in dNTP 5 μm ol/L, 10 μm ol/L, 15 μm ol/L, 20 μm ol/L
Between scope, any number and arbitrarily in archaeal dna polymerase 0.2U/ μ L, 0.25U/ μ L, 0.3U/ μ L, 0.4U/ μ L, 0.5U/ μ L
Scope between numerical value, in Tris-HCL (pH7.5-9.0) 1mM, 2mM, 3mM, 4mM, 5mM between any number and any number
Scope, scope between any number and any number in KCL0.6mM, 1mM, 2mM, 3mM, 4mM, 5mM.
It is highly preferred that final concentration of in PCR reaction system of the component included by above-mentioned dose of box:
Percent by volume is that 2XEasyTaq PCR SuperMix (+dye) of 40-60% is (preferably purchased from the full formula in Beijing gold
Bioisystech Co., Ltd), each 0.2-0.8mM of forward and reverse primer；In above-mentioned PCR reaction system, also include final concentration of 1-
The template DNA of 3.75ng/ μ L.
It is further preferred that final concentration of in PCR reaction system of the component included by above-mentioned dose of box:
Percent by volume is 2X EasyTaq PCR SuperMix (+dye) of 50%, each 0.625mM of forward and reverse primer；
Preferably, in above-mentioned PCR reaction system, also include the template DNA of final concentration of 1.25ng/ μ l；
Such as: the reaction system of 20 μ L includes: 2X EasyTaq PCR SuperMix (+dye) 10 μ L, 5mM's is positive and negative
To each 2.5 μ L of primer, 10ng/ μ l template DNA 2.5 μ L, ddH2O is enough.
Exemplarily, the final concentration of each component in above-mentioned reaction system can be: template DNA 1ng/ μ L, 1.5
In ng/ μ L, 2 ng/ μ L, 2.5 ng/ μ L, 3 ng/ μ L, 3.5 ng/ μ L, 3.75 ng/ μ L between any number and any number
Scope, any number and Arbitrary Digit in each 0.2 mM of two primers of primer centering, 0.4 mM, 0.5 mM, 0.6 mM, 0.8 mM
Scope between value, the percent by volume of 2X EasyTaq PCR SuperMix (+dye) is 40%, 45%, 50%, 55%,
Scope between any number and any number in 60%.
Component included by the PCR kit of the present invention, the final concentration in PCR reaction system can also be: volume hundred
Proportion by subtraction is that the PCR premixed liquid of 85-95% (specially holds up the PCR premixed liquid of section's new industry TSE101-gold medal Mix (1.1X), purchased from north
Jing Qingkexin industry Bioisystech Co., Ltd), each 0.1-1.5 μM of forward and reverse primer；In this PCR reaction system, also include 5-
The template DNA of 20ng/ μ L；
Preferably, final concentration of in PCR reaction system of this component included by agent box: percent by volume is 91%
PCR premixed liquid (specially holds up the PCR premixed liquid of section's new industry TSE101-gold medal Mix (1.1X)), each 0.9 μM of forward and reverse primer；
In this PCR reaction system, also include the template DNA of 13.6ng/ μ L；
Such as: the reaction system of 11 μ L includes: percent by volume be 91% PCR premixed liquid (specially hold up the new industry of section
The PCR premixed liquid of TSE101-gold medal Mix (1.1X)) 10 μ L, the forward and reverse primer mixed liquor 0.5 μ L of 20 μMs (including just,
Each 20 μMs of reverse primer)；In this PCR reaction system, also include the template DNA 0.5 μ L of 300ng/ μ L.
Exemplarily, the final concentration of each component in above-mentioned reaction system can be: template DNA 5ng/ μ L, 10ng/ μ
Scope between any number and any number in L, 15ng/ μ L, 20ng/ μ L, each 0.1 μM of two primers of primer centering, 0.5 μ
M, 0.8 μM, 1 μm M, 0.2 μM, scope between any number and any number in 1.5 μMs, PCR premixed liquid (specially holds up section new
The PCR premixed liquid of industry TSE101-gold medal Mix (1.1X)) percent by volume be in 85%, 88%, 90%, 92%, 95% appoint
Scope between meaning numerical value and any number.
Fourth aspect, the present invention provides a kind of method differentiating lily cultivar Huang Tempo, and the method comprises the following steps:
Step one, pcr amplification reaction: utilize above-mentioned PCR primer to or above-mentioned PCR kit, with Plant Genome to be measured
For template, carry out pcr amplification reaction, obtain pcr amplification product；
The response procedures of this pcr amplification reaction is:
Degeneration: 94-95 DEG C 2-5 minute (exemplarily, can be in 2min, 3min, 4 min, 5 min any number and
Scope between any number)；Amplification: 94-95 DEG C of 10-50 second (exemplarily, can be 10s, 20s, 30s, 35s, 40s,
Scope between any number and any number in 45s, 50s), 54-60 DEG C (can be exemplarily, 54 DEG C, 55 DEG C, 56 DEG C,
57 DEG C, scope between any number and any number in 58 DEG C) the 15-50 second (exemplarily, can be 15s, 30s, 35s,
Scope between any number and any number in 40s, 45s, 50s), 72 DEG C of 25-50 seconds (exemplarily, can be 25s,
30s, 35s, 40s, 45s, 50s) in scope between any number and any number, 30-35 altogether (exemplary ground, permissible
Be 30,31,32,33,34, scope between 35 middle any number and any number) circulation；Extend: 72 DEG C
4-10 minute；
Preferably, the response procedures of this pcr amplification reaction is:
Degeneration: 95 DEG C 3 minutes；Amplification: 95 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 40 seconds, 30 circulations altogether；Extend: 72 DEG C
7 minutes.
Or:
Degeneration: 98 DEG C 2 minutes；Amplification: 98 DEG C 10 seconds, 58 DEG C 15 seconds, 72 DEG C 25 seconds, 30 circulations altogether；Extend: 72 DEG C
4 minutes.
Step 2, detection: detected by pcr amplification product, if, with primer to SEQ ID NO:1 and SEQ ID
NO:2 amplification has obtained specific band SEQ ID NO:5, and/or utilizes primer to expand SEQ ID NO:3 and SEQ ID NO:4
Increase and obtained specific band SEQ ID NO:6；, then treat that measuring plants is lily cultivar Huang Tempo.
By the following examples the detailed description of the invention of the present invention is described in detail.
Embodiment 1
The present embodiment is the acquisition methods of the PCR primer pair differentiating lily cultivar Huang Tempo.The present embodiment has selected 77 altogether
Individual molecular marker is in order to make a distinction lily cultivar Huang Tempo and timber on a molecular scale, and finishing screen is selected 3 amplifications and produced
The RAPD labelling that thing obvious difference, repeatability are good.Utilize these three labelling, in 37-47 DEG C of interval, at three kinds of different brands
PCR instrument on all timber and yellow Tempo can be clearly distinguished；By two specific amplifieds of pair of primers therein (BD3)
Product checks order, and devises 6 pairs of primers altogether according to sequencing result, wherein have 2 pairs of primers can clearly by yellow Tempo with other 5
Common OT type Bulbus Lilii commodity kind distinguishes.Details are as follows:
One, material and method:
1, material:
OT type Bulbus Lilii Huang Tempo (Manissa), timber (Conca d ' or), shady deal (Black out), bursting point
(Flashpoint), be pleasantly surprised (Shocking), Luo Binna (robina) plants ball and is purchased from Beijing Pu Langte gardening company limited；This
6 commodity kinds are imported from Holland.
2, method:
2.1, Bulbus Lilii extracting genome DNA:
Win the blade of above-mentioned 6 kinds ,-40 DEG C of freezings more than 1 day, it is subsequently placed in vacuum drying instrument (CoolSafe
Dehydration in 55-4)；
Take 20 μ g powdered blades again, extract DNA according to CTAB method, comprise the following steps that；
. in described powdered blade, add extract with CTAB liquid (2%CTAB, 1.4mM NaCl, 100mM Tris-HCl
PH8.0,20mM EDTA pH8.0,1%PVP-40,0.2% beta-mercaptoethanol), mixing, in 65 DEG C of water-baths 0.5 hour, obtain
Mixture A；
. mixture A is stopped water-bath, adds isopyknic chloroform/isoamyl alcohol (24:1) and extract twice, obtain supernatant
A；
. in supernatant A, add 2/3 volume isopropanol for precipitating DNA；Wash with lavation buffer solution (75% ethanol) again
Once, dry up, then add TE buffer (10mM Tris-HCl, 1mM EDTA, pH7.4) dissolving, obtain solution A；
. in solution A, add RNase A so that it is final concentration reaches 100 μ g/mL, then mixes 37 DEG C of water-baths 1 hour；Use again
Equal-volume chloroform/isoamyl alcohol (24:1) extracts once, obtains supernatant B；
. to taking, supernatant B adds 1/10 volume 3M sodium acetate pH7.5,2 times of volume dehydrated alcohol, obtain DNA and sink
Form sediment；
. by 75% washing with alcohol DNA precipitation, air-dry, add appropriate ddH2O dissolving DNA, obtains material genomic DNA；
With micro-spectrophotometer with OD260Value (NanoDrop2000) measures concentration, DNA working solution concentration is adjusted to
10ng/μL。
2.2, primer screening:
Have chosen 77 primers of total and carry out PCR amplification (table one), wherein primer 1-60 is Beijing SBS Genetech gene technology
Company limited's RAPD primer, primer 61-77 select from respectively delivered document (Zhao Xiangyun, 1995, Yamagishi, 1995,
ANUSHRI, 2001, Zuo Zhirui, 2005), Beijing SBS Genetech gene technology company limited synthesize.
PCR reaction system is 20 μ L, includes: 10 μ L 2X EasyTaq PCR SuperMix (+dye) (Quan Shijin),
2.5 μ L 5mM primers, 2.5 μ L template DNAs.Amplification program: 95 DEG C of degeneration 3 minutes, amplified reaction be 95 DEG C 30 seconds, 41 DEG C 40
Second, 72 DEG C 50 seconds, 35 circulations altogether, 72 DEG C extension 7 minutes.
Table one: primer information
Above-mentioned document is:
Zhao Xiangyun, 1995: Zhao Xiangyun, Chen Xinlu, Fang Hai, Zhang Yunfang.1995.Evaluate between lily cultivar with R A PD labelling
Genetic affinity.Beijing Agricultural College's journal, 10 (2): 58-63；
Zuo Zhirui, 2005: Zuo Zhirui, Mu Ding, Gao Junping, Liu Chun.2005.Bulbus Lilii genetic diversity and sibship
RAPD analyzes.Gardening journal, 32 (3): 468-472；
ANUSHRI VARSHNEY(2001)Establishment of genetic findelity of in vitro-
raised Lilium bulblets thorough RAPD markers.In Vitro Cell.Dev.Biol.-Plant
37:227-231；
Yamagishi (1995TAG): Detection of section-specific random amplified
polymorphic DNA(RAPD)marker in Lilium.Theor Appl.Genet.91:830-835；
2.3, primer amplification stability test:
2.3.1, the different annealing temperature impact on amplified production
The primer screened in said method 2.2 is selected to carry out expanding stability test.Use thermoelectricity Veriti
Grads PCR instrument, annealing temperature is arranged 6 gradients altogether by 37 DEG C to 47 DEG C, and the temperature difference between adjacent two gradients is 2 DEG C and expands
Increase.PCR reaction system is with described in said method 2.2, and other PCR reaction condition is also identical with said method 2.2.
2.3.2, the different brands PCR instrument impact on amplified production
Using gene 9700, Bole C1000, the PCR instrument of tri-manufacturers of thermoelectricity Veriti, annealing temperature is 41 DEG C, primer
For drawing 43, draw 49, drawing 52, PCR reaction system with described in said method 2.2, other PCR reaction condition also with said method
2.2 it is identical.
2.4, RAPD specific amplified product reclaims, clones and check order
PCR reaction system is 20 μ L, includes: 10 μ L 2X EasyTaq PCR SuperMix (+dye) (Quan Shijin),
2.5 μ L 5mM primers, 2.5 μ L template DNAs.Amplification program: 95 DEG C of degeneration 3 minutes, amplified reaction be 95 DEG C 30 seconds, 41 DEG C 40
Second, 72 DEG C 50 seconds, 35 circulations altogether, 72 DEG C extension 7 minutes.1.4% agarose gel electrophoresis.Length is about 1400bp and
Two specific band cuttings of 900bp are reclaimed.
Operating according to Quan Shi King Company " pEASY-T1 Cloning Kit " test kit description, screening obtains the positive
Clone.Positive colony is selected to entrust AudioCodes prosperous bio tech ltd in Beijing to check order respectively.According to sequencing result profit
By 3 sections of software designs 6 pairs of primers (tables two), Sheng Gong bio-engineering corporation is transferred to synthesize.
Table two: specific primer sequences information
The applying detection of 2.5 special primers
PCR reaction system is 20 μ L, comprises 10 μ L 2X EasyTaq PCR SuperMix (+dye) (Quan Shijin), 2.5 μ
L 5mM special primer (table two), 2.5 μ L template DNAs.Amplification program: 95 DEG C of degeneration 5 minutes, amplified reaction be 95 DEG C 30 seconds, 58
DEG C 40 seconds, 72 DEG C 50 seconds, 35 circulations altogether, 72 DEG C extend 10 minutes.
Two, result:
2.1, primer screening
(numeral represents primer sequence number, letter representation kind, a: yellow Tempo as shown in Figure 1；B: timber), repeat through 1 time
After, two kinds of Bulbus Liliies AFLP system in 16 primers has larger difference, and they are: draw 27, draw 43, draw 46, draw 49, draw 52,
Draw 55, draw 61, draw 63,64, draw 66, draw 68, draw 70-74, draw 77.
Find after repeating for three times, draw 27, to draw 61 amplified bands weak, unstable, is eliminated, draw 46, draw 55, draw 63, draw
64, draw 70, draw 71, draw 73 amplified bands and change greatly, be sometimes difficult to distinguish two parts of materials, therefore these 7 primers also washed in a pan
Eliminate.
Two kinds of Bulbus Liliies drawing 43, draw 49, draw 52, draw 68, draw 74, the comparison in difference that draws in 77 notable, stable.Due to wood
Door drawing 68, draw 74, the amplified band number that draws in 77 more, therefore choose and draw 43, draw 49, draw 52 and carry out expanding stability examination
Test.
2.2, primer amplification stability test
Use thermoelectricity Veriti grads PCR instrument, along with annealing temperature is by 37 DEG C to 47 DEG C step up, draw 43 in kind
In yellow Tempo and timber, the band number of amplified production is also gradually increased, wherein in 6 temperature stable amplification be 1 about
For 1400bp band (as in figure 2 it is shown, odd number swimming lane is " yellow Tempo " amplified production, even number swimming lane is " timber " amplified production,
Rightmost side swimming lane is DNA marker；Primer BD3 is for drawing 43, and BD9 is for drawing 49, and BD12 is for drawing 52).
2.3, RAPD specific amplified product reclaims, clones and check order
(1) time as in figure 2 it is shown, annealing temperature is between 37-47 DEG C, primer BD3 (i.e. drawing 43) have 4 more stable
Amplified production, their size is about 1400bp, 900bp, 750bp and 500bp respectively.Above-mentioned size is only selected to respectively may be about
1400bp, 900bp band sends to the reason of order-checking: the sequencing result of each the most each 100bp in band two ends is insecure, because of
The reliable data of this 750bp and 500bp only have 550bp and 300bp respectively.To select at least one pair of suitable in this interval
Primer, 300bp probability is the lowest, 550bp although it is possible to, but the most final amplified production only has 100-400bp.If only
100bp, amplified production is difficult to distinguish with primer dimer.And just can be seen that from the brightness of DNAmarker, same concentrations
Under, fragment is the least, and brightness is the least, and this point is the most clearly.
(2) two band of a length of 1400bp and 900bp are reclaimed, clone, convert, and random 10 bacterium colonies of picking enter
The detection of row positive colony.As it is shown on figure 3, what wherein D1-D5 inserted is the large fragment of 1400bp, that X1-X5 inserts is 900bp
Small fragment.
Wherein, code name be respectively as follows: the sequencing result of bacterium colony of D1, D4, X1, X3 tetra-order-checking be respectively 1401bp,
1401bp、1463bp、954bp.Owing to X1 sequencing result differs relatively big with expection, so being omitted during design primer.D1、D4
Although meeting expection, and length is 1401bp, but has 156 bases there are differences, and ratio is 11.13%, and this illustrates to survey
The poor accuracy of sequence result.For this respectively with the sequencing result of D1, X3, D4 as foundation, every sequencing result has separately designed 2
Right, 6 pairs of primers (table two) altogether.Find with D1 for the primer sequence according to design simultaneously to be present in X1 simultaneously.
Wherein, above-mentioned D1 bacterium colony sequencing result is following (SEQ ID NO:7): 1401bp altogether；Wherein primer SEQ ID NO:
1,2 all use square frame labelling, primer and between sequence size be 1020bp (being SEQ ID NO:5), primer SEQ ID
NO:3,4 all use underscore labelling, primer and between sequence size be 1209bp (being SEQ ID NO:6)；Oblique with overstriking
Bases different in same position between expression bacterium colony D1 and D4 of body word mark:
Above-mentioned X1 sequencing result is following (SEQ ID NO:8): 1463bp altogether；Wherein primer SEQ ID NO:1,2 all with side
Collimation mark is remembered, primer and between sequence size be 1020bp (being SEQ ID NO:5), primer SEQ ID NO:3,4 all use
Underscore labelling, primer and between sequence size be 1209bp (being SEQ ID NO:6):
TCGAATTCGCGTGTCGCCCTTGAGCCTGCGATATGGCTATGA
GGAGTTTACGGCTTGTGCATTCTCCGTCCTTGAGTGGTATGATCAGACTCTGACCACTCACAAGCAGTCTTTGTGCA
GTTCTTGTGTGGAAGAGTGGAAGGATGACCTGGATAAGGAGATTACATAGCTTCAGAAGAATCATACATGAGAGTTG
GTGACACTTCCAAGAGGCAAGCGGTCGATCGGGTGCAAATGGGTATTCTCGGTGAAGGACGGTGCTTCGGTTTCCAA
AGGTACTAGCGCTGGTATACACTATAAGGCAATATTGGTGGCAAAAGGGTATGCACAAAGGGAGGGTAAAGACTACA
ATGAGGAATTTTCTCCTGCGGTCAAGCACACCTCTATCCGGGTCCTTTTGGCACTAGTTGTGCATTTTGACATGAAG
CTCGAGCAAATGAATGTCAAGACCGCATTCTTGCATAGAGACTTGAAGGAGGATATTTACATGTCACAGCCCGAAGG
TTATGCTGTTGCTGGGAAGGAAGATCAGGTGTGCACGCCTCAGAAATCACTGTACGGGTTGAAGCAGGCTTCAAGGC
AGTGGTACAAGAGGTTTGACAATTTCATGATCGAACATGGGTATGCGAGGAGCTCTTTTGATCCCTGTATGTACAGT
AGGCTGCTCGATGATGGATCCCTTGTGTGTCTCTTGCTCTATGTTGATGACATGCTGATTGCTGTGAGGAGCATGGT
GGAGATTGATAAGTTGAAGTCGATGTTCAGCAGGGAGTTTGAGATGAAGGATCTAGGTTCCGTGAGGAAGATACTCG
GGATGGAGATTGTCAGAGATAGAGGTGAGGGCAGACTGTCTTTGTCACAGCGGGGGTACTTAGAGAAGTTGCTTTGT
CGGTTTGAAATGGATGCAGCTAAGCCGGTGTCTACTCCCCTAGCTGCACACTTCAAACTCTCGGTGATAGCATCTCC
TAGCACTGACGAGGACAGGGAGTACATGGCTAGTGTTCCC
TTTGATATATGCCATGGTTTGTACTCGGCCGGATATCGCACATACGGTTAGTCTCGTGTATCGGTATATGGCGAATC
CGGGAAGGCAGTAATGGCAGGCAGTCAAGTGGATATTGAGATATCTTTGGGGGATGCTAAATAGGAGCATTATCTAT
TAGAGAGGAGCTGGTTCTGATATGGTACAGGGATACATTAACTCTGATTATGCTGGAGATCTTGATAGGTGCCGATC
TACTACAAGTTTCTGTTTTACCTTGACACATGGTCCGGTGAGCTGAAAGTCTCAGTTACAGTCTATTGTTGCTCTAT CCACGACGGAGGCGGAGTACATGGCTCTGACGGAGGCGATAAAGGAAACCATCTGGCTTCGGGGCTCAAGGGCGACA
CGCGAATTCGATATCAAGCTCAGACGAGGCAA。
(3) above-mentioned 6 pairs of primers are carried out PCR amplification with yellow Tempo complete genome DNA for template respectively.As shown in Figure 4, draw
The amplification of thing M1, M2 meets expection；Primer S1, B1, B2 do not have an amplified production, primer S2 amplified production and faint.By
The two lily cultivar can be clearly distinguished in primer M1 and M2, thus selected primer M1 and M2 as specific primer to
In distinguishing lily cultivar Huang Tempo.
Embodiment 2:
The present embodiment is to (M1:SEQ ID NO:1 and SEQ ID NO:2, M2:SEQ by above-mentioned two group-specific primerses
ID NO:3 and SEQ ID NO:4) it is applied to distinguish lily cultivar Huang Tempo and other 5 common OT type lily cultivar, inspection
Survey in its genome and whether contain specific band.
Detection method comprises the following steps:
(1) Plant Genome to be measured is extracted: identical with the method for 2.1 in embodiment 1；Obtain 6 kinds and treat measuring plants (Huang Tian
Despot, timber, shady deal, bursting point, be pleasantly surprised, Luo Binna) genome.
(2) PCR reaction: respectively with M1 and M2 as primer, with above-mentioned 6 kinds of genomes treating measuring plants as template, carry out PCR
Reaction, obtains pcr amplification product.
This PCR reaction system is 20 μ L, includes: 2X EasyTaq PCR SuperMix (+dye) is (purchased from the full formula in Beijing
Gold Bioisystech Co., Ltd) each 2.5 μ L of forward and reverse primer, 10ng/ the μ l template DNA 2.5 μ L, ddH of 10 μ L, 5mM2O foot
Amount.
PCR response procedures is:
Degeneration: 94-95 DEG C 3-5 minute；Amplification: 94-95 DEG C of 30-50 second, 54-60 DEG C of 30-50 second, 72 DEG C of 30-50 seconds, altogether
Count 30-35 circulation；Extend: 72 DEG C 4-10 minute.
" 2.2, primer amplification stability test " of embodiment 1 selected 41 DEG C, to be because initially use be random few core
Nucleotide sequence, annealing region is 37-47 DEG C；The overall banding pattern comprehensive consideration obtained when 41 DEG C is optimal, and characteristic bands exists
All stable existence between 37-47 DEG C；Design, synthesize, use 2 to special primer after, annealing temperature increases, and interval is 54-
60 DEG C, general employing 58 DEG C；In view of the difference of each laboratory monitoring, so the primer selected is all that stability is good；If
2 pairs of primers of meter are high due to specificity, so requiring more relaxed to PCR reaction, can obtain under conditions of the most wide in range
Stable result.
As preferred technical scheme, PCR response procedures is: degeneration: 95 DEG C 3 minutes；Amplification: 95 DEG C 30 seconds, 58 DEG C 30
Second, 72 DEG C 40 seconds, 30 circulations altogether；Extend: 72 DEG C 7 minutes.
(3) electrophoresis detection: by this amplified production with the agarose gel of 1.2%, 0.5h is separated by electrophoresis in 90V invariable power,
Under uviol lamp, photograph is observed afterwards；
As shown in Figure 5: A1-A6 primer be M1, B1-B6 primer be M2.A1, B1 template DNA is yellow Tempo；A2, B2 template
DNA is timber；A3, B3 template DNA is bursting point；A4, B4 template DNA is for being pleasantly surprised；A5, B5 template DNA is Luo Binna；A6、B6
Template DNA is shady deal), during with M1 for primer, yellow Tempo has 1 target stripe, and range estimation size is more bigger than 1Kbp, and reality is
1020bp(SEQ ID NO:5)；Timber, bursting point, being pleasantly surprised does not has any amplified production；Luo Binna indistinctly has 1 near 800bp
Bar weak band, shady deal has 2 bands, and one is less than 1Kbp, other one significantly more than 1200bp.During with M2 for primer, yellow Tempo tool
Having 1 target stripe, range estimation size to be about 1200bp, reality is 1209bp (SEQ ID NO:6)；Timber, bursting point, be pleasantly surprised and
Luo Binna does not has any amplified production；Shady deal has 2 bands, a treaty 1100bp, a treaty 1700bp.
As can be seen here, owing to the amplified production of yellow Tempo having specific amplified band, primer M1 or M2 is utilized
Can distinguish significantly, accurately by the Bulbus Lilii of yellow Tempo with other 5 kinds common OT types.
Embodiment 3:
The present embodiment is to (M1:SEQ ID NO:1 and SEQ ID NO:2, M2:SEQ by above-mentioned two group-specific primerses
ID NO:3 and SEQ ID NO:4) it is applied to distinguish lily cultivar Huang Tempo and timber, detect and whether its genome contains spy
Opposite sex band.
Detection method comprises the following steps:
(1) Plant Genome to be measured is extracted: identical with the method for 2.1 in embodiment 1；Obtain 2 kinds and treat measuring plants (Huang Tian
Despot, timber) genome.
(2) PCR reaction: respectively with M1 and M2 as primer, with above-mentioned 2 kinds of genomes treating measuring plants as template, carry out PCR
Reaction, obtains pcr amplification product.
The cumulative volume of this PCR reaction system is 11 μ L, wherein comprises " holding up section's new industry TSE101-gold medal Mix (1.1X) " PCR
Premixed liquid 10 μ L (purchased from Beijing Qing Kexin industry Bioisystech Co., Ltd), primer mixed liquor (containing each 20 μMs of forward and reverse primer)
0.5 μ L, 300ng/ μ L DNA0.5 μ L；
The program of pcr amplification reaction is: degeneration: 98 DEG C 2 minutes；Amplification: 98 DEG C 10 seconds, 58 DEG C 15 seconds, 72 DEG C 25 seconds, altogether
Count 30 circulations；Extend: 72 DEG C 4 minutes.
Pcr amplification reaction in the present embodiment significantly reduces the time of PCR reaction.
(3) electrophoresis detection: by this amplified production with the agarose gel of 1.2%, 0.5h is separated by electrophoresis in 90V invariable power,
Under uviol lamp, photograph is observed afterwards；See Fig. 6: wherein the template DNA of M1 is timber, does not contains specific band；The template of H4, H5
DNA is yellow Tempo, during with M1 for primer, has 1 range estimation specific band more bigger than 1Kbp, and reality is 1020bp (SEQ
ID NO:5), during with M2 for primer, there is the specific band that 1 range estimation is about 1200bp, reality is 1209bp (SEQ ID
NO:6)。
As can be seen here, owing to the amplified production of yellow Tempo having specific amplified band, primer M1 or M2 is utilized
Can distinguish significantly, accurately by timber Bulbus Lilii similar to outward appearance for yellow Tempo.
Obviously, the above-mentioned embodiment of the present invention is only by clearly demonstrating the citing that the present invention is done, and is not
Restriction to embodiments of the present invention.For those of ordinary skill in the field, on the basis of the above description also
Change or the variation of other multi-forms can be made.Here cannot all of embodiment be given exhaustive.Every belong to this
What the technical scheme of invention was extended out obviously changes or changes the row still in protection scope of the present invention.
Claims (10)
1. the PCR primer pair differentiating lily cultivar Huang Tempo, a primer therein has the SEQ ID in sequence table
The nucleotide sequence of NO:1, another primer has the nucleotide sequence of the SEQ ID NO:2 in sequence table；Or
A primer therein has the nucleotide sequence of the SEQ ID NO:3 in sequence table, and another primer has sequence table
In the nucleotide sequence of SEQ ID NO:4.
2. differentiate a specific band for lily cultivar Huang Tempo, primer described in claim 1 react carrying out PCR
Arrive.
3. differentiate the specific band of lily cultivar Huang Tempo as claimed in claim 2, there is the SEQ ID NO:5 in sequence table
Nucleotide sequence；Or
There is the nucleotide sequence of SEQ ID NO:6 in sequence table.
4. the PCR kit differentiating lily cultivar Huang Tempo, it is characterised in that: including: PCR draws as claimed in claim 1
Thing pair.
The PCR kit of discriminating lily cultivar Huang Tempo the most according to claim 4, it is characterised in that: also include dNTP,
Magnesium chloride, archaeal dna polymerase and PCR buffer.
PCR kit the most according to claim 5, it is characterised in that: the component that described test kit includes is in PCR reaction system
In final concentration of: two each 0.2-0.8mmol/L of primer of described primer centering, described dNTP 5-20 μm ol/L, described chlorination
Magnesium 0.1-2mmol/L, described archaeal dna polymerase 0.2-0.5U/ μ L, Tris-HCL (pH7.5-9.0) 1-5mmol/L and KCL0.6-
5mmol/L is as described PCR buffer.
PCR kit the most according to claim 4, it is characterised in that: the component that described test kit includes is in PCR reaction system
In, the percent by volume of PCR premixed liquid is 85-95%, two primer each 0.1-1.5 μm ol/L of described primer centering.
8. the method differentiating lily cultivar Huang Tempo, it is characterised in that: said method comprising the steps of:
PCR reactions steps: utilize PCR primer described in claim 1 to or claim 2-4 according to any one of PCR reagent
Box, with Plant Genome to be measured as template, carries out PCR reaction, obtains pcr amplification product；
Detecting step: detected by described pcr amplification product, as contained specific band, then institute in described pcr amplification product
State and treat that measuring plants is described lily cultivar Huang Tempo.
Method the most according to claim 8, it is characterised in that: the response procedures of described pcr amplification reaction is:
Degeneration: 94-95 DEG C 2-5 minute；Amplification: 94-95 DEG C of 10-50 second, 54-60 DEG C of 15-50 second, 72 DEG C of 25-50 seconds, amounts to
30-35 circulation；Extend: 72 DEG C 4-10 minute.
Method the most according to claim 8 or claim 9, it is characterised in that:
When described PCR primer to for: a primer therein has the nucleotide base sequence of the SEQ ID NO:1 in sequence table
Row, when another primer has the nucleotide base sequence of the SEQ ID NO:2 in sequence table, described specific band has sequence
The nucleotide sequence of the SEQ ID NO:5 in list；
When described PCR primer to for: a primer therein has the nucleotide base sequence of the SEQ ID NO:3 in sequence table
Row, when another primer has the nucleotide base sequence of the SEQ ID NO:4 in sequence table, described specific band has sequence
The nucleotide sequence of the SEQ ID NO:6 in list.
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| 中华人民共和国农业部: "百合品种鉴定技术规程SSR分子标记法", 《中华人民共和国农业行业标准》 * |
| 陈名红等: "适于百合遗传多样性分析的ＲAPD 引物筛选", 《种子》 * |
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