CN110514495A - The measuring method of nucleus extraction buffer and Piper Plant Genome size - Google Patents
The measuring method of nucleus extraction buffer and Piper Plant Genome size Download PDFInfo
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- CN110514495A CN110514495A CN201910815313.5A CN201910815313A CN110514495A CN 110514495 A CN110514495 A CN 110514495A CN 201910815313 A CN201910815313 A CN 201910815313A CN 110514495 A CN110514495 A CN 110514495A
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- 239000011536 extraction buffer Substances 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 9
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims abstract description 8
- 241000196324 Embryophyta Species 0.000 claims abstract description 7
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 claims abstract description 7
- 241000009298 Trigla lyra Species 0.000 claims abstract 4
- 239000000725 suspension Substances 0.000 claims description 12
- 239000012224 working solution Substances 0.000 claims description 11
- 238000000605 extraction Methods 0.000 claims description 8
- 238000004043 dyeing Methods 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 5
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 claims description 5
- 102000005891 Pancreatic ribonuclease Human genes 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 4
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 claims description 4
- 229910052786 argon Inorganic materials 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 244000299906 Cucumis sativus var. sativus Species 0.000 claims 1
- 230000003139 buffering effect Effects 0.000 claims 1
- 239000012634 fragment Substances 0.000 abstract description 12
- 210000004940 nucleus Anatomy 0.000 description 53
- 244000203593 Piper nigrum Species 0.000 description 26
- 235000008184 Piper nigrum Nutrition 0.000 description 24
- 235000002566 Capsicum Nutrition 0.000 description 23
- 239000006002 Pepper Substances 0.000 description 23
- 235000016761 Piper aduncum Nutrition 0.000 description 23
- 235000017804 Piper guineense Nutrition 0.000 description 23
- 241000722363 Piper Species 0.000 description 19
- 238000005259 measurement Methods 0.000 description 19
- 241000671737 Piper flaviflorum Species 0.000 description 13
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 10
- 210000003855 cell nucleus Anatomy 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 239000007983 Tris buffer Substances 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 240000008067 Cucumis sativus Species 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 description 3
- 240000008384 Capsicum annuum var. annuum Species 0.000 description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 235000007516 Chrysanthemum Nutrition 0.000 description 2
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 2
- 241000218922 Magnoliophyta Species 0.000 description 2
- 241000758706 Piperaceae Species 0.000 description 2
- 241001529246 Platymiscium Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 244000080767 Areca catechu Species 0.000 description 1
- 235000006226 Areca catechu Nutrition 0.000 description 1
- 244000025352 Artocarpus heterophyllus Species 0.000 description 1
- 235000008725 Artocarpus heterophyllus Nutrition 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 208000015220 Febrile disease Diseases 0.000 description 1
- 241001112537 Gesneriaceae Species 0.000 description 1
- 240000000950 Hippophae rhamnoides Species 0.000 description 1
- 235000003145 Hippophae rhamnoides Nutrition 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000209477 Nymphaeaceae Species 0.000 description 1
- 240000005534 Piper auritum Species 0.000 description 1
- 235000008180 Piper betle Nutrition 0.000 description 1
- 240000008154 Piper betle Species 0.000 description 1
- 241000746610 Piper laetispicum Species 0.000 description 1
- 240000005546 Piper methysticum Species 0.000 description 1
- 241000746611 Piper wallichii Species 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- -1 argon ion Chemical class 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
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- 239000000470 constituent Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 210000003783 haploid cell Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000001175 peptic effect Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000005586 smoking cessation Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1493—Particle size
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- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to molecular genetics and Study on Evolution field, the in particular to measuring method of nucleus extraction buffer and Piper Plant Genome size.The nucleus extraction buffer is grouped as by following group: 40~50mmol/L Tris-HCl, 0.8v/v%~1.5v/v%Triton X-100,45~50mmol/L NaHSO3, 0.3w/v%~0.5w/v%PVP, water supplies.The nucleus extraction buffer that the present invention uses can determine the Genome Size of 40 parts of Piper resources, and CV value all controls in error range；And other nucleus extraction buffers are only capable of detecting 2-3 part in 4 parts of germplasm to be measured, and fragment is more compared with the nucleus extraction buffer that the present invention uses, peak is wider.Nucleus extraction buffer provided by the invention solves the detector efficiency at Piper plant signal peak, improves accuracy.
Description
Technical field
The present invention relates to molecular genetics and Study on Evolution field, in particular to nucleus extraction buffer and pepper platymiscium
The measuring method of Genome Size.
Background technique
Piper (Piper L.) plant is the important general tropical constituent of Piperaceae (Piperaceae), is base portion quilt
One of maximum category in sub- plant.The subtropical and tropical zones such as South Asia, South America, Central America are distributed mainly on, there are about 1000
Multiple kinds.Piper has the characteristics that rate of differentiation is fast, is to carry out flowering plant evolution quickly to break up machine with Basal angiosperms
Manage the good example of research.There are many resources with economic value and potentiality to be exploited in Piper.Pepper platymiscium is for passing
System medicine is with a history of thousands of years, and in China, India, Latin America and the West Indies is civil that pepper, slips is widely used
Pepper, Caulis et Folium piperis etc. are used as medicine.In the Asia southeast, people often help digest betel and betel nut together with lime chewing.
Pepper (Piper nigrum L.) is Piper perennial evergreen liana, is spice important in the world
One of crop is known as the good reputation of " king of fragrance ".Originate in India, pepper introduces China from nineteen forty-seven, and cultivated area is up to 3 at present
Ten thousand hectares, gross annual output amount is more than 30,000 tons, occupies the 5th, the world.Pepper is the favorite flavouring of people, in modern pharmaceutical, recklessly
Green pepper uses as peptic and antipyretic, also can be widely used to the fields such as smoking cessation, drug rehabilitation and military affairs.China's pepper growing and cultivation
Kind is drawn with heat based on No. 1 (P.nigrum c.v.Reyin-1), be there is a problem of that cultivar is single and resistance is weak, is influenced
The expansion of China pepper planting scale and the extension of pepper industry, it would be highly desirable to carry out excavating resource and innovation in wild relatives
The work such as utilize.
Pepper wild relatives are numerous, and part resource has merit, such as PIPER FLAVIFLORUM (P.flaviflorum) has
There are stronger anti-seasonal febrile diseases, great Ye Betel (P.laetispicum) has longer flower spike, and Caulis et Folium piperis (P.wallichii) has
Stronger winter resistance etc..But Genome Size is different between Piper difference wild relatives, and many kinds of genome is big
It is small also unclear, it causes to hinder to using wild relatives creative utilization, therefore, carries out the estimation of Piper resource Genome Size and grind
Reference can be provided for germplasm innovation by studying carefully.
Genome Size (C-value) refers to haploid cell core in certain biology or the DNA content in gametophyte, commonly uses
Weight pik (10-12G) or nucleotide base indicates quantity (base pair/Gb).Genome Size both with the life of plant
There is stronger correlations for object character, and have certain relationship with the evolution position of species.The side of Genome Size measurement
There are many methods, and traditional having inspires confidence in the micro- development method of your root and chemical analysis, and new technology flow cytometry (Flow
Cytometry, FCM) have the characteristics that accurately analyze a large amount of cells in moment, make its surmounted it is traditional micro- and
Chemical analysis technology is a kind of measurement Genome Size rapidly and effectively method.Clinic of the flow cytometry in humans and animals
It is widely used in basic research, still, due to containing the secondary metabolites such as a large amount of polysaccharide, polyphenol in plant, sternly
The fluorescent staining intensity for affecting PI dyestuff again reduces the accuracy of measurement Genome Size.Therefore clearly it is suitble to measurement object
The nucleus extraction liquid and measuring method of kind flow cytometry are most important to the species gene group size research.
CN104075983A Chinese patent discloses a kind of measuring method suitable for Gesneriaceae Genome Size,
CN107449717A Chinese patent discloses a kind of method for measuring nymphaeaceae plant Genome Size, but in this two pieces patent
Nucleus extraction buffer is used to all form complete signal peak when Piper Plant Genome size.
Summary of the invention
In view of this, the present invention provides nucleus extraction buffer and the measurement sides of Piper Plant Genome size
Method.The nucleus extraction buffer can determine the Genome Size of 40 parts of Piper resources, and CV value is all controlled in error model
In enclosing, the detector efficiency at Piper plant signal peak is improved, accuracy is improved.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of nucleus extraction buffers, are grouped as by following group:
40~50mmol/L Tris-HCl, 0.8v/v%~1.5v/v%Triton X-100,45~50mmol/L
NaHSO3, 0.3w/v%~0.5w/v%PVP, water supplies.
Preferably, being grouped as by following group:
50mmol/L Tris-HCl, 1.0v/v%Triton X-100,50mmol/L NaHSO3, 0.5w/v%PVP, water
It supplies.
Preferably, the pH value of nucleus extraction buffer is 7.0~7.5.
Preferably, the pH value of nucleus extraction buffer is 7.0.
The present invention also provides a kind of measuring methods of Piper Plant Genome size, include the following steps:
(1) Piper plant leaf blade is taken, using cucumber young leaflet tablet as internal reference, is pre-processed；
(2) step (1) pretreated blade is placed in the nucleus extraction buffer of pre-cooling, is shredded, stood, mistake
Filter obtains nucleus suspension；
(3) nucleus extraction buffer, RNase A and PI working solution are added in the nucleus suspension that step (2) obtain,
30~50min of dyeing is protected from light after mixing, the nucleus suspension after being dyed；
(4) it using the nucleus suspension after flow cytomery dyeing, is analyzed through data, obtains Piper plant gene
Group size.
Preferably, Piper plant leaf blade is 1 leaf.
Preferably, pretreatment are as follows: rinse blade well, then rinsed 2~3 times with water, impregnate 5~10min.
In specific embodiment provided by the invention, pretreatment are as follows: blade is rinsed well, then is rinsed 2 times with water, is impregnated
5min is cleaned blade surface moisture with paper.
Preferably, chopping is using the double-edged razor blade with a thickness of 0.08mm.
Preferably, the time stood is 4~6min；The pore size of filtering is 400~600 mesh.
Preferably, the time of standing is 5min；The pore size of filtering is 400 mesh.
Preferably, the concentration of propidium iodide is 0.8~1.2mg/mL in PI working solution.
Preferably, the concentration of propidium iodide is 1mg/mL in PI working solution.
Preferably, nucleus extraction buffer used in blade, step (2), nucleus extraction used in step (3) buffer
The ratio of liquid, RNase A and PI working solution are as follows: the μ of 20~40mg:0.4~0.6mL:1.8~2.2mL:90~110 g:18~22 μ
L。
Preferably, blade, nucleus extraction buffer used in nucleus extraction buffer, step (3) used in step (2),
The ratio of RNase A and PI working solution are as follows: 30mg:0.5mL:2mL:100 μ g:20 μ L.
Preferably, a length of 488nm of excitation light wave of the argon laser transmitter of flow cytometer, sense channel are
The channel FL2 590/50 [488].
In the present invention, first use 3 μm of normalized particles of Calibration Beads by instrument testing to best shape before detection
State, and the coefficient of variation is within 3%；It is collected using flow cytometer software CyFlow Cube 15-CFG at least 5000 thin
Born of the same parents；Each sample replication five times；Utilize FCS Express V3 software analysis data.With standard specimen Genome Size and appearance
Position is control, goes out peak position according to Piper plant leaf blade measurement sample and calculates its Genome Size；Measure going out for sample
Peak position error is no more than ± 6%.
The present invention provides nucleus extraction buffer and the measuring methods of Piper Plant Genome size.The nucleus
Extraction buffer is grouped as by following group: 40~50mmol/L Tris-HCl, 0.8v/v%~1.5v/v%Triton X-
100,45~50mmol/L NaHSO3, 0.3w/v%~0.5w/v%PVP, water supplies.Present invention has the advantage that
(1) the nucleus extraction buffer that the present invention uses can determine the Genome Size of 40 parts of Piper resources, and
CV value all controls in error range；And other nucleus extraction buffers are only capable of detecting 2-3 part in 4 parts of germplasm to be measured,
And fragment is more compared with the nucleus extraction buffer that uses of the present invention, peak is wider.Nucleus extraction provided by the invention is slow
Fliud flushing improves the detector efficiency at Piper plant signal peak, improves accuracy.
(2) experimental implementation of the present invention includes: blade chopping, filtering, dyeing and examination with computer, needs to operate on ice with other
Compared with the method for centrifugation it is easy to operate, it is time saving easy to learn, coefficient of variation CV be lower than 6%, it is reproducible, be Piper plant germplasm
Distant hybridization and creative utilization provide reliable technical support.
Detailed description of the invention
Fig. 1 be using nucleus extraction buffer of the present invention measurement heat draw No. 1 pepper, PIPER FLAVIFLORUM, jalapeno and
The peak figure of kawakaw；
Fig. 2 is the peak figure using nucleus extraction buffer of the present invention measurement standard specimen cucumber；
Fig. 3 be using nucleus extraction buffer of the present invention measurement heat draw No. 1 pepper, PIPER FLAVIFLORUM, jalapeno and
The peak figure of kawakaw branch Different sites of leaf (the next, superior leaf and 1 leaf)；
Fig. 4 is the peak for drawing No. 1 pepper using Otto, PI kit, Tris and nucleus extraction buffer of the present invention measurement heat
Figure；
Fig. 5 is the peak figure that PIPER FLAVIFLORUM is measured using Otto, PI kit, Tris and nucleus extraction buffer of the present invention；
Fig. 6 is the peak that jalapeno is measured using Otto, PI kit, Tris and nucleus extraction buffer of the present invention
Figure；
Fig. 7 is the peak figure that kawakaw is measured using Otto, PI kit, Tris and nucleus extraction buffer of the present invention；
Fig. 8 is to measure heat using nucleus extraction buffer disclosed in CN104075983A to draw No. 1 pepper, chrysanthemum recklessly
The peak figure of green pepper, jalapeno and kawakaw；
Fig. 9 is to measure heat using nucleus extraction buffer disclosed in CN107449717A to draw No. 1 pepper, chrysanthemum recklessly
The peak figure of green pepper, jalapeno and kawakaw.
Specific embodiment
The invention discloses nucleus extraction buffer and the measuring method of Piper Plant Genome size, this field skills
Art personnel can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replacements and
Change apparent to those skilled in the art, they are considered as being included in the present invention.Method of the invention and
Using being described by preferred embodiment, related personnel can obviously not depart from the content of present invention, spirit and scope
It is interior that method described herein and application are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The present invention provides a kind of methods suitable for Piper Plant Genome size measurement, comprising:
(1) 1mg propidium iodide powder is dissolved in 1mL deionized water, is configured to the PI working solution of 1mg/mL, protected in 4 DEG C
It deposits；
(2) Piper plant leaf blade 20-40mg is taken, first rinses blade to remove surface dirt with tap water well, then
Use ddH2O is rinsed 2 times, and is impregnated 5-10 minutes；Surface moisture is blotted with filter paper, is placed in the culture dish that diameter is 35mm, it will
Blade is totally submerged in the nucleus extraction buffer of 0.5ml pre-cooling, after quickly shredding with a thickness of 0.08mm double-edged razor blade
Extracting solution and blade fragment are scraped to culture dish side and tiltedly put and stands 5min, then obtains cell with 400-600 mesh membrane filtration
Core suspension is into the dedicated loading pipe of flow cytometer；Standard specimen nucleus suspension preparation method is as described above；
(3) be slowly added into nucleus suspension 2ml nucleus extraction buffer of the present invention, 20ulPI working solution and
6ulRNase A (final concentration of 40ug/ml), mixing is gently blown and beaten with liquid-transfering gun, is protected from light 30~50min of dyeing；
(4) Partec company, Germany is usedCube6 flow cytometer is detected and analyzed, argon ion
The excitation wavelength of laser emitter is adopted as 488nm, and sense channel is the channel FL2 590/50 [488]；It is first used before detection
Calibration Beads 3um normalized particle is by instrument testing to optimum state, and the coefficient of variation is within 3%；Utilize stream
Formula cell instrument carries software CyFlow Cube15-CFG at random and collects at least 5000 cells；Each sample replication five times；
(5) FCS Express V3 software analysis data is utilized, is control with standard specimen Genome Size and out peak position, according to
Sample goes out peak position and calculates its Genome Size according to surveying and determination；The appearance location error for measuring sample is no more than ± 6%.
Preferably, Piper plant leaf blade described in the step (2) is 1 leaf.
Preferably, nuclear isolation buffer component described in the step (2) or (3) are as follows: 40-50mmol/L
Tris-HCl, 0.8-1.5%Triton X-100,45-50mmol/L NaHSO3,0.3-0.5%PVP (W/V), pH value are
7.0-7.5。
Examination used in the measuring method of nucleus extraction buffer and Piper Plant Genome size provided by the invention
Agent or instrument are available on the market.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1
Draw No. 1 pepper (P.nigrum cv Reyin1), PIPER FLAVIFLORUM with the different Piper germplasm heat in 4 parts of source places
(P.flaviflorum), for jalapeno (P.auritum) and kawakaw (P.methysticum), acquisition heat draws 1
Number pepper, PIPER FLAVIFLORUM, jalapeno, kawakaw and each 30mg of cucumber tender leaf, using cucumber as standard specimen；
Prepare nucleus extraction buffer, formula are as follows: 50mmol/L Tris-HCl, 1.0v/v%Triton X-100,
50mmol/L NaHSO3, 0.5w/v%PVP, pH value 7.0.
Blade is put into the culture dish that joined 0.5mL nucleus extraction buffer of the present invention, with sharp two-edged knife
Heat is quickly drawn No. 1 pepper respectively by piece, PIPER FLAVIFLORUM, jalapeno, kawakaw and cucumber tender leaf shred, then with 400
Mesh membrane filtration obtains nucleus suspension into the dedicated loading pipe of flow cytometer；
2mL nucleus extraction buffer, 20 μ LPI working solutions and 6 μ LRNase A (final concentration of 40 μ g/mL) are added, use
Liquid-transfering gun gently blows and beats mixing, is protected from light dyeing 30min；Using German Partec companyCube6 flow cytometer
It is detected, using 488nm excitation, FL2 Air conduct measurement fluorescence intensity 590/50；It is carried at random using flow cytometer
Software CyFlow Cube 15-CFG collects at least 5000 cells；Each sample replication five times；Utilize FlowJo-V10
Software analysis data is control with standard specimen Genome Size and out peak position, goes out peak position according to measurement sample and calculates its base
Because of a group size；
By Fig. 1 and Fig. 2 can get heat draw No. 1 pepper, PIPER FLAVIFLORUM, jalapeno, kawakaw Genome Size
Mean value be respectively (845.76 ± 10.03) Mb, (527.06 ± 7.75) Mb, (536.56 ± 13.15) Mb and (1127.36 ±
18.38) Mb, CV mean value are respectively 4.62 ± 0.27,4.09 ± 0.59,3.64 ± 0.43 and 4.43 ± 0.33.
Comparative example 1:
This comparative example is substantially the same manner as Example 1, and sampling blade is respectively inferior leaf, superior leaf and leaf, other steps
Rapid same as Example 1, the peak figure tested is shown in Fig. 3.From figure 3, it can be seen that 4 parts of Piper germplasm use inferior leaf and upper
When the leaf detection of position, minority cannot form complete signal peak, and majority forms complete signal peak, but the signal with leaf
Peak is compared, and the signal peak fragment that inferior leaf and superior leaf are formed is more, and peak is wider, and cell nucleus collection is on the low side.
Comparative example 2:
This comparative example is substantially the same manner as Example 1, and changing cell nucleus Extraction buffer into Otto, (Yang Zhuanying waits different
Strain jackfruit ploidy and Genome Size analyze Journal of Fruit Science, 2015,32 (4): 567-571.), Tris (Zhou Xiang
It is gorgeous, the comparison Gansu Agriculture University journal of 4 kinds of Flow cytometry extraction sea-buckthorn core DNA methods is waited, 2012,47 (4):
155-160.) and three kinds of buffers of PI kit (Guangzhou Ji Yuan Biotechnology Co., Ltd), other steps and 1 phase of embodiment
Together, the peak figure tested is shown in Fig. 4-7.
From fig. 4, it can be seen that being unable to shape using Otto and PI Kits A cell core extracting solution when measurement heat draws No. 1 pepper
At complete signal peak, complete signal peak is formd using Tris nucleus extraction liquid, but fragment is more, peak is wider, and adopts
Complete signal peak is also formed with nucleus extraction buffer in the present invention, fragment is relatively fewer, and peak is relatively narrow, cell nucleus collection
It is more.
From fig. 5, it can be seen that when measurement PIPER FLAVIFLORUM, not using Otto, PI kit and Tris nucleus extraction liquid
Complete signal peak can be formed, and nucleus extraction buffer in the present invention is used to form complete signal peak, fragment is opposite
Less, peak is relatively narrow, and cell nucleus collection is more.
From fig. 6, it can be seen that when measurement jalapeno, using nucleus in Otto, PI kit, Tris and the present invention
Extracting solution can form complete signal peak, but using the signal peak fragment of nucleus extraction buffer in the present invention obviously compared with
Few, peak is relatively narrow, and cell nucleus collection is more.
From figure 7 it can be seen that cannot all be formed completely when measurement kawakaw using Otto and Tris nucleus extraction liquid
Signal peak, complete signal peak is formd using PI Kits A cell core extracting solution, but fragment is more, peak is wider, and uses
Nucleus extraction buffer also forms complete signal peak in the present invention, and fragment is relatively fewer, and peak is relatively narrow, cell nucleus collection compared with
It is more.
Comparative example 3:
This comparative example is substantially the same manner as Example 1, and cell nucleus Extraction buffer is changed into CN104075983A and is disclosed
The nucleus extraction buffer (formula of LB01 are as follows: 15mM Tris, 2mM Na2EDTA, 0.5mM spermine.4HC1,
80mM KCl, 20mM NaCl, 15mM β-mercaptoethanol, 0.1% (v/v) Triton X-100, pH7.5), other steps
Rapid same as Example 1, the peak figure tested is shown in Fig. 8.
From figure 8, it is seen that being capable of forming complete letter when measurement heat draws No. 1 pepper, jalapeno and kawakaw
Number peak, but compared with nucleus extraction buffer of the present invention, signal peak fragment is slightly more, and peak is wider.But when measuring PIPER FLAVIFLORUM,
Signal peak cannot be formed.
Comparative example 4:
This comparative example is substantially the same manner as Example 1, and cell nucleus Extraction buffer is changed into CN107449717A and is disclosed
Nucleus extraction buffer (6. WPB buffer:0.2M Tris-HCl, 4mM MgCl2.6H2O, 2mM Na2EDTA, 86mM
NaCl, 10mM sodium pyrosulfite, 1%PVP-10, l%V/V Triton X-100 adjust pH to 7.5, the filter of 0.22 μm of filtering
Film；7. mGb buffer:45mM MgCl2.6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40,0.2%V/V
Triton X-100,10mM Na2EDTA, 20 μ L/mL β-mercaptoethanol adjust pH to 7.0, the filter of 0.22 μm of filtering
Film), other steps are same as Example 1, and the peak figure tested is shown in Fig. 9.
From fig. 9, it can be seen that being capable of forming complete letter when measurement heat draws No. 1 pepper, jalapeno and kawakaw
Number peak, but compared with nucleus extraction buffer of the present invention, signal peak fragment is more, and peak is wider.But when measuring PIPER FLAVIFLORUM,
Signal peak cannot be formed.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of nucleus extraction buffer, which is characterized in that be grouped as by following group:
40~50mmol/L Tris-HCl, 0.8v/v%~1.5v/v%Triton X-100,45~50mmol/L NaHSO3,
0.3w/v%~0.5w/v%PVP, water are supplied.
2. nucleus extraction buffer according to claim 1, which is characterized in that be grouped as by following group:
50mmol/L Tris-HCl, 1.0v/v%Triton X-100,50mmol/L NaHSO3, 0.5w/v%PVP, water supplies.
3. nucleus extraction buffer according to claim 1 or 2, which is characterized in that the nucleus extraction buffer
PH value be 7.0~7.5.
4. nucleus extraction buffer according to claim 3, which is characterized in that the pH of the nucleus extraction buffer
Value is 7.0.
5. a kind of measuring method of Piper Plant Genome size, which comprises the steps of:
(1) Piper plant leaf blade is taken, using cucumber young leaflet tablet as internal reference, is pre-processed；
(2) step (1) pretreated blade is placed in the nucleus extraction buffer of pre-cooling, is shredded, stood, filtering obtains
Obtain nucleus suspension；
(3) nucleus extraction buffer, RNase A and PI working solution are added in the nucleus suspension that step (2) obtain, mixes
After be protected from light 30~50min of dyeing, the nucleus suspension after being dyed；
(4) it using the nucleus suspension after flow cytomery dyeing, is analyzed through data, it is big to obtain Piper Plant Genome
It is small.
6. measuring method according to claim 5, which is characterized in that the Piper plant leaf blade is 1 leaf.
7. measuring method according to claim 5, which is characterized in that the pretreatment are as follows: rinse blade well, then use
Water rinses 2~3 times, impregnates 5~10min.
8. measuring method according to claim 5, which is characterized in that the time of the standing is 4~6min；The filtering
Pore size be 400~600 mesh.
9. measuring method according to claim 5, which is characterized in that the concentration of propidium iodide is in the PI working solution
0.8~1.2mg/mL, nucleus extraction buffering used in nucleus extraction buffer, step (3) used in the blade, step (2)
The ratio of liquid, RNase A and PI working solution are as follows: the μ of 20~40mg:0.4~0.6mL:1.8~2.2mL:90~110 g:18~22 μ
L。
10. measuring method according to claim 5, which is characterized in that the argon laser of the flow cytometer emits
The a length of 488nm of the excitation light wave of device, sense channel are the channel FL2 590/50 [488].
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