CN104316373A - Extraction method for cell nucleuses of eggplant leaves suitable for flow cytometry - Google Patents

Extraction method for cell nucleuses of eggplant leaves suitable for flow cytometry Download PDF

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CN104316373A
CN104316373A CN201410566300.6A CN201410566300A CN104316373A CN 104316373 A CN104316373 A CN 104316373A CN 201410566300 A CN201410566300 A CN 201410566300A CN 104316373 A CN104316373 A CN 104316373A
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eggplant
minutes
flow cytometry
extraction
nucleus
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周晓慧
鲍生有
刘军
庄勇
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Jiangsu Academy of Agricultural Sciences
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Jiangsu Academy of Agricultural Sciences
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Abstract

The invention provides an extraction method for cell nucleuses of eggplant leaves suitable for a flow cytometry, belonging to the technical field of biology. The extraction method comprises the following steps: preparing cell nucleus extraction buffer solution, sampling, preparing cell nucleus suspension liquid and analyzing by a flow cytometry, wherein the cell nucleus extraction buffer solution comprises the following components: CPW salt solution, 0.5M mannitol, 3mmol/L HEPES, 0.25% of PEG, 0.5% of TritonX-100, and 0.25% of mercaptoethanol, and the pH of the ell nucleus extraction buffer solution is 6.5-7.0. The extraction method further solves the problems of low yield and low purity of cell nucleus extraction of eggplant leaves by determining proper quantity of samples and through multiple times of filtering, the extraction time is short, the obtained purity of the cell nucleuses of the eggplant leaves is high, the extraction method is suitable for analyzing by the flow cytometry, peak of the obtained stream map is high, and slightly influenced by peak of impurities, the DNA ploidy of a to-be-tested material can be easily identified, and thus foundation is laid for large-batch ploidy identification of eggplant material.

Description

A kind of eggplant leaf nucleus extraction method being suitable for flow cytometry
Technical field
The invention provides a kind of eggplant leaf nucleus extraction method being suitable for flow cytometry analysis, belong to biological technical field.
Background technology
Flow cytometer is the novel high-tech instrument be integrated with laser technology, photoelectric measurement technology, computer technology, hydromechanics technology, electronics physics, cellular immunofluorescence chemical technology and monoclonal antibody technique.Various tiny organism particle is as cell, nucleus or chromosome quick streamlined flow in sheath fluid, one by one by a branch of incident beam, and record scattered light and various fluorescence signal with high sensitivity detector, multiparameter, fast quantitative test and sorting are carried out to the nucleus in sheath fluid or other particulates.The research of flow cytometer in higher plant is started late, and what be most widely used is utilize cells were tested by flow cytometry plant cell dna content, ploidy analysis and cell cycle analysis.But there is cell membrane due to plant, and individual complete in order to prepare, not assemble and a large amount of nucleus is more difficult for the single-cell suspension liquor ratio of flow cytometry analysis, thus hinder the application of flow cytometer in plant research.
Utilize flow cytometer can identify the ploidy of each Plants rapidly and accurately, but the concentration of the examined sample cell core extraction of the result of Analysis and Identification and impurities affect.If the concentration of sample cell core suspending liquid is too low, then the peak value occurred in analysis chart can be very low, makes result judge; If the impurity in sample core suspending liquid is more, then peak figure is wider, impurity peak height, equally cannot accurate judged result, and therefore, the sample cell core suspending liquid obtaining high concentration and purity is the key utilizing flow cytometry plant ploidy.
Eggplant (Solanum melongena L.) originates from torrid areas, Southeast Asia, Asia, and ancient Gingko is for tame ground the earliest.The with a long history of eggplant is cultivated by China, it is generally acknowledged that China is eggplant second area of origin.Eggplant is that one of wider solanaceous vegetables is cultivated by China, it not only has higher nutritive value, and eating method is also varied, be a kind of can fresh dry-mate connection, year-round supply, economical and practical bulk vegetable, the dark welcome by numerous producers and consumers.Especially in recent years along with the development of industrialized agriculture, eggplant facility cultivation obtains significant progress, and becomes the important component part of high-efficiency agriculture, plays an important role in guarantee market supply and promotion increasing peasant income.
Along with the development of Plant Biotechnology, plant anther cultivation and pollen cultures technology are that the seed selection of New Eggplant Varieties opens a new way.Utilize eggplant flower pesticide and pollen cultures can create the material of eggplant Different Ploidy, Innovation Germplasm resource, Eggplant Varieties improvement is had great importance.Therefore, how the ploidy of Rapid identification material is one of important content in eggplant ploidy studies.Although chromosome counting method accuracy is high, complicated operation, the Ploidy Identification for lot of materials is still quite difficult.And use cells were tested by flow cytometry vegetable cell nuclear dna content to identify that its ploidy is quick, an easy and approach that accuracy is higher.Therefore, find the preparation method of the eggplant nuclear particulate being applicable to flow cytometry analysis, use flow cytometer research eggplant ploidy is had great importance.
At present, on vegetable crop, the Tris of employing damping fluid extracts nucleus more, and its component is: 15mmol/L Tris-HCl, 80mmol/L KCl, 20mmol/L NaCl, 20mmol/L EDTA-Na2,15mmol/L mercaptoethanol and 0.1% (v/v) TritonX-100, pH7.5.Also Activities of Some Plants is had to adopt Otto damping fluid and MgSO 4damping fluid, but because different vegetable material is because of the difference of its structure and component, the nucleus preparation method being applicable to a Plants might not be suitable for all plants.Inventor also utilizes these three kinds of damping fluids of forefathers and extracting method to prepare the nucleus suspension of eggplant leaf, but does not obtain good Detection results, there is the problem that extraction efficiency is not enough and purity is low, cannot meet the detection analysis of flow cytometer.Thus for these problems and by the experience of forefathers, inventors performed the optimization of nucleus extraction formula of liquid and the improvement of extracting method, be intended to the accuracy and the success ratio that increase sample detection, reduce the concentration of impurity in core suspension, reduce the impact that impurity flow cytometer is analyzed.
Summary of the invention
Technical matters the object of this invention is to provide a kind of nucleus extraction method being suitable for the eggplant leaf of flow cytometry, the method can increase accuracy and the success ratio of sample detection, reduce the concentration of impurity in nuclei suspension, reduce the impact of impurity flow cytometric analysis.The method can be used for flow cytometry eggplant Different Ploidy material, also for the flow cytometry ploidy studies of other crops provides reference.
Technical scheme
In order to realize the object of the invention, the invention provides a kind of nucleus extraction method being suitable for the eggplant leaf of flow cytometry, its nucleus extraction buffer composition used is:
CPW salt solusion (KH 2pO 427.2mg/L, KNO 3101.0mg/L, CaCl 2.2H2O 1480.0mg/L, MgSO 4.7H 2o 246.0mg/L, KI 0.16mg/L, CuSO 4.5H 2o 0.025mg/L, pH5.8), 0.5M sweet mellow wine (mannitol), the 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) of 3mmol/L, 0.25% (w:v) polyglycol (PEG), 0.5% (v:v) Triton X-100,0.25% (v:v) mercaptoethanol, pH6.5-7.0.
Colouring method used in nucleus extraction method of the present invention for first adding after RNaseA digests 10 minutes in nucleus extraction liquid, then adds PI (propidium iodide) and dyes.
Nucleus extraction method of the present invention, concrete steps are as follows:
1) sample: get 0.5 ~ 1 gram of tender leaf from healthy and strong plant the morning in fine day, be placed on 4 DEG C of low temperature refrigerators with self-sealing plastic bag splendid attire and preserve stand-by.
2) nuclei suspension is prepared: the nucleus extraction damping fluid get 0.5 ~ 1 gram of eggplant tender leaf being added 5 ~ 10 milliliters, i.e. sample size: damping fluid consumption=1:10 (w/v), be filtered in centrifuge tube after being shredded with sharp single-edge blade under 4 DEG C of conditions, 4 DEG C of stationary incubation 10 ~ 15 minutes.1000r/min, after centrifugal 10 minutes, abandons supernatant, and precipitation adds the flushing of nucleus Extraction buffer and once filters afterwards, namely obtains nuclei suspension.
3) dye: in the nuclei suspension prepared, first add the endonuclease (RnaseA) that final concentration is 50ug/ml, digest after 10 minutes, add the DNA fluorescent dye propidium iodide (PI) that final concentration is 50ug/ml, 4 DEG C of dyeing are after more than 30 minutes, refilter once with 500 object nylon wires, sample detection.
4) flow cytometry analysis: carry out sample detection on the flow cytometer of U.S. company BD, use low speed collecting cell core, and showing FL2 histogram (DNA content histogram), horizontal ordinate is with the area of relative dna content reflection fluorescence curve, and ordinate is cell check figure.
Described step 2) in, with 300 object nylon net filters after being shredded with sharp single-edge blade under 4 DEG C of conditions.Precipitation adds the flushing of nucleus Extraction buffer and once uses 500 object nylon net filters afterwards.
Beneficial effect
Good effect of the present invention:
1. present invention employs a kind of damping fluid of applicable eggplant leaf nucleus extraction, use the CPW salt solusion of conventional washing bioplast, and in damping fluid, add sweet mellow wine, polyglycol and 4-hydroxyethyl piperazine ethanesulfonic acid, be conducive to maintaining nuclear integrality, obtain the nuclei suspension that concentration is higher, to reach the level of flow cytomery.
2. in nuclei suspension leaching process provided by the invention, 10 minutes are digested by first adding RnaseA, add PI dyeing again, instead of add RnaseA and PI simultaneously, and increase after dyeing and once filter, can reduce cell fragment, the peak value in the streaming collection of illustrative plates of acquisition is high, impact by impurity peaks is little, can extract eggplant leaf nucleus easily and fast and efficiently for flow cytometry analysis.
Accompanying drawing explanation
Fig. 1 eggplant nuclei suspension extracts process flow diagram
Fig. 2 adopts damping fluid of the present invention and extracting method to carry out the collection of illustrative plates of flow cytomery
Fig. 3 adopts existing Tris damping fluid and extracting method to carry out the collection of illustrative plates of flow cytomery
Fig. 4 adopts existing MgSO4 damping fluid and extracting method to carry out the collection of illustrative plates of flow cytomery
Embodiment
With Eggplant Varieties " No. 3, Su Qi " for material carries out flow cytometer ploidy identification, purchased from Jiang Shu seedling company limited of Jiangsu Province.The present invention is further illustrated by reference to the accompanying drawings with embodiment.Implementation process is as follows:
Nucleus extraction damping fluid of the present invention, component is: CPW salt solusion (KH 2pO 427.2mg/L, KNO 3101.0mg/L, CaCl 2.2H 2o 1480.0mg/L, MgSO 4.7H 2o 246.0mg/L, KI 0.16mg/L, CuSO 4.5H 2o 0.025mg/L, pH5.8), 0.5M sweet mellow wine, the HEPES of 3mmol/L, 0.25% (w:v) PEG, 0.5% (v:v) TritonX-100,0.25% (v:v) mercaptoethanol, pH6.5-7.0.
Extraction concrete steps are as follows:
1) sample: get 0.5 gram of tender leaf from healthy and strong plant the morning in fine day, be placed on 4 DEG C of low temperature refrigerators with self-sealing plastic bag splendid attire and preserve stand-by.
2) nuclei suspension is prepared: the nucleus extraction damping fluid of the present invention get 0.5 gram of eggplant tender leaf being added 5 milliliters, shredded with sharp single-edge blade under 4 DEG C of conditions, 300 object nylon net filters in centrifuge tube, 4 DEG C of stationary incubation 10 ~ 15 minutes.1000r/min, after centrifugal 10 minutes, abandons supernatant, and precipitation adds nucleus Extraction buffer and rinses once, and with 500 object nylon net filters, namely obtains nuclei suspension.
3) dyeing and flow cytometry analysis: first add the RnaseA (final concentration is 50ug/ml) that final concentration is 50ug/ml in the nuclei suspension prepared, after 10 minutes, add PI (final concentration is 50ug/ml), 4 DEG C of dyeing are after more than 30 minutes, refilter once with 500 object nylon wires, sample detection.
4) flow cytometry analysis: carry out sample detection on the FACSCalibur flow cytometer of U.S. company BD, use low speed collecting cell core, and show FL2 histogram (DNA content histogram), horizontal ordinate is with the area of relative dna content reflection fluorescence curve, and ordinate is cell check figure.
Three kinds of made in nuclear buffer conventional in prior art have Tris damping fluid, Otto damping fluid and MgSO 4damping fluid.
A:Tris damping fluid: 15mmol/L Tris-HCl, 80mmol/L KCl, 20mmol/L NaCl, 20mmol/L EDTA-Na 2, 15mmol/L mercaptoethanol and 0.1% (v/v) Triton X-100, pH value 7.5.
B:Otto damping fluid: Otto I:100mmol/L citric acid, 0.5% (v/v) Tween20, pH value 2 ~ 3, Otto II:400mmol/L Na 2hPO 412H 2o, pH value 7 ~ 8.
C:MgSO 4damping fluid: 10mmol/LMgSO 47H 2o, 50mmol/L KCl, 5mmol/L4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), 0.3mmol/L dithiothreitol (DTT) (DTT), 0.25% (v/v) TritonX-100, pH value 7.5.
Three kinds of nucleus extraction damping fluid extracting method are:
1) sample: get 0.5 gram of tender leaf from healthy and strong plant the morning in fine day, be placed on 4 DEG C of low temperature refrigerators with self-sealing plastic bag splendid attire and preserve stand-by.
2) nuclei suspension is prepared: the nucleus extraction damping fluid get 0.5 gram of eggplant tender leaf being added 5 milliliters.Shredded with sharp single-edge blade under 4 DEG C of conditions, 300 object nylon net filters in centrifuge tube, 4 DEG C of stationary incubation 10 ~ 15 minutes.1000r/min, after centrifugal 10 minutes, abandons supernatant, and precipitation adds nucleus Extraction buffer and rinses once, and with 500 object nylon net filters, namely obtains nuclei suspension.
3) dyeing and flow cytometry: add RnaseA (final concentration is 50ug/ml) and PI (final concentration is 50ug/ml) in the nuclei suspension prepared.4 DEG C dyeing more than 30 minutes after, sample detection.
The nucleus extraction damping fluid of the invention described above and extracting method and existing made in nuclear buffer and extracting method is adopted to carry out flow cytomery (table 1) to Eggplant Varieties " No. 3, Su Qi " respectively.Result shows: the eggplant nucleus DNA peak figure adopting nucleus extraction damping fluid of the present invention and extracting method to obtain is thin and tall, and impurity is less, and peak width is very narrow, is easy to analyze (Fig. 2) obtained result.
And the eggplant nuclear DNA content peak width adopting Tris Extraction buffer and extracting method to obtain and impurity is many, be difficult to data analysis (Fig. 3).The peak figure impurity adopting MgSO4 damping fluid and extracting method to obtain is a lot, almost cannot separate with impurity peaks, thus cannot analyze accurately (Fig. 4) the nuclear content extracted.Adopt Otto damping fluid and extracting method then cannot obtain effective nucleus, flow cytometer almost can't detect effective signal, thus peak figure can not be formed.
Table 1 different nucleus extraction damping fluid extracts the nuclear flow cytomery effect of eggplant leaf

Claims (5)

1. be suitable for an eggplant leaf nucleus extraction method for flow cytometry, it is characterized in that, its nucleus extraction buffer composition used is:
CPW salt solusion: KH 2pO 427.2mg/L, KNO 3101.0mg/L, CaCl 2.2H 2o 1480.0mg/L, MgSO 4.7H 2o 246.0mg/L, KI 0.16mg/L, CuSO 4.5H 2o 0.025mg/L, pH 5.8;
4-hydroxyethyl piperazine ethanesulfonic acid HEPES, the 0.25%(w:v of 0.5M sweet mellow wine mannitol, 3mmol/L) PEG, 0.5%(v:v) Triton X-100,0.25%(v:v) mercaptoethanol, pH6.5-7.0.
2. method according to claim 1, is characterized in that, it comprises the steps:
1) sample: get 0.5 ~ 1 gram of tender leaf from healthy and strong plant the morning in fine day, be placed on 4 DEG C of low temperature refrigerators with self-sealing plastic bag splendid attire and preserve stand-by;
2) prepare nuclei suspension: the nucleus extraction damping fluid get 0.5 ~ 1 gram of eggplant tender leaf being added 5 ~ 10 milliliters, shredded with sharp single-edge blade under 4 DEG C of conditions, be filtered in centrifuge tube, 4 DEG C of stationary incubation 10 ~ 15 minutes; 1000r/min, after centrifugal 10 minutes, abandons supernatant, and precipitation adds the flushing of nucleus Extraction buffer and once filters afterwards, namely obtains nuclei suspension;
3) dye: in the nuclei suspension prepared, first add the endonuclease RnaseA that final concentration is 50ug/ml, digest after 10 minutes, add DNA fluorescent dye propidium iodide PI, 4 DEG C of dyeing are after more than 30 minutes, refilter once with 500 object nylon wires, sample detection.
3. method according to claim 2, it is characterized in that, in step 3), sample detection refers to carries out sample detection on the flow cytometer of U.S. company BD, use low speed collecting cell core, and showing FL2 histogram, horizontal ordinate is with the area of relative dna content reflection fluorescence curve, and ordinate is cell check figure.
4. according to the method in claim 2 or 3, it is characterized in that, in step 3), in the nuclei suspension prepared, first add the endonuclease RnaseA that final concentration is 50ug/ml, digest after 10 minutes, then add DNA fluorescent dye propidium iodide PI.
5. according to the method in claim 2 or 3, it is characterized in that, in step 3), 4 DEG C of dyeing, after more than 30 minutes, refilter once with 500 object nylon wires.
CN201410566300.6A 2014-10-22 2014-10-22 Extraction method for cell nucleuses of eggplant leaves suitable for flow cytometry Pending CN104316373A (en)

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CN105606518A (en) * 2015-12-21 2016-05-25 武汉大学 Rapid determination method of konjac genome content value
CN105606519A (en) * 2016-01-08 2016-05-25 南京林业大学 Method for rapid identification of ploidy of salicaceae plant
CN106092864A (en) * 2016-06-07 2016-11-09 中国林业科学研究院热带林业研究所 A kind of method of tail alpine ash flow cytometer detection
CN106248561A (en) * 2016-07-21 2016-12-21 西南大学 Be suitable to buffer and preparation, the using method utilizing flow cytometer that various plants carries out Ploidy Identification
CN110146431A (en) * 2019-06-05 2019-08-20 福建农林大学 A kind of preparation method and cell lysis buffer solution of the flow cytometry sample suitable for Calanthe plant
CN110749699A (en) * 2019-10-10 2020-02-04 南京农业大学 Method for screening triploid new germplasm from white loquat seedling
CN110823654A (en) * 2019-11-06 2020-02-21 四川省自然资源科学研究院 Method for identifying ploidy of kiwi fruits by adopting flow cytometer, dissociation liquid and preparation method
CN110849795A (en) * 2019-11-08 2020-02-28 河北省农林科学院经济作物研究所 Method for rapidly detecting Chinese cabbage ploidy by using flow cytometer
CN111257085A (en) * 2020-03-31 2020-06-09 中国科学院植物研究所 Cell nucleus extraction system and method
CN111504746A (en) * 2020-04-29 2020-08-07 扬州大学 Soybean cell nucleus dissociation liquid suitable for flow cytometer analysis and application thereof
CN111562209A (en) * 2020-05-12 2020-08-21 广西大学 Method for rapidly identifying tetraploid eggplant rootstock
CN112458036A (en) * 2020-12-10 2021-03-09 上海交通大学 Preparation and instantaneous transformation method of eggplant protoplast
CN114279786A (en) * 2021-12-24 2022-04-05 河南省农业科学院经济作物研究所 Preparation method of sample suitable for cyperus esculentus flow cytometry and cell lysate

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CN105606518A (en) * 2015-12-21 2016-05-25 武汉大学 Rapid determination method of konjac genome content value
CN105606519A (en) * 2016-01-08 2016-05-25 南京林业大学 Method for rapid identification of ploidy of salicaceae plant
CN105606519B (en) * 2016-01-08 2018-04-24 南京林业大学 A kind of method of Rapid identification Salicaceous Plants ploidy
CN106092864A (en) * 2016-06-07 2016-11-09 中国林业科学研究院热带林业研究所 A kind of method of tail alpine ash flow cytometer detection
CN106248561B (en) * 2016-07-21 2020-07-10 西南大学 Buffer solution suitable for ploidy identification of various plants and preparation and use methods thereof
CN106248561A (en) * 2016-07-21 2016-12-21 西南大学 Be suitable to buffer and preparation, the using method utilizing flow cytometer that various plants carries out Ploidy Identification
CN110146431A (en) * 2019-06-05 2019-08-20 福建农林大学 A kind of preparation method and cell lysis buffer solution of the flow cytometry sample suitable for Calanthe plant
CN110749699A (en) * 2019-10-10 2020-02-04 南京农业大学 Method for screening triploid new germplasm from white loquat seedling
CN110823654A (en) * 2019-11-06 2020-02-21 四川省自然资源科学研究院 Method for identifying ploidy of kiwi fruits by adopting flow cytometer, dissociation liquid and preparation method
CN110849795A (en) * 2019-11-08 2020-02-28 河北省农林科学院经济作物研究所 Method for rapidly detecting Chinese cabbage ploidy by using flow cytometer
CN111257085A (en) * 2020-03-31 2020-06-09 中国科学院植物研究所 Cell nucleus extraction system and method
CN111504746A (en) * 2020-04-29 2020-08-07 扬州大学 Soybean cell nucleus dissociation liquid suitable for flow cytometer analysis and application thereof
CN111562209A (en) * 2020-05-12 2020-08-21 广西大学 Method for rapidly identifying tetraploid eggplant rootstock
CN112458036A (en) * 2020-12-10 2021-03-09 上海交通大学 Preparation and instantaneous transformation method of eggplant protoplast
CN114279786A (en) * 2021-12-24 2022-04-05 河南省农业科学院经济作物研究所 Preparation method of sample suitable for cyperus esculentus flow cytometry and cell lysate

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