CN104007053A - Method for detecting cell number of arabidopsis leaf - Google Patents
Method for detecting cell number of arabidopsis leaf Download PDFInfo
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- CN104007053A CN104007053A CN201410238677.9A CN201410238677A CN104007053A CN 104007053 A CN104007053 A CN 104007053A CN 201410238677 A CN201410238677 A CN 201410238677A CN 104007053 A CN104007053 A CN 104007053A
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Abstract
The invention provides a method for detecting the cell number of an arabidopsis leaf. The method comprises the steps of enzymolysis of the plant leaf; cell lysis and cell nucleus collection; cell nucleus dyeing and counting, and the like. The cell number of the arabidopsis leaf is determined by detecting the cell nucleus number with a flow cytometry. Compared with a manner that the cell number of the plant leaf is observed and counted by a cell counting plate, the method is capable of counting the cell number of the whole plant leaf, and has the advantages of being convenient to operate, objective and precise.
Description
Technical field
The present invention relates to biological technical field, specifically, relate to a kind of method that detects accurately, objectively Arabidopsis leaf cell number.
Background technology
At present, statistics plant leaf blade cell number is mainly observed by cell counting count board, under equal sample preparation condition, enzymolysis time is restive, and the too short cell separation that will cause of enzymolysis time is incomplete, and adhesion body is many, and enzymolysis time is long, bioplast easily breaks, and the intact cell ratio of acquisition is not high, causes statistical error large.When sampling counting, bioplast is skewness in enzyme liquid, and while making to count, repeatability is not high, and subjectivity is strong.
Summary of the invention
The object of this invention is to provide a kind of method that detects accurately, objectively Arabidopsis leaf cell number.
In order to realize the object of the invention, a kind of method that detects Arabidopsis leaf cell number of the present invention, comprises the following steps:
1) enzymolysis of plant leaf blade: get 1 Arabidopsis leaf, shear the fritter of growth 0.5-1.0mm, wide 0.1-0.2mm, then add blade enzymolysis liquid 0.5-1mL, in 20-22 ℃, the dark enzymolysis 10-12 hour of 30-40rpm is complete to blade enzymolysis;
2) lysis and nuclear collection:
By enzymolysis completely blade enzymolysis liquid cross 300-400 mesh sieve, then, with the centrifugal 10-12min of 1400g-1500g, abandon supernatant, add solution I dissolution precipitation, the rifle head that cuts off top is installed on liquid-transfering gun to pressure-vaccum precipitation for several times, until precipitation is dissolved completely; Then by solution in the centrifugal 10-12min of 1400g-1500g, abandon supernatant, in precipitation, add the cell lysis buffer solution of precooling, again the rifle head that cuts off top is installed on liquid-transfering gun to pressure-vaccum precipitation for several times, until mix, after ice bath 5-10min, 4 ℃ of centrifugal 10-12min of 1400g-1500g, abandon supernatant, and gained precipitation is dissolved by the cell lysis buffer solution of precooling again, and repeat above-mentioned steps 1-3 time, until precipitation is grey black;
3) nuclear dyeing and counting:
In the above-mentioned precipitation that is grey black, add cell lysis buffer solution, the rifle head that cuts off top is installed on to pressure-vaccum precipitation several on liquid-transfering gun, until precipitation is dissolved completely, then add isopyknic dye liquor II, mix, after ice bath 30-40min, with aperture 300-400 object nylon membrane, filter, with flow cytometer, detect cell nuclei in filtrate, thereby obtain blade cell number.
Wherein, step 1), the formula of blade enzymolysis liquid is: the 20mM MES of 1.5% cellulase, 0.4% macerozyme, 20mM KCl, pH5.7,0.4M sweet mellow wine, 10mMCaCl
2, 5mM beta-mercaptoethanol and 0.1%BSA, with water, prepare;
Step 2) in, the formula of solution I is: the 0.03%MES of pH5.8,154mM NaCl, 125mM CaCl
2, 5mM KCl and 5mM glucose, with water, prepare;
Step 2) and 3) formula of cell lysis buffer solution described in is: 15mM Tris, 2mMNa
2eDTA, 0.5mM tetra-hydrochloric acid spermine, 80mM KCl, 20mM NaCl, 0.5%TritonX-100 and 15mM beta-mercaptoethanol, pH7.5, prepares with water;
Step 3) formula of the II of dye liquor described in is: 100 μ g/mL PI, 100 μ g/mL RNase and 0.1%TritonX-100, prepare with phosphate buffer; The compound method of described phosphate buffer is: get 7.65g NaCl, 0.725g Na
2hPO
4with 0.212g KH
2pO
4, add 800mL water, adjust pH to 7.4, be then settled to 1L.
The compound method of the enzymolysis liquid of blade step 1) is: take in proportion cellulase, macerozyme, KCl and sweet mellow wine, be dissolved in water, then add wherein the pre-MES prior to 70 ℃ of preheating 5min, in 55 ℃ of heating water bath 10min, then be cooled to room temperature, add CaCl
2, beta-mercaptoethanol and BSA, mix, obtain.
Preferably, the method for detection Arabidopsis leaf cell number of the present invention comprises the following steps:
1) enzymolysis of plant leaf blade: get 1 Arabidopsis leaf, shear the fritter of growth 0.5-1.0mm, wide 0.1-0.2mm, then add blade enzymolysis liquid 1mL, in 22 ℃, the dark enzymolysis of 30-40rpm 12 hours, complete to blade enzymolysis;
2) lysis and nuclear collection:
By enzymolysis completely blade enzymolysis liquid cross 400 mesh sieves, then, with the centrifugal 10min of 1500g, abandon supernatant, add solution I dissolution precipitation, the rifle head that cuts off top is installed on liquid-transfering gun to pressure-vaccum precipitation for several times, until precipitation is dissolved completely; Then by solution in the centrifugal 10min of 1500g, abandon supernatant, in precipitation, add the cell lysis buffer solution of precooling, again the rifle head that cuts off top is installed on liquid-transfering gun to pressure-vaccum precipitation for several times, until mix, after ice bath 5-10min, 4 ℃ of centrifugal 10min of 1500g, abandon supernatant, and gained precipitation is dissolved by the cell lysis buffer solution of precooling again, and repeat above-mentioned steps 1-3 time, until precipitation is grey black;
3) nuclear dyeing and counting:
In the above-mentioned precipitation that is grey black, add cell lysis buffer solution, the rifle head that cuts off top is installed on to pressure-vaccum precipitation several on liquid-transfering gun, until precipitation is dissolved completely, then add isopyknic dye liquor II, mix, after ice bath 30min, with aperture 400 object nylon membranes, filter, with flow cytometer, detect cell nuclei in filtrate, thereby obtain blade cell number.
The present invention has the following advantages:
Compare with adopting cell counting count board observation statistics plant leaf blade cell number, this method can be counted the cell number of whole plant leaf blade, has easy to operate, objective, accurate advantage.
Accompanying drawing explanation
Fig. 1 is detection and the cell count result of flow cytometer to wild type (Col) and the 3rd, 4 true leaf core DNA of rfc3-1 mutant in the embodiment of the present invention.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
Embodiment detects the method for Arabidopsis leaf cell number
1 test material and reagent
1.1 test material
Take the environmental wild type plant (WT) of arabidopsis Colombia and rfc3-1 mutant as supplying examination material.
1.2 main medicine and reagent
Propidium iodide (PI), four hydrochloric acid spermine are purchased from Sigma company; MES (Methyl Methanesulfonate, methyl mesylate), cellulase, macerozyme and beta-mercaptoethanol are purchased from the biological company limited of distance of travel of roc, and other conventional chemical reagent are all purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
2 test methods
The preparation of 2.1 reagent
1. the preparation of blade enzymolysis liquid
The formula of blade enzymolysis liquid is: the 20mM MES of 1.5% cellulase, 0.4% macerozyme, 20mM KCl, pH5.7,0.4M sweet mellow wine, 10mM CaCl2,5mM beta-mercaptoethanol and 0.1%BSA, prepare with water.
Compound method: take in proportion cellulase, macerozyme, KCl and sweet mellow wine, be dissolved in water, then add wherein the pre-MES prior to 70 ℃ of preheating 5min, in 55 ℃ of heating water bath 10min, be then cooled to room temperature, add CaCl
2, beta-mercaptoethanol and BSA, mix and get final product.
2. the preparation of cell lysis buffer solution (pH7.5)
15mM Tris, 2mM Na
2eDTA, 0.5mM tetra-hydrochloric acid spermine, 80mM KCl, 20mM NaCl, 0.5%TritonX-100 and 15mM beta-mercaptoethanol, pH7.5, prepares with water.Under-20 ℃ of conditions, preserve.
3. the preparation of phosphate buffer
Get 7.65g NaCl, 0.725g Na
2hPO
4with 0.212g KH
2pO
4, add 800mL water, adjust pH to 7.4, be then settled to 1L.
4. the preparation of solution I
The 0.03%MES of pH5.8,154mM NaCl, 125mM CaCl
2, 5mM KCl and 5mM glucose, with water, prepare.
5. the preparation of propidium iodide (PI) dye liquor
100 μ g/mL PI, 100 μ g/mL RNase and 0.1%TritonX-100, with above-mentioned 3. middle phosphate buffer preparation.
2.2 detecting step
2.2.1 the enzymolysis of plant leaf blade
Get respectively about 4-5 week of growth, the arabidopsis Col wild type (WT) that growth conditions is good, the 1st, 2,3,4 true leaves of rfc3-1 mutant, by putting into 10-20 sheet in 10mL blade enzymolysis liquid, with blade, blade is evenly cut into the fritter of long 0.5-1.0mm, wide 0.1-0.2mm.Freshly prepared blade enzymolysis liquid is divided in the round bottom centrifuge tube that installs to 2mL, wrap up shading immediately with tinfoil, be placed under 22 ℃ of conditions, on horizontal shaking table, (30-40rpm) dark enzymolysis is 12 hours, until all blade enzymolysis are complete.
2.2.2 vegetable cell cracking and nuclear collection
After blade enzymolysis liquid filters with 400 order steel sieves completely by enzymolysis, in the centrifugal 10min of 1500 * g, abandon supernatant, add solution I dissolution precipitation.Meanwhile, by the liquid-transfering gun pressure-vaccum precipitation that the 200 μ l rifle heads that top cuts off 1-2cm are housed repeatedly, until precipitation is dissolved completely, is mixed.The solution mixing after centrifugal 10min, is abandoned to supernatant under 1500 * g, leave green precipitate and be Leaves Protoplast.Then the cell lysis buffer solution that adds precooling, Leaves Protoplast nucleus is discharged, again by the liquid-transfering gun pressure-vaccum precipitation that the 200 μ l rifle heads that top cuts off 1-2cm are housed repeatedly, until mix, after ice bath 5-10min, 4 ℃ of centrifugal 10min of 1500 * g, abandon supernatant, gained precipitation is dissolved with the lysis of precooling again, and repeats above-mentioned steps 1-3 time, until precipitation is grey black.
2.2.3 nuclear dyeing and counting
In above-mentioned precipitation, add cell lysis buffer solution, by the liquid-transfering gun pressure-vaccum precipitation that the 200 μ l rifle heads that top cuts off 1-2cm are housed repeatedly, until precipitation is dissolved completely, add isopyknic PI dye liquor, mix, after ice bath 30min, with 400 object nylon membranes, filter, with flow cytometer, detect cell nuclei in filtrate, thereby obtain blade cell number.
3. assay and analysis
By detecting with flow cytometer of wild type and the 1st, 2,3,4 true leaf nucleus suspending liquid of rfc3-1 mutant, result as shown in Figure 1, obtain preferably DNA content analysis chart, and the CV value of wild type and mutant rfc3-1 (variation value) is all less than 6%, be respectively 5.9% and 3.9%, illustrate that data are in normal range of variation.
By nuclei count, find cell number that RFC3 gene mutation causes the 1st, 2 true leaves of plant than the minimizing of wild type 1.6 times, the cell number of the 3rd, 4 true leaves than the minimizing of wild type 2.6 times (table 1).
The nuclei count of table 1 wild type (Col) and the 1st, 2,3,4 true leaves of rfc3-1 mutant
This is also unanimously (to be respectively 5.28 * 10 with observe the wild type (Col) of statistics and the 1st, 2 of rfc3-1 mutant plant and 3,4 true leaf bioplast number ratios by cell counting count board
4/ 2.77 * 10
4=1.9 times and 1.18 * 10
5/ 4.53 * 10
4=2.6 times).But when cell counting count board is observed statistics plant leaf blade cell number, because protoplasm easily breaks, the error of statistics is too large, subjectivity is strong.Therefore, this method can be counted the cell number of whole plant leaf blade, has easy to operate, objective, accurate advantage.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (3)
1. a method that detects Arabidopsis leaf cell number, is characterized in that, comprises the following steps:
1) enzymolysis of plant leaf blade: get 1 Arabidopsis leaf, shear the fritter of growth 0.5-1.0mm, wide 0.1-0.2mm, then add blade enzymolysis liquid 0.5-1mL, in 20-22 ℃, the dark enzymolysis 10-12 hour of 30-40rpm is complete to blade enzymolysis;
2) lysis and nuclear collection:
By enzymolysis completely blade enzymolysis liquid cross 300-400 mesh sieve, then, with the centrifugal 10-12min of 1400g-1500g, abandon supernatant, add solution I dissolution precipitation, the rifle head that cuts off top is installed on liquid-transfering gun to pressure-vaccum precipitation for several times, until precipitation is dissolved completely; Then by solution in the centrifugal 10-12min of 1400g-1500g, abandon supernatant, in precipitation, add the cell lysis buffer solution of precooling, again the rifle head that cuts off top is installed on liquid-transfering gun to pressure-vaccum precipitation for several times, until mix, after ice bath 5-10min, 4 ℃ of centrifugal 10-12min of 1400g-1500g, abandon supernatant, and gained precipitation is dissolved by the cell lysis buffer solution of precooling again, and repeat above-mentioned steps 1-3 time, until precipitation is grey black;
3) nuclear dyeing and counting:
In the above-mentioned precipitation that is grey black, add cell lysis buffer solution, the rifle head that cuts off top is installed on to pressure-vaccum precipitation several on liquid-transfering gun, until precipitation is dissolved completely, then add isopyknic dye liquor II, mix, after ice bath 30-40min, with aperture 300-400 object nylon membrane, filter, with flow cytometer, detect cell nuclei in filtrate, thereby obtain blade cell number;
Wherein, step 1), the formula of blade enzymolysis liquid is: the 20mM MES of 1.5% cellulase, 0.4% macerozyme, 20mM KCl, pH5.7,0.4M sweet mellow wine, 10mMCaCl
2, 5mM beta-mercaptoethanol and 0.1%BSA, with water, prepare;
The formula of solution I step 2) is: the 0.03%MES of pH5.8,154mMNaCl, 125mM CaCl
2, 5mM KCl and 5mM glucose, with water, prepare;
Step 2) and 3) formula of cell lysis buffer solution described in is: 15mM Tris, 2mMNa
2eDTA, 0.5mM tetra-hydrochloric acid spermine, 80mM KCl, 20mM NaCl, 0.5%TritonX-100 and 15mM beta-mercaptoethanol, pH7.5, prepares with water;
Step 3) formula of the II of dye liquor described in is: 100 μ g/mL PI, 100 μ g/mL RNase and 0.1%TritonX-100, prepare with phosphate buffer; The compound method of described phosphate buffer is: get 7.65g NaCl, 0.725g Na
2hPO
4with 0.212g KH
2pO
4, add 800mL water, adjust pH to 7.4, be then settled to 1L.
2. method according to claim 1, is characterized in that, comprises the following steps:
1) enzymolysis of plant leaf blade: get 1 Arabidopsis leaf, shear the fritter of growth 0.5-1.0mm, wide 0.1-0.2mm, then add blade enzymolysis liquid 1mL, in 22 ℃, the dark enzymolysis of 30-40rpm 12 hours, complete to blade enzymolysis;
2) lysis and nuclear collection:
By enzymolysis completely blade enzymolysis liquid cross 400 mesh sieves, then, with the centrifugal 10min of 1500g, abandon supernatant, add solution I dissolution precipitation, the rifle head that cuts off top is installed on liquid-transfering gun to pressure-vaccum precipitation for several times, until precipitation is dissolved completely; Then by solution in the centrifugal 10min of 1500g, abandon supernatant, in precipitation, add the cell lysis buffer solution of precooling, again the rifle head that cuts off top is installed on liquid-transfering gun to pressure-vaccum precipitation for several times, until mix, after ice bath 5-10min, 4 ℃ of centrifugal 10min of 1500g, abandon supernatant, and gained precipitation is dissolved by the cell lysis buffer solution of precooling again, and repeat above-mentioned steps 1-3 time, until precipitation is grey black;
3) nuclear dyeing and counting:
In the above-mentioned precipitation that is grey black, add cell lysis buffer solution, the rifle head that cuts off top is installed on to pressure-vaccum precipitation several on liquid-transfering gun, until precipitation is dissolved completely, then add isopyknic dye liquor II, mix, after ice bath 30min, with aperture 400 object nylon membranes, filter, with flow cytometer, detect cell nuclei in filtrate, thereby obtain blade cell number.
3. method according to claim 1 and 2, it is characterized in that, the compound method of the enzymolysis liquid of blade step 1) is: take in proportion cellulase, macerozyme, KCl and sweet mellow wine, be dissolved in water, then add wherein the pre-MES prior to 70 ℃ of preheating 5min, in 55 ℃ of heating water bath 10min, be then cooled to room temperature, add CaCl
2, beta-mercaptoethanol and BSA, mix, obtain.
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| CN106244513A (en) * | 2016-07-26 | 2016-12-21 | 北京林业大学 | A kind of vacuole that separates is distributed and the method for motion with albumen on observation tonoplast |
| CN111060696A (en) * | 2019-12-27 | 2020-04-24 | 湖南农业大学 | Method for reducing false positive rate of plant small molecule signal peptide |
| CN111323284A (en) * | 2020-01-14 | 2020-06-23 | 北京林业大学 | Mitochondrial fluorescence labeling method for tobacco leaf and protoplast |
| CN113444678A (en) * | 2020-03-26 | 2021-09-28 | 武汉华大医学检验所有限公司 | Method for preparing single cell nuclear suspension, single cell sequencing method and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111323284A (en) * | 2020-01-14 | 2020-06-23 | 北京林业大学 | Mitochondrial fluorescence labeling method for tobacco leaf and protoplast |
| CN113444678A (en) * | 2020-03-26 | 2021-09-28 | 武汉华大医学检验所有限公司 | Method for preparing single cell nuclear suspension, single cell sequencing method and kit |
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