CN1818057A - Method for extracting pear pollen protoplast by enzymolysis - Google Patents
Method for extracting pear pollen protoplast by enzymolysis Download PDFInfo
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- CN1818057A CN1818057A CN 200510134904 CN200510134904A CN1818057A CN 1818057 A CN1818057 A CN 1818057A CN 200510134904 CN200510134904 CN 200510134904 CN 200510134904 A CN200510134904 A CN 200510134904A CN 1818057 A CN1818057 A CN 1818057A
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- enzymolysis
- protoplastis
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Abstract
The invention is about the extracting method for the protoplast of the pear flower powder and the pollen tube. The process includes: first to collect the ripe pollen, culture in the pollen medium for 3-3.5h, then to enzymolysis for 50min and centrifugate. So the active protoplast would be get. It can be as the material for the studying of the physiplogical-biochemical characteristic and the cell signal transduction of the pollen and the pollen tube.
Description
Technical field
The present invention is the method that pear pollen and pollen tube protoplastis extract, and relates to plant cell wall breaking and obtains the method with vigor protoplastis, belongs to biological chemistry and Food science field.
Background technology
Plant pollen (pollen) not only produces offspring, enlarges aspects such as genetic composition and distribution range and plays effect of crucial importance plant, and its nutrition is extremely abundant.The Recent study surface: pollen contains nutritive ingredients such as miscellaneous carbohydrate, organic acid, protein, nucleic acid, enzyme and coenzyme, pigment, VITAMIN and mineral substance.Wherein Protein content is very abundant, reaches 7%-40%, surpasses casein.Contain 18 seed amino acids in the pollen, comprising 8 seed amino acids of needed by human, its content is more than 5-7 times of milk, egg, and especially the content of Methionin is abundant especially, its favourable upgrowth and development of children.Pollen also contains vitamin A, vitamins B group (B
1, B
2, B
6, B
12), 10 multivitamins, particularly vitamin-E such as vitamins C, vitamins D, vitamin-E, vitamin P and pantothenic acid, folic acid, nicotinic acid, C, P can strengthen immune function of human body, promotes to promote longevity vessel softening, prevention of arterial sclerosis and coronary heart disease.Pollen also contains more than 90 kind of ferment acid element, enzyme and coenzyme, and the in store natural vigour of these enzymes is the important substance in the vital movement process, is a kind of natural senior nutrition, thereby, also for being described as " nutritional complete food products " (perfect foods) in the world.
China soil is vast, kind surplus spermatophyte has 30,000.Though pollen is little, it is very surprising that plant produces pollen quantity, and a strain corn can produce 5,000 ten thousand pollen.China ploughs 1.000 5 hundred million hm approximately
2(1,500,000,000 mu) so pollen resource is very abundant, occupy first place in the world.About 7,000,000 groups of honeybees are supported in the whole nation, if every group of honeybees were gathered 10Kg pollen in 1 year, will produce 7,000 ten thousand Kg so every year, if add artificial or machine collection pollen, that will be a kind of considerable potential food resource.But development and use at present are less than 1/200, so study, develop, use pollen, the happiness existence to the mankind has profound significance.
Though the edible nourishing of pollen and pharmaceutical use are very big, but because the structure of pollen wall, composition is comparatively special, structure is hard, if do not remove wall, its nutrition is difficult to effectively to be absorbed by human body, and particularly more should to select the pollen protoplast behind the wall for use be raw material for pollen oral solution beverage etc.For this reason, in the Food science field, there are many scholars to carry out relevant development research, existing pollen goes the method for wall can be summed up as following three classes: 1) physical method: this method is also more common, pollen is carried out drying or freezing, and then pollen ground, this method is used for foodstuffs industry more, is used to extract the content of pollen; 2) physiology method: with the solution soaking pollen of certain osmotic pressure, allow protoplastis expand, burst pollen wall, and acquisition protoplastis or fovilla, but it is less that this method is used, 3) fermentation method: directly utilize all enzymes of pollen itself and microorganism, make pollen reach the purpose of breaking wall by fermentation.Though there have been these to go the method for wall, but, there are a lot of problems in these methods, subject matter has: the first not only loses original biologos with the pollen protoplast that these methods obtained, and also can bring certain destructiveness to nutritive ingredient, reduce the nutritive value that is had to a great extent; It two is that these methods of obtaining pollen protoplast can not be suitable for all plant pollens, such as, can't obtain the protoplastis of Rosaceae fruit tree pollen such as pears, apple with these methods.
Summary of the invention
Technical problem the purpose of this invention is to provide the extracting method of rosaceous plant pollen protoplast such as pears, isolate effectively have vigor, preserve original nutrient and active pollen protoplast.This method other Rosaceae fruit trees (apple, peach, apricot, Lee etc.) pollen protoplast that can be used for separate purifying, also the separation for the protoplastis of other histoorgans provides reference.
Technical scheme the invention provides the broken wall of new plant pollen and pollen tube and obtains the method for protoplastis, be to use pollen to cultivate the method for obtaining pollen and pollen tube protoplastis with the two-step approach of enzymolysis, its process is that pollen is cultivated earlier, again enzymolysis and obtain the complete protoplastis of pollen granule and pollen tube.
Adopt pollen to cultivate ' two-step approach ' that combines the protoplastis that obtains pollen and pollen tube with enzymolysis, comprise the following steps:
1) collection of pollen: gather open pollen on the same day, allow the pollen thorough drying then;
2) preparation of substratum (on the basis of BK substratum, adjusting):
Substratum one: sucrose (sucrose) 10%, Ca (NO
3)
24H
2O 0.01%, 250mM MES/Tris[alpha-sulfo formicester sodium sulfonate/three (methylol) aminomethane; PH6.0-6.2).
Substratum two: sucrose (sucrose) 15%, Ca (NO
3)
24H
2O 0.05%, MgSO
47H
2O 0.02%, KNO
30.01%, 0.01%H
3BO
3, 250mM MES/Tris[alpha-sulfo formicester sodium sulfonate/three (methylol) aminomethane, pH6.0-6.2]; According to the difference of purpose, substratum one and substratum two can be chosen one of them use wantonly;
3) dried pollen is got in the cultivation of pollen, puts into pollen substratum (above-mentioned two kinds of substratum can be chosen any one kind of them), and under dark condition, 25～27 ℃, 80rpm cultivates 3～3.5h.When selecting first kind of substratum to cultivate pollen, incubation time can shorten to 2.5h, enzymolysis pollen again, and this method mainly is the protoplastis that is used to obtain pollen granule.Selecting second kind of substratum to cultivate pollen, remove wall through enzymolysis again, mainly is the protoplastis that obtains pollen tube.
4) pollen after the cultivation cleans pollen 2-3 time with centrifugal method, and 1200rpm, scavenging solution are the enzyme mother liquor.
5) preparation of enzyme mother liquor: Mannitol (N.F,USP MANNITOL) 0.6mM, KNO
3100mM, KH
2PO
420mM, MgSO
4100mM, CaCl
2100mM, KI 100mM, CuSO
40.1mM, MES (alpha-sulfo formicester sodium sulfonate) 250mM, Vc 0.5mM, pH 5.3 (Tris-HCI).
6) preparation of enzyme liquid: the female 1.5%Cellulase of adding of the enzyme R-10[cellulase R-10 that gets 100ml, be baby company of German society product (German Serva)], 0.7%Pectolyase Y-23[polygalacturonase Y-23, for the product (Seishin of new company gives birth in Japan, Japan)] and the 1%BSA[bovine serum albumin, west gram sign indicating number company's product (Sigma)].The enzyme liquid for preparing preferably carries out packing, preserves under-20 ℃ of freezing low temperature.
7) enzymolysis: the pollen suspension 100 μ l after draw cleaning, the enzyme liquid of putting into 900 μ l carries out enzymolysis, and the condition of enzymolysis is: dark, 29～31 ℃, 100rpm, enzymolysis time are 50min.
8) behind the enzymolysis, clean 3 times with centrifugal method, 500rpm, the osmotic pressure of scavenging solution will be more than 900mOsM.
Beneficial effect
Positively effect of the present invention:
1) from Rosaceae fruit tree pear pollen and pollen tube, isolates protoplastis first, can be used for various relevant scientific researches, especially the basic research of pear pollen physio-biochemical characteristics with vigor; Such as, pollen protoplast is suitable for the experiment material as the molecular structure of research vegetable cell;
2) use the protoplastis that method of the present invention is extracted with vigor, cross-breeding field plant, bigger application prospect is arranged, the mutual fusion of the protoplastis by same source or different sources with vigor, can develop into new plant again, this method is called somatic hybridization, is the important means of innovation germplasm;
3) the present invention is based on the working foundation to characteristic research such as pear pollen and pollen tube Physiology and biochemistry and cell signallings.Pollen protoplast have haploidy, simple in structure, grow synchronously and advantage such as colony's quantity is big, be considered to " the remarkable system of genetic manipulation and mutation research ", " research pollen physiology and ontogenetic new way " is provided.In addition, in research plant fertilization physiology, many important physical biochemical reactions all are to occur in the pollen tube, as self-incompatibility response etc.; Therefore, obtaining the pollen tube protoplastis is the important prerequisite of carrying out these scientific theory researchs.In a word, present method has the solid theories foundation, and science is strong, method is easier, easy handling and practical application.
4) cultivate with this pollen---the two-step approach of enzymolysis is extracted the protoplastis of pollen and pollen tube, the time by the control enzymolysis, can obtain the complete pollen tube or the protoplastis of pollen granule; The protoplastis that obtains still keeps original vigor; Use the pollen protoplast that this method is obtained, be very suitable for the experiment material of aspect research such as Physiology and biochemistry, cell signalling characteristic as pollen and pollen tube.
Description of drawings
Fig. 1 enzyme process obtains the schema of protoplastis
Fig. 2 sprout three each and every one hour pear pollen.Length is greatly about about 1100 μ m.
Fig. 3 is after enzymolysis, and the pollen tube protoplastis comes out from disruptive pollen tube tip.Under the effect of osmotic pressure, the pollen tube protoplastis is easy to form several spherical parts.In addition, near the protoplastis at most advanced and sophisticated position, volume is big more.
The protoplastis of Fig. 4 pollen tube.Prolong the time of enzymolysis, the pollen tube protoplastis can spin off from pollen tube fully.
The comparison of Fig. 5 pollen tube protoplastis and pollen protoplast.From scheming interior institute target numeral as can be seen, 1-3 is the prolongation of pollen protoplast along with enzymolysis time, comes out from pollen wall gradually, and finally breaks away from extine.4-7 is the pollen tube protoplastis, from figure, may find out, the pollen granule protoplastis is bigger than pollen tube protoplastis.So can distinguish these two kinds of protoplastiss from volume.
Fig. 6 white dyes is identified and is gone the wall effect
Annotate: the arrow indication be protoplastis behind the wall.White dyes is the single-minded cellulosic material that dyes.After white dyes dyeing, under fluorescent microscope, almost can't see cell, illustrate that this cell is successfully sloughed cell walls.
The pollen protoplast of Fig. 7 TTC dyeing 4h
Annotate: the red protoplastis of being dyed by TTC of arrow indication is activated protoplastis.And red color is dark more, illustrates that activity is strong more.
The pollen tube protoplastis that Fig. 8 enzymolysis obtains later.The protoplastis that has also connects together with pollen tube, the pollen tube of disengaging that has.
Fig. 9 left side figure is electrode and cytolemma sealing-in synoptic diagram.Right figure is the sealing-in check, all above 1 gigohm (G Ω).
The Ca that the full cell mode of Figure 10 pear flower tube cell protoplastis writes down
2+Electric current.
The plasma membrane Ca that A stepped start-stop system mode writes down
2+Electric current; The plasma membrane Ca of the gradual record of B
2+Electric current.
Embodiment
Extracting plant pollen (pipe) protoplastis, is one of the prerequisite of studying physiology, biochemical characteristic and the function thereof of pollen (pipe).We use method of the present invention, obtained pear flower tube cell protoplastis with vigor, utilize patch clamp directly to identify the calcium channel of pollen tube and calcium gradient and the correlation function thereof in the pollen tube, for the electrochemical properties of widening and further investigate pollen tube lays the foundation.As everyone knows, obtaining propollen (pipe) protoplastis is the prerequisite of carrying out patch clamp experiments research, and so far, all be to adopt the laser ablation cell walls at the rhizoid of root hair, algae and mycelia, this method needs comparatively expensive experimental equipment, secondly, at present active and complete protoplastis are arranged, all be to be material, and can only gather once for 1 year with the tender and fresh pollen of children about obtaining pollen, and be not report of sophisticated pollen, as pears, apple, peach etc.More not about how obtaining the report of pollen tube protoplastis.
This example just with pear pollen as material, adopt the inventive method to obtain pollen tube protoplastis, and utilize patch clamp successfully to identify the calcium channel of pear pollen tip end with vigor.Experimentation is as follows:
1. at 4-5 between the month, sophisticated pear pollen is gathered in the land for growing field crops, and-20 ℃ of freezing low temperature in air-dry back are preserved down.
2. the pollen of going bail for and depositing, the 2h that thaws at normal temperatures earlier cultivates pollen on above-mentioned substratum, and temperature is 25 ℃, dark condition, 80rpm cultivates 3h (Fig. 2).
3. the pollen after sprouting with enzyme mother liquor eccentric cleaning, 2-3 time, 1200rpm.
4. draw 100 μ L precipitation, add enzyme liquid, put into the constant temperature shaking table immediately, 100rpm, dark condition, 30 ℃, 50min.
5. behind the enzymolysis, put into cryogenic refrigerator earlier, stop enzyme reaction.
6. after enzyme reaction stops, carrying out eccentric cleaning 2-3 time, 500rpm, scavenging solution are extracellular fluid, and the osmotic pressure of extracellular fluid should be 900mOsM.After the cleaning, precipitation is protoplastis, the face shaping of protoplastis, feature such as Fig. 3～and shown in Figure 8.
7. utilize patch clamp technique, the mode (Fig. 9) with full cell has successfully recorded hyperpolarization activated pulse feature inward electric current (Figure 10), for the inward electric current that guarantees record is the outer Ca of born of the same parents
2+Interior stream, must accomplish following 3 points: (1) protoplastis will have activity, and cytolemma is not affected in going the process of wall; (2) electrode and film can form high resistant sealing-in (＞1G Ω); (3) be used to various ionic combinations of the electrode solution of testing and extracellular fluid and concentration and all before experiment, passed through conscientiously design.Extracellular fluid only contains the CaCl of 10mmol/L except that containing the MES and an amount of N.F,USP MANNITOL as the osmoregulation material that is useful on adjust pH
2And the composition of electrode solution simulates plasmasol as possible, adds the GTP and the ATP that keep the normal physiological status of cell, and electrode solution adds Cs simultaneously
+, in order to blocking-up K
+Passage is convenient to Ca
2+Electric current is separated separately and is measured.Ca
2+Electric current as shown in Figure 4, by stepped start-stop system mode (step) and gradual (slow-ramp) mode, when command potential surpass-opening entry writes down less than electric current at 0mV to inward electric current during 100mV.
Claims (1)
1, the extracting method of pollen protoplast comprises the following step:
1) collection of pollen:
Gather open pollen on the same day, allow the pollen thorough drying then;
2) preparation of substratum and enzymolysis solution:
The preparation of substratum: can be with following two kinds of substratum wherein a kind of:
(1) sucrose 10%, Ca (NO
3)
24H
2O 0.01%, 250mM alpha-sulfo formicester sodium sulfonate/three (methylol) aminomethane; PH6.0-6.2;
(2) sucrose 15%, Ca (NO
3)
24H
2O 0.05%, MgSO
47H
2O 0.02%, KNO
30.01%, 0.01%H
3BO
3, 250mM alpha-sulfo formicester sodium sulfonate/three (methylol) aminomethane; PH6.0-6.2; The preparation of enzyme mother liquor: N.F,USP MANNITOL 0.6mM, KNO
3100mM, KH
2PO
420mM, MgSO
4100mM, CaCl
2100mM, KI 100mM, CuSO
40.1mM, alpha-sulfo formicester sodium sulfonate 250mM, Vc0.5mM, pH 5.3 (Tris-HCI);
The preparation of enzyme liquid: get the female 1.5% cellulase R-10 of adding of enzyme of 100ml, 0.7% polygalacturonase Y-23 and 1% bovine serum albumin are preserved under-20 ℃ of freezing low temperature after the enzyme liquid packing for preparing;
3) process of abolishing of pollen wall:
(1) cultivation of pollen: get dried pollen, put into the pollen substratum, cultivate under the condition of 25～27 ℃ of dark, temperature, the time is 3～3.5h;
(2) pollen after the cultivation cleans pollen 2-3 time with whizzer, and 1200rpm, scavenging solution are the enzyme mother liquor;
(3) enzymolysis: draw the pollen suspension 100 μ l after cleaning, put into the enzyme liquid of 900 μ l, beginning enzymolysis, 29～31 ℃ of temperature, 100rpm, dark condition, time 50min;
(4) behind the enzymolysis, clean 3 times with whizzer, 500rpm, scavenging solution is an extracellular fluid, after the cleaning, precipitation is protoplastis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510134904 CN1818057A (en) | 2005-12-30 | 2005-12-30 | Method for extracting pear pollen protoplast by enzymolysis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510134904 CN1818057A (en) | 2005-12-30 | 2005-12-30 | Method for extracting pear pollen protoplast by enzymolysis |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967670A (en) * | 2017-05-19 | 2017-07-21 | 江苏省农业科学院 | A kind of preparation method of birch-leaf pear protoplast |
| CN108642166A (en) * | 2018-05-18 | 2018-10-12 | 南京农业大学 | Utilize the unicellular method for carrying out genome haplotype assembling of pear pollen |
| CN110846269A (en) * | 2019-11-14 | 2020-02-28 | 浙江农林大学 | Method for extracting pear pollen tube membrane |
| CN112461632A (en) * | 2020-09-10 | 2021-03-09 | 南京农业大学 | Method for extracting cellulose from pollen tube |
-
2005
- 2005-12-30 CN CN 200510134904 patent/CN1818057A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967670A (en) * | 2017-05-19 | 2017-07-21 | 江苏省农业科学院 | A kind of preparation method of birch-leaf pear protoplast |
| CN108642166A (en) * | 2018-05-18 | 2018-10-12 | 南京农业大学 | Utilize the unicellular method for carrying out genome haplotype assembling of pear pollen |
| CN110846269A (en) * | 2019-11-14 | 2020-02-28 | 浙江农林大学 | Method for extracting pear pollen tube membrane |
| CN110846269B (en) * | 2019-11-14 | 2021-04-30 | 浙江农林大学 | Method for extracting pear pollen tube membrane |
| CN112461632A (en) * | 2020-09-10 | 2021-03-09 | 南京农业大学 | Method for extracting cellulose from pollen tube |
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Open date: 20060816 |