CN109468263A - A method of the unicellular sample of Activities of Some Plants is prepared with piezo technology - Google Patents
A method of the unicellular sample of Activities of Some Plants is prepared with piezo technology Download PDFInfo
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- CN109468263A CN109468263A CN201811557180.8A CN201811557180A CN109468263A CN 109468263 A CN109468263 A CN 109468263A CN 201811557180 A CN201811557180 A CN 201811557180A CN 109468263 A CN109468263 A CN 109468263A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention discloses a kind of method for preparing the unicellular sample of Activities of Some Plants with piezo technology, operating environment preparation, plant tissue are extracted, handle and are observed, plant tissue is fixed and plant monocyte separation；The beneficial effects of the present invention are, first, this method initiative generates adjustable mechanical vibrotechnique using piezoelectric ceramics, solves the problems, such as the unicellular preparation of Activities of Some Plants, the destruction prepared in phytoplasm body running for structures and function such as plant cell inside cell walls is avoided, maintains the original form of plant cell and activity to the full extent as far as possible；Secondly, this method carries out parameters adjustment according to different plant tissue situations and records, practical operation is facilitated, ensure that the success rate of experiment, improves the efficiency of experiment.
Description
Technical field
The present invention relates to field of biomedicine technology more particularly to a kind of the unicellular sample of Activities of Some Plants is prepared with piezo technology
The method of product.
Background technique
Now with the fast development of unicellular sequencing technologies, also progress is rapid therewith for singe-cell PCR technology, wherein slender
Born of the same parents' isolation technics is the basis of these technologies, and method not very good in the fields such as plant research application obtains complete list
Cell is had to carry out various plant tissues by various enzymatic isolation methods (pectase, cellulase etc.), be destroyed between plant cell just
Normal adhesion structure, but plant cell wall because its constituent structure be similar to intercellular structural, therefore separate it is single celled during
Also it is removed together, the cell of this removal plant cell wall is referred to as protoplast, and existing method is with this single plasm
The method of body separation sequencing represents the protoplast that the unicellular separation of full plants is sequenced, and obtains after the processing of this enzymolysis, digestion
All molecular physiology states of complete plant monocyte whether can be represented is strictly problem, therefore is currently being risen
It is to have to head that unicellular become of the full plants containing cell wall how is obtained in the work of various plant monocyte separation sequencings
The problem first solved.
Summary of the invention
1. technical problems to be solved
Aiming at the problems existing in the prior art, part plant is prepared with piezo technology the purpose of the present invention is to provide a kind of
The method of the unicellular sample of object, to reach separating plant cell adhesion, while with protecting the mesh of plant cell wall again, so i.e.
Plant cell has been separated, and has maintained the complete structure of plant cell and form, it is original to maintain plant cell as much as possible
Activity.
2. technical solution
To solve the above problems, the present invention adopts the following technical scheme that.
A method of the unicellular sample of Activities of Some Plants being prepared with piezo technology, is comprised the following steps:
Step 1, several debugging drops (operation drop) and right are prepared in culture dish with 100uL pipettor and mating pipette tips
Compare drop；
Step 2, with sterilization ophthalmology minor operation tweezer plants bud pointed tip separate living tissue, only retain 1-2 piece and contain
The phyllopodium of growing point is transferred to debugging drop well prepared in advance in culture dish (operation drop) after the pretreatment such as over cleaning
In；
Step 3, observation of plant bud pointed tip separate living tissue structural form under the microscope；
Step 4, adjust and test electric rupture of membranes instrument function；
Step 5, plant sprout pointed tip separate living tissue, and fixed part separate living tissue is unfolded using microneedle；
Step 6, it according to actual conditions under mirror, adjusts the running parameter of electric rupture of membranes instrument and starts piezoelectric vibration, by micro-
It operates needle and handles plant sprout pointed tip separate living tissue, until plant monocyte falls off from separate living tissue；
Step 7, the microneedle for being fixed on part separate living tissue is unclamped, and repeats step in separate living tissue other parts
5- step 6.
Preferably, the step 4 includes the following steps:
Step 4.1, the heavy oil of 5uL or so is packed into microneedle with 10uL pipettor and micro loading needle；
Step 4.2, heavy oil is shifted onto the head of microneedle with hydraulic type microinjection instrument；
Step 4.3, microneedle is fallen into the debugging drop (operation drop) and tests piezoelectricity rupture of membranes instrument function, and
Micromanipulation needle section is cleaned with debugging drop (operation drop), until pressure balance.
Preferably, the culture dish of the step 1 is 60mm culture dish.
Preferably, the microneedle of the step 5 is Piezo TransferTip ES microneedle.
Preferably, the micro loading needle of the step 4.1 is the micro loading needle of Microloader.
Preferably, the heavy oil of the step 4.1 is Sigma FC-770 heavy oil.
Preferably, the running parameter of the electric rupture of membranes instrument of the step 6 includes intensity, speed, number.
Preferably, the running parameter of the electric rupture of membranes instrument of the step 6 is intensity 46, and 20 time/second of speed, number is nothing
It is poor big.
3. beneficial effect
In conclusion the beneficial effects of the present invention are:
(1) it the problem of preparation unicellular this method solve Activities of Some Plants, avoids and prepares in phytoplasm body running
It is original to maintain plant cell to the full extent as far as possible for destruction for structures and function such as plant cell script cell walls
Form and activity；
(2) this method carries out parameters adjustment according to different plant tissue situations and records, and facilitates practical operation, mentions
The high working efficiency of experiment, guarantees and improves the success rate of experiment.
Detailed description of the invention
Fig. 1 is that the arabidopsis bud pointed tip separate living tissue piezoelectricity auxiliary that two microneedles under microscope carry out is slender
Born of the same parents separate photo.
Specific embodiment
The embodiment of the present invention is further illustrated with arabidopsis shoot tip meristem.
Laboratory apparatus
A full set of Germany Eppendorf micromanipulation system is selected, basic configuration is as follows:
1.TransferMan 4r micromanipulation instrument x2 (for precisely mobile microneedle)；
2.CellTram 4r Air hand-driven pressure microinjection instrument x1 is (for drawing and discharging plant cell or routine
Unicellular sample)；
3.CellTram 4r Oil hand oil-pressure formula microinjection instrument x1 is (routinely unicellular for drawing and discharging plant
Sample or single plant stem cell etc.)；
The manual inverted microscope x1 of 4.Leica DMRIB (is used for accurate observation plant monocyte sample, various conventional inversions
Mirror can use)；
5.PiezoXpert piezoelectric type rupture of membranes instrument x1 is (suitable under the cooperation of the above equipment and consumptive material, generating one
Vibration, be transmitted on connected microneedle, cause plant cell to vibrate by operating needle, the plant containing cell wall is single
Cell is fallen from tissue vibration；Can then cooperate above equipment choose it is mobile and select required for discharging containing intact cell wall
Plant monocyte)；
6. 2 pipettor x1 of 10uL reference, several (for that will generate the mating heavy of piezoelectric vibration containing mating pipette tips
Oil is added in micromanipulation needle tubing, and is used to prepare the micro- debugging drop of various 10uL or less (operation drop))；
7. 2 pipettor x1 of 100uL reference, (it is micro- to be used to prepare various 10-100uL containing mating pipette tips are several
Debug drop (operation drop))；
Micromanipulation instrument (is installed on microscope) by 8.Adapter adapter.
Test consumptive material
1. 60mm Tissue Culture Dish: several (to take its ware lid as carrier, prepare plant tissue processing and separated with unicellular
Drop and blank control drop carry out various linked groups' cell micromanipulations)；
The micro loading needle of 2.Microloader: several (microneedle is added in the mating heavy oil for that will give birth to piezoelectric vibration
In pipe)；
3.Piezo TransferTip ES microneedle: several (vibrations generated for receiving following piezoelectricity rupture of membranes instrument
It is dynamic, and it is transmitted to plant tissue, tissue vibration is caused, the plant monocyte containing cell wall is fallen from tissue vibration；It then can be with
Cooperation above equipment is chosen mobile and discharges the required plant monocyte containing intact cell wall selected).
Experiment reagent
1. plant cell culturing liquid: several；
2.Sigma FC-770 heavy oil: (micromanipulation needle tubing is added with the mating micro loading needle of 10uL pipettor in 200uL
In, cooperation generates this controllable proper strength high frequency microvibration).
Experimental procedure
Step 1, in several debugging of 60mm culture dish middle preparation and drop is compareed with 100uL pipettor and mating pipette tips；
Step 2, with sterilization ophthalmology minor operation tweezer living body arabidopsis bud pointed tip separate living tissue, only retain 1-2 piece
Phyllopodium containing growing point is transferred to debugging drop well prepared in advance in culture dish (operation drop) after the pretreatment such as over cleaning
In；
Step 3, arabidopsis bud pointed tip separate living tissue structural form is observed in focusing under the microscope；
Step 4, the heavy oil of 5uL or so is packed into microneedle with 10uL pipettor and micro loading needle, is loaded onto aobvious
Operation needle is fallen into tune by the head that heavy oil is shifted onto after micro OS with the hydraulic type microinjection instrument of connection microneedle
Function of test piezoelectricity rupture of membranes instrument in test solution drop (operation drop), and with debugging drop (operating drop) cleaning operation needle section,
After final pressure balance, prepare formal experiment；
Step 5, left side micromanipulation instrument is operated, adjustment operation needle is unfolded arabidopsis bud pointed tip separate living tissue, then presses
Firmly a part of arabidopsis bud pointed tip separate living tissue carries out tissue and fixes, and operation the right micromanipulation instrument drives micromanipulation
Needle is moved on arabidopsis bud pointed tip separate living tissue, and pushes down another part tissue；
Step 6, start piezoelectric vibration, microneedle is driven to carry out the place of neighbouring arabidopsis bud pointed tip separate living tissue
Reason, can see that arabidopsis is unicellular under the microscope here and separate from tissue block one after another；
Step 7, the suitable parameters of piezoelectricity rupture of membranes instrument are adjusted according to actual conditions under mirror, including intensity, speed, secondary
Number etc. (this experiment setup parameter, intensity 46, speed is 20 times/, number be it is infinitely great, that is, step on i.e. generation after foot pedal
Vibration unclamps foot-operated i.e. stopping vibration after reaching effect), reach unicellular under being detached from, and with keeping the mesh of cell wall, such as
Fruit is satisfied to separating resulting, and a full set of parameter combination can be stored by program writing function；
Step 8, the two sides operation needle being pressed on arabidopsis bud pointed tip separate living tissue is unclamped, the quasi- of next position is carried out
Southern mustard cell piezoelectricity is detached from operational motion.
Experimental result
As shown in Figure 1, microscopically observation arrive carry out arabidopsis bud pointed tip separate living tissue with two microneedles
Piezoelectricity assist unicellular separation, therein one fixes plant living tissue, another microneedle according to microneedle
Piezoelectricity rupture of membranes instrument is connected, when also touching plant living tissue, starts piezoelectric vibration, plant monocyte is shaken from plant living tissue
It falls.Visible upper end plant living tissue does not have piezoelectric vibration in Fig. 1, therefore edge is intact, and the tissue not separated dissipates
Out；But the processed plant living tissue of piezoelectricity microneedle below Fig. 1, because organizing to be dissipated by shake, many unicellular points
It separating out and, living tissue rounded edges have been destroyed, and many vibrations that surrounding is spread get off to be dispersed in the plant monocyte of surrounding,
These cells shaken are used directly for the plant monocyte separation in downstream, and carry out subsequent singe-cell PCR immediately
With unicellular examining order.
The above description is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, all utilizations
The transformation of equivalent product method made by present specification, directly or indirectly uses and is attached to other relative technological products
Technical field, be included within the scope of the present invention.
Claims (8)
1. a kind of method for preparing the unicellular sample of Activities of Some Plants with piezo technology, which is characterized in that comprise the following steps:
Step 1, several debugging drops (operation drop) and contrasting fluid are prepared in culture dish with 100uL pipettor and mating pipette tips
Drop；
Step 2, tweezer plants bud pointed tip separate living tissue retains 1~2 phyllopodium containing growing point, through over cleaning etc.
It is transferred to after pretreatment in the debugging drop (operation drop)；
Step 3, observation of plant separate living tissue structural form under the microscope；
Step 4, adjust and test electric rupture of membranes instrument function；
Step 5, plant tip separate living tissue, and fixed part separate living tissue is unfolded using microneedle；
Step 6, it according to actual conditions under mirror, adjusts the running parameter of electric rupture of membranes instrument and starts piezoelectric vibration, pass through micromanipulation
Needle handles plant tip separate living tissue, until plant monocyte falls off from separate living tissue；
Step 7, the microneedle for being fixed on part separate living tissue is unclamped, and repeats step 5- step in separate living tissue other parts
Rapid 6.
2. a kind of method for preparing the unicellular sample of Activities of Some Plants with piezo technology according to claim 1, feature exist
In the step 4 includes the following steps:
Step 4.1, the heavy oil of 5uL or so is packed into microneedle with 10uL pipettor and micro loading needle；
Step 4.2, heavy oil is shifted onto the head of microneedle with hydraulic type microinjection instrument；
Step 4.3, microneedle is fallen into and tests piezoelectricity rupture of membranes instrument function in the debugging drop (operation drop), and with tune
Test solution drop (operation drop) cleaning micromanipulation needle section, until pressure balance.
3. a kind of method for preparing the unicellular sample of Activities of Some Plants with piezo technology according to claim 1, feature exist
In the culture dish of the step 1 is 60mm culture dish.
4. a kind of method for preparing the unicellular sample of Activities of Some Plants with piezo technology according to claim 1, feature exist
In the microneedle of the step 5 is Piezo TransferTip ES microneedle.
5. a kind of method for preparing the unicellular sample of Activities of Some Plants with piezo technology according to claim 1, feature exist
In the micro loading needle of the step 4.1 is the micro loading needle of Microloader.
6. a kind of method for preparing the unicellular sample of Activities of Some Plants with piezo technology according to claim 1, feature exist
In the heavy oil of the step 4.1 is Sigma FC-770 heavy oil.
7. a kind of method for preparing the unicellular sample of Activities of Some Plants with piezo technology according to claim 1, feature exist
In the running parameter of the electric rupture of membranes instrument of the step 6 includes intensity, speed, number.
8. a kind of method for preparing the unicellular sample of Activities of Some Plants with piezo technology according to claim 7, feature exist
In the running parameter of the electric rupture of membranes instrument of the step 6 is intensity 46, and 20 time/second of speed, number is infinity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811557180.8A CN109468263B (en) | 2018-12-19 | 2018-12-19 | Method for preparing single cell sample of partial plant by piezoelectric technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811557180.8A CN109468263B (en) | 2018-12-19 | 2018-12-19 | Method for preparing single cell sample of partial plant by piezoelectric technology |
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| Publication Number | Publication Date |
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| CN109468263A true CN109468263A (en) | 2019-03-15 |
| CN109468263B CN109468263B (en) | 2021-10-26 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2477644Y (en) * | 2001-04-27 | 2002-02-20 | 中国科学技术大学 | Vibration separator for biological tissue monoplast |
| CN1384186A (en) * | 2001-04-27 | 2002-12-11 | 中国科学技术大学 | Biological tissue unicell vibrating sepaation method and separator |
| CN102135525A (en) * | 2010-12-31 | 2011-07-27 | 重庆工商大学 | Mechanics analysis device and mechanics analysis method for piezomodulated and sensed single cell |
| CN102279255A (en) * | 2010-10-20 | 2011-12-14 | 重庆工商大学 | Mechanical dynamic regulation and control chip for single cell total environment |
-
2018
- 2018-12-19 CN CN201811557180.8A patent/CN109468263B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2477644Y (en) * | 2001-04-27 | 2002-02-20 | 中国科学技术大学 | Vibration separator for biological tissue monoplast |
| CN1384186A (en) * | 2001-04-27 | 2002-12-11 | 中国科学技术大学 | Biological tissue unicell vibrating sepaation method and separator |
| CN102279255A (en) * | 2010-10-20 | 2011-12-14 | 重庆工商大学 | Mechanical dynamic regulation and control chip for single cell total environment |
| CN102135525A (en) * | 2010-12-31 | 2011-07-27 | 重庆工商大学 | Mechanics analysis device and mechanics analysis method for piezomodulated and sensed single cell |
Non-Patent Citations (4)
| Title |
|---|
| MD. HABIBUR RAHMAN等: "Finite Element Analysis of Single Cell Wall Cutting by Piezoelectric-Actuated Vibrating Rigid Nanoneedle", 《IEEE TRANSACTIONS ON NANOTECHNOLOGY》 * |
| ZHAN YU等: "Piezoelectric inkjet assisted rapid electrospray ionization mass spectrometric analysis of metabolites in plant single cells via a direct sampling probe", 《ANALYST》 * |
| 刘亚欣等: "超声振动显微切割工具的设计与研制", 《光学精密工程》 * |
| 田桂中等: "细胞注射中数字化进退针装置的实验研究", 《中国机械工程》 * |
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