CN103898091B - The tissue fixation process of plant genome DNA, protein is extracted for field sampling - Google Patents
The tissue fixation process of plant genome DNA, protein is extracted for field sampling Download PDFInfo
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- CN103898091B CN103898091B CN201310378734.9A CN201310378734A CN103898091B CN 103898091 B CN103898091 B CN 103898091B CN 201310378734 A CN201310378734 A CN 201310378734A CN 103898091 B CN103898091 B CN 103898091B
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Abstract
The invention discloses a kind of tissue fixation process extracting plant genome DNA, protein for field sampling.Tissue sample is cut into cross section and is not more than 0.5cm by the method
2stick put into after histologic fixatives soaks and to spend the night preservations, the sample liquid-transfering gun after fixing is removed most of fixing agent and is added the tissue mixed a little and fixes dry powder and then proceed to 4 DEG C of Refrigerator store several months or-20 DEG C of refrigerators are preserved for a long time.Plant tissue after simple and quick process is carried out to the plant tissue in field work, can be used for DNA, proteins extraction and later experiments.Both facilitate the work flow of field acquisition plant tissue, greatly save again the labour cost of field acquisition plant tissue.The present invention is the method utilizing histologic fixatives to carry out plant species organization material to be fixed, and provides a kind of simple effective method for scientific worker processes fixing plant tissue, particularly field sample of plucking a plant.
Description
Technical field
The invention belongs to agricultural biological technical field, be specifically related to a kind of tissue fixation process extracting plant genome DNA, protein for field sampling.
Background technology
Effective components in plants comprises the compositions such as plant nutrient ingredient, medicinal ingredients, essence, natural pigment, also comprises some biomacromolecules as DNA, RNA and protein etc.The extraction of this several composition such as general medicinal ingredients is carried out after needing to carry out oven dry pre-treatment to material, and such plant tissue is just easy to preserve, and there is not the problem of effective ingredient degradation, generally also there is not very large impact to follow-up experiment.
But when extracting some activeconstituents (enzyme, vitamins C or biomacromolecule are as DNA, RNA or protein etc.) in vegetable material, sometimes need to use fresh plant sample.The acquisition and processing of fresh plant specimen material is the link of crucial importance in Plant Molecular Biology.Should note fresh-keeping during sampling, the extraction usually should carrying out component to be measured after sampling is immediately degraded to prevent effective constituent; Also be placed on-80 DEG C of Ultralow Temperature Freezers after can adopting liquid nitrogen flash freezer to preserve for a long time; If just extract plant genome DNA, sample also can be placed in 0 ~ 4 DEG C of refrigerator and preserve a middle or short term.Take fresh plant materials sample from field, the following period of time before formal mensuration, how correctly appropriate preservation and process are very important, and it has influence on follow-up a series of relevant experiment, is also related to the accuracy of measurement result.
Usual plant sampling returns process immediately or cryopreservation, but often there are all difficulties, return after the meeting from field or field sampling sometimes, due to other things delay or plucked Multi-example and return to process immediately, so just can cause degraded or the loss of sample effective constituent.And there is certain distance in general field and laboratory, some is even separated by very far away, particularly cannot process at short notice especially after field study sampling and keep sample.So how to make plant tissue fix, ensureing that the effective constituent of plant sample is not degraded is a difficulty greatly.
Summary of the invention
The object of this invention is to provide a kind of tissue fixation process extracting plant genome DNA, protein safely and effectively for field sampling, the method safely and effectively by the plant tissue after field sampling, can extract genomic dna and the protein of requirement of experiment.
To achieve these goals, technical scheme of the present invention is: be provided for the tissue fixation process that plant genome DNA, protein are extracted in field sampling, comprise the following steps:
(1) histologic fixatives preparation
A, get 500 ~ 550g (NH
4)
2sO
4, 2.5gTris alkali, 3.7gNa
2above-mentioned three kinds of compositions are mixed by EDTA, obtain the fixing dry powder of tissue;
B, be mixed after tissue fix dry powder and add the hot boiling water of 700 ~ 800mL and fully stir and dissolve, 3 DEG C ~ 4 DEG C are cooled to saturated solution and have Precipitation, and PH is 7.0 ~ 7.4; Obtain histologic fixatives;
(2) Plant tissue samples process
A, Plant tissue samples is cut into cross section is not more than 0.5cm
2plant tissue samples stick;
B, Plant tissue samples stick is put into freshness protection package, load the histologic fixatives of 5 ~ 10 times of volumes toward freshness protection package, tighten after the complete excluding air of freshness protection package, put shady and cool place and fix 12 ~ 24 hours;
C, fixing after Plant tissue samples, remove the histologic fixatives liquid of 3/5 ~ 4/5 ratio in freshness protection package with liquid-transfering gun, and add the fixing dry powder of 1 ~ 5g tissue;
D, then proceed to 4 DEG C of Refrigerator store several months or-20 DEG C of refrigerators are preserved for a long time.
Present invention process flow process is simple, after plant sample is put into the histologic fixatives prepared, the effectively fixing of plant tissue can be realized and preserve, take fresh plant materials to extract plant genome DNA sample, protein and follow-up experiment for field and safe and efficient material is provided, reach the preservation object of the plant tissue materials after save energy, low carbon ring guarantor and harvesting, achieve the combination of ecology, economic and social benefit three.
Accompanying drawing explanation
For CTAB method, DNA extraction is carried out to cassava blade in Fig. 1, adopt the agarose gel electrophoresis of 0.8% to be separated, imaging schematic diagram under ultraviolet;
Fig. 2 adopts the reliability of primer pair transgenosis cassava genomic dna of fatty acid desaturase gene to carry out PCR checking, by the template of plasmid as positive control, adopts the agarose gel electrophoresis with 1.0% to be separated in imaging results schematic diagram under ultraviolet; In figure 1-7 respectively corresponding 1-7 plant sample DNA be the PCR result of template, wherein M is marker; Positive control take plasmid as template; Negative control take water as template;
Figure A in Fig. 3 is 2-D result after extracting directly plant sample albumen; Figure B be fix plant sample with histologic fixatives of the present invention after extract the 2-D result after albumen.
Embodiment
In order to describe technology contents of the present invention, structural attitude in detail, coordinate accompanying drawing to be described further below in conjunction with embodiment.
A kind of tissue fixation process extracting plant genome DNA, protein for field sampling of the present invention, comprises histologic fixatives preparation, Plant tissue samples process two steps:
(1), histologic fixatives preparation
A, get 500 ~ 550g (NH
4)
2sO
4, 2.5gTris alkali, 3.7gNa
2above-mentioned three kinds of compositions are mixed and obtain the fixing dry powder of tissue by EDTA;
B, be mixed after tissue fix dry powder and add the hot boiling water of 700 ~ 800mL and fully stir and dissolve, 3 DEG C ~ 4 DEG C are cooled to saturated solution and have Precipitation, and PH is 7.0 ~ 7.4; Obtain histologic fixatives;
(2), Plant tissue samples process
A, Plant tissue samples is cut into cross section is not more than 0.5cm
2plant tissue samples stick;
B, Plant tissue samples stick is put into freshness protection package, load the histologic fixatives of 5 ~ 10 times of volumes toward freshness protection package, tighten after the complete excluding air of freshness protection package, put shady and cool place and fix 12 ~ 24 hours (fixedly spending the night);
C, fixing after Plant tissue samples, available liquid-transfering gun removes most histologic fixatives liquid in freshness protection package, and adds the fixing dry powder of 1 ~ 5g tissue; The histologic fixatives liquid removed accounts for 60 ℅ ~ 80 ℅ of original histologic fixatives liquid;
D, then proceed to 4 DEG C of Refrigerator store several months or-20 DEG C of refrigerators are preserved for a long time.
For excess moisture or the excessive Plant tissue samples of volume, can adopt do not cut rear direct by sample the mode be embedded in histologic fixatives preserve.
The checking of field transgenosis cassava DNA extraction and template thereof
Cassava leaves agreement that contracts a film or TV play to an actor or actress 0.2 ~ 0.3g after above-mentioned fixing agent fixedly spends the night cleans, CTAB method is adopted to carry out DNA extraction to cassava blade again, the agarose gel electrophoresis of 0.8% is adopted to be separated, under ultraviolet, imaging results as shown in Figure 1, there is not the changes such as degraded in the cassava genomic dna fixing the extraction of rear CTAB method with histologic fixatives, can find out the integrity of DNA.
The reliability of the primer pair transgenosis cassava genomic dna of this gene (fatty acid desaturase gene) is adopted to carry out PCR checking, by the template of plasmid as positive control, under adopting the agarose gel electrophoresis of 1.0% to be separated in ultraviolet, imaging results as shown in Figure 2, the object band that this gene can be detected at 1200bp place can be seen, illustrate that the cassava DNA carried can use through checking.
The extraction of field protein cassava matter
Cassava blade 0.5 ~ 1g after above-mentioned fixing agent fixedly spends the night cleans, the protein of phenol extraction to cassava blade is adopted to extract again, quantitatively polyacrylamide gel 2-D result is run afterwards as shown in Figure 3 B by microplate reader, do not changing qualitatively with the protein that the method is extracted, can be used for doing the subsequent experimental such as 2-D.
As can be seen from the above results, carry out plant tissue materials with histologic fixatives of the present invention to be fixed and to extract plant genome DNA and protein afterwards, do not affect the quality of genomic dna and protein, follow-up experiment is not also made a difference, for field sample of plucking a plant provides one very simple effective method.
Tissue fixation process for field sampling extraction plant genome DNA, protein of the present invention is applicable to any plant sample tissue and fixes, and is not limited only to the field transgenosis cassava in the embodiment of the present invention.
Above disclosedly be only preferred embodiment of the present invention, certainly can not limit the interest field of the present invention with this, therefore according to the equivalent variations that the claims in the present invention are done, still belong to the scope that the present invention is contained.
Claims (1)
1., for a plant tissue fixing means for field sampling, this fixing plant tissue, for extracting plant protein or genomic dna, is characterized in that comprising the following steps:
(1) histologic fixatives preparation
A, get 500 ~ 550g (NH
4)
2sO
4, 2.5gTris alkali, 3.7gNa
2above-mentioned three kinds of compositions are mixed by EDTA, obtain the fixing dry powder of tissue;
B, be mixed after tissue fix dry powder and add the hot boiling water of 700 ~ 800mL and fully stir and dissolve, 3 DEG C ~ 4 DEG C are cooled to saturated solution and have Precipitation, and PH is 7.0 ~ 7.4, obtain histologic fixatives;
(2) Plant tissue samples process
A, Plant tissue samples is cut into cross section is not more than 0.5cm
2plant tissue samples stick;
B, Plant tissue samples stick is put into freshness protection package, load the histologic fixatives of 5 ~ 10 times of volumes toward freshness protection package, tighten after the complete excluding air of freshness protection package, put shady and cool place and fix 12 ~ 24 hours;
C, fixing after Plant tissue samples, remove the histologic fixatives liquid of 3/5 ~ 4/5 ratio in freshness protection package with liquid-transfering gun, and add the fixing dry powder of 1 ~ 5g tissue;
D, then proceed to 4 DEG C of Refrigerator store several months or-20 DEG C of refrigerators are preserved for a long time.
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|---|---|---|---|
| CN201310378734.9A CN103898091B (en) | 2013-08-27 | 2013-08-27 | The tissue fixation process of plant genome DNA, protein is extracted for field sampling |
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|---|---|
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| CN103898091B true CN103898091B (en) | 2016-03-23 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87107763A (en) * | 1986-11-04 | 1988-06-22 | 绮特娜公司 | Preserve the method for plant as the composition of preserving plant and with it |
| CN101636649A (en) * | 2007-02-27 | 2010-01-27 | 恰根有限公司 | Fixation of a biological material |
-
2013
- 2013-08-27 CN CN201310378734.9A patent/CN103898091B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87107763A (en) * | 1986-11-04 | 1988-06-22 | 绮特娜公司 | Preserve the method for plant as the composition of preserving plant and with it |
| CN101636649A (en) * | 2007-02-27 | 2010-01-27 | 恰根有限公司 | Fixation of a biological material |
Non-Patent Citations (1)
| Title |
|---|
| 常见动物植物标本石蜡切片在组织处理上的改进;蒋金芳 等;《农垦医学》;20071031;第29卷(第5期);391-393页 * |
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