CN105602885B - A method of isolating and purifying plant tapetal tissue - Google Patents
A method of isolating and purifying plant tapetal tissue Download PDFInfo
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- CN105602885B CN105602885B CN201510797285.0A CN201510797285A CN105602885B CN 105602885 B CN105602885 B CN 105602885B CN 201510797285 A CN201510797285 A CN 201510797285A CN 105602885 B CN105602885 B CN 105602885B
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- anther
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Abstract
The present invention relates to a kind of methods for isolating and purifying plant tapetal tissue, belong to Developmental Biology and cell biology.The present invention passes through the preparation of acquisition, cleaning solution to anther, the preparation of enzymolysis liquid, anther pre-treatment, the separation and collection step for removing anther microspore, tapetal cell.Using suitable enzyme class and concentration enzymatic hydrolysis separation tapetal tissue, then pass through filter centrifugation purified pool tapetum.This method has many advantages, such as that step is simple and convenient to operate, is time-consuming shorter, and separative efficiency is good, and obtained tapetal tissue can be used for subsequent experimental.
Description
Technical field
The present invention is a kind of method from plant anther separation tapetal tissue, is related to tapetal tissue separation and obtains,
Belong to Developmental Biology and cell biology.
Background technique
Tapetal cell is present on the inside of plant anther, necessary nutriment is provided for microspore development, in microspore
It degrades after reaching maturity.Tapetum takes part in numerous physiology courses such as microspore development, pollen and column cap identification, is development biology
Etc. Related Research Domains research emphasis.
Currently, the specifically expressing of research related gene in the tissue is international research trend.But tapetum is present in flower
Inside medicine, to carry out correlative study firstly the need of separation tapetal tissue, and have no the relevant technologies at present.In order to solve above-mentioned ask
Topic, we have invented a kind of rapid enzymolysis technologies, and are successfully separated out anther tapetum tissue with the technology.In this technology
In, the anther that we utilize 20 buds of Chinese cabbage to obtain obtains enough tapetal tissues after digesting, filtering, and utilizing should
Tissue further extracts the tissue DNA and RNA.
Summary of the invention
The object of the present invention is to provide easy, quick separating anther tapetum methods, exist for further research related gene
Expression regulation lays the foundation in tapetal tissue.
A kind of method from plant anther separation tapetal tissue of the present invention, including anther is handled, enzymatic hydrolysis, separation, is collected
And etc., finally less impurity tapetal tissue.
Specifically include the following steps (Fig. 1):
1) acquisition of anther: 20, bud of flower first three days are acquired, chemical drug is collected；This step has determined needed for separation anther
Minimum number of flowers.
2) preparation of cleaning solution: containing 50mM Hepes, 0.5M sucrose, and pH value is adjusted to 7.5 with KOH；This step is true
Determine tapetum and separate suitable buffer concentration and sugared concentration, guarantees that histocyte will not come to harm in separation process；
3) preparation of enzymolysis liquid: it is 5% cellulase RS and 0.1% pectase Y23 that ultimate density is added into cleaning solution；
This step has determined tapetum separation suitable enzymes type and concentration；
4) anther pre-treatment: anther both ends are removed with scalpel, and are cut among remaining position so as to tapetal cell
Extraction；This step solves the problems, such as that anther microspore removes, and guarantees that enzymolysis liquid comes into full contact with anther tissue；
5) microspore removes: by treated, anther is put into 1.5ml centrifuge tube, and cleaning solution 1ml, vortex 20min is added,
Filtering abandons after filtering comprising microspore solution, other positions of anther are still stored in centrifuge tube, repeats above-mentioned clear
It washes, separating step；The waiting of anther remainder is further processed；
6) 600 μ l enzymolysis liquids: being added to the centrifuge tube that remaining anther part is stored after filtering by the separation of tapetal cell,
Vortex 12min, by the way that supernatant is obtained by filtration, tapetal tissue is contained in supernatant；
7) collection of tapetal cell: supernatant is centrifuged 15min under the conditions of 4 DEG C, 12000g, precipitating is tapetum
(Fig. 2).
The beneficial effects of the present invention are embodied in:
1) tapetal cell is isolated from plant anther for the first time, can be used for studying tapetal tissue characteristic；
2) application method of the invention separates tapetal cell, can be further used for tapetal cell DNA, RNA extraction, grind
Study carefully the expression and regulation with pollen development, identification related gene in tapetum；
3) operation of the present invention is simple, and time-consuming short, separation tapetal tissue impurity is few, and purity is high can be widely applied to difference
The separation of plant tapetal tissue, purifying.
Detailed description of the invention
Fig. 1 anther tapetum isolates and purifies flow chart.
Fig. 2 isolated tapetal tissue.T: tapetum；M: microspore.Scale=500 μm.It can be seen from the figure that
Isolated tapetum purity is higher.
Fig. 3 SP11 is in different tissues expression analysis.SP11 gene specifically expressing in Chinese cabbage anther.G: genomic DNA；
T: tapetum RNA reverse transcription obtains cDNA；D: the RNA reverse transcription of other anther tissues obtains cDNA in separation process.
Fig. 4 SP11 promoter region is S in S genotype8S44DNA methylation rate is analyzed in Chinese cabbage.
Specific embodiment
Separating plant tapetal tissue is research microspore development, pollen and column cap identification related gene in tapetum
One of specifically expressing, prerequisite of regulation.We apply method of the invention, obtain the plant carpet of enough, few impurity
Layer tissue lays the foundation to carry out correlative study.Related blank is also filled up in the foundation of our correlation techniques.
3 days buds isolate tapetal cell before 20 cabbage rolls of this example, and experimentation is following (Fig. 1):
1. collecting 20, first three days Chinese cabbage bud of flower, anther is taken out；
2. cutting off anther both ends, and remainder is cut from centre, convenient for removal microspore；
3. by treated, anther is put into 1.5ml centrifuge tube, and cleaning solution 1ml, vortex 20min, filtering, comprising small is added
Spores solution abandons after filtering, other positions of anther are still stored in centrifuge tube, repeats above-mentioned cleaning, separating step；
The waiting of anther remainder is further processed；
4. 600 μ l enzymolysis liquids to be added to the centrifuge tube for storing remaining anther part after filtering, vortex 12min passed through
Filter obtains supernatant, and tapetal tissue is contained in supernatant；
5. supernatant is centrifuged 15min under the conditions of 4 DEG C, 12000g, precipitating is tapetum (Fig. 2)；
RNA and DNA are further extracted using tapetum is obtained, for detecting BrSP11 expression (Fig. 3) after RNA reverse transcription；
DNA is for detecting BrSP11-S44Promoter region methylation status detects (Fig. 4).
Claims (1)
1. a kind of method for isolating and purifying plant tapetal tissue, it is characterised in that include the following steps:
1) acquisition of anther: 20, bud of flower first three days are acquired, anther is collected；
2) preparation of cleaning solution: containing 50mM Hepes, 0.5M sucrose, and pH value is adjusted to 7.5 with KOH；
3) preparation of enzymolysis liquid: it is 5% cellulase RS and 0.1% pectase Y23 that ultimate density is added into cleaning solution；
4) anther pre-treatment: anther both ends are removed with scalpel, and cut mentioning so as to tapetal cell among remaining position
It takes；
5) microspore removes: by treated, anther is put into 1.5ml centrifuge tube, and cleaning solution 1ml, vortex 20min, mistake is added
Filter abandons after filtering comprising microspore solution, other positions of anther are still stored in centrifuge tube, the above-mentioned cleaning of repetition,
Separating step；The waiting of anther remainder is further processed；
6) separation of tapetal cell: 600 μ l enzymolysis liquids are added to the centrifuge tube for storing remaining anther part after filtering, are vortexed
12min, by the way that supernatant is obtained by filtration, tapetal tissue is contained in supernatant；
7) collection of tapetal cell: supernatant is centrifuged 15min under the conditions of 4 DEG C, 12000g, precipitating is tapetum.
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| CN201510797285.0A CN105602885B (en) | 2015-11-18 | 2015-11-18 | A method of isolating and purifying plant tapetal tissue |
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| CN105602885A CN105602885A (en) | 2016-05-25 |
| CN105602885B true CN105602885B (en) | 2018-12-11 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107653221B (en) * | 2017-10-11 | 2019-06-25 | 宁夏大学 | Tapetal cell separation and collection method suitable for protein science research |
| CN107522771A (en) * | 2017-10-19 | 2017-12-29 | 宁夏大学 | Tapetal cell total protein extraction method |
| CN108611313B (en) * | 2018-05-09 | 2021-03-05 | 扬州大学 | Method for separating mastoid cells from turnip stigma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687404A (en) * | 2005-05-25 | 2005-10-26 | 中国科学院植物研究所 | Method for separating intime of gymnosperm |
| CN101421402A (en) * | 2006-01-11 | 2009-04-29 | 分子作物育种代理有限公司 | Method of producing transgenic graminaceous cells and plants |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687404A (en) * | 2005-05-25 | 2005-10-26 | 中国科学院植物研究所 | Method for separating intime of gymnosperm |
| CN101421402A (en) * | 2006-01-11 | 2009-04-29 | 分子作物育种代理有限公司 | Method of producing transgenic graminaceous cells and plants |
Non-Patent Citations (3)
| Title |
|---|
| 侧柏小孢子囊壁绒毡层和中层细胞的发育;曹玉芳等;《西北植物学报》;20011231;第21卷(第3期);第546-550页 * |
| 甘蓝染色体图谱的构建及SI基因的定位研究;王永;《中国博士学位论文全文数据库 农业科技辑》;20100815(第08期);D048-2 * |
| 金鱼草珠被绒毡层壁囊的分离与鉴定;杨弘远;《植物学报》;19891231;第31卷(第11期);第817-823页 * |
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