CN105602885A

CN105602885A - Method for separating and purifying plant tapetum tissue

Info

Publication number: CN105602885A
Application number: CN201510797285.0A
Authority: CN
Inventors: 王春雷; 高季平; 叶蕴灵; 赵贝贝; 张盼盼
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2015-11-18
Filing date: 2015-11-18
Publication date: 2016-05-25
Anticipated expiration: 2035-11-18
Also published as: CN105602885B

Abstract

The invention relates to a method for separating and purifying a plant tapetum tissue, and belongs to the fields of developmental biology and cell biology. The method comprises the steps of collection of anthers, preparation of a cleaning liquid, preparation of an enzymolysis liquid, pretreatment of the anthers, removal of anther microspores, and separation and collection of tapetum cells. Suitable enzyme species and concentration are used for enzymolysis separation of a tapetum tissue, and then the tapetum is collected through filtration, centrifugation and purification. The method has the advantages of simple steps, convenient operation, shorter consumed time and the like, the separation efficiency is good, and the obtained tapetum tissue can be used for subsequent experiments.

Description

A kind of method of separation and purification plant fine fibre carpet veneer tissue

Technical field

The present invention is a kind of method that separates tapetum tissue from plant anther, relates to tapetum tissue and separates and obtain, and belongs to and sends outEducate biology and cell biology field.

Background technology

Tapetal cell is present in plant anther inner side, for microspore development provides necessary nutriment, becomes at microspore developmentRipe rear degraded. Tapetum has participated in numerous physiology courses such as microspore development, pollen and column cap identification, is the phases such as Developmental BiologyClose research field research emphasis.

At present, the specifically expressing of research related gene in tissue is international research trend. But tapetum is present in flower pesticide inside,Carrying out correlative study first needs to separate tapetum tissue, and has no at present correlation technique. In order to address the above problem, weInvent a kind of rapid enzymolysis technology, and used this technology successfully to isolate anther tapetum tissue. In this technology, weThe flower pesticide that utilizes 20 buds of Chinese cabbage to obtain, after enzymolysis, filtration, obtains enough tapetum tissues, and utilizes this tissue to enterOne step is extracted this tissue DNA and RNA.

Summary of the invention

The object of this invention is to provide easy, separate anther tapetum method fast, for further research related gene is at tapetumIn tissue, expression regulation lays the foundation.

A kind of method that separates tapetum tissue from plant anther of the present invention, comprises the steps such as flower pesticide processing, enzymolysis, separation, collectionSuddenly, finally the tapetum tissue of less impurity.

Specifically comprise the following steps (Fig. 1):

1) collection of flower pesticide: 20, the bud of collection first three day of flower, collect chemical drug; This step has been determined separation flower pesticide instituteNeed minimum number of flowers.

2) preparation of cleaning fluid: contain 50mMHepes, 0.5M sucrose, and regulate pH value to 7.5 with KOH;This step determines that tapetum separates suitable buffer concentration and sugared concentration, ensures in separation process that histocyte is notCan come to harm;

3) preparation of enzymolysis liquid: be 5% cellulase RS and 0.1% pectase Y23 to adding ultimate density in cleaning fluid;This step has determined that tapetum separates suitable enzymes kind and concentration;

4) flower pesticide pre-treatment: remove flower pesticide two ends with scalpel, and cut so that tapetal cell in the middle of residue positionExtract; This step solves the problem that flower pesticide sporidiole is removed, and ensures that enzymolysis liquid fully contacts with anther tissue;

5) sporidiole is removed: flower pesticide after treatment is put into 1.5ml centrifuge tube, add cleaning fluid 1ml, and vortex 20min,Filter, comprise sporidiole solution and abandon after filtering, other positions of flower pesticide are still stored in centrifuge tube, heavyMultiple above-mentioned cleaning, separating step; The processing of flower pesticide remainder products for further;

6) separation of tapetal cell: 600 μ l enzymolysis liquids are joined and filter the rear centrifuge tube that stores residue flower pesticide part,Vortex 12min, obtains supernatant by filtration, and tapetum tissue is contained in supernatant;

7) collection of tapetal cell: by supernatant centrifugal 15min under 4 DEG C, 12000g condition, precipitation is carpetLayer (Fig. 2).

Beneficial effect of the present invention is embodied in:

1) from plant anther, isolate first tapetal cell, can be for research tapetum tissue characteristics;

2) apply method of the present invention and separate tapetal cell, can be further used for tapetal cell DNA, RNA and extract, grindStudy carefully and pollen development, expression and the regulation and control of identification related gene in tapetum;

3) the present invention is simple to operate, consuming time short, separates tapetum and organizes impurity few, and purity is high, can be widely used in different plantsTapetum tissue separates, purifying.

Brief description of the drawings

Fig. 1 anther tapetum separation and purification flow chart.

Fig. 2 separates the tapetum tissue obtaining. T: tapetum; M: sporidiole. Scale=500 μ m. As can be seen from the figure,The tapetum purity separating is higher.

Fig. 3 SP11 is at different tissues expression analysis. SP11 gene is specifically expressing in Chinese cabbage flower pesticide. G: genomic DNA; T: suedeCarpet veneer RNA reverse transcription obtains cDNA; D: in separation process, other anther tissues RNA reverse transcription obtains cDNA.

Fig. 4 SP11 promoter region is S in S genotype₈S₄₄In Chinese cabbage, DNA methylation rate is analyzed.

Detailed description of the invention

Separating plant tapetum tissue is research microspore development, pollen and column cap identification related gene special table in tapetumOne of prerequisite that reaches, regulates and controls. We apply method of the present invention, have obtained the plant fine fibre carpet veneer tissue of enough, few impurity,Lay the foundation for carrying out correlative study. The foundation of our correlation technique, also fills up relevant blank.

This example is isolated tapetal cell with 20 cabbage rolls bud of first 3 days, experimentation following (Fig. 1):

1. collect 20, first three day Chinese cabbage bud of flower, take out flower pesticide;

2. excision flower pesticide two ends, and remainder is cut from centre, be convenient to remove sporidiole;

3. flower pesticide after treatment is put into 1.5ml centrifuge tube, add cleaning fluid 1ml, vortex 20min, filters, and comprises little sporeSub-solution abandons after filtering, and other positions of flower pesticide are still stored in centrifuge tube, repeat above-mentioned cleaning, separating step;The processing of flower pesticide remainder products for further;

4. 600 μ l enzymolysis liquids are joined to the centrifuge tube that stores residue flower pesticide part after filtering, vortex 12min, obtains by filtrationSupernatant, tapetum tissue is contained in supernatant;

5. by supernatant centrifugal 15min under 4 DEG C, 12000g condition, precipitation is tapetum (Fig. 2);

Utilize acquisition tapetum further to extract RNA and DNA, after RNA reverse transcription, express (Fig. 3) for detection of BrSP11; DNAFor detection of BrSP11-S₄₄The promoter region situation that methylates detects (Fig. 4).

Claims

1. a method for separation and purification plant fine fibre carpet veneer tissue, is characterized in that comprising the following steps:

1) collection of flower pesticide: 20, the bud of collection first three day of flower, collect chemical drug;

2) preparation of cleaning fluid: contain 50mMHepes, 0.5M sucrose, and regulate pH value to 7.5 with KOH;

3) preparation of enzymolysis liquid: be 5% cellulase RS and 0.1% pectase Y23 to adding ultimate density in cleaning fluid;

4) flower pesticide pre-treatment: with scalpel removal flower pesticide two ends, and cut so that the extraction of tapetal cell in the middle of residue position;

5) sporidiole is removed: flower pesticide after treatment is put into 1.5ml centrifuge tube, add cleaning fluid 1ml, vortex 20min, mistakeFilter, comprise sporidiole solution through filtration after abandon, other positions of flower pesticide are still stored in centrifuge tube, repeat above-mentioned cleaning,Separating step; The processing of flower pesticide remainder products for further;

6) separation of tapetal cell: 600 μ l enzymolysis liquids are joined and filter the rear centrifuge tube that stores residue flower pesticide part, vortex12min, obtains supernatant by filtration, and tapetum tissue is contained in supernatant;

7) collection of tapetal cell: by supernatant centrifugal 15min under 4 DEG C, 12000g condition, precipitation is tapetum.