CN111426531A - Method for separating substances in and out of iron membrane on surface of wetland plant root - Google Patents

Method for separating substances in and out of iron membrane on surface of wetland plant root Download PDF

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Publication number
CN111426531A
CN111426531A CN202010260630.8A CN202010260630A CN111426531A CN 111426531 A CN111426531 A CN 111426531A CN 202010260630 A CN202010260630 A CN 202010260630A CN 111426531 A CN111426531 A CN 111426531A
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roots
iron
substances
root
centrifuge tube
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迟浩淳
袁永强
王诗忠
仇荣亮
汤叶涛
晁元卿
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Sun Yat Sen University
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Sun Yat Sen University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method for separating substances on the inner layer and the outer layer of an iron membrane on the surface of wetland plant roots, which comprises the steps of S1, taking the roots, shaking out the clear soil, and then washing the roots with sterile water; s2, cutting roots into root segments, placing the root segments into a centrifuge tube, adding PBS-S phosphate buffer solution, swirling, taking the roots out of the centrifuge tube, centrifuging the solution, and extracting the microbial DNA on the outer layer of the iron membrane on the surface of the roots, wherein solid matters in suspension in the centrifuge tube are outer-layer matters of the iron membrane on the surface of the roots; s3, placing the vortexed roots into another new centrifugal tube, adding 0.5M hydroxylamine-0.5M hydrogen chloride solution and sterile glass beads into each centrifugal tube, manually shaking the centrifugal tube up and down for 5min until the surfaces of the roots become white, and obtaining solid substances in suspension in the centrifugal tube, namely the substances in the inner layer of the iron membrane of the root surface. The invention can analyze the circulation process of the inner and outer layers of substances and the change condition of microorganisms, and is convenient for better revealing the influence of the oxygen concentration gradient on the rhizosphere iron-sulfur circulation.

Description

Method for separating substances in and out of iron membrane on surface of wetland plant root
Technical Field
The invention relates to the technical field of material separation, in particular to a method for separating substances in and out of an iron membrane on the surface of wetland plant roots.
Background
Post-mining wastelands typically contain large amounts of iron sulfides (e.g., pyrite, FeS)2) The oxidation acidification can cause the problems of acid wastewater generation, soil acidification, heavy metal release and the like, and meanwhile, due to insufficient supply of nutrient elements such as nitrogen, phosphorus, potassium and the like, common plants are difficult to fix. The wetland plant can transport oxygen in the atmosphere to the root surface through the root aeration tissue, so that the rhizosphere is in a relatively oxidized state, a micro-oxidation environment is formed in the rhizosphere of the plant, and a reducing substance Fe existing in the soil under the flooding condition2+Oxidation occurs to generate a root surface iron film with the main component of iron oxide. Oxidative (aerobic), reductive (anaerobic) microorganisms associated with the iron-sulfur cycle may be present in the wetland plant root surface micro-oxidative environment. The oxygen secretion of the root system causes the oxygen concentration of the rhizosphere to gradually decrease from the surface of the root to the outside, so that the oxygen concentration gradient is generated between the inner layer and the outer layer of the iron membrane on the surface of the root, and the material composition and the microbial community structure of the inner layer and the outer layer of the iron membrane on the surface of the root are different. Therefore, the separation of the substances in the inner layer and the outer layer of the iron membrane on the surface of the root is a precondition for exploring the difference between the structure of the substances in the inner layer and the outer layer of the iron membrane on the surface of the root and the difference between the microbial population, and the substances in the inner layer and the outer layer of the iron membrane on the surface of the root are dissolved step by adopting a chemical method, so that the method support can be provided for the subsequent substance analysis and microbial sequencing and the revealing of the biogeochemical cycle.
In the prior art, the scheme for collecting the iron membrane on the surface of the root is that the root of the wetland plant is shaken and washed by deionized water, after rhizosphere soil is removed, the root section of the plant is placed in PBS-S solution (phosphate buffer solution: NaCl: 17.60 g/L, Na)2HPO4:2.51g/L,NaH2PO40.47 g/L, Silwet-77:0.2 ml/L), and centrifuging for 20-30min to obtain the substances of the entire surface iron membrane of the roots.
The prior art has the following disadvantages: according to the prior art, all substances of the root surface iron membrane can be extracted at one time, substances in and out of the root surface iron membrane cannot be distinguished, the consumed time is long, the extraction is not thorough, the iron membrane is more in residue, and the extraction efficiency is low.
Disclosure of Invention
The invention aims to overcome the existing defects and provide a method for separating substances on the inner layer and the outer layer of the iron film on the surface of wetland plant roots, which provides feasibility for separating the substances on the inner layer and the outer layer of the iron film on the surface of the roots, shortens the extraction time, reduces the residue of the iron film and improves the extraction efficiency of the iron film.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for separating substances in and out of iron membranes on wetland plant roots comprises the following steps:
s1, shearing a proper amount of roots of the wetland plants by using scissors, shaking out the clear soil, and then washing the roots by using sterile water;
s2, cutting the washed roots into 3.5-4.5cm root segments, placing the root segments into a centrifuge tube, adding PBS-S phosphate buffer solution into the centrifuge tube, vortexing for 5min, taking the roots out of the centrifuge tube, centrifuging the solution in the centrifuge tube, extracting the microbial DNA of the outer layer of the surface iron membrane of the roots, obtaining solid substances in suspension in the centrifuge tube, namely the outer layer substances of the surface iron membrane of the roots, and filtering, collecting or storing the outer layer substances of the surface iron membrane of the roots;
s3, placing the vortexed roots into another new centrifugal tube, adding 0.5M hydroxylamine-0.5M hydrogen chloride solution and sterile glass beads with the diameter of 3mm into the centrifugal tube, manually shaking the centrifugal tube up and down for 5min until the root surface becomes white, wherein solid matters in the suspension obtained in the centrifugal tube in the step are inner-layer matters of the epilemia radicata, and then filtering, collecting or storing the inner-layer matters of the epilemia radicata.
Further, in step S1, the number of times of flushing is 3.
Further, in the step S2, the phosphate buffer solution includes 17.60 g/L g of NaCl and 2.51 g/L g of Na2HPO40.47 g/L of NaH2PO40.2 ml/L of Silwet-77.
The method is characterized in that different solutions are used for collecting substances on the inner layer and the outer layer of the iron film on the root surface on the premise of not influencing the properties of the inner layer and the outer layer of the iron film on the root surface.
Compared with the prior art, the invention has the beneficial effects that:
the invention firstly proposes that the iron membrane on the surface of the root is separated into an inner layer and an outer layer according to the oxygen concentration gradient, so that the circulation process of the substances and the change condition of microorganisms on the inner layer and the outer layer can be respectively analyzed, and the influence of the oxygen concentration gradient on the rhizosphere iron-sulfur circulation can be better revealed.
Drawings
FIG. 1 is an image of a root electron microscope after DI water rinsing in step S1 according to the present invention;
FIG. 2 is an image of a root electron microscope after being washed with PBS-S solution in step S2 according to the present invention;
FIG. 3 is an SEM image of 0.5M hydroxylamine hydrochloride and glass beads after being washed in step S3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A method for separating substances in and out of an iron membrane on the surface of cattail roots of cattail leaves comprises the following steps:
shearing a proper amount of roots of typha latifolia with scissors, clamping and shaking off the clear soil with tweezers, washing with sterile water for 3 times, placing the washed soil serving as rhizosphere soil in a centrifuge tube, and washing with deionized water to obtain an imaging picture of a root surface electron microscope shown in figure 1;
cutting the roots washed for 3 times into root segments of about 4cm, placing the root segments into another centrifugal tube, adding a PBS-S solution (phosphate buffer solution: NaCl: 17.60 g/L, Na2HPO4:2.51 g/L2 PO4:0.47 g/L, Silwet-77:0.2 ml/L) into the centrifugal tube, whirling for 5min, taking the roots out of the centrifugal tube, centrifuging the solution in the tube, extracting the microbial DNA on the outer layer of the epilayer of the iron, and obtaining solid substances in the suspension in the tube, wherein an imaging picture of an electron microscope of the roots cleaned by the PBS-S solution is shown in figure 2;
the vortexed roots were placed in a new centrifuge tube. A0.5M hydroxylamine-0.5M hydrogen chloride solution and sterile glass beads 3mm in diameter were added to the centrifuge tubes. And then manually shaking the centrifugal tube up and down for 5min until the root surface becomes white and clean, wherein the solid matter in the suspension obtained in the centrifugal tube in the step is the substance in the inner layer of the iron membrane on the root surface. An image of the root surface of the washed glass beads and 0.5M hydroxylamine hydrochloride is shown in FIG. 3.
The electron microscope images of the residual substance on the root surface after the three steps of washing shown in fig. 1-3 show that the three steps of washing respectively obtain the substances in different areas of the root surface. The processes of fig. 1 to 2 are the extraction of the substance on the outer layer of the iron membrane of the root surface, and fig. 2 to 3 are the extraction of the substance on the inner layer of the iron membrane of the root surface, and it can be seen from fig. 3 that the root surface is completely cleaned and the tissue on the root surface is clearly visible, so that the iron membrane of the root surface is completely collected.
The adhesion condition of the iron film on the root surface is shown by an electron microscope photo, rhizosphere soil is washed by deionized water, and the electron microscope photo shows that the iron film is integrally adhered to the root surface; washing down the outer layer substance of the iron film by PBS-S, and observing the inner layer substance of the iron film attached to the root surface by an electron microscope photo; and finally, washing and punching down the inner layer substance of the iron membrane, wherein the root surface is cleaned in an electron microscope picture, and the tissue form of the root surface can be seen without the adhesion of the iron membrane.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (3)

1. A method for separating substances in and out of iron membranes on wetland plant roots is characterized by comprising the following steps:
s1, shearing a proper amount of roots of the wetland plants by using scissors, shaking out the clear soil, and then washing the roots by using sterile water;
s2, cutting the washed roots into 3.5-4.5cm root segments, placing the root segments into a centrifuge tube, adding PBS-S phosphate buffer solution into the centrifuge tube, vortexing for 5min, taking the roots out of the centrifuge tube, centrifuging the solution in the centrifuge tube, and extracting the microbial DNA on the outer layer of the surface iron membrane of the roots, wherein the solid matter in the suspension in the centrifuge tube is the outer layer matter of the surface iron membrane of the roots;
s3, placing the vortexed roots into another new centrifugal tube, adding 0.5M hydroxylamine-0.5M hydrogen chloride solution and sterile glass beads with the diameter of 3mm into the centrifugal tube, and manually shaking the centrifugal tube up and down for 5min until the surfaces of the roots become white, wherein solid substances in the suspension liquid obtained in the centrifugal tube in the step are inner-layer substances of the iron membrane of the roots.
2. The method according to claim 1, wherein in the step S1, the number of times of washing is 3.
3. The method according to claim 1, wherein the phosphate buffer solution in step S2 comprises 17.60 g/L g NaCl and 2.51 g/L g Na2HPO40.47 g/L of NaH2PO40.2 ml/L of Silwet-77.
CN202010260630.8A 2020-04-03 2020-04-03 Method for separating substances in and out of iron membrane on surface of wetland plant root Pending CN111426531A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112501023A (en) * 2020-12-10 2021-03-16 湖南省烟草公司永州市公司 Method for obtaining plant root surface microbiome

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104160931A (en) * 2014-07-08 2014-11-26 华南农业大学 Quick induction and identification method for reddish brown iron films on plant root surfaces
CN107179223A (en) * 2017-04-18 2017-09-19 中山大学 Root border cells/microbial film generation analogue means and method
CN110295163A (en) * 2019-06-17 2019-10-01 广东省生态环境技术研究所 The extraction reagent and extracting method of The Rhizosphere of Rice iron membrane micro DNA

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104160931A (en) * 2014-07-08 2014-11-26 华南农业大学 Quick induction and identification method for reddish brown iron films on plant root surfaces
CN107179223A (en) * 2017-04-18 2017-09-19 中山大学 Root border cells/microbial film generation analogue means and method
CN110295163A (en) * 2019-06-17 2019-10-01 广东省生态环境技术研究所 The extraction reagent and extracting method of The Rhizosphere of Rice iron membrane micro DNA

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAOCHUN CHI，ET.AL.: ""Variation of the Bacterial Community in the Rhizoplane Iron Plaque of theWetland Plant Typha latifolia"", 《INT. J. ENVIRON. RES. PUBLIC HEALTH》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112501023A (en) * 2020-12-10 2021-03-16 湖南省烟草公司永州市公司 Method for obtaining plant root surface microbiome

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Application publication date: 20200717