CN109425596A - Application of the Fluoresceincarboxylic acid ester in detection phloem sap in the transport and distribution in different tissues vascular bundle - Google Patents
Application of the Fluoresceincarboxylic acid ester in detection phloem sap in the transport and distribution in different tissues vascular bundle Download PDFInfo
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- CN109425596A CN109425596A CN201710788645.XA CN201710788645A CN109425596A CN 109425596 A CN109425596 A CN 109425596A CN 201710788645 A CN201710788645 A CN 201710788645A CN 109425596 A CN109425596 A CN 109425596A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The present invention provides application of the Fluoresceincarboxylic acid ester in detection phloem sap in the transport and distribution in plant different tissues and organ.The Fluoresceincarboxylic acid ester is one kind of fluorescein derivative, there is higher fluorescent absorption under the spectrum of 488nm, has preferable fluorescent yield and good water solubility.The present invention also provides a kind of methods of transport and distribution of detection phloem sap in plant different tissues and organ, comprising: 1) marker introduced plant is marked；2) transporting direction for observing marker and the distribution in different vascular bundles.The present invention will be in marker introduced plant by the method for artificial drainage, transporting direction using confocal microscopy marker and the distribution in different vascular bundles, the method provide important real value in the distribution of library organ for the correlation of the research each histoorgan of plant, assimilation quotient.
Description
Technical field
The present invention relates to a kind of labeling methods, and in particular to Fluoresceincarboxylic acid ester is in detection phloem sap in plant difference
Application in tissue and transport and distribution in organ.
Background technique
For vascular plant, vascular bundle is broadly divided into xylem and bast, and xylem is mainly plant from soil
Moisture is absorbed in earth is transported up supply plant water sources, and the nutriments such as bast prevailing traffic inorganic salts are for the growth of plant
Development.Vascular bundle participates in the exchange transport of plant substance in vivo, and support and the life for consolidating plant through entire plant
It is long.Most of photoassimilates that plant carries out photosynthesis generation are all transported in the organ of library for its growth and development, and are determined
It is again many to the factor of library organ transportation to determine photosynthate, including photosynthetic efficiency, the phloem loading of source, photoassimilates
Long-distance transportation and after the unloading and unloading of library organ bast transport etc..Therefore photoassimilates transport for
Plant products and quality have important decisive action.In order to study the transport of photoassimilates and other substances in cell
And distribution, we are using the fluorescent dye being commonly esterified in medicine, botany and microbiological research at present as mark
Remember object, is observed in conjunction with laser confocal scanning microscope to be analyzed.Fluoresceincarboxylic acid ester as a kind of fluorescent dye,
It is a kind of " film impermeability " probe, it can carry out long-distance transportation in bast.In bast, Fluoresceincarboxylic acid ester can
To be degraded into Fluoresceincarboxylic acid by endogenous esterase, so that the light that is excited, which inspires fluorescence, facilitates observation.Although Fluoresceincarboxylic acid ester
Have application in the research of plant unloading manner, but using Fluoresceincarboxylic acid ester as marker to entire plant different tissues
There is presently no reports for the research of Translocation of Assimilates and distribution in bast.
Summary of the invention
The object of the present invention is to provide Fluoresceincarboxylic acid esters to detect phloem sap in plant different tissues and organ
Transport and distribution in application.
The Fluoresceincarboxylic acid ester is one kind of fluorescein derivative, there is higher fluorescent absorption under the spectrum of 488nm,
With preferable fluorescent yield and good water solubility.Its structural formula is as shown in Equation 1:
The present invention also provides the sides of transport and distribution of a kind of detection phloem sap in plant different tissues and organ
Method.
The method of transport and distribution of the detection phloem sap provided by the present invention in plant different tissues and organ,
Include the following steps:
1) marker introduced plant is marked；
2) transporting direction for observing marker and the distribution in different vascular bundles.
In the method, use Fluoresceincarboxylic acid ester as marker.
The Fluoresceincarboxylic acid ester is one kind of fluorescein derivative, there is higher fluorescent absorption under the spectrum of 488nm,
With preferable fluorescent yield and good water solubility.Its structural formula is as shown in Equation 1:
In the method, the concentration of the Fluoresceincarboxylic acid ester used is 50mM/L-100mM/L, uses dimethyl sulfoxide
(DMSO) dissolution obtains.
The plant designed in the method can be cucumber.
The kind of the cucumber is the close thorn of state's agriculture 25 or Xintai City.
In the method, marker can be introduced from blade surface.Wherein the blade carries out photosynthesis for mature
Blade.
In the method, can from the positions such as petiole, stem, carpopodium, melon handle and fruit marker.
In the method, observation can be marked and sampled on sunny daytime.
In the method, using the transporting direction of confocal microscopy marker and in different vascular bundles
Distribution.
It is marked in blade surface, petiole and stem is observed after 2-3 hour.
It is marked in blade surface, carpopodium, melon handle and fruit is observed after 4-5 hour.
The concrete operations of the detection can carry out as steps described below: sunny when 1) introducing Fluoresceincarboxylic acid ester from blade
Under weather, selection growth first is normal, the blade reached maturity, and then slightly rubs three to the four of blade with the sand paper of 400 mesh
A different zones (as shown in Figure 1) draw Fluoresceincarboxylic acid ester solution with pipettor and drip to three to four different zones, Ge Gequ
The Fluoresceincarboxylic acid ester solution of 100 μ l is added dropwise in domain, that is, is engraved in processing region and covers one layer of preservative film to prevent Fluoresceincarboxylic acid ester
The volatilization of solution, and one layer of masking foil is covered to prevent Fluoresceincarboxylic acid ester from decomposing.Normal illumination handle 2-3 hour it
Afterwards, take the petiole of labeled leaf with the stem tissue being connected to laboratory is taken back on ice, free-hand section is micro- in confocal scanning later
It is observed under mirror；Or
2) when introducing Fluoresceincarboxylic acid ester from blade, under fair weather, selection growth first is normal, the leaf reached maturity
Then piece is slightly rubbed three to four different zones (as shown in Figure 1) of blade with the sand paper of 400 mesh, draws carboxylic with pipettor
Base fluorescein ester solution drips to three to four different zones, and each region is added dropwise the Fluoresceincarboxylic acid ester solution of 100 μ l, that is, is engraved in
Processing region covers one layer of preservative film to prevent the volatilization of Fluoresceincarboxylic acid ester solution, and covers one layer of masking foil to prevent carboxylic
Base fluorescein ester decomposes.After normal illumination handles 4-5 hour, same section carpopodium is taken, melon handle and fruit to taking back on ice
Laboratory is observed under confocal scanning microscope after free-hand section.
The present invention, by marker introduced plant, utilizes confocal microscopy marker by the method for artificial drainage
Transporting direction and the distribution in different vascular bundles, the method be to study the correlation of each histoorgan of plant, same
Compound provides important real value in the distribution of library organ.
Detailed description of the invention
Fig. 1 is the method schematic diagram that Fluoresceincarboxylic acid ester is introduced from blade.
Fig. 2 is Fluoresceincarboxylic acid ester after blade introducing, to the observation figure of Fluoresceincarboxylic acid in petiole and stem.
Fig. 3 is Fluoresceincarboxylic acid ester after blade introducing, carpopodium, melon and fruit in observation to Fluoresceincarboxylic acid
Figure.
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, biomaterial etc., are commercially available unless otherwise specified.
In following embodiments, Fluoresceincarboxylic acid ester is purchased from Sigma company, trade name 5 (6)-
Carboxyfluorescein diacetate。
In following embodiments, experimental material is cucumber, and kind is the close thorn of state's agriculture 25 or Xintai City.
In following embodiments, mark substance is Fluoresceincarboxylic acid ester, and concentration 50mM/L, marker dimethyl sulfoxide is dilute
It releases.
In following embodiments, configured Fluoresceincarboxylic acid ester solution must be wrapped up with masking foil, prevent solution degradation.
Embodiment 1, Fluoresceincarboxylic acid ester are introduced from blade, the observation in the vascular bundle of petiole and stem to Fluoresceincarboxylic acid
Cucumber seeds are seeded in the pot for growing seedlings of 9cm × 9cm, using turf: vermiculite=2:1 matrix formulations, to seedling
Length to three leaves are wholeheartedly transplanted to China Agricultural University's heliogreenhouse, Routine Management, single climing training.In fair weather, to complete exhibition
Open, the blade reached maturity carries out Fluoresceincarboxylic acid ester label, after marking 2-3 hour, take the petiole of labeled leaf be connected
Stem tissue, at least three times biology repeat.
Concrete operation step are as follows: when introducing Fluoresceincarboxylic acid ester from blade, under fair weather, selection growth first is normal,
Then the blade reached maturity slightly is rubbed three to four different zones (as shown in Figure 1) of blade with the sand paper of 400 mesh, is used
Pipettor draws Fluoresceincarboxylic acid ester solution and drips to three to four different zones, and the Fluoresceincarboxylic acid of 100 μ l is added dropwise in each region
Ester solution is engraved in processing region and covers one layer of preservative film to prevent the volatilization of Fluoresceincarboxylic acid ester solution, and covers one layer
Masking foil is to prevent Fluoresceincarboxylic acid ester from decomposing.Normal illumination handle 2-3 hour after, take the petiole of labeled leaf be connected
Stem tissue, be put in and take back laboratory on ice, observed under confocal scanning microscope after free-hand section.
Fluoresceincarboxylic acid ester Fluirescence observation: it after taken tissue free-hand section, spreads to the glass slide of 2.5cm × 7.5cm
On, with 80% glycerite mounting, the coverslip of 2.5cm × 5.0cm is covered, it is burnt to be placed in the copolymerization that exciting light is 488nm
It is observed under flying-spot microscope.
After Fluoresceincarboxylic acid ester index blade, the petiole of labeled leaf and the cross section for the stem tissue being connected are observed, it can be with
Green fluorescence is seen in the vascular bundle in petiole and stem cross section, as shown in Figure 2.
Using Fluoresceincarboxylic acid ester index blade, Fluoresceincarboxylic acid can be observed and transported from blade vascular bundle to petiole and stem
Different vascular bundles, this just implys that the phloem sap in blade can be freely transported along vascular bundle to petiole and connected
In stem tissue, and there is distribution in each vascular bundle of petiole and stem.
Embodiment 2, Fluoresceincarboxylic acid ester from blade introduce, carpopodium, melon in the vascular bundle with fruit to Fluoresceincarboxylic acid
Observation
Cucumber seeds are seeded in the pot for growing seedlings of 9cm × 9cm, using turf: vermiculite=2:1 matrix formulations, to seedling
Length to three leaves are wholeheartedly transplanted to China Agricultural University's heliogreenhouse, Routine Management, single climing training.It is long to there is the same day of blooming to plant
Fruit when, in fair weather, to being fully deployed, the blade reached maturity carries out Fluoresceincarboxylic acid ester label, marks 4-5 hour
Later, the carpopodium, melon handle and fruit of the same section of labeled leaf are taken, at least biology repeats three times.
Concrete operation step are as follows: fine when plant length to the fruit for having the same day of blooming when introducing Fluoresceincarboxylic acid ester from blade
Under bright weather, selection growth first is normal, the blade reached maturity, and is then slightly rubbed and the three of blade is arrived with the sand paper of 400 mesh
Four different zones (as shown in Figure 1) draw Fluoresceincarboxylic acid ester solution with pipettor and drip to three to four different zones, each
The Fluoresceincarboxylic acid ester solution of 100 μ l is added dropwise in region, that is, is engraved in processing region and covers one layer of preservative film to prevent Fluoresceincarboxylic acid
The volatilization of ester solution, and one layer of masking foil is covered to prevent Fluoresceincarboxylic acid ester from decomposing.4-5 hour is handled in normal illumination
Later, the carpopodium, melon handle and fruit for taking the same section of labeled leaf, are put in and take back laboratory on ice, burnt in copolymerization after free-hand section
It is observed under flying-spot microscope.
Fluoresceincarboxylic acid ester Fluirescence observation: it after taken tissue free-hand section, spreads to the glass slide of 2.5cm × 7.5cm
On, with 80% glycerite mounting, the coverslip of 2.5cm × 5.0cm is covered, it is burnt to be placed in the copolymerization that exciting light is 488nm
It is observed under flying-spot microscope.
After Fluoresceincarboxylic acid ester index blade, the carpopodium of observing the same section of labeled leaf, melon, can the longitudal section with fruit
With in the carpopodium of same section, melon is green fluorescence is seen in the vascular bundle with the longitudal section of fruit, as shown in Figure 3.
Using Fluoresceincarboxylic acid ester index blade, Fluoresceincarboxylic acid can be observed and transported from blade vascular bundle to same section
Carpopodium, melon the vascular bundle different with fruit, this just imply that the phloem sap in blade can freely be transported along vascular bundle to
With in the carpopodium, melon handle and fruit of section, and the carpopodium of same section, melon and each vascular bundle of fruit in have distribution.
Claims (9)
1. application of the Fluoresceincarboxylic acid ester in detection phloem sap in the transport and distribution in plant different tissues and organ.
2. application according to claim 1, it is characterised in that: the structural formula of the Fluoresceincarboxylic acid ester is as shown in Equation 1:
。
3. a kind of method of transport and distribution of detection phloem sap in plant different tissues and organ, including following steps
It is rapid:
1) marker introduced plant is marked；
2) transporting direction for observing marker and the distribution in different vascular bundles.
4. according to the method described in claim 3, it is characterized by: using Fluoresceincarboxylic acid ester as label in the method
Object；
The structural formula of the Fluoresceincarboxylic acid ester is as shown in Equation 1:
。
5. according to the method described in claim 4, it is characterized by: in the method, the concentration of the Fluoresceincarboxylic acid ester used
For 50mM/L-100mM/L, obtained with dmso solution.
6. the method according to any one of claim 3-5, it is characterised in that: in the method, introduced from blade surface
Marker；
Wherein the blade carries out photosynthetic blade for mature.
7. the method according to any one of claim 3-6, it is characterised in that: in the method, from petiole, stem, carpopodium,
Marker is observed at melon handle and fruit position；
In the method, observation is marked and sampled on sunny daytime.
8. the method according to any one of claim 3-7, it is characterised in that: burnt micro- using copolymerization in the method
The transporting direction of sem observation marker and the distribution in different vascular bundles.
9. the method according to any one of claim 3-8, it is characterised in that: in the method, marked in blade surface,
Petiole and stem are observed after 2-3 hour；Or
It is marked in blade surface, carpopodium, melon handle and fruit is observed after 4-5 hour.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710788645.XA CN109425596A (en) | 2017-09-04 | 2017-09-04 | Application of the Fluoresceincarboxylic acid ester in detection phloem sap in the transport and distribution in different tissues vascular bundle |
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| CN201710788645.XA CN109425596A (en) | 2017-09-04 | 2017-09-04 | Application of the Fluoresceincarboxylic acid ester in detection phloem sap in the transport and distribution in different tissues vascular bundle |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011550A (en) * | 2020-08-17 | 2020-12-01 | 中国农业大学 | Method for blocking exchange of petiole phloem identification signals |
| CN112268731A (en) * | 2020-09-25 | 2021-01-26 | 江苏大学 | Low-damage plant xylem juice sampling method |
| CN112461572A (en) * | 2020-10-20 | 2021-03-09 | 江苏大学 | Method for rapidly extracting plant phloem juice |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10215894A (en) * | 1997-02-06 | 1998-08-18 | Mitsubishi Chem Corp | Detection of live cell |
| CN102495037A (en) * | 2011-12-07 | 2012-06-13 | 南京农业大学 | Fluorescent tracing method of assimilation substance unloading path of phloem in pear fruit |
| CN102680670A (en) * | 2012-05-22 | 2012-09-19 | 北京农学院 | Method for introducing fluorescence tracer into radish fleshy root and application of method |
| CN102942553A (en) * | 2012-10-15 | 2013-02-27 | 石平 | Preparation method for 6-carboxylfluorescein |
-
2017
- 2017-09-04 CN CN201710788645.XA patent/CN109425596A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10215894A (en) * | 1997-02-06 | 1998-08-18 | Mitsubishi Chem Corp | Detection of live cell |
| CN102495037A (en) * | 2011-12-07 | 2012-06-13 | 南京农业大学 | Fluorescent tracing method of assimilation substance unloading path of phloem in pear fruit |
| CN102680670A (en) * | 2012-05-22 | 2012-09-19 | 北京农学院 | Method for introducing fluorescence tracer into radish fleshy root and application of method |
| CN102942553A (en) * | 2012-10-15 | 2013-02-27 | 石平 | Preparation method for 6-carboxylfluorescein |
Non-Patent Citations (1)
| Title |
|---|
| ALISON G.ROBERTS: "Phloem Unloading in Sink Leaves of Nicotiana benthamiana:Comparison of a Fluorescent Solute with a Fluorescent Virus", 《THE PLANT CELL》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011550A (en) * | 2020-08-17 | 2020-12-01 | 中国农业大学 | Method for blocking exchange of petiole phloem identification signals |
| CN112268731A (en) * | 2020-09-25 | 2021-01-26 | 江苏大学 | Low-damage plant xylem juice sampling method |
| CN112268731B (en) * | 2020-09-25 | 2023-02-17 | 江苏大学 | Low-damage plant xylem juice sampling method |
| CN112461572A (en) * | 2020-10-20 | 2021-03-09 | 江苏大学 | Method for rapidly extracting plant phloem juice |
| CN112461572B (en) * | 2020-10-20 | 2023-01-17 | 江苏大学 | Method for rapidly extracting plant phloem juice |
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Application publication date: 20190305 |