CN106289907A - Quickly prepare small-scale marine fishes or the method for larva and juvenile division phases chromosome - Google Patents
Quickly prepare small-scale marine fishes or the method for larva and juvenile division phases chromosome Download PDFInfo
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- CN106289907A CN106289907A CN201610656985.2A CN201610656985A CN106289907A CN 106289907 A CN106289907 A CN 106289907A CN 201610656985 A CN201610656985 A CN 201610656985A CN 106289907 A CN106289907 A CN 106289907A
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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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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Abstract
A kind of method quickly preparing small-scale marine fishes or larva and juvenile division phases chromosome, belong to field of marine biotechnology, described method is by the improvement to existing seawater fish method of chromosome preparation, substantial amounts of dispersion preferable division phases chromosome is prepared, to meet the demand of the research such as various small-scale marine fishes or larva and juvenile Idioplasm identification, ploidy analysis, chromosome fluorescence in-situ hybridization analysis, sex controll with minimal amount of Experimental fish body surface organization.The inventive method can overcome test fish quantity little restriction few, individual, from the beginning of processing test fish, within 6～9 hours, result i.e. be can be observed, simple and convenient, preparation process can be adjusted flexibly and process intensity, can carry out at any time in cultivation nursery base or production unit, after preparing chromosome, basic guarantee test fish survives.
Description
Technical field:
The invention belongs to field of marine biotechnology, be specifically related to one and quickly prepare small-scale marine fishes or larva and juvenile
The method of division phases chromosome.
Background technology:
Chromosome is the carrier of hereditary material, the research to Fishes Chromosomes, is possible not only to inquire into its classification position and be
System develops, it helps the qualification of nearly edge species and colony assay, exploitation and genetic breeding to resource have important
Meaning.Existing Fish have kind more than 22000 in the whole world, are to be distributed the widest, the monoid of most species in vertebrates, embody multiple many
The biological characteristics of sample, has great economic worth.But in vertebrates, the chromosome of Fish is less, and number is on the high side, has
The progress closing chromosome research is relatively slow, and before and after 2005, the Fish of existing karyotype report only have about 2100 kinds,
Accounting for the 10% of sum, these Fish having chromosome to record are concentrated mainly on Cypriniformes and Perciformes, and great majority are fresh water
Fish (Gao Wen, Advances in study of fish chromosome, Ningde Journal of Teachers College (natural science edition), 2005,17 (1): 15-17), relevant
The report that seawater fish chromosome is recorded is also few, and this is not inconsistent with seawater fish aquatic breeding fast development.
From (Ojima Y S, Hitotsumachi S, Makino.Cytogentic studies in such as Ojima in 1966
Lower vertebrates.Proc Jap Acad, 1966,42 (1): 62-66) use Hypotonic treatment and airing first
Film-making, carry out Fishes Chromosomes research successful after, current Fishes Chromosomes film-making is all adopted in this way, but uses this
The method of kind to prepare a large amount of division phases and chromosome image slice, thin piece clearly is more difficult, it is impossible to meet chromosome correlation analysis
Requirement.It has been recognized that fish cell di is low, auxiliary is needed to use PHA (phytohemagglutinin) (Yamamoto
K,Ojima Y.A PHA-culture method for cells from tissue of teleosts.Japan J
Genet, 1973,48 (3): 235-238) promote cell division, to obtaining the best split coil method, but this dye
The program that colour solid preparation method processes because of many PHA, causes fish body more and once injures, and injects PHA and Colchicine
Method required time is longer, and some often failed because test fish is dead up to more than 30 hours.The different tissues of different Fish
Cell division phase index is not quite similar, and at most fish apoplexy due to endogenous wind, hemopoietic organ is that kidney (head-kidney), especially freshwater fish are making
Renal tissue is all used during chromosome sectioning.According to custom renal tissue film-making, the renal tissue of some Fish is not the most very
Prosperity, can not get enough split coil method (the group type analysis of Mao Lianju, Li Ya beautiful .5 kind seawater fish chromosome, Dalian Fisheries Sciences
Institute's journal, 2002,17 (2): 108-113).Ye Youqu Fish gill tissue and spleen make chromosome sectioning, but chromosome
Split coil method number is different because of fish, and reported success also only has Hemitripterus and continuous spleen, and spleen is only the fish of high monoid
Apoplexy due to endogenous wind is the most flourishing.For small fishes and larva and juvenile, solution to cut their kidney, spleen for preparing dyeing
Body is by no means easy.
Chromosome preparation need to take into full account experiment purpose and the characteristic of test material itself, marine fishes from the two poles of the earth to red
Road, from seashore to ocean, from top layer to myriametre about abyss have distribution.The multiformity of living environment, facilitates marine fish
The multiformity of class.But owing to life style is identical, marine fishes in different poses and with different expressions have a series of common feature: have breathing water
The gill of middle dissolved oxygen, fin-shaped be easy to hydrogymnastic limbs, can secreting mucus to reduce the skin of water movement resistance.Ocean
Environment is again to hide crisis everywhere, and directly the fish body of contact water body needs the various stimulations constantly adapting in water body, so
Body surface organization's cell is typically all in the state of vigorous division and proliferation.Prepare a large amount of split coil method chromosome, it is necessary to assure examination
The taken histiocyte testing fish is in the state of division and proliferation in a large number.
Fish breeding work has many kinds to relate to chromosome set operation, such as gynogenesis and multiploid induction etc..Educate
The test fish that kind of initial stage obtains all is of great rarity, but scientific research personnel wants to detect their chromosome composition and is not willing and kills test
Fish.Small fishes and fry cannot use conventional methods especially prepares chromosome, traditional Fishes Chromosomes preparation method
The longest, it is necessary to putting to death test fish, cost is the most heavy.
Summary of the invention:
The technical problem to be solved in the present invention is to provide one quickly to prepare small-scale marine fishes or larva and juvenile divides mid-term
The method splitting phase chromosome, described method is by the improvement to existing seawater fish method of chromosome preparation, with minimal amount of reality
Test and prepare substantial amounts of dispersion preferable division phases chromosome with fish body surface organization, with meet various small-scale marine fishes or
The demand of the research such as larva and juvenile Idioplasm identification, ploidy analysis, chromosome fluorescence in-situ hybridization analysis, sex controll.This
Inventive method can overcome test fish quantity little restriction few, individual, from the beginning of processing test fish, within 6～9 hours, knot i.e. be can be observed
Really, simple and convenient, preparation process can be adjusted flexibly and process intensity, can open at any time in cultivation nursery base or production unit
Exhibition, after preparing chromosome, basic guarantee test fish survives.
The present invention completes according to following operational approach:
A kind of method quickly preparing small-scale marine fishes or larva and juvenile division phases chromosome, described method includes:
1) it is fully understood by testing biological characteristics and the cleaning of slide of fish；2) small-scale test Fish or larva and juvenile are put into Colchicine
Solution went swimming；3) gill tissue or the isozyme that take test fish carry out chromosome and prepare.
Described is fully understood by testing biological characteristics and the cleaning of slide of fish: understand the test physical characteristic of fish, battalion
Support composition, swimming model of action and life style, to determine sampling point and testing program, determine sampling point and testing program
Standard as follows: the short and small person of fin ray fin ray free-end sample, the transparent keratin of fin ray such as cicada's wings or cutin then be not suitable for sample,
Changing to take the gill filament, what fin ray fat content was high should strengthen hypotonic dynamics, selects 0.0375M or 0.05M KCl hypotonic medium, gill cover opening
Spending the big gill filament free-end that takes, the fierce test fish swimming water body of motion requires more than 1.5L, actionless test fish fish trip
Swimming water body requires 100-200ml；The microscope slide of chromosome sectioning requires totally without greasy dirt, does not hang water, clear before prepared by chromosome
Wash clean.
Described colchicine solution went swimming that small-scale test Fish or larva and juvenile are put into: preparation containing 0.005% or
The sea water of 0.01% (g/ml) Colchicine, allows test fish swim wherein 4-6 hour, and the water temperature of swimming water body is more former supports water temporarily
Temperature improves or reduces 2-3 DEG C, to stimulate the ability strengthening test fish body surface organization division and proliferation；
Described take the test a small amount of gill tissue of fish or isozyme carries out chromosome and prepares: take 1-8mm3Gill tissue or fin ray
Tissue, is immediately placed in hypotonic 50min in 0.0375M, 0.05M or 0.075M KCl solution, transfers to Fresh pre-cooling
Ka Nuoshi fixative is fixed, each step process is required for jiggle the test tube processing sample, makes effect more abundant；So
Rear employing heat drips sheet method film-making.
Further, described Ka Nuoshi fixative is the mixed liquor that volume ratio is 3:1 of methanol and glacial acetic acid.
Further, the described fixing means in Ka Nuoshi fixative, for changing fixative 3 times, fixes 15 points every time
Clock.
Present invention beneficial effect compared with the prior art:
1. the present invention prepares the method simple and fast of small-sized fish division phases chromosome, it is simple to operation, prepares chromosome
Test fish survives rear basic guarantee, can be repeated several times preparation, and required Experimental fish can lack to 1, it is adaptable to various oceans
Fish.Only need to understand physiology and the life characteristic of test fish, determine that specimen locations is the gill or fin.
2. using this method can ensure that laboratory sample is fresh, cytoactive is good, the division phases chromosome morphology of preparation
Good, good dispersion degree, it is simple to observe and count.
Accompanying drawing explanation
Fig. 1 pomfret parr division phases chromosome: a, the first tail juvenile fish chromosome, b, the second tail juvenile fish chromosome.
Fig. 2 star column division phases chromosome: a, body colour blackout juvenile fish chromosome, b, body colour turn white juvenile fish dyeing
Body.
Detailed description of the invention:
The present invention is described in detail to combine accompanying drawing below by specific embodiment, the following is most preferred embodiment and specifically grasps
Making technical process, protection scope of the present invention is not by any pro forma restriction of embodiment.
Embodiment 1:
The method quickly preparing silvery pomfret division phases chromosome, its step includes: 1) be fully understood by the biology of silvery pomfret
Characteristic and the cleaning of slide；2) pomfret parr colchicine solution went swimming；3) take a small amount of gill tissue of fish body and carry out the system that dyes
Standby.Complete aforesaid operations process concrete operations as follows:
On July 8th, 2016, take 1 age Stromateoides argenteus 2 tail, the long 5-6cm of body, raise with special " Stromateoides argenteus milk cream " in 20L filtering sea
In, pomfret has a lively disposition, and does not stop swimming, afraid of one's shadow and break the water.Its fin ray matter such as cicada's wings, cell is difficult to dissociate.Cause
This decision takes the gill as the material preparing chromosome.The microscope slide of chromosome sectioning requires totally without greasy dirt, does not hang water, in dyeing
Clean up before body preparation.Microscope slide first use in concentration potassium dichromate washing liquid (concentrated sulphuric acid 200ml, distilled water 200ml, dichromic acid
Potassium 20g) soak 24 hours, pure water repeatedly rinses after rushing completely to the greatest extent to washing liquid and drains moisture, is dipped in dehydrated alcohol 12 little
Time, after taking-up, alcohol burner is fired, and after cooling, mounted box is standby, is sure not to touch slide surface with hands and causes grunge pollution.
The Colchicine mother solution filtering sea that the mass ratio of configuration is 0.4% is diluted to Colchicine final concentration of
The working solution 2L of 0.01%, working solution water temperature improves 2-3 DEG C compared with silvery pomfret former breeding water body temperature, it is also possible to reduces 2-3 DEG C, allows children
Stromateoides argenteus was working solution went swimming 5 hours, owing to silvery pomfret natural disposition is timid, afraid of one's shadow broke the water or abdominal part flatulence is beaten very, scaring
Excessively can cause death, therefore during swimming, need to block above container, container surroundings ensures that peace and quiet are shaken with reducing the shadow as far as possible
Dynamic.Take a small amount of gill tissue, with the KCL solution of 0.075mol/L hypotonic 50 minutes, then with the freshly prepared Ka Nuoshi of pre-cooling
(Carnoy) liquid (methanol: glacial acetic acid=3:1) is the most fixing, changes fixative 3 times, fixes 15 minutes every time.Then heat is used
Drip sheet method film-making, during film-making first with the glacial acetic acid solution of 50% dissociate gill tissue become cell suspension, by play wall or suction pipe piping and druming
Mode, make cell from tissue agglomerate dissociate out, be placed on pre-cooling in 4 DEG C of refrigerators, microscope slide be placed on smooth slabstone or gold
Preheating on genus face, slabstone or metal covering can draw the cell suspension dissociated, from the eminence of 80cm with heating by electric cooker to 55-60 DEG C
Drip 2-3 and drop on the microscope slide of preheating, quickly blot dropping liquid with the wedge angle of the 3 metafiltration paper fragments being cut into, and move to side dripping sheet
While dry.Commercially available Jim Sa working solution dyes 30 minutes, and tap water flowing water rinses the unnecessary dyeing liquor of slide surface about 10 seconds
Clock, gets final product microscopy after drying, observe chromosome morphology.Division phases is many, and chromosome morphology and dispersion are all fine, see Fig. 1.
Embodiment 2:
The method quickly preparing cabrilla division phases chromosome, its step includes: 1) be fully understood by lithosporic fry
Biological characteristics and the cleaning of slide；2) star column colchicine solution went swimming；3) take a small amount of isozyme of fish body to enter
Prepared by row chromosome.Complete aforesaid operations process concrete operations as follows:
In October, 2011, taking 6 monthly age lithosporic fry 2 tails, be respectively the new germ plasm cultivated, a long 5cm of urosome, body colour is sent out
Black, a long 7cm of urosome, body colour is turned white, and meticulously raises in 20L filtering sea.Star column happiness swimming, but activity is the most acute
Strong, tail fin and dorsal fin are flourishing, cell legibility from, therefore determine to take dorsal fin or tail fin setup action prepare the material of chromosome.Dye
The microscope slide of colour solid film-making requires totally without greasy dirt, does not hang water, cleans up before prepared by chromosome.Microscope slide first use in concentration
Potassium dichromate washing liquid is soaked 24 hours, and the compound method of described potassium dichromate washing liquid is by the dense sulfur of 200ml in 400ml solution
Acid, the distilled water of 200ml, 20g potassium dichromate composition, pure water repeatedly rinses after rushing completely to the greatest extent to washing liquid and drains moisture, soaks
In dehydrated alcohol 12 hours, after taking-up, alcohol burner was fired, and after cooling, mounted box is standby, is sure not to touch slide surface with hands and causes
Grunge pollution.
The Colchicine mother solution filtering sea that the mass ratio of configuration is 0.4% is diluted to Colchicine final concentration of
The working solution 1L of 0.005%, working solution water temperature improves 2-3 DEG C compared with cabrilla former breeding water body temperature, it is also possible to reduce 2-3 DEG C,
Allow children's cabrilla working solution went swimming 4 hours, owing to effect water body is less than normal, for ensureing juvenile fish activity, preferably with little during swimming
Gas stone is inflated.Take a small amount of fin tissue, with the KCL solution of 0.0375mol/L hypotonic 50 minutes, then with the freshly prepared card of pre-cooling
Nuo Shi (Carnoy) liquid (methanol is that 3:1 mixes with the volume ratio of glacial acetic acid) is the most fixing, changes fixative 3 times, fixes every time
15 minutes.Then use heat to drip sheet method film-making, be first organized into cell suspension with the glacial acetic acid solution of 50% fin that dissociates during film-making, lead to
Crossing and play wall or the mode of suction pipe piping and druming, make cell dissociate out from tissue agglomerate, be placed on pre-cooling in 4 DEG C of refrigerators, microscope slide is put
Preheating on smooth slabstone or on metal covering, slabstone or metal covering can be drawn the cell dissociated hang with heating by electric cooker to 55-60 DEG C
Liquid, drips 2-3 from the eminence of 70cm and drops to the microscope slide of preheating, quickly blots with the wedge angle of the layer 2-4 filter paper fragment being cut into and drips
Liquid, and move to side dry dripping a sheet.Commercially available Jim Sa working solution dyes 30 minutes, and it is many that tap water flowing water rinses slide surface
Remaining dyeing liquor about 10 seconds, gets final product microscopy after drying, observe chromosome morphology.Article two, the division phases of fish is all a lot, dye
Colour solid form and dispersion are all fine, see Fig. 2.
By the method for this quick preparation marine fishes division phases chromosome of our invention, also it is prepared for half sliding
The division phases chromosome of the Fish such as tongue sole, turbot and Paralichthys lethostigma, Idioplasm identification, heredity for these marine fishes are educated
The research such as kind, sex controll provides heredity foundation.
Claims (3)
1. the method quickly preparing small-scale marine fishes or larva and juvenile division phases chromosome, it is characterised in that described side
Method includes: 1) be fully understood by testing biological characteristics and the cleaning of slide of fish；2) small-scale test Fish or larva and juvenile are put into
Colchicine solution went swimming；3) gill tissue or the isozyme that take test fish carry out chromosome and prepare；
Described is fully understood by testing biological characteristics and the cleaning of slide of fish: understand the test physical characteristic of fish, nutrition group
One-tenth, swimming model of action and life style, to determine sampling point and testing program, determine the mark of sampling point and testing program
Accurate as follows: the short and small person of fin ray samples at fin ray free-end, the transparent keratin of fin ray such as cicada's wings or cutin are then not suitable for sampling, change to take
The gill filament, what fin ray fat content was high should strengthen hypotonic dynamics, selects 0.0375M or 0.05M KCl hypotonic medium, and gill cover opening degree is big
Take gill filament free-end, the fierce test fish swimming water body of motion requires more than 1.5L, actionless test fish fish swimming water
Body requires 100-200ml；The microscope slide of chromosome sectioning requires totally without greasy dirt, does not hang water, cleans dry before prepared by chromosome
Only；
Described puts into small-scale test Fish or larva and juvenile colchicine solution went swimming: preparation is containing 0.005% or 0.01%
(g/ml) sea water of Colchicine, allows test fish swim wherein 4-6 hour, and the more former water temperature of supporting temporarily of the water temperature of swimming water body improves
Or reduce 2-3 DEG C, to stimulate the ability strengthening test fish body surface organization division and proliferation；
Described take the test a small amount of gill tissue of fish or isozyme carries out chromosome and prepares: take 1-8mm3Gill tissue or isozyme,
It is immediately placed in hypotonic 50min in 0.0375M, 0.05M or 0.075M KCl solution, transfers to the Ka Nuoshi of Fresh pre-cooling
Fixative is fixed, each step process is required for jiggle the test tube processing sample, makes effect more abundant；Then use
Heat drips sheet method film-making.
Method the most according to claim 1, it is characterised in that the described body that Ka Nuoshi fixative is methanol and glacial acetic acid
Long-pending than the mixed liquor for 3:1.
Method the most according to claim 1, it is characterised in that the described fixing means in Ka Nuoshi fixative is more
Change fixative 3 times, fix 15 minutes every time.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115656169A (en) * | 2022-10-14 | 2023-01-31 | 宁波市海洋与渔业研究院 | Method for making scomberomorus niphonius chromosome karyotype diagram |
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2016
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| CN101344466A (en) * | 2007-07-11 | 2009-01-14 | 中国科学院海洋研究所 | A sample preparation method for determination of sex hormone levels in juvenile flounder fish |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115656169A (en) * | 2022-10-14 | 2023-01-31 | 宁波市海洋与渔业研究院 | Method for making scomberomorus niphonius chromosome karyotype diagram |
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