CN110050736A - A kind of dissolving method of dwarf clam egg membrane - Google Patents
A kind of dissolving method of dwarf clam egg membrane Download PDFInfo
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- CN110050736A CN110050736A CN201910328736.4A CN201910328736A CN110050736A CN 110050736 A CN110050736 A CN 110050736A CN 201910328736 A CN201910328736 A CN 201910328736A CN 110050736 A CN110050736 A CN 110050736A
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- Prior art keywords
- egg
- egg membrane
- dwarf
- clam
- ovum
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/54—Culture of aquatic animals of shellfish of bivalves, e.g. oysters or mussels
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a kind of methods of dwarf clam egg membrane dissolution, it is characterized in that 3~the 7min of ovum collected with 0.25% trypsin treatment of seawater dilution configuration, it carries out neutralizing trypsase with cow's serum simultaneously to terminate reaction, then pure ovum is obtained by the method for filtration washing, and the degree of egg membrane dissolution is detected by dissolution experiment after micro- sem observation and egg membrane separation, ovum is carried out by precision processing simultaneously, detection egg membrane dissolves the influence developed to ovum fertilization, it was found that rate of fertilization reaches as high as 88%, cleavage rates reach as high as 53%, D type incidence reaches as high as 30%.The present invention realizes the dissolution of dwarf's clam egg membrane, and treated that ovum can continue to develop by the present invention, this studies ovums such as dwarf's clam transgenic researches the meaning of tool positive important.
Description
Technical field
The invention belongs to marine organisms egg membrane research fields, and in particular to a kind of dissolving method of dwarf clam egg membrane.
Background technique
Model animal systems are mainly derived from terrestrial animal, such as mouse, drosophila, zebra fish at present.Crown gear animal conduct
One of three big primary animal monoids on the earth, which represent primary animal types in ocean, but so far without model organism, seriously
Constrain the further investigation in the field, it would be highly desirable to break through.
Dwarf clam (Mulinia lateralis) NATURAL DISTRIBUTION belongs to soft in North America the Atlantic coast and Caribbean sea area
Body animal door, curtain clam mesh, clam section are that the shellfish that dwells is buried in the small-sized bivalve of one kind.It has development breeding cycle short (2 months left sides
The right side, zebra fish are about 3 months), the features such as small, egg laying amount is big, early development shell is transparent, as diving for mode shellfish
Power is recognized in the world.But the egg cell diameter of dwarf clam, between 40~50 microns, egg cell is smaller, and egg membrane is thicker,
And it is fitted closely with cytoplasma membrane, this will be that dwarf clam is developed as model organism, carries out gene editing, RNAi, mutant and lures
It leads equal genetics research and brings very big puzzlement, conventional soda acid and physical method processing are also difficult to realize while egg membrane removes also
It can guarantee the integrality and fertility of egg cell.To solve this problem, a kind of mild method is needed, to take into account dwarf clam
The holding of the dissolution of egg membrane and developmental potency of being fertilized, this verifies the meaning with positive important to shellfish gene function is carried out.
Summary of the invention
The object of the present invention is to provide a kind of dissolving methods of dwarf clam egg membrane, can realize that dwarf clam egg membrane is instant fastly
While solution, the subsequent development ability of egg cell is taken into account, to make up the deficiencies in the prior art.
The dissolving method of dwarf's clam egg membrane of the invention, comprises the following steps that
1) trypsase dissolves: 0.25% trypsin solution is configured with the dilution of 30 ‰ seawater, at 22~23 DEG C of room temperature
Immersion treatment egg cell handles 3~7min；
2) cow's serum neutralizes: the egg cell solution of step 1) trypsin treatment is neutralized with isometric cow's serum
Then trypsase uses antiseptic sea water filtration washing egg cell, completes the dissolution of egg membrane.
The present invention uses trypsin treatment egg membrane, relative to traditional soda acid method, specific can be directed to egg membrane
Peptide backbone carries out high-efficiency dissolution, not will cause egg membrane and is seriously crushed so that egg cell is difficult to carry out fertilization development；Pancreas simultaneously
For protease relative to protease such as protease E and Proteinase Ks, action condition is relatively mild, and can be with containing trypsase
The cow's serum specificity of inhibitor terminates reaction, effectively controls the degree of egg membrane dissolution；Simultaneously relative to reagents such as sperm solvents
Processing, trypsase are easy to obtain, and cost is more cheap.
Detailed description of the invention
Fig. 1: under the conditions of different disposal, the morphologic observation figure of egg cell；
Fig. 2: under the conditions of different disposal, the morphologic observation figure of egg membrane.
Specific embodiment
Below by way of concrete operations to technical solution of the present invention further instruction
Embodiment 1:
The good female dwarf clam of gonad development is selected, temperature-raising method is hastened parturition by drying in the shade, and specially sets dwarf clam
In dry shady and cool environment 1.5h, (5~6 DEG C higher than normal cultivation temperature), about 1h are then placed in 26~27 DEG C of high-temperature water
Left and right can lay eggs.Then the egg cell that the discharge of dwarf clam is collected with the sterile bolting silk of 500 purposes, uses the filtering sea of sterilizing
(used sterilizing seawater salinity is 30 ‰, and temperature is 22~23 DEG C) slowly rinses 2~3 times, and egg cell is transferred to
It (can store in 4 DEG C of refrigerators) in 50ml sterile tube.Then egg cell is transferred in six sterile orifice plates, each Kong Lijia
Enter the 0.25% trypsin solution processing egg cell that 30 ‰ seawater of 1ml dilute configuration, slowly mixes, after handling 3~7min
It is added with the isometric cow's serum of solution and neutralizes trypsase.Then egg cell solution is transferred to 20um steril cell sieve
On, with antiseptic sea water filtration washing, egg cell collection is cleaned spare.Egg cell is taken to be observed under the microscope to detect egg membrane
Dissolution degree.As shown in Figure 1, relative to control group, the egg cell surface after handling 3min locally occurs recessed under 20 times of visuals field
It falls into, and after handling 7min, there is obvious hole in egg cell surface, while having broken egg membrane disperse around egg cell,
Show that trypsin solution has preferable solute effect to egg membrane.
For the more intuitive dissolution phenomena for observing egg membrane, untreated ovum is taken out from the ovum of 4 DEG C of preservations, is passed through
The broken method of hypotonic combination physics isolates the egg membrane of normal ovum, handles ovum using the trypsin solution of same concentrations
Film tracks the course of dissolution of egg membrane under 20 times of visuals field of microscope, as shown in Fig. 2, egg membrane presents more complete when untreated
Form, after trypsin solution handles 3min, it is broken that part occurs in egg membrane, but still keeps relatively complete form, is locating
After managing 7min, apparent loose structure is presented in egg membrane, shows that the peptide backbone of egg membrane is difficult to support the normal form of egg membrane, this from
Side demonstrates trypsin solution to the stronger solvability of egg membrane.
In order to further examine processing after egg cell fertility and developmental potency, take the egg cell of different disposal to be placed in
Antiseptic sea water (salinity is 30 ‰, and temperature is 22~23 DEG C) is added in different clean culture dishes, while fresh sperm is added
Guarantee to be fertilized, added semen volume is determined by the amount of sperm concentration and egg cell, guarantee that each surrounding oocytes there are 2~3
Mirror, in the hatching process of egg cell, the rate of fertilization, cleavage rates and D-shaped incidence for counting different disposal and control group come
Influence of the trypsin treatment to egg cell subsequent development is evaluated, the results are shown in Table 1,
Table 1: under the conditions of different disposal, the fertilization and developmental potency of egg cell
Control group is compared, after tryptose processing, the fertilization of egg cell and developmental potency are significantly affected, but are still had
After part egg cell has developmental potency, such as 3min processing, there are 30% or so fertilized eggs that can develop D-shaped, in 7min
After processing, still there is 5% can develop D-shaped, it is thin that this illustrates that trypsin solution can also take into account ovum while dissolving egg membrane
The fertilization of born of the same parents and subsequent development, this has positive important meaning to the subsequent experimental of egg cell.
Claims (3)
1. a kind of dissolving method of dwarf clam egg membrane, which is characterized in that the method comprises the following steps that
1) trypsase dissolves: it is configured to trypsin solution with seawater dilution, in 22~23 DEG C of immersion treatment egg cells of room temperature,
Handle 3~7min；
2) cow's serum neutralizes: the egg cell solution of step 1) trypsin treatment being carried out to neutralize pancreas egg with isometric cow's serum
Then white enzyme uses antiseptic sea water filtration washing egg cell, completes the dissolution of egg membrane.
2. the method as described in claim 1, which is characterized in that the salinity of seawater is 30 ‰ in the step 1).
3. the method as described in claim 1, which is characterized in that the concentration of trypsin solution is in the step 1)
0.25%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910328736.4A CN110050736A (en) | 2019-04-23 | 2019-04-23 | A kind of dissolving method of dwarf clam egg membrane |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910328736.4A CN110050736A (en) | 2019-04-23 | 2019-04-23 | A kind of dissolving method of dwarf clam egg membrane |
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| CN110050736A true CN110050736A (en) | 2019-07-26 |
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| CN201910328736.4A Pending CN110050736A (en) | 2019-04-23 | 2019-04-23 | A kind of dissolving method of dwarf clam egg membrane |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110055213A (en) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | A kind of separation method of dwarf clam egg membrane |
| CN110720411A (en) * | 2019-11-27 | 2020-01-24 | 海南晨海水产有限公司 | Method for removing egg membrane in grouper hatching process |
| CN114561343A (en) * | 2022-02-18 | 2022-05-31 | 河南师范大学 | Batch preparation method of membrane-removed living fish eggs |
| CN115521904A (en) * | 2022-09-29 | 2022-12-27 | 中国海洋大学 | Method for dissolving dwarf clam egg membrane |
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| US4532883A (en) * | 1983-10-14 | 1985-08-06 | Monterey Abalone Farms | Process for controlling the microbial environment in abalone mariculture |
| CN1528131A (en) * | 2003-09-26 | 2004-09-15 | �й���ѧԺ�����о��� | Method for preparing sterile transgenic fish |
| CN101647419A (en) * | 2009-09-18 | 2010-02-17 | 湖北省水产科学研究所 | Separation method of fries and roes after hatching of loach fertilized roes |
| CN105265353A (en) * | 2015-06-07 | 2016-01-27 | 肖义军 | Pearl nucleus having mantellum cell hydrogel layer at surface and preparation method therefor |
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2019
- 2019-04-23 CN CN201910328736.4A patent/CN110050736A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4532883A (en) * | 1983-10-14 | 1985-08-06 | Monterey Abalone Farms | Process for controlling the microbial environment in abalone mariculture |
| CN1528131A (en) * | 2003-09-26 | 2004-09-15 | �й���ѧԺ�����о��� | Method for preparing sterile transgenic fish |
| CN101647419A (en) * | 2009-09-18 | 2010-02-17 | 湖北省水产科学研究所 | Separation method of fries and roes after hatching of loach fertilized roes |
| CN105265353A (en) * | 2015-06-07 | 2016-01-27 | 肖义军 | Pearl nucleus having mantellum cell hydrogel layer at surface and preparation method therefor |
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| 楼允东: "《鱼类育种学》", 31 July 2001, 中国农业出版社 * |
| 覃扬: "《医学分子生物学实验教程》", 31 March 2004, 中国协和医科大学出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110055213A (en) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | A kind of separation method of dwarf clam egg membrane |
| CN110720411A (en) * | 2019-11-27 | 2020-01-24 | 海南晨海水产有限公司 | Method for removing egg membrane in grouper hatching process |
| CN114561343A (en) * | 2022-02-18 | 2022-05-31 | 河南师范大学 | Batch preparation method of membrane-removed living fish eggs |
| CN115521904A (en) * | 2022-09-29 | 2022-12-27 | 中国海洋大学 | Method for dissolving dwarf clam egg membrane |
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