WO2020192174A1

WO2020192174A1 - Method for identifying individual female sea cucumbers having ovaries in development for holothuria scabra

Info

Publication number: WO2020192174A1
Application number: PCT/CN2019/123192
Authority: WO
Inventors: 陈廷; 任春华; 胡超群; 李小敏; 吴菲菲; 江晓
Original assignee: 中国科学院南海海洋研究所
Priority date: 2019-11-22
Filing date: 2019-12-05
Publication date: 2020-10-01
Also published as: CN110720413A; JP2021519575A; CN110720413B; JP6970996B2

Abstract

A method for identifying individual female sea cucumbers having ovaries in development for Holothuria scabra, comprising: extracting pseudocoelom fluid of a Holothuria scabra; performing centrifugal separation to separate out vitellin; performing SDS-PAGE electrophoresis and dyeing; on the basis of the electrophoresis result, distinguishing female sea cucumbers having ovaries in development, male sea cucumbers, and Holothuria scabras having undeveloped reproductive glands. Samples of individual female sea cucumbers having ovaries in development exhibit characteristic bands of 200 to 250kD, and samples of both individuals having undeveloped reproductive glands and individual male sea cucumbers exhibit no characteristic bands. Individual female sea cucumbers having ovaries in development can be identified by extracting pseudocoelom fluid samples while not harming Holothuria scabras, providing application values to the selection of parent sea cucumbers during artificial seedling development.

Description

Method for identifying individual female ginseng during ovarian development stage of sea cucumber

Technical field

The invention belongs to the technical field of mariculture biology, and specifically relates to a method for identifying individual female ginseng during ovarian development stage of sea cucumber.

Background technique

Holothuriascabra (Holothuriascabra), also known as white ginseng and Adenophora, belongs to the phylum Echinoderm, Trepange, Cladophyta, Laceta, Trepangaceae, and Trepang genus. Distributed in the west from Port Natal to the Red Sea, east to the Caroline Islands, Fiji Islands, north to Japan, south to Australia, and China's Guangxi, Guangdong, Hainan and other places, generally living on the edge of coastal reefs and Sandy bottom with strong currents and seagrass. Rough sea cucumber has thicker body wall, rich collagen, smooth and elastic taste, not only has high nutritional value, but also has high medicinal value. Sea cucumber is an important tropical sea cucumber breeding species and high-end aquatic product for export. It has the characteristics of rapid growth, simple and diverse breeding methods, fewer diseases, and large market demand. It has great potential for breeding development in the coastal areas of South my country.

The breeding period of sea cucumbers in South China is from May to September every year, and the parent ginseng is usually induced to produce artificially by the method of dry and flowing water stimulation. Only testis-developed male ginseng and ovarian-developed female ginseng can be used to artificially induce labor, and ovarian-developed female ginseng is difficult to obtain. The appearance of ovarian-developed female ginseng individuals is the same as that of undeveloped gonads and male ginseng individuals, which brings difficulties to the selection of parent ginseng during artificial breeding of sea cucumber. Therefore, there is an urgent need to develop a method that can identify female ginseng during ovarian development without damaging the sea cucumber.

The ultracentrifugal sedimentation method refers to the method of using centrifugal force to gradually settle the dispersed particles in the dispersion system to separate them. When the particles in the solution are subjected to strong centrifugation, if the density of the particles is greater than the density of the solvent, the particles will sink.

Summary of the invention

The purpose of the present invention is to provide a method for identifying individual female ginseng during the ovarian developmental stage of sea cucumber. The method of the present invention can identify the individual female ginseng during the ovarian development stage of sea cucumber, so that the body cavity can be extracted without damaging sea cucumber. Liquid samples are used to identify female ginseng individuals during ovarian development, and have application value for the selection of parent ginseng during artificial seedlings.

The present invention finds that the body cavity fluid of female ginseng during the ovarian development stage is rich in yolk protein, while the individuals with undeveloped gonads and male ginseng individuals do not contain the protein. However, because the body cavity fluid also contains contaminated proteins with a molecular weight close to that of the vitellin, it is difficult to directly identify the vitellin in the body cavity fluid by SDS-PAGE protein electrophoresis. Vitellin is an apolipoprotein that forms a lipoprotein complex with lipids. The present invention can firstly separate lipoproteins (yolk protein) and other non-lipid-bound contaminated proteins through the ultracentrifugation method, thereby removing contaminated proteins that have a molecular weight similar to that of egg yolk protein in the previous SDS-PAGE protein electrophoresis. Then the lipoproteins were taken for SDS-PAGE electrophoresis and Coomassie brilliant blue staining, and the ovarian developmental female ginseng, male ginseng and undeveloped gonads were distinguished by electrophoresis results.

Therefore, the method for identifying individual female ginseng during the ovarian development stage of sea cucumber of the present invention includes the following steps:

The body cavity fluid of the sea cucumber was extracted, and the vitellin was separated by centrifugation, and then subjected to SDS-PAGE electrophoresis and staining. The electrophoresis results distinguished female ginseng, male ginseng and undeveloped sea cucumber during ovarian development. Specifically, there are obvious bands at 200-250kDa for ovarian developmental female ginseng individuals, but undeveloped gonadal individuals and male individuals do not have this characteristic band, thus identifying individual female ginsengs during ovarian developmental period.

Preferably, said centrifugal separation of vitellin is to separate vitellin by ultracentrifugation. After high-speed centrifugation of the body cavity fluid of sea cucumber, its vitellin is located in the upper part of the supernatant, and the separated vitellin can be obtained by sucking the upper part of the supernatant. The specific plan is: add 4 mL of KBr with a concentration of 1 mM PMSF and a density of 1.215 g/mL to the bottom of the ultracentrifuge tube, and add 1 mL of the body cavity fluid of sea cucumber to the upper part of the ultracentrifuge tube, and use the Himac CP 100MX ultracentrifuge. Centrifuge the P100AT-0132 rotor at 180,000g for 24 hours at 10°C, and take the upper 300μL liquid, which is the solution containing the vitellin.

The staining is Coomassie brilliant blue staining.

Preferably, the said extraction of the body cavity fluid of the sea cucumber is to use a syringe pre-added with the protease inhibitor PMSF solution through the body wall of the sea cucumber and insert it to the body cavity to extract the body cavity fluid.

Preferably, the protease inhibitor PMSF solution is a solution of 100 mM PMSF.

In the process of ultracentrifugation, due to the combination of apolipoprotein and lipids, the density is lower than that of the solvent, and it is above the solvent after centrifugation; while other non-lipid-bound proteins have a higher density than the solvent and settle below the solvent after centrifugation.

In the present invention, the yolk protein is separated from other miscellaneous proteins by an ultracentrifugation method, and then the yolk protein is subjected to SDS-PAGE protein electrophoresis and Coomassie brilliant blue staining, and then the female ginseng in the ovarian development stage is identified by the characteristic strip of the yolk protein. individual. The analysis results showed that the individual samples of female ginseng during ovarian development showed a characteristic band of 200-250kD, while the samples of undeveloped gonads and individual samples of male ginseng showed no such characteristic band. The invention can identify the individual female ginseng during the ovarian development stage by sampling the body cavity fluid without damaging the sea cucumber, and has application value for the selection of the parent ginseng during artificial seedling breeding.

Description of the drawings

Figure 1 shows the results of SDS-PAGE electrophoresis in the body cavity fluid of female ginseng individuals, underdeveloped gonads and male ginseng individuals during ovarian development of sea cucumber;

Figure 2 shows the result of ultracentrifugation of the body cavity fluid of sea cucumber. The left picture shows the ultrachao liquid with Sudan black dye, and the right picture shows the ultrachao liquid without Sudan black dye;

Figure 3 shows the SDS-PAGE electrophoresis results of the upper liquid of the body cavity fluid of the female ginseng individuals, the undeveloped gonad individuals, and the male ginseng individuals during the ovarian development stage of the sea cucumber.

detailed description

In order to make the objectives, technical solutions and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the embodiments described in this specification are only for explaining the present invention, but not for limiting the present invention. The parameters, ratios, etc. of the embodiments can be selected according to local conditions and have no substantial influence on the results. Except for special instructions, the examples are all conventional reagents and method steps in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

Example 1:

1. SDS-PAGE electrophoresis of sea cucumber body cavity fluid

(1) Preparation of 100mM protease inhibitor PMSF (phenylmethylsulfonyl fluoride)

Weigh 174mg PMSF and dissolve it in 1mL ethanol to prepare 174mg/mL stock solution. Then use 9mL PBS to dilute PMSF with a concentration of 174mg/mL to 100mM PMSF working solution.

(2) Extraction and centrifugation of body cavity fluid of sea cucumber

Select 20 sea cucumbers with body length greater than 20cm and weight greater than 500g into 20 pieces. Use a 2 mL syringe pre-added with 20 μL of 100 mM PMSF through the body wall of the sea cucumber, insert 2 cm to the body cavity, and extract the body cavity fluid. Centrifuge the extracted body cavity fluid in a refrigerated centrifuge at 1,000 g rotating speed at 4°C for 15 min. Discard the precipitate and transfer the supernatant to a new centrifuge tube for later use.

(3) Selection and SDS-PAGE electrophoresis of sea cucumber body cavity fluid samples

After taking the body cavity fluid, the sea cucumber was dissected to identify the male and female and gonadal development of the sea cucumber. Select 3 female ginsengs in the ovarian development stage, 3 undeveloped sea cucumbers and 3 male ginsengs, and 9 corresponding body cavity fluid samples (supernatant obtained after centrifugation of the body cavity fluid in step (2)) Perform SDS-PAGE electrophoresis. Take 20uL spare samples of the above 9 individual cavity fluids respectively, add an equal volume of 2× Loading buffer, boil in boiling water for 3-5 minutes, and then use 8% SDS-PAGE for protein electrophoresis and Coomassie brilliant blue staining . The results of electrophoresis showed that after SDS-PAGE electrophoresis in the 9 selected sea cucumber body cavity fluid samples, obvious bands were found at >200kDa (Figure 1), indicating that this method could not be used to isolate the individual female ginseng and gonadal in the ovarian developmental stage of sea cucumber. The developmental individual and the male ginseng individual are distinguished.

2. Separation of lipoproteins by ultracentrifugation

The preparation of the ultracentrifugation liquid and the separation method of lipoproteins are carried out with reference to the general method of ultracentrifugation and purification of human plasma lipoproteins.

(1) Preparation of ultracentrifugation liquid

Weigh EDTA-Na ₂ 186g, 950 mL of deionized water, pH adjusted with NaOH to 8.0, add water to 1L, formulated in 0.5M EDTA solution (pH8.0).

Weigh 11.40g of NaCl, add 0.5mL 0.5M EDTA solution, add deionized water to 1L, and prepare solution A.

Weigh 33.496g KBr and dissolve it in 100mL solution A, add 100mM PMSF 1mL to a final concentration of 1mM, and prepare a separation solution with a density of 1.215g/mL.

(2) Ultracentrifugation

Add a total of 4 mL of KBr solution (separation solution) with a concentration of 1 mM PMSF and a density of 1.215 g/mL to the bottom of the ultracentrifuge tube, and 1 mL of body cavity fluid [see step (2) in step 1.] into the upper part of the ultracentrifuge tube. In the Himac CP 100MX ultracentrifuge, the P100AT-0132 rotor is used to centrifuge at 180,000g at 10°C for 24 hours.

(3) Identification of lipoprotein

After ultracentrifugation, 1.2‰ Sudan black solution (lipid stain) was added to the body cavity fluid of sea cucumber for staining. The results showed that the upper liquid of the ultracentrifuge was stained black by Sudan black (Figure 2), indicating that lipoprotein (yolk protein) was located on the top of the ultracentrifuge tube. Since Sudan black staining will affect the subsequent SDS-PAGE electrophoresis, the Sudan black staining method is not used in the actual operation.

3. Identification of female individuals during ovarian development of sea cucumber

After ultracentrifugation, pipette 300 μL of liquid (including lipoprotein) from the upper part of the centrifuge tube to a new centrifuge tube for later use. Take 20 μL of the stock solution, add an equal volume of 2×Loading buffer, and boil it in boiling water for 3-5 minutes, and then use 8% SDS-PAGE for protein electrophoresis and Coomassie brilliant blue staining of the processed protein sample. The results of electrophoresis showed that samples from female ginseng individuals during ovarian development had obvious bands (presumed as vitellin) at 200-250kDa, while samples from undeveloped gonads and male individuals did not have this characteristic band. Therefore, the lipoprotein can be separated from other proteins by ultracentrifugation, and then the lipoprotein is subjected to SDS-PAGE protein electrophoresis, and then the female ginseng individuals in the ovarian development stage are identified by the characteristic strip of vitellin.

Claims

A method for identifying individual female ginseng during the ovarian development stage of sea cucumber, characterized by comprising the following steps:

The body cavity fluid of the sea cucumber was extracted, and the vitellin was separated by centrifugation, and then subjected to SDS-PAGE electrophoresis and staining. The electrophoresis results distinguished female ginseng, male ginseng and undeveloped sea cucumber during ovarian development.
The method according to claim 1, wherein the difference between female ginseng, male ginseng and undeveloped sea cucumber during ovarian development by electrophoresis results is that there is a clear band at 200-250kDa The female ginseng individuals during the ovarian development stage, but neither the undeveloped gonadal individuals nor the male individuals have this characteristic band. Thus, the female ginseng individuals during the ovarian development stage of Sea Cucumber were identified.
The method according to claim 1, wherein the centrifugation to separate the vitellin is to separate the vitellin by ultracentrifugation.
The method according to claim 3, wherein the separation of vitellin by ultracentrifugation is to add 4 mL of a KBr solution containing 1 mM PMSF and a density of 1.215 g/mL to the bottom of the ultracentrifuge tube, and 1 mL of sea cucumber The body cavity fluid is added to the upper part of the ultracentrifuge tube, in a Himac CP 100MX ultracentrifuge, using a P100AT-0132 rotor, centrifuged at 180,000g at 10°C for 24h, and 300μL of the upper part of the liquid is taken, which is the solution containing vitellin.
The method according to claim 1, wherein the staining is Coomassie brilliant blue staining.
The method according to claim 1, wherein the extraction of the body cavity fluid of the sea cucumber is by using a syringe pre-added with a protease inhibitor PMSF solution through the body wall of the sea cucumber, inserted into the body cavity, and extracting the body cavity fluid.
The method of claim 6, wherein the protease inhibitor PMSF solution is a 100 mM PMSF solution.