Method for establishing fish germplasm resource library

Abstract

The invention relates to a method for establishing a fish germplasm resource library, and belongs to the technical field of biological germplasm resource protection. The invention discloses a method for establishing a fish germplasm resource library. The method comprises the following steps: investigating fish germplasm resources and habitats, determining target fishes, determining sub-library types of a fish germplasm resource library, determining the minimum sample storage amount of the sub-libraries of the fish germplasm resource library, collecting samples, treating the samples, storing the samples, and regularly detecting the quality of the samples. And establishing a fish germplasm resource database and managing fish germplasm resource data to establish a complete fish germplasm resource database, and storing and sharing fish germplasm resource information. According to the invention, digitalization and informatization of fish germplasm resources can be realized, hierarchical, multi-dimensional and full-coverage management is carried out on information data of the fish germplasm resources, query, exchange and sharing of the resources are facilitated, resource utilization efficiency is improved, transparent exchange and scientific research cooperation of information are promoted, and economic benefits are increased. And a technical support is provided for seed source safety and seed industry revitalizing construction in China.

Description

Technical Field

[0001] The invention relates to a method for establishing a fish germplasm resource bank, and belongs to the technical field of biological germplasm resource protection. Background Art

[0002] At present, with the increasing interference of human activities, climate and environmental changes, and changes in agricultural breeding methods, the risk of local varieties disappearing has increased, the number of populations and regional distribution have changed greatly, and wild resources have decreased sharply. Once they disappear and become extinct, the excellent genes and culture they contain will also disappear, and biodiversity will also be affected, and the loss is difficult to estimate. As an important strategic resource, aquatic germplasm resources have an important impact on people's lives and social and economic development. Protecting aquatic germplasm resources is conducive to protecting aquatic biodiversity and maintaining the sustainable development of the aquaculture industry, and it has important significance and role.

[0003] The construction of fish germplasm resource banks is an important strategic measure to protect biodiversity, especially for rare and endangered species. In recent years, the country has comprehensively accelerated the construction of rare and endemic fish germplasm resource banks. However, in terms of fish resource bank display and sample management, there are problems such as incomplete library construction, unclear data information, unsystematic processes, low display, low retrieval efficiency, and insufficient management level. Summary of the invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a method for establishing a fish germplasm resource bank, which can collect and protect fish germplasm resources in a river basin at multiple levels, and construct fish germplasm resource banks at different levels such as living banks, specimen banks, cell banks and nucleic acid banks. Moreover, the present invention can further strengthen the standardized construction of resource banks and their information display and management.

[0005] To achieve the above object, the technical solution provided by the present invention is: a method for establishing a fish germplasm resource bank, comprising the following steps:

[0006] (1) Investigate fish germplasm resources and habitats: Conduct a survey of fish germplasm resources and habitats in the basin by combining field investigation with reference to relevant data, and obtain information on fish species diversity, population structure, and temporal and spatial distribution; understand the living habits and hydrological characteristics of fish through historical data analysis and on-site field surveys, and obtain an overview of the habitat distribution of fish spawning grounds, feeding grounds, and wintering grounds;

[0007] (2) Determine target fish: select target fish for the fish germplasm resource bank based on the species protection level, representativeness and relative importance index of fish in the waters;

[0008] (3) Determine the type of fish germplasm resource bank sub-bank: Determine the type of germplasm resource bank sub-bank corresponding to each target fish species based on the endangered status of each target fish species;

[0009] (4) Determine the minimum sample storage volume of the fish germplasm resource bank sub-banks: Considering the difficulty of constructing each sub-bank, calculate the minimum sample storage volume of the seven sub-banks for each target species;

[0010] (5) Sample collection: Collect the fish samples selected in step (2) accordingly; collect fish samples through wild fishing, aquatic origin, breeding farms, seed breeding farms and farms;

[0011] (6) Sample processing: Processing the samples collected in step (5) according to the different requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank to meet the sample preservation requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank and obtain corresponding information;

[0012] A. Sample processing of living body bank:

[0013] a. Transportation and transfer: Use appropriate containers and water conditions to safely transport the collected fish individuals to the live bank, avoid overcrowding and long transportation times, and reduce stress and mortality during transportation;

[0014] b. Temporary holding: Temporarily place the collected fish individuals in suitable temporary holding facilities, ensuring that appropriate water conditions, diet and living space are provided to promote individual adaptation and health;

[0015] c. Health assessment and screening: Conduct health assessment on each individual to check whether there is disease, injury or other health problems, screen out healthy individuals as the final samples for storage, and avoid introducing sick or unsuitable individuals into the living bank;

[0016] d. Adaptability assessment before entry: Before individuals enter the living bank, an adaptability assessment is conducted to ensure that they can adapt to the new environmental conditions and social structure. The adaptability assessment at least includes behavioral observation, food intake and social interaction monitoring of the individuals;

[0017] e. Entry and identification: Individuals that meet the criteria are formally introduced into the living body bank and identified. The living body bank identification includes at least the individual number, collection information and health status;

[0018] f. Health monitoring: Regularly check and evaluate the health and reproductive status of individuals in the live bank, and adjust the feeding and management strategies based on the monitoring results to maximize the survival and reproductive success rate of individuals in the live bank;

[0019] B. Sample processing of specimen library:

[0020] There are many types of fish specimens, mainly including immersed specimens, skinned specimens, skeleton specimens and transparent specimens. Immersed specimens are specimens that are directly placed in various chemicals for preservation; skinned specimens refer to specimens that retain the fish skin through various methods and fill it with various materials to restore it to its original state; skeleton specimens refer to specimens that remove the internal organs and muscle tissue of the fish as a whole or in part through various methods, and only retain the bones, and then use chemicals to degrease, bleach and reshape the bones in series; transparent skeleton specimens refer to small fish that have been degreased, transparentized and stained with chemicals, so that their hard bones and cartilage tissues can be clearly observed; the processed specimens are placed in the specimen library and marked, and the specimen library mark includes at least the specimen number, specimen type and specimen fish species;

[0021] C. Tissue Bank Sample Processing:

[0022] a. After putting on disposable rubber gloves, the collector first uses flame to burn the front end of the scissors that have been cleaned with alcohol, and then waits for it to cool down before cutting the fins or muscle tissue of the specimen;

[0023] b. Then use tweezers cleaned with alcohol to place it into a 2ml centrifuge tube prepared in advance, screw on the tube cap and place it in a freezing box; the centrifuge tube contains a label number and more than 95% ethanol;

[0024] c. Repeat cleaning of tools before removing the next portion of molecular tissue material to avoid cross contamination;

[0025] D. Gene bank sample processing:

[0026] a. Sample sampling: For the aquatic organisms for which the genomic DNA library is to be constructed, the number of samples to be sampled shall be determined in accordance with the provisions of GB / T18654.2;

[0027] b. Extraction of genomic DNA: Use traditional genomic DNA extraction methods or commercial genomic DNA extraction kits to extract genomic DNA of the species;

[0028] c. Treatment of genomic DNA: Depending on the selected vector, the genomic DNA is digested with an appropriate restriction endonuclease to produce ends that match the vector;

[0029] d. Detect and recover DNA enzyme slices of appropriate size: perform agarose gel electrophoresis on the DNA fragments obtained by cleavage, observe with a gel imager, and recover the enzyme-cut segments of appropriate size with low melting point agarose gel;

[0030] e. Vector selection: Select the appropriate vector based on the size of the target region of the genome, the difficulty of screening the library, and the copy number of the vector;

[0031] f. Ligation of genomic DNA fragments and vectors: Use ligase to connect the above DNA fragments of appropriate size into the vector;

[0032] g. Transformation of recombinant vector into host cells and identification of positive clones: Transform the recombinant vector into competent host cells and screen positive clones using the blue-white color of the colonies;

[0033] E. Sperm bank sample processing:

[0034] a. Use a towel or paper towel to wipe the moisture off the abdomen and genital area of ​​the mature male fish, gently press the abdomen to expel urine, collect the discharged semen with a dry straw, and place it in a clean, dry and graduated glass bottle or test tube. Avoid direct sunlight during operation and transportation;

[0035] b. Use a microscope to examine three times and take the average. Semen with a sperm survival rate of more than 80% can be used for cryopreservation; the sperm survival rate is the percentage of motile sperm to all sperm;

[0036] c. According to the different fish species, choose the appropriate cryopreservation diluent and antifreeze agent. After completing semen collection and microscopic examination, add the cryopreservation diluent. After mixing the semen of different fish with the pre-cooled diluent in proportion, place it in a 4℃ refrigerator for 20min to 30min to balance.

[0037] d. Add the balanced semen and diluent to 1.0 mL to 1.5 mL in 2 mL cryotubes, and freeze in liquid nitrogen according to the three-step freezing mode;

[0038] e. When thawing, first take out the cryopreservation tube from the liquid nitrogen and place it in the liquid nitrogen vapor for 2-3 minutes to balance, then take it out from the liquid nitrogen tank and put it in a 37°C water bath for rapid thawing or thaw it at room temperature; after the frozen sperm is thawed, dry insemination is performed with fresh eggs, pour an appropriate amount of frozen semen on the eggs, gently stir and mix, add fresh water or sea water equivalent to 2-3 times the amount of eggs to activate sperm, let it stand for 10 minutes, add 5-10 times the amount of water to wash away dead eggs and excess sperm, and place the fertilized eggs in a thaw device for thaw;

[0039] F. Cell bank sample processing:

[0040] a. Wash the collected fish tissue blocks with PBS at least 3 times in a clean bench;

[0041] b. Transfer the tissue into a penicillin vial containing L-15 medium, cut the tissue into 1 mm pieces with scissors, and wash with PBS three times;

[0042] c. Soak with a small amount of L-15 complete medium and inoculate evenly on a 25cm 2Culture flask, record cell information on the side wall of the culture flask, including tissue type, time and culture generation, and place it upside down in an incubator at a suitable temperature for culture;

[0043] d. After 10-12 hours, add 3 ml of complete culture medium and start upright culture. During the primary culture period, change the culture medium every 2-3 days;

[0044] e. Subculture requires the first subculture when the cells grow to 95% of the bottom of the cap by adhering to the wall. The first subculture is carried out in the original bottle, and the subsequent subcultures are subcultured at a ratio of 1 to 2. The original culture medium is aspirated, and after washing with PBS, an appropriate amount of pancreatic enzyme is added to digest the cells. When the cells shrink and become round and are about to detach from the wall, the pancreatic enzyme is aspirated, and the culture medium is added and pipetted several times until the cells detach from the wall.

[0045] (7) Sample storage: The method for storing samples in a living body bank is to build a fish pond for ex situ conservation; the method for storing samples in a specimen bank is to place the specimen or organ in a 10% formalin solution, replace the solution several times until it is fixed, then place it in a 5% formalin solution for another 2-3 weeks, and finally place it in a glass specimen bottle with a preservation solution, and seal it with vaseline or paraffin to prevent the fixative from evaporating or leaking; the method for storing samples in a tissue bank is to place the collected tissue sample in a 2 ml centrifuge tube containing 95% or more ethanol and store it at -20°C or -70°C; the method for storing samples in a gene bank is to dissolve the genomic DNA that has been tested to meet the requirements in TE with a pH value of 8.0 Preservation; The sperm bank sample storage method is to add the balanced semen and diluent to 1.0mL~1.5mL and dispense it into 2mL cryopreservation tubes, and then put it into liquid nitrogen for freezing preservation according to the three-step cooling and freezing mode; The cell bank sample storage method is to aspirate the culture medium, rinse the cells with phosphate buffer, add an appropriate amount of trypsin for digestion, and when the cells growing on the wall are small and round and about to detach from the wall, aspirate the trypsin, add an appropriate amount of culture medium and blow to detach the cells; mix the cell suspension after blowing with an equal volume of cell cryopreservation culture medium, put it into a cryopreservation tube, first place it in a 4℃ refrigerator for 30min, then place it in a -80℃ refrigerator for 2h~4h, and finally store the cell cryopreservation tube in liquid nitrogen;

[0046] (8) Regular testing of sample quality: After samples are put into storage, three samples are randomly selected from each batch for testing. The quality of samples is regularly evaluated and identified, and samples that do not meet the requirements are eliminated and replenished;

[0047] Live stock library: Regularly test the health status, survival rate and growth rate of live fish. If fish are sick, the samples in that batch will be removed and replenished;

[0048] Specimen library: regularly take out specimens from the specimen tank and observe the changes in specimen morphology. If the morphology changes greatly or the specimens are seriously decayed, the batch of samples will be discarded and replenished;

[0049] Tissue bank: Regularly check whether the tissue samples in the refrigerator are sealed, whether they are stored at -20℃ or -70℃, and whether they are frozen and thawed multiple times; multiple freeze-thaw cycles will result in smaller fragments of the obtained genomic DNA and reduced extraction quality; if they are not sealed, the temperature does not meet the standard, or the number of freeze-thaw cycles exceeds 5 times, the batch of samples should be discarded and replenished;

[0050] Gene bank: DNA samples are taken out of the refrigerator regularly to test concentration, purity and integrity;

[0051] The benzene ring structure of the bases on the DNA chain has strong absorption in the violet region, and its absorption peak is at 260nm; by detecting the absorbance values ​​of the DNA sample at 260nm and 280nm:

[0052] A260 / A280 ratio of 1.8-2.0 indicates good purity; A260 / A280 < 1.8 indicates that protein impurities remain in the nucleic acid extraction or the sample concentration is very low; 260 / A280 > 2.0 indicates that the DNA is partially degraded or there is RNA contamination;

[0053] The ratio of A260 / A230 of pure nucleic acid should be greater than 2.0. The DNA concentration can be calculated based on the purity test. Generally, a DNA sample concentration of 100-300 ng / uL is conducive to long-term storage of DNA.

[0054] The integrity of DNA was analyzed by gel electrophoresis. There was a bright band near the sample well, without impurities or tailing, indicating that the DNA molecule was intact and not degraded. If there was a bright band in the sample well, it indicated that there was protein residue in the DNA solution. If there was diffusion and tailing in the lane, it indicated that the DNA was degraded.

[0055] If the DNA sample concentration is lower than 100ng / uL, it will be removed and replenished; if the A260 / A280 in the DNA sample is > 2.0, it will be removed and replenished; if the integrity of the DNA sample is poor, and there are diffusion and tailing in the lane, it will be removed and replenished;

[0056] Sperm bank: regularly take out sperm samples from liquid nitrogen tanks for thawing and recovery, and test the vitality of sperm after freezing and recovery. If the recovery rate is less than 50%, the batch of samples will be discarded and replenished;

[0057] Cell bank: regularly take out cell samples from the liquid nitrogen tank for thawing and resuscitation. The resuscitation method is to randomly select three samples of each generation of cells for resuscitation culture. After the cells divide again, observe the degree of cell deformity of the passaged cells under a microscope. If all of them are deformed, all samples of the generation are removed and replenished.

[0058] (9) Establishing a fish germplasm resource database: Based on the various data obtained in steps (1) to (7), a complete fish germplasm resource database is established. The fish germplasm resource database is constructed with fish germplasm resource spatial distribution data, fish habitat spatial distribution data and target fish germplasm resource bank data as the first-level categories. The specific construction method is as follows:

[0059] S1. Collect basic data on fish germplasm resources and habitats: Set the geographical area to be studied for the fish germplasm resources database to be constructed, and obtain basic data on fish germplasm resources and habitats within the geographical area through actual survey;

[0060] S2. Establish spatial distribution data of fish germplasm resources: pre-process the fish germplasm resources data, form a basic database with spatial data as the main body, and separate and obtain spatial distribution data of fish germplasm resources;

[0061] The spatial distribution data of fish germplasm resources are extracted according to the data type of the spatial distribution data of the first-level fish germplasm resources and refined into the second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into abundance and biomass data according to the biological data type; the data structure of the spatial distribution data of fish germplasm resources set based on the basic database includes the name of the administrative region, the administrative region code, the type and code of fish germplasm resources, the second-level type and code of fish germplasm resources, the third-level type and code of the spatial distribution data of fish germplasm resources and the number of catch per unit fishing effort;

[0062] S3. Establish spatial distribution data of habitats: pre-process the fish habitat data, form a basic database with spatial data as the main body, and separate and obtain spatial distribution data of fish habitats;

[0063] The spatial distribution data of fish habitats are extracted according to the first-level fish habitat spatial distribution data type and refined into the second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into spawning grounds, feeding grounds and wintering grounds according to the habitat data type; the data structure of the spatial distribution data of fish habitats set based on the basic database includes the name of the administrative region, the administrative region code, the spatial distribution data and coding of fish habitats, the second-level type and coding of the spatial distribution data of fish habitats, the third-level type and coding of the spatial distribution data of fish habitats and the area of ​​the map;

[0064] S4. Establish target fish germplasm resource bank data: process and analyze target fish germplasm resource bank data to obtain fish habitat spatial distribution data;

[0065] The spatial distribution data of fish habitats are extracted according to the data type of the first-level target fish germplasm resource bank and refined into the second-level and third-level data types. The second-level data types include living bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank, and the third-level data types are divided into sample marking information, sample processing information and sample genetic information.

[0066] (10) Fish germplasm resource data management: Based on the database in step (9), a fish germplasm resource data management system is established, including a container management system, a sample management system, a data management system and a security management system; the container management system is used to manage the equipment data for storing samples; the sample management system at least includes managing the increase, decrease and status of different types of samples, automating the sample information entry and maintenance process through fish wireless radio frequency identification chip technology, recording the sample usage and maintenance history, real-time monitoring of the resource library status, recording the increase and decrease changes, and providing an interface for querying various resource library information; the data management system implements the processing and storage logic of genetic and information data, designs data distribution and access interfaces, and ensures the security and integrity of data; implements structured and persistent storage of data, and designs and develops the logical functions of an online resource sharing platform; the security management system determines the security risks and protection requirements of the system through a comprehensive analysis of the information system architecture, the business it carries and the system classification, and determines the security protection measures based on the corresponding protection requirements and the balance between security, cost and efficiency.

[0067] The further improvement of the above scheme is as follows:

[0068] The type of germplasm resource bank sub-bank corresponding to each target species in step (3) is as follows:

[0069] Extinct species: building a specimen library;

[0070] Extinct species in the wild: building a specimen library;

[0071] Critically endangered species: building a specimen library;

[0072] Endangered species: build specimen and tissue banks;

[0073] Vulnerable species: construct specimen banks, nucleic acid banks, and tissue banks;

[0074] Low-risk species: construct specimen banks, nucleic acid banks, tissue banks, sperm banks, and cell banks;

[0075] Least-endangered species: construct specimen banks, nucleic acid banks, tissue banks, sperm banks, cell banks, and living body banks;

[0076] In step (4), except for extinct species, species extinct in the wild, and critically endangered species, the minimum sample storage volume of the germplasm resource bank sub-banks of other species is based on the minimum sample quantity of the specimen bank to calculate the minimum sample quantity corresponding to other banks. The specific formula is:

[0077] The number of samples in the tissue bank = the number of samples in the specimen bank * 10; the number of samples in the gene bank = the number of samples in the specimen bank * 10; the number of samples in the sperm bank = the number of samples in the specimen bank; the number of samples in the cell bank = the number of samples in the specimen bank; the minimum number of parents and backup parents in the live bank of the endangered species is the minimum production capacity requirement of the provincial aquatic original breeding farm;

[0078] When processing tissue bank samples to obtain materials for transcriptome experiments in step (6), first use a scalpel or scissors combined with tweezers to quickly remove the gills, brain, heart and liver of the anesthetized or recently killed fish specimens and place them in pre-cooled cryovials, and then temporarily store them in a small liquid nitrogen tank, a -80°C freezer or dry ice. The entire process is performed under strict sterile conditions and on dry ice or ice cubes. The obtained molecular experimental materials should be promptly placed in a -80°C refrigerator for long-term storage.

[0079] The process of generating the three-level class data in step (9) is as follows:

[0080] 1) Living body bank:

[0081] Sample labeling information of the living body bank: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the biological characteristics of each sample, including at least sex, age and reproductive status; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0082] Sample processing information of the living bank: record the environment type and water quality when the samples were collected, water environment conditions during transportation and transfer, health status before storage, adaptability assessment data before storage, health monitoring data after storage, water quality management after storage, feed type and feeding frequency, disease inspection and nutrition regulation methods;

[0083] Genetic information of living bank samples: record genetic characteristics, breeding rules and reproduction strategy information;

[0084] 2) Specimen library:

[0085] Sample labeling information of the specimen library: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0086] Sample processing information of the specimen library: record the type of environment and water quality when the sample was collected; record the method of specimen collection and preparation;

[0087] Genetic information of specimen bank samples: record the taxonomic status, morphological characteristics and habitat preference of fish;

[0088] 3) Tissue Bank:

[0089] Tissue bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the fish sample part information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0090] Tissue bank sample processing information: record the type of environment and water quality when the sample was collected; record the tissue collection and cryopreservation methods;

[0091] Genetic information of tissue bank samples: record fish tissue structure, pathological signs and tissue-specific expression information;

[0092] 4) Gene Bank:

[0093] Gene bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record genotype information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0094] Gene bank sample processing information: record the fish length, weight, age, gonad development, environment type and water quality when the samples were collected; record the gene sample collection and freezing storage methods;

[0095] Genetic information of gene bank samples: record gene library information;

[0096] 5) Sperm Bank:

[0097] Sperm bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the sperm storage volume of each sample; record the minimum sample storage volume data; record the actual sample storage volume data;

[0098] Sperm bank sample processing information: record the fish length, weight, age, gonadal development, environment type and water quality at the time of sample collection; record sperm collection and cryopreservation methods;

[0099] Genetic information of sperm bank samples: record the normal sperm morphology rate, sperm motility, fertilization rate, hatching rate and sperm membrane integrity rate before and after cryopreservation;

[0100] 6) Cell Bank:

[0101] Cell bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record cell type information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0102] Cell bank sample processing information: record the fish length, weight, age, gonad development, environment type and water quality at the time of sample collection; record cell collection, cell culture and cryopreservation methods;

[0103] Genetic information of cell bank samples: record the morphological characteristics, cell growth characteristics and chromosome karyotype of fish cells.

[0104] The container management system in step (10) at least includes daily data management of ultra-low temperature refrigerators, liquid nitrogen tanks and freezing tubes; monitoring the status, temperature, humidity and liquid level of storage devices; managing and allocating storage space; real-time status query and abnormal status alarm.

[0105] The specific solution for the security management system security in step (10) is as follows:

[0106] 1) Authentication and authorization: Use the Web Token mechanism to implement user authentication and authorization to ensure that only legitimate users can access system functions;

[0107] 2) HTTPS: Use the HTTPS protocol in front-end and back-end communications to encrypt data transmission and prevent man-in-the-middle attacks;

[0108] 3) Input validation and SQL injection prevention: Strict validation of user input to prevent malicious input and SQL injection attacks;

[0109] 4) Cross-site scripting (XSS) and cross-site request forgery (CSRF) protection: Implement content security policy (CSP) to limit the way browsers load and execute external resources, effectively preventing XSS attacks; include a unique CSRF token for each user request, and the server verifies the token when receiving the request to ensure the legitimacy of the request, effectively preventing CSRF attacks; ensure user data and session security through the above methods;

[0110] 5) Regular security reviews: Perform regular security reviews on the system to ensure that the system is consistent with the latest security standards and best practices.

[0111] In step (10), the fish germplasm resource data management system selects a multi-layer architecture, each layer is responsible for a specific function, and communicates with other layers through clearly defined interfaces, thereby improving the modular maintainability and scalability of the system;

[0112] The system is divided into five levels in the order of functional interaction;

[0113] 1) Access layer:

[0114] The user access device and the interaction between the information library and the system belong to the access layer. The components used in this layer include API interface and ID card authentication;

[0115] Users can access the system through multiple terminals, and administrators can manage and set various system functions through the management background;

[0116] The samples in the information library are generated into QR codes by the system according to the specified coding rules. After printing, the printing device pastes it on the corresponding sample container. When sampling, the QR code can be scanned by the scanning device to obtain the sample information;

[0117] 2) Gateway layer:

[0118] The information base transmits data through the local gateway and transmits the data to the server for processing;

[0119] The monitoring indicator data of the information database is transmitted to the server through the local gateway for comparison. If the monitoring data is abnormal, an early warning reminder will be issued in time;

[0120] After users visit the web page, they interact with the server through the restful API to separate the front-end and back-end.

[0121] 3) WEB layer:

[0122] The Web layer is the user interface layer in the system architecture. It is mainly responsible for processing requests from users and presenting information to users. It includes components such as the front-end framework, page routing, and template engines, providing a good user experience and a friendly user interface.

[0123] 4) Service layer:

[0124] The service layer is responsible for implementing the core business logic, including at least database data retrieval and storage, access control logic, monitoring data recording, logging, message queue data processing and data analysis;

[0125] 5) Data layer:

[0126] The data layer is responsible for storing and retrieving data, and includes at least databases, caches, and communication methods:

[0127] The database is used to store the persistent data of the system, using the relational database PostgreSQL or MySQL;

[0128] Cache uses Redis to store frequently accessed data to improve system performance;

[0129] The communication method adopts a front-end and back-end separation method, and the front-end and back-end interact through a restful API to enhance the maintainability and scalability of the system; the information of the monitoring equipment is communicated with the server in a timely manner through a message queue to ensure the timeliness and stability of the transmitted data.

[0130] According to the above technical scheme, the method for establishing a fish germplasm resource bank provided by the present invention collects fish germplasm resource information in a river basin at multiple levels, constructs fish germplasm resource banks at different levels such as living body banks, specimen banks, cell banks and nucleic acid banks, and uses fish germplasm resource banks at different levels to construct a fish germplasm resource management system to store and share fish germplasm resource information. Compared with the prior art, the present invention has the following advantages:

[0131] (1) Because the technical solution adopted by the present invention collects fish germplasm resource information at multiple levels within the watershed, the present invention can ensure the comprehensiveness and diversity of resources and provide richer and more accurate original data for research and protection.

[0132] (2) Because the technical solution adopted by the present invention constructs a comprehensive fish germplasm resource library including a living library, a specimen library, a cell library and a nucleic acid library, which diversifies the resource preservation forms, the present invention can cope with different research needs and preservation conditions, thereby increasing the safety and availability of resources.

[0133] (3) Because the technical solution adopted by the present invention constructs a cell bank and a nucleic acid bank, the present invention can achieve long-term stable preservation of fish germplasm. At the same time, with the help of modern biotechnology, resources can be restored and regenerated, providing an effective means for the protection of rare and endangered fish species.

[0134] (4) Because the technical solution adopted by the present invention constructs a fish germplasm resource data management system, the present invention can realize the digital and information management of fish germplasm resources, facilitate the query, exchange and sharing of resources, improve the efficiency of resource utilization, and promote transparent exchange of information and scientific research cooperation. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 Flow chart of the establishment method of fish germplasm resource bank;

[0136] Figure 2 Schematic diagram of the multi-layer architecture of fish germplasm resource data management system; DETAILED DESCRIPTION

[0137] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the following embodiments. In the technical solution provided by the present invention, a method for establishing a fish germplasm resource bank is shown in the flow chart. Figure 1 As shown, the following steps are included:

[0138] (1) Investigate fish germplasm resources and habitats: Conduct a survey of fish germplasm resources and habitats in the basin by combining field investigation with reference to relevant data, and obtain information on fish species diversity, population structure, and temporal and spatial distribution; understand the living habits and hydrological characteristics of fish through historical data analysis and on-site field surveys, and obtain an overview of the habitat distribution of fish spawning grounds, feeding grounds, and wintering grounds;

[0139] (2) Determine target fish: select target fish for the fish germplasm resource bank based on the species protection level, representativeness and relative importance index of fish in the waters;

[0140] In this embodiment, the specific calculation method for selecting the target fish is as follows:

[0141] X = fish species score = fish species protection level score + fish representativeness score + fish relative importance index score. Fish with X ≥ 6 are listed as target fish;

[0142] Species protection level score: 5 points for national level 1, 4 points for national level 2, and 3 points for provincial or autonomous region level;

[0143] Representativeness score: 3 points for species endemic to China and 3 points for species endemic to the basin;

[0144] Relative importance index score: IRI = (N + W) * F;

[0145] Where: N is the percentage of the number of tails of a species to the total number of tails; W is the percentage of the biomass of the species to the total biomass; F is the percentage between the station frequency of the corresponding species and the total number of surveyed stations; when IRI ≥ 10 is 1 point, 1 ≤ IRI < 10 is 2 points, and IRI < 1 is 3 points;

[0146] In the process of calculating X, the highest score is taken for the fish species protection level score, and the cumulative score is taken for the fish representativeness score.

[0147] (3) Determine the type of fish germplasm resource bank sub-bank: Determine the type of germplasm resource bank sub-bank corresponding to each target fish species based on the endangered status of each target fish species;

[0148] In this embodiment, the types of germplasm resource bank sub-banks corresponding to each target species are as follows:

[0149] Extinct species: building a specimen library;

[0150] Extinct species in the wild: building a specimen library;

[0151] Critically endangered species: building a specimen library;

[0152] Endangered species: build specimen and tissue banks;

[0153] Vulnerable species: construct specimen banks, nucleic acid banks, and tissue banks;

[0154] Low-risk species: construct specimen banks, nucleic acid banks, tissue banks, sperm banks, and cell banks;

[0155] Least-endangered species: construct specimen banks, nucleic acid banks, tissue banks, sperm banks, cell banks, and living body banks;

[0156] The above endangered situations are determined according to national standards.

[0157] (4) Determine the minimum sample storage volume of the fish germplasm resource bank sub-banks: Considering the difficulty of constructing each sub-bank, calculate the minimum sample storage volume of the seven sub-banks for each target species;

[0158] In this embodiment, except for extinct species, species extinct in the wild, and critically endangered species, the minimum sample storage volume of the germplasm resource bank sub-bank of other species is based on the minimum sample quantity of the specimen bank to calculate the minimum sample quantity corresponding to other banks. The specific formula is:

[0159] The number of samples in the tissue bank = the number of samples in the specimen bank * 10; the number of samples in the gene bank = the number of samples in the specimen bank * 10; the number of samples in the sperm bank = the number of samples in the specimen bank; the number of samples in the cell bank = the number of samples in the specimen bank; the minimum number of parents and reserve parents in the living bank of least concern species is the minimum production capacity requirement of the provincial aquatic breeding farm.

[0160] (5) Sample collection: Collect the fish samples selected in step (2) accordingly; collect fish samples through wild fishing, aquatic origin, breeding farms, seed breeding farms and farms;

[0161] (6) Sample processing: Processing the samples collected in step (5) according to the different requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank to meet the sample preservation requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank and obtain corresponding information;

[0162] A. Sample processing of living body bank:

[0163] a. Transportation and transfer: Use appropriate containers and water conditions to safely transport the collected fish individuals to the live bank, avoid overcrowding and long transportation times, and reduce stress and mortality during transportation;

[0164] b. Temporary holding: Temporarily place the collected fish individuals in suitable temporary holding facilities, ensuring that appropriate water conditions, diet and living space are provided to promote individual adaptation and health;

[0165] c. Health assessment and screening: Conduct health assessment on each individual to check whether there is disease, injury or other health problems, screen out healthy individuals as the final samples for storage, and avoid introducing sick or unsuitable individuals into the living bank;

[0166] d. Adaptability assessment before entry: Before individuals enter the living bank, an adaptability assessment is conducted to ensure that they can adapt to the new environmental conditions and social structure. The adaptability assessment at least includes behavioral observation, food intake and social interaction monitoring of the individuals;

[0167] e. Entry and identification: Individuals that meet the criteria are formally introduced into the living body bank and identified. The living body bank identification includes at least the individual number, collection information and health status;

[0168] f. Health monitoring: Regularly check and evaluate the health and reproductive status of individuals in the live bank, and adjust the feeding and management strategies based on the monitoring results to maximize the survival and reproductive success rate of individuals in the live bank;

[0169] B. Sample processing of specimen library:

[0170] There are many types of fish specimens, mainly including immersed specimens, skinned specimens, skeleton specimens and transparent specimens. Immersed specimens are specimens that are directly placed in various chemicals for preservation; skinned specimens refer to specimens that retain the fish skin through various methods and fill it with various materials to restore it to its original state; skeleton specimens refer to specimens that remove the internal organs and muscle tissue of the fish as a whole or in part through various methods, and only retain the bones, and then use chemicals to degrease, bleach and reshape the bones in series; transparent skeleton specimens refer to small fish that have been degreased, transparentized and stained with chemicals, so that their hard bones and cartilage tissues can be clearly observed; the processed specimens are placed in the specimen library and marked, and the specimen library mark includes at least the specimen number, specimen type and specimen fish species;

[0171] C. Tissue Bank Sample Processing:

[0172] a. After putting on disposable rubber gloves, the collector first uses flame to burn the front end of the scissors that have been cleaned with alcohol, and then waits for it to cool down before cutting the fins or muscle tissue of the specimen;

[0173] b. Then use tweezers cleaned with alcohol to place it into a 2ml centrifuge tube prepared in advance, screw on the tube cap and place it in a freezing box; the centrifuge tube contains a label number and more than 95% ethanol;

[0174] c. Repeat cleaning of tools before removing the next portion of molecular tissue material to avoid cross contamination;

[0175] Among them, the molecular materials of large fish can be taken in a small amount, as long as they can be completely immersed in the preservation solution; for small specimens, several tissue parts or the whole specimen are directly placed in a centrifuge tube filled with 95% or more ethanol solution;

[0176] When processing tissue bank samples to obtain materials for transcriptome experiments, first use a scalpel or scissors combined with tweezers to quickly remove the gills, brain, heart and liver of anesthetized or recently killed fish specimens and place them in pre-cooled cryotubes. Then place them in a small liquid nitrogen tank, a -80℃ freezer or dry ice for temporary storage. The entire process is carried out under strict sterile conditions and on dry ice or ice cubes. The molecular experimental materials obtained should be promptly placed in a -80℃ refrigerator for long-term storage.

[0177] D. Gene bank sample processing:

[0178] a. Sample sampling: For the aquatic organisms for which the genomic DNA library is to be constructed, the number of samples to be sampled shall be determined in accordance with the provisions of GB / T18654.2;

[0179] b. Extraction of genomic DNA: Use traditional genomic DNA extraction methods or commercial genomic DNA extraction kits to extract genomic DNA of the species;

[0180] c. Treatment of genomic DNA: Depending on the selected vector, the genomic DNA is digested with an appropriate restriction endonuclease to produce ends that match the vector;

[0181] d. Detect and recover DNA enzyme slices of appropriate size: perform agarose gel electrophoresis on the DNA fragments obtained by cleavage, observe with a gel imager, and recover the enzyme-cut segments of appropriate size with low melting point agarose gel;

[0182] e. Vector selection: Select the appropriate vector based on the size of the target region of the genome, the difficulty of screening the library, and the copy number of the vector;

[0183] f. Ligation of genomic DNA fragments and vectors: Use ligase to connect the above DNA fragments of appropriate size into the vector;

[0184] g. Transformation of recombinant vector into host cells and identification of positive clones: Transform the recombinant vector into competent host cells and screen positive clones using the blue-white color of the colonies;

[0185] E. Sperm bank sample processing:

[0186] a. Use a towel or paper towel to wipe the moisture off the abdomen and genital area of ​​the mature male fish, gently press the abdomen to expel urine, collect the discharged semen with a dry straw, and place it in a clean, dry and graduated glass bottle or test tube. Avoid direct sunlight during operation and transportation;

[0187] b. Use a microscope to examine three times and take the average. Semen with a sperm survival rate of more than 80% can be used for cryopreservation; the sperm survival rate is the percentage of motile sperm to all sperm;

[0188] c. According to the different fish species, choose the appropriate cryopreservation diluent and antifreeze agent. After completing semen collection and microscopic examination, add the cryopreservation diluent. After mixing the semen of different fish with the pre-cooled diluent in proportion, place it in a 4℃ refrigerator for 20min to 30min to balance.

[0189] d. Add the balanced semen and diluent to 1.0 mL to 1.5 mL in 2 mL cryotubes, and freeze in liquid nitrogen according to the three-step freezing mode;

[0190] e. When thawing, first take out the cryopreservation tube from the liquid nitrogen and place it in the liquid nitrogen vapor for 2-3 minutes to balance, then take it out from the liquid nitrogen tank and put it in a 37°C water bath for rapid thawing or thaw it at room temperature; after the frozen sperm is thawed, dry insemination is performed with fresh eggs, pour an appropriate amount of frozen semen on the eggs, gently stir and mix, add fresh water or sea water equivalent to 2-3 times the amount of eggs to activate sperm, let it stand for 10 minutes, add 5-10 times the amount of water to wash away dead eggs and excess sperm, and place the fertilized eggs in a thaw device for thaw;

[0191] F. Cell bank sample processing:

[0192] a. Wash the collected fish tissue blocks with PBS at least 3 times in a clean bench;

[0193] b. Transfer the tissue into a penicillin vial containing L-15 medium, cut the tissue into 1 mm pieces with scissors, and wash with PBS three times;

[0194] c. Soak with a small amount of L-15 complete medium and inoculate evenly on a 25cm 2 Culture flask, record cell information on the side wall of the culture flask, including tissue type, time and culture generation, and place it upside down in an incubator at a suitable temperature for culture;

[0195] d. After 10-12 hours, add 3 ml of complete culture medium and start upright culture. During the primary culture period, change the culture medium every 2-3 days;

[0196] e. Subculture requires the first subculture when the cells grow to 95% of the bottom of the cap by adhering to the wall. The first subculture is carried out in the original bottle, and the subsequent subcultures are subcultured at a ratio of 1 to 2. The original culture medium is aspirated, and after washing with PBS, an appropriate amount of pancreatic enzyme is added to digest the cells. When the cells shrink and become round and are about to detach from the wall, the pancreatic enzyme is aspirated, and the culture medium is added and pipetted several times until the cells detach from the wall.

[0197] (7) Sample storage: The method for storing samples in a living body bank is to build a fish pond for ex situ conservation; the method for storing samples in a specimen bank is to place the specimen or organ in a 10% formalin solution, replace the solution several times until it is fixed, then place it in a 5% formalin solution for another 2-3 weeks, and finally place it in a glass specimen bottle with a preservation solution, and seal it with vaseline or paraffin to prevent the fixative from evaporating or leaking; the method for storing samples in a tissue bank is to place the collected tissue sample in a 2 ml centrifuge tube containing 95% or more ethanol and store it at -20°C or -70°C; the method for storing samples in a gene bank is to dissolve the genomic DNA that has been tested to meet the requirements in TE with a pH value of 8.0 Preservation; The sperm bank sample storage method is to add the balanced semen and diluent to 1.0mL~1.5mL and dispense it into 2mL cryopreservation tubes, and then put it into liquid nitrogen for freezing preservation according to the three-step cooling and freezing mode; The cell bank sample storage method is to aspirate the culture medium, rinse the cells with phosphate buffer, add an appropriate amount of trypsin for digestion, and when the cells growing on the wall are small and round and about to detach from the wall, aspirate the trypsin, add an appropriate amount of culture medium and blow to detach the cells; mix the cell suspension after blowing with an equal volume of cell cryopreservation culture medium, put it into a cryopreservation tube, first place it in a 4℃ refrigerator for 30min, then place it in a -80℃ refrigerator for 2h~4h, and finally store the cell cryopreservation tube in liquid nitrogen;

[0198] (8) Regular testing of sample quality: After samples are put into storage, three samples are randomly selected from each batch for testing. The quality of samples is regularly evaluated and identified, and samples that do not meet the requirements are eliminated and replenished;

[0199] Live stock library: Regularly test the health status, survival rate and growth rate of live fish. If fish are sick, the samples in that batch will be removed and replenished;

[0200] Specimen library: regularly take out specimens from the specimen tank and observe the changes in specimen morphology. If the morphology changes greatly or the specimens are seriously decayed, the batch of samples will be discarded and replenished;

[0201] Tissue bank: Regularly check whether the tissue samples in the refrigerator are sealed, whether they are stored at -20℃ or -70℃, and whether they are frozen and thawed multiple times; multiple freeze-thaw cycles will result in smaller fragments of the obtained genomic DNA and reduced extraction quality; if they are not sealed, the temperature does not meet the standard, or the number of freeze-thaw cycles exceeds 5 times, the batch of samples should be discarded and replenished;

[0202] Gene bank: DNA samples are taken out of the refrigerator regularly to test concentration, purity and integrity;

[0203] The benzene ring structure of the bases on the DNA chain has strong absorption in the violet region, and its absorption peak is at 260nm; by detecting the absorbance values ​​of the DNA sample at 260nm and 280nm:

[0204] A260 / A280 ratio of 1.8-2.0 indicates good purity; A260 / A280 < 1.8 indicates that protein impurities remain in the nucleic acid extraction or the sample concentration is very low; 260 / A280 > 2.0 indicates that the DNA is partially degraded or there is RNA contamination;

[0205] The ratio of A260 / A230 of pure nucleic acid should be greater than 2.0. The DNA concentration can be calculated based on the purity test. Generally, a DNA sample concentration of 100-300 ng / uL is conducive to long-term storage of DNA.

[0206] The integrity of DNA was analyzed by gel electrophoresis. There was a bright band near the sample well, without impurities or tailing, indicating that the DNA molecule was intact and not degraded. If there was a bright band in the sample well, it indicated that there was protein residue in the DNA solution. If there was diffusion and tailing in the lane, it indicated that the DNA was degraded.

[0207] If the DNA sample concentration is lower than 100ng / uL, it will be removed and replenished; if the A260 / A280 in the DNA sample is > 2.0, it will be removed and replenished; if the integrity of the DNA sample is poor, and there are diffusion and tailing in the lane, it will be removed and replenished;

[0208] Sperm bank: regularly take out sperm samples from liquid nitrogen tanks for thawing and recovery, and test the vitality of sperm after freezing and recovery. If the recovery rate is less than 50%, the batch of samples will be discarded and replenished;

[0209] Cell bank: regularly take out cell samples from the liquid nitrogen tank for thawing and resuscitation. The resuscitation method is to randomly select three samples of each generation of cells for resuscitation culture. After the cells divide again, observe the degree of cell deformity of the passaged cells under a microscope. If all of them are deformed, all samples of the generation are removed and replenished.

[0210] (9) Establishing a fish germplasm resource database: Based on the various data obtained in steps (1) to (7), a complete fish germplasm resource database is established. In this embodiment, the fish germplasm resource database is constructed with fish germplasm resource spatial distribution data, fish habitat spatial distribution data and target fish germplasm resource library data as the first-level categories. The codes and definitions of the first-level categories are shown in Table 1.

[0211] Table 1 Codes and definitions of the first-level categories

[0212]

[0213] The specific construction method is as follows:

[0214] S1. Collect basic data on fish germplasm resources and habitats: Set the geographical area to be studied for the fish germplasm resources database to be constructed, and obtain basic data on fish germplasm resources and habitats within the geographical area through actual survey;

[0215] S2. Establish spatial distribution data of fish germplasm resources: pre-process the fish germplasm resources data, form a basic database with spatial data as the main body, and separate and obtain spatial distribution data of fish germplasm resources;

[0216] The spatial distribution data of fish germplasm resources are extracted according to the data type of the spatial distribution data of the first-level fish germplasm resources and refined into the second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into abundance and biomass data according to the biological data type, see Table 2; the data structure of the spatial distribution data of fish germplasm resources set based on the basic database includes the name of the administrative region, the administrative region code, the type and code of fish germplasm resources, the second-level type and code of fish germplasm resources, the third-level type and code of the spatial distribution data of fish germplasm resources and the number of catches per unit fishing effort, see Table 3;

[0217] Table 2 Codes and definitions of spatial distribution data of fish germplasm resources at all levels

[0218]

[0219] Table 3. Data structure of spatial distribution of fish germplasm resources

[0220]

[0221]

[0222] S3. Establish spatial distribution data of habitats: pre-process the fish habitat data, form a basic database with spatial data as the main body, and separate and obtain spatial distribution data of fish habitats;

[0223] The spatial distribution data of fish habitats are extracted according to the data type of the first-level fish habitat spatial distribution data and refined into the second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into spawning grounds, feeding grounds and wintering grounds according to the habitat data type, see Table 4; the data structure of the spatial distribution data of fish habitats set based on the basic database includes the name of the administrative region, the administrative region code, the spatial distribution data and code of fish habitats, the second-level type and code of the spatial distribution data of fish habitats, the third-level type and code of the spatial distribution data of fish habitats and the area of ​​the map;

[0224] Table 4 Codes and definitions of fish habitat spatial distribution data at all levels

[0225]

[0226]

[0227] Table 5. Data structure of spatial distribution of fish habitats

[0228]

[0229] S4. Establish target fish germplasm resource bank data: process and analyze target fish germplasm resource bank data to obtain fish habitat spatial distribution data;

[0230] The spatial distribution data of fish habitats are extracted according to the data type of the first-level target fish germplasm resource bank and refined into the second-level and third-level data types. The second-level data type includes living bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank. The third-level data type is divided into sample marking information, sample processing information and sample genetic information, see Table 6;

[0231] Table 6 Codes and definitions of fish habitat spatial distribution data at all levels

[0232]

[0233] In this embodiment, the three-level class data generation process is as follows:

[0234] 1) Living body bank:

[0235] Sample labeling information of the living body bank: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the biological characteristics of each sample, including at least sex, age and reproductive status; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0236] Sample processing information of the living bank: record the environment type and water quality when the samples were collected, water environment conditions during transportation and transfer, health status before storage, adaptability assessment data before storage, health monitoring data after storage, water quality management after storage, feed type and feeding frequency, disease inspection and nutrition regulation methods;

[0237] Genetic information of living bank samples: record genetic characteristics, breeding rules and reproduction strategy information;

[0238] 2) Specimen library:

[0239] Sample labeling information of the specimen library: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0240] Sample processing information of the specimen library: record the type of environment and water quality when the sample was collected; record the method of specimen collection and preparation;

[0241] Genetic information of specimen bank samples: record the taxonomic status, morphological characteristics and habitat preference of fish;

[0242] 3) Tissue Bank:

[0243] Tissue bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the fish sample part information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0244] Tissue bank sample processing information: record the type of environment and water quality when the sample was collected; record the tissue collection and cryopreservation methods;

[0245] Genetic information of tissue bank samples: record fish tissue structure, pathological signs and tissue-specific expression information;

[0246] 4) Gene Bank:

[0247] Gene bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record genotype information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0248] Gene bank sample processing information: record the fish length, weight, age, gonad development, environment type and water quality when the samples were collected; record the gene sample collection and freezing storage methods;

[0249] Genetic information of gene bank samples: record gene library information;

[0250] 5) Sperm Bank:

[0251] Sperm bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the sperm storage volume of each sample; record the minimum sample storage volume data; record the actual sample storage volume data;

[0252] Sperm bank sample processing information: record the fish length, weight, age, gonadal development, environment type and water quality at the time of sample collection; record sperm collection and cryopreservation methods;

[0253] Genetic information of sperm bank samples: record the normal sperm morphology rate, sperm motility, fertilization rate, hatching rate and sperm membrane integrity rate before and after cryopreservation;

[0254] 6) Cell Bank:

[0255] Cell bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record cell type information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0256] Cell bank sample processing information: record the fish length, weight, age, gonad development, environment type and water quality at the time of sample collection; record cell collection, cell culture and cryopreservation methods;

[0257] Genetic information of cell bank samples: record the morphological characteristics, cell growth characteristics and chromosome karyotype of fish cells.

[0258] (10) Fish germplasm resource data management: Based on the database in step (9), a fish germplasm resource data management system is established, including a container management system, a sample management system, a data management system and a security management system; the container management system is used to manage the equipment data for storing samples; the sample management system at least includes managing the increase, decrease and status of different types of samples, automating the sample information entry and maintenance process through fish wireless radio frequency identification chip technology, recording the sample usage and maintenance history, real-time monitoring of the resource library status, recording the increase and decrease changes, and providing an interface for querying various resource library information; the data management system implements the processing and storage logic of genetic and information data, designs data distribution and access interfaces, and ensures the security and integrity of data; implements structured and persistent storage of data, and designs and develops the logical functions of an online resource sharing platform; the security management system determines the security risks and protection requirements of the system through a comprehensive analysis of the information system architecture, the business it carries and the system classification, and determines the security protection measures based on the corresponding protection requirements and the balance between security, cost and efficiency.

[0259] In this embodiment, the container management system includes at least daily data management of ultra-low temperature refrigerators, liquid nitrogen tanks and freezing tubes; monitoring the status, temperature, humidity and liquid level of storage devices; managing and allocating storage space; real-time status query and abnormal status alarm.

[0260] The specific solutions for the security aspects of the safety management system are as follows:

[0261] 1) Authentication and authorization: Use the Web Token mechanism to implement user authentication and authorization to ensure that only legitimate users can access system functions;

[0262] 2) HTTPS: Use the HTTPS protocol in front-end and back-end communications to encrypt data transmission and prevent man-in-the-middle attacks;

[0263] 3) Input validation and SQL injection prevention: Strict validation of user input to prevent malicious input and SQL injection attacks;

[0264] 4) Cross-site scripting (XSS) and cross-site request forgery (CSRF) protection: Implement content security policy (CSP) to limit the way browsers load and execute external resources, effectively preventing XSS attacks; include a unique CSRF token for each user request, and the server verifies the token when receiving the request to ensure the legitimacy of the request, effectively preventing CSRF attacks; ensure user data and session security through the above methods;

[0265] 5) Regular security reviews: Perform regular security reviews on the system to ensure that the system is consistent with the latest security standards and best practices.

[0266] like Figure 2 As shown in the figure, the fish germplasm resources data management system adopts a multi-layer architecture, each layer is responsible for a specific function, and communicates with other layers through clearly defined interfaces, which improves the modular maintainability and scalability of the system;

[0267] In this embodiment, the fish germplasm resource data management system is divided into five levels in the order of functional interaction;

[0268] 1) Access layer:

[0269] The user access device and the interaction between the information library and the system belong to the access layer. The components used in this layer include API interface and ID card authentication;

[0270] Users can access the system through multiple terminals, and administrators can manage and set various system functions through the management background;

[0271] The samples in the information library are generated into QR codes by the system according to the specified coding rules. After printing, the printing device pastes it on the corresponding sample container. When sampling, the QR code can be scanned by the scanning device to obtain the sample information;

[0272] 2) Gateway layer:

[0273] The information base transmits data through the local gateway and transmits the data to the server for processing;

[0274] The monitoring indicator data of the information database is transmitted to the server through the local gateway for comparison. If the monitoring data is abnormal, an early warning reminder will be issued in time;

[0275] After users visit the web page, they interact with the server through the restful API to separate the front-end and back-end.

[0276] 3) WEB layer:

[0277] The Web layer is the user interface layer in the system architecture. It is mainly responsible for processing requests from users and presenting information to users. It includes components such as the front-end framework, page routing, and template engines, providing a good user experience and a friendly user interface.

[0278] 4) Service layer:

[0279] The service layer is responsible for implementing the core business logic, including at least database data retrieval and storage, access control logic, monitoring data recording, logging, message queue data processing and data analysis;

[0280] 5) Data layer:

[0281] The data layer is responsible for storing and retrieving data, and includes at least databases, caches, and communication methods:

[0282] The database is used to store the persistent data of the system, using the relational database PostgreSQL or MySQL;

[0283] Cache uses Redis to store frequently accessed data to improve system performance;

[0284] The communication method adopts a front-end and back-end separation method, and the front-end and back-end interact through a restful API to enhance the maintainability and scalability of the system; the information of the monitoring equipment is communicated with the server in a timely manner through a message queue to ensure the timeliness and stability of the transmitted data.

Claims

1. A method for establishing a fish germplasm resource bank, characterized in that: The following steps are involved: (1) Investigate fish germplasm resources and habitats: Conduct a survey of fish germplasm resources and habitats in the basin by combining field investigation with reference to relevant data, and obtain information on fish species diversity, population structure, and temporal and spatial distribution; understand the living habits and hydrological characteristics of fish through historical data analysis and on-site field surveys, and obtain an overview of the habitat distribution of fish spawning grounds, feeding grounds, and wintering grounds; (2) Determine target fish: select target fish for the fish germplasm resource bank based on the species protection level, representativeness and relative importance index of fish in the waters; (3) Determine the type of fish germplasm resource bank sub-bank: Determine the type of germplasm resource bank sub-bank corresponding to each target fish species based on the endangered status of each target fish species; (4) Determine the minimum sample storage volume of the fish germplasm resource bank sub-banks: Considering the difficulty of constructing each sub-bank, calculate the minimum sample storage volume of the seven sub-banks for each target species; (5) Sample collection: Collect the fish samples selected in step (2) accordingly; collect fish samples through wild fishing, aquatic origin, breeding farms, seed breeding farms and farms; (6) Sample processing: Processing the samples collected in step (5) according to the different requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank to meet the sample preservation requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank and obtain corresponding information; A. Sample processing of living body bank: a. Transportation and transfer: Use appropriate containers and water conditions to safely transport the collected fish individuals to the live bank, avoid overcrowding and long transportation times, and reduce stress and mortality during transportation; b. Temporary holding: Temporarily place the collected fish individuals in suitable temporary holding facilities, ensuring that appropriate water conditions, diet and living space are provided to promote individual adaptation and health; c. Health assessment and screening: Conduct health assessment on each individual to check whether there is disease, injury or other health problems, screen out healthy individuals as the final samples for storage, and avoid introducing sick or unsuitable individuals into the living bank; d. Adaptability assessment before entry: Before individuals enter the living bank, an adaptability assessment is conducted to ensure that they can adapt to the new environmental conditions and social structure. The adaptability assessment at least includes behavioral observation, food intake and social interaction monitoring of the individuals; e. Entry and identification: Individuals that meet the criteria are formally introduced into the living body bank and identified. The living body bank identification includes at least the individual number, collection information and health status; f. Health monitoring: Regularly check and evaluate the health and reproductive status of individuals in the live bank, and adjust the feeding and management strategies based on the monitoring results to maximize the survival and reproductive success rate of individuals in the live bank; B. Sample processing of specimen library: There are many types of fish specimens, mainly including immersed specimens, skinned specimens, skeleton specimens and transparent specimens. Immersed specimens are specimens that are directly placed in various chemicals for preservation; skinned specimens refer to specimens that retain the fish skin through various methods and fill it with various materials to restore it to its original state; skeleton specimens refer to specimens that remove the internal organs and muscle tissue of the fish as a whole or in part through various methods, and only retain the bones, and then use chemicals to degrease, bleach and reshape the bones in series; transparent skeleton specimens refer to small fish that have been degreased, transparentized and stained with chemicals, so that their hard bones and cartilage tissues can be clearly observed; the processed specimens are placed in the specimen library and marked, and the specimen library mark includes at least the specimen number, specimen type and specimen fish species; C. Tissue Bank Sample Processing: a. After putting on disposable rubber gloves, the collector first uses flame to burn the front end of the scissors that have been cleaned with alcohol, and then waits for it to cool down before cutting the fins or muscle tissue of the specimen; b. Then use tweezers cleaned with alcohol to place it into a 2 ml centrifuge tube prepared in advance, screw on the tube cover and place it in a freezing box; The centrifuge tube contains the label number and more than 95% ethanol; c. Repeat cleaning of tools before removing the next portion of molecular tissue material to avoid cross contamination; D. Gene bank sample processing: a. Sample sampling: For the aquatic organisms for which the genomic DNA library is to be constructed, the number of samples to be sampled shall be determined in accordance with the provisions of GB / T18654.2; b. Extraction of genomic DNA: Use traditional genomic DNA extraction methods or commercial genomic DNA extraction kits to extract genomic DNA of the species; c. Treatment of genomic DNA: Depending on the selected vector, the genomic DNA is digested with an appropriate restriction endonuclease to produce ends that match the vector; d. Detect and recover DNA enzyme slices of appropriate size: perform agarose gel electrophoresis on the DNA fragments obtained by cleavage, observe them with a gel imager, and recover the enzyme-cut segments of appropriate size with low-melting-point agarose gel; e. Vector selection: Select the appropriate vector based on the size of the target region of the genome, the difficulty of screening the library, and the copy number of the vector; f. Ligation of genomic DNA fragments and vectors: Use ligase to connect the above DNA fragments of appropriate size into the vector; g. Transformation of recombinant vector into host cells and identification of positive clones: Transform the recombinant vector into competent host cells and screen positive clones using the blue-white color of the colonies; E. Sperm bank sample processing: a. Use a towel or paper towel to wipe the moisture off the abdomen and genital area of ​​the mature male fish, gently press the abdomen to expel urine, collect the discharged semen with a dry straw, and place it in a clean, dry and graduated glass bottle or test tube. Avoid direct sunlight during operation and transportation; b. Use a microscope to examine three times and take the average. Semen with a sperm survival rate of more than 80% can be used for cryopreservation; the sperm survival rate is the percentage of motile sperm to all sperm; c. According to the different fish species, choose the appropriate cryopreservation diluent and antifreeze agent. After completing semen collection and microscopic examination, add the cryopreservation diluent. After mixing the semen of different fish with the pre-cooled diluent in proportion, place it in a 4℃ refrigerator for 20min to 30min to balance. d. Add the balanced semen and diluent to 1.0 mL to 1.5 mL in 2 mL cryotubes, and freeze in liquid nitrogen according to the three-step freezing mode; e. When thawing, first take out the cryopreservation tube from the liquid nitrogen and place it in the liquid nitrogen vapor for 2-3 minutes to balance, then take it out from the liquid nitrogen tank and put it in a 37°C water bath for rapid thawing or thaw it at room temperature; after the frozen sperm is thawed, dry insemination is performed with fresh eggs, pour an appropriate amount of frozen semen on the eggs, gently stir and mix, add fresh water or sea water equivalent to 2-3 times the amount of eggs to activate sperm, let it stand for 10 minutes, add 5-10 times the amount of water to wash away dead eggs and excess sperm, and place the fertilized eggs in a thaw device for thaw; F. Cell bank sample processing: a. Wash the collected fish tissue blocks with PBS at least 3 times in a clean bench; b. Transfer the tissue into a penicillin vial containing L-15 medium, cut the tissue into 1 mm pieces with scissors, and wash with PBS three times; c. Soak with a small amount of L-15 complete medium and inoculate evenly on a 25cm 2 Culture flask, record cell information on the side wall of the culture flask, including tissue type, time and culture generation, and place it upside down in an incubator at a suitable temperature for culture; d. After 10-12 hours, add 3 ml of complete culture medium and start upright culture. During the primary culture period, change the culture medium every 2-3 days; e. Subculture requires the first subculture when the cells grow to 95% of the bottom of the cap by adhering to the wall. The first subculture is carried out in the original bottle, and the subsequent subcultures are subcultured at a ratio of 1 to 2. The original culture medium is aspirated, and after washing with PBS, an appropriate amount of pancreatic enzyme is added to digest the cells. When the cells shrink and become round and are about to detach from the wall, the pancreatic enzyme is aspirated, and the culture medium is added and pipetted several times until the cells detach from the wall. (7) Sample storage: The method for storing samples in a living body bank is to build a fish pond for ex situ conservation; the method for storing samples in a specimen bank is to place the specimen or organ in a 10% formalin solution, replace the solution several times until it is fixed, then place it in a 5% formalin solution for another 2-3 weeks, and finally place it in a glass specimen bottle with a preservation solution, and seal it with vaseline or paraffin to prevent the fixative from evaporating or leaking; the method for storing samples in a tissue bank is to place the collected tissue sample in a 2 ml centrifuge tube containing 95% or more ethanol and store it at -20°C or -70°C; the method for storing samples in a gene bank is to dissolve the genomic DNA that has been tested to meet the requirements in TE with a pH value of 8.0 Preservation; The sperm bank sample storage method is to add the balanced semen and diluent to 1.0mL~1.5mL and dispense it into 2mL cryopreservation tubes, and then put it into liquid nitrogen for freezing preservation according to the three-step cooling and freezing mode; The cell bank sample storage method is to aspirate the culture medium, rinse the cells with phosphate buffer, add an appropriate amount of trypsin for digestion, and when the cells growing on the wall are small and round and about to detach from the wall, aspirate the trypsin, add an appropriate amount of culture medium and blow to detach the cells; mix the cell suspension after blowing with an equal volume of cell cryopreservation culture medium, put it into a cryopreservation tube, first place it in a 4℃ refrigerator for 30min, then place it in a -80℃ refrigerator for 2h~4h, and finally store the cell cryopreservation tube in liquid nitrogen; (8) Regular testing of sample quality: After samples are put into storage, three samples are randomly selected from each batch for testing. The quality of samples is regularly evaluated and identified, and samples that do not meet the requirements are eliminated and replenished; Live stock library: Regularly test the health status, survival rate and growth rate of live fish. If fish are sick, the samples in that batch will be removed and replenished; Specimen library: regularly take out specimens from the specimen tank and observe the changes in specimen morphology. If the morphology changes greatly or the specimens are seriously decayed, the batch of samples will be discarded and replenished; Tissue bank: Regularly check whether the tissue samples in the refrigerator are sealed, whether they are stored at -20℃ or -70℃, and whether they are frozen and thawed multiple times; multiple freeze-thaw cycles will result in smaller fragments of the obtained genomic DNA and reduced extraction quality; if they are not sealed, the temperature does not meet the standard, or the number of freeze-thaw cycles exceeds 5 times, the batch of samples should be discarded and replenished; Gene bank: DNA samples are taken out of the refrigerator regularly to test concentration, purity and integrity; The benzene ring structure of the bases on the DNA chain has strong absorption in the violet region, and its absorption peak is at 260nm; by detecting the absorbance values ​​of the DNA sample at 260nm and 280nm: A260 / A280 ratio of 1.8-2.0 indicates good purity; A260 / A280 < 1.8 indicates that protein impurities remain in the nucleic acid extraction or the sample concentration is very low; 260 / A280 > 2.0 indicates that the DNA is partially degraded or there is RNA contamination; The ratio of A260 / A230 of pure nucleic acid should be greater than 2.

0. The DNA concentration can be calculated based on the purity test. Generally, a DNA sample concentration of 100-300 ng / uL is conducive to long-term storage of DNA. The integrity of DNA was analyzed by gel electrophoresis. There was a bright band near the sample well, without impurities or tailing, indicating that the DNA molecule was intact and not degraded. If there was a bright band in the sample well, it indicated that there was protein residue in the DNA solution. If there was diffusion and tailing in the lane, it indicated that the DNA was degraded. If the DNA sample concentration is lower than 100ng / uL, it will be removed and replenished; if the A260 / A280 in the DNA sample is > 2.0, it will be removed and replenished; if the integrity of the DNA sample is poor, and there are diffusion and tailing in the lane, it will be removed and replenished; Sperm bank: regularly take out sperm samples from liquid nitrogen tanks for thawing and recovery, and test the vitality of sperm after freezing and recovery. If the recovery rate is less than 50%, the batch of samples will be discarded and replenished; Cell bank: regularly take out cell samples from the liquid nitrogen tank for thawing and resuscitation. The resuscitation method is to randomly select three samples of each generation of cells for resuscitation culture. After the cells divide again, observe the degree of cell deformity of the passaged cells under a microscope. If all of them are deformed, all samples of the generation are removed and replenished. (9) Establishing a fish germplasm resource database: Based on the various data obtained in steps (1) to (7), a complete fish germplasm resource database is established. The fish germplasm resource database is constructed with fish germplasm resource spatial distribution data, fish habitat spatial distribution data and target fish germplasm resource bank data as the first-level categories. The specific construction method is as follows: S1. Collect basic data on fish germplasm resources and habitats: Set the geographical area to be studied for the fish germplasm resources database to be constructed, and obtain basic data on fish germplasm resources and habitats within the geographical area through actual survey; S2. Establish spatial distribution data of fish germplasm resources: pre-process the fish germplasm resources data, form a basic database with spatial data as the main body, and separate and obtain spatial distribution data of fish germplasm resources; The spatial distribution data of fish germplasm resources are extracted according to the data type of the spatial distribution data of the first-level fish germplasm resources and refined into the second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into abundance and biomass data according to the biological data type; the data structure of the spatial distribution data of fish germplasm resources set based on the basic database includes the name of the administrative region, the administrative region code, the type and code of fish germplasm resources, the second-level type and code of fish germplasm resources, the third-level type and code of the spatial distribution data of fish germplasm resources and the number of catch per unit fishing effort; S3. Establish spatial distribution data of habitats: pre-process the fish habitat data, form a basic database with spatial data as the main body, and separate and obtain spatial distribution data of fish habitats; The spatial distribution data of fish habitats are extracted according to the first-level fish habitat spatial distribution data type and refined into the second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into spawning grounds, feeding grounds and wintering grounds according to the habitat data type; the data structure of the spatial distribution data of fish habitats set based on the basic database includes the name of the administrative region, the administrative region code, the spatial distribution data and coding of fish habitats, the second-level type and coding of the spatial distribution data of fish habitats, the third-level type and coding of the spatial distribution data of fish habitats and the area of ​​the map; S4. Establish target fish germplasm resource bank data: process and analyze target fish germplasm resource bank data to obtain fish habitat spatial distribution data; The spatial distribution data of fish habitats are extracted according to the data type of the first-level target fish germplasm resource bank and refined into the second-level and third-level data types. The second-level data types include living bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank, and the third-level data types are divided into sample marking information, sample processing information and sample genetic information. (10) Fish germplasm resource data management: Based on the database in step (9), a fish germplasm resource data management system is established, including a container management system, a sample management system, a data management system and a security management system; the container management system is used to manage the equipment data for storing samples; the sample management system at least includes managing the increase, decrease and status of different types of samples, automating the sample information entry and maintenance process through fish wireless radio frequency identification chip technology, recording the use and maintenance history of samples, monitoring the status of the resource library in real time, recording the increase and decrease changes, and providing an interface for querying the information of various resource libraries; the data management system implements the processing and storage logic of genetic and information data, designs the data distribution and access interface, and ensures the security and integrity of the data; Realize structured and persistent storage of data, design and develop the logical functions of the online resource sharing platform; the security management system determines the security risks and protection requirements of the system through a comprehensive analysis of the information system architecture, the business it carries and the system classification, and determines the security protection measures based on the corresponding protection requirements, balancing the relationship between security, cost and efficiency.

2. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that The type of germplasm resource bank sub-bank corresponding to each target species in step (3) is as follows: Extinct species: building a specimen library; Extinct species in the wild: building a specimen library; Critically endangered species: building a specimen library; Endangered species: build specimen and tissue banks; Vulnerable species: construct specimen banks, nucleic acid banks, and tissue banks; Low-risk species: construct specimen banks, nucleic acid banks, tissue banks, sperm banks, and cell banks; Least-endangerment species: construct specimen banks, nucleic acid banks, tissue banks, sperm banks, cell banks, and living organism banks.

3. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that: In step (4), except for extinct species, species extinct in the wild, and critically endangered species, the minimum sample storage volume of the germplasm resource bank sub-banks of other species is based on the minimum sample quantity of the specimen bank to calculate the minimum sample quantity corresponding to other banks. The specific formula is: The number of samples in the tissue bank = the number of samples in the specimen bank * 10; the number of samples in the gene bank = the number of samples in the specimen bank * 10; the number of samples in the sperm bank = the number of samples in the specimen bank; the number of samples in the cell bank = the number of samples in the specimen bank; the minimum number of parents and reserve parents in the living bank of least concern species is the minimum production capacity requirement of the provincial aquatic breeding farm.

4. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that: When processing tissue bank samples to obtain materials for transcriptome experiments in step (6), first use a scalpel or scissors combined with tweezers to quickly remove the gills, brain, heart and liver of the anesthetized or recently killed fish specimens and place them in pre-cooled cryovials, and then temporarily store them in a small liquid nitrogen tank, a -80°C freezer or dry ice. The entire process is performed under strict sterile conditions and on dry ice or ice cubes. The obtained molecular experimental materials should be promptly placed in a -80°C refrigerator for long-term storage.

5. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that The process of generating the three-level class data in step (9) is as follows: 1) Living body bank: Sample labeling information of the living body bank: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the biological characteristics of each sample, including at least sex, age and reproductive status; record the minimum sample storage volume data and actual sample storage volume data of each sample; Sample processing information of the living bank: record the environment type and water quality when the samples were collected, water environment conditions during transportation and transfer, health status before storage, adaptability assessment data before storage, health monitoring data after storage, water quality management after storage, feed type and feeding frequency, disease inspection and nutrition regulation methods; Genetic information of living bank samples: record genetic characteristics, breeding rules and reproduction strategy information; 2) Specimen library: Sample labeling information of the specimen library: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record the minimum sample storage volume data and actual sample storage volume data for each sample; Sample processing information of the specimen library: record the type of environment and water quality when the sample was collected; record the method of specimen collection and preparation; Genetic information of specimen bank samples: record the taxonomic status, morphological characteristics and habitat preference of fish; 3) Tissue Bank: Tissue bank sample labeling information: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record the fish sample part information; Record the minimum sample storage volume data and actual sample storage volume data for each sample; Tissue bank sample processing information: record the type of environment and water quality when the sample was collected; Document tissue collection and cryopreservation methods; Genetic information of tissue bank samples: record fish tissue structure, pathological signs and tissue-specific expression information; 4) Gene Bank: Gene bank sample identification information: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record genotype information; record the minimum sample storage volume data and actual sample storage volume data for each sample; Gene bank sample processing information: record the fish length, weight, age, gonad development, environment type and water quality when the samples were collected; record the gene sample collection and freezing storage methods; Genetic information of gene bank samples: record gene library information; 5) Sperm Bank: Sperm bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record the sperm storage volume of each sample; record the minimum sample storage volume data; Record actual sample storage volume data; Sperm bank sample processing information: record the fish length, weight, age, gonadal development, environment type and water quality at the time of sample collection; record sperm collection and cryopreservation methods; Genetic information of sperm bank samples: record the normal sperm morphology rate, sperm motility, fertilization rate, hatching rate and sperm membrane integrity rate before and after cryopreservation; 6) Cell Bank: Cell bank sample labeling information: assign a unique identifier to each sample for easy identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record cell type information; record the minimum sample storage volume data and actual sample storage volume data for each sample; Cell bank sample processing information: record the fish length, weight, age, gonad development, environment type and water quality at the time of sample collection; record cell collection, cell culture and cryopreservation methods; Genetic information of cell bank samples: record the morphological characteristics, cell growth characteristics and chromosome karyotype of fish cells.

6. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that: The container management system in step (10) at least includes daily data management of ultra-low temperature refrigerators, liquid nitrogen tanks and freezing tubes; monitoring the status, temperature, humidity and liquid level of storage devices; managing and allocating storage space; real-time status query and abnormal status alarm.

7. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that The specific solution for the security management system security in step (10) is as follows: 1) Authentication and authorization: Use the Web Token mechanism to implement user authentication and authorization to ensure that only legitimate users can access system functions; 2) HTTPS: Use the HTTPS protocol in front-end and back-end communications to encrypt data transmission and prevent man-in-the-middle attacks; 3) Input validation and SQL injection prevention: Strict validation of user input to prevent malicious input and SQL injection attacks; 4) Cross-site scripting (XSS) and cross-site request forgery (CSRF) protection: Implement content security policy (CSP) to limit the way browsers load and execute external resources, effectively preventing XSS attacks; include a unique CSRF token for each user request, and the server verifies the token when receiving the request to ensure the legitimacy of the request, effectively preventing CSRF attacks; ensure user data and session security through the above methods; 5) Regular security reviews: Perform regular security reviews on the system to ensure that the system is consistent with the latest security standards and best practices.

8. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that: In step (10), the fish germplasm resource data management system selects a multi-layer architecture, each layer is responsible for a specific function, and communicates with other layers through clearly defined interfaces, thereby improving the modular maintainability and scalability of the system; The system is divided into five levels in the order of functional interaction; 1) Access layer: The user access device and the interaction between the information library and the system belong to the access layer. The components used in this layer include API interface and ID card authentication; Users can access the system through multiple terminals, and administrators can manage and set various system functions through the management background; The samples in the information library are generated into QR codes by the system according to the specified coding rules. After printing, the printing device pastes it on the corresponding sample container. When sampling, the QR code can be scanned by the scanning device to obtain the sample information; 2) Gateway layer: The information base transmits data through the local gateway and transmits the data to the server for processing; The monitoring indicator data of the information database is transmitted to the server through the local gateway for comparison. If the monitoring data is abnormal, an early warning reminder will be issued in time; After users visit the web page, they interact with the server through the restful API to separate the front-end and back-end. 3) WEB layer: The Web layer is the user interface layer in the system architecture. It is mainly responsible for processing requests from users and presenting information to users. It includes components such as the front-end framework, page routing, and template engines, providing a good user experience and a friendly user interface. 4) Service layer: The service layer is responsible for implementing the core business logic, including at least database data retrieval and storage, access control logic, monitoring data recording, logging, message queue data processing and data analysis; 5) Data layer: The data layer is responsible for storing and retrieving data, and includes at least databases, caches, and communication methods: The database is used to store the persistent data of the system, using the relational database PostgreSQL or MySQL; Cache uses Redis to store frequently accessed data to improve system performance; The communication method adopts a front-end and back-end separation method, and the front-end and back-end interact through a restful API to enhance the maintainability and scalability of the system; the information of the monitoring equipment is communicated with the server in a timely manner through a message queue to ensure the timeliness and stability of the transmitted data.

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