74 results about "Paralichthys" patented technology

A fish allogenous frozen-sperm induced gynogenesis method comprises a frozen storage method of the allogenous fish sperm, a genetic inactivation method, an induced method for the gynogenesis diploid fry and an identification method for the gynogenesis fry. The present invention establishes the method adopting the frozen-sperm of the sea bass to induce the egg gynogenesis of a plurality of sea fishes for the first time, which selects the cold-shock starting time, cold-shock temperature and the cold-shock durance time that are separately suitable for fish eggs of the strip-spotted flounder, the turbot and the canine-tooth paralichthys olivaceus to establish a micro-satellite marking identification method of the gynogenesis fry. The percentage rate adopting the present invention to acquire the gynogenesis fry of the strip-spotted flounder, the turbot and the canine-tooth paralichthys olivaceus is 7.8 percentages to 40.7 percentages, 26 percentages to 34 percentages and 10 percentages to 25 percentages respectively. The established method of the present invention is characterized in advancing, high efficiency, accuracy and reliability, which has an important application value in fish gender controlling and the production of the all-female fries and has a broad popularization and application prospect on fish cultivation and fish breeding.

The invention disclosesbacillus amyloliquefaciens M001 and an application thereof. The bacillus amyloliquefaciens M001 is preserved in the CGMCC(China General Microbiological Culture Collection Center)of CCCCM(China Committee for Culture Collection of Microorganisms), the preservation date is Nov. 4th, 2013, and the preservation number is CGMCC No.8433. Thestrain is isolated and screened from the intestinal tract of paralichthysolivaceus, and can obviously improve the nonspecific immune level of cultured fish and enhance the capacity of resisting bacterial pathogen infection of the cultured fish.

The invention relates to a selective breeding method for disease-resistant superior strains of paralichthys olivaceus, which comprises group selective breeding and family selective breeding of the paralichthys olivaceus and is characterized by also comprising molecular marker-assisted selective breeding, manual gynogenesis selective breeding or inbreeding selective breeding of a disease-resistant paralichthys olivaceus family. Multiple kinds of biotechnology such as group selective breeding, intraspecific hybridization, family selective breeding, molecular marker-assisted selective breeding, manual gynogenesis selective breeding, inbreeding and the like is comprehensively adopted; a selective breeding method for the disease-resistant superior strains of marine fishes in China is established for the first time by taking the paralichthys olivaceus as a material; the superior strains of the paralichthys olivaceus of which the vibrio anguillarum resistance is obviously improved are bred; and the breeding survival rate is improved by 20 to 47 percent and the growth rate is improved by over 20 percent. The method opens up a new technical approach for the selective breeding of the disease-resistant strains of the fishes, is suitable for popularization and application to all cultured fishes, has great significance and application value for breeding the disease-resistant superior strains of the cultured fishes, and can generate great economic and social benefits.

The invention relates to a traditional Chinese medicine preparation for preventing and curing paralichthys olivaceus and sea cucumber scuticoci liatlda diseases. The preparation comprises the following traditional Chinese medicine components in percentage by weight: 5-20 percent of abrotanum, 5-20 percent of azedarach, 5-20 percent of cyrtomium fortunei, 5-40 percent of stemona, 5-20 percent of golden cypress, 5-20 percent of betel nut and 5-30 percent of omphalia, and the sum of the weight percentages are 100. According to the invention, medicines with components restraining or killing pathogens are selected to make a formula on the basis of traditional Chinese theories, breeding bait is added and absorbed by the host of a parasite, scuticoci liatldas parasitizing in the body or on the body surface of the host of the parasite are completely killed in a body fluid absorbing and supplying way, so that the traditional Chinese medicine preparation has the advantages that medicine feeding is simple, the medicine effect is quick, the insert killing effect is good, the recovery of the physique of the host of the parasite is quick, the survival rate is high, side effects and medicine residue are avoided, as a result, economical loss caused by scuticoci liatlda infection during the production processes of breeding and growing.

The invention relates to a marine fish cell culture technology, in particular to an aralichthys olivaceus muscle satellite cell line establishing method, a specific primer for identifying a paralichthys olivaceus muscle satellite cell marker gene and application of the specific primer. The aralichthys olivaceus muscle satellite cell line establishing method comprises the following steps: performing primary culture and subculture of aralichthys olivaceus muscular tissues, and then establishing a cell line; after establishing, performing freezing preservation, resuscitation and identification, wherein a cell culture fluid adopted to primary culture and subculture is obtained through adding fetal calf serum and a human basic fibroblast growth factor into the cell culture fluid. The built aralichthys olivaceus muscle satellite cell line is in the format of fusiform or spindle-shaped monocytes, allows continuous passage, can provide a great number of aralichthys olivaceus muscle satellite cells, and not only can be directly used for researches on aralichthys olivaceus muscle development related functional genes to provide rich cell sources for researches on the molecular mechanism of a fish muscle differentiation pathway, but also is expected to provide a research platform for researches and application of muscle character improvement during aralichthys olivaceus culture production.

The invention discloses a method for preparing pathogenicity aeromonas hydrophila bacteria inactivating vaccine, which comprises the following steps: firstly, inoculating the pathogenicity aeromonas hydrophila bacteria separated from the brown paralichthys olivaceus with abdominal dropsy through PCR detecting to the common LB culture medium to be cultured so as to reach a certain density; secondly, inactivating the bacteria by using formaldehyde solution; thirdly, collecting the thalli by using centrifugal culture and then diluting the thalli by using physiological saline so as to get the pathogenicity aeromonas hydrophila bacteria inactivating vaccine. The preparing method is rapid, simple and convenient in operation, and easy in batch preparing; the gotten vaccine is a pure biological agent which does not has any toxic and side effect and does not cause pollution to the meat quality of the paralichthys olivaceus and the surrounding water. The immunity protecting rate reaches 80 percent by using the vaccine prepared by the method to inoculate brown paralichthys olivaceus so as to radically curb the spreading of the pathogeny. The invention provides new approach for preventing and curing the epidemic abdominal dropsy of brown paralichthys olivaceus.

The invention relates to an induction and detection method for androgenesis dihaploid of Paralichthys olivaceus. The induction and detection method provided by the invention has the beneficial effects that nuclear genetic materials in eggs can be deactivated by only carrying out cold-shock treatment on the fertilized eggs for a certain time, the effect the same as that achieved by ray irradiation deactivation in the traditional androgenesis induction techniques is achieved; and hydrostatic treatment is adopted, the androgenesis dihaploid of the Paralichthys olivaceus can be rapidly prepared.

The invention discloses a paralichthys olivaceus transportation method of paralichthys olivaceus capable of keeping paralichthys olivaceus alive without water, including the following steps: (1) temporary culturing: choosing and putting healthy and energetic paralichthys olivaceus into seawater with an alive keeping agent and performing temporary culturing for 1-2d; (2) gradient cooling: cooling the seawater, when the water temperature is above 10 DEG C, the cooling rate is 2-4 DEG C/h, when the water temperature is 1-10 DEG C, the cooling rate is 0.8-1 DEG C/h, and when the water temperature is below 1 DEG C, the cooling rate is 0.3-0.5 DEG C/h, reducing the seawater temperature to -0.5-1.5 DEG C according to the cooling rate; (3) packaging and oxygen filling: taking the paralichthys olivaceus out of the seawater, and quickly putting the paralichthys olivaceus in an anhydrous double-layer inflatable bag, filling pure oxygen, and sealing the bag; and (4) temperature controlling and transporting: putting the anhydrous double-layer inflatable bags into -0.5-1.5 DEG C environment, and transporting the bags. The inventive method has the advantages of easy operation, low transportation cost, no pollution and high quality, is suitable for long-distance transportation, and is beneficial to improving the economic value of paralichthys olivaceus.

The invention relates to the technical field of biology, disclosing a method for fast detecting the reverse transcription-polymerase chain reaction of paralichthys rhabdovirus. The method comprises the following steps of: designing a special primer; extracting RNA of a sample to be detected; having the reverse transcription-polymerase chain reaction; and judging a result by means of agarose gel electrophoresis. The method has the advantages of high speed, good accuracy and specificity and high sensitivity, is suitable for fast testing and quarantining the paralichthys rhabdovirus, and is applicable to aquafarms and disease inspection bureaus, thereby having a wide application prospect.

The invention discloses a paralichthys olivaceus Chinese herbal medicine attractant and a preparation method. The paralichthys olivaceus Chinese herbal medicine attractant is prepared from the following raw materials in parts by weight: 8-18 parts of medicated leaven, 7-15 parts of pericarpium citri reticulatae, 3-10 parts of fructus aurantii, 1-5 parts of anise, 1-5 parts of foeniculum vulgare, 5-10 parts of portulaca oleracea, 8-15 parts of cordate houttuynia and 3-11 parts of eclipta prostrata. The preparation method comprises the following steps: mixing the raw materials, decocting, filtering, concentrating and then obtaining the attractant through the spray drying technology. The medicated leaven, the pericarpium citri reticulatae, the fructus aurantii, the anise and the foeniculum vulgare in the attractant have traditional Chinese medicine fragrance, the palatability of the feed can be improved, and the gastric juice secretion, intestinal juice secretion and salivary secretion are promoted, and the absorption of the organism to the bait is strengthened; the portulaca oleracea contains niacin saponin, oxalic acid, vitamin A, vitamin B, vitamin C and tannic acid, has good attraction effect to the paralichthys olivaceus and has the effect of treating dysentery; the cordate houttuynia and the eclipta prostrate have strong fishlike smell to stimulate the taste bud of the paralichthys olivaceus so as to promote the feeding; furthermore, the cordate houttuynia has inhibiting effect on the microorganisms such as saccharomycetes and the like.

The invention discloses a paralichthys olivaceus fertility related SNP molecular marker and a screening method and application thereof. The screening method comprises the following steps: separately carrying out cyp21a gene re-sequencing on fertile paralichthys olivaceus and infertile DH paralichthys olivaceus at first to preliminarily screen difference SNP sites; verifying the SNP sites on a large scale by using a fertile population capable of spawning normally in a breeding season and DH infertile population incapable of spawning; carrying out parting on the SNP sites, which are obtained instep (1), of the paralichthys olivaceus by using a multiple SNaPshot SNP parting method; and carrying out statistics on genotype frequency of the SNP sites which are successful in parting by using SPSS software, and carrying out difference significance analysis by using chi-square test to screen out SNP sites related to paralichthys olivaceus fertility. In the invention, a paralichthys olivaceus fertility research model is established, a method for rapidly establishing a large number of 21OHD animal models is provided, and SNP sites of fertility related cyp21a genes are obtained by screening.

The invention relates to an industrial culture device capable of simulating a flounder paralichthys wild benthic environment. The industrial culture device comprises a culture pond, wherein a general blow-off hole is formed in the middle of the bottom of the culture pond; a hollow water level column for adjusting a water level is mounted on the general blow-off hole; a horizontal porous carrying platform for segmenting the bottom of the culture pond to form a lower blow-off layer space is arranged above the bottom of the culture pond; a sand filtering thin film covers on the upper surface of the porous carrying platform; a natural sea sand layer is arranged on the sand filtering thin film; an upper air guide pipe, which is connected with an air pump, is mounted on a peripheral side wall of the culture pond on the upper surface of the natural sea sand layer; and airflow blown out from an air guide hole of the upper air guide pipe passes through the upper surface of the natural sea sand layer, so that the natural sea sand layer is stirred. By adopting the industrial culture device provided by the invention, separation of blow-off and a habitat environment is realized and the quality of the habitat environment on an upper layer of a pond bottom is guaranteed, so that the condition of blind side melanism of flounder paralichthys is greatly reduced.

The invention discloses a genetic engineering vaccine of lymphocystis disease of flounder paralichthys olivaceus and a production method thereof, which is characterized in that the method for producing the genetic engineering vaccine comprises the following steps: inserting gene MCP segments (size: 0.6 KB) of nucleocapsid proteins of lymphocystis disease virus (LCDV) into an eukaryotic expression vector pEGFP-N2 so as to form recombinant plasmids pEGFP-N2-LCDV-MCP (size: 0.6 KB); translating the recombinant plasmids into escherichia coli so as to prepare production strains, lining the production strains to a Mi nca agar plate so as to separate and purify the production strains and then prepare and determine qualified level-1 strains; then inoculating the level-1 strains in an agar flat bottle, after cultivating the level-1 strains for 24h-48h at a temperature of 37 DEG C, identifying the obtained level-1 strains and determining qualified level-2 strains; carrying out fermentation and cultivation on the obtained level-2 strains so as to obtain fermentative level-2 strain culture solution; carrying out bacteria collection and centrifugation as well as plasmid DNA extraction and purification on the fermentative level-2 strain culture bacterium solution, then carrying out collection, centrifugation, plasmid DNA extraction, purification and routine inspection on the cultured bacterium solution, if qualified, the genetic engineering vaccine is obtained. The genetic engineering vaccine of the invention has the advantages of complete and simple production process, good stability and low cost; and compared with current inactivated vaccines, attenuated vaccines, polypeptide vaccines, subunit vaccines and recombinant polypeptide vaccines and the like, the genetic engineering vaccine of the invention has good immunization effect and no adverse effect.

The invention belongs to the field of aquaculture, and particularly relates to an artificial control method for inducing the synchronous maturing of the gonads of male paralichthys dentatus parent and female paralichthys olivaceus parent. In the method, the male paralichthys dentatus parent is subjected to full-illumination environmental control in autumn and winter to suppress the gonad development of the male paralichthys dentatus parent; and in the spring of the next year, the full-illumination environment is changed into short-illumination environment, and the male paralichthys dentatus parent and the female paralichthys olivaceus parent are subjected to gonad development control at the same time to realize the gonad maturing of the male paralichthys dentatus parent. The method provided by the invention can obtain mature sperms and ova at the same time, prolongs the germ production period of the male paralichthys dentatus parent, increases the sperm quantity, and lays a technological base for obtaining batch high-quality sperms and ova in the large-scale offspring seed production of hybrid flounder.

The invention provides a paralichthys olivaceus streptococcus iniae GAPDH series-connection multi-epitope polypeptide and application thereof. The provided series-connection multi-epitope polypeptideis formed by binding four antigen epitope peptides through peptide joints, wherein the amino acid sequences of the four antigen epitope peptides are shown in SEQID NO:1-4 respectively. One specific amino acid sequence of the series-connection multi-epitope polypeptide is SEQID NO:8. The invention further provides a streptococcus iniae GAPDH multi-epitope vaccine prepared by using the series-connection multi-epitope polypeptide as an antigen and a vaccine adjuvant. The multi-epitope vaccine can provide strong immune protection for paralichthys olivaceus resistance to S.iniae infection, and theeffect is significantly higher than that of a GAPDH recombinant protein subunit vaccine and a S.iniae inactivated vaccine.

The invention belongs to the technical field of marine organisms, particularly a method for carrying out interspecies cross artificial insemination on an egg of paralichthys olivaceus and a semen of paralichthys dentatus, which is frozen for preservation. The semen of paralichthys dentatus, which is frozen for preservation at an ultralow temperature for a long time, is dissolved in water by two steps; after the semen of paralichthys dentatus is dissolved, diluent of which the volume is three times of that of the frozen semen of paralichthys dentatus is added into the frozen semen of paralichthys dentatus; after the frozen semen of paralichthys dentatus and the diluent are mixed, centrifugation is carried out for 5min; then supernate is removed to obtain frozen semen suspension with the same volume with the frozen semen of paralichthys dentatus before the frozen semen of paralichthys dentatus is diluted; the frozen semen and natural seawater are uniformly mixed according to the proportion of a volume ratio of 1:40 and the mixture is activated; after the mixture is activated, the egg of paralichthys olivaceus is injected and the mixture and the egg of paralichthys olivaceus are slightly and uniformly mixed to enable the egg of paralichthys olivaceus and the frozen semen of paralichthys dentatus to be sufficiently in contact; standing is carried out for 5 to 10min and egg washing is carried out to obtain a fertilized egg to carry out incubation. The invention establishes a cryopreservation technology for semen of paralichthys dentatus and a method for interspecies cross artificial insemination on the egg of paralichthys olivaceus, overcomes the difficulty of asynchrony of development of parental fishes of paralichthys dentatus and paralichthys olivaceus and provides a novel method and a novel idea for seawater fish cross breeding.

The invention discloses an inactivated pentavaccine for flounder paralichthys and a preparation method of the inactivated pentavaccine. The inactivated pentavaccine consists of the following formulated components in parts by weight: 5-8 parts of glycine, 5-15 parts of complex vitamins, 7-9 parts of aspartic acid, 13-15 parts of vibrio anguillarum, 8-10 parts of vibrio scophthalmi, 2-4 parts of vibrio alginolyticus, 2-4 parts of an astragalus polysaccharide adjuvant, 12-13 parts of medicinal starch, 4-6 parts of vibrio vulnificus, 4-5 parts of edwardsiella tarda liquid, 10-20 parts of maltodextrin, 1-3 parts of vibrio harveyi and 0.5-0.7 part of a mildew preventive. The inactivated pentavaccine provided by the invention has multiple immune effects; and the inactivated pentavaccine, as being inoculated once, can effectively prevent infection of five bacterial pathogenic bacteria of the flounder paralichthys; therefore, a basis is provided for the research and development of inactivated multivalent vaccines for marine fish.

Paralichthys lymphocystis virus strain, LCDV011126, with the preservation number of CCTCC No. V202007, is prepared through taking Paralichthys tumor tissue of lymphocystis virus, crushing, adding phosphoric acid buffering liquid for homogenation, adding amphitolerant, freezing, thawing, centrifugating to obtain coarse virus extracting liquid, filtering, inoculation to fresh water fish cell for further culture at constant temperature, observing cell virus, collecting cell, fixing, superthin slicing, and dyeing for observation. The process is simple, easy to regulate and control and low in cost, and may be used in amplifying great amount of lymphocystis virus and providing required virus material.

The invention discloses a cytology division method of a scophthalmus maximus spermiogenesis phase. The method comprises the following steps: fixing, pre-dyeing, dehydrating and embedding, dyeing by uranyl acetate, dyeing by lead citrate, and slicing preparing and analysis. A scophthalmus maximus spermiogenesis process is divided into 8 phases: comprising sperm cell I-VII stage and mature sperm. The method has the beneficial effects that the cytology division method overcomes the problem that during common histological slice observation, sperm cells at different metamorphosis periods cannot bedistinguished due to limited magnification times, pre-dyeing is helpful for increasing dyeing uniformity, tissue structure definition, and tissue dyeing contrast grade, dyeing effect can be optimized,and image definition of the tissue structure of the sperm cells can be obviously enhanced; the cytology division method can fill the research blank of phase division for the flounder paralichthys spermiogenesis, and can be used for cytology division of the spermiogenesis phase of the other seawater fish.

The invention discloses a paralichthys olivaceus cultivating method. The paralichthys olivaceus cultivating method comprises the following steps of mixedly cultivating male and female parent paralichthys olivaceus to obtain paralichthys olivaceus fertilized eggs; hatching the paralichthys olivaceus fertilized eggs to obtain paralichthys olivaceus fry; washing and immersing a cultivation system by fresh water after sterilizing the cultivation system; processing natural sea water, enabling the natural sea water to reach a preset cultivation index, obtaining cultivation sea water, and injecting the cultivation sea water in the cultivation system; putting the paralichthys olivaceus fry in the cultivation system for cultivating, carrying out bait feeding and pest control and prevention, and monitoring the water quality of the cultivation sea water in real time; processing cultivation waste water, and enabling the cultivation waste water to reach the preset cultivation index so as to be used as the cultivation sea water which can be recycled; injecting the cultivation sea water into the cultivation system. The paralichthys olivaceus cultivating method disclosed by the invention is relatively environment-friendly and has smaller pollution on the environment; in addition, the paralichthys olivaceus can be cultivated by utilizing the natural sea water which can reach the preset cultivation index after being processed, the cultivation risk is lower, the cultivation period is shorter, the cultivation efficiency is higher, and the cultivation quality is better.