46 results about "Pacific oyster" patented technology

The invention discloses an oyster polysaccharide (alpha-1, 4; 1, 6-gluglucosan and beta-1, 6-gluglucosan with relative molecular weight at 4000-6500Da) and making method and application in the cosmetics, which comprises the following steps: washing Dalian bay oyster or Atlantic oyster through water at 0-10 deg.c; boiling oyster at 70-80 deg.c under 0.3-0.5 Mpa for 20-45min; filtering; collecting supernatant; centrifuging to remove sediment; adjusting pH value of supernatant to 5-6 through acetic acid; acidolyzing for 1-3h; intercepting material with molecular weight less than 30000Da through film separator; adjusting pH value of filtrate over 30000Da to 6-8 through NaOH; centrifuging to remove sediment; dialyzing supernatant in the distilled water; passing tomographic column of Sephadex G-100 molecular sieve; collecting material at adsorbing peak with OD at 280 nm; freezing; drying.

The invention discloses a screening method of Pacific oyster EST micro satellite label, which comprises the following steps: finding micro satellite site with Pacific oyster EST sequence announced by GeneBank and micro satellite searching software SSRhunter; screening and separating 2-6 base repeat unit micro satellite fragment (repeat times >5); getting repeat ESTs sequence with micro satellite; designing primer at two sides flank sequence of micro satellite repeat sequence; detecting optimization primer as the micro satellite label further; proceeding overall merit for micro satellite repeatability, stability, polymorphism; getting micro satellite label. The invention can be used for Pacific oyster species resource and genetic heterogeneity, molecular population genetics, species resource determination, genetic atlas construct, functional gene research, Pacific oyster breeding and cultivation.

The invention discloses an SNP marker related to rapid growth of pacific oysters and an identification method as well as application thereof, and belongs to the field of molecular biological DNA marking technology and application. By applying a high-resolution melting curve analytic technique and utilizing 108 SNP loci mined from an EST common data base to perform genetic typing on pacific oyster rapid growth population after 5 generation breeding selection, 3 alleles closely related to the growth trait of pacific oysters are obtained via correlation analysis. The 3 SNP marker preponderant alleles can be applied to selection of rapid growth parent pacific oysters, and further provide a scientific auxiliary means for breeding selection of rapid growth pacific oysters. Compared with the conventional breeding method, the method has the advantages of strong purposefulness and direct action effect, besides, the operation is simple, the detection is rapid, the cost is low, and extensive popularization and application is facilitated.

The invention discloses a method for preparing an oyster hydrolyzate through microbial fermentation, which is characterized in that a pacific oyster is fermented under the action of Aspergillus oryzae to prepare an oyster hydrolyzate. The preparation process comprises the following steps: taking fresh oyster meat, cleaning, homogenizing, and inoculating with the Aspergillus oryzae for fermentation; and after the fermentation is finished, carrying out filtration sterilization, and concentrating the filtrate to obtain the oyster hydrolyzate. By adopting a liquid fermentation mode, the method has the advantages of reasonable design of process parameters, high operability, low cost and high total nitrogen extraction rate and glycogen extraction rate, avoids the use of finished proteases and amylases, can make full use of oyster resources, and has better industrial application potential.

A method for cryopreserving bivalve oocytes, particularly those of Pacific oysters, by preparing a solution of a cryoprotectant in water, combining this with the oocytes, carrying out a first cooling step to a temperature of about −4° C. to −20° C., and carrying out a second cooling step to a temperature of about −25°°C. to −55° C.

The invention provides a seed production method of a black-shell pacific oyster new strain. The seed production method comprises the following steps: firstly, two preliminarily screened shells are black with black mantles and high rate of flesh in body, and a group with a regular shell shape is taken as a core population; pacific oyster individuals with black shells, high rate of flesh in body and black mantles are selected out as an optimal population by adopting a size selection method and carry out offspring cultivation according to a conventional shellfish breeding method as a reproductive population to obtain a black-shell new strain F1; through a family line breeding mode, the black shell character is further purified to form a second-generation breeding family line F2; and finally, through continuous purification of a plurality of generations, a new strain with stable characters is obtained. According to the seed production method, the black-shell pacific oyster new strain with stable genetic character can be obtained, and the mantle melanin and the rate of flesh in body of obtained oysters in the new strain are both greatly increased.

The invention discloses a method applied to identification of pacific oyster family and belongs to the technical field of genomic molecular markers. The method comprises the following steps: (1) searching type I and type II SNP mutation sites in an SNP database acquired by an Illumina GoldenGate method; (2) establishing genotype database files of filial generations and parents, and acquiring genetic parameters of each SNP site by utilizing Cervus 3.0 software; (3) performing family simulation identification and analysis according to the allele frequency of each site by utilizing the Cervus 3.0 software, performing actual family identification and analysis, calculating a LOD value of a candidate parental pair, and selecting parents with the highest LOD value being positive value as real parents. According to the invention, an SNP marking method applied to identification of pacific oyster family is developed, and the reaction system and polymerase chain reaction (PCR) amplification conditions are optimized. The experiments prove that the primers have the characteristics of high repeatability, high stability, high solubility curve resolution and the like, and a foundation is laid for breeding of the pacific oyster family.

The invention discloses a pacific oyster no-damage sampling method, comprising the following steps of: a, firstly soaking pacific oyster for 10-15 hours with MgCl2 sea water solution with the concentration of 3-5%; b, then disinfecting a scalper or dissecting scissors with 70% alcohol, and scissoring pallium or gill of the pacific oyster; and c, washing the pacific oyster subjected to sampling operation in the steps a and b for 3-4 times with sea water, and putting the pacific oyster back into a breeding box for breeding. According to the method, a pacific oyster no-damage sampling method is established in a pacific oyster selective breeding process, an ideal parent can be provided for pacific oyster quality character family breeding, the method disclosed by the invention also can be applied to polyploidy breeding, and no-damage ploidy detection can be carried out on a polyploidy. High-quality varieties with good meat quality, fast growth speed and strong stress resistance are bred through selective breeding, or a triploid is obtained by adopting a polyploidy breeding technology, thus product quality of pacific oyster in China is improved.

The invention relates to a method for authenticating oyster population, in particular to a novel method for authenticating Pacific oyster population, comprising the steps of sample collection, genome extraction, primer design, PCR amplification, and EST-SSRs specific site authentication. In the step of sample collection, sample individuals from different populations of ostrea cucullata, Pacific oyster and crassostrea rivularis are collected; in the step of genome extraction, genomes of a plurality of samples of each oyster population are extracted; in the step of primer design, EST-SSRs primers are designed according to an expressed sequence tag (EST) sequence containing microsatellites; in the step of PCR amplification, the EST-SSRs primers are used to carry out the PCR amplification to obtain the PCR product with a target segment; and in the step of EST-SSRs specific site authentication, agarose gel electrophoresis is firstly carried out on the PCR product to obtain the PCR product with clear and stable bands, and then polyacrylamide gelelectrophoresis is carried out to detect polymorphic sites so as to authenticate the allele Pacific oyster population having the allele sites with specific EST microsatellite sequence. In the invention, an EST-SSRs molecule marker technology is utilized, the primers are reasonably designed, and two-step electrophoresis detections are adopted so as to achieve the purpose of authenticating the Pacific oyster population. The invention has the advantages of simple method, time saving and accurate analysis.

The invention belongs to the technical field of marine organisms, and concretely relates to a method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms. The method comprises the following steps: carrying out illumination stimulation on male pacific oysters with the full growth sexual glands, dissecting the sexual glands, and filtering by a bolting silk net to obtain a large number of high-quality pacific oyster sperms; mixing a seminal fluid with an antifreeze liquid according to an appropriate ratio, packaging the obtained mixture in 2 ml or 5 ml cryopreserved tubes; carrying out balance incubation and programmed cooling, and preserving the obtained substances in liquid nitrogen for a long term; and carrying out water bath unfreezing, and carrying out natural water activation to obtain sperms with the frozen sperm vitality of above 70% and the fertilization rate of above 95%. The method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms has great significance to artificial breeding, germplasm preservation, genetic diversity and sustainable farming.

The invention provides a cultivation method for a new strain of pure white shell pacific oysters. The cultivation method includes the steps that a class group which has two white and fan-shaped shells and is high in meat yield is preliminarily screened to serve as core groups; 10% of white shell class group is optimized from the group through a head cutting selection method, wherein the white shell class group has double pure white and fan-shaped shells and a creamy white pallium, grows fast and is high in meat yield, and the white shell class group serves as an optimized class group and a propagation group to obtain the new strain F1 with the pure white shells; by means of the family selection mode, the pure white shell character is further purified to form a second generation selection family F2; finally the new strain with stable characters is obtained through continuous purification of multiple generations. The strain of the pure white shell pacific oysters has the advantages of stable inheritance, the high meat yield, high commodity value and huge cultivation and popularization prospects, and the appearance is as white as polished jade.

The invention discloses a gene recombinant protein for a pacific oyster IgSF molecule CgCAICP1. An amino acid sequence of the gene recombinant protein is shown as SEQ ID NO.1. The preparation method sequentially comprises the following steps: performing PCR amplification on coding region fragments of the pacific oyster IgSF molecule CgCAICP1 by using specific primers P1 and P2, wherein a DNA sequence of the primer P1 is shown as SEQ ID NO.2, and a DNA sequence of the primer P2 is shown as SEQ ID NO.3; performing NcoI and Hind III enzyme digestion on the PCR amplification product and a pET32a vector, connecting the PCR amplification product and the vector by T4 ligase, transforming, and sequencing and identifying the recon; transferring the recon into an Escherichia coli Transetta (DE3) expression strain for performing induction culture; purifying and renaturating, thereby obtaining the gene recombinant protein. The gene recombinant protein for the pacific oyster IgSF molecule CgCAICP1disclosed by the invention can be applied to preparation of medicines for inhibiting Gram-negative bacteria.

The invention discloses a method for inducing ocean seashell polyploidy by using sodium chloride. The method is characterized by comprising the following steps: selecting the seashell to grow the shell to respectively obtain sperms and ovum; carrying out artificial insemination; continuously observing the developing condition of zygotes through microscopic examination to determine the number of the water body for culturing the zygotes; adding sodium chloride into the culture sea water containing the zygotes according to a proportion that 20-40 go of sodium chloride is added in 1000ml of culture seat water when 40-50 percent of zygotes are observed to release a first polar body or a first zygote is observed to occur the first polar body, so as to prevent the release of a second polar body or the first polar body of the zygotes; evenly stirring and treating for 10-15 min; and transferring the zygotes to normal sea water to incubate . The ocean seashell comprises Pacific oyster, chlamys farreri, Argopecten irradians, Crassostrea rivularis, clam, Scapharca broughtonii and the like. The seashell zygotes are treated by using the sodium chloride and the initiating agent so as to inhibit the release of the polar body and produce the shell polyploidy with no toxicity, efficiency, simple operation and extremely low cost.

The invention provides a parent crassostrea nippona breeding method for promoting maturity and spawning. The method comprises the steps of firstly, selecting and placing parent crassostrea nippona in a nursery pond for maturity-promoting breeding; feeding the parent crassostrea nippona with crassostrea nippona monodus subterraneus bait, and conducting maturity-promoting breeding on the crassostrea nippona by raising the temperature step by step, so that gonad of the parent crassostrea nippona achieves maturity; drying the parent crassostrea nippona achieving gonad maturity in the shade, and then placing the parent crassostrea nippona into a spawning pond; picking out female mature pacific oysters through microscopic examination, taking out ova through dissection, filtering the ova with a screen, and removing tissue fragments and pacific oyster ova, so that tissue filtered fluid is obtained; adding the tissue filtered fluid into the spawning pond, and inducing the parent crassostrea nippona to spawn and discharge seminal fluid; finally, moving away the parent crassostrea nippona, so that fertilized ova of artificial breeding are obtained. By means of the method, the problems that it is difficult for parent crassostrea nippona bred through indoor artificial maturation-promoting breeding to get mature and spawn can be solved, the parent crassostrea nippona spawns quickly, spawn quality is good, the artificial breeding production requirements for the crassostrea nippona are satisifed, and the foundation is laid for large-scale production of the crassostrea nippona.

The invention provides a northern high-temperature-resistant pacific oyster new strain culture method. Firstly, pacific oyster and crassostrea nippona parents are collected, synchronous maturity promoting culture of the parents is performed, male and female oysters are distinguished by dissection and microscopy after sexual glands of the pacific oyster and crassostrea nippona parents are synchronously mature, mature eggs of pacific oysters and semen of crassostrea nippona are acquired, cross fertilization is performed to obtain hybrid generations of the two parents, offspring is cultured after cross groups are obtained, the first hybrid generations are determined as pacific oyster and crassostrea nippona first hybrid generations by a microsatellite molecular marker technique method, the hybrid generations are self-reproduced by a butt selection method, and a plurality of generations are continuously bred to obtain a high-temperature-resistant pacific oyster new strain. The obtained pacific oyster new strain has the advantages of high temperature resistance, high survival rate, growing rapidness, high dressing percentage and the like.

The invention discloses a preparation method of Pacific oyster neutral polysaccharide, comprising the steps of (1) defatting dried powder of Pacific oyster with acetone, and drying to obtain the defatted oyster powder; (2) dissolving the defatted oyster powder in water, treating with an ultrasonic microwave extractor, centrifuging the treated solution, concentrating supernate by evaporating to obtain oyster polysaccharide crude extract; (3) adding the oyster polysaccharide crude extract into ethanol-(NH4)2SO4 double-water phase system to carry out extraction separation, wherein the extracted bottom phase is oyster neutral polysaccharide crude extract; (4) dialyzing the oyster neutral polysaccharide crude extract to obtain oyster polysaccharide fine extract, and concentrating and drying the oyster polysaccharide fine extract to obtain the Pacific oyster neutral polysaccharide. The preparation method has the advantages that the extraction process is simple, has low cost and is suitable for intermittent and large-scale production of high-purity high-yield high-bioactivity oyster neutral polysaccharide, the prepared oyster neutral polysaccharide is suitable for long-term storage and has high purity, immunological activity of mouse spleen lymphocytes can be improved, and the activity of Hepg-2 hepatoma cells is inhibited.

The invention relates to an induction method for a 'Haida 2#' new variety tetraploid of crassostrea gigas. The induction method comprises the steps of selecting a 'Haida 2#' new variety of crassostreagigas as parent oysters, carrying out manual maturity-promoting culture until the parent oysters are mature, selecting female and male individuals with good gonad development as parent oysters, dissecting for fetching ova, filtering, putting the ova into filtered seawater for maturation, enabling the maturated ova to be fertilized with sperms in the filtered seawater, processing fertilized ova byvirtue of cytochalasin B so as to inhibit the discharging of first polar bodies, after the processing, collecting the fertilized ova, transferring the fertilized ova into an ethanol solution for soaking, finally transferring the fertilized ova into a culture container, and carrying out incubation and larva culture. According to the induction method, the discharging of the first polar bodies of the fertilized ova of the ''Haida 2#'' new variety of a diploid is inhibited by virtue of the cytochalasin B, and the living 'Haida 2#' new variety tetraploids of crassostrea gigas are directly induced,so that the survival rate is greatly increased, and a seedling culturing foundation is provided for the amplified breeding of the 'Haida 2#' new variety tetraploid of crassostrea gigas.

The invention discloses a pacific oyster DM9-containing structural domain protein CgDM9CP-4 of which the amino sequence is as shown in SEQ ID NO.1. A preparation method comprises the following steps:performing PCR (Polymerase Chain Reaction) amplification on a pacific oyster CgDM9CP-4 gene coding region segment by using specific primers P1 and P2, wherein the DNA (Deoxyribonucleic Acid) sequenceof the primer P1 is as shown in SEQ ID NO.2, and the DNA sequence of the primer P2 is as shown in SEQ ID NO.3; performing Nde I and Xho I digestion on the PCR amplification product and a pET-30a vector, connecting through a connection enzyme, converting, and sequencing and identifying recons; transferring the recons into an escherichia coli Transetta (DE3) expression strain, performing induced culture, and further performing purification and renaturation, thereby obtaining a recombinant protein with amino sequences of a sequence table SEQ ID NO.1. The pacific oyster DM9-containing structural domain protein CgDM9CP-2 can be adopted to prepare medicines for inhibiting pichia pastoris and vibrio anguillarum.

The invention discloses a method for producing allotetraploid by hybridizing crassostrea angulata diploid and crassostrea gigas triploid. According to the method, the crassostrea gigas triploid is used as a female parent, the full weight is used as a target, the crassostrea angulata diploid is preferably selected as a male parent, and the oyster allotetraploid is produced through drug-induced hybridization. The oyster allotetraploid obtained by the method has the eurythermic characteristic, and the oyster triploid produced by hybridizing the oyster allotetraploid with the common crassostrea angulata or crassostrea gigas diploid is suitable for coastal aquaculture of the south and north in China , and has wide popularization and application prospects.

The invention relates to a method for screening maternal pacific oysters with high glycogen content and a related SNP (Single Nucleotide Polymorphism) primer pair thereof. By virtue of genome-wide association study of 486 individuals, 67 SNP signals in significant correlation with glycogens located on chromosome 6 of the pacific oyster are identified. A corresponding detection method is developed aiming at one SNP site at the base scaffold 1243_53489 of the area, and the result shows that the SNP site can be simply and rapidly detected by utilizing the method. Meanwhile, the CC genotype individual at the site is compared with the TT genotype individual. CC genotype maternal oysters are screened to be used for guiding oyster breeding. A nucleotide sequence of the upstream / downstream 500kb of the pacific oysters at the site is shown as SEQ ID No.1. The method disclosed by the invention has the beneficial effects that the maternal oysters can be subjected to genotype identification before fry breeding, and the glycogen content of the offspring is improved. The study result is as follows: the obtained SNP marker has high reliability, and the effect is stable.

The invention discloses a novel shellfish opsonin DCP (DM9domain-containing protein) capable of promoting the phagocytosis of lymphocytes. The amino acid sequence of the opsonin DCP is shown in SEQ ID NO. 2. According to the invention, DCP is isolated from crassostrea gigas for the first time; function analysis and identification show that the DCP of the crassostrea gigas can be bound with surface molecules of vibrio parahaemolyticus, can greatly promote the phagocytosis of lymphocytes on the vibrio parahaemolyticus, and is a novel shellfish opsonin. As the DCP can greatly promote the phagocytosis of lymphocytes, an important theory and practice basis are provided for disease prevention and control of shellfish.

The invention discloses primers and probe for detecting amount of gene expression of interleukin 17-5 of a pacific oyster by PCR. According to primer and probe nucleotide sequences, a forward primer is 5'-tccctggtccaccatcatg-3', a reverse primer is 5'-ggaacaccactgaggacttgg-3', and the probe is 5'-cgcgtcagctacc-3'. A fluorescent group added to the probe is 6-carboxyfluorescein. The accuracy and specificity of the primers and the probe are high, so the digital PCR technology is used for detecting the sequential variation of the interleukin 17-5 of the pacific oyster, and the result is used as anindex to evaluate the health status of the farmed pacific oyster. An established evaluation system has potential application value in early warning and prediction of diseases of farmed shellfishes, acontrol group or an internal control gene is not required, and the application limitation of a traditional fluorescence real-time quantitative PCR detection method is broken through.

The invention discloses a screening method of Pacific oyster EST micro satellite label, which comprises the following steps: finding micro satellite site with Pacific oyster EST sequence announced by GeneBank and micro satellite searching software SSRhunter; screening and separating 2-6 base repeat unit micro satellite fragment (repeat times >5); getting repeat ESTs sequence with micro satellite; designing primer at two sides flank sequence of micro satellite repeat sequence; detecting optimization primer as the micro satellite label further; proceeding overall merit for micro satellite repeatability, stability, polymorphism; getting micro satellite label. The invention can be used for Pacific oyster species resource and genetic heterogeneity, molecular population genetics, species resource determination, genetic atlas construct, functional gene research, Pacific oyster breeding and cultivation.

The invention provides a parent crassostrea nippona breeding method for promoting maturity and spawning. The method comprises the steps of firstly, selecting and placing parent crassostrea nippona in a nursery pond for maturity-promoting breeding; feeding the parent crassostrea nippona with crassostrea nippona monodus subterraneus bait, and conducting maturity-promoting breeding on the crassostrea nippona by raising the temperature step by step, so that gonad of the parent crassostrea nippona achieves maturity; drying the parent crassostrea nippona achieving gonad maturity in the shade, and then placing the parent crassostrea nippona into a spawning pond; picking out female mature pacific oysters through microscopic examination, taking out ova through dissection, filtering the ova with a screen, and removing tissue fragments and pacific oyster ova, so that tissue filtered fluid is obtained; adding the tissue filtered fluid into the spawning pond, and inducing the parent crassostrea nippona to spawn and discharge seminal fluid; finally, moving away the parent crassostrea nippona, so that fertilized ova of artificial breeding are obtained. By means of the method, the problems that it is difficult for parent crassostrea nippona bred through indoor artificial maturation-promoting breeding to get mature and spawn can be solved, the parent crassostrea nippona spawns quickly, spawn quality is good, the artificial breeding production requirements for the crassostrea nippona are satisifed, and the foundation is laid for large-scale production of the crassostrea nippona.

The invention discloses a fertility recovery method for oyster distant hybrid sterility. The fertility recovery method includes by a distant hybridization mode, performing synchronous gonad treatment on Hongkong oysters and pacific oysters, and performing interspecific oyster hybridization to acquire a highly-sterile filial generation F1; during maturity seasons of filial generation F1 hybrids, analyzing fertility of the hybrids and screening out fertile individuals; by a family or group reproduction mode, performing self-reproduction of the fertile individuals of the hybrids F1 to acquire a secondary filial generation F2; through molecular markers, screening out individuals relatively similar with the hybrids F1 in terms of genotype from completely-fertile hybrids F2 to acquire completely-fertile groups with characteristics of the hybrids F1 so as to realize complete hybrid fertility recovery. By the fertility recovery method, the problem of failure to continue follow-up breeding work due to oyster intersterility frequently caused during distant hybridization is solved, data and references are provided for distant backcross, multiple cross, potential estimation on hybrid offspring breeding and the like of oysters, and possibility is also provided for research and development of new species of the oysters.

Disclosed in the present invention is a preparation method for a Pacific oyster neutral polysaccharide, comprising the following steps: (1) defatting Pacific oyster dry powder by means of acetone and drying the same to obtain defatted oyster powder; (2) dissolving the defatted oyster powder in water and treating the same using an ultrasonic microwave extractor, centrifuging a solution which is obtained after treatment, and then evaporating and concentrating a supernatant to obtain a crude oyster polysaccharide extract; (3) adding the crude oyster polysaccharide into an ethanol-(NH4)2SO4 dual aqueous phase system to perform extraction and isolation, an extracted lower phase being a crude liquid oyster neutral polysaccharide extract; (4) dialyzing the crude liquid oyster neutral polysaccharide extract to obtain a refined oyster polysaccharide liquid extract, wherein an oyster neutral polysaccharide may be obtained after concentration and drying. The present invention has a simple extraction process, is low cost, and is suitable for intermittent and large-scale production of high purity, high yield and high bio-activity oyster neutral polysaccharide products, while the prepared oyster neutral polysaccharide may be stored for a long period of time, has high purity, may increase the immune activity of mouse splenic lymphocytes and inhibit the activity of Hepg-2 liver cancer cells.

The invention provides a cultivation method for a new strain of pure white shell pacific oysters. The cultivation method includes the steps that a class group which has two white and fan-shaped shells and is high in meat yield is preliminarily screened to serve as core groups; 10% of white shell class group is optimized from the group through a head cutting selection method, wherein the white shell class group has double pure white and fan-shaped shells and a creamy white pallium, grows fast and is high in meat yield, and the white shell class group serves as an optimized class group and a propagation group to obtain the new strain F1 with the pure white shells; by means of the family selection mode, the pure white shell character is further purified to form a second generation selection family F2; finally the new strain with stable characters is obtained through continuous purification of multiple generations. The strain of the pure white shell pacific oysters has the advantages of stable inheritance, the high meat yield, high commodity value and huge cultivation and popularization prospects, and the appearance is as white as polished jade.