93 results about "Scylla paramamosain" patented technology

The invention relates to genetic engineering of Scylla paramamosain, and provides a Scylla paramamosain anti-lipopolysaccharide factor, and a preparation method and application thereof. The preparation method comprises the following steps: building a recombinant expression vector for the Scylla paramamosain anti-lipopolysaccharide factor; introducing the recombinant expression vector into host cells, and carrying out induced expression on the host cells to obtain an expression product; and separating and purifying the expression product to obtain a recombinant protein, namely the Scylla paramamosain anti-lipopolysaccharide factor. The Scylla paramamosain anti-lipopolysaccharide factor has obvious growth inhibition and sterilization effects on Gram-negative bacteria such as Shigella flexneri, Vibrio alginolyiicus, Vibrio parahaemolyticus, Pseudomonas stutzeri and Pseudomonas fluorescens and Gram-positive bacteria such as Corynebacterium glutamicum, and has a potential application value in the preparation of antibacterial medicaments and antimicrobial additives for animal feed.

The invention relates to a method for establishing a microsatellite molecular marker of scylla paramamosain. The method comprises the following steps of: extracting genome DNA of the scylla paramamosain; performing enzyme digestion on the genome DNA, jointing and pre-amplifying; hybridizing an amplification product, a probe and a magnetic bead and performing secondary PCR amplification; cloning and sequencing the amplification product; analyzing the sequence and designing a microsatellite specific primer; performing PCR amplification on the genome DNA of different geographic groups or individuals in the group; and performing 6 percent modified polyacrylamide gel electrophoresis to detect the PCR amplification product so as to obtain a polymorphic map of genetic variation of the scylla paramamosain. The method has a good application prospect in the fields such as genetic variation analysis of the scylla paramamosain, germplasm resource protection and management, genetic linkage map establishment, marker-assisted breeding and the like.

Scylla paramamosain antiviral type anti-lipopolysaccharide factor, its preparation method and application. The invention relates to a scylla paramamosain antiviral type anti-lipopolysaccharide factor and provides the gene sequence and amino acid sequence of the scylla paramamosain antiviral type anti-lipopolysaccharide factor Sp-ALF2, and a preparation method of the scylla paramamosain antiviral type anti-lipopolysaccharide factor Sp-ALF2 and its application. The preparation method provided by the invention comprises the following steps of: constructing a recombinant expression vector of the scylla paramamosain antiviral type anti-lipopolysaccharide factor Sp-ALF2; introducing the recombinant expression vector into a host cell, and carrying out inducible expression on the host cell to obtain an expression product; separating and purifying the product to obtain a recombinant protein, namely Sp-ALF2. The scylla paramamosain antiviral type anti-lipopolysaccharide factor Sp-ALF2 has obvious functions of inhibiting and killing WSSV and a plurality of pathogens, and has a wide application value of preparing novel antiviral drugs and being used as an animal antiviral feed additive.

The invention discloses a scylla paramamosain anti-bacterial polypeptide Sp-NPFin and its application. A molecular formula is C227H373N71O62S1, and its amino acid sequence is shown as SEQ ID NO: 01. The scylla paramamosain anti-bacterial polypeptide Sp-NPFin has strong antibacterial activity and antifungal activity, good antibacterial effect, wide antibacterial spectrum and fast sterilization rate. The scylla paramamosain anti-bacterial polypeptide Sp-NPFin is derived from crustaceans and can be applied to aquatic feed additives, can be developed to anti-mold preservatives, antibacterial agents and antibacterial drugs, and has broad application prospects.

The invention relates to SNP sites for sex determination of Scylla paramamosain and an identification method thereof. A base sequence is SEQ ID No. 1 or SEQ ID No. 2 and has a length of 285 bp; and the SNP sites are site 62, site 70, site 74, site 145, site 147, site 163, site 186 and site 194 in the SEQ ID No. 1 or SEQ ID No. 2. The identification method comprises the following steps: extraction of the genome DNAs of male and female individuals; construction of a reduced genome library and high-flux sequencing; sequence analysis and screening of candidate SNP markers linked with sexes; and large-sample verification of the candidate SNP markers. According to the invention, the sex-linked molecular markers of Scylla paramamosain are identified for the first time and have great guidance significance to the sex determination and differentiation mechanism of Scylla paramamosain and to research on unisexual breeding. The method provided by the invention has the advantages of accuracy, sensitivity, reliability and the like, has good universality, does not need to learn about genome information in advance and is applicable to a great number of species without reference genomes, so the method has good promotion and application potential.

The invention relates to scylla paramamosain soft crab mixed feed and a preparation method thereof. The mixed feed comprises 35-40 parts of fish meal, 25-30 parts of peeled bean pulp, 4-6 parts of shrimp head powder, 4-6 parts of fish dissolved pulp, 10-15 parts of pregelatinized starch, 4-5 parts of fish oil, 0.5-2 parts of soybean lecithin, 0.5-2 parts of cholesterol, 3-4 parts of mixed vitamins, 2-3 parts of mixed minerals, 0.5-1 part of choline chloride, 1-2 parts of monocalcium phosphate, 4-6 parts of sodium alginate and 0.1-0.2 part of eclosion hormones. The mixed feed is particularly designed for scylla paramamosain soft crabs, is balanced in nutrient component, good in palatability and stable in raw material source, and a function of promoting eclosion of scylla paramamosain is achieved after the shrimp head powder, the eclosion hormones as well as the soybean lecithin and the cholesterol of certain ratios are used together; and the LC-PUFA (Long-Chain Polyunsaturated Fatty Acid) of a high ratio, particularly n-3LC-PUFA, is beneficial to increase of the survival rate and the eclosion rate, and nutrition requirements of the scylla paramamosain at specific growth stages can be met; as an adhesive, the sodium alginate is capable of improving the feed water stability and reducing the feed loss rate and can be taken in for 100% by the scylla paramamosain, the culture successrate of 'soft crabs' is 100%, the culture time is shortened by more than 10 days when being compared with that of natural bait or commercial crab feed, and the mixed feed is simple to prepare and very good in popularization value.

The invention provides a coding gene of scylla paramamosain allergenic protein and an application thereof. The coding gene of the allergenic protein is composed of a nucleotide sequence shown in SEQ ID NO: 1. The invention also discloses an expression vector and a recombinant strain containing the gene, a method for preparing the allergenic protein and the application of the allergenic protein. The coding gene of the scylla paramamosain allergenic protein is cloned into a prokaryotic expression vector, recombinant expression plasmid is constructed, and transferred into host bacteria and inducing expression is carried out; the scylla paramamosain allergenic protein rScy p 9 can be obtained, the IgE binding capacity of the scylla paramamosain allergenic protein rScy p 9 and serum of a crustacean allergic patient is measured, a reference basis can be provided for clinical diagnosis of allergic symptoms and detailed screening of allergens, and prevention, control and treatment of allergicdiseases of the Scy p 9 are timely and accurate.

The invention discloses a compounded feed for factory circulating-water three-dimensional culture of scylla paramamosain and belongs to the technical field of aquaculture. The feed for the scylla paramamosain, disclosed by the invention, contains the following raw materials in parts by weight: 250-350 parts of fish meal, 150-250 parts of soybean meal, 50-100 parts of shrimp shell meal, 200-300 parts of corn starch, 20-30 parts of squid meal, 20-30 parts of fish oil, 5-10 parts of choline, 10-20 parts of lecithin, 5-10 parts of cholesterol, 5-10 parts of sodium carboxymethyl cellulose, 10-20 parts of sodium alginate, 10-20 parts of multi-dimensional multi-mineral premix and 5-10 parts of calcium dihydrogen phosphate. According to the feed disclosed by the invention, the formula is scientific and reasonable, the preparation method is simple, convenient and quick, the processing cost is low, the performance is good, the food calling performance is high, the growth and shelling of the scylla paramamosain can be effectively promoted, the survival rate is increased, and the pollution to environments caused by culture excrements is alleviated.

The invention relates to an artificial seeding raising method for scylla paramamosain in a small water body, which comprises the following steps: gradually increasing aeration quantity from the scylla paramamosain zoea cultivation phase and the megalopa cultivation phase to the young crab cultivation phase; collocating corresponding baits in each phase; from the day before differentiated II-phase zoea, changing the water every day, and adding algae to adjust water color, thus realizing the artificial seeding raising for scylla paramamosain in the small water body. The method has the advantages of low application requirements to the seeding raising, low investment, high efficiency, and easiness in understanding, can be quickly understood and handled by the farmers, is fit for artificial seeding raising in the inbred line breeding process, and has an excellent application prospect.

The invention relates to a quick detection method for Scylla paramamosain by microsatellite markers from functional genes, which comprises the following steps: (1) extracting genomic DNA of the Scylla paramamosain; (2) acquiring a gene sequence containing microsatellite repeats according to a functional gene sequence of the Scylla paramamosain in a GeneBank database; (3) designing microsatellite marker primers; (4) performing PCR amplification on the genomic DNA of different geographic population or different individuals in the population of the Scylla paramamosain; and (5) detecting a PCR product by polyacrylamide gel electrophoresis. The detection method has the advantages of quickness, accuracy, sensitivity and the like, and can intuitively detect genotypes of different individuals of the Scylla paramamosain so as to quickly acquire a polymorphic map for hereditary variation at functional gene microsatellite loci of the Scylla paramamosain; and the microsatellite markers can be applied to the fields such as hereditary variation analysis of the Scylla paramamosain, germ plasm resource protection and management, genetic linkage map construction and the like.

The invention relates to an artificial cultivation brooding and incubating method for scylla paramamosain. The method comprises four steps of preparation of a seeding crab cultivation pond, selection of seeding crabs and placement in the pond, enhanced cultivation of the seeding crabs and cultivation of brood crabs. The method has the advantages of large brood amount, high incubation rate, strong larva vigor, safety and easiness in handle. The method can be quickly understood and handled by farmers, is fit for artificial breeding of offspring seeds at a large scale, and is expected to facilitate health and fast development of the artificial breeding work of scylla paramamosain domestically.

The invention relates to a whole genome DNA of scylla paramamosain mitochondria and a testing method. The DNA molecule which is in a closed ring structure has a length of 15824 bp; and the testing method comprises the steps of: (1) collecting relative gene sequences of the scylla paramamosain mitochondria and comparing the relative gene sequences of the scylla paramamosain mitochondria with whole genome sequences of mitochondria of sibling species; (2) designing a primer and carrying out PCR (Polymerase Chain Reaction) amplification; and (3) sequencing PCR products and assembling the sequences. According to the invention, the whole genome DNA sequences of the scylla paramamosain mitochondria are obtained. The testing method disclosed by the invention has the advantages of being fast in speed, low in cost, easy to master and the like; and the obtained whole genome DNA of the mitochondria can be applied to the fields such as heritable variation analysis of the scylla paramamosain, genetic resource authorization and protection, phylogeny and the like.

The invention relates to a method for screening a scylla paramamosain microsatellite molecular marker. The method comprises the following steps of: extracting scylla paramamosain genome DNA (Deoxyribonucleic Acid) and diluting the DNA for later use; designing a 5' anchor primer and performing PCR (Polymerase Chain Reaction) amplification; cloning an amplification product and sequencing; performing sequence analysis and designing a microsatellite specific marker; performing PCR amplification on the genome DNA of different geographical populations of scylla paramamosain or individuals in the populations by using the primer; detecting a PCR amplification product by using 6 percent denaturing polyacrylamide gel electrophoresis; and determining the genotype of each individual according to different migration distances of the amplification product to obtain a polymorphic map of the genetic variance of scylla paramamosain. The method is easy to operate, is safe and has a high positive rate and a true and reliable result. By adopting the method, the polymorphic map of the genetic variance of scylla paramamosain can be obtained quickly. The method is mainly applied to genetic variance analysis of scylla paramamosain, protection and management of the germ plasm resource, establishment of a genetic linkage map and genetic breeding.

The invention relates to an artificial directional mating method of Scylla paramamosain. The artificial directional mating method of Scylla paramamosain includes: firstly, selecting black barrels as mating sites, spreading sand 10-15cm in thickness at the bottom of each black barrel, setting an air filling device, burying a nozzle of the air filling device in the sand, injecting fresh sea water, and keeping each black barrel in the dark; secondly, selecting healthy female and male crabs weighing 180-220g as candidate parents, cleaning the crabs with the fresh sea water, and putting two male crabs and two or three female crabs in each black barrel for breeding; thirdly, using Sinonovacula constricta as feed, changing all the water in black barrel every day, and taking the female carbs which finish mating for continual breeding. The artificial directional mating method of Scylla paramamosain has the advantages of simplicity in operation, high success rate, low input cost and the like, selective artificial directional mating can be achieved, and the artificial directional mating method of Scylla paramamosain is applicable to artificial large-scale propagation and selection of Scylla paramamosain.

The invention relates to a trypsin gene cDNA sequence of Scylla paramamosain and a cloning method and application thereof. The trypsin gene cDNA sequence of Scylla paramamosain has the sequence shown in SEQ ID NO:1; the cloning thereof comprises the following steps: extracting total RNA from the hepatopancreas of the Scylla paramamosain; building a cDNA plasmid library; carrying out authentication and sequencing analysis on the cDNA plasmid library; and according to the BLAST comparison, obtaining the sequence shown in SEQ ID NO:1. The trypsin gene cDNA sequence of the Scylla paramamosain can forecast feeding amount and exuviation cycle in the germchit and reproductive process of blue crabs so that people can master the life cycle of the blue crabs and select and use in inheritance breeding, reduce the bait consumption, improve the germchit survival rate, thereby optimizing the process of grow seedlings.

The invention provides a domestication method for adapting scylla paramamosain seedlings to saline-alkali soil water area culture. The method comprises the following steps of: temporarily culturing selected scylla paramamosain seedlings in a culture pond and then reducing the salinity of seawater to be not higher than 1%; and putting the scylla paramamosain seedlings into the seawater with a pH value of 7.0-8.5 and the carbonate alkalinity of 2.5-3.5 mM, and raising the pH value of the culture seawater to 9-10 and the carbonate alkalinity to 9.0-9.5 mM within not less than 3 days, thereby completing domestication. According to the method, the adaptability of scylla paramamosain to saline-alkali soil water areas can be effectively domesticated, the survival rate of the scylla paramamosain put into saline-alkali soil is greatly increased, and economic benefits of culture are increased.

The invention provides a scylla paramamosain sensitizing protein Scy p 1 derivative and an application thereof. The amino acid sequence of the derivative is shown as SEQ ID NO:1. According to the scylla paramamosain sensitizing protein Scy p 1 derivative, an N terminal, a C terminal and two key linear epitope regions of a wild type Scy p 1 amino acid sequence are deleted. Amplifying is carried outthrough an overlap extension PCR technology to obtain an Scy p 1 derivative gene sequence; connecting and transforming into a BL21 host cell are carried out, and inducible expression is carried out;the expressed derivative is purified by a Ni column, and the influence of Scy p 1 on mast cell degranulation before and after transformation is analyzed. Compared with wild-type Scy p 1, the Scy p 1 derivative can significantly reduce release of beta-aminohexosidase in LAD2 cells, is expected to be used for immunotherapy or immunological prevention, and reduces side effects in immunotherapy so asto improve the safety index of treatment.

A PCR-based rapid genetic sex identification method for Scylla Paramamosain is disclosed. The method includes (1) designing control primers, and designing female-specific primers by utilizing a base mismatch process; (2) extracting genome DNA of a Scylla Paramamosain individual to be tested; (3) by using the control primers and female-specific primers respectively, and by using the genome DNA of Scylla Paramamosain as a template, performing PCR amplification at an annealing temperature of 65 DEG C to obtain corresponding PCR products; and (4) detecting the PCR products through electrophoresiswith agarose gel having a concentration of 1.5% to determine whether the individual is male or female. According to the method, the base mismatch process of the sex-specific SNP site is utilized to design the female-specific primers, amplification is performed through an optimized PCR reaction system and a PCR reaction program, and then electrophoresis with agarose gel is performed for detection to achieve genetic sex identification of the Scylla Paramamosain. The method has advantages of simple operation, a high accuracy rate, a low cost, good repeatability and high practicability, and can beused for large-scale rapid identification for Scylla Paramamosain in the early period of growth and development.

The invention discloses a device for artificial rapid mating of scylla paramamosain, which comprises an aquaculture glass tank, a catchment tank, a circulating pipe, a water pump and a stainless steelsupport for placing the catchment tank. The catchment tank is located above the aquaculture glass tank, the bottom of the catchment tank is provided with a water flow pipe, and an opening at the bottom of the water flow pipe faces the inside of the aquaculture glass tank; the bottom end of the circulating pipe is connected with the water pump located in the aquaculture glass tank, and the top ofthe circulating pipe extends to the upper part of the catchment tank; a stone layer is laid at the bottom of the catchment tank, sand bags are laid at the upper part of the stone layer, an activated carbon layer is laid at the upper parts of the sand bags, and filter biochemical cotton is laid at the upper part of the activated carbon layer. Through the use of the device, the pair mating process of the scylla paramamosain is efficient, fast, stable and controllable, the mating efficiency of the scylla paramamosain is improved, the workload of the staff is reduced, and the condition that the scylla paramamosain is susceptible to pathogenic bacteria in the mating process is effectively avoided.

The present invention relates to a cSNP molecular marker detection kit and a detection method for scylla paramamosain disease-resistant characters, relates to the technical field of molecule auxiliary disease resistance breeding in aquatic livestock, and particularly relates to a single nucleotide polymorphism cSNP molecular marker, capable of influencing disease resistance of blue crabs, in a cDNA sequence of scylla paramamosain SpALF6, as well as a detection kit and a detection method thereof. On a basis that a disease resistance factor SpALF6 of the scylla paramamosain is identified, by performing sequence comparison, it is found that 215th nucleotide in the cDNA sequence of SpALF6 and 222th nucleotide G of a variant of the SpALF6, namely SpALF6-V, are nonsense mutation for each other. In the invention, the mutation site is used as the basis, a convenient and practical molecular marker detection kit is built, and a matched RT-PCR detection method is provided; detection results can be used as important indicators for judging the disease resistance of the blue crabs, and the single nucleotide polymorphism cSNP molecular marker has the great importance in predicting of the disease resistance of the blue crabs and molecule auxiliary disease resistance breeding.

The invention discloses a method for improving the survival rate of crab seedlings of scylla paramamosain.The method comprises the following steps: (1) preparing a crab seedling pond: preparing two cement ponds with one used as a reservoir for adjusting sea water and facilitating water change, and the other one used as the crab seedling pond for desalinating the crab seedlings; (2) preparing the crab seedlings; (3) disinfecting the crab seedlings; (4) performing desalination culture on the crab seedlings; (5) packaging and transporting the crab seedlings; (6) putting the crab seedlings into abreeding pond. By utilizing the method disclosed by the invention, through the desalination culture of the scylla paramamosain before stocking, the survival rate of the fed scylla paramamosain is greatly improved, and the economic benefit of breeding is increased.

The invention relates to a method for microsatellite marker-based genealogy authentication of scylla paramamosain. The method comprises the following steps of 1, genomic DNA extraction, 2, microsatellite primer PCR amplification, 3, denaturing polyacrylamide gel electrophoresis of amplification products, and 4, family discrimination. The method provided by the invention can overcome physical marker defects, can realize accurate and effective discrimination of scylla paramamosain individuals from different families, is suitable for identifying mix-bred different individuals from multiple families in family breeding, can promote healthy and fast development of artificial breeding of scylla paramamosain, and has a good application prospect.

The invention discloses a scylla paramamosain C type lectin Spctl-2. The amino acid sequence of the scylla paramamosain C type lectin Spctl-2 is SEQ ID No: 1. The invention also discloses a gene for coding the scylla paramamosain C-type lectin Spctl-2. The nucleotide sequence of the antibacterial protein Spctl-2 gene is SEQ ID No: 2; the invention further discloses a recombinant antibacterial protein rSpctl-2 based on the C-type lectin Spctl-2, The invention also discloses an application of the recombinant antibacterial protein rSpctl-2 in preparation of medicines for preventing and controlling Vibrio fluvialis infection. The invention also discloses an application of the recombinant antibacterial protein rSpctl-2 in preparation of medicines for preventing and controlling micrococcus luteus. The invention also discloses a preparation method of the recombinant antibacterial protein rSpctl-2. The recombinant protein is produced by using escherichia coli, has the advantage of secretory expression, is easy to industrially produce and provides more choices for designing the dosage form of the immunopotentiator.

The invention relates to a scylla paramamosain peroxy gene Sp-PX, an amino acid sequence encoded by using the scylla paramamosain peroxy gene Sp-PX and a clone method. The overall length of a cDNA sequence of the gene is obtained by primer design, PCR (Polymerase Chain Reaction) amplification reaction and company sequence measurement, the nucleotide sequence of the scylla paramamosain peroxy gene Sp-PX has 2331bp, and the amino acid sequence encoded by using the scylla paramamosain peroxy gene Sp-PX has 776 amino acid residues. A research on the scylla paramamosain peroxy gene Sp-PX has important significance for further perfecting the theoretical basis of innate immune systems of invertebrates and can also provide theoretical foundation for finding immunization strategy of reinforcing the antibacterial ability and anti-virus capability of the scylla paramamosain.

The invention discloses a scylla paramamosain double-stranded specific deoxyribonuclease as well as a preparation method and an application thereof and relates to double-stranded specific deoxyribonucleases. The preparation method comprises the steps of extracting total RNA (Ribose Nucleic Acid) of hepatopancreas of scylla paramamosain, carrying out reverse transcription on the total RNA by using an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) technology to obtain cDNA (complementary Deoxyribose Nucleic Acid), obtaining a new DSN (Duplex-Specific Nuclease) gene by utilizing a SMART II-RACE method, constructing a prokaryotic expression recombinant plasmid of the scylla paramamosain by utilizing a genetic recombination method and carrying out high-efficiency expression and purification in escherichia coli to obtain recombinant scylla paramamosain DSN with biological activity. The invention provides a recombinant vector, comprising the gene sequence of the scylla paramamosain double-stranded specific deoxyribonuclease. The invention provides a biological agent which contains the scylla paramamosain double-stranded specific deoxyribonuclease and is applied to degrading double-stranded DNA.