Biotechnology research means that people use the knowledge of modern biology, engineering and other basic disciplines to control and remold organisms according to their pre-designed or simulated organisms and their functions to develop commercial processing, product production and social services. Emerging technology areas. In the past decade or so, the rapid development of biotechnology has been on the rise. At present, many biotechnology achievements have been applied in fisheries. For example, China's cattle and sheep genetic fish technology has reached the international advanced level; the tilapia and rainbow trout genetic chain map preparation and the positioning of some quantitative trait loci (sex, growth, body color) for these The traits provided a good basis for marker-assisted breeding of DNA molecules, and Japan, the United States, and Canada have also cloned growth hormone genes, prolactin genes, and antifreeze protein genes from Hemp, Rainbow Trout, Tilapia, and Quercus. Successfully expressed in microorganisms produced growth hormones and other important proteins in some fish. So far, foreign countries have succeeded in obtaining the polyploid-staining sports species engineering technology on more than 20 kinds of fish, shrimps and shellfish such as grass carp, clam, rainbow trout, tilapia, gingiva, scallops, oysters, prawns, and abalone. The polyploidy new varieties have a significantly faster growth rate than their parents; the gynogenetic lines of rainbow trout, hard-headed sturgeon, Atlantic salmon, and red seabream obtained in France, Japan, Norway, etc. are artificially induced. The broad prospects; China's shrimp white spot disease gene decoding, for its disease prevention and control technology provides a reliable basis for drug screening; the development of aquatic disease detection technology also provides necessary measures to control the occurrence of disease; through the immune means to control aquatic animals Disease has become a hot spot in current research. In addition, the use of biotechnology to protect the aquatic environment and control pollution is also an important biological engineering method for the ecological protection of water bodies and the sustainable development of their industries.
1 Gene manipulation technology
1. l Gene cloning This is a cloning method based on the special function of proteins, and it is also one of the strategies adopted earlier in gene cloning. First, a gene or cDNA expression library is constructed, and then the expression library is divided into several sub-libraries to transfect the cells respectively, and the function of sub-library expression products is detected through some specific sensitive physiological and biochemical indicators or antibody immune reactions, and a positive sub-library is detected. Afterwards, one way is to further subdivide the sub-library and repeat the screening until the final monoclonal function gene is obtained; the other way is to use the in-situ immunohistochemistry method to isolate the single cells that are positive and then separate. Corresponding functional genes. In addition, there are many new gene cloning techniques such as genome mismatch screening (GMS), representative difference analysis (RDA), exon amplification technology, and mRNA differential display technology also have a wide range of application potential. Complete sequencing of the genomes of representative aquatic organisms (including fish, shrimp, shellfish, and pathogenic microorganisms and viruses) while performing specific functional genes such as drug genes, enzyme genes, hormone polypeptide genes, disease resistance genes, and salt tolerance genes The cloning and functional analysis of clones are the focus of research. The genes that have been identified and cloned include: Penaeus vannamei antibacterial peptide genes, oyster allergen genes, Atlantic salmon and Atlantic salmon antibody genes, rainbow trout Vasa genes, herring genetically modified P53 genome genes, and diflagellates. Factor 5A gene. The streptococcus GTH (gonadotropin) receptor cDNA, Abactor actin gene, cyanobacterial pyruvate kinase gene, salmon rhodopsin gene regulation series, and gingival lysozyme gene, and the like.
1.2 Gene transfer Gene transfer as an effective technical means for the improvement of biological genetics, the cultivation of fast-growing and stress-tolerant varieties, has become the focus of applied technology research and development. High-volume, high-efficiency transgenesis is the focus of gene transfer studies. In addition to traditional microinjection, particle bombardment, and sperm-carrier methods, retrovirus-mediated, electroporation, and transposon-mediated methods have been developed. And embryonic cell-mediated methods. In recent years, research focused on target gene screening, such as disease resistance genes, insulin growth factor genes, and green fluorescent protein genes as target genes. Hayat et al. (1991) successfully injected human and salmon growth hormone into eggs of salmon and salmon; Rahman (1992) injected the metallothionein-binding protein gene and mouse growth hormone into single-cell stage. In tilapia eggs, about 20% of the integration rate was obtained; Gross (1992) transferred the bovine growth hormone gene to pike medium. Most of the above studies use exogenous genes. From the aspects of safety, expression intensity of foreign genes, and generational transmission, the use of fish's own "whole fish gene" may be ideal. Du (1992) first used the “whole fish gene†to transfer the growth hormone gene to Atlantic salmon fertilized eggs. The individual weight of the transgenic fish obtained was 38 times larger than that of the control group. The average weight of the offspring of the transgenic fish was 5.2 times greater than that of the control group. In the category of transgenic research, there has been a gradual expansion from economically farmed fish to cultured shrimp, shellfish and some ornamental fish.
1.3 Gene chip microarray technology, also known as gene microarray technology, refers to the immobilization of a large number (usually higher than 400 cells per square centimeter) of nucleic acid probe molecules onto a carrier (slide or membrane) and labeled sample molecules. (mRNA, cDNA, genomic DNA, etc.) are hybridized, and the number and sequence information of the sample molecules are obtained by detecting the hybridization signal intensity of each probe molecule. The biggest advantage of this technology is that it can detect and analyze a large number of sample sequences at a time. Currently, gene chip technology has been widely used in gene expression assays by designing different combinations of probe arrays and using specific analysis methods. Mutation detection, polymorphism analysis, genomic library mapping, and hybridization sequencing. The prerequisite for the application of the gene chip technology is to have a large number of probe molecules of known sequence, and the number of aquatic genes that have been determined so far is very limited, which greatly limits the application of the gene chip technology in the study of functional genomics of aquatic organisms.
l. 4 Genetic marker restriction endonuclease (RFLP) technology, random amplified polymorphism (RAPD) technology, DNA fingerprinting technology, amplified fragment length polymorphism (AFPL) and other molecular markers for gene diversity analysis. Provides basis for germplasm identification and genetic breeding. Knox et al. (1991) used plasmid DNA restriction fragment length polymorphisms to discriminate between the populations of migratory and terrestrial Atlantic salmon and the wild populations of Norway and Scotland. Moran (1996) used the variation of plasmid DNA to distinguish between artificial hatching and wild locust populations in the Spanish rivers and found the relationship between population size and plasmid DNA variation. Carter (1991) et al. and Gross (1994) used DNA fingerprinting techniques to identify different groups of female genital tilapia and the different populations of smallmouth bass. Sun Xiaowen et al. (2000) combined RAPD technology with SSLP (Simple Sequence Length Polymorphism) technology to initially establish genetic linkage maps of common carp. Wang Zhiyong et al. (2002) used AFLP technology to study the genetic variation and divergence of the true sea bream population in coastal China.
2 Cell and genomic technologies
2. l Polyploid induction usually involves two groups of plant and animal cell genomes. Animals with more than 2 sets of chromosomes formed by natural environmental factors or artificial special treatment are called polyploids. The main purpose of artificial induction is to use triploids. Triploids have the characteristics of fast growth, high net meat rate, good meat quality, and long life cycle; the triploid also contributes to population control and has high resistance to disease. And stress resistance. Valenti (1975) cold-processed the fertilized eggs of Australian tilapia, induced 75% of polyploids and hatched as high as 90%. Hong Yunhan (1990) heat-treated the eggs before the first cleavage and obtained 56.3% of tetraploids. Myers (1991) compared two types of triploid hard-headed pupae obtained from normal fertilized eggs by heat treatment and from tetraploid and diploid hybrids, and found that the former had poor growth in the early stages, and the latter had high survival rate but fertilization rate. Lower. Guo et al. (1996) used diploid and tetraploid Pacific oysters to produce 100% triploid oysters, which individuals were 13% to 51% larger than normal diploids. The results of nearly 4 months of culture in the estuary indicate that the triploid adductor muscle is 73% heavier than the control and the average body tissue wet weight is 36% higher. Most of the triploid shellfish are immature during the summer, and the weight and glycogen content of the closed shells are significantly higher in the growth period than in the control group.
2.2 Gynecological Development Gynogenetic development refers to the fact that the egg requires the activation of sperm but the sperm does not provide a parthenogenetic manner of genetic material. In nature, there is a parthenogenetic aquatic animal, artificial induction means can also make unfertilized eggs for gynogenetic reproduction, but the majority of the resulting individuals are haploid and hard to survive, and need to be followed by a special treatment of diploidization. In addition to speeding up the establishment of breeding lines and control of sex, gynogenetic development can also allow some rare recessive alleles to appear and produce good traits, allowing dominant genes with important economic traits to become homozygous. Developmental offspring can also be used to identify inbreeding decline in fish. Jiang Yisi (1982) et al. and Wu Qingjiang (1981) had successfully used gamma rays to irradiate semen from fish and induced gynogenetic reproduction of red salmon eggs and salmon eggs. Cai Nai-er (1995) first reported the artificial induction of gynogenetic development in Chinese shrimps using a four-step induction method similar to that used to study artificially induced gynogenetic development in fish. Three batches of gynogenetic individuals were bred, but large-scale There are still some difficulties in production. Li Shengzhong et al. (1997) induced the development of diploid gynoecium in rainbow trout by heat shock, and all three experiments were successful. In order to limit the reproduction of transplanted grass carp and silver carp, the spermatozoa treated with ultraviolet rays induce the gynogenesis of ferret eggs, and then undergo temperature shock treatment to obtain gynogenetic fishes, but also fertilization rate and hatching rate. Low problem.
2.3 Nuclear Transplantation Cell nuclear transfer technology is the transfer of a cell nucleus to another enucleated unfertilized egg or intracellular biotechnology. The famous Chinese biologist Tong Dijie and others took the lead in carrying out nuclear transfer of the same species of fish in goldfish and fish. Subsequently, nuclear transfer between different subfamilies was performed between these two species of fish, and a variety of transplanted nuclear-matrix fish were obtained. Yan et al. (1985) successfully performed nuclear transfer between two different subfamily species in the bonefish, namely transplanting the nuclei of the grass carp to the cytoplasm of the crustacean, obtaining a nuclear hybrid fish. Its appearance characteristics are similar to that of grass carp, indicating that the expression of genetic information is mainly affected by the nuclei of the transferred fish, and the growth rate of nucleocytoplasmic hybrid fish is slightly faster than that of grass carp, but much faster than that of short-nosed pupae. The sperm produced by it can be The grass carp eggs are backcrossed and the offspring are obtained. Lin et al. (1996) transplanted somatic, carp, and Nile tilapia somatic nuclei into the mature enucleated eggs of carps to obtain embryos and juveniles at different stages of development. At present, China is a world leader in the theoretical research and practical application of fish cell nuclear transfer technology.
2.4 Cell culture technology Cell culture technology is a technique that separates, cultures, induces, or reproduces cells of organisms. At present, this technology has been widely used in the culture of fish cells, pearl shell mantle epithelial cells, and algae cells. Aquaculture animal cell culture starts with fish, and it is systematically developed with the study of fish cell culture. There are also many established fish cell lines. These cell lines have many uses. They can replace live animals for virus research, can be used for genetic studies such as chromosome production, are used for making cell vaccines, are sources of donor cell nuclei for nuclear transfer and breeding research, and are used for toxicity and water quality monitoring. The indicator organism (cells are more sensitive to toxic substances than fish) and so on. Zhang Nianci et al. (1981) established a diploid ZC-7901 cell line in the snail's snout end tissue. Chen Minrong et al established the aneuploid cell line CAB-80 in 1985. Zuo Wengong et al established the CIK cell line of grass carp kidney in 1986. , Tong Sangliang et al established the rainbow trout macrophage cell line in 1989. Compared with fish, studies on cell culture of shellfish, shrimps and other aquatic animals have been less reported at home and abroad. For example, Hanson et al. established a freshwater snail embryo cell line in 1976, and Xu Yali recently established PMO cell line of P. monodon.
3 Disease Diagnosis Technology
3. Monoclonal antibody technology Monoclonal antibody technology is a technology developed by K and M in 1975 that utilizes hybridoma cells to produce a large number of specific antibodies directed against an antigenic determinant. Compared with conventional serum antibodies, monoclonal antibodies have strong specificity, can recognize single antigenic determinants, and are easy to prepare. They can keep cell lines repeatedly and obtain the same antibodies. Therefore, monoclonal antibodies are widely used in the detection of aquaculture pathogens. For example, it is applied to diagnosing scallops of scallops, diagnosis of lymphocytes combined with fish and oysters, and detection of Vibrio philippinarum P1 caused by the brown ringworm disease in the Philippines. In addition, in recent years, using this technology, monoclonal antibodies against exotoxicity to the hydrophila of M. viridis have been prepared.
3.2 Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (EL1SA) is a technique that combines the antigen-host immune response with the enzyme's catalytic reaction. The basic principle is that the antigen or antibody is adsorbed on the solid phase carrier, and then is combined with the enzyme-labeled antigen or antibody, and the substrate is hydrolyzed and colored under the participation of an appropriate substrate to show the antigen and antibody specificity through the presented color change. The existence of the reaction. ELISA has the characteristics of high sensitivity, strong specificity, and rapid response, and the results can be quantified. It can also be used for localization analysis of antigens, antibodies, and antigen-antibody complexes. ELISA has been used in the detection of IPNV, VHSV, CCV, PERV, SVCV, GCHV, and IHNV. With the continuous updating and improvement of technical methods, especially the application of monoclonal antibodies, the detection sensitivity and heterosexuality are greatly improved (Ristow et al., 1991). Davis (1994) combined with cell culture technology to detect IPNV, greatly enhanced the detection of specificity, sensitivity and objectivity.
3.3 Nucleic Acid Hybridization Techniques Nucleic acid hybridization techniques are the use of specific labeled DNA or RNA probes to hybridize with target nucleotide sequences complementary to probes in pathogen organisms to determine whether the host carries a class of pathogenic molecules. Biological techniques can be divided into Southern hybridization, Northern hybridization and nucleic acid in situ hybridization. This technology has advantages such as high sensitivity, high specificity, and rapid detection. In recent years, it has been favored in the detection of aquaculture virus pathogens such as shrimp viruses. Subramanian (1993) used the synthesized CDNA probe to successfully detect the dsDNA of water and Reovirus in infected cells and tissues by nucleic acid hybridization test; Lupinai et al. (1993) used RNA probes prepared by RNA-RNA imprinting. A new virus, Aqua Reovirus A, was isolated and identified by hybridization. Dopazo (1994) et al. used the prepared cDNA probes for the detection and diagnosis of IPNV. IPNV was detected from the cultured cells inoculated with virus for 4 to 8 hours.
3.4 PCR technology Polymerase chain reaction (PCR) is a method for rapid amplification of specific DNA fragments by enzymatic synthesis in vitro. The application of PCR technology in the diagnosis of animal diseases such as fish has only just begun, and it has shown its great potential and broad application prospects (Davies etaL 1994). Arakawa (1990) used PCR to detect rainbow trout IH-NV, and identified the specificity of the product with biotin-labeled oligonucleotide probes. Boyle (1991) used PCR to detect CCV DNA with a sensitivity of 0. Lpg, and successfully detected CCV carriers. Lastra (1994) applied reverse transcriptase polymerase chain reaction (RT-PCR) to detect highly purified viruses in cultured cells with a sensitivity of lpg; in addition, they also used silver salmon and salmon in tissues. IPNV is detected and amplified. For the detection of other aquatic animal viruses, as early as 1990 there was a report on the detection of nuclear polyhedrosis virus from shrimp tissues using PCR technology (Vickers, 1990). Chang et al. successfully used PCR for the first time to perform amplification of shrimp viruses. Wang et al. (1996) used PCR to detect shrimp baculovirus and obtained the expected amplification product. In addition, PCR-based techniques can also be used to detect viruses or bacteria that are enriched in shellfish enterovirus and water or aquatic organisms.
4 Immune control technology
4.1 Immunostimulants include specific and non-specific immune enhancers, but more to enhance non-specific immune function. Its main function is to display the body's defensive effect by activating its own immune function, and it has a wide range of functions and high safety. The function of the immunopotentiator is various. In the crustacean, it can activate the phagocytic cells in the hemolymph, increase the ability to phagocytose the pathogen, stimulate the production or increase of the antibacterial and bacteriolytic activity in the hemolymph, and activate the protooxygenase system. Identify signals and mediate phagocytosis. In fish, it can activate the phagocytosis of neutrophils and macrophages; activate lymphocytes to produce or secrete lymphokines to coordinate humoral and cellular immunity; stimulate the production of antibodies and the production of complement. The most commonly used immunostimulants are dextran, lipopolysaccharides, bacterial peptides, levuximab, chitin, egg products, vitamins, and hormones. Hardie et al. (1991) reported that the level of complement activity was increased in fish given large amounts of vitamin C. Atlantic salmon, after injection of yeast glucan, also showed increased complement activity (Engstad et al. 1992); the use of immunostimulants can also affect the activity of lysozyme, a hydrolase that exists in the mucus of fish. In fish, serum and macrophages, they can kill pathogenic microorganisms and protect them (Engstad et al. 1992). In addition, NK cells can also be activated by growth hormone (Kajita et al. 1992) and Kajita et al. (1990).
4.2 DNA vaccines In the prevention and treatment of aquatic animal diseases, the traditional vaccine is the use of inactivated bacterial vaccines or weakly toxic bacterial vaccines, the former often have little effect, while the latter can still cause minor infections or the risk of reverting to mutations in pathogenic strains. . DNA vaccine, also known as gene vaccine or nucleic acid vaccine, separates gene fragments responsible for translating antigenic proteins from viruses or microorganisms, connects with carriers in vitro to form recombinant DNAs, and then transfers them into host cells to express antigenic proteins in host cells. To achieve the effect of immunity. The preparation method of this vaccine is simple, low in cost, suitable for large-scale production, it can be expressed in vivo for a long time, and continuously stimulates the body's immune system, can form a multivalent vaccine, that is, a vaccine can induce immunity against multiple epitopes. Protective effects. The DNA vaccine for fish is mainly aimed at viral fish diseases. Currently used for research are hemorrhagic septic virus (VHSV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), and salmonid virus ( EVA, EVEX), grass carp reovirus (FRV), scorpion virus and other vaccines. Lorenzen (1999) constructed a DNA vaccine against the VHSV G protein that induced 70% of rainbow trout to produce a higher level of immunoprotection. Traxler et al. (1999) used a naked DNA encoding the IHNV G protein (PCMV4-G) to immunize salmon. After 8 weeks, they were challenged with the IHNV virus and the protection rate reached 40% to 100%. Of course, as a new generation of vaccines, DNA vaccines are still incomplete. The most interesting issue is the safety issue. People are worried that DNA vaccines will be integrated into the chromosomes of the host and cause insertion mutations. However, they have not yet been found in experiments. this phenomenon.
4.3 Antisense technology Antisense technology is the general term for antisense nucleic acid technology and ribozyme technology developed in the past more than a decade. It can be used to specifically block the function of the virus. Antisense nucleic acids can be classified into antisense RNA technology and antisense DNA technology, but generally referred to as antisense RNA technology. Antisense RNAs are RNAs and their derivatives that are synthesized using the sense strand of DNA as a template. They can block their functions by complementary binding to the corresponding mRNA. In 1985, for the first time, the antisense nucleic acid and expression vector of the enzyme gene or structural gene necessary for virus survival were connected and transferred into the cell or the antisense nucleic acid was directly injected into the cell. The discoverer had an inhibitory effect on the virus. At present, the use of artificially synthesized antisense RNA has successfully inhibited the expression of a variety of viral genes in prokaryotic and eukaryotic cells. Ribozymes are catalytic RNAs that recognize each other by base pairing with the target RNA. It binds and catalyzes the hydrolysis of target RNA, so it has both antisense RNA and sequence-specific cleavage of RNA. Experiments have shown that ribozymes can also inhibit the replication and expression of viruses, and that the transferred genes can be passaged. Numerous studies have shown that ribozymes can be used to cleave viral target RNAs. However, there is no report on the successful application of antisense technology for the treatment of aquaculture plant and animal viral diseases. However, as a highly specific detection technology, antisense technology has great potential for development.
5 Environmental bioremediation technology The deterioration of the aquatic environment has caused great threats to fisheries. The outbreak of diseases is mainly caused by environmental pollution. Environmental bioremediation technology is a new research and development field. The use of bioremediation technology in foreign countries to improve the aquatic environment has made new progress. The so-called environmental bioremediation technology is the use of biological agents to remove hazardous waste in the aquatic environment, the application of microbial mechanisms, degradation of a variety of pollutants, improve the aquatic environment. Bioremediation is an environmental biotechnology that is more extensive than biodegradation and focuses on biodegradation. The method includes using living organisms or their production products to degrade pollutants, reduce toxicity or convert them into non-toxic products, enrich and fix toxic substances (including heavy metals, etc.), and large-scale bioremediation also includes ecological regulation in ecosystems, etc. . This kind of biotechnology can be applied to large-scale aquaculture and industrial aquaculture, oil pollution, heavy metal pollution, urban sewage and other marine waste (water) treatment. The dynamic mechanism of microbial reaction to the environment, the biochemical mechanism of degradation process, the symbiotic relationship and mutual benefit mechanism between biosensors, marine microorganisms and other organisms, and the research on the separation and purification of anti-adhesive substances need to be further studied. . Researches that have been conducted include: Studying the ability of heavy metal-sulfur protein gene algae to adsorb heavy metals in seawater environments; The feasibility and application potential of five oil-degrading microorganisms in repairing water environments contaminated by five oils; Marine magnetic bacteria in removal and Potential applications for recovery of heavy metals in seawater environments; removal of nitrogen from wastewater from fish farms using rod-shaped bacteria; microalgae used as marine feed diets screened by molecular techniques; cold-resistant decane-degrading bacteria were isolated and studied in marine environments Aromatized hydrocarbon microbial degradation technology.
6 Conclusion In conclusion, aquaculture is an applied subject, and its development does not only depend on the technology of the subject, but more importantly, it requires comprehensive biotechnology, including: genetics, nutrition, pathology, endocrinology, and bioengineering. When solving the key technical issues of a certain breeding object, they all involve high-tech integrated biotechnology. The application of all these biotechnologies will undoubtedly have a profound and profound epoch-making significance for the development of fisheries.
Author: Zhao Hongxia Zhan Zi-Rong Xu Yong
1 Gene manipulation technology
1. l Gene cloning This is a cloning method based on the special function of proteins, and it is also one of the strategies adopted earlier in gene cloning. First, a gene or cDNA expression library is constructed, and then the expression library is divided into several sub-libraries to transfect the cells respectively, and the function of sub-library expression products is detected through some specific sensitive physiological and biochemical indicators or antibody immune reactions, and a positive sub-library is detected. Afterwards, one way is to further subdivide the sub-library and repeat the screening until the final monoclonal function gene is obtained; the other way is to use the in-situ immunohistochemistry method to isolate the single cells that are positive and then separate. Corresponding functional genes. In addition, there are many new gene cloning techniques such as genome mismatch screening (GMS), representative difference analysis (RDA), exon amplification technology, and mRNA differential display technology also have a wide range of application potential. Complete sequencing of the genomes of representative aquatic organisms (including fish, shrimp, shellfish, and pathogenic microorganisms and viruses) while performing specific functional genes such as drug genes, enzyme genes, hormone polypeptide genes, disease resistance genes, and salt tolerance genes The cloning and functional analysis of clones are the focus of research. The genes that have been identified and cloned include: Penaeus vannamei antibacterial peptide genes, oyster allergen genes, Atlantic salmon and Atlantic salmon antibody genes, rainbow trout Vasa genes, herring genetically modified P53 genome genes, and diflagellates. Factor 5A gene. The streptococcus GTH (gonadotropin) receptor cDNA, Abactor actin gene, cyanobacterial pyruvate kinase gene, salmon rhodopsin gene regulation series, and gingival lysozyme gene, and the like.
1.2 Gene transfer Gene transfer as an effective technical means for the improvement of biological genetics, the cultivation of fast-growing and stress-tolerant varieties, has become the focus of applied technology research and development. High-volume, high-efficiency transgenesis is the focus of gene transfer studies. In addition to traditional microinjection, particle bombardment, and sperm-carrier methods, retrovirus-mediated, electroporation, and transposon-mediated methods have been developed. And embryonic cell-mediated methods. In recent years, research focused on target gene screening, such as disease resistance genes, insulin growth factor genes, and green fluorescent protein genes as target genes. Hayat et al. (1991) successfully injected human and salmon growth hormone into eggs of salmon and salmon; Rahman (1992) injected the metallothionein-binding protein gene and mouse growth hormone into single-cell stage. In tilapia eggs, about 20% of the integration rate was obtained; Gross (1992) transferred the bovine growth hormone gene to pike medium. Most of the above studies use exogenous genes. From the aspects of safety, expression intensity of foreign genes, and generational transmission, the use of fish's own "whole fish gene" may be ideal. Du (1992) first used the “whole fish gene†to transfer the growth hormone gene to Atlantic salmon fertilized eggs. The individual weight of the transgenic fish obtained was 38 times larger than that of the control group. The average weight of the offspring of the transgenic fish was 5.2 times greater than that of the control group. In the category of transgenic research, there has been a gradual expansion from economically farmed fish to cultured shrimp, shellfish and some ornamental fish.
1.3 Gene chip microarray technology, also known as gene microarray technology, refers to the immobilization of a large number (usually higher than 400 cells per square centimeter) of nucleic acid probe molecules onto a carrier (slide or membrane) and labeled sample molecules. (mRNA, cDNA, genomic DNA, etc.) are hybridized, and the number and sequence information of the sample molecules are obtained by detecting the hybridization signal intensity of each probe molecule. The biggest advantage of this technology is that it can detect and analyze a large number of sample sequences at a time. Currently, gene chip technology has been widely used in gene expression assays by designing different combinations of probe arrays and using specific analysis methods. Mutation detection, polymorphism analysis, genomic library mapping, and hybridization sequencing. The prerequisite for the application of the gene chip technology is to have a large number of probe molecules of known sequence, and the number of aquatic genes that have been determined so far is very limited, which greatly limits the application of the gene chip technology in the study of functional genomics of aquatic organisms.
l. 4 Genetic marker restriction endonuclease (RFLP) technology, random amplified polymorphism (RAPD) technology, DNA fingerprinting technology, amplified fragment length polymorphism (AFPL) and other molecular markers for gene diversity analysis. Provides basis for germplasm identification and genetic breeding. Knox et al. (1991) used plasmid DNA restriction fragment length polymorphisms to discriminate between the populations of migratory and terrestrial Atlantic salmon and the wild populations of Norway and Scotland. Moran (1996) used the variation of plasmid DNA to distinguish between artificial hatching and wild locust populations in the Spanish rivers and found the relationship between population size and plasmid DNA variation. Carter (1991) et al. and Gross (1994) used DNA fingerprinting techniques to identify different groups of female genital tilapia and the different populations of smallmouth bass. Sun Xiaowen et al. (2000) combined RAPD technology with SSLP (Simple Sequence Length Polymorphism) technology to initially establish genetic linkage maps of common carp. Wang Zhiyong et al. (2002) used AFLP technology to study the genetic variation and divergence of the true sea bream population in coastal China.
2 Cell and genomic technologies
2. l Polyploid induction usually involves two groups of plant and animal cell genomes. Animals with more than 2 sets of chromosomes formed by natural environmental factors or artificial special treatment are called polyploids. The main purpose of artificial induction is to use triploids. Triploids have the characteristics of fast growth, high net meat rate, good meat quality, and long life cycle; the triploid also contributes to population control and has high resistance to disease. And stress resistance. Valenti (1975) cold-processed the fertilized eggs of Australian tilapia, induced 75% of polyploids and hatched as high as 90%. Hong Yunhan (1990) heat-treated the eggs before the first cleavage and obtained 56.3% of tetraploids. Myers (1991) compared two types of triploid hard-headed pupae obtained from normal fertilized eggs by heat treatment and from tetraploid and diploid hybrids, and found that the former had poor growth in the early stages, and the latter had high survival rate but fertilization rate. Lower. Guo et al. (1996) used diploid and tetraploid Pacific oysters to produce 100% triploid oysters, which individuals were 13% to 51% larger than normal diploids. The results of nearly 4 months of culture in the estuary indicate that the triploid adductor muscle is 73% heavier than the control and the average body tissue wet weight is 36% higher. Most of the triploid shellfish are immature during the summer, and the weight and glycogen content of the closed shells are significantly higher in the growth period than in the control group.
2.2 Gynecological Development Gynogenetic development refers to the fact that the egg requires the activation of sperm but the sperm does not provide a parthenogenetic manner of genetic material. In nature, there is a parthenogenetic aquatic animal, artificial induction means can also make unfertilized eggs for gynogenetic reproduction, but the majority of the resulting individuals are haploid and hard to survive, and need to be followed by a special treatment of diploidization. In addition to speeding up the establishment of breeding lines and control of sex, gynogenetic development can also allow some rare recessive alleles to appear and produce good traits, allowing dominant genes with important economic traits to become homozygous. Developmental offspring can also be used to identify inbreeding decline in fish. Jiang Yisi (1982) et al. and Wu Qingjiang (1981) had successfully used gamma rays to irradiate semen from fish and induced gynogenetic reproduction of red salmon eggs and salmon eggs. Cai Nai-er (1995) first reported the artificial induction of gynogenetic development in Chinese shrimps using a four-step induction method similar to that used to study artificially induced gynogenetic development in fish. Three batches of gynogenetic individuals were bred, but large-scale There are still some difficulties in production. Li Shengzhong et al. (1997) induced the development of diploid gynoecium in rainbow trout by heat shock, and all three experiments were successful. In order to limit the reproduction of transplanted grass carp and silver carp, the spermatozoa treated with ultraviolet rays induce the gynogenesis of ferret eggs, and then undergo temperature shock treatment to obtain gynogenetic fishes, but also fertilization rate and hatching rate. Low problem.
2.3 Nuclear Transplantation Cell nuclear transfer technology is the transfer of a cell nucleus to another enucleated unfertilized egg or intracellular biotechnology. The famous Chinese biologist Tong Dijie and others took the lead in carrying out nuclear transfer of the same species of fish in goldfish and fish. Subsequently, nuclear transfer between different subfamilies was performed between these two species of fish, and a variety of transplanted nuclear-matrix fish were obtained. Yan et al. (1985) successfully performed nuclear transfer between two different subfamily species in the bonefish, namely transplanting the nuclei of the grass carp to the cytoplasm of the crustacean, obtaining a nuclear hybrid fish. Its appearance characteristics are similar to that of grass carp, indicating that the expression of genetic information is mainly affected by the nuclei of the transferred fish, and the growth rate of nucleocytoplasmic hybrid fish is slightly faster than that of grass carp, but much faster than that of short-nosed pupae. The sperm produced by it can be The grass carp eggs are backcrossed and the offspring are obtained. Lin et al. (1996) transplanted somatic, carp, and Nile tilapia somatic nuclei into the mature enucleated eggs of carps to obtain embryos and juveniles at different stages of development. At present, China is a world leader in the theoretical research and practical application of fish cell nuclear transfer technology.
2.4 Cell culture technology Cell culture technology is a technique that separates, cultures, induces, or reproduces cells of organisms. At present, this technology has been widely used in the culture of fish cells, pearl shell mantle epithelial cells, and algae cells. Aquaculture animal cell culture starts with fish, and it is systematically developed with the study of fish cell culture. There are also many established fish cell lines. These cell lines have many uses. They can replace live animals for virus research, can be used for genetic studies such as chromosome production, are used for making cell vaccines, are sources of donor cell nuclei for nuclear transfer and breeding research, and are used for toxicity and water quality monitoring. The indicator organism (cells are more sensitive to toxic substances than fish) and so on. Zhang Nianci et al. (1981) established a diploid ZC-7901 cell line in the snail's snout end tissue. Chen Minrong et al established the aneuploid cell line CAB-80 in 1985. Zuo Wengong et al established the CIK cell line of grass carp kidney in 1986. , Tong Sangliang et al established the rainbow trout macrophage cell line in 1989. Compared with fish, studies on cell culture of shellfish, shrimps and other aquatic animals have been less reported at home and abroad. For example, Hanson et al. established a freshwater snail embryo cell line in 1976, and Xu Yali recently established PMO cell line of P. monodon.
3 Disease Diagnosis Technology
3. Monoclonal antibody technology Monoclonal antibody technology is a technology developed by K and M in 1975 that utilizes hybridoma cells to produce a large number of specific antibodies directed against an antigenic determinant. Compared with conventional serum antibodies, monoclonal antibodies have strong specificity, can recognize single antigenic determinants, and are easy to prepare. They can keep cell lines repeatedly and obtain the same antibodies. Therefore, monoclonal antibodies are widely used in the detection of aquaculture pathogens. For example, it is applied to diagnosing scallops of scallops, diagnosis of lymphocytes combined with fish and oysters, and detection of Vibrio philippinarum P1 caused by the brown ringworm disease in the Philippines. In addition, in recent years, using this technology, monoclonal antibodies against exotoxicity to the hydrophila of M. viridis have been prepared.
3.2 Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (EL1SA) is a technique that combines the antigen-host immune response with the enzyme's catalytic reaction. The basic principle is that the antigen or antibody is adsorbed on the solid phase carrier, and then is combined with the enzyme-labeled antigen or antibody, and the substrate is hydrolyzed and colored under the participation of an appropriate substrate to show the antigen and antibody specificity through the presented color change. The existence of the reaction. ELISA has the characteristics of high sensitivity, strong specificity, and rapid response, and the results can be quantified. It can also be used for localization analysis of antigens, antibodies, and antigen-antibody complexes. ELISA has been used in the detection of IPNV, VHSV, CCV, PERV, SVCV, GCHV, and IHNV. With the continuous updating and improvement of technical methods, especially the application of monoclonal antibodies, the detection sensitivity and heterosexuality are greatly improved (Ristow et al., 1991). Davis (1994) combined with cell culture technology to detect IPNV, greatly enhanced the detection of specificity, sensitivity and objectivity.
3.3 Nucleic Acid Hybridization Techniques Nucleic acid hybridization techniques are the use of specific labeled DNA or RNA probes to hybridize with target nucleotide sequences complementary to probes in pathogen organisms to determine whether the host carries a class of pathogenic molecules. Biological techniques can be divided into Southern hybridization, Northern hybridization and nucleic acid in situ hybridization. This technology has advantages such as high sensitivity, high specificity, and rapid detection. In recent years, it has been favored in the detection of aquaculture virus pathogens such as shrimp viruses. Subramanian (1993) used the synthesized CDNA probe to successfully detect the dsDNA of water and Reovirus in infected cells and tissues by nucleic acid hybridization test; Lupinai et al. (1993) used RNA probes prepared by RNA-RNA imprinting. A new virus, Aqua Reovirus A, was isolated and identified by hybridization. Dopazo (1994) et al. used the prepared cDNA probes for the detection and diagnosis of IPNV. IPNV was detected from the cultured cells inoculated with virus for 4 to 8 hours.
3.4 PCR technology Polymerase chain reaction (PCR) is a method for rapid amplification of specific DNA fragments by enzymatic synthesis in vitro. The application of PCR technology in the diagnosis of animal diseases such as fish has only just begun, and it has shown its great potential and broad application prospects (Davies etaL 1994). Arakawa (1990) used PCR to detect rainbow trout IH-NV, and identified the specificity of the product with biotin-labeled oligonucleotide probes. Boyle (1991) used PCR to detect CCV DNA with a sensitivity of 0. Lpg, and successfully detected CCV carriers. Lastra (1994) applied reverse transcriptase polymerase chain reaction (RT-PCR) to detect highly purified viruses in cultured cells with a sensitivity of lpg; in addition, they also used silver salmon and salmon in tissues. IPNV is detected and amplified. For the detection of other aquatic animal viruses, as early as 1990 there was a report on the detection of nuclear polyhedrosis virus from shrimp tissues using PCR technology (Vickers, 1990). Chang et al. successfully used PCR for the first time to perform amplification of shrimp viruses. Wang et al. (1996) used PCR to detect shrimp baculovirus and obtained the expected amplification product. In addition, PCR-based techniques can also be used to detect viruses or bacteria that are enriched in shellfish enterovirus and water or aquatic organisms.
4 Immune control technology
4.1 Immunostimulants include specific and non-specific immune enhancers, but more to enhance non-specific immune function. Its main function is to display the body's defensive effect by activating its own immune function, and it has a wide range of functions and high safety. The function of the immunopotentiator is various. In the crustacean, it can activate the phagocytic cells in the hemolymph, increase the ability to phagocytose the pathogen, stimulate the production or increase of the antibacterial and bacteriolytic activity in the hemolymph, and activate the protooxygenase system. Identify signals and mediate phagocytosis. In fish, it can activate the phagocytosis of neutrophils and macrophages; activate lymphocytes to produce or secrete lymphokines to coordinate humoral and cellular immunity; stimulate the production of antibodies and the production of complement. The most commonly used immunostimulants are dextran, lipopolysaccharides, bacterial peptides, levuximab, chitin, egg products, vitamins, and hormones. Hardie et al. (1991) reported that the level of complement activity was increased in fish given large amounts of vitamin C. Atlantic salmon, after injection of yeast glucan, also showed increased complement activity (Engstad et al. 1992); the use of immunostimulants can also affect the activity of lysozyme, a hydrolase that exists in the mucus of fish. In fish, serum and macrophages, they can kill pathogenic microorganisms and protect them (Engstad et al. 1992). In addition, NK cells can also be activated by growth hormone (Kajita et al. 1992) and Kajita et al. (1990).
4.2 DNA vaccines In the prevention and treatment of aquatic animal diseases, the traditional vaccine is the use of inactivated bacterial vaccines or weakly toxic bacterial vaccines, the former often have little effect, while the latter can still cause minor infections or the risk of reverting to mutations in pathogenic strains. . DNA vaccine, also known as gene vaccine or nucleic acid vaccine, separates gene fragments responsible for translating antigenic proteins from viruses or microorganisms, connects with carriers in vitro to form recombinant DNAs, and then transfers them into host cells to express antigenic proteins in host cells. To achieve the effect of immunity. The preparation method of this vaccine is simple, low in cost, suitable for large-scale production, it can be expressed in vivo for a long time, and continuously stimulates the body's immune system, can form a multivalent vaccine, that is, a vaccine can induce immunity against multiple epitopes. Protective effects. The DNA vaccine for fish is mainly aimed at viral fish diseases. Currently used for research are hemorrhagic septic virus (VHSV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), and salmonid virus ( EVA, EVEX), grass carp reovirus (FRV), scorpion virus and other vaccines. Lorenzen (1999) constructed a DNA vaccine against the VHSV G protein that induced 70% of rainbow trout to produce a higher level of immunoprotection. Traxler et al. (1999) used a naked DNA encoding the IHNV G protein (PCMV4-G) to immunize salmon. After 8 weeks, they were challenged with the IHNV virus and the protection rate reached 40% to 100%. Of course, as a new generation of vaccines, DNA vaccines are still incomplete. The most interesting issue is the safety issue. People are worried that DNA vaccines will be integrated into the chromosomes of the host and cause insertion mutations. However, they have not yet been found in experiments. this phenomenon.
4.3 Antisense technology Antisense technology is the general term for antisense nucleic acid technology and ribozyme technology developed in the past more than a decade. It can be used to specifically block the function of the virus. Antisense nucleic acids can be classified into antisense RNA technology and antisense DNA technology, but generally referred to as antisense RNA technology. Antisense RNAs are RNAs and their derivatives that are synthesized using the sense strand of DNA as a template. They can block their functions by complementary binding to the corresponding mRNA. In 1985, for the first time, the antisense nucleic acid and expression vector of the enzyme gene or structural gene necessary for virus survival were connected and transferred into the cell or the antisense nucleic acid was directly injected into the cell. The discoverer had an inhibitory effect on the virus. At present, the use of artificially synthesized antisense RNA has successfully inhibited the expression of a variety of viral genes in prokaryotic and eukaryotic cells. Ribozymes are catalytic RNAs that recognize each other by base pairing with the target RNA. It binds and catalyzes the hydrolysis of target RNA, so it has both antisense RNA and sequence-specific cleavage of RNA. Experiments have shown that ribozymes can also inhibit the replication and expression of viruses, and that the transferred genes can be passaged. Numerous studies have shown that ribozymes can be used to cleave viral target RNAs. However, there is no report on the successful application of antisense technology for the treatment of aquaculture plant and animal viral diseases. However, as a highly specific detection technology, antisense technology has great potential for development.
5 Environmental bioremediation technology The deterioration of the aquatic environment has caused great threats to fisheries. The outbreak of diseases is mainly caused by environmental pollution. Environmental bioremediation technology is a new research and development field. The use of bioremediation technology in foreign countries to improve the aquatic environment has made new progress. The so-called environmental bioremediation technology is the use of biological agents to remove hazardous waste in the aquatic environment, the application of microbial mechanisms, degradation of a variety of pollutants, improve the aquatic environment. Bioremediation is an environmental biotechnology that is more extensive than biodegradation and focuses on biodegradation. The method includes using living organisms or their production products to degrade pollutants, reduce toxicity or convert them into non-toxic products, enrich and fix toxic substances (including heavy metals, etc.), and large-scale bioremediation also includes ecological regulation in ecosystems, etc. . This kind of biotechnology can be applied to large-scale aquaculture and industrial aquaculture, oil pollution, heavy metal pollution, urban sewage and other marine waste (water) treatment. The dynamic mechanism of microbial reaction to the environment, the biochemical mechanism of degradation process, the symbiotic relationship and mutual benefit mechanism between biosensors, marine microorganisms and other organisms, and the research on the separation and purification of anti-adhesive substances need to be further studied. . Researches that have been conducted include: Studying the ability of heavy metal-sulfur protein gene algae to adsorb heavy metals in seawater environments; The feasibility and application potential of five oil-degrading microorganisms in repairing water environments contaminated by five oils; Marine magnetic bacteria in removal and Potential applications for recovery of heavy metals in seawater environments; removal of nitrogen from wastewater from fish farms using rod-shaped bacteria; microalgae used as marine feed diets screened by molecular techniques; cold-resistant decane-degrading bacteria were isolated and studied in marine environments Aromatized hydrocarbon microbial degradation technology.
6 Conclusion In conclusion, aquaculture is an applied subject, and its development does not only depend on the technology of the subject, but more importantly, it requires comprehensive biotechnology, including: genetics, nutrition, pathology, endocrinology, and bioengineering. When solving the key technical issues of a certain breeding object, they all involve high-tech integrated biotechnology. The application of all these biotechnologies will undoubtedly have a profound and profound epoch-making significance for the development of fisheries.
Author: Zhao Hongxia Zhan Zi-Rong Xu Yong
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