2011 Annual Report
1a.Objectives (from AD-416)
1: Develop homozygous clonal lines of rainbow trout resistant to bacterial and viral pathogens from previously developed transgenic trout. Test disease resistance in outcrosses of different genetic background to evaluate protection.
•1.a. Develop homozygous clonal lines of disease resistant rainbow trout.
•1.b. Identify the expression profiles of genes related to innate immunity and adaptive immunity pathways in homozygous clonal lines of transgenic fish resistant to bacterial, parasitic, and viral infections.
2: Assess whether transgenic rainbow trout resistant to bacterial and viral pathogens are also resistant to infection by parasites (e.g., Ceratomyxa shasta and Myxbolus cerebralis).
3: Develop transgenic rainbow trout capable of accumulating astaxanthin in the flesh and identifying and characterizing factors affecting this trait.
1b.Approach (from AD-416)
With the rapid growth of the human population and the increasing consumption of seafood for health considerations, the world demand for seafood products is mounting rapidly. To this end many countries have turned their attention to various forms of intensive aquaculture production. However, intensive aquaculture has brought about several negative impacts such as increasing disease outbreak incidences and decreasing the quality and nutritional value of fish flesh, resulting in significant economic losses. The application of modern molecular biological technologies including genomics and proteomics will enhance efforts to resolve these problems. We have developed transgenic trout carrying disease resistance genes known as cecropins: these fish exhibit resistance to infection by pathogens affecting aquaculture production including Aeromonas salmonicida and infectious hepatic necrosis virus (IHNV). Using disease resistant transgenic fish and new strains capable of accumulating higher levels of astaxanthin in the flesh as experimental models, we propose to identify and characterize specific pathways and genes leading to increased disease resistance and improved flesh color and nutritional quality. Genes identified from these studies will be characterized for their potential in developing superior broodstock through selective breeding.
Efforts to generate transgenic models to study biochemical pathways affecting traits of interest fall under NP106 Components 2, 4, and 5. Progress under Objectives 1 and 2 included breeding the following clonal lines of cecropin P1 transgenic fish: S7-342-F695, S8-505-G231, S7-375-F180, S9-746-F509, S9-659-F073, S9-638-F297, U6-768-G410, A12-944, and A13-831). The disease resistance characteristic of each clonal line has also been confirmed by challenge studies against Aeromonas salmonicida and IHNV. These are all male fish and the sperm samples collected from multiple individuals of each line were cryo-preseved. Tissues of kidney, liver, and spleen from several clonal lines have been collected and RNA extracted. DNA microarray analysis is under way to determine the profile of gene expression of innate immunity and adapted immunity related genes. Heterozygous fish from several clonal lines carrying pig cecropin P1 gene have been established. Challenge studies with parasite Ceratomyxa shasta will be conducted in the spring 2012. To date nine clonal lines of disease resistance transgenic trout have been produced. The sperm samples of these fish have been cryo-preserved.
Progress towards Objective 3 (Component.
5)included attempts to create transgenic fish capable of accumulating astaxanthin in the flesh. Although crtW and crtZ genes obtained from our collaborator from South Korea were cloned into transgene vector and transformed into CHSE cells, synthesis of astaxanthin was not detected. After many nucleotide sequence determination attempts, it was concluded that both crtW and crtZ genes were incorrect. For this reason, we were not able to carry out gene transfer into rainbow trout. Now we have re-isolated crtW and crtZ genes from Paracocus haeundaensis. A di-cistronic transgene containing crtW and crtZ genes has been constructed. This di-cistronic transgene will be introduced into rainbow trout in the new fiscal year.