원문정보
초록
영어
Loss of L-gulonolactone oxidase (GULO), which catalyzes the last step of the ascorbic acid (AA) biosynthesis pathway, results in a complete lack of AA in several Osteichthyes fish species, including zebrafish. In this study, sGULO, the active GULO gene from cloudy catshark (Scyliorhinus torazame) was cloned into zebrafish using the Gateway cloning method. The resulting Tg(b-actin:sGULO:mCherry) fish were analyzed for the effects of a reestablished AA pathway. Fluorescent microscopy and PCR were used to analyze the integration of the construct into the zebrafish genome. Catalytic activity of sGULO, AA production, growth-related characteristics, and gene expression were investigated to evaluate the effects of AA production in Tg fish. The mCherry fluorescent protein indicated the proper integration and expression of the sGULO construct in zebrafish. The sGULO gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in Tg fish. The growth of Tg fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in Tg fish compared with wild type. Expression of the AA transporter slc23a1 was significantly downregulated in Tg homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.
목차
INTRODUCTION
MATERIALS AND METHODS
Zebrafish Husbandry
Bioinformatics Characterization and Isolation of Cloudy Catshark sGULO
Assembling the Expression Construct
Construction of Transgenic Zebrafish
Establishment of Transgenic Zebrafish
GULO Assay and AA Quantification
Feed Preparation
Growth Assay
Gene Expression Analysis
Statistical Analysis
RESULTS
Bioinformatics Analysis
Tg(b-actin:sGULO:mCherry) F0, F1, and F2 Generations
sGULO Enzyme Activity and Endogenous AA Production
Physiological Difference
Gene Expression Analysis
DISCUSSION
CONCLUSION
REFERENCES