AUTHOR=Shanaka K. A. S. N. , Jung Sumi , Janson N. D. , Jayasingha J. R. P. , Madushani K. P. , Kim Myoung-Jin , Lee Jehee 

TITLE=Growth and Antioxidant-Related Effects of the Reestablished Ascorbic Acid Pathway in Zebrafish (Danio rerio) by Genomic Integration of L-Gulonolactone Oxidase From Cloudy Catshark (Scyliorhinus torazame)

JOURNAL=Frontiers in Physiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2021.685595

DOI=10.3389/fphys.2021.685595

ISSN=1664-042X

ABSTRACT=<p>Loss of <sc>L</sc>-gulonolactone oxidase (<italic>GULO</italic>), 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, <italic>sGULO</italic>, the active <italic>GULO</italic> gene from cloudy catshark (<italic>Scyliorhinus torazame</italic>) was cloned into zebrafish using the Gateway cloning method. The resulting <italic>Tg(b-actin:sGULO:mCherry)</italic> 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 <italic>Tg</italic> fish. The mCherry fluorescent protein indicated the proper integration and expression of the <italic>sGULO</italic> construct in zebrafish. The <italic>sGULO</italic> gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in <italic>Tg</italic> fish. The growth of <italic>Tg</italic> fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in <italic>Tg</italic> fish compared with wild type. Expression of the AA transporter <italic>slc23a1</italic> was significantly downregulated in <italic>Tg</italic> homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.</p>