CONSTRUCTING A TRANSGENIC ZEBRAFISH WHICH EXPRESSES L-GULONO-GAMMA-LACTONE OXIDASE, IN ATTEMPT TO REVOKE VITAMIN C BIOSYNTHESIS AND BOOST THE TELEOST GROWTH

The  L-Gulono-γ-lactone oxidase (GULO ) is the enzyme which catalyzes the last enzymatic reaction of vitamin C biosynthesis in animals. GULO catalyze the oxidation of L-gulonolactone into ascorbic acid. Therefore, this key enzyme plays a significant role in vitamin C biosynthesis. The animal kingdom has an ability to synthesis vitamin C in their body except for some exceptions. Some vertebrates including guinea pigs, some bat species, certain passerine birds, anthropoid primates and teleost fish have lost the ability of vitamin C biosynthesis due to the lack of functional GULO gene in their genome. Throughout the vertebrate evolution, GULO activity has fluctuated, and thus far, it has been a paradox in science. The animals which have lost the synthesis of vitamin C synthesis ability should consume the vitamin C by their diet, and vitamin C deficiency would lead to severe conditions such as scurvy, impaired wound healing, reduction of growth and immunity.

Zebrafish also have lost the GULO entirely from their genome and should consume dietary vitamin C. Our study was focused on expressing the lost GULO gene in zebrafish and examining the growth difference with the wild type. In this study, we isolated a characterized GULO (Sgulo) gene from Cloudy catshark (Scyliorhinus torazame ) and constructed an expression vector which consists b-actin promoter, Sgulo, and mcherry reporter using Tol2 based gateway cloning. The expression clone and the transposase mRNA were co-micro injected to the one-cell stage zebrafish embryo. F0 generation was selected after 24-hour post-injection by the fluorescent microscopy. Subsequently, the F1 and F2 generations were made by crossing with the wild type and F1 fish.

The Transgenic (Tg ) and the wildtype embryos were nurtured in a controlled environment and provided the same diet for three months to measure the growth (body weight) and procollagen expression . In this research, we were able to integrate Sgulo into the zebrafish genome and construct a transgenic zebrafish which express a foreign GULO gene. As results indicate the expression in Tg zebrafish shows a significant growth increment compared to wildtype fish. Collectively these results suggest us the possible vitamin C biosynthesis in Tg(b-actin:Sgulo:mcherry) fish,  and  it opens the  potentials to conduct further studies.