Genetic engineering of IP6 (inositol hexaphosphate) metabolism in the Japanese medaka (Oryzias latipes).

摘要

One of the major limitations of aquaculture has been the inefficient digestion of phytate-bound phosphorus in plant-based diets. The enzyme phytase has been found to degrade phytate into a form that can be utilized by non-ruminant animals. The goal of this research was to produce transgenic Japanese medaka (Oryzias latipes) capable of utilizing phosphorus in the phytate form and reducing phosphorus excretion. In order to achieve this goal, the efficacy and functionality of several promoters were examined through cell culture techniques. The phytase gene from Aspergillus niger was cloned and used in conjunction with the functional promoters to produce four constructs for phytase expression. These constructs were initially tested in cell culture for expression and secretion of the phytase enzyme, and were later microinjected or electroporated into Japanese medaka embryos. Putative transgenic fish were tested for germ-line transmission of the transgene. Fish positive for the transgene were back-crossed to the wild type population of fish for two generations to produce the BC₂ population and reduce genetic background. BC₂ fish were used to test for transgene copy number, localization of transgene expression, and ability to utilize phytate as the only phosphorus form. Although there was much variation between individual fish, the OnMT-PHY-GH1t-OnMT-EGFP construct did produce significantly (P 0.05) heavier fish than their non-transgenic siblings when fed a test diet in which phytate served as the only phosphorus source. Transgenic fish with this construct were capable of utilizing the phosphorus from phytate. Survival of transgenic fish was up to 4-fold higher than non-transgenic for the CMV-PHY-GH1t-CMV-EGFP, OnH3-PHY-GH1t-OnH3-EGFP, and OnMT-PHY-GH1t-OnMT-EGFP constructs, indicating that poor phytate utilization by non-transgenic fish resulted in greater mortality. This data also suggested that through the degradation of phytate by fish transgenic for the phytase gene, many of the anti-nutritional factors of phytate could be overcome.