Abstract
Matrix proteins play critical roles in regulating the prismatic and nacreous layer formation in the shell. However, due to the dearth of in vivo experiments, their specific roles during shell formation are still unclear. In this study, a new method to detect the content of Sr in the nacreous layer (DCSr-NL), which can semiquantitatively measure the nacreous growth rate, has been proposed. In vitro experiments show that during in vitro crystallization, the Sr element can replace Ca partially, resulting in isomorphism. In vivo experiments show that the best labeling conditions are when the Sr/Ca in seawater is 0.3, at 24 °C, and at 4 days of culture. Although a surface morphological difference in the inner layer of nacre is seldom detected by scanning electron microscopy (SEM), knockdown of the classical gene nacrein or unknown gene NU9, combined with DCSr-NL, shows that both significantly decrease the nacreous layer formation rate. The knockdown of the classical gene Pif177 or unknown genes NU3 or MRPN affects the surface morphology and decreases the nacreous layer formation rate. In general, thanks to DCSr-NL, we can efficiently analyze the growth rate of the nacre with or without morphological changes by SEM, and it is of considerable significance for exploring the target gene's function in forming the nacre in vivo.