Novel Genes Participating in the Formation of Prismatic and Nacreous Layers in the Pearl Oyster as Revealed by Their Tissue Distribution and RNA Interference Knockdown

Daisuke Funabara,Kikuhiko Okamoto,Kiyohito Nagai,Shigeharu Kinoshita,Shugo Watabe,Yuki Osakabe,Satoshi Kanoh,Fumito Ohmori,Hiroki Koyama,Ayaka Ota,Shuichi Asakawa,Kaoru Maeyama,Saeri Mizutani,Daniel John Jackson

doi:10.1371/journal.pone.0084706

Daisuke Funabara, Kikuhiko Okamoto + Show 12 more

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https://doi.org/10.1371/journal.pone.0084706

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Abstract

In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411 (MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers.

Highlights

The shells of pearl oysters comprise two layers, termed the prismatic and nacreous layers, formed by secreting organic materials from epithelial cells in the mantle [1]
We identified 12 novel genes that are responsible for shell formation, using RNA interference (RNAi) methods, Reverse transcription (RT)-PCR and in situ hybridization
The fact that the RNAi knockdown of each gene resulted in abnormal shell formation phenotypes confirmed that our previous screening of gene candidates for shell formation based on the expression levels of genes in the expressed sequence tag (EST) data [8] was successful

Summary

Introduction

The shells of pearl oysters comprise two layers, termed the prismatic and nacreous layers, formed by secreting organic materials from epithelial cells in the mantle [1]. The prismatic layer is thought to be formed by the secretion of materials from the edge region of the mantle (the mantle edge) and the nacreous layer is formed from the inner part of the mantle (the pallium). The main components of shells are calcium carbonate and organic substances, such as chitin and various proteins. Crystal structures are different between the two layers: calcite is present in the prismatic layer and aragonite is found in the nacreous layer. The accumulation of calcium carbonate as calcite and aragonite crystals was thought to be regulated by proteins secreted from the mantle. The first protein to be isolated from the nacreous layer, exists in both the nacreous and prismatic layers and, is thought to be involved in the formation of the whole shell [2,3,4]

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