Identification of Genes Directly Involved in Shell Formation and Their Functions in Pearl Oyster, Pinctada fucata

Abstract

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the ‘aragonitic line’. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P.fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the ‘aragonitic line’, and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.

Novel Basic Protein, PfN23, Functions as Key Macromolecule during Nacre Formation

“…Isolation and Bioinformatic Analysis of PfN23-Based on the expression sequence tag in the suppression subtractive hybridization library of the D-shape stages (33) in P. fucata, we obtained a 1220-bp transcript including a 5Ј-untranslated region of 77 bp, an open reading frame of 702 bp encoding a deduced 233 amino acid protein, and a 3Ј-untranslated region of 518 bp (GenBank TM accession number JN995665). The deduced mature protein had a calculated molecular mass of 23.4 kDa, and it was referred to as PfN23.…”

“…The gene was expressed during the D-shape stage of development (33), so the function of PfN23 was clarified using morpholinos. Two translational-blocking morpholinos were designed to block the translation of PfN23 (Fig.…”

“…In this study, a serine-rich basic protein, PfN23, was cloned from a previously constructed suppression subtractive hybridization library (33). A combination of genetic and biochemical analyses showed that PfN23 was found in the EDTA-soluble B, activities of peptides.…”

“…Three suppression subtractive hybridization libraries were previously constructed to isolate genes expressed during important developmental stages in shell formation (33). We retrieved expression sequence tags from these libraries to identify genes involved in nacre formation.…”

A History of the Cultured Pearl Industry

“…Parallel studies in the field of life science using genetic research have recently made rapid progress. In pearl research, genome analysis is also making rapid progress (Shen and Morse, 1997;Kono et al, 2000;Zhang and Zhang, 2003;Wang et al, 2008Wang et al, , 2009, and there have been many significant studies in the field of biomineralization, such as the elucidation of the nacre-forming mechanism (Suzuki et al, 2009;Jackson et al, 2010;Joubert et al, 2010;Kinoshita et al, 2011;Fang et al, 2011;Gardner et al, 2011;Montagnani et al, 2011;Isowa et al, 2012;Marie et al, 2012;Wang et al, 2012). An important milepost was reached in 2012 when, in a world first, a group including the Okinawa Institute of Science and Technology Graduate University and others performed draft genome sequencing of the Japanese Akoya oyster (Takeuchi et al, 2012).…”