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.
Highlights
Induced or controlled mineralization, known as biomineralization, plays an essential role in a majority of metazoan taxa [1]
Changes of crystals and the microstructure of the shell were correlated with biomineralization-related sequences in the D–T library that were involved in amorphous calcium carbonate (ACC) formation, sequences in the U– D library were involved in aragonite formation, and sequences in the J–U library were involved in the formation of the microstructure of the nacreous and prismatic layers
Bioinformatics analysis showed that only a small number of sequences in the three libraries were with associated gene ontology (GO) terms
Summary
Induced or controlled mineralization, known as biomineralization, plays an essential role in a majority of metazoan taxa [1]. Several molluscan shell matrix proteins were separated and identified, and their distributions and functions in shell formation were extensively investigated in pioneering work on this topic [6,7,8,9,10], despite considerable efforts the detailed molecular mechanisms operating in shell biomineralization remain poorly understood. This is attributable to problems in protein separation and purification owing to a highly acidic amino acid content and protein complexation with minerals, as well as the absence of an effective experimental system [11]
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