Guiju Huang

Highly expressed EGFR in pearl sac may facilitate the pearl formation in the pearl oyster, Pinctada fucata.

Epidermal growth factor receptor (EGFR) plays an important role in cell growth, proliferation, differentiation and migration. Yet whether it functions in pearl formation or not is not reported. In this study, EGFR was cloned from the pearl oyster Pinctada fucata (named as Pf-EGFR) and its expression profiles were investigated. The cDNA was 2156bp long with an ORF of 1017bp encoding 338 amino acid residues. The deduced polypeptide contained an L domain and a cysteine-rich domain, consistent with the characteristics of ErbB family. The sequence of Pf-EGFR shared 22.78-56.71% identity with other EGFRs. The genomic sequence of Pf-EGFR consisted of six exons and five introns, being 5190bp in total length, and expressed in all the tested tissues with a higher expression level in the pearl sac (P<0.05). In situ hybridization showed that Pf-EGFR was specifically expressed on both the inner side of the outer fold and the outer side of the inner fold of the mantle, as well as on the whole pearl sac including the connective tissues. In addition, Pf-EGFR was markedly increased at larval metamorphosis, significantly higher than other development periods (P<0.05). These results indicated that the Pf-EGFR may facilitate pearl formation as well as larval metamorphosis.

Differential Gene Expression during Larval Metamorphic Development in the Pearl Oyster, Pinctada fucata, Based on Transcriptome Analysis

P. fucata experiences a series of transformations in appearance, from swimming larvae to sessile juveniles, during which significant changes in gene expression likely occur. Thus, P. fucata could be an ideal model in which to study the molecular mechanisms of larval metamorphosis during development in invertebrates. To study the molecular driving force behind metamorphic development in larvae of P. fucata, transcriptomes of five larval stages (trochophore, D-shape, umbonal, eyespots, and spats) were sequenced using an Illumina HiSeq™ 2000 system and assembled and characterized with the transcripts of six tissues. As a result, a total of 174,126 unique transcripts were assembled and 60,999 were annotated. The number of unigenes varied among the five larval stages. Expression profiles were distinctly different between trochophore, D-shape, umbonal, eyespots, and spats larvae. As a result, 29 expression trends were sorted, of which eight were significant. Among others, 80 development-related, differentially expressed unigenes (DEGs) were identified, of which the majority were homeobox-containing genes. Most DEGs occurred among trochophore, D-shaped, and UES (umbonal, eyespots, and spats) larvae as verified by qPCR. Principal component analysis (PCA) also revealed significant differences in expression among trochophore, D-shaped, and UES larvae with ten transcripts identified but no matching annotations.

Characterization of transcriptome and identification of biomineralization genes in winged pearl oyster (Pteria penguin) mantle tissue

The winged pearl oyster Pteria penguin is a commercially important marine pearl oyster species, with pearls that are quite different from those of other pearl oysters. Among such species, mantle tissue is the main organ responsible for shell and pearl formation, a biomineralization process that is regulated by a series of genes, most of which remain unknown. In this study, we sequenced and characterized the transcriptome of P. penguin mantle tissue using the HiSeq 2000 sequencing platform. A total of 93,204 unique transcripts were assembled from 51,580,076 quality reads, with a mean length of 608bp, and 40,974 unigenes were annotated. The sequence data enabled the identification of 79,702 potential single nucleotide polymorphism loci and 4345 putative simple sequence repeat loci. A total of 71 unique transcripts were identified homologous to known biomineralization genes, including mantle gene, nacrein, pearlin, pif, chitinase, and shematrin, of which only 3 were previously reported in P. penguin. qPCR analysis indicated that 10 randomly selected biomineralization genes were much more highly expressed in mantle tissue than in the other tissues. In addition, 30 unique sequences were identified as highly expressed, with FPKM values of >3000, and most of these were biomineralization-related genes, including shematrin family genes, a jacalin-related lectin synthesis gene, calponin-2, and paramyosin. These findings will be useful for future studies of biomineralization in P. penguin, as well as in other Pteria species.

Identification of twenty novel polymorphic microsatellite DNA markers from transcripts of the pearl oyster Pinctada fucata using next-generation sequencing approach

The pearl oyster, Pinctada fucata (Gould 1850) is a commercially important marine shellfish cultured for producing saltwater pearls mainly in China and Japan (Yu and Chu 2006). It is common in most areas of tropical and subtropical oceans and seas in the Pacific and Indian regions. In 1965, this specie was successfully propagated and reared under artificial conditions in Guangxi province in southern China and expanded rapidly to the neighbouring Guangdong and Hainan provinces subsequently (Meng et al. 1996). The pearls produced by the animals are referred to as ‘South China Sea Pearl’, accounting for over 90% production of the total marine pearls produced in China. For the last few years, some traits of P. fucata appear to have degenerated, due to overfishing, coastal water pollution and artificial propagation of years without recording their background, which hampered the advance of the pearl industry. Genetic improvement and culture of elite varieties should be carried out to prevent slowdown of the growth rate because of inbreeding depression and deterioration of the pearl quality. Microsatellite DNA markers have proved to be a useful tool for evaluating the level of genetic variation of natural populations in many fishery animals because of the high polymorphism, abundance, neutrality and codominance (Liu and Cordes 2004). Polymorphicmicrosatellite loci have been frequently applied in the analysis of genetic diversity of populations. In spite of some microsatellite loci in this species were reported (Tong et al. 2007; Kuang et al. 2009; Shi et al. 2009; Qu et al. 2010; You et al. 2012; Wu et al.

The complete mitochondrial genome of the hybrid grouperEpinephelus fuscoguttatus(♀)×Epinephelus lanceolatus(♂)

Abstract In this study, we reported the complete mitochondrial DNA sequence of the hybrid grouper Epinephelus fuscoguttatus (♀)×Epinephelus lanceolatus (♂). The full-length of the mitochondrial genome consisted of a 16,644 bp fragment, with the base composition of A (29.21%), C (26.84%), G (15.65%) and T (28.29%). It contained 2 rRNA genes, 13 protein-coding genes, 22 tRNA genes, and a major non-coding control region (D-loop region). The composition and order of these genes were identical to most other vertebrates. All the protein initiation codons were ATG, except that COX1 began with GTG and ATP-6 was not determined. The complete mitogenome of the hybrid E. fuscoguttatus (♀)×E. lanceolatus (♂) provided an important data set for the study in genetic mechanism of the hybridization.

The complete nucleotide sequence of Malabar grouper (Epinephelus malabaricus) mitochondrial genome.

Abstract In this study, we reported the complete mitochondrial DNA sequence of the Epinephelus malabaricus. The full-length of the mitochondrial genome consisted of a 16,423 bp fragment, with the base composition of A (28.70%), T (26.55%), G (15.92%) and C (28.83%). It contained 2 rRNA genes, 13 protein-coding genes, 22 tRNA genes and a major non-coding control region (D-loop region). The composition and order of these genes were identical to most of other vertebrates. All the protein initiation codons were ATG, except that COX1 began with GTG, ATP-6 and ND6 was not determined, respectively. The complete mitogenome of the Epinephelus malabaricus provided an important data set for the study in genetic mechanism of the hybridization.

Expression, Purification, and Functional Activity of Shell Matrix Protein Pearlin from the Pearl Oyster Pinctada fucata

ABSTRACT Nacre, also known as mother of pearl, contains factors that can promote osteogenesis. In this study, pearlin, a protein from the mantle tissue of the pearl oyster Pinctada fucata, was expressed and purified, and its functional activity analyzed using two mineralogenic cell lines, MRC-5 (fibroblasts) and MC3T3-E1 (preosteoblasts). The open reading frame of pearlin was subcloned into the expression vector pET32a(+) and used to transform Escherichia coli BL21 (DE3) strain. The expression of the recombinant protein (molecular weight: 34.19 kDa) was induced by isopropyl-β-D-thiogalactopyranoside in the form of inclusion bodies which were solubilized in 8 M urea. The recombinant protein was renatured by stepwise dialysis, purified by Ni-NTA affinity chromatography, and analyzed for the effects on cell proliferation by the MTT assay and osteoblastic differentiation by alkaline phosphatase (ALP) activity. The recombinant pearlin promoted proliferation ofMRC-5 andMC3T3-E1 cells at 10 µg/mL and increased their ALP activity at 2.5 µg/mL and 5 µg/mL, respectively. The present study showed that the recombinant pearlin exerted functional effects similar to those of the natural protein, laying a foundation for large-scale production of pearlin in a prokaryotic system.