Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii

Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460 bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a “QPS” and an invariant “WND” motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.     

A Novel Matrix Protein Hic31 from the Prismatic Layer of Hyriopsis Cumingii Displays a Collagen-Like Structure

In this study, we clone and characterize a novel matrix protein, hic31, from the mantle of Hyriopsis cumingii. The amino acid composition of hic31 consists of a high proportion of Glycine residues (26.67%). Tissue expression detection by RT-PCR indicates that hic31 is expressed specifically at the mantle edge. In situ hybridization results reveals strong signals from the dorsal epithelial cells of the outer fold at the mantle edge, and weak signals from inner epithelial cells of the same fold, indicating that hic31 is a prismatic-layer matrix protein. Although BLASTP results identify no shared homology with other shell-matrix proteins or any other known proteins, the hic31 tertiary structure is similar to that of collagen I, alpha 1 and alpha 2. It has been well proved that collagen forms the basic organic frameworks in way of collagen fibrils and minerals present within or outside of these fibrils. Therefore, hic31 might be a framework-matrix protein involved in the prismatic-layer biomineralization. Besides, the gene expression of hic31 increase in the early stages of pearl sac development, indicating that hic31 may play important roles in biomineralization of the pearl prismatic layer.     

三角帆蚌Hc-GSK3β基因鉴定及内壳色性状相关SNP筛选

为了探究三角帆蚌(Hyriopsis cumingii)糖原合成激酶-3β(GSK3β)基因对壳色的影响,研究采用RACE技术获得Hc-GSK3β基因cDNA全长1867 bp,其中包含1261 bp的ORF区编码420个氨基酸, ORF中含有一个S＿TKc结构域,该结构域序列高度保守。组织差异表达分析发现Hc-GSK3β基因在紫色蚌鳃、斧足、内脏团和边缘膜组织中表达量高于白色蚌的表达量(P<0.05),且在斧足和边缘膜表达差异水平达到极显著(P<0.01),而在紫色蚌闭壳肌组织中表达量显著低于白色蚌(P<0.05)。原位杂交(ISH)实验结果显示在三角帆蚌外套膜的外褶、中褶、內褶、背膜区和腹膜区均有阳性信号产生,且在外褶的信号表达较强烈。该基因经重测序比较,共鉴定出6个SNP位点,其中在C+185A位点的CA基因型在紫色蚌的分布频率显著高于白色三角帆蚌(P<0.05);在紫色蚌中, T+341G位点TT基因型三角帆蚌内壳颜色参数b值显著低于TG基因型(P<0.05)。研究表明, Hc-GSK3β基因参与了三角帆蚌壳色形成,筛选的SNP标记可用于三角帆蚌壳...     

Analysis of a silkworm F1 hybrid with yellow cocoon generated by crossing two white-cocoon strains: Further evidences for the roles of Cameo2 and CBP in formation of yellow cocoon

In this report, we examined the gene expression related to carotenoid transport for a silkworm F₁ hybrid with yellow cocoon generated by crossing two white-cocoon strains, Qiubai and 12-260. Our results showed that, in Qiubai, Cameo2, a transmembrane protein gene belonging to the CD36 family genes, was expressed normally in the silk gland, but no intact carotenoid-binding protein (CBP) mRNA (only the truncated CBP mRNA) was detected in the midgut. In 12-260, we detected the intact CBP mRNA expression in the midgut, but no Cameo2 expression in the silk gland. Regarding the F₁ hybrid from crossing Qiubai and 12-260, both Cameo2 and intact CBP mRNA expressed normally in the silk gland and midgut. HPLC detection confirmed that in the F₁ hybrid the carotenoids could be absorbed from dietary mulberry leaves through the midgut and transferred to silk gland via the hemolymph, which eventually colored cocoons into yellow. We also identified four CBP mRNA isoforms expressed in the midgut of the F₁ hybrid, subsequently named as variants 5–8. Our results provide further evidences for the roles of Cameo2 and CBP in the formation of yellow cocoon of silkworm.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Molecular characterization of Hydra acetylcholinesterase and its catalytic activity

A full-length cDNA encoding an acetylcholinesterase (AChE) from Hydra magnipapillata was isolated. All of the important aromatic residues that line a catalytic gorge in cholinesterases of other species were conserved, but the sequences of peripheral anionic and choline binding sites were not. Hydra AChE, expressed in Xenopus oocytes, showed AChE activity. The gene was expressed in both ectodermal and endodermal epithelial cells except for the tentacles and basal disk. AChE gene expression was not detected in the regenerating tips in either the head or the foot, indicating that regeneration is controlled by the non-neuronal cholinergic system in Hydra.     

水稻金属耐受蛋白基因OsMTP2生物信息学及表达分析

为了解水稻(Oryza sativa)响应重金属污染及对重金属积累的机制,利用生物信息学手段对水稻金属耐受蛋白(metal tolerance protein, MTP) OsMTP2的结构特征进行预测,并分析OsMTP2基因的表达特征。结果表明,该蛋白由415个氨基酸组成,具有很强的疏水性。mRNA原位杂交结果表明,OsMTP2在叶片输导组织中有较强的表达,但在根、花药中没有检测到明显的杂交信号。这说明水稻金属耐受蛋白基因OsMTP2主要在输导组织中表达,并在金属离子运输中发挥重要作用。     

Molecular characterization and differential expression of the myostatin gene in Coilia nasus

Estuarine tapertail anchovy (Coilia nasus, junior synonym C. ectenes) is a widely distributed and commercially important aquaculture species, although its growth in aquaculture settings is so slow as to pose a serious practical problem. In order to understand the molecular mechanisms of growth, we cloned the myostatin gene in C. nasus (CnMSTN) by homologous cloning methods. Its full-length cDNA is 2252 bp, with a 1125-bp open reading frame (ORF) that encodes a 374-amino acid protein. The CnMSTN protein is predicted to contain domains typical of MSTN, including a TGFb-propeptide domain and a TGFB domain. Gene expression patterns were detected by RT-qPCR. CnMSTN is expressed strongly in the muscle and brain, and comparatively lower in the gills, liver, spleen, intestine, trunk kidney and head kidney. The effects of stress on the muscle and brain MSTN levels were evaluated by RT-qPCR. CnMSTN in the muscle was positively regulated by loading and transport stress, but brain CnMSTN expression was not affected. We found NaCl could reduce the death rate caused by loading and transporting stress, and in this group, CnMSTN mRNA expression in the muscle revealed increased, but decreased in the brain. Further, in the fasting experiment, the CnMSTN mRNA revealed decrease in the muscle, on the contrary, it showed increase in the brain. Selection upon variants of the MSTN gene has shown great potential in breeding work for mammals, and our results provide the basic knowledge for breeding of C. nasus.     

Molecular characterization,sexually dimorphic expression,and functional analysis of 3′-untranslated region of vasa gene in half-smooth tongue sole (Cynoglossus semilaevis)

Vasa is a highly conserved ATP-dependent RNA helicase expressed mainly in germ cells. The vasa gene plays a crucial role in the development of germ cell lineage and has become an excellent molecular marker in identifying germ cells in teleosts. However, little is known about the structure and function of the vasa gene in flatfish. In this study, the vasa gene (Csvasa) was isolated and characterized in half-smooth tongue sole (Cynoglossus semilaevis), an economically important flatfish in China. In the obtained 6425-bp genomic sequence, 23 exons and 22 introns were identified. The Csvasa gene encodes a 663-amino acid protein, including highly conserved domains of the DEAD-box protein family. The amino acid sequence also shared a high homology with other teleosts. Csvasa expression was mainly restricted to the gonads, with little or no expression in other tissues. Real-time quantitative polymerase chain reaction analysis revealed that Csvasa expression levels decreased during embryonic and early developmental stages and increased with the primordial germ cell proliferation. A typical sexually dimorphic expression pattern of Csvasa was observed during early development and sex differentiation, suggesting that the Csvasa gene might play a differential role in the proliferation and differentiation of male and female primordial germ cells (PGCs). Csvasa mRNA expression levels in neomales were significantly lower than those in normal males and females, indicating that the Csvasa gene might be implicated in germ cell development after sex reversal by temperature treatment. In addition, medaka (Oryzias latipes) PGCs could be transiently labeled by microinjection of synthesized mRNA containing the green fluorescence protein gene and 3′-untranslated region of Csvasa, which confirmed that the Csvasa gene has the potential to be used as a visual molecular marker of germ cells and laid a foundation for manipulation of PGCs in tongue sole reproduction.     

Intestinal tube formation in Caenorhabditis elegans requires vang-1 and egl-15 signaling

Understanding how epithelial organs form during morphogenesis is a major problem in developmental biology. In the present paper, we provide a detailed analysis of vang-1, the only homolog of the planar cell polarity protein Strabismus/Van Gogh in Caenorhabditis elegans. We demonstrate that during organogenesis of the intestine, (i) VANG-1 specifically interacts with PDZ 2 domain of DLG-1 (Discs large) and becomes phosphorylated by the kinase domain of the FGF-like receptor tyrosine kinase EGL-15; (ii) VANG-1 is predominantly restrained to the cell cortex but relocates to the apical junction; and (iii) in vang-1 embryos epithelial cells of the intestine are not correctly arranged along the anterior-posterior axis. To investigate what determines the disposition of the VANG-1 protein, either truncated protein forms were expressed in the intestine or RNAi was used to remove the functions of gene products previously shown to be involved in apical junction formation. Removal of the VANG-1 PDZ binding motif “− ESAV” and depletion of dlg-1 or let-413 gene functions interferes with the localization of VANG-1. In addition, egl-15 embryos show a premature relocation of VANG-1 to the apical junction, causing defects that resemble those observed in mutant vang-1 embryos and after intestine-specific overexpression of full-length vang-1. Finally, the localization of VANG-1 depends on DSH-2, a homolog of the planar cell polarity protein Dishevelled and depletion phenocopies vang-1 and egl-15 phenotypes in the embryonic intestine.     

Seasonal patterns of metabolism and the heat shock response (HSR) in farmed mussels Mytilus galloprovincialis

Mussels (Mytilus galloprovincialis) are the main bivalves cultured in the Mediterranean Sea and in the Thermaikos gulf North of Greece. Global warming might affect these cultures since increases in the sea temperatures especially during summertime and may cause mass mortalities. Thus projections of the effects of global warming and the knowledge of the mechanisms of thermal limitation and adaptation are considered important in this context. In the present work we studied the seasonal expression of Hsp70 and Hsp90 of aquacultured mussels in nature in an effort to examine whether mussels are seasonally thermally stressed. Moreover we determined the activity of the key glycolytic enzyme PK in order to examine seasonal changes in the metabolic profile. The present data showed a biphasic seasonal pattern in the expression of Hsps in the mantle and PAM of M. galloprovincialis. The expression of inducible Hsp72 and Hsp90 increased between February and mid April, and remained constant or decreased slightly by the end of May. Hsps expression started again in early June leading to a gradual increase by mid July and remained by the end of August. The pattern of the HSR indicates increased protein turnover or protein damage in the tissues of aquacultured M. galloprovincialis between winter and summer seasons. The induction temperature is probably limited, and beyond some upper limits there is no further change in the HSR induction temperatures and the HSR sets in. PK activity in the mantle was significantly higher in winter than in spring. Early May saw a reactivation of PK to higher than spring values. Similar to mantle PK, PK activity in PAM was significantly higher in winter than in spring. A slight reactivation was observed by the end of August.