Cloning and characterization of a trypsin-like serine protease gene, a novel regeneration-related gene from Apostichopus japonicus.

Trypsin-like serine protease (TLS) plays an important role in many physiological processes including wound healing, phlogosis reaction, blood clotting, regeneration etc. In this paper, a 1216 bp full-length cDNA sequence of TLS including 39 bp 5' UTR and 355 bp 3'UTR coding for a theoretical 273 amino acids protein was cloned from Apostichopus japonicus by means of the RACE technique for the first time. Bioinformatic analysis revealed that the gene with a 20 residues N-terminal signal peptide and a conserved C-terminal domain belongs to the trypsin-like serine protease superfamily. His78, Asp130 and Ser223 are the principal residues of the catalytic center. In-situ hybridization (ISH) analysis revealed that the TLS gene was widely distributed in different tissues. The expression patterns during different regeneration stages of the TLS gene in the body wall, intestine and respiratory trees were investigated using real-time quantitative PCR. The results show that there was a remarkable and temporary up-regulation of TLS gene expression in the body wall within 1h and subsequent down-regulation of TLS similar to intestine and respiratory trees. With the recovery of tissues, the expression level of the TLS gene was gradually up-regulated and finally reached normal levels. TLS was regulated during different regeneration stages suggesting that TLS is important in the regeneration process of A. japonicus.

Molecular cloning, characterization and expression analysis of a C-type lectin (AJCTL) from the sea cucumber Apostichopus japonicus.

For the sea cucumber Apostichopus japonicus, a C-type lectin (AJCTL) was identified using rapid amplification of cDNA ends (RACE) PCR techniques. The full-length cDNA of AJCTL is composed of 710bp with a 618bp open reading frame (ORF) that encodes a polypeptide of 205 amino acids with a N-terminal signal peptide and a C-terminal C-type lectin domain (CTLD). The calculated molecular mass of the whole protein is 22.5kDa and its predicted isoelectric point is 5.59. AJCTL belongs to the group VII of regulatory proteins and it might function as a Ca(2+)-dependent monosaccharide binding lectin specifically and recognizing mannose-type ligands. In situ hybridization demonstrated that the expression of AJCTL was located in the body wall, longitudinal muscles, intestinum and respiratory tree. This became apparent especially in the cytoplasm of epidermal cells and granular haemocytes. Real-time PCR data suggested that AJCTL was mostly synthesized in the longitudinal muscles and intestinum and less pronounced in the respiratory tree and body wall of adults. After 12h stimulation by Vibrio harveyi, at increasing bacterial concentration gradient, the expression of AJCTL in sea cucumber increased as well. This indicated that CTL is related to an innate immune response.

Molecular cloning and its expression of trachealess gene (As-trh) during development in brine shrimp, Artemia sinica.

Basic helix-loop-helix-PAS (bHLH-PAS) family transcription factors are implicated in multiple developmental and physiological regulatory processes. Herein, a full-length cDNA encoding a bHLH-PAS domain transcription factor trachealess gene (designated as As-trh) was cloned and characterized from brine shrimp (Artemia sinica) for the first time. The full-length cDNA of As-trh was 2,698 bp with a 2,319 bp open reading frame encoding a deduced protein of 772 amino acid polypeptide with a calculated molecular mass of 86.02 kDa and an isoelectric point of 5.87. Sequence alignment revealed that As-trh had high homology with other species trh gene, including the D-trh gene in Drosophila melanogaster and Bm-trh in Bombyx mori. The early and persistent expression of As-trh in the naupliar stages by whole-mount embryonic in situ hybridization and immunohistochemistry suggest that As-trh functions very early in the salt gland and may be required continuously in this tissue. Later in development, expression of As-trh begins to decrease and disappear in salt gland of the older nauplius and appears in the thoracic epipods of the sub-adult Artemia. These results indicated that As-trh might play an important role in osmoregulatiory organ development from the larvae stages through adult stages.

Cloning and the expression pattern of Spätzle gene during embryonic development and bacterial challenge in Artemia sinica.

Spätzle gene codes for a NGF-like protein, it involves in the embryonic development and innate immune response of insects and other invertebrate. In dorsal ventral axis differentiation, proSpätzle is activated by serine endoproteases Easter and then binds to the Toll receptor in ventral axis of oocyte which initiates the ventral axis development. Besides, it could also be activated by another protease named Spätzle-processing enzyme (SPZ) to mediate Toll pathway which involves in innate immune response in fungal and Gram-positive bacterial infection of invertebrate. In this paper, a full-length cDNA of Spätzle was firstly isolated from Artemia sinica which belonged to Spätzle-4 family. The expression of Spätzle was investigated at various stages during the embryonic development of A. sinica using real-time PCR and immunohistochemistry assays. The result showed that the high expression level of Spätzle appeared at 7 and 10 days of the embryo. A gradual increased level of Spätzle transcript occurred after being challenged with Gram-positive bacteria. Immunohistochemistry assay showed that Spätzle was mainly expressed in the cephalothorax and on the alimentary canal surface during embryonic development. This new Spätzle member showed a constitutive and regional expression during the embryonic development of A. sinica. It may play an important role in dorsal-ventral differentiation at the early development stages and in immune response pathway at the pseudoadult and adult stage, as well as during infection.

Molecular analysis and its expression of a pou homeobox protein gene during development and in response to salinity stress from brine shrimp, Artemia sinica.

Brine shrimps of the genus Artemia are aquatic species of economic importance because of their important significance to aquaculture and are used as a model species in physiology and developmental biology. Research on Artemia POU homeobox gene function will enhance our understanding of the physiological and developmental processes of POU homeobox gene in animals. Herein, a full-length cDNA encoding an Artemia POU homeobox protein gene 1 (APH-1) from Artemia sinica (designated as As-APH-1) was cloned and characterized by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. The As-APH-1 gene encoded a protein of 388 amino acid polypeptide with a calculated molecular mass of 42.85kDa and an isoelectric point of 6.90 and the protein belongs to the POU III family. Multiple sequence alignments revealed that A. sinica As-APH-1 protein sequence shared a conserved POU homeobox domain with other species. The early and persistent expression of As-APH-1 in the naupliar stages by semi-quantitative RT-PCR and whole-mount embryonic immunohistochemistry suggest that As-APH-1 functions very early in the salt gland and may be required continuously in this organ. Later in development, expression of As-APH-1 begins to dramatically decrease and disappear in salt gland of the sub-adult Artemia. In addition, we also discovered that As-APH-1 increased obviously as the salinity increased, indicating that As-APH-1 might be used as a good indicator of salinity stress. In summary, we are the first to identify the As-APH-1 gene and to determine its gene expression patterns in early embryogenesis stages and in different salinity stress in brine shrimp, A. sinica. The result of expression of As-APH-1 affected by salinity changes will provide us further understanding of the underlying mechanisms of osmoregulation in Artemia early embryonic development.

Expression pattern of a Gram-negative bacteria-binding protein in early embryonic development of Artemia sinica and after bacterial challenge.

Gram-negative bacteria-binding proteins (GNBPs) are important pattern recognition receptors (PRRs) in invertebrate innate immune systems. These immune proteins are capable of identifying and binding to specific targets on the surface of microorganisms, and thereby trigger a variety of defense reactions through the activation of protease cascades and intracellular immune signaling pathways. In this paper, a full-length cDNA of GNBP coding for a conserved ß-glucan-binding domain was isolated from Artemia sinica. Bioinformatic analysis showed that it belonged to a new member of the GNBP family. The expression of GNBP was investigated at various stages during the embryonic development of A. sinica using real-time PCR and immunohistochemistry assay. The result showed that the highest expression level was on the 10th day of the embryo. The larvae showed a remarkable down-regulation of GNBP transcript after challenged with Gram-negative bacteria. In contrast, the level of GNBP transcript in the larvae increased gradually after challenged with Gram-positive bacteria. Immunohistochemistry assay showed that during embryonic development, GNBP protein was mainly expressed in the head, thorax, and on the alimentary canal surface. This new GNBP member showed a constitutive and regional expression during the early embryonic development of A. sinica, and may therefore play an important role at the adult stage, as well as during infection.

Mitochondrial functions on oocytes and preimplantation embryos.

Oocyte quality has long been considered as a main limiting factor for in vitro fertilization (IVF). In the past decade, extensive observations demonstrated that the mitochondrion plays a vital role in the oocyte cytoplasm, for it can provide adenosine triphosphate (ATP) for fertilization and preimplantation embryo development and also act as stores of intracellular calcium and proapoptotic factors. During the oocyte maturation, mitochondria are characterized by distinct changes of their distribution pattern from being homogeneous to heterogeneous, which is correlated with the cumulus apoptosis. Oocyte quality decreases with the increasing maternal age. Recent studies have shown that low quality oocytes have some age-related dysfunctions, which include the decrease in mitochondrial membrane potential, increase of mitochondrial DNA (mtDNA) damages, chromosomal aneuploidies, the incidence of apoptosis, and changes in mitochondrial gene expression. All these dysfunctions may cause a high level of developmental retardation and arrest of preimplantation embryos. It has been suggested that these mitochondrial changes may arise from excessive reactive oxygen species (ROS) that is closely associated with the oxidative energy production or calcium overload, which may trigger permeability transition pore opening and subsequent apoptosis. Therefore, mitochondria can be seen as signs for oocyte quality evaluation, and it is possible that the oocyte quality can be improved by enhancing the physical function of mitochondria. Here we reviewed recent advances in mitochondrial functions on oocytes.