Dataset Information

Erk2 in ovarian development of green mud crab Scylla paramamosain.

ABSTRACT: We identified extracellular signal-regulated kinase 2 (erk2) from green mud crab, Scylla paramamosain, in this article. It was originally identified from an expressed sequence tag fragment from a normalized gonadal cDNA library. 5' Rapid amplification of cDNA end (RACE) technique was used to obtain the 5' untranslated region (UTR). The full-length cDNA of Sp-erk2 is 1516 bp, including a 5'-terminal UTR of 19 bp, an open-reading frame of 1098 bp, and a 3'-terminal UTR of 399 bp. The translated protein is 365 amino acids in length with a predicted molecular weight of 42 kDa, which is the same as other species. It is the first time that the expression of Sp-erk2 in different stages of ovary development of crustacean was analyzed, and the result showed that the expression of Sp-erk2 increased gradually with ovarian development, with a peak in the mature phase. In situ hybridization histochemistry was used to clarify the detail of expression. Positive signals illustrated that Sp-erk2 mRNA is present in follicular cells when the ovary is in early stages, and in both follicular cells and oocytes when it is in mature phases. All above suggest that Sp-erk2 is important for ovarian development.

SUBMITTER: Ma A

PROVIDER: S-EPMC3391492 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Erk2 in ovarian development of green mud crab Scylla paramamosain.

Ma Ani A Wang Yilei Y Zou Zhihua Z Fu Mingjun M Lin Peng P Zhang Ziping Z

DNA and cell biology 20120306 7

We identified extracellular signal-regulated kinase 2 (erk2) from green mud crab, Scylla paramamosain, in this article. It was originally identified from an expressed sequence tag fragment from a normalized gonadal cDNA library. 5' Rapid amplification of cDNA end (RACE) technique was used to obtain the 5' untranslated region (UTR). The full-length cDNA of Sp-erk2 is 1516 bp, including a 5'-terminal UTR of 19 bp, an open-reading frame of 1098 bp, and a 3'-terminal UTR of 399 bp. The translated pr ...[more]

PMID: 22394010

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Project description:BackgroundInfection of bacterial Vibrio parahaemolyticus is common in mud crab farms. However, the mechanisms of the crab's response to pathogenic V. parahaemolyticus infection are not fully understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs that function as regulators of gene expression and play essential roles in various biological processes. To understand the underlying mechanisms of the molecular immune response of the crab to the pathogens, high-throughput Illumina/Solexa deep sequencing technology was used to investigate the expression profiles of miRNAs in S. paramamosain under V. parahaemolyticus infection.Methodology/principal findingsTwo mixed RNA pools of 7 tissues (intestine, heart, liver, gill, brain, muscle and blood) were obtained from V. parahaemolyticus infected crabs and the control groups, respectively. By aligning the sequencing data with known miRNAs, we characterized 421 miRNA families, and 133 conserved miRNA families in mud crab S. paramamosain were either identical or very similar to existing miRNAs in miRBase. Stem-loop qRT-PCRs were used to scan the expression levels of four randomly chosen differentially expressed miRNAs and tissue distribution. Eight novel potential miRNAs were confirmed by qRT-PCR analysis and the precursors of these novel miRNAs were verified by PCR amplification, cloning and sequencing in S. paramamosain. 161 miRNAs (106 of which up-regulated and 55 down-regulated) were significantly differentially expressed during the challenge and the potential targets of these differentially expressed miRNAs were predicted. Furthermore, we demonstrated evolutionary conservation of mud crab miRNAs in the animal evolution process.Conclusions/significanceIn this study, a large number of miRNAs were identified in S. paramamosain when challenged with V. parahaemolyticus, some of which were differentially expressed. The results show that miRNAs might play some important roles in regulating gene expression in mud crab under V. parahaemolyticus infection, providing a basis for further investigation of miRNA-modulating networks in innate immunity of mud crab.

Project description:BACKGROUND: The green mud crab (Scylla paramamosain) is the most prevalent crustacean on the southeast coast of China. The molecular regulatory mechanism of sex determination and gonadal differentiation in this species has received considerable attention in recent years because of the huge differences--both biological and economic--between male and female crabs. In this study, next-generation sequencing technology was used to develop deep-coverage transcriptomic sequencing data for the testis and ovary of S. paramamosain. RESULTS: A total of 365,116 reads (testis 171,962, ovary 193,154) with an average sequence length of 285 bp were produced from testis and ovary cDNA libraries. After filtering out contaminating reads, the clean reads were assembled, producing a total of 21,791 isotigs and leaving 22,814 reads as singlets. Using the BLASTX program, 3,471 unique sequences (2,275 isotigs and 1,196 singletons) were annotated with known protein sequences from the NCBI non-redundant (Nr) protein sequence database. The Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses allowed the 224 unique sequences that were annotated with enzyme code (EC) numbers to be mapped into 174 KEGG pathways. After comparing the ovary and testis libraries, 4,021 gonad-differentially, 10,522 ovary-specifically, and 19,013 testis-specifically expressed genes were identified. Moreover, 33 ovary-specific, 14 testis-specific, and 34 gonad-differential transcripts were confirmed by semi-quantitative PCR and quantitative real-time PCR. In addition, 8,610 putative simple sequence repeats (SSRs) and 23,879 potential single nucleotide polymorphisms (SNPs) were identified. CONCLUSION: This is the first large-scale RNA sequencing of S. paramamosain to be reported. We have identified many important functional genes and made a preliminary attempt to construct the regulatory network involved in the gonadal development of crustaceans. The annotated transcriptome data will provide fundamental support for future research into the reproduction biology of S. paramamosain. A large number of candidate SSRs and SNPs were detected, which could be used as genetic markers for population genetics and functional genomics in this species.

Project description:Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. In this study, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV) but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at a 3.5-Å resolution reveals three major capsid proteins (VP1 to VP3) organized into a pseudo-T=3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at a 3.3-Å resolution reveals a T= 3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus.IMPORTANCE Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Here we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High-resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The three-dimensional (3D) structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined in this study could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.

Dataset Information

Erk2 in ovarian development of green mud crab Scylla paramamosain.

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Erk2 in ovarian development of green mud crab Scylla paramamosain.

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