Abstract

Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway.

Highlights

  • The sea cucumber (Apostichopus japonicus) has become one of the most important aquaculture species in China, achieving valuable profits [1]

  • To obtain an overview of the sea cucumber coelomocytes transcriptome, a cDNA library was generated from an equal mixture of RNA from nine individuals of the three groups using Illumina Hiseq2500 platform

  • Among the annotated representative contigs to the Nr database, 6724 (46.7%) representative contigs were matched to Strongylocentrotus purpuratus, 1497 (10.4%) to Saccoglossus kowalevskii, 849 (5.9%) to Branchiostoma floridae, 230 (1.6%) to Nematostella vectensis, 230 (1.6%) to Crassostrea gigas, 202 (1.4%) to Capitella teleta, 173 (1.2%) to Aplysiacali fornica, 158 (1.1%) to Homo sapiens, 158 (1.1%)

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Summary

Introduction

The sea cucumber (Apostichopus japonicus) has become one of the most important aquaculture species in China, achieving valuable profits [1]. Etiological studies [3,4,5] indicated that a bacterial species (Vibrio/Pseudoalteromonas/Aeromonas spp.), a parasite (Parasitic nematode) and virus (a spherical virus) are the major pathogens for this serious disease, including. Various studies have been conducted to investigate the immunity of sea cucumber, trying to identify the immune factors, clone immune-related genes, and obtain immune-related expressed sequence tags (EST) [13,14,15,16,17,18]. The sea cucumbers were divided into a disease-resistant group and susceptibility group according to a series of symptoms of skin ulcer syndrome [4]. Putative disease-resistant genes and susceptibility genes of sea cucumber were screened. Gene-associated markers were screened for potential genetic research

Illumina Sequencing and Assembly
Gene Annotation
Nr Annotation
GO Annotation
COG Annotation
KEGG Annotation
Selecting Disease-Resistant and Susceptibility Genes
MAPK Signaling Pathway
ERBB Signaling Pathway
Lysosomes
Endocytosis
Chemokine Signaling Pathway
Differential Expression Verification of Putative Genes
Sea Cucumber and Microbial Challenge
Total RNA Extraction and cDNA Library Construction
Sequencing and Assembly
Annotation of Representative Contig
SNPs and SSRs Detection
Validation of Illumina Sequencing Results by qRT-PCR
Conclusions
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