Transcriptome Sequencing Of Coelomocyte And Identification And Expression Of Immune-related Genes After Vibrio Splendidus Infection

Sea cucumber (Apostichopus japonicus) is one of Chinese traditional precious seafood species, which has the highest value due to its nutritional and supported medicinal properties among Holothuroidea. Sea cucumber farming has become a vigorous industry in China. However, with rapid expansion and intensification of sea cucumber farming, a series of problems including diseases and offspring degradation, seriously hinder sustainable development of sea cucumber industry. In the study we established gene expression profiles of sea cucumber using transcriptome sequencing, and then obtained differentially expressing genes from which we screened out immune-related genes. Furthermore, RT-PCR technology was used for verification of these genes expression.The sea cucumbers from the same family are infected by Vibrio splendidusand are divided into disease-resistant(A), susceptibility(S), negative control (C)and blank control group(K). Total coelomic fluid RNA are extracted and sequenced via Illumina platform and digital gene expression profile. The Illumina RNA-seq analysis generated319455942 clean reads.186658 transcripts were produced from 201 to 15051 with a N50 length of 1245 and 89891 unigenes from 201 to 15051 with a N50 length of 791 by de novo assembly analysis. Among these, 89891 non-redundant unigenes were classified into three major functional categories-biological process(9574), cellular component 11078) and molecular function(10994). A total of 18887 unigenes were consequently classified into specific pathways,2972 unigenes are annotated in Enzyme Commission (EC) and 4005 unigenes took participate in 254 pathways, and 13126 unigenes were categorized into25KOG classifications. Furthermore, our results revealed that 358 gene in K&A and 102 genesin K&S were differentially expressed in Vibrio splendidus treatment experiment, among which 30 genes and 19 genes are respectively potential disease-resistant and susceptibility genes that could be further confirmed their functions in sea cucumber.RT-PCR primers of 24 disease-resistant and 15 susceptibility genes were respectively designed, standard curves of them were constructed and relative expression levels were detected, the results showed that amplification results are good. Standard curves of A1-A20 and S1-S5 genes are built, amplification efficiency was 0.92-1.11, R2 was 0.990-0.999.The results of relative expression levels showed expression levels of A8 were upregulated and disagreed with sequencing results, however, results of other genes were consistent.In this study, we selected randomly a family of sea cucumber and obtained cDNA after Vibrio splendidus infection. Relative expression levels of these genes were detected and the results were demonstrated, except for SI7, expression levels of other genes were consistent with sequencing results. Moreover, a few genes were involved in immune signaling pathway, such as Lysosome, Endocytosis, Chemokine signaling pathway, MAPK signaling pathway and ERBB signaling pathway. FOXO, AC, STAT5, NFkB took part in chemokine signaling pathway, SGSH, DNase II, ABCA2, AP-3, NEU1 and AP-1 took part in lysosome, RTK, AP-2, PAR6, rabaptinS, CHMP5, VPS37 took part in endocytosis, JNK, NCK, STATS, ERBB-1 took part in ERBB signaling pathway, JNK, MEKK4, FLNA, NFkB, EGFR took part in MAPK signaling pathway.