Cloning And Characterization Of MicroRNAs From Vibrio Splendidus Challenged Sea Cucumber Apostichopus Japonicus

Skin ulceration syndrome has become the major obstacle in the development ofsea cucumber Apostichopus japonicas industry because of its higher spreading speed,mortality rates and distribution.Nowadays, rare information was available on howthe pathogen escaped from host immune system surveillance and suceesfullyreproduction in the host. MicroRNAs (miRNAs) constitute a family of small RNAspecies around21-25nt which have been demonstrated to be one of key effectors inmediating host-pathogen interaction.In this study, two haemocytes miRNA libraries were constructed with deepsequenced by illumina Hiseq2000from healthy (L1) and skin ulceration syndrome A.japonicus (L2). Sone candidated miRNAs were selected for expression analysis by q-PCR. The results were indicated as follows:The high throughput solexa sequencing resulted in9,579,038and7,742,558clean data from L1and L2, respectively. After screening by reference genome,miRase and Rfam databases,302,399and222,605reads were obtained in the endfrom L1and L2, respectively. Sequences analysis revealed that40conservedmiRNAs were found in both libraries, in which let-7and mir-125were speculated tobe clustered together and expressed accordingly. Eighty-six miRNA candidates werealso identified by reference genome search and stem-loop structure prediction.Importantly, mir-31and mir-2008displayed significant differential expressionbetween the two libraries according to FPKM model, which might be considered aspromising targets for elucidating the intrinsic mechanism of skin ulcerationsyndrome outbreak in the species. Real-time quantitative PCR (RT-qPCR)experiments were preformed for6miRNAs like mir-31, mir-34, mir-242, mir-375,mir-2008and mir-2012of the two samples. The up-regulated expression profileswere detected in mir-242,mir-2008,mir-31and mir-375, and the down-regulated werein the mir-34and mir-2012. Our results suggest that elucidation of the molecularmechanisms responsible for miRNA regulation of the host’s innate immune system should help with the development of new control strategies to prevent A. japonicusinfections.