THE PRECURSOR CLONING AND IMMUNE RESPONSE ANALYSIS OF MIR-200A AND MIR-200B IN CYNOGLOSSUS SEMILAEVIS

In this study, we cloned the precursor sequences of miR-200a and miR-200b from C. semilaevis miR-200 family by PCR. The precursor sequences of miR-200a and miR-200b were 82 bp and 88bp in length respectively. We analyzed the secondary structure and homology of the precursor sequence using the mfold Web Server and Clustalx1.83. The results showed that both precursors had typical stem-ring structure and shared highly homology with other species. The qRT-PCR results indicated that miR-200a and miR-200b were expressed in all 13 tissues (liver, intestine, spleen, head kidney, metanephros, gill, blood, brain, skin, muscle, stomach, heart and ovary) of C. semilaevis, and that miR-200a highly expressed in head kidney and lowly in blood, and miR-200b highly expressed in liver and lowly in muscle. Vibro anguillarum infection initially induced and then decreased the expression f miR-200a and miR-200b in 4 immune tissues (liver, intestine, spleen and head kidney) of C. semilaevis with various peak at each time point. miR-200a has the highest level in the liver and spleen at 6h and in intestine and head kidney at 12h respectively after infecttion by V. anguillarum. miR-200b had the highest level in the intestine, spleen and head kidney at 12h after infection by V. anguillarum. miR-200a and miR-200b were expressed up-regulated in liver cell of C. semilaevis (HTLC) when stimulated using pathogen analogs (LPS, PGN, WGP and PolyI:C). miR-200a was obviously up-regulated in HTLC after treatment by PolyI:C and the expression level achieved 9 times at 6h than at 0 h. miR-200b was obviously up-regulated quickly in HTLC after stimulated by WGP, compared with 0 h, the expression level increased 9 times at 2h. In a word, the results of this study would provide the theory basis for the research of immune-related miRNA in C. semilaevis.