水生生物学报2017，Vol.41Issue(2)：314-320,7.DOI:10.7541/2017.38
半滑舌鳎miR-200a和miR-200b前体克隆及其免疫应答分析
THE PRECURSOR CLONING AND IMMUNE RESPONSE ANALYSIS OF MIR-200A AND MIR-200B IN CYNOGLOSSUS SEMILAEVIS
摘要
Abstract
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, in-testine, 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-regu-lated in HTLC after treatment by PolyI:C and the expression level achieved 9 times at 6h than at 0 h. miR-200b was ob-viously 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.关键词
半滑舌鳎/miR-200a/miR-200b/前体克隆/荧光实时定量PCR/免疫应答Key words
Cynoglossus semilaevis/miR-200a/miR-200b/Precursor cloning/qRT-PCR/Immune response分类
生物科学引用本文复制引用
沙珍霞,陈学杰,陈亚东,颜慧..半滑舌鳎miR-200a和miR-200b前体克隆及其免疫应答分析[J].水生生物学报,2017,41(2):314-320,7.基金项目
国家自然科学基金(31572644和31172439) （31572644和31172439）
鳌山科技创新计划(2015ASKJ02-03)资助 [Supported by the National Natural Science Foundation of China (31572644和31172439) （2015ASKJ02-03）
Aoshan Science and Technology Innovation Project (2015ASKJ02-03)] （2015ASKJ02-03）
