摘要
采用生物信息学方法对斑点叉尾(Ictalurus punctatus)ipu-miR-143的靶基因进行预测,并对预测到的ipu-miR-143靶基因进行生物学鉴定。生物信息学预测结果显示,斑点叉尾微管相关蛋白基因RP/EB家族EB1基因的3′-UTR区具有ipu-miR-143的潜在作用位点。结合对几种近缘物种中RP/EB家族EB1基因和miR-143的进化保守性进行分析,推测ipu-miR-143在斑点叉尾中可以通过靶向EB1基因的3′-UTR区而发挥其调控功能。因此,本研究将斑点叉尾EB1基因的3′-UTR区(含有miR-143靶位点)构建到pMIR-REPORTTM Luciferase载体的下游,通过双荧光素酶报告基因检测系统对ipu-miR-143的靶基因进行鉴定。采用HEK293和CCK两种细胞进行细胞转染,两种细胞中转染miR-143 mimics组荧光相对活性与对照组相比均表现为显著性降低(P<0.05)。共转染miR-143 inhibitors后,CCK细胞中荧光素酶相对活性(8.27±1.02)与对照组相比(5.44±1.55)显著上调(P<0.05);HEK293细胞中荧光素酶相对活性(6.30±1.19)与对照组(4.26±0.84)相比有所上升,但无显著性差异(P>0.05)。初步研究结果提示,miR-143可以通过靶向EB1基因的3′-UTR区而发挥其调控功能。本研究旨为后续深入研究斑点叉尾EB1基因的转录后调控机制提供基础依据。
The potential target of ipu-miR-143 was predicted and identified using a luciferase reporter gene assay with HEK293 and CCK cell lines in vitro. Bioinformatic analyses indicated that the 3′untranslated region （3＆#39;-UTR） of the microtubule-associated protein RP/EB family member 1 gene （EB1） contains an ipu-miR-143 target site, which per-fectly complements the seed region （positions 2-8） of the mature ipu-miR-143 sequence. Therefore, we speculate that the EB1 gene might act as a direct regulatory target of miR-143 in channel catfish. The pMIR-EB1 report vector con-taining the ipu-miR-143 complementary sequence was constructed to investigate the target of ipu-miR-143 in channel catfish. Then, pMIR-EB1 and miR-143 mimics were co-transfected into the HEK293 and CCK cells to detect the bio-logical activity of ipu-miR-143. In comparison with the control groups, the miR-143 mimic groups showed significantly lower levels of luciferase expression in the two cell lines （P〈0.05）. Luciferase activity of the ipu-miR-143 inhibitor groups was significantly higher than that of the control groups in the CCK cell line （P〈0.05）. Contrarily, there were no significant differences between the inhibitors and control groups in the HEK293 cell line （P〉0.05）. The results demon-strate that the EB1 gene could be a target gene of ipu-miR-143 and provide valuable information for further research on the post-transcriptional mechanisms of ipu-miR-143 in channel catfish.
出处
《中国水产科学》
CAS
CSCD
北大核心
2014年第2期283-290,共8页
Journal of Fishery Sciences of China
基金
江苏省自然科学基金面上研究项目(BK2011864)
