刺参补体AjC3活性相关miRNA的筛选与初步研究

Screening and study of miRNA related to complement AjC3 in sea cucumber Apostichopus japonicus

为了从miRNA调控方面探索刺参Apostichopus japonicus的免疫机制,用脂多糖刺激刺参,在第0、6、12 h取样(记为样本A00、A06、A12),以海胆Strongylocentrotus purpuratus基因组为参照基因,运用高通量测序和相关生物信息学方法筛选与刺参补体(AjC3)相关的miRNAs,并通过实时定量PCR验证相关miRNA和AjC3 mRNA的表达变化。结果表明:经比对筛选出刺参体腔细胞的miRNA共41个;通过靶基因预测和差异表达分析,共得到靶基因为补体AjC3的miRNA有3个,分别为spu-miR-133、spu-miR-137、spu-miR-2006;3种miRNA和AjC3 mRNA实时定量PCR显示,spu-miR-133表达量呈先升高后降低的趋势,12 h达到最高值,spu-miR-137表达量呈先降低后升高的趋势,9 h达到最低值,12 h达到最高值,spu-miR-2006表达量呈先升高后降低的趋势,9 h达到最高值,AjC3 mRNA表达量呈先升高后降低的趋势,9 h达到最高值。研究表明,spu-miR-133、spu-miR-137可能共同抑制调控补体AjC3基因表达,spu-miR-2006在免疫反应中对其他靶基因进行调控。

MiRNAs influencing complement C3 in sea cucumber Apostichopus japonicus were screened in the sea cucumber challenged with LPS,referenced to genome of sea urching Strongylocentrotus purpuratus,41 miRNAs by software,0,6,and 12 h after challenge by high-throughput sequencing and bioinformatics methods,and content change in related miRNAs and complement AjC3 mRNA was detected by real-time quantitative PCR to evaluate the immunity in the sea cucumber.Three miRNA（spu-miR-133,spu-miR-137 and spu-miR-2006） was shown to affect their target genes,the sea cucumber complement C3.The RT-PCR showed that the expression of spu-miR-133 was increased from 0 h to 12 h,with the peak value at 12 h,while the expression of spu-miR-137 was decreased from 0 h to 9 h and increased from 9 h to 12 h.The spu-miR-2006 had the peak expression value at 9 h and then decreased in expression.The expression of AjC3 mRNA was found to be increased from 0 h to 9 h and decreased from 9 h to 12 h.It was speculated that spu-miR-133 and spu-miR-137 inhibited the translation of complement AjC3 mRNA directly and that spu-miR-2006 regulated the expression of other genes in immune response.The findings provide a theoretical basis for the immune research.