凡纳滨对虾低温差异表达miRNA的分析鉴定及靶基因的验证

为了解析miRNA及靶基因在凡纳滨对虾低温适应过程中的分子调控机制, 研究开展了凡纳滨对虾常温(28℃)及低温锻炼(16℃, 6d)下肝胰腺小RNA文库的测序和分析。从常温对虾的小RNA文库测序获得18—32 nt的高质量序列10690259条, 鉴定出已知的成熟miRNAs 57条; 而从低温锻炼对虾的小RNA文库获得18—32 nt的高质量序列序列8587144条, 鉴定出已知的成熟miRNAs 48条。分析获得25个在低温锻炼下呈显著差异表达的miRNAs。运用qRT-PCR验证了3条低温下差异极显著的miRNAs的表达模式。结果显示, 3条miRNAs的表达模式与高通量测序结果基本一致。预测低温差异表达miRNAs的靶基因, 并与转录组分析获得的低温显著差异表达基因进行比对, 从二者重合的基因集中挑选出4个基因, 即nuclear export mediator factor Nemf-lik、synapse-associated protein、seleno proteins 以及DEAD-box RNA helicase Variant 1, 运用qRT-PCR验证其在不同条件低温锻炼凡纳滨对虾肝胰腺中的表达规律, 为解析miRNAs及其靶基因在对虾低温适应过程中的分子调控机制提供了基础数据。

IDENTIFICATION AND ANALYSIS OF DIFFERENTIALLY EXPRESSED MIRNA UNDER LOW TEMPERATURE AND VALIDATION OF TARGET GENES IN LITOPENAEUS VANNAMEI

To examine the molecular regulatory mechanism of microRNAs and target genes in Litopenaeus vannamei during cold adaptation, we conducted microRNAs (miRNAs) analysis on the hepatopancreas of L. vannamei under normal temperature of 28℃ and cold acclimation (16℃ for 6 days) by using Solexa sequencing. In total, 10690259 and 8587144 unique sequences of 18—32 nt length were obtained from small RNA libraries at room temperature and low temperature, including 57 and 48 known mature miRNAs, respectively. Expression analysis revealed 25 significantly differential expressed miRNAs between groups, and three of them were further confirmed by PCR. Moreover, we also observed that cold regulated target genes of significantly differential expressed miRNAs including the nuclear export mediator factor Nemf-lik, synapse-associated protein, seleno proteins and DEAD-box RNA helicase Variant 1. These results suggest that miRNAs and their target genes may mediate the cold adaption in L. vannamei.