虹鳟热应激相关miRNAs及其靶基因鉴定和转录后调控机理研究

Rainbow trout (Oncorhynchus mykiss) is a cold-water fish of salmonid, which is cultivated as the main freshwater specie in China, but its poor resistance to heat seriously restricts the development of aquaculture. So far, the active genes and their regulation mechanisms during the heat-stress response still remain unclear in rainbow trout, especially, the study of post-transcriptional regulation is presently lacking. MicroRNAs (miRNAs) are a class of short endogenously non-coding RNAs that regulate genes expression at the post-transcription. In this project, experiments will be performed on female fish of full-sib. The whole blood and liver samples from 10 individuals that reared in different temperature (18℃, five fish, and 24℃ of heat-stress, five fish ) will be taken, and a total of 20 miRNAs sequencing-libraries from the whole blood and liver will be constructed. Then the identification of differentially expressed miRNAs of the whole blood/liver under different treatment temperature will be executed based on sRNA-Seq and bioinformatics analysis, and afterward, more than five miRNAs showed significantly different expression levels in the whole blood/liver will be confirmed by quantitative real-time PCR (qRT-PCR). Subsequently we will use bioinformatic approaches to predict the possible target genes of each miRNA which expressed differently. Then the GO and KEGG analysis to target genes will be carried, and the prediction to representative target genes will be verified by luciferase reporter gene assay, as well as their functions will be confirmed by overexpression and inhibition test in cultured hepatocytes of rainbow trout. These results will reveal the mechanisms of post-transcriptional regulation during the heat-stress response and contribute to heat resistant technology in rainbow trout.

虹鳟是我国淡水养殖的主要冷水性鱼类，但对高温耐受性差严重制约其养殖业发展。迄今，对虹鳟应答高温刺激的基因及其调控机理仍不清楚，尤其对转录后调控缺少研究。miRNA是真核生物中一类内源性非编码小分子RNA(sRNA)，通过作用于相应的靶基因(mRNA)在转录后水平发挥调控作用。本研究以虹鳟全同胞雌鱼为材料，利用正常水温和高温胁迫下各5尾虹鳟的全血、肝脏组织，构建20个sRNA测序文库，通过第二代测序技术，鉴定热应激下虹鳟全血、肝脏中差异表达的miRNAs，选择个别miRNAs通过qRT-PCR对差异表达进行验证后，以生物信息学预测差异表达miRNAs的靶基因，对靶基因进行功能和信号通路分析，在此基础上选择主要通路上的核心靶基因，用荧光素酶报告基因检测试验验证预测结果，通过离体肝细胞miRNAs 过表达/抑制试验进行功能验证，最终揭示虹鳟热应激的转录后调控机理，为发展虹鳟抗热应激技术奠定基础。

虹鳟是典型的冷水性鱼类，对较高水温的耐受能力差，随着全球气候变暖，夏季高温对虹鳟养殖的威胁越来越严重，鉴定虹鳟应答高温刺激的相关基因，在基因表达的不同层次上理解热应激的响应策略和调控机理，可以为虹鳟耐高温遗传选育和改良、发掘虹鳟抗高温的各种措施提供理论基础。microRNA（miRNA）是一类对基因进行转录后调控的单链小分子RNA。目前，热应激下虹鳟miRNA的研究还未见文献报道。本研究选取全同胞家系的虹鳟作为试验材料，取对照组（18℃）和热处理组（24℃）各3尾雌鱼的肝脏和头肾，用llumina HiSeq 2500测序平台和生物信息学方法，对虹鳟肝脏和头肾组织miRNAs进行系统性分析，鉴定热应激下差异表达的miRNAs，并揭示新的虹鳟特异miRNAs；接着分别从肝脏和头肾RNA-seq和small RNA-seq结果中筛选出稳定表达的mRNAs（肝脏5个，头肾5个）和miRNAs（肝脏4个，头肾5个），通过geNorm，NormFinder，BestKeeper和Comparative ΔCt四种方法同时评估这些基因和四个常用参考基因和一个小的非编码RNA (U6)的表达稳定性，筛选出热应激下虹鳟肝脏和头肾的内参mRNA和内参miRNA，随后用筛选出的内参miRNA对small RNA-seq结果进行RT-qPCR验证；同时，为了研究miRNA在热应激过程中对虹鳟的调控作用，本研究从small RNA-seq结果中筛选出差异表达的6个miRMAs，预测其靶基因，并采用双荧光素酶报告基因检测系统对其靶向关系进行验证。对有靶向关系的4组miRNA-mRNA，选取两组在细胞水平上进行了功能验证。通过以上研究，初步揭示了虹鳟热应激转录后调控机制。