铜离子胁迫下凡纳滨对虾免疫相关MicroRNA的筛选鉴定与功能研究

Our previous study found that hemocyte apoptosis ratio significantly increased and relative mRNA expressions of immune- and apoptosis-related genes were also changed in Litopenaeus vannamei exposed to copper. We hypothesize that these processes may be a protection strategy via physiological accommodation for environmental stress. MicroRNAs (miRNAs) are short nucleotide RNA molecules that negative regulators of gene expression in a variety of organisms. The research of miRNA will directly reflect the molecular mechanism of stress response in shrimp. Based on our previous transcriptome database for hemocytes of L.vannamei, we will construct the miRNA library in hemocyte of control and copper-exposed shrimp. The immune-related miRNA will be screened and will characterize their expression using qPCR and northern blot. The relationship between miRNA and its target genes will be identified by western blot. Furthermore, we will explore its role and mechanism in the regulation of immune response by interference the miRNAs expression in vivo. This study would not only provide new insights into the mechanism of immune regulation, but also pave the way for further defining the function of miRNA in immune response and other various biological functions in L. vannamei.

我们的前期工作表明，在铜胁迫下，凡纳滨对虾血细胞的凋亡率有上升的趋势，凋亡和免疫相关基因的表达量也发生改变，推测细胞凋亡的发生和基因表达量的改变可能是虾类抗胁迫的生理调节保护策略。MicroRNA（miRNA）是广泛存在于生物体细胞内，具有强大的调控基因表达作用，miRNA的研究能够更直观地体现对虾胁迫响应的分子机制。本项目在前期转录组的基础上构建凡纳滨对虾铜离子胁迫前后的miRNA文库，从中分析免疫相关的miRNA，用qPCR和northern blot 鉴定特异性miRNA的表达，通过western blot鉴定其与靶基因的靶向关系，并在虾体内通过干预 miRNA表达来验证特异性miRNA对胁迫响应的调控作用及调控机制。本研究将是凡纳滨对虾免疫调控机制研究新的切入点，为阐明miRNA调控免疫相关基因的级联机制奠定基础，对于解释凡纳滨对虾的其它各种生物功能提供新思路。

工业化和集约化养殖的发展，使我国水域重金属污染日趋严重，给水生生物的生存和发育构成严重的威胁。在生产实践中，普遍使用硫酸铜等铜类化合物进行病害防治和清塘灭藻，使其中的铜离子沉淀积累在池塘中，从而导致了水体环境的恶化，严重制约了虾类养殖业的健康发展。我们的前期工作表明，在铜胁迫下，凡纳滨对虾血细胞的凋亡率有上升的趋势，凋亡和免疫相关基因的表达量也发生改变，推测细胞凋亡的发生和基因表达量的改变可能是虾类抗胁迫的生理调节保护策略。MicroRNA（miRNA）广泛存在于生物体细胞内，具有强大的调控基因表达作用，miRNA的研究能够更直观地体现对虾胁迫响应的分子机制。本项目在前期研究的基础上构建了凡纳滨对虾铜离子胁迫前后的miRNA文库，分析胁迫前后差异表达的miRNA，从中筛选免疫相关的miRNA，初步构建miRNA通过调节靶基因参与免疫反应的调控网络，并在虾体内通过干预 miRNA的表达来验证特异性miRNA对胁迫响应的调控作用及调控机制。本研究将是凡纳滨对虾免疫调控机制研究新的切入点，为阐明miRNA调控免疫相关基因的级联机制奠定基础，对虾类养殖业的健康发展具有重要的现实意义.