mRNA剪接前导序列在海洋桡足类中的普适性与特异性研究

Copepods as the critical link in marine food-web, the variation of their abundance, population and community structure control the primary production and the fishery resource dynamics. Gene expression study on copepods is crucial for the understanding of molecular mechanisms regulating their growth and development, physiological functions and response to environmental changes. However, the lack of efficient and lineage-specific tool impedes the gene expression study in copepods. Splice leader (SL), the lineage-specific conserved sequence at 5’-end of the mRNA molecule, offers a powerful tool for transcript isolation, gene expression profiling and research on in situ gene expression. In the proposed study, we will investigate SL trans-splicing for species throughout major orders of copepods including Calanoida, Cyclopoida and Harpacticoida. The present of SL will be verified by analyzing the 5’-end of cDNA, RNA structure and genomic organization. The ubiquity and specificity of SL will be examined by comparing SL sequences among species. The prevalence of SL in mRNA will be investigated by analyzing the functional diversity of cDNA amplified by SL. The outcome of this project will shed light on the SL trans-splicing in copepod, provide support to develop a copepod-specific genetic tool, and lay a foundation for the further studies on in situ gene expression of this ecologically important lineage.

桡足类在海洋食物网中起着承上启下的作用，其种群和群落结构及丰度变化控制着初级生产，并直接影响渔业资源动态。在基因水平上揭示桡足类生长发育、生理功能以及生态适应机制，对于阐明桡足类在生态系统中的作用机理具有重要意义。然而，对于这类个体小、数量大的浮游动物，目前尚难以高效、准确捕获其特异性mRNA，限制了对其自然状态下原位基因表达的认知。剪接前导序列(spliced leader, SL)，作为mRNA 5’端的基因标签，是捕获特异性mRNA的有力工具。本项目拟以哲水蚤目、剑水蚤目和猛水蚤目的代表种为研究对象，从序列和结构两方面确立桡足类SL；通过分析SL同源性及基于SL序列扩增构建cDNA文库，系统地论证桡足类SL的普遍性、特异性及适用性。本研究结果将为阐明桡足类RNA反式剪接模式、开发其转录本基因标签提供理论依据，并为这类海洋生物资源的原位基因表达、种群及群落分子生态学研究奠定基础。

本项目以桡足类哲水蚤目、剑水蚤目、猛水蚤目及管口水虱目代表种类为研究对象，初步论证了桡足类反式剪接前导序列（spliced leader, SL）的普遍性、特异性及作为基因标签的适用性。系统验证了哲水蚤目SL在该目不同种类、性别、发育阶段和功能基因中的普遍性及SL序列的目特异性（order-specific），在管口水虱目发现了潜在的目特异性SL，但尚未在剑水蚤目和猛水蚤目中发现SL。从序列和结构两方面确立哲水蚤目特异性SL，克隆测序了两种哲水蚤的SL基因（SL DNA）及其转录本（SL RNA）、管口水虱目SL RNA，比较分析其基因排列及RNA二级结构特征。建立了基于SL构建哲水蚤目桡足类全长cDNA文库的新方法，并首次应用于研究有机污染物对桡足类毒性效应的分子机制，同时将SL扩增与高通量扩增子测序技术相结合，优化了文库构建方法。本项目研究成果可为阐明桡足类反式剪接模式的进化机制提供理论依据，根据验证的SL普遍性、特异性及适用范围，开发的类群特异性基因标签大大提高了哲水蚤目桡足类基因表达研究的效率，为研究桡足类在其他污染物或影响因子胁迫下的分子响应机制提供了重要工具，并为这类海洋生物资源的原位基因表达、种群及群落分子生态学研究奠定基础。