病原相关媒介蜱类涎腺小RNA特征表达谱分析及其在蜱媒病预警中的应用研究

As a critical organ,salivary gland of tick faciliates pathogens transmission with mechanisms remaining to determined.Small RNAs in salivary meditates the process of interplay between pathogen and host cells through sensing pathogen associated molecular pattern(PAMP)and pattern recognition receptor(PRR)and triggering the regulation of target genes expression. Due to the specific PAMP and PRR associated specific pathogen,different pathogen invading to salivary might result in specifc small RNA profilings with massive parallel sequences, which evolves as increasing prominent and logical alternative for pathogen characters and indicates the complicated interaction process between pathogen and salivary cells. To explore the potential applications in predicting unrecognized tick borne pathogens, small RNA expression profilings in the infection and invading process are eager to be monitored and analysed with second generation deep sequencing techniques. The massive parallel sequences from these small RNAs are planed to align,assembly, clone, identify and annotate for their functions in the sensing and regulation pathways between pathogens and salivary cells. Compared to different pathogens, the specific characters of small RNAs profilings are expected to distinguish from the others and be slaved as limestone to discover unrecognized pathogens in tick. In the project, we aim a novel predicting techniques for tick borne disease and choose Lyme spirochetes, Babesia microti and novel Bunyarvirus related to severe fever with thrombocytopenia syndrome discovered in central China in 2009 as representatives to monitor the specific small RNAs profilings during the infection and invading process in tick salivary. The expected characters of small RNAs profilings obtained from different pathogens invading and infection experimentally might pave a new way to discover novel tick borne pathogens and benefit the prevention and control strategies of tick borne disease in the future.

小RNA（small RNA）是介导蜱类涎腺细胞和病原体相互作用的效应分子。小RNA表达谱反映着涎腺细胞与病原体相互作用的信息特征，指示着蜱媒病原的传播过程。对病原入侵过程中小RNA的认识，不仅有助于了解病原与宿主相互关系，而且可以从涎腺反应和病原体调控的角度认知病原体，这也是发现新的蜱媒病原体的重要手段之一。本项目拟选择莱姆病螺旋体、人巴贝西原虫、新布尼亚病毒为代表，利用深度测序技术动态监测病原体感染传播过程中的涎腺小RNA表达谱的变化情况，明确与病原体感染相关的小RNA特征表达谱，分析原体感染过程中涎腺活性成分的信号传导过程和表达调控通路，明确蜱媒病原体与涎腺相互作用的分子基础，探索利用小RNA表达谱建立新型蜱媒病预警技术的可行性，为科学防控蜱媒疾病提供基础数据和技术支撑。

蜱类涎腺是病原体感染、定居和扩散的重要器官，涎腺与病原体的相互作用是揭示病原体克服蜱类先天免疫和宿主免疫胁迫的重要手段。为了分析病原体感染对蜱类涎腺编码、具有调节功能小RNA（miRNA）表达的影响，探讨利用miRNA特征表达谱作为蜱媒病原预警技术的可能性和可行性。项目组以我国广泛分布的莱姆病螺旋体Borrelia garinii和全沟硬蜱Ixodes persulcatus分别代表媒介和病原体，利用高通量测序技术和miRNA分析技术，获得了全沟硬蜱涎腺特征性miRNA表达谱。结果表明，感染前后，全沟硬蜱miRNA文库共获得长度在18-30个核苷酸的小片段4,579,483和4,021,389个；感染后的涎腺小RNA明显受到抑制。感染前，全沟硬蜱共表达1848个进化保守miRNA；而感染后则只有1326个表达。表达丰度前十位miRNA在莱姆病螺旋体感染前后有6 个稳定表达，包括miR-1、iR-375、bantam、miR-184、miR-739和 miR-263a. 莱姆病感染前后，全沟硬蜱涎腺miRNA的表达谱呈现显著变化，其中，136 miRNA上调表达，上调显著的6个miRNA包括miR-let-7a、miR-184、 miR-315、miR-71、miR-2001 和 miR-301；而252 个miRNA下调表达， 下调显著的5个miRNA包括 miR-96、 miR-219a、 miR-5332、 miR-5316b 和miR-307。本项目初步获得莱姆病螺旋体感染后全沟硬蜱涎腺特征性的miRNA表达谱，为进一步研究这些小RNA的功能提供了技术，同时也为基于涎腺miRNA表达谱开展蜱媒病预警和控制工作提供科学基础和技术支持。