腐食酪螨microRNA及其对过敏原基因表达调控作用研究

The mold mite Tyrophagus putrescentiae (Astigmata: Acaridae) resides in a wide variety of stored products, causing economic losses, but is also found in human living environments, where it is associated with asthma, perennial rhinitis, atopic dermatitis, eczema, and other allergic disorders. The allergenicity of mites like T. putrescentiae can be affected by the expression of genes encoding their allergens. Mounting evidence demonstrates the critical roles of small non-coding microRNAs (miRNAs) in regulation of gene expression across various species. To date, however, no miRNAs have been identified from house dust mites and storage mites. We hypothesize that miRNAs are critical to the expression of allergen-encoding genes from T. putrescentiae. Here, we propose to collect the eggs, larva, nymph, and adults of T. putrescentiae for next-generation sequencing and bioinformatics analyses that will enable development of transcriptome libraries to resolve gene expression profiles. miRNA libraries will also be constructed to resolve miRNAs expression profiles. We will predict the associations between these microRNAs and their target genes, particularly encoding allergens, using bioinformatics tools. To clarify the regulatory roles of these miRNAs on allergen expression, molecular tools including culture of mites, silencing of miRNAs, and qRT-PCR to measure gene expression will be applied. The successful completion of the proposed studies will identify the allergen composition and miRNAs of T. putrescentiae for the first time. Such findings will be helpful for understanding how miRNAs regulate expression of allergen-encoding genes. Further, this knowledge can provide a basis for the design of targeting molecules that may alter allergen expression and for the production of recombinant allergens to diagnose and treat allergic diseases.

腐食酪螨既是一种重要的储藏物害螨，也是室内过敏原的重要来源螨种之一。miRNAs已报道于30余种节肢动物体内，具有调控生长发育、参与宿主与病原体的交互作用、参与宿主免疫反应等功能。经检索miRBase 21.0，尚未收录房舍和储藏物螨类miRNAs。本研究计划收集腐食酪螨卵、幼螨、若螨、成螨，分别建立miRNAs文库并测序，获得其miRNA表达谱；分别建立转录组文库并测序，获得其基因表达谱，包括所有过敏原基因；通过生物信息学软件预测miRNAs与过敏原基因的相互关系后，用螨细胞培养、基因沉默miRNAs、qRT-PCR等手段验证miRNAs对过敏原基因靶向调控作用。研究有望全面解析腐食酪螨miRNAs表达谱、过敏原组成，深化人们对miRNAs功能和作用的认识，为针对miRNAs设计抑制剂防制螨类以及制备重组过敏原诊治过敏性疾病等奠定基础。

腐食酪螨(Tyrophagus putrescentiae)是全球分布的储藏物螨类，它可以引起特应性体质者患上IgE介导的过敏反应性疾病，是继屋尘螨、粉尘螨、热带无爪螨之后又一重要的致敏螨种。本课题：（1）分别收集卵、幼螨、若螨、成螨四个发育时期样本，通过miRNAs文库建立与测序，鉴定出793条miRNAs，其中有169条在不同发育时期表达差异具有显著性（P<0.01），在全球首次解析腐食酪螨miRNAs表达谱。（2）取腐食酪螨纯净的成螨螨体，进行线粒体组测序，组装获得线粒体基因组大小为13290bp，GC含量为29.85%；用全基因组鸟枪法构建不同插入片段的文库经Illumina Hiseq二代和Pacbio三代测序，组装获得的基因组全长为118,292,147bp；取不同发育时期的腐食酪螨，分别构建转录组文库并测序，获得200829条unigene。在全球首次解析腐食酪螨全基因组和转录组信息。（3）整合多组学数据，功能注释获得腐食酪螨过敏原38个组分编码基因信息，通过比较卵、幼、若、成螨转录组学数据获得此38个组分编码基因表达量的不同，从而首次解析腐食酪螨过敏原谱。（4）克隆、表达、纯化了部分过敏原单组分，其中Tyr p 6、11、12、13、15、20、28、29、32、35、36均为新发现过敏原，为腐食酪螨过敏性疾病临床诊断、治疗制品的研发奠定了基础。（5）通过不同发育时期转录组学数据和miRNAs表达谱的关联分析，预测了miRNAs和过敏原基因表达的相关性，并经双荧光素酶报告基因系统检测证明了PC-5P-5534223_1和 PC-5p-5698441_1)对过敏原Tyr p 24、PC-5P-5698441_1对过敏原Tyr p 3、PC-5P-7050653_1对过敏原Tyr p 4具有调控作用，在全球首次证实螨虫miRNAs对螨虫过敏原基因表达的调控作用，为后续针对miRNAs设计抑制剂或采用miRNAs阻断剂抑杀螨类提供科学依据。