基于DNA条形码重要医学媒介螨虫的DNA分类与系统发育研究

DNA barcoding technique is a fast and accurate species identification technique that uses a short section of DNA from a standardized region of the genome. That DNA sequence can be used to identify different species, in the same way a supermarket scanner identifies purchases using the familiar black stripes of the UPC barcode. Since its introduction in 2003, the technique has been developing rapidly in the past decade. Motivated by the present inclusion of "DNA barcode program of important vectors and its closely related species" in the priority list of the International Barcode of Life (IBOL) project, we propose a novel DNA barcode study for three types of mites that are medically important to humans, namely Demodex mites, Sarcoptic mite and dust mite. The major contributions of our project are threefold. Firstly, the sequences of mites will be obtained and aligned to screen target genes, such as mitochondrial CO1, 16S, 12S, ribosomal 28S and ITS. Secondly, ROC curve analysis will be conducted to confirm the appropriate DNA barcode sequences for molecular identification of mites and to establish the DNA barcoding identification platform. The analysis can compensate for the defects of morphological identification and achieve the goals of classifying the mites based on DNA, identifying phenotypic differences, discovering cryptic species and identifying incomplete mite bodies. The findings can help improve the current understanding of Acariformes from the perspective of DNA barcodes research. Finally, the phylogenetic trees will be reconstructed to clarify the phylogenetic relationship. A successful launching of the project with the support of the grants will greatly enhance the research of DNA barcode of medical mites in China and bring the quality of research in this field to the world frontier. Our findings and methodology will lay a solid foundation for DNA barcode research of diverse vectors across Acari and DNA diagnose of acariasis in the future.

DNA条形码技术是基于现代商品零售业条形码系统、以DNA序列为检测对象而创建的一种快速准确的生物身份识别系统，短短十余年得到了飞速发展。在目前"医学媒介种类和近缘物种DNA条形码计划"已被列为"国际生命条形码计划（IBOL）"优先启动行列的背景下，本项目提出对重要医学媒介螨虫蠕形螨、疥螨和尘螨进行DNA条形码研究，通过获取螨虫序列，比对筛选线粒体CO1、16S、12S和核糖体28S、ITS等目的基因，利用ROC曲线分析，确定适合螨虫分子鉴定的DNA条形码，建立DNA条形码分类鉴定平台，旨在弥补形态学鉴定的缺陷，实现螨虫的DNA分类、表型差异鉴别、隐存种的发现以及不完整螨体的鉴定，改善真螨目在DNA条形码研究领域滞后的现状；构建系统进化树，以阐明其系统发育关系；推动我国在医学媒介螨虫DNA条形码研究走在世界前列，为后续对多种医学媒介节肢动物DNA条形码研究和螨源性疾病的DNA诊断打好基础。

在当前“医学媒介种类和近缘物种DNA条形码计划”已被列为“国际生命条形码计划（IBOL）”优先启动行列，而螨类DNA条形码研究几乎空白的背景下，本项目经过四年的不懈努力共获得重要医学螨虫九科17种，基于传统的形态分类，结合生物信息学手段顺利完成了分子鉴定与DNA条形码筛选，取得了以下两个突破性结论：一是不同螨类分类群在科及以下低阶元分子鉴定的DNA条形码不同，mtDNA 12S或rDNA 28S D5是蠕形螨科的DNA条形码；mtDNA COI是疥螨科和肉食螨科的DNA条形码；mtDNA 16S是粉螨科、麦食螨科、食甜螨科和皱皮螨科的DNA条形码；rDNA ITS区或rDNA ITS2是痒螨科和皮刺螨科的DNA条形码。二是发现rDNA 28S D5作为螨类通用的DNA条形码，不仅适用于真螨目33科186种的分子鉴定，而且适用于寄螨目7科39种的分子鉴定。另外，由于螨类RNA水平研究的滞后直接限制了螨类种群致病性差异与功能基因的研究，为此本项目添加了螨类在RNA水平的研究，取得了四个开拓性进展：一是首次建立了螨类RNA提取方法，完成了六科9种螨虫的RNA提取和cDNA文库构建；二是基于转录组测序完成了兔痒螨、犬疥螨、柏氏禽刺螨、马六甲肉食螨、蠕形螨和粉尘螨六科8种螨虫的功能注释和重要功能基因筛选鉴定；三是完成了粉尘螨、兔痒螨、柏氏禽刺螨和马六甲肉食螨4种螨虫的热应激与冷应激差异功能基因筛选、CDS序列确认和RT-qPCR表达量检测，尤其是确认了热休克蛋白家族（HSPs）和粉尘螨蛋白家族（DFPs）是粉尘螨热胁迫与冷胁迫应激调节的两个关键基因家族；四是建立了适合粉尘螨RNA沉默的干扰方法—非侵入性浸泡法。本项目发表论文12篇，SCI收录10篇；向GenBank投递基因序列474条，转录组数据24×6Gb；参加会议交流10人次，特邀讲座2次；培养研究生8人，指导省级大学生创新实验3期；受邀陕西台科普宣讲3次。本项目取得的研究成果，不仅改善了螨类在DNA条形码研究领域滞后的现状，推动我国在医学媒介螨虫DNA条形码的研究走在世界前列，而且对于揭示螨源性疾病的致病机制，消除螨类对人类造成的危害都具有非常重要的意义。