使用三代测序技术研究线粒体DNA非编码区域对其DNA复制和转录的调控

Mitochondrial DNA(mtDNA) replication and transcription have long been a hot topic in evolutionary biology. It is well established that mtDNA is replicated through a mechanism called D-loop in nearly all species. However, recent studies have demonstrated that mtDNA in Caenorhabditis elegans is replicated by a unique mechanism called “rolling cycle”. We have recently assembled the full-length of mtDNAs for 14 Caenorhabditis species using existing NGS data. Surprisingly, we found multiple novel Non-Coding Regions (NCRs) in the mtDNAs of a subset of the 14 species which are near in position, suggesting a vital role of these NCRs in mtDNA replication or transcription. To test the hypothesis, capability of capturing full-length mtDNA and its derived RNAs would be critical for pinpointing the roles of NCRs in DNA replication and transcription. We propose to address the question using a third-generation sequencing technology called Nanopore, which produces sequencing reads of unusual length up to tens of kilo base pairs, leading to a possibility of capturing DNA or primary or mature transcripts in full length. The roles of the NCRs would emerge by comparing their positioning in the intermediates of newly replicated DNA or synthesized primary or mature transcripts. Comparative analysis of the expression of mitochondrial related genes between C. briggsae and its sister species C. nigoni and their hybrids may provide insight into the role of the NCRs in hybrid incompatibility between the two species.

线粒体的复制和转录机制一直是进化生物学研究的热点。几乎所有物种的线粒体DNA都采用置换模式进行复制。而最近有研究表明隐杆线虫线粒体DNA的复制机制却采用罕见的滚环模式。申请人在前期工作中用二代测序重构了14个线虫物种的线粒体DNA序列，发现以C. briggsae为代表的部分隐杆线虫，其线粒体DNA存在额外的非编码序列。由于这些序列位置相近且结构类似，暗示其可能在线粒体DNA复制或转录中起重要作用。为验证这样的假说，申请人拟使用以可以产生超长读长的Nanopore测序技术，鉴别特定物种中线粒体DNA的复制中间体和初始转录本。通过确定这些非编码序列在复制中间体和初始转录本中的相对位置，判断其是否在DNA复制起始或转录过程中起关键作用。 同时，通过比较C. briggsae及其姐妹物种C. nigoni之间线粒体相关基因的表达，阐明线粒体DNA中新非编码序列的形成对物种间杂交不亲和性的影响。

线粒体的复制和转录机制一直是进化生物学研究的热点。几乎所有物种的线粒体DNA都采用置换模式进行复制。而最近有研究表明隐杆线虫线粒体DNA的复制机制却采用罕见的滚环模式。..申请人通过构建相应的生物信息学分析流程（mitovar），用二代测序重构了14个线虫物种的线粒体DNA序列，发现以C. briggsae为代表的部分隐杆线虫，其线粒体DNA存在额外的非编码序列。这样的情况不仅出现在C. briggsae中，在C. elegans数据的挖掘中，我们同样在多个天然品系中证实了这一现象。这些非编码序列存在位置保守性，都位于tRNA cluster中，但是缺乏序列保守性，但是包括一定的串联重复序列。这些位置相近且结构类似的非编码序列，可能在线粒体DNA复制或转录中起重要作用。..为验证这样的假说，申请人使用以可以产生超长读长的Nanopore测序技术，鉴别了C. elegans, C. briggsae和C. nigoni三个物种中线粒体DNA的复制中间体和初始转录本。 通过确定这些非编码序列在复制中间体和初始转录本中的相对位置，归纳总结了相应的复制和转录中间体模式。 同时，通过比较C.briggsae及其姐妹物种C. nigoni两个方向杂交产生的F1个体中线粒体复制和转录中间体的不同，推断线粒体DNA的复制和转录模式在两物中杂交的核质不亲和中起了重要作用。