六足动物线粒体rRNA经验进化模型的建立及系统发育分析应用

The mitochondrial ribosomal RNA genes are one of the most frequently used molecular markers in phylogenetic analyses. Paired regions in rRNA molecular evolve via selectively neutral substitutions in the form of compensatory mutations to maintain energetically stable secondary structures. Ribosomal RNA loop and stem regions are therefore subject to very different selection regimes, which can hamper the use of rRNA genes for phylogenetic purposes. In particular, correlated variation of nucleotides in stem regions has been suggested to corrupt phylogenetic analyses as these covariation patterns of paired sites do not display independent phylogenetic signal which assumed in Markov chain models. Here, we plan to contribute 30 new sequences both for the mitochondrial 12S and 16S rRNA (mt rRNA) genes, and determine the sequences of both ends of these two mt rRNA genes by 5' and 3' RACE, and circularization and RT-PCR methods. In total about 230 mt rRNA sequences will be aligned through different methods to predicted the consensus mt rRNA secondary structure models. Using secondary structural information, each sequence in the alignments be converted into a 20-symbol code: one symbol for each of the four individual bases, and one symbol for each of the 16 ordered pairs. Substitutions in the coded sequences are defined in the natural way, as observed changes between two sequences at any particular site. For given ranges (windows) of sequence divergence, substitution frequency matrices obtained for the coded sequences. Using a technique originally developed for modeling amino acid substitutions, we were able to estimate the actual evolutionary distance for each window. The actual evolutionary distances were used to derive instantaneous rate matrices, and from these we selected a universal rate matrix. Finally, the phylogenetic utility and performance of empirical substitution models will be evaluated and compareed with other methods. The results of study have signifacent roles for interpretations of the evolution of RNA sequence and its application in phylogeny analysis.

线粒体rRNA是分子系统发育研究中最为常用的分子标记之一。由于rRNA二级结构茎区配对的碱基间存在协同进化关系，违背了一些基于马尔科夫链为基础的进化模型假设，造成基于线粒体rRNA序列所构建的系统发育关系可信度较差。本项目在测定30余种六足动物线粒体两种rRNA全长序列和采用RACE技术确定3-5种线粒体rRNA基因准确的起始和终止位点的基础上，对230多条六足纲的rRNA序列采用多种比对策略和方法进行准确比对，在此基础上建立六足动物线粒体rRNA二级结构模型，对于给定范围的序列分歧，采用氨基酸经验模型估计方法计算序列之间替换频率矩阵，以估计每个窗口中的实际进化距离并用于估计瞬时速率矩阵，由此获得六足动物线粒体12S和16S rRNA序列的经验进化模型，将其应用于重建节肢动物主要类群的系统发育关系并评价其有效性。项目对于研究RNA序列的进化及系统发育应用有重要的意义。

项目执行期间，项目组成员先后赴陕西、安徽、山西、湖北省、河南、广西、湖南、海南、西藏等省区进行了实验材料收集进行了收集完成本项目所需的实验材料；并通过GenBank数据库下载六足总纲线粒体rRNA全长序列。依照项目研究计划，首先在六足总纲目级水平构建近缘物种间线粒体rRNA二级结构，其次比较总目或更高级阶元分类群中二级结构的共同点或保守结构域。通过比较研究发现，昆虫线粒体rRNA保守的区域在碱基组成方面倾向于更高的G+C组成。多新翅类昆虫线粒体rRNA序列在序列结构方面较副新翅类和寡新翅类昆虫更为保守。此外，我们还对隆额网翅蝗A. coreana全线粒体基因组序列测定并对其基本特征进行了描述，对tRNA、rRNA以及控制区内的茎环结构进行了预测，并发现了控制区内与其他蝗亚目昆虫相似的保守基序“TATTTwATryAyAAA”。测定短角外斑腿蝗Xenocatantops brachycerus（KC542806）、比氏蹦蝗Sinopodisma pieli、突额无齿蝗Asseratus eminifrontus Huang、青海痂蝗Bryodema miramae、黑胫钩额螽Ruspolia lineosa和瘦露螽Phaneroptera gracilis Burmeister全线粒体基因组序列，描述其序列特征。通过对长尾山雀属7种丛山雀线粒体全基因组的测定及控制区重复序列的结构分析，探究控制区序列重复的演化模式，推测平行同源控制区之间的同源重组极有可能是该区域重复序列之间协同进化的机制。对黄眉林雀15个个体线粒体控制区及其他基因位点单倍型网络、系统发育分析以及这5个基因序列位点祖先状态重建发现，控制区中串联重复单元相同数目的重复为多元起源，通过滑链错配的逆转模型能够很好的解释这一现象；而串联重复单元的起源则有可能通过重组插入发生。另外完成蜻蜓目昆虫山西金光伪蜻Somatochlora shanxiensis、褐尾绿综蟌 Megalestes distans，螳螂目Amorphoscelis sp.、Caliris sp.、Ceratomantis sp.、Decimiana sp.、Eremiaphila sp.膜翅目小黄家蚁Monomorium pharaonis测序注释工作。目前发表学术论文5篇，均为SCI论文，8名研究生先后参与了本项目的研究工作，并毕业4名