蝗亚目三种昆虫线粒体基因组测序与蝗总科系统发育分析

【作者】 孙慧敏；

【导师】 郑哲民；

【作者基本信息】 陕西师范大学 ， 动物学， 2009， 博士

【摘要】 线粒体在细胞新陈代谢、疾病、成熟衰老的过程中起重要作用,是氧化磷酸化及许多重要生化反应进行的场所。线粒体基因组是独立于染色体基因组之外的细胞器基因组,具有独立的复制、转录系统,通常是一个含有37个基因的双链、环状的DNA分子,因其特殊的遗传方式而常用于分子系统学的研究。线粒体基因组作为研究基因组结构、功能和进化的最佳模型,对基因组学研究的探索具有重要理论价值和实践意义。本研究采用L-PCR及二次PCR技术对蝗总科2种蝗虫(北极翘尾蝗Primnoaarctica(Zhang & Jin,1985)和红拟棒角蝗Gomphocerippus rufus(Linnaeus,1758))及1种蚱(秦岭微翅蚱Alulatettix qinlingensis)进行全线粒体基因组测序、拼接和注释;结合已经测出的直翅目蝗总科8科8种昆虫线粒体基因组序列,从结构基因组学、比较基因组学与进化基因组学的角度总结了蝗总科昆虫的线粒体基因组结构组成以及序列进化等方面的一般特征,丰富了直翅目昆虫的线粒体基因组数据,为进一步开展昆虫线粒体谱系基因组学的研究奠定了基础;联合NCBI上已经发表及该项目组其他成员所测出来的20种蝗总科昆虫的全线粒体基因组,以东方蝼蛄(Gryllotalpa orietalis)和摩门螽蟖(Anabrus simplex)作为外群,对蝗总科进行了系统发育关系重建;本研究还基于线粒体基因组蛋白编码基因联合序列对蝗总科8种昆虫及蚱总科的秦岭微翅蚱和蜢总科的变色乌蜢对蝗亚目进行系统发育重建。主要获得以下结果:1.所测得的3种昆虫全线粒体基因组的基因组成及各基因次序和转录方向与飞蝗(Locusta migratoria)线粒体基因组一致,各包含13个蛋白编码基因,22个tRNA基因,2个rRNA基因和一个A+T丰富区。tRNAK和tRNAD的位置与六足动物祖先线粒体基因组的基因排列方式相比发生了互换。2.对直翅目内各种昆虫线粒体基因组A+T含量进行比较,发现蝗总科内锥头蝗科较其他各科基因组的A+T含量低;蝗亚目中各种昆虫线粒体基因组A+T含量与螽亚目内各种昆虫线粒体基因组A+T含量相比普遍较高。3.蝗总科各科昆虫蛋白编码基因密码子使用具有偏向性,第三位点为A或T的密码子使用频率较高。4.蝗总科8科昆虫各种基因间隔区最长之处皆位于tRNASer-UCN与ndl基因之间,在atp8/atp6和nad4/nad4L之间均存在7bp的碱基重叠。基因间隔区和基因重叠区的数目、长度在各种蝗虫中均较为相似。5.ATG是蝗总科各科昆虫的蛋白编码基因中使用最多的起始密码子,其次是ATT,使用较少的起始密码子是ATA。使用最多的终止密码子是TAA。多数直翅目昆虫线粒体基因组的nd5基因以不完全终止密码子T/TA作为终止信号。6.在所测定昆虫的tRNA二级结构中,均存在一定数目的碱基错配现象,而且其中绝大部分为G-U错配。7.在北极翘尾蝗和红拟棒角蝗线粒体基因组的A+T丰富区中均含有一个茎环结构,其结构与Saito(2005)在飞蝗线粒体基因组中发现的茎环结构十分相似,并且在该二级结构茎的5′端均有一个十分保守的“TA”。8.本研究基于线粒体基因组对蝗总科8科进行系统发育重建,结果支持夏氏系统中的斑翅蝗科和斑腿蝗科的单系性;发现剑角蝗科、网翅蝗科都不是单系群,建议将剑角蝗科、网翅蝗科和槌角蝗科三科合并为一科,与斑腿蝗科和网翅蝗科并列;支持蝗总科的锥头蝗科和瘤锥蝗科合并成一个科,这个分支是蝗总科中较早分离出来的一个分支;发现癞蝗科与斑腿蝗科具有较近的亲缘关系。同时本研究支持Otte系统中的斑翅蝗亚科和大足蝗亚科的单系性,但是不能支持斑腿蝗亚科的单系性。斑腿蝗亚科、黑蝗亚科和稻蝗亚科有较近的亲缘关系。9.通过基于线粒体基因组对蝗总科系统发育重建研究得出的结果,推测夏氏系统蝗总科中8科的进化关系是锥头蝗科+瘤锥蝗科→斑翅蝗科→癞蝗科→斑腿蝗科→剑角蝗科→槌角蝗科+网翅蝗科。10.本研究基于线粒体基因组得出的蝗总科的系统进化关系可以将分布在中国的蝗总科昆虫分成2个分支,锥头蝗科和瘤锥蝗科组成一个分支,位于系统进化树的基部,斑腿蝗科、斑翅蝗科、剑角蝗科、槌角蝗科、网翅蝗科和癞蝗科形成另一个分支。这种划分方式更符合Otte系统,不同之处在于癞蝗科没能从后一分支中分离出来。更多还原

【Abstract】 Mitochondrial play a central role in metabolism,disease and aging.They are the site of oxidative phosphorylation,as well as a variety of other biochemical functions. Mitochondrial genome is a genome that separate from the nuclear chromatin,with its own systems for DNA replication,transcription,and commonly is a double-strand closed-circular molecule,and contains 37 genes.Mitochondrial genome is commonly used in studies of molecular phylogenetics,and is the best model for the studies of genome structure,function and evolution.The complete mitochondrial genome sequences for three species of Caelifera, Primnoa arctica,Gomphocerippus rufus and Alulatettix qinlingensis,were sequenced, assembled and annotated.Complete mitogenomes of eight families were compared. The common features of genome structure and organization,sequence and evolution were summarized,these enriches mitochondrial genome data of Orthoptera. Phylogenetic of Acridoidea was reconstructed utilizing 20 insects from Acridoidea and phylogenetic of Cealifera was reconstructed utilizing 10 insects.The conclusions of these studies showed as following:1.The genes arrangement of mitochondrial genome in Primnoa arctica, Gomphocerippus rufus and Alulatettix qinlingensis were identical to that of Locusta migratoria,and were different from ancient insects in that the translocation of tRNAK-tRNAD was rearrangement as tRNAD-tRNAK.This rearrangement was common in mitogenome of Acridoidea.2.Average A+T content of mitogenome from Acridoidea was found higher than that of Tettigonioidea.3.The usage of synonymous codons was markedly correlated with the nucleotide composition at the third codon position.Generally,the codons whose third position are A or T were frequently used.4.The longest space of genes in mitogenome of Acridoidea was usually locates between tRNASer-UCN and ndl.There are 7bp long overlapping regions located between atp8/atp6 and nad4/nad4L.The interspaces and overlapping regions were similar at the feature of number and length in mitogenome of species.5.ATG is the most frequently used starting codon,ATT is the second frequently used one,ATA is the one that being used lessly.TAA is the most frequently used termination codon.Most of nd5 in mitogenome of Orthoptera utilize incomplete termination codon(T/TA) as terminate singnal.6.The mismatched base pairs occur in the tRNA genes,and the majority of these are G-U pairs.7.There was a stem and loop structure located in both A+T rich regions of mitochondrial genome of Primnoa arctica and Gomphocerippus rufus.And the structure was similar with the structure that Saito(2005) found on the same region of L. migratoria.They each has a conserved structure of"TA" on the 5’ of the stem and loop structure.8.Phylogenetic analyses confirmed the monophyly of Oedipodidae and Catantopidae of Xia’s system,and the monophyly of Acrididae and Arcypteridae were not supported.Monophyly of Oedipodinae and Gomphocerinae in One’s system were supported,while monophyly of Catantopinae was not suppoted.Catantopinae, Melanoplinae and Oxyinae formed a sister group.9.The result of reconstruction of systemtic evolution relationship of eight families in Acridoidea concluded that the relationship of evolution is Pyrgomorphidae+Chrotogonidae→Oedipodidae→Pamphagidae→Catantopidae→Acrididae→Gomphoceridae+Arcypteridae.10.This phylogenetic analyses based on mitogenome separated Acridoidea distributing in China into two clades.The first clade is consisted of Chrotogonidae and Pyrgomorphidae,which formed the basic of the tree;the second and large clade is consisted of Catantopidae,Oedipodidae,Acrididae,Gomphoceridae,Arcypteridae and Pamphagidae.These clades are coincident with that in Otte’s classification system,and the difference was that Pamphagidae in our study could not be separated from the clade of Acridoidea.更多还原