中国地鼠线粒体基因组的序列分析与分子进化

目的 获得中国地鼠线粒体基因组序列,为线粒体疾病模型提供分子数据.方法 参照近缘物种的线粒体基因组序列,设计27对特异引物,采用TD-PCR及测序技术获得了中国地鼠的线粒体全基因组序列,分析了其基因组特点和各基因的定位.还结合GenBank中已发表的其他5种啮齿类动物的线粒体基因组序列,探讨啮齿类动物不同科间的系统进化关系.结果 中国地鼠线粒体基因组全长为16 283 bp,碱基组成为33.53％A、30.50％T、12.98％G、22.80％C,包括13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码基因控制区.中国地鼠和金黄地鼠亲缘关系最近.结论 中国地鼠线粒体基因组各基因长度、位置与典型的啮齿类动物相似,其编码蛋白质区域和rRNA基因与其他啮齿类动物具有很高的同源性,显示线粒体基因组在进化上十分保守.5种动物的分子系统进化树与传统分类地位一致.     

缅甸陆龟线粒体全基因组的测序及分析

本文参照近缘物种的线粒体基因组序列,设计17对特异引物,采用LD-PCR、PCR及测序技术获得了我国广西产缅甸陆龟的线粒体全基因组序列,分析了其基因组特点和各基因的定位。结果表明:缅甸陆龟线粒体基因组全长为16813bp,碱基组成为35.30%A、26.47%T、12.09%G、26.14%C,包括13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码基因控制区(D-Loop区)。缅甸陆龟线粒体基因组各基因长度、位置与典型的脊椎动物相似,其编码蛋白质区域和rRNA基因与其它脊椎动物具有很高的同源性,显示龟类线粒体基因组在进化上十分保守。将缅甸陆龟的线粒体基因组序列提交到GenBank,获得的检索号为DQ656607。本文还结合GenBank中已发表的其它16种龟鳖类动物的线粒体基因组序列,探讨龟鳖类动物不同科间的系统进化关系。     

短尾蝮蛇线粒体基因组全序列分析

参照近缘物种的线粒体基因序列设计并筛选得到8对引物,结合TA克隆和步移测序获得了全长17227bp的短尾蝮蛇线粒体基因组全序列.与多数蛇类线粒体基因组类似,其共编码包括13个蛋白、2个rRNA和22个tRNA在内的37个基因,另外还包含2个非编码的富含AT的控制区.基因间排列紧凑,多数基因间间隔极短甚至发生重叠.除nad1、cox1和nad3外,多数蛋白编码基因均以ATG作为起始密码子,终止密码子的使用则存在TAA、AGA、AGG和不完全的T4种情况.基于合并的19个tRNA基因序列组合数据采用NJ、MP和ME3种算法对21种蛇进行了初步的系统发育分析,结果表明,各主要分类单元之间的亲缘关系与前人基于形态学、线粒体12SrRNA和cytb基因序列研究的结论完全一致,这证实了基于合并的线粒体tRNA基因序列进行蛇类物种DNA分子系统学研究的可行性.     

广西华珊瑚蛇线粒体基因组全序列测定与分析

本研究对眼镜蛇科广西华珊瑚蛇（Sinomicrurus peinani）线粒体基因组序列进行测定与分析,并探究其与近缘种的系统发育关系。结果表明,广西华珊瑚蛇线粒体基因组是一条全长19 477 bp的环状DNA,基因组碱基构成为A（33.4%）、T（28.1%）、C（26.6%）和G（11.9%）。共编码38个基因,包含2个核糖体RNA（rRNA）基因、22个转移RNA（tRNA）基因、13个蛋白质编码基因及1个线粒体基因控制区（D-loop）。13个蛋白质编码基因均采用AUG作为起始密码子,UAA和UGA作为终止密码子;蛋白质编码基因编码频率较高的氨基酸分别为亮氨酸（Leu）、异亮氨酸（Ile）、苏氨酸（Thr）和丝氨酸（Ser）;相对密码子使用度（RSCU）频率最高的4个密码子依次是CGA、UGA、CUA和CCA。22个tRNA,除tRNASer（一臂两环）外其他均可形成典型三叶草结构。基于眼镜蛇科线粒体基因组系统发育分析结果表明,与广西华珊瑚蛇关系最密切的是中华珊瑚蛇（Sinomicrurus macclellandi）,其次是孟加拉眼镜蛇（Naja kaouthia）与眼镜王蛇（Ophiophagus hannah）。     

中国狼（Canis lupus chanco）线粒体全基因组序列分析

参照近缘物种线粒体全基因组序列,设计14 对特异引物,采用PCR产物直接测序法测得中国狼线粒体基因组全序列,并分析其基因组特点和各基因的定位.用pDRAW32软件,预测12种限制性酶的酶切图谱.结果表明,中国狼线粒体基因组全长16 774 bp, 包括13 个蛋白质编码基因、2 个rRNA 基因、22 个tRNA 基因和1个非编码控制区.除ND2、 ND3和ND5 基因以ATA作为起始密码子外,其它基因的起始密码子均为ATG.除COXⅢ、 ND4、 ND3基因的终止密码子分别为不完全的T,T,TA;ND2,COXⅡ,Cytb基因的终止密码子分别是TAG、TAG、AGA外;其余基因均以TAA作为终止密码子,而欧亚狼和狗COXⅡ基因则以TAA终止.基于近缘哺乳动物15种近缘物种的线粒体基因组的12S rRNA 和 16S rRNA 基因全序列,用邻近法、最大简约法和最大似然法构建系统进化树,系统进化关系与传统的系统分类基本一致.并在已有文献的基础上,探讨了中国狼的进化地位.     

枣食芽象甲线粒体基因组全序列测定与系统发育分析

【目的】从线粒体基因组水平上探讨枣食芽象甲Scythropus yasumatsui与近缘种的系统发育关系。【方法】利用Illumina MiSeq测序平台对枣食芽象甲线粒体基因组进行测序,对基因组序列进行拼装、注释和特征分析;利用贝叶斯法和最大似然法构建基于象甲科13个物种的线粒体基因组13个蛋白质编码基因核苷酸序列的系统发育树。【结果】结果表明,枣食芽象甲线粒体基因组全长为16 472 bp (GenBank登录号: MF807224),包含13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和2个非编码控制区,37个基因的排列顺序与祖先昆虫的线粒体基因排列顺序一致。13个蛋白质编码基因的起始密码子为ATN,其中除了cob和nad1基因的完全终止密码子为TAG外,其余11个基因的完全终止密码子为TA(A)。22个tRNA基因中除了trnS1缺少DHU臂,反密码子由GCT变为TCT外,其余均能形成典型的三叶草结构。基于13个蛋白质编码基因序列构建的系统发育树结果显示,象甲科8个亚科系统发育关系为：(((隐喙象亚科(Cryptorhynchinae)+(象虫亚科(Curculioninae)+魔喙象亚科(Molytinae)))+长小蠹亚科(Platypodinae))+(粗喙象亚科(Entiminae)+Cyclominae亚科))+隐颏象亚科(Dryophthorinae)+小蠹亚科(Scolytinae))。【结论】在13种象甲科昆虫物种中,同属于粗喙象亚科的枣食芽象甲与南美果树象甲Naupactus xanthographus在系统发育树中聚为同一分支,表明基于线粒体基因组全序列的分子系统发育结果与传统的形态分类结果是一致的。     

中华鳖线粒体基因组序列分析

参照近源物种线粒体基因组序列,设计17对特异引物,采用PCR产物直接测序法测得中华鳖线粒体基因组全序列.初步分析其基因组特点和各基因的定位,用pDRAW32软件预测12种限制性酶对其的酶切图谱.结果表明,中华鳖线粒体基因组全长17364bp,核苷酸组成为35.23%A、27.26%T、25.73%C、11.78%G,包括13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码控制区.基于线粒体基因组编码的13个蛋白质的氨基酸序列,用NJ法和MP法构建系统进化树,分析6种龟鳖类动物之间的亲缘关系,与传统的系统分类基本一致,初步确定淡水龟科与海龟科的亲缘关系比与龟科的亲缘关系要近.     

乌龟线粒体全基因组序列和结构分析

龟鳖类同其它类群脊椎动物的系统进化关系一直存在争论。为进一步从分子水平上探讨这一问题,本文参照近源物种的线粒体基因组,设计了16对特异引物,采用PCR产物直接测序法测得了乌龟线粒体基因组全序列。结果表明:乌龟线粒体基因组序列全长16576bp,包括2个rRNA基因、22个tRNA基因、13个蛋白质编码基因和1个非编码控制区。乌龟线粒体基因组结构和基因排列顺序与其它龟鳖类相同,在“WANCY区”包含一个“stemloop”结构,ND3基因174位点存在一个额外插入的腺苷酸(A)。本文通过比较分析结构基因在主要脊椎动物类群中的排列顺序,探讨了龟鳖类与其它主要脊椎动物类群的系统进化关系     

小麂、赤麂、黑麂mtDNA序列变异性及反映的进化关系

利用已测定的鹿科麂亚科动物小麂、赤麂、黑麂的线粒体全基因组序列,统计它们各自连接在一起的13个蛋白编码基因、22个tRNA基因、2个rRNA基因和1个控制区序列的碱基长度和组成,计算rRNA基因遗传距离,估算分歧时间,比较蛋白编码基因的碱基水平和氨基酸水平上的差异,基于连接在一起的13个氨基酸序列,以羊为外群,通过邻位相连法和最大简约性法构建进化树,探讨小麂、赤麂、黑麂的进化关系。结果表明,小麂是较原始的物种,赤麂和黑麂较为近缘,是从类似小麂的祖先演化而来。     

眼镜王蛇线粒体基因组全序列分析

参照近缘物种线粒体基因序列共设计和合成了8对引物, 结合Ex Taq-PCR、TA克隆和步移测序技术, 文章首次获得眼镜王蛇线粒体基因组全序列(GenBank登录号: EU_921899)。该基因组全长17 267 bp, 共编码13个蛋白、2个rRNA、23个tRNA-- 其中tRNA-Ile基因发生了复制, 属于一种新的蛇类物种线粒体基因排列模式, 另外还含有2个非编码的富含AT的控制区。除了8个tRNA基因和1个蛋白基因由L链编码外, 其余均由H链编码, 其中H链编码基因的A和T含量接近, 而L链上A的含量则明显高于T。基于21种蛇合并的“12S＋16S”rRNA基因序列的系统发育分析表明, 眼镜王蛇属与眼镜蛇属亲缘关系较近, 两者与环蛇属共同构成一个单系群。作为国内外眼镜王蛇线粒体基因组全序列的首次报道, 上述结果对于蛇类物种分子系统发育和进化研究具有重要意义。     

白腹鼠属几个相似种的差异探讨

到目前为止,安氏白腹鼠、川西白腹鼠、社鼠、针毛鼠作为独立种没有多大的争议。但在形态上,安氏白腹鼠与川西白腹鼠、川西白腹鼠与社鼠、社鼠与针毛鼠具有较大的相似性,差异微小,有大量的中间过渡类型。因此,要对这些种进行准确鉴定非常困难。本文首先对这4个种的阴茎形态进行了比较,发现它们确实是独立的种。它们之间的主要区别在于尿道小瓣形态、位置及阴茎骨形态和长短比例上：（1）安氏白腹鼠,外环层有明显的乳突,尿道小瓣分两叉,位置很低,阴茎骨近支显著长于远支;（2）川西白腹鼠,外环层光滑,尿道小瓣位置较高,阴茎骨近支很细而直,近支明显长于远支;（3）社鼠,尿道小瓣较小,位置很低,呈舌状,阴茎骨近支较粗,显著向腹面弯曲,远支粗大且较长,近支长略大于远支;（4）针毛鼠,尿道小瓣位置较高,呈舌状,阴茎骨近支较细弱,显著向腹面弯曲,远支粗大且较长。采用单因素方差分析发现,川西白腹鼠与社鼠在眶间宽、上齿列长和下齿列长等方面差异显著;社鼠和针毛鼠在后头宽、眶间宽和听泡长等方面差异显著;安氏白腹鼠与其他3个种均具有显著的差异。     

Phylogenetic relationships in genus Niviventer (Rodentia: Muridae) in China inferred from complete mitochondrial cytochrome b gene

Chinese species of the genus Niviventer, predominantly distributed in the southeastern Tibetan Plateau and in Taiwan, are a diverse group and have not yet received a thorough molecular phylogenetic analysis. Here, we reconstructed the phylogenetic relationships of 32 specimens representing nine Chinese species of Niviventer, based on sequences of the complete mitochondrial cytochrome b gene. Maximum parsimony, maximum likelihood and Bayesian analysis resulted in three consistent trees, each supported by high bootstrap values. The results showed that the Niviventer species included here are monophyletic. The nine species were classified into three distinct clades: clade A with Niviventer brahma, N. confucianus, N. coxingi, N. culturatus, N. eha and N. fulvescens; clade B with N. andersoni and N. excelsior; clade C with N. cremoriventer. Our results also suggested that N. culturatus should be a valid species rather than a subspecies of N. confucianus. Divergence times among species were calibrated according to the middle-late Pleistocene (1.2-0.13 Mya) fossil records of N. confucianus. The results demonstrated that the first radiation event of the genus Niviventer occurred in early Pleistocene (about 1.66 Mya), followed by the divergence of clades A and B at about 1.46 Mya. Most of the extant Niviventer species appeared during early to middle Pleistocene (about 1.29-0.67 Mya). These divergence times are coincidental with the last uplift events of the Tibetan Plateau, Kun-Huang movement, Pleistocene glaciations and the vicariant formation of Taiwan Strait. Consequently geographical events and Pleistocene glaciations have played a great role in the diversification of Niviventer.     

The complete mitochondrial genome sequence of the black-capped capuchin (Cebus apella)

The phylogenetic relationships of primates have been extensively investigated, but key issues remain unresolved. Complete mitochondrial genome (mitogenome) data have many advantages in phylogenetic analyses, but such data are available for only 46 primate species. In this work, we determined the complete mitogenome sequence of the black-capped capuchin (Cebus apella). The genome was 16,538 bp in size and consisted of 13 protein-coding genes, 22 tRNAs, two rRNAs and a control region. The genome organization, nucleotide composition and codon usage did not differ significantly from those of other primates. The control region contained several distinct repeat motifs, including a putative termination-associated sequence (TAS) and several conserved sequence blocks (CSB-F, E, D, C, B and 1). Among the protein-coding genes, the COII gene had lower nonsynonymous and synonymous substitutions rates while the ATP8 and ND4 genes had higher rates. A phylogenetic analysis using Maximum likelihood and Bayesian methods and the complete mitogenome data for platyrrhine species confirmed the basal position of the Callicebinae and the sister relationship between Atelinae and Cebidae, as well as the sister relationship between Aotinae (Aotus) and Cebinae (Cebus/Saimiri) in Cebidae. These conclusions agreed with the most recent molecular phylogenetic investigations on primates. This work provides a framework for the use of complete mitogenome information in phylogenetic analyses of the Platyrrhini and primates in general.     

五种啮齿类动物mtDNA蛋白编码基因序列变化的比较

目的利用本实验室测定的中国地鼠、金黄地鼠和GenBank中田鼠、小鼠、大鼠的线粒体全基因组序列,比较分析五种啮齿类动物的mtDNA蛋白编码基因序列的变异,探讨其分子进化关系。方法将五种动物各自的13个蛋白编码基因分别连接成一个序列,用DNAstar-EditSeq分析软件计算每个序列的碱基长度和组成,计算蛋白编码基因的碱基和氨基酸的差异。以人为外群,基于连接在一起的13个蛋白编码基因的氨基酸序列,用MEGA4.0软件通过最大简约性法(MP)和非加权成对平均数法(UPGMA)构建进化树。结果在五种啮齿动物的13个蛋白基因序列中,A、T、C、G碱基的平均含量为32.4%、29.6%、26.2%和11.9%,中国地鼠mtDNA各蛋白编码序列以及其编码的氨基酸序列与其他物种相比,与金黄地鼠的相应序列差异最小,与大鼠mtDNA各蛋白编码序列以及其编码的氨基酸序列差异较大。分子进化树也显示中国地鼠和金黄地鼠的亲缘关系最近,与小鼠、大鼠存在的差异相对大。结论五种动物的碱基组成的百分比中显示G的相对缺乏,相互之间的进化关系与传统的分类地位基本吻合。     

高桥仁蚧线粒体全基因组测序与分析

【目的】测定和分析半翅目(Hemiptera)仁蚧科(Aclerdidae)首个线粒体全基因组——高桥仁蚧Aclerda takahashii线粒体全基因组序列,并探讨与其他蚧虫类群的系统发育关系。【方法】基于Illumina测序技术进行高桥仁蚧线粒体全基因组测序,并进行生物信息学分析;基于已报道的15科31种半翅目昆虫的线粒体全基因组序列运用最大似然法(ML)和贝叶斯推断法(BI)构建半翅目系统发育树。【结果】高桥仁蚧线粒体基因组全长16 599 bp, AT含量高达84.51%。在该线粒体基因组中,缩减tRNA非常普遍, 10个tRNA缺失二氢尿嘧啶(DHU)臂或TΨC臂, tRNAser(S1)和tRNAser(S2)缺失DHU臂和TΨC臂。系统发育树显示, 与仁蚧科亲缘关系最近的是蚧科(Coccidae)。【结论】本研究报道了首个仁蚧科的线粒体基因组, 发现在高桥仁蚧线粒体基因组中存在普遍的tRNA缺臂现象, 为进一步系统地研究蚧虫线粒体基因组提供了数据支持。     

The complete mitochondrial genome sequence of the little grebe (<Emphasis Type="Italic">Tachybaptus ruficollis</Emphasis>)

Podicipediformes comprises one family (Podicipedidae) including 6 genera, 22 species, and the phylogenetic placement of this order was still in debate. In this study, we sequenced the complete mitochondrial genome (mitogenome) of little grebe (Tachybaptus ruficollis) in Podicipediformes, and explored the phylogenetic position of this order with mitogenome sequences of 21 species from ten families in seven orders. The genome was 16,688 bp in length, and contained 37 genes typical to avian mitogenomes and one control region. The gene organization and characters were similar with other two mitogenomes available in Podicipediformes to date. Phylogenetic tree was constructed with Bayesian method based on mitogenome sequences excluding the control regions. The results supported the closest relationship between Podicipediformes and Phoenicopteriformes, and the topology of our tree was generally similar with the conclusions of previous molecular systematic investigations. Our results furtherly proved the validity of mitogenome data in taxonomic and phylogenetic studies.     

Cross-species transmission in the speciation of the currently known murinae-associated hantaviruses

To gain more insight into the phylogeny of Dabieshan virus (DBSV), carried by Niviventer confucianus and other Murinae-associated hantaviruses, genome sequences of novel variants of DBSV were recovered from Niviventer rats trapped in the mountainous areas of Wenzhou, China. Genetic analyses show that all known genetic variants of DBSV, including the ones identified in this study, are distinct from other Murinae-associated hantaviruses. DBSV variants show geographic clustering and high intraspecies diversity. The data suggest that DBSV is a distinct species in the genus Hantavirus. Interestingly, DBSV shows the highest sequence identity to Hantaan virus (HTNV), with a >7% difference in the sequences of the N, GPC, and L proteins, while N. confucianus is more closely related to Rattus norvegicus (the host of Seoul virus [SEOV]) than to Apodemus agrarius (the host of HTNV and Saaremaa virus [SAAV]). Further genetic analyses of all known Murinae-associated hantaviruses (both established and tentative species) show that many of them, including DBSV, may have originated from host switching. The estimation of evolutionary rates and divergence time supports the role of cross-species transmission in the evolution of Murinae-associated hantaviruses. The detection of positive selection suggests that genetic drift may contribute to the speciation of Murinae-associated hantaviruses and that adaptation has a role as well.     

毛茛科和芍药科叶绿体基因组密码子特征和系统发育比较

为研究和比较毛茛科和芍药科叶绿体基因组密码子使用模式和系统进化关系,以完成测序的毛茛科33种植物、芍药科7种植物叶绿体基因组为材料,采用分析软件CodonW在线软件CUSP和R软件对叶绿体基因进行密码子特征分析。用MAFFT 软件,MEGA软件进行系统发育分析。研究结果表明芍药科植物叶绿体基因组和毛茛科植物（耧斗菜属除外）叶绿体基因组高频密码子一致性高,具有29个高频密码子,基本偏向与于A/U结尾,但最优密码子存在差异。毛茛科和芍药科叶绿体基因组密码子偏好性的形成因素主要受自然选择的影响,且芍药科叶绿体基因组密码子偏好性受自然选择的影响大于毛茛科。基于叶绿体基因组全序列和基于叶绿体基因组CDS序列的系统进化关系表明,芍药科基于叶绿体基因组全序列和基于叶绿体基因组CDS序列的系统进化关系虽然部分不同,但都可以被划分为芍药组和牡丹组。毛茛科基于叶绿体基因组的系统进化关系不符合中国植物志分类关系,但支持把毛茛科划分为4亚科14族。系统进化分析结果也支持芍药科独立于毛茛科和毛茛目,被划分到虎耳草目,同时证明了叶绿体基因组作为超级DNA条形码的可行性。     

Complete mitochondrial genome of Oxuderces dentatus (Perciformes, Gobioidei)

The complete mitochondrial genome (mitogenome) of Oxuderces dentatus was determined first. The genome was 17,116?bp in length and consisted of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions [the control region (CR) and the origin of the light strand replication], the gene composition and order of which was similar to most other vertebrates. The overall base composition of the heavy strand was T 27.9%, C 26.8%, A 30.2%, and G 15.1%, with a slight A+T bias of 58.1%. In addition to the discrete and conserved sequence blocks, unusual long tandem repeat unit (three 150-bp tandem repeat units and an incomplete copy of 146?bp) was also detected within CR. This mitogenome sequence data would play an important role in population genetics and phylogenetic analysis of the Gobioidei.     

Mitogenome phylogenetics: the impact of using single regions and partitioning schemes on topology, substitution rate and divergence time estimation

The availability of mitochondrial genome sequences is growing as a result of recent technological advances in molecular biology. In phylogenetic analyses, the complete mitogenome is increasingly becoming the marker of choice, usually providing better phylogenetic resolution and precision relative to traditional markers such as cytochrome b (CYTB) and the control region (CR). In some cases, the differences in phylogenetic estimates between mitogenomic and single-gene markers have yielded incongruent conclusions. By comparing phylogenetic estimates made from different genes, we identified the most informative mitochondrial regions and evaluated the minimum amount of data necessary to reproduce the same results as the mitogenome. We compared results among individual genes and the mitogenome for recently published complete mitogenome datasets of selected delphinids (Delphinidae) and killer whales (genus Orcinus). Using Bayesian phylogenetic methods, we investigated differences in estimation of topologies, divergence dates, and clock-like behavior among genes for both datasets. Although the most informative regions were not the same for each taxonomic group (COX1, CYTB, ND3 and ATP6 for Orcinus, and ND1, COX1 and ND4 for Delphinidae), in both cases they were equivalent to less than a quarter of the complete mitogenome. This suggests that gene information content can vary among groups, but can be adequately represented by a portion of the complete sequence. Although our results indicate that complete mitogenomes provide the highest phylogenetic resolution and most precise date estimates, a minimum amount of data can be selected using our approach when the complete sequence is unavailable. Studies based on single genes can benefit from the addition of a few more mitochondrial markers, producing topologies and date estimates similar to those obtained using the entire mitogenome.