鞘翅目昆虫线粒体基因组研究进展

鞘翅目（Coleoptera）是世界上最具多样性的类群,具有很高的生态和形态多样性,这些多样性吸引了很多进化生物学家和分类学家的关注。随着分子生物学的发展,分子生物学技术广泛应用于鞘翅目系统学的研究,但随着研究的深入,简单的分子片段已经不能满足研究的需求,需要发掘更新的分子标记。近年来,线粒体全基因组已经成为鞘翅目分子系统学研究中很重要的分子标记之一,并广泛地应用于鞘翅目昆虫各个阶元的研究中。本文就鞘翅目线粒体全基因组的概况、研究进展及存在问题进行了总结和讨论。目前,鞘翅目线粒体基因组的研究主要包括物种线粒体基因组组成与结构、分子系统学和分子进化等方面。线粒体基因组在解决系统发育和进化方面表现出了很多的优越性,然而也存在着一些缺点,如序列难获得、基因类型单一、各基因进化速率不同、应用较局限等。     

昆虫比较线粒体基因组学研究进展

动物线粒体基因组因其基因组成稳定、基因排列相对保守、普遍为母系遗传、极少发生重组等而被广泛应用于进化与系统发育等研究。目前,昆虫中已有356个线粒体基因组序列被测定,代表了33个目中的28个目。大量比较基因组学研究使得我们对昆虫线粒体基因组的特征与进化方式有了较为清晰的认识。本文对昆虫线粒体基因组的测序进展、基因组的结构特征、碱基组成、控制区的特征、基因重排及其机理、进化速率及其在昆虫系统发育研究中的应用等方面的研究进展进行介绍。     

异刺草螽线粒体基因组序列分析

本研究采用高通量测序技术对异刺草螽的基因组进行测序,并组装得到了完整的线粒体基因组序列。结果显示:异刺草螽线粒体基因组序列全长16 038 bp,包含13个蛋白质编码基因、22个t RNA基因、2个r RNA基因和1个控制区。异刺草螽线粒体基因组的总碱基组成如下:A为37.3%、C为15.4%、G为10.3%、T为36.9%,A+T含量较高,为74.2%。异刺草螽线粒体基因的排列与祖先序列相同,该线粒体基因组序列为直翅目螽斯科的系统发生和进化研究提供了重要的分子基础。     

龟鳖类线粒体全基因组的比较研究

在基因组水平上,比较分析了已登录GenBank的19种龟鳖类线粒体全基因组的结构特征．结果表明：1）除平胸龟、扁陆龟外,其余17种龟鳖类线粒体基因组结构、基因排列顺序均与典型的脊椎动物相似,显示龟鳖类线粒体基因组在进化上十分保守：2）19种龟鳖类线粒体全基因组和各部分的碱基组成均表现出高AT、低G含量的偏向,在控制区中表现尤为明显：3）除中华鳖和白腹摄龟外,其余种类的某些蛋白编码基因中都存在一个或多个额外插入的核苷酸：4）除侧颈龟亚目的非洲侧颈龟外,其余18种曲颈龟线粒体DNA的“WANCY”区中都存在轻链复制起始点（OL）,且它们的二级结构、核苷酸组成高度保守,推测该结构可能是曲颈龟亚目的一个共同特征：5）部分龟鳖类线粒体基因组控制区3’端存在大片段（200～450bp）的重复序列,某些龟鳖类中有由（AT）构成的微卫星序列,并且这些拷贝序列在种间表现出一定的差异,其可作为特异的分子标记,对于龟鳖类动物系统学的研究、亲缘关系的鉴定、物种多样性的保护和研究等方面具有重要的参考价值．     

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     

糙刺参（Stichopus horrens）线粒体基因组及一种新的基因排列顺序

线粒体基因组序列在后口动物进化研究中有着重要的作用.本研究使用PCR方法获得糙刺参（Stichopus horrens）线粒体全基因组序列.该序列全长16257bp,包含13个蛋白编码基因,22个tRNA基因和2个rRNA基因.除了一个蛋白编码基因（nad6）和5个tRNA基因（tRNASer（UCN）,tRNAGln,tRNAAla,tRNAVal,tRNAAsp）在轻链上编码外,其余的基因都在重链上编码.糙刺参线粒体重链碱基有30.8%A,23.7%C,16.2%G,和29.3%T构成.假定的线粒体控制区长675bp.基因间间隔碱基长1～50bp,共227bp.11个基因间有重叠碱基,长1～10bp,共25bp.13个蛋白编码基因起始密码子均为ATG,终止密码子大多为TAA（nad3和nad4的为TAG）.亮氨酸编码频率最高（16.29%）,随后是丝氨酸（10.34%）和苯丙氨酸（8.37%）.所有的22个tRNA基因均能折叠成三叶草结构.通过和其他4种海参的线粒体基因排列顺序比较,本研究发现糙刺参的线粒体基因排列顺序是一种新的排列方式.     

短尾猴陈旧粪便中DNA的提取

分子粪便学（Molecular scatology）是一门将传统粪便分析方法与分子生物学技术相结合,以动物粪便为实验材料进行多领域研究的学科（魏辅等,2001）。虽然该方法已在野生濒危动物保护遗传学和分子生态学研究中发挥了很大作用（Kohnand Wayne,1997）,但目前大多数分子粪便学研究中使用的材料是新鲜粪便,从保存时间很长的陈旧粪便中很难提取到高质量的DNA用于PCR扩增以及序列分析,严重制约了分子粪便学的广泛应用（Wasser et al．,1997;Constable et al．,2001;Murphy et al．．2002）。     

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.     

多位点DNA指纹技术在保加利亚普通田鼠中的应用探讨

DNA指纹是一种重要的现代分子遗传学标记技术（Jeffreys et al．,1985）,它所揭示的是生物体大量的、无遗传编码信息的、具有高度多态性的卫星DNA（Chen,1996）。这些DNA序列往往占据了生物体基因组总量的80％以上,由于它不编码蛋白基因,在系统发育过程中,通常不被自然选择和人工选择,使得生物变异积累形成个体基因组间的巨大差异。因此,DNA指纹受到生物学家的青睐,以用于生物个体和群体的基因组分析（Burke and Bruford,1987;Buitmap et al．,1991;Weising et al．,1995）。     

稻纵卷叶螟线粒体基因组及细胞色素C氧化酶基因分析

线粒体基因是研究生物系统进化的重要分子标记,本文对迁飞昆虫稻纵卷叶螟Cnaphalocrocis medinalis线粒体DNA进行扩增及注释,结果表明:稻纵卷叶螟线粒体基因组全长15377bp,AT含量为82%,基因组结构为鳞翅目所特有的CR-M-I-Q结构。以细胞色素C氧化酶基因(COX)为分子标记,对有翅类昆虫22个物种进行系统树构建,发现与昆虫翅的发生高度吻合。对存在于PCGs终止密码子判定的争议予以阐明并提出注释建议,有利于今后动物线粒体基因组序列注释的统一规范。     

Nucleotide sequence of the Aspergillus nidulans mitochondrial gene coding for the small ribosomal subunit RNA: homology to E. coli 16S rRNA.

The complete primary structure of the 1437 bp gene coding for mitochondrial 15S rRNA and its flanking regions was determined by Maxam-Gilbert sequencing of cloned HindIII fragment H3 of A. nidulans mtDNA. The gene product reveals significant homology (59%) to E. coli 16S rRNA, and the potential secondary structures of both rRNA molecules are very similar, except that the hairpin structures 7, 8 and 30 of the Brimacombe 16S rRNA model are deleted, and that two sequences of 8 and 31 nucleotides are inserted in the mitochondrial species.     

Molecular identification of crocodile species using novel primers for forensic analysis

All crocodilians are under varying degrees of threat due to over exploitation and these species have been listed in Appendix I or II of CITES. The lack of molecular techniques for the identification of confiscated samples makes it difficult to enforce the law. Conclusive forensic identification of species requires a complete gene sequence which is difficult in case of degraded samples. We have developed two novel sets of primers to amplify two partial cytochrome b gene sequences of six crocodile species i.e. Crocodylus palustris, Crocodylus porosus, Crocodylus siamensis, Crocodylus niloticus, Gavialis gangeticus and Caiman crocodilus. These partial sequences were edited to give a complete cyt b gene sequence, which can be used as an effective tool for forensic authentication of crocodile species. A phylogeny of crocodile species was reconstructed using these sequences. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these ancient species.     

Crocodilian phylogeny inferred from twelve mitochondrial protein-coding genes, with new complete mitochondrial genomic sequences for Crocodylus acutus and Crocodylus novaeguineae

We report complete mitochondrial genomic sequences for Crocodylus acutus and Crocodylus novaeguineae, whose gene orders match those of other crocodilians. Phylogenetic analyses based on the sequences of 12 mitochondrial protein-coding genes support monophyly of two crocodilian taxonomic families, Alligatoridae (genera Alligator, Caiman, and Paleosuchus) and Crocodylidae (genera Crocodylus, Gavialis, Mecistops, Osteolaemus, and Tomistoma). Our results are consistent with monophyly of all crocodilian genera. Within Alligatoridae, genus Alligator is the sister taxon of a clade comprising Caiman and Paleosuchus. Within Crocodylidae, the basal phylogenetic split separates a clade comprising Gavialis and Tomistoma from a clade comprising Crocodylus, Mecistops, and Osteolaemus. Mecistops and Osteolaemus form the sister taxon to Crocodylus. Within Crocodylus, we sampled five Indopacific species, whose phylogenetic ordering is ((C. mindorensis, C. novaeguineae), (C. porosus, (C. siamensis, C. palustris))). The African species C. niloticus and New World species C. acutus form the sister taxon to the Indopacific species, although our sampling lacks three other New World species and an Australian species of Crocodylus.     

湾鳄(Crocodylus porosus)线粒体基因组全序列结构分析与鳄类系统发生

该文测序了湾鳄的线粒体基因组全序列,全长为16,917bp。湾鳄mtDNA结构与其他脊椎动物相似,由22个tRNA,2个rRNA和13个蛋白质编码基因及1个非编码的控制区(D-loop)所组成。除NADH6和tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer(UCN)、tRNAGlu、tRNAPro在L-链上编码之外,其余基因均在H-链编码。基因排列顺序与已测序的鳄类一致,这显示了鳄类线粒体基因排列顺序上的保守性。但鳄类线粒体基因排列顺序与脊椎动物的典型排列方式相比,有较大的差异,尤其是tRNAPhe基因的重排、tRNASer-tRNAHis-tRNALeu基因族的排列方式等。湾鳄mtDNA和已测序的鳄类一样,缺失轻链复制起始点(OLR)。基于17种鳄mtDNA控制区保守区,采用PAUP4.0最大简约法(Maximumparsimony,MP)构建MP树,邻接法(Neighbor-joiningmethod,NJ)构建NJ树,结果显示:食鱼鳄(Gavialisgangeticus)和假食鱼鳄(Tomistomaschlegelii)聚为一支后再与鳄科(Crocodylidae)的其他物种形成姐妹群,这与基于食鱼鳄和假食鱼鳄的线粒体全序列的分析结果一致,支持将食鱼鳄并入鳄科的观点。结果还支持非洲窄吻鳄(Crocodyluscataphractus)与鳄属(Crocodylus)构成姐妹群,可以单独划分为属的观点。     

A phylogenetic hypothesis for Crocodylus (Crocodylia) based on mitochondrial DNA: evidence for a trans-Atlantic voyage from Africa to the New World

The phylogenetic relationships among extant species of Crocodylus (Crocodylia) have been inconsistently resolved by previous systematic studies. Here we used nearly complete mitochondrial (mt) genomes (～16,200 base pairs) for all described Crocodylus species, eight of which are new to this study, to derive a generally well-supported phylogenetic hypothesis for the genus. Model-based analyses support monophyly of all Asian+Australian species and paraphyly of Crocodylus niloticus (Nile crocodile) with a monophyletic New World clade nested within this species. Wild-caught Nile crocodiles from eastern populations group robustly with the four New World species to the exclusion of Nile crocodiles from western populations, a result that is also favored by parsimony analyses and by various subpartitions of the overall mt dataset. The fossil record of Crocodylus extends back only to the Late Miocene, while the earliest fossils assigned to C. niloticus and to New World Crocodylus are Pliocene. Therefore, in combination with paleontological evidence, mt DNA trees imply a relatively recent migration of Crocodylus from Africa to the Americas, a voyage that would have covered hundreds of miles at sea.     

Nucleotide sequence and evolution of the 18S ribosomal RNA gene in maize mitochondria.

The nucleotide sequence of the gene coding for the 18S ribosomal RNA of maize mitochondria has been determined and a model for the secondary structure is proposed. Dot matrix analysis has been used to compare the extent and distribution of sequence similarities of the entire maize mitochondrial 18S rRNA sequence with that of 15 other small subunit rRNA sequences. The mitochondrial gene shows great similarity to the eubacterial sequences and to the maize chloroplast, and less similarity to mitochondrial rRNA genes in animals and fungi. We propose that this similarity is due to a slow rate of nucleotide divergence in plant mtDNA compared to the mtDNA of animals. Sequence comparisons indicate that the evolution of the maize mitochondrial 18S, chloroplast 16S and nuclear 17S ribosomal genes have been essentially independent, in spite of evidence for DNA transfer between organelles and the nucleus.     

Mitochondrial genomes and divergence times of crocodile newts: inter-islands distribution of Echinotriton andersoni and the origin of a unique repetitive sequence found in Tylototriton mt genomes

Crocodile newts, which constitute the genera Echinotriton and Tylototriton, are known as living fossils, and these genera comprise many endangered species. To identify mitochondrial (mt) genes suitable for future population genetic analyses for endangered taxa, we determined the complete nucleotide sequences of the mt genomes of the Japanese crocodile newt Echinotriton andersoni and Himalayan crocodile newt Tylototriton verrucosus. Although the control region (CR) is known as the most variable mtDNA region in many animal taxa, the CRs of crocodile newts are highly conservative. Rather, the genes of NADH dehydrogenase subunits and ATPase subunit 6 were found to have high sequence divergences and to be usable for population genetics studies. To estimate the inter-population divergence ages of E. andersoni endemic to the Ryukyu Islands, we performed molecular dating analysis using whole and partial mt genomic data. The estimated divergence ages of the inter-island individuals are older than the paleogeographic segmentation ages of the islands, suggesting that the lineage splits of E. andersoni populations were not caused by vicariant events. Our phylogenetic analysis with partial mt sequence data also suggests the existence of at least two more undescribed species in the genus Tylototriton. We also found unusual repeat sequences containing the 3' region of cytochrome apoenzyme b gene, whole tRNA-Thr gene, and a noncoding region (the T-P noncoding region characteristic in caudate mtDNAs) from T. verrucosus mtDNA. Similar repeat sequences were found in two other Tylototriton species. The Tylototriton taxa with the repeats become a monophyletic group, indicating a single origin of the repeat sequences. The intra-and inter-specific comparisons of the repeat sequences suggest the occurrences of homologous recombination-based concerted evolution among the repeat sequences.     

Are crocodiles really monophyletic?--Evidence for subdivisions from sequence and morphological data

Recently, the phylogenetic placement of the African slender snouted crocodile, Crocodylus cataphractus, has come under scrutiny and herein we address this issue using molecular and morphological techniques. Although it is often recognized as being a "basal" form, morphological studies have traditionally placed C. cataphractus within the genus Crocodylus, while molecular studies have suggested that C. cataphractus is very distinct from other Crocodylus. To address the relationship of this species to its congeners we have sequenced portions of two nuclear genes (C-mos 302bp and ODC 294bp), and two mitochondrial genes (ND6-tRNA(glu)-cytB 347bp and control region 457bp). Analyses of these molecular datasets, both as individual gene sequences and as concatenated sequences, support the hypothesis that C. cataphractus is not a member of Crocodylus or Osteolaemus. Examination of 165 morphological characters supports and strengthens our resurrection of an historic genus, Mecistops (Gray 1844) for cataphractus.     

Mitochondrial 16S rRNA sequence diversity of hominoids

We determined nucleotide sequences of the 16S rRNA gene of mitochondrial DNA (mtDNA) (about 1.6 kb) for 35 chimpanzee, 13 bonobo, 10 gorilla, 16 orangutan, and 23 gibbon individuals. We compared those data with published sequences and estimated nucleotide diversity for each species. All the ape species showed higher diversity than human. We also constructed phylogenetic trees and networks. The two orangutan subspecies were clearly separated from each other, and Sumatran orangutans showed much higher nucleotide diversity than Bornean orangutans. Some gibbon species did not form monophyletic clusters, and variation within species was not much different from that among species in the subgenus Hylobates.     

Recombination sequences in plant mitochondrial genomes: diversity and homologies to known mitochondrial genes.

Several plant mitochondrial genomes contain repeated sequences that are postulated to be sites of homologous intragenomic recombination (1-3). In this report, we have used filter hybridizations to investigate sequence relationships between the cloned mitochondrial DNA (mtDNA) recombination repeats from turnip, spinach and maize and total mtDNA isolated from thirteen species of angiosperms. We find that strong sequence homologies exist between the spinach and turnip recombination repeats and essentially all other mitochondrial genomes tested, whereas a major maize recombination repeat does not hybridize to any other mtDNA. The sequences homologous to the turnip repeat do not appear to function in recombination in any other genome, whereas the spinach repeat hybridizes to reiterated sequences within the mitochondrial genomes of wheat and two species of pokeweed that do appear to be sites of recombination. Thus, although intragenomic recombination is a widespread phenomenon in plant mitochondria, it appears that different sequences either serve as substrates for this function in different species, or else surround a relatively short common recombination site which does not cross-hybridize under our experimental conditions. Identified gene sequences from maize mtDNA were used in heterologous hybridizations to show that the repeated sequences implicated in recombination in turnip and spinach/pokeweed/wheat mitochondria include, or are closely linked to genes for subunit II of cytochrome c oxidase and 26S rRNA, respectively. Together with previous studies indicating that the 18S rRNA gene in wheat mtDNA is contained within a recombination repeat (3), these results imply an unexpectedly frequent association between recombination repeats and plant mitochondrial genes.