基于18S rRNA和线粒体16S rRNA基因序列的柳蚕进化分析

为探讨柳蚕Actias selene Hübner与鳞翅目昆虫的系统发育关系,本研究利用PCR扩增获得了柳蚕核糖体18S rRNA和线粒体16S rRNA基因的部分序列,长度分别为391bp和428bp。并采用邻近距离法(NJ)、最大简约法(MP)、类平均聚类法(UPGMA)构建系统进化树。结果表明,柳蚕线粒体16SrRNA基因序列与大蚕蛾科昆虫的16SrRNA基因序列均表现出偏好于碱基AT的倾向。柳蚕与所研究的其它蚕类的遗传距离介于0.016至0.140之间,其中与温带柞蚕Antheraea roylii的遗传距离最小,与野桑蚕Bombyx mandarina的遗传距离最大。而基于鳞翅目昆虫18S rRNA基因部分序列的进化分析显示,柳蚕与柞蚕Antheraea pernyi之间的遗传距离最小(0.010),与蓖麻蚕Samia ricini的遗传距离最大(0.017)。     

基于16S rRNA基因序列探讨中国粉蛉科的系统发育关系

本研究测定了粉蛉科Coniopterygidae9属15种昆虫16SrRNA基因部分序列,对序列的碱基组成、转换/颠换比率、遗传距离、变异位点等进行分析。基于16SrRNA基因序列数据,分别采用邻接法（NJ）、最大简约法（MP）和最大相似法（ML）建立粉蛉科分子系统发育关系。研究结果表明：粉蛉亚科的粉蛉属Coniopteryx与重粉蛉属Semilalis是姊妹群,（虫齿）粉蛉属Conwentzia较前二者原始;囊粉蛉亚科的卷粉蛉属Helicoconis和隐粉蛉属Cryptoscenea的亲缘关系较近。曲粉蛉属Coniocompsa和异粉蛉属Heteroconis聚类在一起,但自展值较低。瑕粉蛉属Spiloconis和囊粉蛉属Aleuropteryx的位置在囊粉蛉亚科中不够稳定。     

基于线粒体COⅠ基因的齿小蠹属昆虫DNA条形码研究

齿小蠹属（鞘翅目： 小蠹科）昆虫是植物检疫中经常截获的类群, 为探讨线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因的特定区段作为DNA条形码快速准确鉴定齿小蠹种类的可行性, 以齿小蠹属昆虫为研究对象, 测定分析了线粒体COⅠ基因462 bp碱基序列。序列分析结果显示： 变异位点为259个, 保守位点203个, 简约信息位点181个, 自裔位点78个。所有位点中, A, G, C和T碱基平均含量分别为30.7%, 16.5%, 17.0%和35.8%。A+T含量较高, 为66.5%, 明显高于G+C含量, 表现明显的A+T碱基偏嗜, 且A与T含量相当, 符合昆虫线粒体基因碱基组成的基本特征。转换与颠换结果显示： 该段序列未达到饱和, 可以得到准确的进化分析。利用Kimura 2-parameter模型分析遗传距离得到, 同物种间的遗传距离介于0.002~0.007之间, 不同种间的遗传距离介于0.056~0.431间, 平均遗传距离为0.199, 说明该段序列能够区分不同物种。基于COⅠ基因序列构建的邻接法系统发育树（NJ树）显示, 同一物种聚为同一小支, 且分支自展值均为100%; 近缘种能聚集在一起, 且置信度很高（≥97%）。结果表明应用基于COⅠ基因片段的DNA条形码进行齿小蠹属昆虫分类鉴定具有可行性。     

琥珀蚕线粒体全基因组测序及序列分析

【目的】分析昆虫的线粒体基因组能很好地指示昆虫物种的亲缘关系。本研究旨在探索琥珀蚕Antheraea assama线粒体基因组并在线粒体水平上了解大蚕蛾科(Saturniidae)属及种间的分子系统进化关系。【方法】采用PCR步移法并结合克隆测序的策略,测定了珍稀绢丝昆虫琥珀蚕的线粒体基因组全序列,分析其结构特点和碱基组成;采用邻近距离法(NJ)构建大蚕蛾科及外群共14种昆虫线粒体蛋白质编码基因的系统发育树,并分析琥珀蚕在大蚕蛾科中的系统发育关系。【结果】琥珀蚕线粒体基因组序列全长15 312 bp(Gen Bank登录号:KU301792),包含13个蛋白质编码基因、22个tRNA基因、2个核糖体rRNA基因和一段332 bp的A+T富集区,呈现典型的鳞翅目昆虫线粒体基因组的核苷酸组成及基因排布顺序。分析结果表明,琥珀蚕线粒体基因组中A+T含量高达80.18%,13个蛋白质编码基因中,除了COX1以CGA为起始密码子,其他均为典型的起始密码子ATN。COX1、COX2和ND5均以不完整的T为终止密码子,其余基因都是以典型的TAA或TAG为终止密码子。预测的22个tRNA二级结构中,除tRNASer(AGN)缺乏DHU臂外,其他21个tRNA均能形成典型的三叶草结构。由线粒体蛋白质基因串联序列构建的NJ系统发育树表明,琥珀蚕与柞蚕Antheraea pernyi、天蚕Antheraea yamamai、明目大蚕Antheraea frithi构成鳞翅目大蚕蛾科柞蚕属Antheraea这一分支。在9种大蚕蛾科昆虫中,琥珀蚕与柞蚕属的天蚕亲缘关系最近,与巨大蚕蛾属Attacus的乌桕大蚕Attacus atlas亲缘关系较远。【结论】琥珀蚕线粒体基因组的基因排列方式同其他已测定的鳞翅目昆虫的完全相同。基于线粒体基因组的大蚕蛾科昆虫系统发育关系与传统的形态分类学结果一致,即琥珀蚕隶属于柞蚕属Antheraea。     

基于线粒体ND2基因的中国斑翅蝗科部分种类分子系统学研究（直翅目：蝗总科）

本文的目的是通过对斑翅蝗科部分种类的线粒体ND2基因进行分析,重建斑翅蝗科昆虫的系统发育关系,并探讨分子系统发育关系和传统分类结果的异同。扩增并测定了我国斑翅蝗科10属16种蝗虫的线粒体ND2全基因1 023 bp的序列,对序列的碱基组成、转换颠换、系统发育信号等进行了分析。并基于ND2全基因序列数据,分别采用邻接法（NJ）、最简约法（MP）、最大似然法（ML）和贝叶斯法重建了10属16种蝗虫的系统发育关系。结果表明：斑翅蝗科蝗虫ND2全基因A+T含量平均为74.6%;痂蝗亚科和异痂蝗亚科没能得到区分,建议合并为一个亚科;而斑翅蝗亚科和飞蝗亚科的分类地位还存在争议。     

基于线粒体COⅠ基因序列的小萤叶甲属部分种类分子系统学研究（鞘翅目：叶甲科：萤叶甲亚科）

本文目的是通过对小萤叶甲属部分种类的线粒体COⅠ基因进行比较,探讨小萤叶甲属昆虫进化与寄主植物之间的关系,同时对几种分类地位模糊的昆虫进行分析和归类。测定了我国菱角萤叶甲Galerucella birmanica Jacoby和褐背小萤叶甲Galerucella grisescens Joannis以及小猿叶甲Phaedon brassicae Baly线粒体COⅠ基因720 bp序列,并调用GenBank中小萤叶甲属等其他8种昆虫的同源序列,对序列的碱基组成、转换颠换、遗传距离等进行了分析。并以小猿叶甲为外群,分别采用邻接法（NJ）、最大简约法（MP）和贝叶斯推论法（BI）建立这些种的分子系统发育关系。序列分析结果表明：小萤叶甲属昆虫COⅠ基因A+T含量平均为71.8％,存在较强的A+T含量偏向性,氨基酸的变异率为18.3％; 小萤叶甲属与外群之间的遗传距离(0.169～0.198)远远大于属内种间的距离(0.001～0.134)。依据分子系统树结果我们推测小萤叶甲属昆虫的进化与寄主植物之间有着显著的关系,在传统分类学上曾隶属于其他属的几种昆虫与小萤叶甲昆虫有着更近的亲缘关系。     

基于12S rRNA基因序列探讨崇安地蜥的分类地位

为探讨崇安地蜥Platyplacous sylvaticus的分类地位,测定了崇安地蜥线粒体12S rRNA基因全序列,并从GenBank中下载了东亚产10种草蜥、3种地蜥的同源序列进行分析,采用Mega V2.1软件的NJ法和ME法、PAUP4.0软件的MP法构建分子系统树.结果表明:崇安地蜥线粒体12S rRNA基因全序列(952 bp)中T、C、A、G碱基含量分别为23.1%、22.9%、35.9%、18.1%;与其它同源序列比对后有978 bp,发现321个位点出现变异,占总位点数32.8%,其中199个简约信息位点,为总变异位点的62%;转换/颠换之比平均为2.16.构建的分子系统树中,NJ树和ME树完全一致,与MP树略有差异.3种构树法中崇安地蜥与南台草蜥Takydromus sauteri、峨眉地蜥P.intermedius、先岛地蜥P.dorsalis均聚为一支,崇安地蜥与先岛地蜥亲缘关系最近.本实验结果支持将地蜥属并入草蜥属和取消地蜥亚属的观点.     

云南猕猴mtDNA控制区全序列测定

目的测定云南猕猴线粒体DNA控制区全序列,对其进行鉴定及进化分析。方法利用PCR技术扩增猕猴线粒体DNA控制区全序列,结合GenBank中下载的猕猴参考序列（AY612638）,采用多个生物学软件对序列碱基组成、同源性、转换／颠换比等遗传信息进行分析,并基于邻接法（NJ）和最小进化法（ME）构建系统进化树。结果云南猕猴线粒体DNA控制区全长为（1084-1089）bp,A、T、G和c四种碱基平均含量分别为29．9％、26．9％、12．3％和30．9％,A＋T含量（56．8％）高于G＋C含量（43．2％）。所分析序列间的同源性为91．5％-99．5％,平均核苷酸变异率为4．5％,变异类型包括转换、颠换、插入和缺失4种形式,转换／颠换比值平均为26．1。进化树显示云南猕猴存在两个平行进化的姐妹分支。结论本研究获得了云南猕猴mtDNA控制区全序列,为猕猴进化关系研究及mtDNA控制区功能研究奠定基础。     

内蒙古11种主要草原蝗虫16S rDNA序列分析

测定了内蒙古11种主要草原蝗虫的线粒体基因组16S rDNA序列,并构建了其分子系统树。在获得的505 pb序列中,G+C占302%,A+T占698%,表现出A/T偏向性。在505 bp个碱基中检测到156个多态性位点,占碱基总数的309%。156个多态性位点中包括50个单变异多态性位点（占总位点数的321%）和106个简约信息位点（占总位点数的679%）。分别采用NJ、ME、MP和UPGMA聚类方法构建系统发生树。结果表明,4科的17种蝗虫共聚为6支,其中槌角蝗科、斑腿蝗科与网翅蝗科3个科的蝗虫先聚在一起,再与斑翅蝗科相聚。4种聚类方法中UPGMA聚类法更为符合传统的以形态学为基础的分类体系。     

应用16S rDNA序列探讨斑腿蝗科的单系性及其亚科的分类地位

本文测定了斑腿蝗科10亚科20种蝗虫和其他蝗科3种蝗虫的线粒体16S rDNA部分序列,并从GenBank中下载了15种蝗亚目昆虫的16S rRNA基因相应序列片段。比对后的序列长度是397 bp,其中有196个变异位点,157个简约信息位点,A+T平均含量为71.7%,C+G平均含量为28.3%。以序列差异比值为横坐标,以碱基转换数和颠换数为纵坐标作散点图,结果表明颠换多于转换,且随着差异程度的增加,转换明显出现了饱和。以蚱总科的日本蚱Tetrix japonica和卡尖顶蚱Teredorus carmichaeli作外群,用ME、等权MP、加权MP及贝叶斯法重建系统发生树。分子系统树表明,斑腿蝗科并非是一单系群,该科的切翅蝗亚科与稻蝗亚科也均不是一单系群;卵翅蝗、伪稻蝗和稻蝗三者有很近的亲缘关系;支持将黑蝗亚科和秃蝗亚科合为一个亚科——秃蝗亚科;现行的稻蝗亚科并非一单系群,而是一多系群。分子系统学研究结果和传统的基于形态特征的斑腿蝗科的分类体系有很大的不同。     

Molecular phylogeny of silk producing insects based on internal transcribed spacer DNA1

Silk moths are the best studied silk secreting insects and belong to the families Bombycidae and Saturniidae. The phylogenetic relationship between eleven silk producing insects was analyzed using the complete DNA sequence of the internal transcribed spacer DNA 1 locus. The PCR amplification and sequence analysis showed variation in length ranging from 138 bp (Antheraea polyphemus) to 911 bp (Hyalopora cecropia). Microsatellite sequences were found and was be used to distinguish Saturniidae and Bombycidae members. The nucleotide sequences were aligned manually and used for construction of phylogenetic trees based on Maximum parsimony and Maximum likelihood methods. The topology in both the approaches yielded a similar tree that supports the ancestral position of the Antheraea assama.     

Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA

Molecular phylogeny of some of the economically important silkmoths was derived using three mitochondrial genes, 12S rRNA, 16S rRNA, and COI, and the control region (CR). Maximum likelihood (ML) analyses showed two distinct clades, one consisting of moths from Bombycidae family and the other from Saturniidae family. The mitochondrial CR showed length polymorphisms with indels. The ML analyses for complete mitochondrial genome sequences of Bombyx mori (strains Aojuku, C108, Backokjam, and Xiafang), Japanese and Chinese strains of B. mandarina (Japanese mandarina and Chinese mandarina) and, Antheraea pernyi revealed two distinct clades, one comprising of B. mori strains and the other with B. mandarina, and A. pernyi forming an outgroup. Pairwise distances revealed that all of the strains of B. mori studied are closer to Chinese than to Japanese mandarina. Phylogenetic analyses based on whole mitochondrial genome sequences, the finding of a tandem triplication of a 126bp repeat element only in Japanese mandarina, and chromosome number variation in B. mandarina suggest that B. mori must have shared its recent common ancestor with Chinese mandarina. Another wild species of the Bombycidae family, Theophila religiosa, whose phylogenetic status was not clear, clustered together with the other bombycid moths in the study. Analysis of the interspecific hybrid, A. proylei gave evidence for paternal inheritance of mitochondrial DNA.     

Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkworm species that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA andcoxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) forcoxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.     

Characterization of the complete mitochondrial genome of the giant silkworm moth, Eriogyna pyretorum (Lepidoptera: Saturniidae)

The complete mitochondrial genome (mitogenome) of Eriogyna pyretorum (Lepidoptera: Saturniidae) was determined as being composed of 15,327 base pairs (bp), including 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The arrangement of the PCGs is the same as that found in the other sequenced lepidopteran. The AT skewness for the E. pyretorum mitogenome is slightly negative (-0.031), indicating the occurrence of more Ts than As. The nucleotide composition of the E. pyretorum mitogenome is also biased toward A + T nucleotides (80.82%). All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 and 2 (cox1 and cox2). Two of the 13 PCGs harbor the incomplete termination codon by T. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA, with the exception of trnS1(AGN) and trnS2(UCN). Phylogenetic analysis among the available lepidopteran species supports the current morphology-based hypothesis that Bombycoidea, Geometroidea, Notodontidea, Papilionoidea and Pyraloidea are monophyletic. As has been previously suggested, Bombycidae (Bombyx mori and Bombyx mandarina), Sphingoidae (Manduca sexta) and Saturniidae (Antheraea pernyi, Antheraea yamamai, E. pyretorum and Caligula boisduvalii) formed a group.     

Comparative genetic diversity and genetic structure of three Chinese silkworm species Bombyx mori L. (Lepidoptera: Bombycidae), Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan (Lepidoptera: Saturniidae)

The genetic diversity and genetic structure of three Chinese silkworm species Bombyx mori L., Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan were comparatively assessed based on RAPD markers. At the species level, A. pernyi and B. mori showed high levels of genetic diversity, whereas S. cynthia ricini showed low level of genetic diversity. However, at the strain level, A. pernyi had relatively highest genetic diversity and B. mori had lowest genetic diversity. Analysis of molecular variance (AMOVA) suggested that 60% and 72% of genetic variation resided within strains in A. pernyi and S. cynthia ricini, respectively, whereas only 16% of genetic variation occurred within strains in B. mori. In UPGMA dendrogram, individuals of A. pernyi and B. mori formed the strain-specific genetic clades, whereas those of S. cynthia ricini were distributed in a mixed way. The implications of these results for the conservation and utilization in breeding programs of three silkworm species are discussed.     

Distinctive presence of peritracheal athrocytes in Bombyx mori L. and Bombyx mandarina M. as compared to their absence in several other lepidopteran species

Pericardial cells are present in a wide variety of insects and are thought to constitute the majority of 'athrocytes (nephrocytes)'. In contrast, peritracheal athrocytes have only been observed in Bombyx mori L. Although peritracheal athrocytes have a distinct morphology, it is unknown whether these cells are common to all lepidopterans. We anatomically compared eight lepidopteran species: Bombyx mori L. and Bombyx mandarina M. (Bombycidae); Samia cynthia ricini D. (Saturniidae); Agrius convolvuli L. (Sphingidae); Spodoptera litura F. and Mythimna separata W. (Noctuidae); Pieris rapae L. (Pieridae); and Glyphodes pyloalis W. (Crambidae). Of these species, only Bombyx mori L. and Bombyx mandarina M. possess peritracheal athrocytes.     

拓扑树间的通经拓扑距离

给出了一种新的系统树间的拓扑距离,使用NJ,MP,UPGMA等3种方法对13种动物的线粒体中14个基因（含组合的）DNA序列数据进行系统树的构建,利用分割拓扑距离和本文给出的通经拓扑距离对这14种系统树这间及其与真树进行比较。结果显示,NJ法对获得已知树的有效率最高,MP法次之,UPGMA法最低。这14种DNA序列所构建的系统树与已知树的拓扑距离基本上是随其DNA序列长度增加而减小,但两者的相关系数并未达到显著水平,分割拓扑距离在总体上可反映树间的拓扑结构差异,但其测度精确度比通经拓扑距离要低。     

The complete mitochondrial genome of the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae)

We determined the complete nucleotide sequence of the mitogenome from Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae). The 15,566 bp circular genome contains atypical gene organization and order for lepidopteran mitogenomes. The mitogenome contains the lowest A+T content (80.16%) among the known lepidopteran mitogenome sequences. An unusual feature is the occurrence of more Ts than As, with a slightly negative AT skewness (−0.021), in the composition of the major genome strand. All protein-coding genes are initiated by ATN codons, except for cytochrome oxidase subunit I, which is proposed by the TTAG sequence as observed in other lepidopterans. All transfer RNAs (tRNAs) have a typical clover-leaf structure of mitochondrial tRNA, except for tRNA ^Ser (AGN) , the DHU arm of which could not form a stable stem-loop structure. Two aligned sequence blocks with a length of more than 50 bp and 90% of the sequence identity were identified in the A+T-rich region of the Saturniidae and Bombycoidae species.