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。     

中华雏蝗（Chorthippus chinensis Tarb）线粒体基因组分析

采用Lon-PCR扩增线粒体全基因组和保守引物步移法结合克隆方法测定并拼接获得了中华雏蝗（Chorthippus chinensis Tarb）线粒体基因组全序列．序列的注释和分析结果表 明,中华雏蝗线粒体基因组序列全长15 599 bp,共有13个编码蛋白质基因、22个tRNA基因、2个rRNA基因和1个A+T富集区．基因顺序与非洲飞蝗（Locusta migratoria）相同,也发生了2个 tRNA Asp(D)和tRNALys(K)的倒置．13个编码蛋白质基因都使用了ATN作为起始密码子．除ND1以TAG和ND5的终止密码子为不完全的T外,其余11个编码蛋白质基因的终止密码子都为完整的TAA．6种直翅类昆虫13个蛋白质的氨基酸序列的联合数据集构建的系统树与形态分类系统一致,中华雏蝗与非洲飞蝗为姐妹群,并与东方蝼蛄构成一单系群．     

云斑车蝗线粒体基因组全序列测定与分析

采用长距 PCR 扩增及保守引物步移法并结合克隆测序测定并注释了云斑车蝗 Gastrimargus marmoratus （Thunberg）的线粒体基因组全序列。结果表明：云斑车蝗线粒体基因组全序列为15 904 bp（GenBank登录号为EU527334）,A+T含量略高于非洲飞蝗Locusta migratoria,为76.04%,包括13个蛋白质编码基因,22个tRNA 基因,2个rRNA基因和一段1 057 bp的A+T富集区。蛋白质基因的起始密码子中,除COⅠ和ND5为TTG以外,均为昆虫典型的起始密码子ATN。ND5基因使用了不完全终止密码子T,其余基因均为典型的TAA或TAG。预测了22个tRNA基因的二级结构,发现tRNA^Ser（AGN）缺少DHU臂, tRNA^Ser（UGY）的反密码子环上有9个碱基。预测了云斑车蝗12S和16S rRNA二级结构,分别包括3个结构域30个茎环和6个结构域44个茎环。A+T富集区含有3个串联重复序列。     

基于线粒体ND2基因的直翅目部分种类分子系统发育分析

测定了39种直翅目昆虫线粒体ND2基因全长序列,联合GenBank中41种直翅目昆虫的ND2基因序列,探讨ND2基因在解决直翅目系统发育分析上的功效,为建立直翅目的主要类群之间稳定的系统发育关系提供更多的数据。研究结果表明,直翅目昆虫的ND2基因序列全长为996～1 029 bp,平均长度为1 020 bp,A+T含量平均为73%。用贝叶斯法(Bayesian,BI)、最简约法(maximum parsimony,MP)和最大似然法(maximum likelihood,ML)构建系统树,SH检验显示,RAxML法构建的ML树似然值最大,与PAUP*的ML法构建的ML树差异显著,而与贝叶斯树和简约树没有明显差异。所有系统树都显示直翅目为单系群;而蝗亚目的剑角蝗科、网翅蝗科、槌角蝗科和斑腿蝗科均不是单系群,锥头蝗科与瘤锥蝗科亲缘关系较近,这与Otte分类系统一致。螽亚目基本由两大分支构成,一支是蝼蛄总科和蟋蟀总科聚集而成,且具有很高的置信度;另一大分支由螽斯总科独自构成。     

基于线粒体Cytb基因序列探讨中国斑翅蝗科部分种类的系统发育关系

基于斑翅蝗科14种的线粒体Cytb基因462 bp序列,使用MEGA2和PAUP4.0b软件包进行分析,显示Cytb基因序列具有明显的高A、T偏向性和距离依赖的TS/TV值.采用邻接法(NJ)、最大似然法(MP)和极似然法(ML)分别构建斑翅蝗科4亚科8属的系统发育树,不同算法构建的系统发育树均支持分为四大分支(亚科):A(绿纹蝗属)、B(束颈蝗属)、C((飞蝗属+车蝗属)+小车蝗属)、D((痂蝗属+异痂蝗属)+皱膝蝗属);红胫小车蝗和黄胫小车蝗作为两个种比较合适;痂蝗亚科和异痂蝗亚科应该合并为一个亚科;飞蝗亚科与斑翅蝗亚科的分类关系尚未解决,需要进一步的研究进行分析验证.     

中国迁粉蝶属(鳞翅目,粉蝶科)基于NDⅠ和COⅠ基因部分序列的系统发育关系

测定了中国迁粉蝶属3种4亚种以及迁粉蝶指名亚种5个生态型的线粒体COⅠ基因和NDⅠ基因部分序列,以灵奇尖粉蝶为外群,分别采用最大简约法、最大似然法和贝叶斯法构建分子系统树,探讨了中国迁粉蝶属3种4亚种间的系统发育关系,并首次明确了迁粉蝶指名亚种5个生态型的亲缘关系。结果显示在测得的COⅠ基因661bp序列和NDⅠ基因472bp序列中,有变异位点227个,简约位点119个,A+T含量(75.5%)明显偏高。迁粉蝶属4亚种分子系统关系为:(镉黄迁粉蝶指名亚种+((梨花迁粉蝶指名亚种+梨花迁粉蝶海南亚种)+迁粉蝶指名亚种)),迁粉蝶指名亚种5个生态型分子系统关系为:(无纹型+((有纹型+红角型)+(银斑型+血斑型)))。     

华南虎、东北虎、孟加拉虎的D-loop和ND5序列及其在系统进化分析中的应用

本文采用PCR和质粒克隆测序方法,获得了华南虎线粒体D-loop区的480 bp序列和东北虎、孟加拉虎线粒体D-loop区的503 bp序列;同时还获得了这三个虎亚种和金钱豹线粒体ND5基因5'端309 bp的部分序列.根据D-loop序列分析,华南虎与孟加拉虎、东北虎的平均距离(p-distance)分别为0.110 88和0.110 87,而东北虎与孟加拉虎间的平均距离为0.009 94;根据ND5序列分析,华南虎与孟加拉虎、东北虎的平均距离分别为0.114 34和0.117 58,而东北虎与孟加拉虎间的平均距离为0.003 24.三个虎亚种的mtDNA D-loop和ND5序列比较表明,华南虎是这三个虎亚种中最为古老的亚种.     

华南虎、东北虎、孟加拉虎的D-100p和ND5序列及其在系统进化分析中的应用

本文采用PCR和质粒克隆测序方法,获得了华南虎线粒体D-loop区的480bp序列和东北虎、孟加拉虎线粒体D-loop区的503bp序列;同时还获得了这三个虎亚种和金钱豹线粒体ND5基因5’端309bp的部分序列。根据D-loop序列分析,华南虎与孟加拉虎、东北虎的平均距离(p-distance)分别为0．11088和O．11087,而东北虎与孟加拉虎间的平均距离为0．00994;根据ND5序列分析,华南虎与孟加拉虎、东北虎的平均距离分别为0．11434和0．11758,而东北虎与孟加拉虎间的平均距离为0．00324。三个虎亚种的mtDNA D-loop和ND5序列比较表明,华南虎是这三个虎亚种中最为古老的亚种。     

郑氏比蜢线粒体基因组全序列的测定与分析

采用长距PCR扩增及保守引物步移法测定并注释了郑氏比蜢(Pielomastax zhengi)的线粒体基因组全序列。郑氏比蜢的线粒体基因组全长15602 bp,A+T含量为71.8%,37个基因位置与飞蝗的一致, 基因间隔序列共计10处47bp, 间隔长度从1～20bp不等; 有14对基因间存在52bp重叠, 重叠碱基数在1～8bp之间。蛋白质基因的起始密码子均为昆虫典型的起始密码子ATN。ND5基因使用了不完全终止密码子T,其余基因均为典型的TAA或TAG。除tRNASer(AGN)的DHU臂缺失外, 其余21个tRNA基因的二级结构均属典型的三叶草结构, 但在郑氏比蜢中有5个tRNA(tRNACys、tRNALys、 tRNAPhe、 tRNAPro tRNAArg)基因变异较大, 无法采用常规算法预测出来, 表现在这5个tRNA二级结构的TψC臂仅有3～4对配对碱基, tRNALys 和 tRNAArg的反密码臂仅有 4 对配对碱基。预测的lrRNA二级结构总共有6个结构域(结构域Ⅲ缺失), 44个茎环结构。预测的srRNA的二级结构包含3个结构域, 30个茎环结构。比较郑氏比蜢、西藏飞蝗(Locusta migratoria tibetensis)和疑钩额螽(Ruspolia dubia)rRNA二级结构后,发现郑氏比蜢与西藏飞蝗的更相似。A+T丰富区中存在一个被认为与复制及转录起始有关的Ploy(T)结构。     

蝗科高级阶元的分子系统发育(英文）

迄今,蝗科内各分类阶元之间的系统发生关系大部分是未知的。本文用来自中国24种蝗科昆虫的12SrDNA和16SrDNA2个基因的联合序列(共795bp)数据,以锥头蝗科的锥头蝗(Pyrgomorpha conica)为外群,重建了分子系统树。研究结果表明,在12SrDNA与16SrDNA组成的联合数据中,转换的替代速率明显比颠换的替代速率高得多,核酸的替代已经发生了饱和。分子系统树表明:斑翅蝗亚科是一单系群,该亚科是一个合法的亚科,但斑腿蝗亚科和蝗亚科都不是单系群;斑翅蝗亚科在蝗科内是一个相对原始的类群,而稻蝗亚科比斑翅蝗亚科相对进化,比蝗科的其他亚科的种类相对原始。     

Use of the dnaJ gene for the detection and identification of all Legionella pneumophila serogroups and description of the primers used to detect 16S rDNA gene sequences of major members of the genus Legionella

We sequenced about 930 bp of the dnaJ gene from 15 Legionella pneumophila serogroups and some other members of the genus Legionella. As L. pneumophila 16S rDNA sequences could not discriminate between all subspecies and serogroups, we assessed the use of dnaJ gene sequences to differentiate between Legionella subspecies as well as between L. pneumophila serogroups. A phylogenetic analysis revealed that dnaJ gene sequences were more variable between the L. pneumophila serogroups than mip gene and 16S rDNA sequences. By studying 61 strains from 41 species of the genus Legionella, as well as other genera, we established a PCR method that could amplify 285 bp of dnaJ gene from all L. pneumophila serogroups. This primer set was more sensitive than mip gene primers and was able to detect 0.25 ng of purified DNA. We also describe the 16S rDNA primers that were used to detect most Legionella genus members.     

Phylogenetic relationships of leaf monkeys (Presbytis; Colobinae) based on cytochrome b and 12S rRNA genes

Little is known about the classification and phylogenetic relationships of the leaf monkeys (Presbytis). We analyzed mitochondrial DNA sequences of cytochrome b (Cyt b) and 12S rRNA to determine the phylogenetic relationships of the genus Presbytis. Gene fragments of 388 and 371 bp of Cyt b and 12S rRNA, respectively, were sequenced from samples of Presbytis melalophos (subspecies femoralis, siamensis, robinsoni, and chrysomelas), P. rubicunda and P. hosei. The genus Trachypithecus (Cercopithecidae) was used as an outgroup. The Cyt b NJ and MP phylogeny trees showed P. m. chrysomelas to be the most primitive, followed by P. hosei, whereas 12S rRNA tree topology only indicated that these two species have close relationships with the other members of the genus. In our analysis, chrysomelas, previously classified as a subspecies of P. melalophos, was not included in either the P. m. femoralis clade or the P. m. siamensis clade. Whether or not there should be a separation at the species level remains to be clarified. The tree topologies also showed that P. m. siamensis is paraphyletic with P. m. robinsoni, and P. m. femoralis with P. rubicunda, in two different clades. Cyt b and 12S rRNA are good gene candidates for the study of phylogenetic relationships at the species level. However, the systematic relationships of some subspecies in this genus remain unclear.     

Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences

The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.     

1株产细菌纤维素芽胞杆菌的分离及鉴定

从残次水果中分离出1株菌株ZF-7,其产物经纤维素特异性染色反应、红外光谱分析及纤维素酶水解实验后,被确定为细菌纤维素。在对ZF-7菌株常规形态学及生理生化特性鉴定的基础上,对部分长度的16S rDNA同源性进行了分析,发现ZF-7菌株与解淀粉芽胞杆菌相似度可达99.5%,现命名为Bacillus amyloliquefaciens ZF-7。对ZF-7菌株在振荡培养和静置培养条件下的发酵性能进行了初步考察,得到细菌纤维素产率分别为6.6和6.2 g/L。     

Taxonomic status of two South American sympatric squat lobsters, Munida gregaria and Munida subrugosa (Crustacea: Decapoda: Galatheidae), challenged by DNA sequence information

We investigated the taxonomic status of two sympatric morphospecies of squat lobsters from southern South America (Beagle Channel, Strait of Magellan, and Burdwood Bank), Munida gregaria and Munida subrugosa , by DNA sequence analysis of three mitochondrial (mt)DNA gene fragments [416 bp of 16S rDNA(165), 566 bp of cytochrome c oxidase subunit I(COI) and 418 bp of NADH dehydrogenase subunit 1 (ND1)]; and the nuclear rDNA internal transcribed spacer (ITS) 1 (883–952 bp). We obtained a total of 79 sequences from 32 individuals. The 16S sequences of all M. gregaria and M. subrugosa were invariant and identical, whereas COI and ND1 showed 12 and 15 variable sites, respectively. These polymorphisms were shared between morphospecies. Interspecific Tamura–Nei distances for COI and ND1 sequences were 0.0024 and 0.0032, respectively, and were not significantly different from intraspecific distances (Kruskal–Wallis tests: P  = 0.58 and P  = 0.69, for COI and ND1, respectively). Similar to the results obtained from the mtDNA sequences, no relationship was found between the ITS1 maximum parsimony tree topology and the morphologic classification of specimens in M. gregaria and M. subrugosa . We conclude that M. gregaria and M. subrugosa from southern South America may either represent a case of a dimorphic species, or a case of incomplete lineage sorting. The fact that these two morphospecies did not show fixed differences over a total of 1947 bp analysed reinforces the hypothesis of a single dimorphic species.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 421–434.     

基于线粒体基因的东方蜜蜂群体亚分化研究

【目的】进一步了解我国境内东方蜜蜂Apis cerana(Fabricius)群体的亚分化状况,为保护和合理利用这一宝贵的蜂种资源提供理论依据。【方法】采用公开的两对引物对中国境内19个地区的东方蜜蜂线粒体tRNAIle~ND2与16S rRNA基因的部分序列进行了扩增、测序,并与其他地区东方蜜蜂的相应序列进行了比对分析。【结果】扩增获得的tRNAIle~ND2基因的部分序列长度为471~474 bp,序列中共13个变异位点;16S rRNA基因的部分序列长度为581~585 bp,序列中共6个变异位点。ND2基因部分蛋白比对结果显示,仅山西沁源东方蜜蜂有一个位点发生变异。【结论】基于两基因部分序列所构建的系统发育树表明,海南东方蜜蜂明显区别于其他地区的东方蜜蜂;阿坝地区的东方蜜蜂可能属于高海拔地区的一种生态型,未支持其单独作为一个亚种的结论;吉林3个地区的东方蜜蜂之间亲缘关系较近,可能属于一个生态型;云南东方蜜蜂的变异比较丰富。     

飞蝗总DNA的抽提及其RAPD分析条件的摸索

通过试验寻求得到一种快速、简便抽提飞蝗（Ｌocusta sp.)总DNA方法,使每头雄性和雌性成虫分别可以得以５０和１００μg的总ＤＮＡ。所得到的总ＤＮＡ ＯＤ２６０／ＯＤ２８０为１．５－２．２,分子量４５kb。为了获得高分子量的ＤＮＡ产品,使ＲＰＡＤ结果具重复性,酚氯仿抽提后的ＤＮＡ沉淀用灭菌Ｔip头挑出,而不用离心收集。对各种分析条件如摸板、Ｔaq酶、dNTP及引物的浓度、不同的ＰＣＲ仪、反应管进行了比较试验,发现在一定的范围内,它们对ＲＡＰＤ结果影响。用优化的试验条件对我国３个飞蝗亚种５个地理种群进行ＲＡＰＤ分析。结果在３个亚种ＵＰＧＭＡ聚类图中,东亚飞蝗和西藏飞蝗珠２个种群以１００％Ｂootstrap分别聚类在一起,亚洲飞蝗与东亚飞蝗的２个种群以６６％的Ｂootstrap聚类在一起,在３个亚种所有个体的ＵＰＧＭＡ聚类图中,亚种内的所有个体都聚类在一起,各自形成独立分支,说明３个飞蝗亚种有明显的区别。西藏飞蝗的２个种群之间,群居型与散居型东亚飞蝗之间在聚类图中混合聚类,说明它们之间存在基因交流。     

Molecular organization of 5S rDNA in bitterlings (Cyprinidae)

Molecular organization and nucleotide sequences of the 5S rRNA gene and NTS were investigated in freshwater fish, bitterlings (Acheilognathinae), including 10 species/subspecies of four genera, Acheilognathus, Pseudoperilampus, Rhodeus, and Tanakia, to understand the evolutionary trait of 5S rDNA arrays. Southern hybridization analysis revealed a general trend with tandem repeats of 5S rDNA in all the examined bitterlings. Sequence analysis demonstrated a conserved 120 bp sequence of the 5S rRNA gene and a short NTS of 56–67 bp with two distinct portions, a conserved (5′-flanking portion; at positions −1 to −38) and a variable part (3′-flanking portion), in 6 of 10 species/subspecies examined. The conserved NTS region was most likely an external promoter so far observed in various vertebrates, whereas the variable NTS region could be divided into two types due to its nucleotide polymorphisms. Molecular phylogeny using the 5S rRNA gene and NTS sequences suggested the occurrence of 5S rDNA duplication before speciation and a concerted evolution for the gene and conserved NTS regions, but a birth-and-death process to maintain the variable NTS region. Thus, the 5S rDNA in the examined bitterlings might have evolved under a mixed process of evolution.