利用12S rRNA、COI基因分子标记鉴定少棘巨蜈蚣干燥体

本研究旨在利用线粒体DNA上的12S rRNA基因、COI基因分子标记鉴定少棘巨蜈蚣(Scolopendra subspinipes mutilans L. Koch)干燥体。通过提取少棘巨蜈蚣干燥体样本DNA,PCR扩增线粒体DNA上的12S r RNA基因、COI基因片段,对PCR产物进行电泳检测及测序分析,测序结果在GenBank上进行BLAST搜索,同源性分析,并用MEGA7.0软件对所有实验样品及少棘巨蜈蚣的近缘物种进行遗传距离分析、构建邻接(NJ)树以验证序列比对结果。结果表明,从少棘巨蜈蚣(Scolopendra subspinipes mutilans L. Koch)干燥体中成功地提取到了基因组总DNA,并成功扩增出了用于动物种属鉴定的12S rRNA、COI基因片段。但所得蜈蚣样本12S rRNA基因片段序列与NCBI的GenBank中的物种的同源性无90%以上的。通过文献调研发现尚无蜈蚣12S rRNA基因片段的相关报道,将该序列作为新的基因序列注册到NCBI基因数据库中,该基因片段序列的GenBank登录号为JN558832.1。这说明利用线粒体DNA上的12S rRNA、COI基因DNA分子标记皆可准确鉴定动物种属。本研究得到的12S rRNA基因片段序列可作为后续准确鉴定少棘巨蜈蚣药材的分子标记参考。     

基于涉案兽类12S rRNA基因的NCBI数据库序列可靠性分析

本研究以常用于动物种属鉴定的12S rRNA基因位点为研究对象,利用所测得的17种常见涉案兽类12S rRNA基因部分片段序列及NCBI数据库中下载的该物种DNA序列及其近缘物种DNA序列,构建系统进化树。根据进化树的聚类情况,判断NCBI数据库中的相关基因序列或物种名称的正确性,并对其中错误序列的登陆号进行标记,以防对后续涉案动物的准确鉴定造成影响。分别从17种常见涉案兽类(共26份样本)中提取线粒体DNA,并利用通用引物扩增线粒体DNA上的12S rRNA基因部分片段并进行测序分析。通过NCBI数据库的Blast比对功能,筛选出与本研究物种同源性由高到低的物种,并从NCBI基因数据库中下载此类近缘物种的12S rRNA基因序列共351条,利用MEGA7.0软件构建该物种及其近缘物种系统进化树。通过比对发现NCBI中登录号为KP202279等3个序列所对应物种拉丁名错误。登录号为AY184436等11个序列所对应物种拉丁名可能存在疑问。GenBank中某些物种拉丁名有同种异名现象。因此,NCBI数据库数据可靠性有待进一步验证,只能作为涉案物种鉴定的参考数据之一,可借助构建系统进化树等方法来确认其结果的准确性。     

12S rRNA和Cyt b基因序列测定在獐乳制品鉴定中的应用

采用改良的蛋白酶K消化和酚/氯仿抽提的方法从脂肪含量很高的动物乳制品及胃组织中提取出基因组总DNA,利用特异引物扩增了线粒体12S rRNA基因和Cyt b基因的部分片段,测定了12S rRNA和Cyt b基因的PCR扩增产物序列,使用BLAST搜索软件将其序列与GenBank中的同源序列进行比对,并利用DNAMAN分析软件分析序列同源性。结果表明3件检材均来源于獐Hydropotes inermis。本研究所用方法在野生动物乳制品鉴定中具有较高的应用价值。     

羊驼线粒体12srRNA/tRNA-Val/16rRNA 基因序列测定及分子进化研究

对GaneBank中登录的部分骆驼科动物线粒体12 srRNA/tRNA-Val/16 rRNA基因序列进行同源性比较,并借助DNAstar软件设计引物,扩增并进行序列分析,旨在揭示其遗传变异的规律。PCR条件优化后成功扩增出长度为1 014 bp的DNA片段,b lastn分析显示,该片断与骆驼科动物的同源性高于95%,所获得片断包括30bp的12 srRNA基因部分序列,71 bp的tRNA-Val基因全序列和913 bp的16 srRNA基因部分序列。利用RNA-structure 4.2 RNA分析软件绘制了部分骆驼科动物的线粒体tRNA-Val推导性二级结构图,比较结果显示,羊驼线粒体tRNA-Val氨基酸臂和反密码子环呈属间特异性遗传。     

人参细胞器核糖体小亚单位DNA的PCR扩增与序列测定

采用CTAB法提取了人参(Panax ginseng)根基因组DNA,根据植物叶绿体16S rDNA和线粒体18SrDNA与细菌16S rDNA序列具有高度同源性,用扩增细菌16S rDNA的一对通用引物(8f,1492r)扩增了人参细胞器核糖体小亚单位DNA.对扩增产物进行了克隆与测序,经多序列比对,扩增片段分别与已知植物叶绿体16S rDNA和线粒体18S rDNA具高度同源性,表明该对引物可以用来扩增绝大多数植物细胞器核糖体小亚单位DNA,可以作为鉴定植物叶绿体16S rDNA和线粒体18S rDNA的一种基本实验技术.     

曲额乳白蚁mtDNA-PCR鉴定

基于白蚁16S rRNA基因序列差异设计一对引物进行曲颚乳白蚁PCR鉴定,该引物可以与曲颚乳白蚁线粒体DNA进行PCR反应,特异性扩增约200bp目的DNA片断,成功构建以曲颚乳白蚁16S rRNA基因序列为基础的mtDNA-PCR鉴定体系。     

复合PCR扩增人mtDNA HVR方法的初探

目的:建立一种高效的扩增线粒体DNA高可变区(mtDNA HVR)的方法.方法:本研究选取5例健康成人静脉血,用人血全基因组DNA试剂盒,提取全基因组DNA,设计引物,用复合PCR方式,对线粒体DNA中的高可变区进行扩增.复合扩增的方式为:用6对套叠引物分开进行两次独立的PCR,扩增mtDNA HVR.第一次扩增用3对引物,目标DNA片段基本涵盖整个线粒体DNA的高可变区,扩增后得到互不重叠的3个短片段,分别为113 bp,126 bp和131bp.第二次复合扩增用其余的3对引物,目标片段基本重叠在第一次扩增所得的目标片段的区域内,扩增得到3个互不重叠的片段为124 bp,133 bp和93bp.所有扩增产物经过纯化后测序.结果:复合PCR方式获得的mtDNA HVR基因序列完整,5个样本均出现特异性条带,电泳结果条带单一、清晰.结论:复合扩增PCR方法对mtDNA HVR区的扩增效率高,测序结果稳定,结合6对套叠引物,不但保证了序列的完整性,另外,两次独立的PCR也减少了PCR反应过程中错配的发生,此法也适用于保存时间较久的古代线粒体DNA短片段的研究.复合扩增PCR还展示出了潜在的高产量的特点,相对传统PCR显示了其更多的优势.     

慈竹叶蝉类害虫DNA条形码分析

叶蝉类昆虫形态结构多样,在农林生态系统的物种多样性和植物保护工作中扮演着重要角色,但其物种的准确鉴定一直是农林植保工作中的难点。DNA条形码技术极大促进了农林生态系统物种的快速、准确鉴定。本研究经过连续2年的野外调查采集慈竹Bambusa emeiensis主要叶蝉种类,扩增了广泛分布于中国慈竹的12种主要叶蝉类害虫的线粒体基因COⅠ和16S rRNA序列片段,并进行了遗传距离、系统发育及矢量Klee-diagram图分析。结果显示:慈竹叶蝉昆虫COⅠ基因序列片段(590 bp)种内遗传距离为0.004,种间遗传距离为0.283;16S rRNA基因序列片段(463 bp)种内遗传距离为0.003,种间遗传距离为0.257;不同种间存在明显的条形码间隔。2个基因序列片段的分子系统发育分析结果与形态学研究谱系关系一致。Klee-diagram图分析结果和分子系统发育结果一致。上述结果表明,COⅠ和16S rRNA基因适用于慈竹叶蝉类昆虫的物种鉴定,可为竹林叶蝉类昆虫的准确快速鉴定提供参考方法。     

基于16S rRNA和rpoB基因的分子系统发育分析在铜绿假单胞菌鉴定中的应用

为对比16S rRNA和rpo B基因分子系统发育分析与传统表型分类法对铜绿假单胞菌的鉴定,评估16S rRNA和rpo B基因序列分析在铜绿假单胞菌鉴定中的应用,用表型分类方法对临床自动微生物鉴定系统鉴定为铜绿假单胞菌的23株分离株进行再鉴定,PCR扩增23株分离株16S rRNA和rpo B基因片段,并测序进行系统发育分析。结果表明,表型再鉴定结果与自动微生物鉴定系统鉴定结果一致。基于两个基因的系统发育分析均显示分离株p22与不动杆菌属序列聚为一枝,其余22株分离株与铜绿假单胞菌序列聚为一枝。因此p22应鉴定为不动杆菌,16S rRNA和rpo B基因序列分析均能准确鉴定铜绿假单胞菌并能较好建立假单胞菌属内种间关系。     

沼蛙(Rana guentheri)种内遗传分化状况初步研究

本文测定了香港特别行政区内沼蛙(Rana guentheri)多个种群的线粒体16S rRNA基因部分DNA序列,并与广州及越南共4个沼蛙基因序列作对比,研究各地区种群遗传分化状况,研究表明三个地区之间的序列遗传分化为0．4％～1．1％,表明沼蛙mtDNA 16S rRNA基因进化速率比较低,其中香港沼蛙10个个体在mtDNA 16S rRNA基因上没有出现变异,共发现5种单倍型,其中香港与广州种群关系最近,越南河内种群与志玲种群关系最近。     

mtDNA标记在几种海关进出口动物产品鉴定中的应用

从海关查获的动物及其制品中提取基因组DNA,通过PCR技术扩增mtDNA细胞色素b(cytochrome b,cyt b)和12S rRNA基因并测定其序列。利用互联网上丰富的基因数据库进行BIAST。搜索,准确地鉴定了动物的种类,从而为海关查获的走私珍稀野生动物的鉴定提供了一条灵敏和可靠的途径。     

Two-step denaturing gradient gel electrophoresis (2S-DGGE), a gel-based strategy to capture full-length 16S rRNA gene sequences

Obtaining full-length 16S rRNA gene sequences is important for generating accurate taxonomy assignments of bacteria, which normally is realized via clone library construction. However, the application of clone library has been hindered due to its limitations in sample throughput and in capturing minor populations (<1?% of total microorganisms). To overcome these limitations, a new strategy, two-step denaturing gradient gel electrophoresis (2S-DGGE), is developed to obtain full-length 16S rRNA gene sequences. 2S-DGGE can compare microbial communities based on its first-round DGGE profiles and generate partial 16S rRNA gene sequences (8-534?bp, Escherichia coli numbering). Then, strain-specific primers can be designed based on sequence information of bacteria of interest to PCR amplify their remaining 16S rRNA gene sequences (515-1541?bps, E. coli numbering). The second-round DGGE can confirm DNA sequence purity of these PCR products. Finally, the full-length 16S rRNA gene sequences can be obtained through combining the two partial DNA sequences. By employing 2S-DGGE, taxonomies of a group of dehalogenating bacteria have been assigned based on their full-length 16S rRNA gene sequences, several of which existed in dehalogenating microcosms as minor populations. In all, 2S-DGGE can be utilized as a medium throughput method for taxonomic identification of interested/minor populations from single or multiple microbial consortia.     

Sequence of the gene for isoleucine tRNA1 and the surrounding region in a ribosomal RNA operon of Escherichia coli

A DNA fragment of about 2000 base pairs carrying the gene for tRNA(1) (Ile) has been cloned from a total Eco RI endonuclease digest of Escherichia coli DNA. Sequence analyses revealed that about the first 850 base pairs from one end of the fragment contain a nucleotide sequence corresponding to that in the 3'-end of 16S rRNA. The gene for tRNA(Ile) follows the 16S rRNA gene and both genes flank a spacer sequence of 68 base pairs. The spacer region contains a repeating, a hair pin and a symmetrical structure when the sequence is viewed in the single stranded form. A notable hair pin structure is also observed in the region adjacent to the 3'-end of the tRNA(1) (Ile) gene. In addition, about 850 base pairs from the other end of the DNA fragment have been found to contain the nucleotide sequence of the 5'-end of 23S rRNA. The presence of the genes for tRNA(1) (Ile), 16S and 23S rRNA and the hybridization to tRNA(1) (Ala) suggest that this cloned DNA is part of one of the E. coli rRNA operons carrying these two tRNA genes as a spacer.Images     

Molecular characterization of trichomonads from feces of dogs with diarrhea

Trichomonads are occasionally observed in the feces of dogs with diarrhea. On the basis of superficial morphological appearance, these infections have been attributed to opportunistic overgrowth of the commensal, Pentatrichomonas hominis. However, molecular characterization of canine trichomonads has never been reported. This study was performed to determine, by means of rRNA gene sequence analysis, the identity of trichomonads observed in feces from dogs with diarrhea. Total DNA was isolated from fecal samples obtained from a 3-mo-old mixed breed dog and litter of German Shepherd puppies having profuse liquid diarrhea containing numerous trichomonads. Total DNA was subject to PCR amplification of partial 18S rRNA gene or 5.8S, ITS1, ITS2, and partial 18S and 28S rRNA genes using species-specific and universal primers, respectively. Products of 642 and 1864 base-pair length were amplified and cloned. On the basis of rRNA gene sequence, the trichomonads observed in the single dog and the litter of puppies shared 100% identity with Tritrichomonas foetus and P. hominis, respectively. The present study is the first to establish the molecular identity of trichomonads infecting dogs with diarrhea. These studies validate the longstanding assumption that canine trichomoniasis may be attributed to P. hominis. Importantly, these studies additionally recognize that canine trichomoniasis may also be caused by infection with T. foetus.     

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。     

12S rRNA 在黑麂和黄麂物种鉴定中的应用

黑麂(Muntiacus crinifons),别名乌獐,其体长为95～108 cm,体重21～26 kg,是我国特有物种,全国资源量约6 000只,为国家I级重点保护的动物[1,2].由于黑麂皮革上乘,且肉嫩味美,故长期被不法分子列为主要的偷猎对象之一[1].黄麂(Muntiacus reevesi),又名小麂,其体长为73～87 cm,体重10～15 kg,为浙江省重点保护的野生物种.由于黄麂的经济价值高,一直为传统的出口商品.正因为如此,黄麂在近年来遭到了人类的大肆猎杀[2].     

Retrieval of nearly complete 16S rRNA gene sequences from environmental DNA following 16S rRNA-based community fingerprinting

16S rRNA-based fingerprinting techniques allow rapid analyses of overall bacterial community structure but suffer from a lack of phylogenetic information hitherto retrievable from the short 16S rRNA gene sequences obtained from excised bands. An approach is presented that allows nearly complete 16S rRNA gene sequences to be retrieved for abundant components of the bacterial community as obtained by the community fingerprint, i.e. those reflected by major fingerprint bands. This was achieved by designing a pair of highly specific primers derived from the sequence of an excised band. Combined with universal 16S rRNA primers, these specific primers were applied directly to environmental DNA serving as template. This procedure allowed the generation of a nearly complete 16S rRNA gene sequence of the target taxon by specific polymerase chain reaction (PCR) followed by cycle sequencing down to a relative abundance of at least 1.5% of the environmental DNA. The procedure was exemplified for an epsilonproteobacterium related to Thiomicrospira denitrificans occurring in the central Baltic Sea. This approach is based only on PCR without any cloning step involved. It allows focussing on specific target taxa and is thus rather efficient. This approach should be applicable in general to 16S rRNA or 16S rRNA gene-based fingerprinting techniques and their respective environmental DNA.