3种色型仿刺参线粒体和核糖体序列片段遗传结构比较研究

仿刺参作为我国重要的海水养殖物种,良种选育是目前研究的重点,其中体色是重要的考虑因素之一。为了比较3种不同体色仿刺参的遗传结构,采用PCR扩增与测序技术对我国3种体色(青色、白色和紫色)仿刺参线粒体16S rRNA和COⅠ与核糖体18S rRNA-ITS-28S rRNA序列片段进行分析,揭示其遗传结构差异及系统发育关系。结果表明, 16S rRNA、COⅠ和18S rRNA-ITS-28S rRNA序列长度分别为812~830 bp、877~915 bp、1 536~1 572 bp。16S rRNA、COⅠ和18S rRNA-ITS-28S rRNA序列在青色、紫色和白色仿刺参中分别检测到单倍型个数分别为4/5/5、5/3/4和4/4/3, COⅠ序列多态性位点数目所比例最大, 16S rRNA序列最小。3种色型仿刺参16S rRNA、COⅠ和18S rRNA-ITS-28S rRNA序列遗传距离数值均比较小,范围分别为0~0.014 7、0~0.021 2和0~0.010 3,没有达到种的分化水平。以紫海胆为外群,基于COⅠ序列构建系统发育树,结果表明白刺参单独聚为一支,但是紫色和青色仿刺参不同个体间相互交叉聚集,无法通过发育树区分。     

Spacer length variation in rice 5S rRNA genes revealed by polymerase chain reaction

Polymerase chain reaction (PCR) was used to amplify 5S rRNA spacer from wild rice (Oryza rufipogon and O. nivara) and cultivated rice (indica and japonica varieties of O. sativa L). The results show that there is spacer length variation within and between species, and the typical indica and japonica varieties have their unique banding patterns of amplified 5S rRNA spacers, whereas intermediate showed no specific amplification profile of spacer regions. The 5S rRNA genes in intermediate are either identical with that of indica variety or that of japonica variety. These data suggest that the spacer length polymorphisms can be used to distinguish between closely ralated species and subspecies. Supported by the National Natural Science Foundation of China Yi Qingming, born in Apr. 1938, Professor     

云台山白云岩表层土可培养细菌多样性

运用纯培养法和基于16S rRNA基因序列的系统发育分析贵州云台山白云岩表层土可培养细菌的多样性.稀释涂布平板分离共获得131株细菌,限制性内切酶HhaⅠ和HaeⅢ对扩增的16S rRNA基因片段进行酶切分型,根据ARDRA酶切图谱划分为40个操作分类单元.16S rRNA基因序列测定和系统发育分析结果显示,这些菌株隶属于包括厚壁菌门(Firmicutes,64.89%)、β-变形菌纲(β-Proterbacteria,3.82%)、γ-变形菌纲(γ-Proterbacteria,17.56%)、放线细菌门(Actinobacteria,11.45%)和拟杆菌门(Bacteroidetes,3.82%)等5大类群,共13个属.优势菌群为芽胞杆菌属(Bacillus,53.44%),亚优势菌群为寡养单胞菌属(Stenotrophomonas,9.16%)、假单胞菌属(Pseudomonas,8.40%)和微小杆菌属(Exiguobacterium,8.40%).15.00%的有效序列与GeneBank已知序列相似性小于等于97%,可能为潜在新类群.研究表明,云台山白云岩喀斯特地区不仅含有较为丰富的细菌物种多样性,且潜藏着许多新的微生物资源.     

银杏与其它裸子植物之间的关系—分子生物学的证据

银杏Ginkgo biloba L.Ls-rRNA 5＇末端188个核苷酸序列已被准确测定。与苏铁Cycas revoluta Thunb.,水松Glyptostrobus pensilis(Staunt.)Koch,南方红豆杉Tax-us mairei(Lemee et Levl.)S.Y.Hu和5种被子植物及1种绿藻的同源序列比较分析,其所构建的rRNA系统树图表明,银杏与苏铁的关系较为密切,与其它裸子植物的关系相对较远。     

Phylogenetic positions of some genera and species of the family Buccinidae (Gastropoda: Mollusca) from China based on ribosomal RNA and COI sequences

A phylogenetic analysis of members of the family Buccinidae was conducted using 18S rRNA gene, 28S rRNA gene and the mitochondrial cytochrome oxidase Ⅰ gene. We studied 18 species of Buccinidae that belong to eight different genera and inhabit the China coastal seas. We analyzed the patterns of divergence between an outgroup and basal ingroup taxa, the monophyly of the genus Neptunea, and the position of one unnamed species within the Buccinidae. A phylogenetic tree (neighbor-joining (NJ) method) was reconstructed based on the sequences of 18S rRNA, 28S rRNA and COI, with Rapana venosa as outgroup. The NJ tree indicated that the 18 species could be divided into five groups. The genus Buccinum was monophyletic, whereas Neptunea was shown to be paraphyletic since it included Siphonalia subdilatata and Neptunea sp., a new species. This novel species otherwise clustered consistently with Neptunea cumingi in three other phylogenetic trees, showing a low genetic distance and divergence percentage of sequences belonging to the genus Neptunea. A smaller genetic distance and a smaller difference of 18S rRNA, 28S rRNA and COI sequences between Neptunea cumingii and Neptunea arthritica cumingii confirmed them to be the same species.     

梭鱼和鲻鱼线粒体16S rRNA和COⅠ基因片段的比较分析

运用PCR技术,扩增了梭鱼和鲻鱼线粒体16S rRNA和COⅠ基因片段,并比较分析了其种间的序列差异。获得16S rRNA基因的540～560 bp碱基序列,出现26个碱基的插入与缺失位点;获得COⅠ基因的602～604 bp碱基序列,出现2个插入缺失位点;16S rRNA和COⅠ基因的序列中G平均含量最低,且(A+T)含量高于(G+C)含量,与其他鱼类中的16S rRNA和COⅠ基因片段研究结果相一致。在16S rRNA基因片段中,梭鱼出现1种单倍型,鲻鱼为2种单倍型;在COⅠ基因片段中,梭鱼样品中检测到3个单倍型,鲻鱼样品中检测到5个单倍型。以Takifugu poecilonotus为外群,构建的NJ系统发育树,基于16S rRNA和COⅠ基因序列获得的NJ系统树基本一致,鲻鱼和梭鱼种内个体分别聚为一支,显示16S rRNA和COⅠ基因适合于梭鱼和鲻鱼的物种鉴定。     

An rpoB gene-based PCR-DGGE method for simultaneous detection of multiple Vibrio species

Using PCR-denaturing gradient gel electrophoresis (DGGE) targeting the RNA polymerase beta subunit (rpoB) gene, a simultaneous detection method for Vibrio species was established. rpoB gene-based PCR-DGGE was carried out with eight Vibrio Reference strains (each from different species), mixed sample (including these Vibrio Reference strains),two non Vibrio strains, four environmental Vibrio strains, and three unidentified environmental strains. For comparison, 16S rRNA gene-based PCR-DGGE of the eight Vibrio Reference strains was performed with universal primers. In addition, three unidentified strains were identified by 16S rRNA and gyrB gene sequencing and API20E system in order to confirm the accuracy of rpoB gene-based PCR-DGGE detection. Results revealed that rpoB-based PCR-DGGE could well discriminate eight Vibrio Reference strains and could not discriminate different strains within the same species. The bands derived from two non Vibrio strains could not match with any bands in Reference marker. Meanwhile, 16S rRNA gene-based DGGE failed to distinguish these Reference strains. Furthermore, four out of eight Vibrio species exhibited heterogenous bands in 16S rRNA gene-based DGGE. Sequencing and API 20E identification of unidentified strains coincided with the detection by rpoB gene-based PCR-DGGE. The results demonstrated that rpoB-based PCR-DGGE provided a rapid and efficient method for simultaneous detection of multiple Vibrio species, which can avoid the limitations inherent in 16S rRNA gene-based PCR-DGGE.     

基于16S rRNA序列的鳃金龟亚科5族种类的系统发育关系

测定了鳃金龟亚科部分种类的线粒体16SrRNA部分序列,运用MEGA 4.0、PAUP*4.0b10和MrBayes等软件,对5个族29个代表种的序列变异和系统关系进行了研究.序列变异分析结果显示:绢金龟族Sericini、哦鳃金龟族Hopliini、鳃金龟族Melolonthini的族内遗传距离分别为7.7%,11.3%,10.6%,族间遗传距离在12.6%～19.2%.最大似然树(ML)和贝叶斯树(BI)结果表明,所有种类分别聚集在所属的族下,哦鳃金龟族、绢金龟族分别为单系.齿爪鳃金龟属Holotrichia、云鳃金龟属Polyphylla、胸突鳃金龟属Hoplosternus、Hilyotrogus、双绺鳃金龟属Amphimallon、Hoplochelus、婆鳃金龟属Brahmina和皱鳃金龟属Trematodes聚在鳃金龟族分支下,没有形成明显的根鳃金龟族Rhizotrogini分支.齿爪鳃金龟属的非单系性与前人研究结果一致.皱鳃金龟属Trematodes和婆鳃金龟属Brahmina的代表种为姐妹种.绢金龟族的绢金龟属Serica和玛绢金龟属Maladera多数种类聚在本属分支下.证明16SrRNA序列可探讨鳃金龟亚科高级阶元的系统发育关系.     

利用12S rRNA基因标记鉴定鱼翅真伪

为检测鱼翅样本真伪及鲨鱼种属,通过提取送检鱼翅、鱼翅羹样本DNA,扩增其线粒体DNA上的用于动物种属鉴定的12S rRNA基因片段,并进行DNA测序和序列分析,测序结果在GenBank上进行BLAST搜索,与数据库中相关物种序列进行同源性分析。结果表明,在送检的23份样本中,18份鱼翅羹样本、2份粉丝状鱼翅、1份翅状鱼翅中均未成功提取到DNA,未扩增出目的基因片段,而从2份鱼翅样本中成功地提取到了基因组总DNA,并成功扩增出了12S rRNA基因片段,这2份样本的12S rRNA基因片段的碱基序列分别与黑边鳍真鲨(Carcharhinus limbatus)、斜锯牙鲨(Rhizoprionodon terraenovae)的同源性达到99%。对鉴定出含有鲨鱼成分的2份样本进行高温泡发处理,高温泡发实验表明,1份样本(22号)有明胶析出。研究证实,12SrRNA基因序列测定技术能准确、快速鉴定待检样本中是否含有鲨鱼成分,结合高温泡发时是否有明胶析出可综合判定鱼翅真伪。     

尖塘鳢属鱼类线粒体16SrRNA基因序列变异及分子系统进化

为探讨尖塘鳢属鱼类的系统发育关系,笔者通过PCR法得到云斑尖塘鳢、线纹尖塘鳢和尖头塘鳢的线粒体16SrRNA基因部分序列（569bp）,并结合从GenBank中下载的平头沙塘鳢、溪鳢及刺盖塘鳢的16SrRNA基因部分同源序列,分析了6种塘鳢科鱼类的系统发育关系．运用MAGA4．0软件分析可知,塘鳢鱼类6个种之间存在142个变异位点,其中简约信息位点57个,转换／颠换之比为1．6,表明塘鳢科鱼类16SrRNA基因序列转换／颠换未达饱和;6种塘鳢科鱼类之间的序列差异在0．054～0．147之间,其中平头沙塘鳢和刺盖塘鳢的序列差异最大（0．147）,线纹尖塘鳢与云斑尖塘鳢的序列差异最小（0．054）,且种间序列差异不大．通过NJ,ME,MP系统树的建立,表明线纹尖塘鳢与云斑尖塘鳢的亲缘关系最近,且尖塘鳢属与塘鳢属鱼类的同源性不高,验证了传统分类学把尖塘鳢独立成属的科学性．