胶州湾浮游植物rbcL基因分子遗传多样性研究

利用聚合酶链式反应扩增胶州湾表层海水浮游植物核酮糖1,5-二磷酸羧化/氧化酶大亚基基因(rbcL)片段,建立了该基因片段变异类型文库.随机测定了28个rbcL片段序列,依此初步分析了胶州湾表层海水浮游植物rbcL基因分子遗传多样性.结果表明,春季胶州湾表层海水浮游植物优势种群为D类rbcL代表的浮游植物,其中隐藻占28.6%、Stramenopies占32.1%、定鞭藻占28.6%、红藻占3.6%.B类rbcL代表的浮游植物为绿藻,占7.1%.根据各操作分类单元丰度计算的分子遗传多样性指数为2.85,根据逆翻译成的氨基酸序列计算的序列多样性为0.20.     

基于线粒体COⅠ基因和控制区序列的辽宁沿海弯棘斜棘(鱼衔)群体遗传多样性和遗传结构分析

为研究辽宁沿海弯棘斜棘(鱼衔)(Repomucenus curvicornis)自然群体的遗传多样性及遗传结构,采用PCR扩增获得辽宁沿海弯棘斜棘(鱼衔)辽东湾群体(n=22)及黄海北部群体(n=18)线粒体的COⅠ及控制区(CR)部分DNA序列片段,进行序列比较及遗传多样性分析.获得弯棘斜棘(鱼衔)COⅠ基因片段624 bp,其A、T、C、G平均含量分别为24.09%、31.04%、25.28%和19.59%;CR片段460 bp,其A、T、C、G平均含量分别为32.96%、32.80%、14.86%和19.38%.基于COⅠ基因和CR序列得到的两群体变异位点数、平均核苷酸差异数、单倍型多样性指数以及核苷酸多样性指数分别为:38、4.67、0.96±0.02和0.0075±0.0042;26、3.35、0.97±0.02和0.0073±0.0043.序列分析结果均显示,辽东湾群体的遗传多样性低于黄海北部群体.分子方差(AMOVA)分析结果显示,基于COⅠ基因片段辽东湾与黄海北部群体间无明显遗传分化(Fst=0.0091,P=0.25)而基于CR序列两群体间具有较小但接近显著的遗传分化(Fst=0.0264,P=0.09).研究表明,线粒体CR序列与COⅠ基因均可作为检测弯棘斜棘(鱼衔)群体遗传多样性的有效分子标记,但CR序列遗传分化的敏感度要高于COⅠ基因,更适合作为弯棘斜棘(鱼衔)群体遗传研究的分子标记.     

基于线粒体COⅠ基因和控制区序列的辽宁沿海弯棘斜棘鳂群体遗传多样性和遗传结构分析

为研究辽宁沿海弯棘斜棘逐(Repomucenus curvicornis)自然群体的遗传多样性及遗传结构,采用PCR扩增获得辽宁沿海弯棘斜棘逐辽东湾群体(n=22)及黄海北部群体(n=18)线粒体的COⅠ及控制区(CR)部分DNA序列片段,进行序列比较及遗传多样性分析。获得弯棘斜棘逐COⅠ基因片段624 bp,其A、T、C、G平均含量分别为24.09%、31.04%、25.28%和19.59%;CR片段460 bp,其A、T、C、G平均含量分别为32.96%、32.80%、14.86%和19.38%。基于COⅠ基因和CR序列得到的两群体变异位点数、平均核苷酸差异数、单倍型多样性指数以及核苷酸多样性指数分别为:38、4.67、0.96±0.02和0.0075±0.0042;26、3.35、0.97±0.02和0.0073±0.0043。序列分析结果均显示,辽东湾群体的遗传多样性低于黄海北部群体。分子方差(AMOVA)分析结果显示,基于COⅠ基因片段辽东湾与黄海北部群体间无明显遗传分化(F_(st)=0.0091,P=0.25)而基于CR序列两群体间具有较小但接近显著的遗传分化(F_(st)=0.0264,P=0.09)。研究表明,线粒体CR序列与COⅠ基因均可作为检测弯棘斜棘逐群体遗传多样性的有效分子标记,但CR序列遗传分化的敏感度要高于COⅠ基因,更适合作为弯棘斜棘逐群体遗传研究的分子标记。     

胶州湾叶绿素a浓度及浮游植物的粒级组成

2008年2、5、8和11月对胶州湾及邻近水域中表层叶绿素a浓度和浮游植物粒级组成进行了调查.结果表明:胶州湾内和湾外表层叶绿素a年平均浓度分别为4.90和2.03 mg·m^-3;叶绿素a浓度的平面分布呈现自东北部及近岸向中部、南部及湾外递减的趋势;叶绿素a浓度季节变化明显,冬季和夏季浓度较高,春季次之,呈现温带海域双峰型的变化趋势.胶州湾浮游植物粒级组成以微型浮游植物为主,平均占叶绿素a总量的60.9%,其次是小型浮游植物,超微型浮游植物所占比例最低,与我国近海浮游植物粒级组成基本一致.与历史资料相比,微型浮游植物所占比例有所增加,超微型浮游植物所占比例降低.     

DNA条形码技术在大型红藻分子鉴定中的应用

本研究对浙江省枸杞岛贻贝养殖区大型海藻资源进行了调查,在形态学观察的基础上,选取93株红藻样品运用DNA分子条形码技术进行分子鉴定分析。采用的3个分子标记分别为rbcL(叶绿体基因组中1,5-二磷酸核酮糖羧化/加氧酶大亚基基因),COI(细胞色C氧化酶亚基I)和UPA(23S rRNA基因片段,通用质体扩增区)基因片段。通过实验,我们共扩增出rbcL基因片段79条,扩增成功率为84.95%;共扩增出COI基因片段63条,扩增成功率为67.74%;共扩增出UPA基因片段76条,扩增成功率为81.72%。3个分子标记的测序成功率分别为:rbcL基因78.49%,COI基因29.03%,UPA基因72.04%。COI基因的扩增效率和测序成功率均低于rbcL和UPA基因。基于3个基因构建(Neighbor-joining,N-J)系统进化树表明,rbcL基因可以将红藻样品归类为5目,6科,7属,12种;COI基因可以将样品归类为4目,5科,5属,5种;UPA基因可以将样品归类为6目,7科,8属,11种。rbcL、COI和UPA 3个基因结合可以将样品归类为7目8科11属21种。研究表明rbcL基因以及UPA基因较适合做为鉴定大型红藻的分子标记,COI基因做为红藻分子标记的潜力有待进一步开发和研究。     

水稻叶绿体16S启动子克隆改造、载体构建及转化研究

利用PCR方法从水稻叶绿体基因组DNA中分离16S启动子,并在其下游加入rbcL基因SD序列,以增强该启动子的翻译能力;序列分析表明,除加入的SD序列外,扩增片段与水稻(Oryza sativa)叶绿体基因组DNA序列16S启动子相应区域同源性为100％。将16S启动子与bar基因和gfp基因的融合基因连接,以psbA基因的3′序列为终止子,并以烟草叶绿体trnH—psbA和trnK为同源片段构建了烟草叶绿体表达载体pRl6S。用基因枪转化烟草,转化植株经Southern、Northern检测及后代遗传学分析,发现：16S启动子具有启动活性,融合基因已在烟草叶绿体中稳定整合并遵循母系遗传规律。     

百合psbA-trnH基因序列变异及遗传多样性分析

目的:通过对叶绿体基因psbA-trnH序列变化差异来研究百合遗传多样性。方法:利用PCR技术扩增百合叶绿体ps-bA-trnH基因片段并测序,再用生物软件Clustalx2、Mega5.0、DnaSP对测序结果进行分析。结果:扩增的psbA-trnH基因片段长度约为469～583bp,GC含量为34.47～35.20%,其核苷酸变异位点27个,信息位点12个,变异位点主要集中在88～107 bp区域,根据这些变异位点供试材料可以分为3类,核苷酸的多样性为0.01150。结论:叶绿体基因trnH-psbA序列可以区分百合种间的差异,可以作为一种很好方法来研究遗传多样性。     

甘薯叶绿体rbcL基因的克隆与序列分析

根据烟草、水稻和菠菜叶绿体的全基因组序列设计引物,以甘薯的叶绿体基因组DNA为模板,PCR扩增包舍甘暑叶绿体rbcL完整基因(GenBank登录号为AY942199)在内的一段序列.序列分析表明:此片段的全长为1 627 bp,包括1 443bp的编码区序列在内.推测编码480个氨基酸,同时构建了此片段的限制性酶切图谱.相似性比较显示,此基因编码区序列与烟草、菠菜、小麦、水稻、玉米、矮牵牛、紫花苜蓿、拟南芥、莨菪、葡萄以及甜菜的rbcL基因核苷酸的同源性为85%～98%,氨基酸的同源性为92%～95%.     

罗氏沼虾不同群体线粒体COI基因的序列差异和遗传标记研究

利用PCR方法扩增获得罗氏沼虾（Macrobrachiumrosenbergii）线粒体DNA的COI基因,测定该基因片段序列。分析了罗氏沼虾缅甸原种F1代、江苏养殖群体和广西选育F2代3个群体共17只个体的序列核苷酸位点差异和遗传多态。结果表明,缅甸原种F1代遗传多样性最为丰富,江苏养殖群体和广西选育群体的遗传多样性相对贫乏。在长度为498bp的基因片段中,共检测到10个多态性核苷酸位点（占2.01%）,17只个体具有5种基因型,3群体各自的平均核苷酸位点差异分别为0.88%、0.07%和0。UPGMA分子系统聚类树显示,江苏养殖群体和广西选育群体的遗传关系最近,其单倍型混杂聚成一支,而缅甸原种F1群体相对独立为另外一支。COI基因可以作为区分两分支群体的遗传标记。     

罗氏沼虾3个群体线粒体COⅠ基因的序列差异和遗传标记研究

利用PCR方法扩增获得罗氏沼虾(Macrobrachium rosenbergii)线粒体DNA的COⅠ基因,测定该基因片段序列.分析了罗氏沼虾缅甸原种F1代、江苏养殖群体和广西选育F2代3个群体共17只个体的序列核苷酸位点差异和遗传多态.结果表明,缅甸原种F1代遗传多样性最为丰富,江苏养殖群体和广西选育群体的遗传多样性相对贫乏.在长度为498 bp的基因片段中,共检测到10个多态性核苷酸位点(占2.01%),17只个体具有5种基因型,3群体各自的平均核苷酸位点差异分别为0.88%、0.07%和0.UPGMA分子系统聚类树显示,江苏养殖群体和广西选育群体的遗传关系最近,其单倍型混杂聚成一支,而缅甸原种F1群体相对独立为另外一支.COⅠ基因可以作为区分两分支群体的遗传标记.     

胶州湾浮游植物水华期群落结构特征

根据2001年8月对胶州湾海域进行的为期2d的大面积调查资料,对浮游植物的群落结构进行了初步研究,结果表明,浮游植物群落主要由沿岸暖水性种类组成,以硅藻为主,共34种;还有少量的甲藻7种和绿藻1种,湾中央水域出现的物种数最多,为37种;湾边缘最少,仅出现10种,细胞数量的密集区出现在湾东部边缘水域,最高值为6．96×10⁸个cell·m^-3;低值区出现在湾口,最低值仅为3．18×10⁶个cell·m^-3调查期间浮游植物的物种多样性及其均匀度的最低值均出现在湾东部边缘水域;高值区出现在湾口和湾中央水域,胶州湾海域水团运动和水体富营养化程度是影响浮游植物群落分布的主要环境因素。     

Diversity of phototrophic phytoplankton in Northern South China Sea indicated by <Emphasis Type="Italic">rbcL</Emphasis> analysis

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) as an important enzyme in photosynthetic process exists in various marine phytoplankton. To investigate the photosynthetic phytoplankton communities, conservative encoding gene of RubisCO large subunit (rbcL) was chosen as a target gene in this study. We constructed 33 clone libraries from samples collected in the north of South China Sea (NSCS) and retained 3173 sequences for further analysis. Results of BLASTp showed Stramenopiles and Haptophyta were predominant taxonomic groups in this area, while only five harmful species of Dinophyta were observed. According to the estimators of biodiversity, the photosynthetic community of N709 had very low genetic diversity and richness, which could be explained by the influence of brackish estuarine environment. Beta diversity showed that all samples could be clustered into three groups and those samples with approximately the same distance to land clustered together. Temperature, depth, and latitude of stations as biogeographic factors were indicated to have a significantly positive or negative relation with biodiversity estimators of the phytoplankton community. We concluded that biogeographic factors could be linked with difference in diversity and population of natural phytoplankton assemblages in horizontal surface of NSCS in summer 2007.     

PLMItRNA,a database on the heterogeneous genetic origin of mitochondrial tRNA genes and tRNAs in photosynthetic eukaryotes

The updated version of PLMItRNA reports information and multialignments on 609 genes and 34 tRNA molecules active in the mitochondria of Viridiplantae (27 Embryophyta and 10 Chlorophyta), and photosynthetic algae (one Cryptophyta, four Rhodophyta and two Stramenopiles). Colour-code based tables reporting the different genetic origin of identified genes allow hyper-textual link to single entries. Promoter sequences identified for tRNA genes in the mitochondrial genomes of Angiospermae are also reported. The PLMItRNA database is accessible at http://bighost.area.ba.cnr.it/PLMItRNA/.     

湛江沿海海洋微藻及其多糖、脂类和蛋白藻株多样性研究

从硇洲岛和徐闻珊瑚礁自然保护区潮间带采集的海水和沉积物样品中分离培养海洋微藻, 筛选其中富含多糖、脂类或蛋白质的藻株。采用形态学观察、18S rDNA序列比较及其系统发育分析法, 对分离培养的海洋微藻及其富含多糖、脂类或蛋白质的藻株进行分类鉴定和生物多样性分析。分离、培养、鉴定并储藏了189株海洋微藻, 归属于65个种, 分布于硅藻门(Bacillariophyta)、绿藻门(Chlorophyta)、定鞭藻门(Haptophyta)和红藻门(Rhodophyta)的9纲、25目、30个科、38个属; 其中多糖含量较高的46株海洋微藻, 分布于25个种, 20个属; 脂类含量较高的46株海洋微藻, 分布于32个种, 15个属; 蛋白含量较高的46株海洋微藻, 分布于28个种, 18个属。结果表明硇洲岛和徐闻珊瑚礁自然保护区潮间带可培养的海洋微藻及其富含多糖、脂类和蛋白质藻株的物种多样性丰富, 在新型药物、活性天然产物、功能食品和饲料及其添加剂的发掘等方面具有良好前景。     

Comparisons of the Fungal and Protistan Communities among Different Marine Sponge Holobionts by Pyrosequencing

To date, the knowledge of eukaryotic communities associated with sponges remains limited compared with prokaryotic communities. In a manner similar to prokaryotes, it could be hypothesized that sponge holobionts have phylogenetically diverse eukaryotic symbionts, and the eukaryotic community structures in different sponge holobionts were probably different. In order to test this hypothesis, the communities of eukaryota associated with 11 species of South China Sea sponges were compared with the V4 region of 18S ribosomal ribonucleic acid gene using 454 pyrosequencing. Consequently, 135 and 721 unique operational taxonomic units (OTUs) of fungi and protists were obtained at 97 % sequence similarity, respectively. These sequences were assigned to 2 phyla of fungi (Ascomycota and Basidiomycota) and 9 phyla of protists including 5 algal phyla (Chlorophyta, Haptophyta, Streptophyta, Rhodophyta, and Stramenopiles) and 4 protozoal phyla (Alveolata, Cercozoa, Haplosporidia, and Radiolaria) including 47 orders (12 fungi, 35 protists). Entorrhizales of fungi and 18 orders of protists were detected in marine sponges for the first time. Particularly, Tilletiales of fungi and Chlorocystidales of protists were detected for the first time in marine habitats. Though Ascomycota, Alveolata, and Radiolaria were detected in all the 11 sponge species, sponge holobionts have different fungi and protistan communities according to OTU comparison and principal component analysis at the order level. This study provided the first insights into the fungal and protistan communities associated with different marine sponge holobionts using pyrosequencing, thus further extending the knowledge on sponge-associated eukaryotic diversity.     

Testing for endemism, genotypic diversity and species concepts in Antarctic terrestrial microalgae of the Tribonemataceae (Stramenopiles, Xanthophyceae)

The genetic diversity of all available culture strains of the Tribonemataceae ( Stramenopiles , Xanthophyceae ) from Antarctica was assessed using the chloroplast-encoded psbA /rbcL spacer region sequences, a highly variable molecular marker, to test for endemism when compared with their closest temperate relatives. There was no species endemic for Antarctica, and no phylogenetic clade corresponded to a limited geographical region. However, species of the Tribonemataceae may have Antarctic populations that are distinct from those of other regions because the Antarctic strain spacer sequences were not identical to sequences from temperate regions. Spacer sequences from five new Antarctic isolates were identical to one or more previously available Antarctic strains, indicating that the Tribonemataceae diversity in Antarctic may be rather limited. Direct comparisons of the spacer sequences and phylogenetic analyses of the more conserved rbcL gene revealed that current morphospecies were inadequate to describe the actual biodiversity of the group. For example, the genus Xanthonema , as currently circumscribed, was paraphyletic. Fortunately, the presence of distinctive sequence regions within the psbA/rbcL spacer, together with differences in the rbcL phylogeny, provided significant autoapomorphic criteria to re-define the Tribonemataceae species.     

Phytoplankton community diversity is influenced by environmental factors in the coastal East China Sea

Surface seawater was collected in four different seasons in the coastal East China Sea adjacent to the Yangtze River Estuary and phytoplankton community diversity was analysed using rbcL genetic markers. Phytoplankton diversity (Shannon Index) was found to be highest in autumn and lowest in summer, which was mainly controlled by seawater temperature, river runoff, the Taiwan Warm Current and possibly other environmental factors. For taxa characterized by Form IAB rbcL, the abundance of Chlorophyta was much greater than those of Proteobacteria and Cyanobacteria throughout the year with the most dominant taxa being Bathycoccus prasinos (Chlorophyta) in spring and Micromonas sp. (Chlorophyta) in other seasons. For taxa identified by Form ID rbcL, Coscinodiscophyceae (diatoms) constituted the largest group (most clones) in the phylogenetic tree. Dinophysis fortii (a dinoflagellate) was found to be the most abundant species in winter and spring and Skeletonema spp. (a diatom) dominated the phytoplankton community in summer and autumn. The seasonal dominance of Dinophysis fortii agreed well with the recently increasing proportion of dinoflagellates in the phytoplankton community in the coastal East China Sea. The abundance of Dinophysis fortii was negatively correlated with seawater temperature, suggesting that harmful algal blooms caused by this species may primarily occur in spring.     

Prasinoxanthin-constaining Prasinophyceae Discovered in Jiaozhou Bay, China

The class Prasinophyceae （Chlorophyta） contains some photosynthetic eukaryotic ultraplankton species characterized by containing prasinoxanthin. The existence and abundance of these organisms can be estimated by the diagnostic pigment. We detected the unique pigments of prasinoxanthin-containing Prasinophyceae in Jiaozhou Bay, China using high performance liquid chromatography （HPLC）. This was the first finding of this kind in Chinese seas. Using the ratio of prasinoxanthin to chlorophyll a, the abundance of prasinoxanthin-containing Prasinophyceae has been calculated. The average contribution of prasinoxanthin-containing Prasinophyceae to the chlorophyll a pool was 8.5% and 17.0% in May and August 2004 in Jiaozhou Bay, and the maximums were 25.9% and 36.3%. Size fractionated pigment analysis suggested that more than 80% of prasinoxanthin were in the fraction of 2-20 μm. According to the results of pigment and morphological analysis, the possible genera of prasinoxanthin-containing Prasinophyceae and the reasons for causing this high abundant phytoplankton in Jiaozhou Bay were discussed. This kind of phytoplankton can not be discovered in traditional biological investigation, but its contribution to the coastal ecosystem is significant enough to be studied further.     

Diversity of microbial eukaryotes in Kongsfjorden,Svalbard

Microbial eukaryote diversity was assessed in Arctic Kongsfjorden (Svalbard), by constructing SSU rDNA clone libraries. Samples were collected from different depths at the outer basin in summer (2006), plus an additional one glacial and one sediment sample. The libraries displayed diversity based on 284 full-length sequences. Four main phyla, namely, Alveolates, Stramenopiles, Cercozoans, and Metazoans were often screened in this fjord. Alveolate occupied the highest percentage of taxa in the library of surface sea water, besides the Metazoan-related clones. Moreover, dinoflagellates, diatoms, and pico-Prasinophytes were detected as prevalent phytoplankton through the analysis of libraries. Questions related to the quantity of these phytoplankton and their roles in the microbial food loop arose from an ecological viewpoint.