Molecular phylogenetic analysis of attached Ulvaceae species and free-floating Enteromorpha from Qingdao coasts in 2007

Based on the sequence data of the nuclear ribosomal DNA internal transcribed spacer(ITS) 1,5.8 S,and ITS 2,the molecular phylogeny was analyzed on Ulvaceae species collected from Qingdao coasts in summer of 2007,including 15 attached Ulva and Enteromorpha samples from 10 locations and 10 free-floating Enteromorpha samples from seven locations.The result supported the monophyly of all free-floating Enteromorpha samples,implying the unialgal composition of the free-floating Enteromorpha,and the attached Ulvaceae species from Qingdao coasts were grouped into other five clades,suggesting that they were not the biogeographic origin of the free-floating Enteromorpha in that season.     

壳聚糖酶高产菌株的筛选和发酵条件的研究

从采集的土样中分离到1株产壳聚糖酶能力较强的菌株OU01,并对OU01进行了16S rDNA序列、形态及生理生化鉴定分析。16S rDNA序列分析表明该菌属于微杆菌属;该菌的形态特征与生理生化鉴定结果表明该菌与Microbacteriumsp.的相似性最高;因此将其初步鉴定为微杆菌属(Microbacterium)。同时对该菌的发酵条件进行了初步研究:该菌的最适培养基组成为(g/L):chitosan 10,(NH4)2SO420,MgSO4.7H2O 1.3,K2HPO4.3H2O 1.4,Glucose 1,Yeast extract 3,NaCl 5,起始pH=6.30。最适发酵温度为30℃,最佳发酵时间为96 h,在上述最优条件下,该菌株产酶达到118 U/mL。Microbacteri-umsp.OU01菌株为壳聚糖酶的研究和应用提供了新的来源。     

Molecular identification of bloom-forming species Phaeocystis globosa (Pryninesiophyta) and its dispersal based on rDNA ITS sequence analysis

The colony-forming Phaeocystis species are causative agents of dense bloom occurrences in coastal waters worldwide. It is difficult to separate them because of the different morphologies associated with their colonial stages. In this study we applied molecular approaches to analyze the genetic variation of Phaeocystis globosa and Phaeocystis pouchetii from several geographic regions, and to assist in tracing the dispersal of bloom-forming Phaeocystis species in coastal waters of China. The sequences of the internal transcribed spacers (ITS1 and ITS2) of rDNA and the 5.8S ribosomal RNA gene of Phaeocystis strains were determined. Sequence comparison shows that P.globosa was the most divergent to P. pouchetii, exhibiting sequence divergence higher than 0.08. However, lower genetic divergences existed between strains of P.globosa. The sequence comparison of the Phaeocystis rDNA ITS clearly shows that the species isolated from the southeast coast of China is identified as P.globosa rather than P. cf. pouchetii or other species. Furthermore, the significance of rDNA variation in distinct global populations of P.globosa suggested it might have had sufficient time to accumulate detectable mutations at the rDNA locus, supporting the hypothesis of ancient dispersal of P.globosa to many areas, meaning that P.globosa blooms in the coastal waters of China are endemic rather than a newly introduced species or a foreign source. Finally, based on the high divergent region of rDNA ITS, a pair of species-specific primers for P.globosa were designed, they could be useful to detect the presence of this species in mixed plankton assemblages or flagellate stages that are recognized with diffculties by means of conventional microscopy.     

魁蚶线粒体16S rRNA和COI基因片段序列测定及其应用前景

魁蚶（Ｓｃａｐｈａｒｃａｂｒｏｕｇｈｔｏｎｉｉ）是蚶科贝类的一种大型经济种类，主要分布于我国、日本和朝鲜半岛及俄罗斯东南部沿海。在我国，主要分布于辽宁、河北和山东沿海，生活在３～５０ｍ（多为２０～３０ｍ）水深的软泥或泥沙质海底，是我国北方沿海重要的经济贝类之一。但但有有关关其其自自然然群群体体的的遗遗传传变变异异及及群群体体间间遗遗传传分分化化等等方方面面的的研研究究不不多多 ;;同同时时，，作作者者和和日日本本研研究究者者发发现现，，中中国国、、韩韩国国和和日日本本魁魁蚶蚶群群体体在在形形态态和…     

Development of a real-time PCR method for Thalassiosira rotula rapid detection

Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ-PCR) method was developed for quantitative detection of T.rotula. The RFQ-PCR assay data showed that the results obtained with the RFQ-PCR quite good agreement with those with the light microscope (LM) counting method, which suggested that the RFQ-PCR could be a useful method for red tide alga detection.     

几株原甲藻核糖体大亚基RNA基因的部分克隆及序列分析

对7株赤潮原甲藻28S rDNA 5'端部分序列进行扩增、克隆和序列测定,并从GeneBank上获取14个原甲藻28S rDNA序列,用NJ法和ME法构建了原甲藻属的系统树,并对序列进行分析.结果表明,7株原甲藻28S rDNA扩增序列长度为950~958 bp,通过NJ法和ME法构建的系统树完全一致.大部分分离自不同海域的同种原甲藻的序列高度保守,而不同种间在序列高变区却有较大的差异.但来自南海海域的海洋原甲藻(Prorocentrum micans)与分离自其他海域的株系序列差异较大,甚至超出了有些种间的差异.由28S rDNA高变区获得的序列,有望成为浮游植物特异性分子探针设计的良好靶区域.     

中国沿海亚历山大藻(Alexandrium)核糖体rDNA部分序列分析及该藻属分子系统进化研究

分析了几株自南海及东海分离的亚历山大藻的rDNA部分序列信息,其中包括核糖体大亚基(LSU)rDNA的5′端D1-D2区序列,以及5.8SrDNA和ITS区序列;同时也对实验室保种的部分来自其它国家和地区的亚历山大藻相关序列进行了测序和分析,并以此作为序列分析中的参考。采用ClustalX及MEGA2软件对所得到的序列信息进行了综合分析与对比。结果表明,分离自南海的塔玛亚历山大藻(Alexandriumtamarense)和分离自东海的链状亚历山大藻(A.catenella),即便是在ITS区和LSUrDNA等高变区,其序列信息也完全一致。与基因库中搜索到的其它亚历山大藻rDNA序列信息相比较,中国沿海的塔玛/链状亚历山大藻序列更接近于塔玛复合种的“亚洲温带”基因型。对于分离自南海的另外两株未定种的亚历山大藻,通过对比序列信息,发现它们与相关亚历山大藻(A.affine)非常接近。分离于我国台湾地区的微小亚历山大藻(A.minutum)在序列上与分离自新西兰的藻株相似,而与分离自欧洲的微小亚历山大藻藻株相差较大。中国沿海亚历山大藻rDNA序列信息的获得为针对有毒藻种设计特异性核酸探针,发展灵敏快速的生物检测技术奠定了基础。     

Molecular analysis of complete ssu to lsu rdna sequence in the harmful dinoflagellatealexandrium tamarense (korean isolate, HY970328M)

New PCR primers (N=18) were designed for the isolation of complete SSU to LSU rDNA sequences from the dinoflagellateAlexandrium tamarense. Standard PCR, employing each primer set selected for amplifications of less than 1.5 kb, successfully amplified the expected rDNA regions of A. tamarense (Korean isolate, HY970328M). Complete SSU, LSU rDNAs and ITS sequences, including 5.8S rDNA, were recorded at 1,800 bp, 520 bp and 3,393 bp, respectively. The LSU rDNA sequence was the first report inAlexandrium genus. No intron was found in the LSU rRNA coding region. Twelve D-domains within the LSU rDNA were put together into 1,879 bp (44.4% G+C), and cores into 1514 bp (42.8% G+C). The core sequence was significantly different (0.0867 of genetic distance, 91% sequence similarity) in comparison withProrocentrum micans (GenBank access. no. X16108). The D2 region was the longest in length (300 bp) and highly variable among the 12 D-domains. In a phylogenetic analysis using complete LSU rDNA sequences of a variety of phytoplankton,A tamarense was clearly separated with high resolution against other species. The result suggests that the sequence may resolve the taxonomic ambiguities ofAlexandrium genus, particularly of the tamarensis complex.     

Molecular identification of bloom-forming species Phaeocystis globosa(Prymnesiophyta)and its dispersal based on rDNA ITS sequence analysis

Abstract-The colony-forming Phaeocystis species are causative agents of dense bloom occurrences incoastal waters worldwide. It is difficult to separate them because of the different morphologies associatedwith their colonial stages. In this study we applied molecular approaches to analyze the genetic variationof Phaeocystis globosa and Phaeocystis pouchetii from several geogrtaphic regions, and to assist in tracingthe dispersal of bloom-forming Phaeocystis species in coastal waters of China. The Sequences of the inter-nal transcribed sacers (ITS1 and ITS2) of rDNA and the 5.8S ribosomal RNA gene of Phaeocystisstrains were determined. Sequence comparison shows that P.globosa was the most divergent toP. pouchetii, exhibiting sequence divergence higher than 0.08. However, lower genetic divergences ex-isted between strains of P.globosa. The sequence comparison of the Phaeocystis rDNA ITS clearlyshows that the species isolated from the southeast coast of China is identified as P. globosa rather thanP. cf. po     

Application of the first internal transcribed spacer(ITS-1) of ribosomal DNA as a molecular marker to population analysis in farrer''s scallop Chlamys farreri

Sequence variation of the first internal transcribed spacer of ribosomal DNA （ ITS - 1 ） was examined and its application to the study of genetic variation was explored in four populations of farter＇ s scallop Chlamys farreri. ITS - 1 fragments, with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS - 1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA （analysis of molecular variance） showed that the majority （96.26%） of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P 〈 0.05 （ = 0.042）, indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.