The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ （COⅡ） gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A＋T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Partial Sequence Analysis of Mitochondrial COI Gene of the Chinese Shrimp, Fenneropenaeus Chinensis

Eight hundred and thirty eight base pair fragment of mitochondrial COI gene of wild and cultured populations (CP1, CP4, CP5 and CP6) of Fenneropenaeus chinensis was amplified and sequenced. The A, T, G and C contents of the sequence were 235bp (28.0% ),.307bp (36.6%), 138bp (16.5%) and 158bp (18.9%), respectively. Furthermore, 556bp fragment of the sequence was used to discuss the phylogenetic relationship among 14 Penaeidae species using Alpheus armillatus as the outgroup. From the molecular phylogenetic tree constructed by neighbor-joining method, we obtained three large shrimp groups: Farfantepenaeus, Litopenaeus and Fenneropenaeus group. The results also indicated that there were a closer genetic relationships between F. aztecus and F. paulensis, L. schmitti and L. setiferus, F. indicus and F. merguiensis, and the genus Farfantepenaeus was closer to Litopenaeus.     

Distribution and elimination of Norfloxacin in Fenneropenaeus chinensis larvae

This study examined the distribution and elimination of Norfloxacin(NFLX) in Fenneropenaeus chinensis ovary and egg and newly hatched larvae.Mature parental shrimp were exposed to 4 or 10 mg L 1NFLX for 2 or 5 d.Ovary and eggs of the shrimp were sampled after spawning in order to detect NFLX residue using high-performance liquid chromatography(HPLC).Results showed that NFLX residue accumulated in F.chinensis eggs after the parental exposure,with the highest residue detected in ovary.To examine the fate of NFLX residue in larvae,we further determined the concentration of NFLX residue in F.chinensis eggs and larvae at 4 different developmental stages after 24-h exposure.From the newly metamorphosed larvae(0 h post-metamorphosis,h.p.m),samples were taken at different time intervals to 72 h.p.m.HPLC assay showed that the concentrations of NFLX residue in zoea exposed to 4 and 10 mg L 1NFLX were the highest at 1.5 h,i.e.,0.332 and 0.454 μg g 1,respectively.At the two NFLX exposure levels,the elimination time of half NFLX(half life) in nauplius was 45.36 and 49.85 h,respectively,followed by that in zoea(31.68 and 33.13 h),mysis larvae(42.24 and 47.28 h) and postlarvae(24.48 and 30.96 h).Both NFLX exposure levels had a germicidal effect.The distribution and elimination of NFLX residue in F.chinensis tissue,eggs and larvae correlated well with the drug exposure level.The disappearance of NFLX residue coincided with the larval growth,and the half-life of NFLX decreased with the larval development.     

A Successful Microbound Diet for the Larval Culture of Chinese Shrimp Fenneropenaeus chinensis

A 13-day feeding trial was carried out to evaluate the effectiveness of a microbound diet for rearing the larvae of Chinese shrimp Fenneropenaeus chinensis in comparison with the live foods that consist of Isochrysis galbana, Chlorella vulgaris, Tetraselrnis chuii, rotifer （Brachionus plicatilis） and Arternia sp. Larvae of 0 to 13d post-hatch （dph） were reared in a temperature-controlled semi-open culture system and stocked at a density of 100 larvae L^-1 in tanks, each containing 50 L sterilized seawater with salinity 30-32. Larvae were manually fed either the live foods or the microbound diet 6 times per day. At 13 dph, the growth of the larvae fed on the microbound diet was approximately 84% of that fed on the live foods. The survival rate of the larvae fed on the microbound diet was 44.29% at 13dph, which was not significantly different from that of larvae fed on live foods （63.55%）. The body length and development index （DI） of the larvae fed on the microbound diet were always lower than those of larvae fed on live foods. However, the differences reached significant levels only at 11 and 13 dph （P〈 0.05）. The mean dry weight loss of the microbound diet was 9.2% after 90 min immersion in seawater, indicating that this diet has a good water stability. The microbound diet contains 52.23% crude protein and 10.27% lipid and is easy to prepare. These characteristics of the diet suggest good potentials for its successful use in the larviculture of other penaeid and fish species.     

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca²⁺, suggesting that the clotting reaction is catalyzed by a Ca²⁺-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.     

Isolation, cloning and sequencing of AFLP markers related to disease-resistance traits in Fenneropenaeus chinensis

Amplified fragment length polymorphisms （AFLP） technique was used to analyze the fingerprinting of four successive generations of Fenneropenaeus chinensis to reveal their disease-resistance traits. Some loci showed quite different genetic frequencies due to artificial selection, which implied that these fragments were putative markers related to the disease-resistance trait. We developed a simple and effective method to further characterize these AFLP fragments. Specific AFLP bands were cut directly from polyacrylamide gels, re-amplified, cloned and sequenced. Eight putative genetic markers were sequenced and their sizes ranged from 63 to 209 bp. The sequences were submitted to dbGSS （database of Genome Sequence Survey）; and the BLAST analysis showed low similarity to the function genes, indicating these markers were tightly linked to a disease-resistance trait but were not functional genes.     

湛江海域文昌鱼(Branchiostoma)线粒体Cyt b基因的全序列分析

测定了邻近湛江的茂名海区和硇洲岛海域文昌鱼各2个样本线粒体Cytb基因全序列,以尾索动物亚门的2种海鞘(Ciona spp.)和脊椎动物亚门的七鳃鳗(Petromyzon marinus)作为外群,利用分子系统学方法与GenBank收录的14个文昌鱼(Branchiostoma)的Cytb基因全序列进行了比较,UPGMA法构建了系统发生树。全序列分析表明:主产于大西洋(批针文昌鱼B.lanceolatum和佛罗里达文昌鱼B.floridae)、厦门文昌鱼(B.belcheri belcheri Gray)和湛江文昌鱼线粒体Cytb基因全序列长度为1143bp,终止密码子同为UAG;主产于青岛文昌鱼(B.belcheri tsingtaunese)和日本文昌鱼为1141bp,以T结尾,转录过程中构成UAA终止密码子。系统分析表明:16个文昌鱼样品体现出太平洋和大西洋、太平洋南北明显分化。确认采自茂名和硇洲岛的文昌鱼为厦门文昌鱼(B.belcheribelcheriGray);支持将白氏文昌鱼青岛亚种(B.belcheri tsingtaunese)提升为种,命名为日本文昌鱼(B.japonicus)。     

Cloning,characterization,and expression analysis of the DEAD-box family genes,Fc-vasa and Fc-PL10a,in Chinese shrimp (Fenneropenaeus chinensis)

RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication,DNA repair,and RNA processing.However,the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level.We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins,Fc-vasa and Fc-PL10a,from the testes of Chinese shrimp,Fenneropenaeus chinensis.The two predicted amino acid sequences contain all the conserved motifs characterized ...     

Construction of a muscle cDNA library of Chinese shrimp <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> and sequence analysis of the troponin I gene

A muscle cDNA library of Chinese shrimp (Fenneropenaeus chinensis) was constructed with the SMART™ cDNA Library Construction Kit. The titer of optimal primary library was 7.7×10⁵ pfu mL⁻¹ and that of the amplified library was 3.0×10⁹ pfu mL⁻¹. The percentages of the recombinant clones of primary and amplified libraries were over 98%. The insert sizes were longer than 400 bp with an average of 1000 bp. A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin I gene. This library provided a useful resource for the functional genomic research of F. chinensis.     

Nitrogen and Phosphorus Budget of a Polyculture System of Sea Cucumber （Apostichopus japonicus）, Jellyfish （Rhopilema esculenta） and Shrimp （Fenneropenaeus chinensis）

The nitrogen(N) and phosphorus(P) budget and the ecological efficiency of a polyculture system of sea cucumber(Apostichopus japonicus), jellyfish(Rhopilema esculenta) and shrimp(Fenneropenaeus chinensis) were studied in a cofferdam, 120.2 ha in size. The nutrients were supplied by spring tide inflow. In total, 139600 kg N yr-1 and 9730 kg P yr-1 input to the system; while 118900 kg N yr-1 and 2840 kg P yr-1 outflowed from the system concurrently, thus the outflow was 85.7%(N) and 29.2%(P) of inflow. The production of N and P was 889.5 kg yr-1 and 49.28 kg yr-1(sea cucumber) and 204 kg yr-1 and 18.03 kg yr-1(jellyfish and shrimp), respectively. The utilization rate of N and P by polycultured animals was 7.8‰ and 6.9‰, respectively, 21.9% and 38% higher than that of monocultured sea cucumber. Our results indicated that the polyculture system was an efficient culture system of animals and a remediation system of coastal environment as well; it scavenged 14.3% and 70.8% of N and P, respectively. Such an ecological efficiency may be improved further by increasing either the stocking density or the size of sea cucumber or both.     

Effects of Salinity Fluctuation Frequency on the Growth, Molting Rate and Hemolymph 20-Hydroxyecdysone Concentration in Juvenile Chinese shrimp, Fenneropenaeus chinensis

This experiment was conducted to examine the effects of salinity fluctuation frequency on the growth, molting rate and hemolymph 20-hydroxyecdysone (20-HE) concentration for juvenile Fenneropenaeus chinensis with an initial wet body weight of 1.115 g±0.012 g. The salinity of the control treatment (represented by D0) was 28 throughout the experiment, whereas treatments D2,D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8d, respectively. The range of salinity fluctuation was 4. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8d, respectively; each original salinity was decreased abruptly to salinity 24, which first lasted for another 2d and was then raised to its initial value 28. This constituted a salinity fluctuation cycle and afterwards the cycle repeated. It was found that: 1) The weight gain of shrimp in treatment D4 was 202.9% and significantly higher than that in treatment D0 (P<0.05). The molting rate in treatment D2 was the lowest, while that in treatment D6 was the highest and significantly higher than those in treatments D0 and D2 (P<0.05). 2) The hemolymph 20-HE concentration of shrimps in all treatments was at low levels and increased first gradually during the inter-molt period and then increased sharply. It reached a peak value during the pre-molt stage, and then decreased abruptly during the post-molt stage when the lowest level occurred. 3) During the post-molt stage, the hemolymph 20-HE concentration in treatment D2 was 7.47pgμL-1 and significantly higher than that in treatment D0(P<0.05). There were, however, no significant differences in hemolymph 20-HE concentration among all five treatments during the inter-molt stage (P>0.05). During the pre-molt stage, the hemolymph 20-HE concentration in treatment D6 was significantly higher than that in treatment D0(P<0.05), whereas the hemolymph 20-HE concentration in treatment D2 was significantly lower than that in other treatments (P<0.05). Adequate salinity fluctuation promoted the molting rate through increasing hemolymph 20-HE concentration at the pre-molt stage.     

Structure Analysis of the Complete Mitochondrial Genome in Cultivation Variety 'Rongfu'

In this report,complete mitochondrial genome sequences of Laminaria cultivation variety 'Rongfu' were obtained.The results showed the length of circular molecule of mtDNA was 37 638 bp(64.7% A+T),encoding three rRNAs(23S,16S and 5S),25 tRNAs,35 known mitochondrial proteins and 3 ORFs.Sequence alignment indicated its mtDNA genome was very similar to that of Laminaria japonica.Phylogenetic trees inferred from concatenated 30 mitochondrial genes showed that 'Rongfu',Laminaria japon-ica,Laminaria longipedalis,L...     

Effects of Salinity Fluctuation Frequency on the Osmolarity, Na^＋-K^＋ -ATPase Activity and HSP70 Expression in Juvenile Chinese Shrimp, Fenneropenaeus chinensis

Experiments were conducted to examine the effects of salinity fluctuation frequency on the osmolarity, Na+-K+-ATPase activity and HSP70 of Chinese shrimp Fenneropenaeus chinensis with initial wet body weight of 1.460 g ± 0.091 g. The salinity in the control group (D0) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8...     

3种星虫线粒体16S rRNA、COI和Cyt b基因片段的序列比较

对裸体方格星虫(Sipunculus nudus)、可口革囊星虫(Phascolosoma esculenta)和澳洲管体星虫(Siphonosoma australe)的线粒体16S rRNA、COI和细胞色素b(Cytb)基因片段序列进行比较,并对其系统发生进行了初步探讨。采用PCR方法得到总长度分别为531～544bp(16S)、652～675bp(COI)和406～453bp(Cytb)的线粒体片段。片段碱基A+T比例较高(16S rRNA基因58.3%,COI基因56.9%,Cytb基因59.5%)。16S rRNA片段存在169个碱基变异位点(其中包括167个简约信息位点)和44个碱基插入/缺失,种内个体间变异较小;COI片段有512个碱基(333个简约信息位点)存在变异,79个碱基插入/缺失;Cytb片段存在347个碱基(318个简约信息位点)变异位点,16个碱基插入/缺失。数据分析结果支持3种星虫和环节动物的分类地位较近,与软体动物较远的分类观点。此外,裸体方格星虫与澳洲管体星虫之间亲缘关系较近(D=0.3159、0.3156、0.2361)。认为3种星虫线粒体16S rRNA、COI和Cytb基因在种间存在明显的多态性,证实了三种基因序列均普遍适用于星虫种及以上阶元的系统学分析。     

星洲银罗非鱼线粒体细胞色素b基因的序列分析

克隆并测定了星洲银罗非鱼(Oreochromis sp.)细胞色素b基因(cyt b)的全序列,得到全长1 141bp的序列用于分析,用MEGA4.0分析软件与GenBank中的线粒体DNA序列进行序列比较,显示星洲银罗非鱼与其他鱼类的cyt b基因的同源性较高,同源度介于83%~93%间;根据星洲银罗非鱼与莫桑比克罗非鱼、尼罗罗非鱼等12种鱼类的cyt b基因序列构建的系统进化树,初步确定尼罗罗非鱼、莫桑比克罗非鱼为星洲银罗非鱼的父母本。     

一株H_5N_1亚型禽流感病毒株NA基因的克隆与序列分析

用RT-PCR方法从1个H5N1亚型禽流感病毒分离株A/Chicken/Guangdong/DH/1997扩增NA基因cDNA片段,将其克隆至pMD18-T载体,获得重组质粒pMD-NA,并对其核苷酸序列进行测定和分析。结果表明,该毒株的NA基因长度为1350bp,编码449个氨基酸,与其它H5N1亚型AIV分离株的核苷酸序列同源性为97.0%～99.4%,氨基酸序列同源性为97.7%～99.1%,提示禽流感病毒NA基因保守性较高。NA基因氨基酸序列的聚类分析表明该毒株与来自香港的A/Pheasant/HK/FY155/01和A/Ch/HK/FY150/01两个分离株处于同一进化枝,亲缘关系较近。     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

1　Introduction　PorphyraisthemainobjectforalgafarmingandplaysaveryimportantroleinChinesemarineindus tries.Recently ,therehasbeenagreatlossinPor phyracultivationduetothedegenerationoftheculti var,sothereisanincreasingdemandforgoodPor phyracultivars .Theprerequisiteofthetraditionalbreed ingandbioengineeringresearchofPorphyraistheconstructionofpurelines .Traditionally ,theclassifi cationofPorphyrawasaccordingtotheirmorphologi calcharacteristics .However ,mostmorphologicalfea turesofPorphyraar…