FIVE RED TIDE SPECIES IN GENUS PROROCENTRUM INCLUDING THE DESCRIPTION OF PROROCENTRUM DONGHAIENSE LU SP. NOV. FROM THE EAST CHINA SEA

A new planktonic dinoflagellate, Prorocentrum donghaiense I_a sp. nov., is described in the present paper. The water sample was collected from the Changjiang Estuary, the East China Sea. The species identification is based on shape, size, surface micro-morphology, ornamentation of thecal plates and the architecture of the pel‘iflagellar area and the intercalary bands as seen by fight and scanning electron microscope. Prorocentrum donghaiense Lu sp. nov. is compared with other prorocentrum species with respect to morphological characteristics and bloom behavior. It is not known whether Prorocentrum donghaiense Lu sp. nov produces phycotoxins like some other Prorocentrum species. Four other red tide species in the family Prorocentraceae (Dinophyceae), namely P. balticum, P. minimum, P.micans, P. triestinum, were examined and identified by light and scanning electron microscope. They have been recorded as bloom-forming species. Some aggregates of Prorocentrum are observed at the end of blooms. An event of strong discoloration caused by P. donghaiense could be detected by satellite sensor in the East China Sea in the late spring, of 1995.     

Construction of cDNA Library of Pyrocystis lunula（Pyrophyta）

1　Introduction　Photosyntheticdinoflagellatesareimportantprima ryproducersandcausesoftoxic‘redtide’ .Theyarefoundinmostaquaticenvironmentsandformamajorpartofthemodernplankton .Dinoflagellatesmaybeimportantindicatorsofenvironmentalstatus (Dale ,1996 ) .…     

Molecular Cloning and Expression ofPoIR2, a Novel Gene Involved in Immune Response in Japanese Flounder （Paralichthys olivaceus）

A novel immune-related gene was expressed in Japanese flounder (Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 (P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed as soluble type.     

Nutrient and chlorophyll a anomaly in red-tide periods of 2003-2008 in Sishili Bay, China

Sishili Bay is the most important aquiculture and tourism area for the city of Yantai, China; however, red tides occurred frequently and have caused huge economic losses in this bay in recent years. To gain a better understanding of the local ecological environments in the bay, we conducted this research between 2003 and 2008 to analyze variations in nutrients and chlorophyll (chl-a) during high frequency red tide period (May to September). The results show that the chl-a concentration increased from 2.70 in 2003 to 7.26 mg/m3 in 2008, while the concentration of total inorganic nitrogen (TIN) and silicate (SiO3-Si) increased lineally from 5.18 and 1.45 μmol/L in 2003 to 18.57 and 9.52 μmol/L in 2008, respectively, and the annual phosphate (PO4-P) varied between 0.15 and 0.46 μmol/L. Special attention was given to a red tide in August 2007 occurred when water temperature was high and nutrient concentrations increased sharply because of a heavy rainfall. Overall, the results show the P limitation in Sishili Bay, and reveal that red tides were caused by eutrophication from terrestrial inputs and local warm weather, particularly during rainy periods. Therefore, to control red tide, greater efforts should be made to reduce sewage discharges into Sishili Bay, particularly during rainfall seasons.     

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.     

Construction of a Muscle cDNA Library of Chinese Shrimp Fenneropenaeus chinensis and Sequence Analysis of the Troponin Ⅰ Gene

A muscle cDNA library of Chinese shrimp (Fenneropenaeus chinensis) was constructed with the SMARTTM cDNA Li- brary 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 ofE chinensis.     

荷那龙罗非鱼两种GHSR基因的克隆与序列分析

用RT-PCR和RACE方法,从荷那龙罗非鱼(Oreochromis hornorum)垂体中克隆到生长激素促分泌素受体(GHSR)cDNA全序列。荷那龙罗非鱼GHSR基因具有GHSR-1a与GHSR-1b两个高度保守cDNA序列。GHSR-1a序列全长1 646 bp,包括225 bp的5′非编码区,266 bp的3′非编码区和1 155 bp的开放阅读框,编码384个氨基酸残基,具有7个跨膜结构域结构(transmembrane domains,TM);GHSR-1b序列全长1 877 bp,包括225 bp的5′非编码区,755 bp的3′非编码区和897 bp的开放阅读框,编码298个氨基酸残基,只具有前5个TM,在第6个TM的第4个氨基酸处开始缺失。将GHSR cDNA序列与基因组序列比较发现,这两种cDNA转录本来自同一个基因的不同变体。     

Variability of Prorocentrum donghaiense response to allelopathic action from Alexandrium pacificum in laboratory culture

The dinoflagellates Alexandrium pacificum and Prorocentrum donghaiense are two wellknown harmful algal blooms(HABs)-forming species,both were usually found in the same sea areas in form of HABs in China.To date,there is no mechanistic model that can fully explain the occurrence of P.donghaiense blooms with A.pacificum.We found that different strains of P.donghaiense had different intrinsic growth rates of 0.107-0.215/d,and these strains exhibited different responses to the allelopathic action from A.pacificum.Some strains of P.donghaiense could grow well despite some degrees of inhibition in a short period,suggesting the two algal species P.donghaiense and A.pacificum could coexist,even if A.pacificum was allelopathic.Our findings may advance the understanding of phenotypes in P.donghaiense and provide a potential mechanism involved in the coexistence of P.donghaiense and A.pacificum in the same area.     

Cloning, Expression and Characterization of Translationally Controlled Tumor Protein （TCTP） Gene from Flatfish Turbot （Scophthalmus maximus）

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 451 bp and an open reading flame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5'UTR of SmTCTP started with a 5'-terminal oligopyrimidine tract (5'-TOP), a typical feature for translationaily controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte-brate TCTPs in a range of 58.8% to 64.1%. The length offish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rer/o) and rohu (Labeo rohita) is 171 aa in length. North-ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves-tigation of the biological function of TCTP in fish.     

Characterization of SNAP-25 gene from marine teleostean, Lateolabrax japonicus

The t-SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) plays an essential role in regulating fusion between the vesicle and plasma membranes during exocytosis. To clone and characterize SNAP-25 gene, the first step in the functional study of SNARE proteins in marine teleostean, was to obtain the cDNA of sea perch SNAP-25 (SPsn25) by RT-PCR and RACE-PCR amplification of a Japanese sea perch. The full-length cDNA of 831bp contains a CDS of 615 bp, coding 204 amino acid residues, and a 5′UTR of 219bp. Bioinformatic analysis revealed that SPsn25 corresponds with SNAP-25a isoform and shares 91.1% identity with SNAP-25a of a goldfish and a zebrafish. The SPsn25 expression in both mRNA and protein levels in the Japanese sea perch had been identified through semi-quantitative RT-PCR and Western Blot assay. Together, these data again confirmed the nerve tissue specificity of the fish SNAP-25 gene expression.     

Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

The myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. We have isolated the cDNA clone encoding MyHC of the grass carp, Ctenopharyngodon idella. The sequence comprises 5 934 bp, including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues. The deduced amino acid sequence showed 69% homology to rabbit fast skeletal MyHC and 73%–76% homology to the MyHCs from the mandarin fish, walleye pollack, white croaker, chum salmon, and carp. The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80% homology to the corresponding regions of other fish MyHCs. The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR. The MyHC gene showed the highest expression in the muscles compared with the kidney, spleen and intestine. Developmentally, there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage. The highest expression was detected in hatching larva. Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.