Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-st...Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.展开更多
Black rockfish(Sebastes schlegelii)has evolved viviparous reproduction mode.Different from the oviparous fishes,the spermatozoa of black rockfish are stored on the surface of ovarian follicle for over four months unti...Black rockfish(Sebastes schlegelii)has evolved viviparous reproduction mode.Different from the oviparous fishes,the spermatozoa of black rockfish are stored on the surface of ovarian follicle for over four months until the egg mature in ovary,while the adaptive evolution of fertilization-related genes remains to be studied.In the present study,based on the genome and transcriptome information of black rockfish,a total of 10 hyaluronidase genes were identified by phylogenetic and sequence analyses,including hyal1,hyal2,hyal3,hyal4,hyal6 and spam1a,spam1b,spam1c and spam1d.The spam1 subfamilies,including spam1a,spam1b,spam1c and spam1d,were significantly expanded compared to other species.The adaptive evolution of hyaluronidase was further investigated by selection pressure analysis of branch model and branch site model.The results showed that only spam1 subfamily was positively selected with a large number of positive selection sites,and the evolution rate was significantly higher thanthose of other teleosts.Two positively selected sites,LYS-171 and GLY-164,were located in the neutral hyaluronidase activity domain,which was pivotal for SPAM1 to participate in enzymatic hydrolysis of the extracellular hyaluronic acid matrix and mediate acrosome reaction in mammals.Furthermore,spam1 was mainly expressed in spermatocytes,secondary spermatocytes,and spermatids of testis,whereas it was not found in metamorphic sperm and mature sperm based on the spatiotemporal expression analysis.All results indicated that spam1 might originate from the testis and might be an adaptation of viviparous trait in black rockfish.展开更多
The leopard coral grouper(Plectropomus leopardus)is a species of significant economic importance.Although artificial cultivation of P.leopardus has thrived in recent decades,the advancement of selective breeding has b...The leopard coral grouper(Plectropomus leopardus)is a species of significant economic importance.Although artificial cultivation of P.leopardus has thrived in recent decades,the advancement of selective breeding has been hindered by the lack of comprehensive population genomic data.In this study,we identified over 8.73 million single nucleotide polymorphisms(SNPs)through whole-genome resequencing of 326 individuals spanning six distinct groups.Furthermore,we categorized 226 individuals with high-coverage sequencing depth(≥14×)into eight clusters based on their genetic profiles and phylogenetic relationships.Notably,four of these clusters exhibited pronounced genetic differentiation compared with the other populations.To identify potentially advantageous loci for P.leopardus,we examined genomic regions exhibiting selective sweeps by analyzing the nucleotide diversity(θπ)and fixation index(FST)in these four clusters.Using these high-coverage resequencing data,we successfully constructed the first haplotype reference panel specific to P.leopardus.This achievement holds promise for enabling high-quality,cost-effectiveimputationmethods.Additionally,we combined low-coverage sequencing data with imputation techniques for a genome-wide association study,aiming to identify candidate SNP loci and genes associated with growth traits.A significant concentration of these genes was observed on chromosome 17,which is primarily involved in skeletal muscle and embryonic development and cell proliferation.Notably,our detailed investigation of growth-related SNPs across the eight clusters revealed that cluster 5 harbored the most promising candidate SNPs,showing potential for genetic selective breeding efforts.These findings provide a robust toolkit and valuable insights into the management of germplasm resources and genome-driven breeding initiatives targeting P.leopardus.展开更多
A better understanding of genetic bases of growth regulation is essential for bivalve breeding,which is helpful to improve the yield of the commercially important bivalves.While previous studies have identified some c...A better understanding of genetic bases of growth regulation is essential for bivalve breeding,which is helpful to improve the yield of the commercially important bivalves.While previous studies have identified some candidate genes accounting for variation in growth-related traits through genotype-phenotype association analyses,seldom of them have verified the functions of these putative,growth-related genes beyond the genomic level due to the difficulty of culturing commercial bivalves under laboratory conditions.Fortunately,dwarf surf clam Mulinia lateralis can serve as a model organism for studying marine bivalves given its short generation time,the feasibility of being grown under experimental conditions and the availability of genetic and biological information.Using dwarf surf clam as a model bivalve,we characterize E2F3,a gene that has been found to account for variation in growth in scallops by a previous genome-wide association study,and verify its function in growth regulation through RNA interference(RNAi)experiments.For the first time,E2F3 in dwarf surf clam,which is termed as MulE2F3,is characterized.The results reveal that dwarf surf clams with MulE2F3 knocked down exhibit a reduction in both shell size and soft-tissue weight,indicating the functions of MulE2F3 in positively regulating bivalve growth.More importantly,we demonstrate how dwarf surf clam can be used as a model organism to investigate gene functions in commercial bivalves,shedding light on genetic causes for variation in growth to enhance the efficiency of bivalve farming.展开更多
The community dynamics and potential carbon ?xation of intertidal macroalgae were investigated monthly from April 2014 to April 2015 in the northwest coast of Yellow Sea. Seasonal variations in biomass and carbon ?xat...The community dynamics and potential carbon ?xation of intertidal macroalgae were investigated monthly from April 2014 to April 2015 in the northwest coast of Yellow Sea. Seasonal variations in biomass and carbon ?xation were presented and showed close relationship with community structure.The carbon ?xation rate ranged from 0.48±0.13 mg C/(g FW ·d) to 4.35±0.12 mg C/(g FW ·d). Sargassum thunbergii, Chondrus ocellatus and Ulva intestinalis were three most in?uential species which contributed27%, 21.9% and 18.5% variation of carbon ?xation rate, respectively. Standing carbon stocks ranged from7.52 g C/m^2 to 41.31 g C/m^2, and estimated carbon stocks varied from 11.77 g C/m 2 to 96.49 g C/m^2. The larger dif ference between estimated and standing carbon stocks implied that more ?xed carbon was exported from the community in summer and autumn than in winter. This study suggested that intertidal macroalgal community could provide a potential function in carbon ?xation of coastal ecosystem.展开更多
Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated (~730 generations,~two days each) to adapt freshwater BG11 medium. A number of freshwater medium adapted colony-derived strains were obtained. The s...Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated (~730 generations,~two days each) to adapt freshwater BG11 medium. A number of freshwater medium adapted colony-derived strains were obtained. The strains were verified phylogenetically to be N. oceanica LAMB0001 based on the 18S ribosomal RNA gene. Freshwater-medium adapted strain (FA1) grew faster in the BG11 medium prepared with freshwater than wild-type N. oceanica grew in f/2 medium prepared with seawater. We assumed that (1) the expression patterns of the genes that expressed differentially between FA1 and the wild-type N. oceanica exposing to the BG11 medium (WT-F) have been reprogrammed;(2) the physiological processes in which these genes involved have been modified;and (3) a Gene Ontology (GO) term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up- or down-regulated if it was enriched simultaneously by up- or down-regulated DEGs between FA1 and WT-F, respectively. Under these assumptions, we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion, membrane and membrane integrity, material transportation, cell movement, and cellular signaling network. These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium. It is feasible to domesticate marine microalgae to a freshwater habitat, which may aid to modify their cultivation performances.展开更多
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 specie...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 by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PLIO subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction （RT-PCR） and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-wasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.展开更多
To investigate sulfide detoxification in Urech＆ unicinctus, oxygen consumption rate and sulfide detoxification products were analyzed during sulfide exposure under controlled laboratory conditions. The results showed...To investigate sulfide detoxification in Urech＆ unicinctus, oxygen consumption rate and sulfide detoxification products were analyzed during sulfide exposure under controlled laboratory conditions. The results showed that oxygen consumption rates were elevated significantly during 3 h sulfide exposure compared to the control （P〈0.05）. The concentration of sulfite in body wall and hindgut of experimental worms increased significantly （P〈0.05） when exposed to 50 μmolL^-1 sulfide, reached a maximum at 24 h and then decreased. Similar result was observed in worms exposed to 150 μmolL^-1 sulfide except that sulfite concentration reached a maximum at 12 h. Contents of thiosulfate in body wall and hindgut of U. unicinctus exposed to sulfide were also significantly higher than that of the control without sulfide exposure. In conclusion, during short-time sulfide exposure U. unicinctus may raise oxygen consumption to detoxify toxic sulfide into sulfite and thiosulfate. Sulfide detoxification was restrained when the exposure time was prolonged or sulfide concentration was increased, which was indicated by decrease of sulfite, the intermediate product of sulfide detoxification.展开更多
The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP ...The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP content assay. Histopathological examination of gill, liver and kidney of exposed fishes showed that gill was a target organ of butachlor. The butachlor seriously impaired the respiration of gills by a series of lesions such as edema, lifting and detachment of lamellar epithelium, breakdown of pillar cells, and blood congestion. The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality. Antioxidant enzyme activity assay of the in vitro cultured flounder gill （FG） ceils exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase （SOD）, catalase （CAT） and glutathione peroxidase （GPX）. Furthermore, along with the decline of antioxidant enzyme activities, ATP content in the exposed FG cells decreased, too. This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP. Similar decrease of ATP content was also observed in the exposed flounder gill tissues. Taken together, as in FG cells, butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells, which in turn resulted in the blood congestion and suffocation of exposed flounder.展开更多
Seven microsatellite markers were used to evaluate the genetic diversity and differentiation of seven stocks of Litopenaeus vannamei, which were introduced from Central and South America to China. All seven microsatel...Seven microsatellite markers were used to evaluate the genetic diversity and differentiation of seven stocks of Litopenaeus vannamei, which were introduced from Central and South America to China. All seven microsatellite loci were polymorphic, with polymorphism information content(PIC) values ranging from 0.593 to 0.952. Totally 92 alleles were identified, and the number of alleles(Na) and effective alleles(Ne) varied between 4 and 21 and 2.7 and 14.6, respectively. Observed heterozygosity(Ho) values were lower than the expected heterozygosity(He) values(0.526–0.754), which indicated that the seven stocks possessed a rich genetic diversity. Thirty-seven tests were detected for reasonable significant deviation from Hardy-Weinberg equilibrium. Fis values were positive at five loci, suggesting that there was a relatively high degree of inbreeding within stocks. Pairwise Fst values ranged from 0.0225 to 0.151, and most of the stock pairs were moderately differentiated. Genetic distance and cluster analysis using UPGMA revealed a close genetic relationship of L. vannamei between Pop2 and Pop3. AMOVA indicated that the genetic variation among stocks(11.3%) was much lower than that within stocks(88.7%). Although the seven stocks had a certain degree of genetic differentiation and a rich genetic diversity, there is an increasing risk of decreased performance due to inbreeding in subsequent generations.展开更多
Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To inves- tigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was con- struct...Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To inves- tigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was con- structed. Single-pass sequencing of the 5＇ ends of 4 081 clones yielded 4 002 high quality expressed sequence tags （ESTs）, which were assembled into 241 contigs and 1 673 singletons, representing 1 914 unigenes. The average length of the ESTs was 582 bp, with sizes ranging from 100-1 500 bp. Basic Local Alignment Search Tool （BLASTX） analysis revealed that 1 664 unigenes had significant homology to known genes in the Na- tional Center for Biotechnology Information （NCBI） non-redundant （nr） database （E-value≤5-10）. Among them, the two most abundant genes encoded metallothionein （157 ESTs） and chlorophyll a/b-binding pro- tein （38 ESTs）, accounting for 7.1% and 1.7% of the total ESTs, respectively. Using Kyoto Encyclopedia of Genes and Genomes （KEGG）, 1 462 unigenes were assigned to 1 161 pathways （E-value≤5-10）. A total of 938 unigenes were assigned Gene Ontology （GO） terms based on the GO hierarchy analysis, and InterProScan searches recognized 1 003 InterPro families. Three genes for metallothionein in Z. marina that belonged to Class II was identified. Results of this study will improve understanding of the molecular mechanisms of saline tolerance in Z. marina.展开更多
Gracilariopsis lemaneiformis is a commercially exploited alga. Its filaceous thallus can be divided into three parts, holdfast, middle segment and tip. The growth and branch forming trend and agar content of these thr...Gracilariopsis lemaneiformis is a commercially exploited alga. Its filaceous thallus can be divided into three parts, holdfast, middle segment and tip. The growth and branch forming trend and agar content of these three parts were analyzed, respectively, in this study. The results showed that the tip had the highest growth rate and branched most, although it was the last part with branch forming ability. The holdfast formed branches earliest but slowly. Holdfast had the highest agar content. We also assessed the difference in protoplast formation and regeneration among three parts. The middle segment displayed the shortest enzymolysis time and the highest protoplast yield; whereas the tip had the strongest vitality of protoplasts formation. Juvenile plants were only obtained from the protoplasts generated from the tip. These results suggested that the differentiation and function of G. lemaneiformis was different.展开更多
Photosynthesis includes the collection of light and a/b-binding （LHC） proteins. In high plants, the LHC gene family constituting the light-harvesting complex ofphotosystems I and II. the transfer of solar energy usi...Photosynthesis includes the collection of light and a/b-binding （LHC） proteins. In high plants, the LHC gene family constituting the light-harvesting complex ofphotosystems I and II. the transfer of solar energy using light-harvesting chlorophyll includes LHCA and LHCB sub-families, which encode proteins Zostera marina L. is a monocotyledonous angiosperm and inhab- its submerged marine environments rather than land environments. We characterized the Lhca and Lhcb gene families of Z. marina from the expressed sequence tags （EST） database. In total, 13 unigenes were annotated as ZmLhc, 6 in Lhca family and 7 in ZmLhcb family. ZmLHCA and ZmLHCB contained the conservative LHC motifs and amino acid residues binding chlorophyll. The average similarity among mature ZmLHCA and ZmLHCB was 48.91% and 48.66%, respectively, which indicated a high degree of diver- gence within ZmLHChc gene family. The reconstructed phylogenetic tree showed that the tree topology and phylogenetic relation- ship were similar to those reported in other high plants, suggesting that the Lhc genes were highly conservative and the classification of ZmLhc genes was consistent with the evolutionary position of Z. marina. Real-time reverse transcription （RT） PCR analysis showed that different members of ZmLhca and ZmLhcb responded to a stress in different expression patterns. Salinity, temperature, light intensity and light quality may affect the expression of most ZmLhca and ZmLhcb genes. Inorganic carbon concentration and acidity had no obvious effect on ZmLhca and ZmLhcb gene expression, except for ZmLhca6.展开更多
Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynoge...Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.展开更多
Nannochloropsis oceanica promises to be an industrial-level producer of polyunsaturated fatty acids. In this study, the fastest and slowest growing N. oceanica mutants were selected through N-methyl-N＇-nitro-N-nitros...Nannochloropsis oceanica promises to be an industrial-level producer of polyunsaturated fatty acids. In this study, the fastest and slowest growing N. oceanica mutants were selected through N-methyl-N＇-nitro-N-nitrosoguanidine mutation, and two mutant strains and the wild type （WT） subjected to transcriptome profiling. It was found that the 0D680 reads at stationary growth phase of both WT and its mutants were proportional to their cell density, thus indicating their division rate and growth speed during culture. This chemical mutation was effective for improving growth performance, and the fast strain divided faster by upregulating the expression of genes functioning in the cell cycle and downregulating genes involved in synthesis of amino acids, fatty acids, and sugars as well as the construction of ribosome and photosynthetic machinery. However, the relationship among the effected genes responsible for cell cycle, metabolism of fatty and amino acids, and construction of ribosome and photosynthetic machinery remained unclear. Further genetic studies are required for clarifying the genetic/metabolic networks underpinning the growth performance ofN. oceanica. These findings demonstrated that this mutation strategy was effective for improving the growth performance of this species and explored a means ofmicroalgal genetic improvement, particularly in species possessing a monoploid nucleus and asexual reproduction.展开更多
Phycoerythrin （PE） is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cyto...Phycoerythrin （PE） is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E.coli BL21 to make two recombinant strains BEX （pGEX-PE） and BGL （pBGL）. PE expressing in BEX （pGEX-PE） was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX （pGEX-PE） cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX （pGEX-PE） and BGL （pBGL） cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX （pGEX-PE） and BGL （pBGL） cell lysates were significantly higher than the negative control BL2 I（pGEX-4T） （P〈0.02）, and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mgmL^-1 BGL （pBGL） products, the scavenging effects of the supernatants of BEX （pGEX-PE） and BGL （pBGL） cell lysates were stronger than that of negative control BL21（pGEX-4T）. However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This research showed that phycoerythrin protein expressing in E. coli has the potential medical and sanitarian value.展开更多
DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. ...DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop(Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at m RNA and protein levels. The c DNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame(ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher(P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.展开更多
Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has ...Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems.展开更多
Aureococcus anophagefferens caused brown tides for three consecutive years from 2009 to 2011 in the coastal waters of Qinhuangdao, China, with numerous, widespread ecological and economic impact on ecosystems. To unde...Aureococcus anophagefferens caused brown tides for three consecutive years from 2009 to 2011 in the coastal waters of Qinhuangdao, China, with numerous, widespread ecological and economic impact on ecosystems. To understand the population dy- namics of nanoplankton during the brown tides, sequences of the V9 region of the 18S rDNA gene, used as a marker, were analyzed by Illumina sequencing to assess nanoplankton biomass, and real-time fluorescence quantitative PCR was performed to analyze spa- tial variation in the 18S rDNA copy concentrations of nanoplankton off the Qinhuangdao coast in July, 2011. The results showed that A. anophagefferens and Minutocellus polymorphus were the dominant species in the local phytoplankton community during the brown tide in July 2011. The highest 18S rDNA copy concentrations of A. anophagefferens and M. polymorphus were detected at stations SHG and FN, respectively. The central area most strongly affected by the brown tide migrated southward from 2011 to 2013. Redundancy analysis (RDA) showed that the decreasing NOx concentration might provide suitable nutrient conditions for the A. anophagefferens outbreak. During the brown tide caused by A. anophagefferens, other phytoplankton, such as diatoms, cryptophytes, chlorophytes, dinoflagellates and other flagellates, could co-occur with it. For zooplankton, due to less selective feeding behavior, Amoebozoa was the most abundant zooplankton at station SHG, while Ciliophora was the most abundant zooplankton at other sta- tions for its more selective feeding.展开更多
Three Gracilariopsis lemaneiformis strains,including wild type and high-temperature-resistant cultivars 981 and 2007,were studied with the changes in their photosynthetic pigment contents and related gene transcriptio...Three Gracilariopsis lemaneiformis strains,including wild type and high-temperature-resistant cultivars 981 and 2007,were studied with the changes in their photosynthetic pigment contents and related gene transcription levels under different light intensities(10,60,100,and 200μmolm^(−2)s^(−1)).The three G.lemaneiformis strains had the following photosynthetic pigments with high-to-low contents:phycoerythrin(PE),phycocyanin(PC),allophycocyanin(APC),and chlorophyll a(Chl a).Among the three strains,cultivar 981 had the highest PE content,followed by cultivar 2007.The PC and APC contents were similar among the three strains,but they were higher in cultivars 981 and 2007 than in the wild type.The Chl a contents in the three G.lemaneiformis strains were equal.A low light intensity(10μmolm^(−2)s^(−1))promoted photosynthetic pigment accumulation in G.lemaneiformis and improved the relative PE gene transcription(peA and peB)in a short period(≤6 d).A high light intensity decreased the PE content.PebA and PebB,which catalyzed phycoerythrobilin synthesis,showed no compensatory upregulation at a low light intensity among the strains except for the wild type.At a high light intensity,transcription levels of pebA and pebB in the three strains were upregulated.This study provided an experimental basis for elucidating the photosynthesis of G.lemaneiformis.As key genes of algal growth,photo-synthesis-related genes served as useful gene markers for screening elite varieties with good traits in breeding.Cultivar 2007 was superior to cultivar 981 in terms of maintaining high pigment levels in a wide range of light intensities,which is the most suitable for aquaculture.展开更多
基金the National Key R&D Program of China(Nos.2018YFD0901506,2018YFD0900305)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018 SDKJ0406-3)。
文摘Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.
基金supported by the National Natural Science Foundation of China(Nos.32273133,31970492).
文摘Black rockfish(Sebastes schlegelii)has evolved viviparous reproduction mode.Different from the oviparous fishes,the spermatozoa of black rockfish are stored on the surface of ovarian follicle for over four months until the egg mature in ovary,while the adaptive evolution of fertilization-related genes remains to be studied.In the present study,based on the genome and transcriptome information of black rockfish,a total of 10 hyaluronidase genes were identified by phylogenetic and sequence analyses,including hyal1,hyal2,hyal3,hyal4,hyal6 and spam1a,spam1b,spam1c and spam1d.The spam1 subfamilies,including spam1a,spam1b,spam1c and spam1d,were significantly expanded compared to other species.The adaptive evolution of hyaluronidase was further investigated by selection pressure analysis of branch model and branch site model.The results showed that only spam1 subfamily was positively selected with a large number of positive selection sites,and the evolution rate was significantly higher thanthose of other teleosts.Two positively selected sites,LYS-171 and GLY-164,were located in the neutral hyaluronidase activity domain,which was pivotal for SPAM1 to participate in enzymatic hydrolysis of the extracellular hyaluronic acid matrix and mediate acrosome reaction in mammals.Furthermore,spam1 was mainly expressed in spermatocytes,secondary spermatocytes,and spermatids of testis,whereas it was not found in metamorphic sperm and mature sperm based on the spatiotemporal expression analysis.All results indicated that spam1 might originate from the testis and might be an adaptation of viviparous trait in black rockfish.
基金supported by the National Key Research and Development Program of China (2022YFD2400501)Key R&D Project of Hainan Province (ZDYF2021XDNY133)+2 种基金Project of Sanya Yazhouwan Science and Technology City Management Foundation (SKJC-2020-02-009)PhD Scientific Research and Innovation Foundation of Sanya Yazhou Bay Science and Technology City (HSPHDSRF-2022-02-007)Young Elite Scientists Sponsorship Program by CAST (2023QNRC001)。
文摘The leopard coral grouper(Plectropomus leopardus)is a species of significant economic importance.Although artificial cultivation of P.leopardus has thrived in recent decades,the advancement of selective breeding has been hindered by the lack of comprehensive population genomic data.In this study,we identified over 8.73 million single nucleotide polymorphisms(SNPs)through whole-genome resequencing of 326 individuals spanning six distinct groups.Furthermore,we categorized 226 individuals with high-coverage sequencing depth(≥14×)into eight clusters based on their genetic profiles and phylogenetic relationships.Notably,four of these clusters exhibited pronounced genetic differentiation compared with the other populations.To identify potentially advantageous loci for P.leopardus,we examined genomic regions exhibiting selective sweeps by analyzing the nucleotide diversity(θπ)and fixation index(FST)in these four clusters.Using these high-coverage resequencing data,we successfully constructed the first haplotype reference panel specific to P.leopardus.This achievement holds promise for enabling high-quality,cost-effectiveimputationmethods.Additionally,we combined low-coverage sequencing data with imputation techniques for a genome-wide association study,aiming to identify candidate SNP loci and genes associated with growth traits.A significant concentration of these genes was observed on chromosome 17,which is primarily involved in skeletal muscle and embryonic development and cell proliferation.Notably,our detailed investigation of growth-related SNPs across the eight clusters revealed that cluster 5 harbored the most promising candidate SNPs,showing potential for genetic selective breeding efforts.These findings provide a robust toolkit and valuable insights into the management of germplasm resources and genome-driven breeding initiatives targeting P.leopardus.
基金supported by the National Natural Science Foundation of China(No.U2106231)the National Key R&D Program of China(No.2022YFD2400303)the Key R&D Project of Shandong Province(No.2022 TZXD003).
文摘A better understanding of genetic bases of growth regulation is essential for bivalve breeding,which is helpful to improve the yield of the commercially important bivalves.While previous studies have identified some candidate genes accounting for variation in growth-related traits through genotype-phenotype association analyses,seldom of them have verified the functions of these putative,growth-related genes beyond the genomic level due to the difficulty of culturing commercial bivalves under laboratory conditions.Fortunately,dwarf surf clam Mulinia lateralis can serve as a model organism for studying marine bivalves given its short generation time,the feasibility of being grown under experimental conditions and the availability of genetic and biological information.Using dwarf surf clam as a model bivalve,we characterize E2F3,a gene that has been found to account for variation in growth in scallops by a previous genome-wide association study,and verify its function in growth regulation through RNA interference(RNAi)experiments.For the first time,E2F3 in dwarf surf clam,which is termed as MulE2F3,is characterized.The results reveal that dwarf surf clams with MulE2F3 knocked down exhibit a reduction in both shell size and soft-tissue weight,indicating the functions of MulE2F3 in positively regulating bivalve growth.More importantly,we demonstrate how dwarf surf clam can be used as a model organism to investigate gene functions in commercial bivalves,shedding light on genetic causes for variation in growth to enhance the efficiency of bivalve farming.
基金Supported by the Public Science and Technology Research Funds Projects of Ocean(No.201305030)the National Natural Science Foundation of China(No.41276137)the China Agriculture Research System(No.CARS-50)
文摘The community dynamics and potential carbon ?xation of intertidal macroalgae were investigated monthly from April 2014 to April 2015 in the northwest coast of Yellow Sea. Seasonal variations in biomass and carbon ?xation were presented and showed close relationship with community structure.The carbon ?xation rate ranged from 0.48±0.13 mg C/(g FW ·d) to 4.35±0.12 mg C/(g FW ·d). Sargassum thunbergii, Chondrus ocellatus and Ulva intestinalis were three most in?uential species which contributed27%, 21.9% and 18.5% variation of carbon ?xation rate, respectively. Standing carbon stocks ranged from7.52 g C/m^2 to 41.31 g C/m^2, and estimated carbon stocks varied from 11.77 g C/m 2 to 96.49 g C/m^2. The larger dif ference between estimated and standing carbon stocks implied that more ?xed carbon was exported from the community in summer and autumn than in winter. This study suggested that intertidal macroalgal community could provide a potential function in carbon ?xation of coastal ecosystem.
基金Supported by the Fundamental Research Funds for the Central Universities(No.201762017)
文摘Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated (~730 generations,~two days each) to adapt freshwater BG11 medium. A number of freshwater medium adapted colony-derived strains were obtained. The strains were verified phylogenetically to be N. oceanica LAMB0001 based on the 18S ribosomal RNA gene. Freshwater-medium adapted strain (FA1) grew faster in the BG11 medium prepared with freshwater than wild-type N. oceanica grew in f/2 medium prepared with seawater. We assumed that (1) the expression patterns of the genes that expressed differentially between FA1 and the wild-type N. oceanica exposing to the BG11 medium (WT-F) have been reprogrammed;(2) the physiological processes in which these genes involved have been modified;and (3) a Gene Ontology (GO) term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up- or down-regulated if it was enriched simultaneously by up- or down-regulated DEGs between FA1 and WT-F, respectively. Under these assumptions, we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion, membrane and membrane integrity, material transportation, cell movement, and cellular signaling network. These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium. It is feasible to domesticate marine microalgae to a freshwater habitat, which may aid to modify their cultivation performances.
基金Supported by the Specialized Research Fund for the Doctoral Program of Higher Education,China (No. 200804230015)the National High Technology Research and Development Program of China (863 Program) (No. 2006AA10A401)
文摘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 by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PLIO subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction （RT-PCR） and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-wasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.
基金supported by the National Natural Science Foundation of China under contract Nos.40776074and 30570223
文摘To investigate sulfide detoxification in Urech＆ unicinctus, oxygen consumption rate and sulfide detoxification products were analyzed during sulfide exposure under controlled laboratory conditions. The results showed that oxygen consumption rates were elevated significantly during 3 h sulfide exposure compared to the control （P〈0.05）. The concentration of sulfite in body wall and hindgut of experimental worms increased significantly （P〈0.05） when exposed to 50 μmolL^-1 sulfide, reached a maximum at 24 h and then decreased. Similar result was observed in worms exposed to 150 μmolL^-1 sulfide except that sulfite concentration reached a maximum at 12 h. Contents of thiosulfate in body wall and hindgut of U. unicinctus exposed to sulfide were also significantly higher than that of the control without sulfide exposure. In conclusion, during short-time sulfide exposure U. unicinctus may raise oxygen consumption to detoxify toxic sulfide into sulfite and thiosulfate. Sulfide detoxification was restrained when the exposure time was prolonged or sulfide concentration was increased, which was indicated by decrease of sulfite, the intermediate product of sulfide detoxification.
基金supported by the National Basic Research Program of China (Grant No 2006CB101805)Undergraduate Innovative Experiment Program of China (Grant Number 091042328)
文摘The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP content assay. Histopathological examination of gill, liver and kidney of exposed fishes showed that gill was a target organ of butachlor. The butachlor seriously impaired the respiration of gills by a series of lesions such as edema, lifting and detachment of lamellar epithelium, breakdown of pillar cells, and blood congestion. The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality. Antioxidant enzyme activity assay of the in vitro cultured flounder gill （FG） ceils exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase （SOD）, catalase （CAT） and glutathione peroxidase （GPX）. Furthermore, along with the decline of antioxidant enzyme activities, ATP content in the exposed FG cells decreased, too. This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP. Similar decrease of ATP content was also observed in the exposed flounder gill tissues. Taken together, as in FG cells, butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells, which in turn resulted in the blood congestion and suffocation of exposed flounder.
基金supported by the National High Technology Development Project of China under contract No. 2012AA10A404the Recommended International Advanced Agricultural Science and Technology Project of China under contract No. 2012-S5
文摘Seven microsatellite markers were used to evaluate the genetic diversity and differentiation of seven stocks of Litopenaeus vannamei, which were introduced from Central and South America to China. All seven microsatellite loci were polymorphic, with polymorphism information content(PIC) values ranging from 0.593 to 0.952. Totally 92 alleles were identified, and the number of alleles(Na) and effective alleles(Ne) varied between 4 and 21 and 2.7 and 14.6, respectively. Observed heterozygosity(Ho) values were lower than the expected heterozygosity(He) values(0.526–0.754), which indicated that the seven stocks possessed a rich genetic diversity. Thirty-seven tests were detected for reasonable significant deviation from Hardy-Weinberg equilibrium. Fis values were positive at five loci, suggesting that there was a relatively high degree of inbreeding within stocks. Pairwise Fst values ranged from 0.0225 to 0.151, and most of the stock pairs were moderately differentiated. Genetic distance and cluster analysis using UPGMA revealed a close genetic relationship of L. vannamei between Pop2 and Pop3. AMOVA indicated that the genetic variation among stocks(11.3%) was much lower than that within stocks(88.7%). Although the seven stocks had a certain degree of genetic differentiation and a rich genetic diversity, there is an increasing risk of decreased performance due to inbreeding in subsequent generations.
基金The Key Science and Technology Program of Shandong Province under contract No. 2012GHY11527Natural Science Foundation of Shandong Province under contract No. Q2007E02+1 种基金Specialized Research Fund for the Doctoral Program of Higher Education (New Teachers) under contract No. 20070423027the Public Science and Technology Research Funds Projects of Ocean, State Oceanic Administration of the People’s Republic of China under contract No. 201105021-8
文摘Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To inves- tigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was con- structed. Single-pass sequencing of the 5＇ ends of 4 081 clones yielded 4 002 high quality expressed sequence tags （ESTs）, which were assembled into 241 contigs and 1 673 singletons, representing 1 914 unigenes. The average length of the ESTs was 582 bp, with sizes ranging from 100-1 500 bp. Basic Local Alignment Search Tool （BLASTX） analysis revealed that 1 664 unigenes had significant homology to known genes in the Na- tional Center for Biotechnology Information （NCBI） non-redundant （nr） database （E-value≤5-10）. Among them, the two most abundant genes encoded metallothionein （157 ESTs） and chlorophyll a/b-binding pro- tein （38 ESTs）, accounting for 7.1% and 1.7% of the total ESTs, respectively. Using Kyoto Encyclopedia of Genes and Genomes （KEGG）, 1 462 unigenes were assigned to 1 161 pathways （E-value≤5-10）. A total of 938 unigenes were assigned Gene Ontology （GO） terms based on the GO hierarchy analysis, and InterProScan searches recognized 1 003 InterPro families. Three genes for metallothionein in Z. marina that belonged to Class II was identified. Results of this study will improve understanding of the molecular mechanisms of saline tolerance in Z. marina.
基金supported by the Twelfth Five-Year Plan in National Science and Technology for the Rural Development in China (2012AA10A411)Public Welfare Project of Ministry of Agriculture of China (200903030)
文摘Gracilariopsis lemaneiformis is a commercially exploited alga. Its filaceous thallus can be divided into three parts, holdfast, middle segment and tip. The growth and branch forming trend and agar content of these three parts were analyzed, respectively, in this study. The results showed that the tip had the highest growth rate and branched most, although it was the last part with branch forming ability. The holdfast formed branches earliest but slowly. Holdfast had the highest agar content. We also assessed the difference in protoplast formation and regeneration among three parts. The middle segment displayed the shortest enzymolysis time and the highest protoplast yield; whereas the tip had the strongest vitality of protoplasts formation. Juvenile plants were only obtained from the protoplasts generated from the tip. These results suggested that the differentiation and function of G. lemaneiformis was different.
基金supported by the Key Science and Technology Program of Shandong Province (Grant no.2012GHY11527)the Public Science and Technology Research Funds Projects of Ocean,State Oceanic Administration of China (Grant no.201105021)
文摘Photosynthesis includes the collection of light and a/b-binding （LHC） proteins. In high plants, the LHC gene family constituting the light-harvesting complex ofphotosystems I and II. the transfer of solar energy using light-harvesting chlorophyll includes LHCA and LHCB sub-families, which encode proteins Zostera marina L. is a monocotyledonous angiosperm and inhab- its submerged marine environments rather than land environments. We characterized the Lhca and Lhcb gene families of Z. marina from the expressed sequence tags （EST） database. In total, 13 unigenes were annotated as ZmLhc, 6 in Lhca family and 7 in ZmLhcb family. ZmLHCA and ZmLHCB contained the conservative LHC motifs and amino acid residues binding chlorophyll. The average similarity among mature ZmLHCA and ZmLHCB was 48.91% and 48.66%, respectively, which indicated a high degree of diver- gence within ZmLHChc gene family. The reconstructed phylogenetic tree showed that the tree topology and phylogenetic relation- ship were similar to those reported in other high plants, suggesting that the Lhc genes were highly conservative and the classification of ZmLhc genes was consistent with the evolutionary position of Z. marina. Real-time reverse transcription （RT） PCR analysis showed that different members of ZmLhca and ZmLhcb responded to a stress in different expression patterns. Salinity, temperature, light intensity and light quality may affect the expression of most ZmLhca and ZmLhcb genes. Inorganic carbon concentration and acidity had no obvious effect on ZmLhca and ZmLhcb gene expression, except for ZmLhca6.
基金supported by the Scientific and Technological Innovation Project of Qingdao National Laboratory for Marine Science and Technology (No. 2015A SKJ02)the National Natural Science Foundation of China (No. 31540063)
文摘Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.
基金Supported by the National Natural Science Foundation of China(No.31270408)the National High Technology Research and Development Program of China(863 Program)(No.2014AA022001)
文摘Nannochloropsis oceanica promises to be an industrial-level producer of polyunsaturated fatty acids. In this study, the fastest and slowest growing N. oceanica mutants were selected through N-methyl-N＇-nitro-N-nitrosoguanidine mutation, and two mutant strains and the wild type （WT） subjected to transcriptome profiling. It was found that the 0D680 reads at stationary growth phase of both WT and its mutants were proportional to their cell density, thus indicating their division rate and growth speed during culture. This chemical mutation was effective for improving growth performance, and the fast strain divided faster by upregulating the expression of genes functioning in the cell cycle and downregulating genes involved in synthesis of amino acids, fatty acids, and sugars as well as the construction of ribosome and photosynthetic machinery. However, the relationship among the effected genes responsible for cell cycle, metabolism of fatty and amino acids, and construction of ribosome and photosynthetic machinery remained unclear. Further genetic studies are required for clarifying the genetic/metabolic networks underpinning the growth performance ofN. oceanica. These findings demonstrated that this mutation strategy was effective for improving the growth performance of this species and explored a means ofmicroalgal genetic improvement, particularly in species possessing a monoploid nucleus and asexual reproduction.
文摘Phycoerythrin （PE） is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E.coli BL21 to make two recombinant strains BEX （pGEX-PE） and BGL （pBGL）. PE expressing in BEX （pGEX-PE） was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX （pGEX-PE） cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX （pGEX-PE） and BGL （pBGL） cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX （pGEX-PE） and BGL （pBGL） cell lysates were significantly higher than the negative control BL2 I（pGEX-4T） （P〈0.02）, and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mgmL^-1 BGL （pBGL） products, the scavenging effects of the supernatants of BEX （pGEX-PE） and BGL （pBGL） cell lysates were stronger than that of negative control BL21（pGEX-4T）. However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This research showed that phycoerythrin protein expressing in E. coli has the potential medical and sanitarian value.
基金supported by the National High Technology Research and Development Program of China (863 Program) (2012AA10A402)the Natural Science Foundation of Qingdao (11-2-4-1(10)-jch)the Key Natural Science Foundation of Shandong Province (Z2008D02)
文摘DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop(Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at m RNA and protein levels. The c DNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame(ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher(P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.
基金Supported by National Natural Science Foundation of China (No. 30600455)the National Basic Research Program of China (973 Program, No.2004CB117402)
文摘Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems.
基金supported by the Public Science and Technology Research Funds Projects of Ocean (No. 201205031)the National Natural Science Foundation of China (No. 41521064)the Scientific and Technological Innovation Project of the Qingdao National Laboratory for Marine Science and Technology (No. 2016ASKJ02)
文摘Aureococcus anophagefferens caused brown tides for three consecutive years from 2009 to 2011 in the coastal waters of Qinhuangdao, China, with numerous, widespread ecological and economic impact on ecosystems. To understand the population dy- namics of nanoplankton during the brown tides, sequences of the V9 region of the 18S rDNA gene, used as a marker, were analyzed by Illumina sequencing to assess nanoplankton biomass, and real-time fluorescence quantitative PCR was performed to analyze spa- tial variation in the 18S rDNA copy concentrations of nanoplankton off the Qinhuangdao coast in July, 2011. The results showed that A. anophagefferens and Minutocellus polymorphus were the dominant species in the local phytoplankton community during the brown tide in July 2011. The highest 18S rDNA copy concentrations of A. anophagefferens and M. polymorphus were detected at stations SHG and FN, respectively. The central area most strongly affected by the brown tide migrated southward from 2011 to 2013. Redundancy analysis (RDA) showed that the decreasing NOx concentration might provide suitable nutrient conditions for the A. anophagefferens outbreak. During the brown tide caused by A. anophagefferens, other phytoplankton, such as diatoms, cryptophytes, chlorophytes, dinoflagellates and other flagellates, could co-occur with it. For zooplankton, due to less selective feeding behavior, Amoebozoa was the most abundant zooplankton at station SHG, while Ciliophora was the most abundant zooplankton at other sta- tions for its more selective feeding.
基金This research was supported by the National Natural Sci-ence Foundation of China(No.31872555)the China Agri-culture Research System(No.CARS-50)the Key Pro-gram of Science and Technology Innovation Ningbo(No.2019B10009).
文摘Three Gracilariopsis lemaneiformis strains,including wild type and high-temperature-resistant cultivars 981 and 2007,were studied with the changes in their photosynthetic pigment contents and related gene transcription levels under different light intensities(10,60,100,and 200μmolm^(−2)s^(−1)).The three G.lemaneiformis strains had the following photosynthetic pigments with high-to-low contents:phycoerythrin(PE),phycocyanin(PC),allophycocyanin(APC),and chlorophyll a(Chl a).Among the three strains,cultivar 981 had the highest PE content,followed by cultivar 2007.The PC and APC contents were similar among the three strains,but they were higher in cultivars 981 and 2007 than in the wild type.The Chl a contents in the three G.lemaneiformis strains were equal.A low light intensity(10μmolm^(−2)s^(−1))promoted photosynthetic pigment accumulation in G.lemaneiformis and improved the relative PE gene transcription(peA and peB)in a short period(≤6 d).A high light intensity decreased the PE content.PebA and PebB,which catalyzed phycoerythrobilin synthesis,showed no compensatory upregulation at a low light intensity among the strains except for the wild type.At a high light intensity,transcription levels of pebA and pebB in the three strains were upregulated.This study provided an experimental basis for elucidating the photosynthesis of G.lemaneiformis.As key genes of algal growth,photo-synthesis-related genes served as useful gene markers for screening elite varieties with good traits in breeding.Cultivar 2007 was superior to cultivar 981 in terms of maintaining high pigment levels in a wide range of light intensities,which is the most suitable for aquaculture.
