Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos

m Background: Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. Results: A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp) in length, with a 342 bp open reading frame (ORF) encoding a putative protein of 113 amino acids (aa). Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH) shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC) cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. Conclusion: Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.

Fast-growing transgenic common carp mounfing compensatory growth

Compensatory growth is a phase of accelerated growth apparent when favourable conditions are restored after a period of growth depression. To investigate if F-2 common 'all-fish' growth hormone gene transgenic common carp (Cyprinus carpio) could mount compensatory growth, a 9 week study at 29 degrees C was performed. The control group was fed to satiation twice a day throughout the experiment. The other two groups were deprived of feed for 1 or 2 weeks, respectively, and then fed to satiation during the re-feeding period. At the end of the experiment, the live masses of fish in the deprived groups were still significantly lower than those of the controls. During the re-feeding period, size-adjusted mean specific growth rates and mean feed intakes were significantly higher in the deprived fish than in the controls, indicating a partial compensatory growth response in these fish. No significant differences were found in food conversion efficiency between the deprived and control fish during re-feeding, suggesting that hyperphagia was the mechanism responsible for increased growth rates. The proximate composition of the deprived fish at the end of the experiment was similar to that of the control fish. This study is, to our knowledge, the first to report that fast-growing transgenic fish can achieve partial compensation of growth following starvation. (c) 2007 The Authors Journal compilation (c) 2007 The Fisheries Society of the British Isles.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

Molecular characterization of common carp (Cyprinus carpio) Sonic Hedgehog and discovery of its maternal expression

Sonic hedgehog (Shh), one of important homologous members of the hedgehog (Hh) family in vertebrates, encodes a signaling molecule that is involved in short- or long-range patterning processes during embryogenesis. In zebrafish, maternal activity of Hh was found to be contributing to the formation of primary motoneurons. However, we found that all of the known Hh members were not maternally expressed in zebrafish. In the present study, full-length cDNA of common carp (Cyprinus carpio) Shh (cShh) was gained by degenerate reverse-transcription PCR (RT-PCR) and rapid amplification of cDNA ends. Sequence comparison shows that cShh coding sequence shares 93.4% identity with zebrafish Shh coding sequence, and their corresponding protein sequences have 91.9% similarity. Comparative analysis of Shh genomic sequences and Hh protein sequences from different species revealed that the genomic structures of Hh are conserved from invertebrate to vertebrate. In contrast to zebrafish Shh, cShh transcripts were detectable from one-cell stage by RT-PCR analysis. Whole mount in situ hybridization verified the maternal expression of Shh in common carp, which is, to our knowledge, the first report of that in vertebrates, suggesting that Shh might be responsible for the maternal Hh activity in common carp.

Molecular cloning and expression pattern of 14 kDa apolipoprotein in orange-spotted grouper, Epinephelus coioides

A novel fish-specific apolipoprotein (apo-14 kDa) has been recently cloned from eel and pufferfish. However, its expression pattern has not been elucidated. in this study, EcApo-14 has been screened from hypothalamic cDNA library of male orange-spotted grouper, which shows 62.9%, 51%, 46.9%, 43.2%, and 31.9% identities to Apo-14 of European flounder, pufferfish, Japanese eel, gibel carp, and grass carp, respectively. RT-PCR analysis reveals that this gene is first transcribed in neurula embryos and maintains a relatively stable expression level during the following embryogenesis. EcApo-14 transcripts are at a very high level during embryonic and early larval development in the yolk syncytial layer (YSL), and decrease in YSL and form intense staining in liver at 3 days after hatching. In adult tissues, EcApo-14 is predominantly expressed in liver and brain. The data suggested that EcApo-14 might play an important role in liver and brain morphogenesis and growth. (c) 2005 Elsevier Inc. All rights reserved.

Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 mu g/mu L. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

Hydrothermal synthesis of Fe and Mn doped SnO2 and its magnetic structures

The dilute magnetic semiconductor of Sn1-x-yMnxFeyO2 (0 <= x <= 0.10, 0 <= y <= 0.10) Were syhthesized with the hydrothermal method using SnCl4, Mn(CH3COO)(2) center dot 4H(2)O and FeCl3 center dot 6H(2)O as the raw materials. The structure, morphologies and magnetic properties of the sample were characterized via X-ray powder diffractometer(XRD), transmission electron microscopy(TEM), Raman spectrum and superconducting and quantum interference device(SQUIT), and Mossbeaur spectrum. No secondary phase was found in the XRD spectrum. The morphology of the samples is affected by the kind or the mount of transition metal. The local vibrating model-of Mn Positioned SnO2 sites was found in Raman spectrum. The measured magnetic results indicate that when x = 0.10, y = 0, the sample exhibits strong magnetization in low-temperature (5 K), but the magnetization decrease rapidly at room. temperature; In contrast, when x = 0, y = 0.1, the sample's magnetization and coercivity are both small, but being temperature independent. Mossbeaur spectra indicates that part of the Fe is ferromagnetic coupled, and the simulating results indicate that the ferromagnetic character is intrinsic.

Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages

Shell formation is one of the important events during larval development and metamorphosis in bivalves. However, the molecular mechanisms and environmental cues regulating shell initiation and growth are unclear. Here, we report that ferritin, a principal protein for biological iron storage and metabolism, might play a role in larval shell development of the bivalve mollusk Meretrix meretrix. A full-length ferritin subunit cDNA, named as MmeFer, was cloned and characterized. The MmeFer mRNA expression in different developmental stages, from trochophore to post larvae, was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). MmeFer mRNA expression in larvae of later developmental stages increased at least 8-fold following trochophores. Moreover, the temporal and spatial expressions of MmeFer mRNA were examined by whole mount in situ hybridization. In the trochophore stage, MmeFer was detectable where it was supposed to be for shell initiation. In the later developmental stages, MmeFer was found near digestive glands and mantle that secret larval shell. MmeFer expression was also detected in larvae cultured in artificial seawater with different iron concentrations ranging from 0 to 100 mu M. These results suggest that ferritin may play a role in the shell formation of mollusks. (C) 2009 Elsevier Inc. All rights reserved.

Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

Identification and expression of the amphioxus Cks1 gene

A full-length Cks1 homologue gene, AmphiCks1, was identified in amphioxus, Branchiostoma belcheri tsingtauense. Sequence characteristics, phylogeny and patterns of expression during embryonic and larval development were established. The protein predicted from AmphiCks1 showed high sequence identity with vertebrate and invertebrate homologues. Protein structural studies and phylogenetic analysis suggested that Cks homologues are evolutionarily conserved. The AmphiCks1 transcript was detected in most early developmental stages by northern blotting and whole-mount in situ hybridization, suggesting a role for the gene in cell division. (c) 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Expression of Bblhx3, a LIM-homeobox gene, in the development of amphioxus Branchiostoma belcheri tsingtauense

Amphioxus Bblhx3 was identified as a LIM-homeobox gene expressed in gastrulae. Structural analysis suggested that it is a member of lhx3 but not of lhx1 gene group. Whole mount in situ hybridization revealed, that expression of Bblhx3 was initiated at the early gastrula stage and continued at least until 10-day larvae. Expression of Bblhx3 first appeared in the vegetal and future dorsal area in initial gastrulae and became restricted to the endoderm during gastrulation. In neurulae and early larvae, Bblhx3 was expressed in the developing neural tube, the notochord and preoral pit lineage. In 10-day larvae, Bblhx3 was expressed only in the preoral pit. This expression pattern is apparently distinct from that of vertebrate lhx3 genes that are not expressed during gastrulation. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

Cloning and expression analysis of myogenin from flounder (Paralichthys olivaceus) and promoter analysis of muscle-specific expression

Myogenin is a bHLH transcription factor of the MyoD family. It plays a crucial role in myoblast differentiation and maturation. We report here the isolation of flounder myogenin gene and the characterization of its expression patterns. Sequence analysis indicated that flounder myogenin shared a similar structure and the conserved bHLH domain with other vertebrate myogenin genes. Flounder myogenin gene contains 3 exons and 2 introns. Sequence alignment and phylogenetic showed that flounder myogenin was more homologous with halibut (Hippoglossus hippoglossus) myogenin and striped bass (Morone saxatilis) myogenin. Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites that give rise to slow muscles, and expanded later to the lateral region of the somite that become fast muscles. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region. Its expression could only be detected in the caudal somites, which was consistent with the timing of somite maturation. Transient expression analysis showed that the 546 bp flounder myogenin promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos. (c) 2007 Elsevier Inc. All rights reserved.

A simple and accurate method for determining accurately dissolved inorganic carbon in seawaters

Dissolved inorganic carbon (DIC) account for more than 95% of total carbon in seawater, so it is necessary to make reliable and precise measurements of DIC to study marine carbon cycling. In order to establish a simple and speed method, an airproof device of gas extraction-absorption was designed. Finally a simple method was developed for the determination of DIC in seawater through a large mount of experiments. The determination procedure is as follows: 100 similar to 150 mL seawater was put into conical flask, then add 10% H3PO4, the DIC in seawater sample was dissolved to form CO2 gas and carried by pure N-2, then the CO2 gas was absorbed by two grades 0.1 mol/L NaOH solution. Finally the absorbed solution was titrated by HCl standard solution of 0.01000 mol/L with the end points detected with the indicator phenolphthalein and bromocresol green-methyl red mixture. The precision and accuracy of the method were satisfied. This method was used to analyse seawater samples from Jiaozhou bay in June, 2003. The result shows that the average DIC in surface seawater is 2066 mumol/L, DIC in bottom seawater is 2075 mumol/L inside bay, but the average DIC in surface seawater is 1949 mumol/L, DIC in bottom seawater is 2147 mumol/L outside bay.

中国东部上第三系硬粘土的工程地质特性及边坡开挖工程环境效应研究

The engineering geological properties of Neogene hard clays and related engineering problems are frontiers in the fields of Engineering Geology, Soil Mechanics and Rock Mechanics. Recently, it has been recognized that Neogene hard clay is the intermediate type of material between the soil and the rock. Many aspects of them, such as sampling, testing, calculating and engineering process, are special, which could not be researched by means of theories and methods of traditional Soil Mechanics of Rock Mechanics. In order to get real knowledge and instruct the engineering practice, intersect studying of multiple disciplines, including Engineering Geology, Soil Mechanics and Rock Mechanics, etc., is necessary. Neogene hard clay is one of the important study objects of regional problem rocks & soils in our country, which extensively distributed in China, especially in Eastern China. Taking the related areas along the middle line of the Project of Transferring Water from the South to the North (e.g. Nanyang basin, Fangcheng-Baofeng area and Handan-Yongnian area), South-west of Shandong, Xu-Huai area and Beijing area, etc. as main study areas, the paper divided Neogene hard clays into reduction environment dominated origin and oxidation environment dominated origin, which distributed on areas western and eastern to Mount Taihangshan respectively. Intermediate types are also existed in some areas, which mainly distribute near the edges of depositional basins; they are usually of transitions between diluvial and lacustrine deposits. As to Neogene hard clays from Eastern China, the clay particle content is high, and montmorillonite or illite/montmorillonite turbostratic mineral is the dominating clay mineral. The content of effective montmorillonite is very high in each area, which is the basis for the undesirable engineering properties of Neogene hard clays. For hard clays from the same area, the content of effective montmorillonite in gray-greenish hard clay is much higher than that in purple-brownish or brown-yellowish hard clay, which is the reason why the gray-greenish hard clay usually has outstanding expansive property. On the other hand, purple-brownish or brown-yellowish hard clay has relatively less montmorillonite, so its property is better. All of these prove that the composition (clay mineral) of Neogene hard clay is the control factor for the engineering properties. Neogene hard clays have obvious properties such as fissured, overconsolidated and expansive, which are the main reasons that many engineering problems and geological harzards usually occur in Neogene hard clays. The paper systematically elaborates the engineering properties of Neogene hard clays from Eastern China, analyses the relationships between engineering properties and basic indexes. The author introduces the ANN method into the prediction of engineering property indexes of hard clays, which provides a new way for quantitatively assessment and prediction of engineering property indexes. During investigation in the field, the author found that there exists obvious seam-sheared zone between different hard clays in Miocene Xiacaowan formation in Xu-Huai area. Similar phenomenon also exists near the borderline between Neogene hard clays and underlying coal measures in the Southwest of Shandong province, which could be observed in the cores. The discovery of seam-sheard zone has important theoretical and practical significance for engineering stability analysis and revealing the origin of fissures in Neogene hard clays. The macrostructure, medium structure and microstructure together control the engineering properties of hard clays. The author analyses and summarizes the structural effects on hard clays in detail. The complex of the strength property of hard clays is mostly related to the characteristics of fissures, which is one of the main factors that affect the choice of shear strength parameters. So structure-control theory must be inseparably combined with composition-control theory during the engineering geological and rock/soil mechanics research of hard clays. The engineering properties, such as fissured, overconsolidated and expansive, control the instability of engineering behaviors of Neogene hard clays under the condition of excavation, i.e. very sensitive to the change of existence environment. Based on test data analysis, the author elaborates the effects of engineering environment change on the engineering properties. Taking Nanyang basin as example, the author utilizes FEM to study the effects of various factors on stability of cutting canal slopes, than sets forth the characteristics, development laws and formation mechanism of the deformation and failure of hard clay canal slopes, summarizes the protection and reinforcement principles, as well as the protection and remedy steps. On the basis of comparison of engineering properties of domestic and foreign Neogene muddy deposits, in the view of whole globe and associated with the geological characteristics of China, the paper demonstrates that the intermediate type of the material between the soil and the rock, named "hard clay/soft rock", which can not be separated abruptly, really exists in China. The author has given a preliminary classification based on its geological origin and distribution law, which is very significant for promoting the mixture of Engineering Geology, Soil Mechanics and Rock Mechanics. In the course of large scales engineering construction in China, many engineering experiences and testing data are gained, summarizing these testing results and automatically managing them with computer technology are very necessary. The author develops a software named "Hard Clay-Soft Rock Engineering Geological Information Management and Analysis System (HRGIMS)", realizes the automatic and visual management of geo-engineering information, on the basis of information management, the functions of test data analysis and engineering property prediction are strengthened. This system has well merits for practice and popularization.