基于转录组测序的卤虫卵生和卵胎生相关基因表达研究

为揭示卤虫(Artemia)不同繁殖模式的发生机制, 文章通过构建孤雌生殖卤虫卵生和卵胎生差异转录组文库并结合生物信息学分析, 对两种繁殖模式间的差异表达基因进行查找筛选, 然后利用qRT-PCR对候选繁殖模式相关基因的表达进行分析。转录组测序显示有1452个差异表达基因, 包括601个上调基因和851个下调基因。根据差异表达基因GO功能分类结果可知, 注释到生物过程、细胞组成和分子功能的unigene分别有1243、306和530个。KEGG富集分析结果显示差异基因显著富集在抗原加工和核糖体通路中。结合转录组分析, 进一步筛选得到6个生殖相关基因, 并针对不同繁殖模式下的卤虫进行qRT-PCR, 结果表明, 6个生殖相关基因在卵生卤虫卵巢中的表达量均显著高于卵胎生卤虫。此外, 对6个候选生殖相关基因编码的蛋白质保守结构域进行预测, 发现均与之前报道的相应基因保守结构域一致。综上所述, 研究所选择的6个基因可能影响参与了卤虫的生殖过程。研究结果为孤雌卤虫繁殖模式分子机制调控的研究提供了基础信息, 有助于完善卤虫的生殖生物学理论。     

Structural and expression analysis of hepatic vitellogenin gene during ovarian maturation in Anguilla japonica

Vitellogenin (Vtg), the precursor molecule for yolk, is synthesized in the liver under estrogenic control. In all oviparous species, including fish, the process of vitellogenesis is crucial to subsequent embryonic development. This study attempted to obtain the cDNA encoding for Vtg from female Japanese eels, Anguilla japonica. Rapid amplification of cDNA ends (RACE) and polymerase chain reaction (PCR) were used to amplify Vtg cDNA prepared from liver extracts. Obtained PCR products were subcloned and sequenced. The overall sequence of eel Vtg cDNA isolated in this study contained 5395 bp nucleotides. This Vtg sequence encodes 1743 amino acids of the precursor molecule, and is entirely composed of the characteristic N-terminal lipovitellin-I region, an internal polyserine domain region, and a c-terminal lipovitellin-II region. The deduced amino acid sequence from these clones shares 34–61% identity with other teleost Vtgs. Northern blot assays of Vtg gene expression following hormonal treatment demonstrated that this Vtg is synthesized in the liver under stimulation by estradiol injection. However, Vtg synthesis may not be enhanced by salmon pituitary homogenate (SPH) induction for the developing ovarian follicles. Notably, the effect of methyltestosterone, following SPH injection, may be more appropriate for the uptake of Vtg by ovarian follicle maturation during the artificial maturation of Japanese female eels.     

Diversity, structure, and expression of the gene for p26, a small heat shock protein from Artemia

p26, a small heat shock protein, is thought to protect Artemia embryos from stress during encystment and diapause. Full-length p26 cDNAs were compared and used to determine phylogenetic relationships between several Artemia species. The alpha-crystallin domain of p26 was the most conserved region of the protein and p26 from each Artemia species contained characteristic amino-terminal WD/EPF and carboxy-terminal VPI motifs. Sequence conservation suggested the importance of p26 to oviparously developing Artemia embryos and indicated common functions for the protein during development and stress resistance, although as shown by modeling some species-specific p26 amino acid substitutions may have adaptive significance. The p26 gene obtained from A. franciscana exhibited a unique sHSP intron arrangement with an intron in the 5'-untranslated region. Computer-assisted analysis revealed heat shock elements and other putative cis regulatory sequences but their role in gene regulation is unknown. In contrast to previous results for which Northern blots were analyzed, p26 gene expression was observed in ovoviviparous embryos by use of PCR-based methodology, but the p26 protein was not detected.     

Deubiquitinating enzyme BAP1 is involved in the formation and maintenance of the diapause embryos of Artemia

The modification of proteins by ubiquitination and deubiquitination plays an important role in various cellular processes. BRCA1-associated protein-1 (BAP1) is a deubiquitinating enzyme whose function in the control of the cell cycle requires both its deubiquitinating activity and nuclear localization. In the present study, a ubiquitin carboxyl-terminal hydrolase belonging to the BAP1 family was identified and characterized from Artemia parthenogenetica, a member of a family of brine shrimp that, under certain conditions, produce and release diapause embryos in which cell division and turnover of macromolecules are arrested. Western blot analysis and in vitro enzyme activity assay revealed ArBAP1 to be a cytoplasmic protein with typical ubiquitin hydrolase activity. Northern blot analysis revealed that ArBAP1 was abundant in the abdomen of Artemia producing diapause-destined embryos. Furthermore, by in situ hybridization, ArBAP1 was located exclusively in the embryos. In vivo knockdown of ArBAP1 by RNA interference resulted in the formation of embryos with split shells and abortive nauplii. The present findings suggest that ArBAP1, the first reported cytoplasmic BAP1, participates in the formation of diapause embryos and plays an important role in the control of cell cycle arrest in these encysted embryos.     

Vitellogenin cleavage products as indicators for toxic stress in zebra fish embryos: a proteomic approach

Vitellogenins (Vtgs) are the major yolk proteins in all oviparous animals. Systematic and regulated processing of these during embryogenesis is crucial for embryonic development. In the present study, toxicant-induced disturbance of Vtg degradation processes during Danio rerio (DR) embryogenesis was analysed to establish a sensitive tool for monitoring toxic stress at the molecular level. A 2-DE-based proteomic approach for whole DR embryos was established to study Vtg cleavage products (lipovitellin (Lv) derivatives). Ethanol was chosen as a positive control for a toxicity related change in the proteome of whole zebra fish embryos. Protein extracts from embryos treated with two ethanol concentrations, 0.5 and 2% v/v, showing either no or very strong visible effects, like absent heartbeat and blood circulation, were examined. Significant changes in the Lv pattern were detected for both conditions. The results are interpreted as scope for the use of the high abundant Lv derivatives as sensitive stress indicators in zebra fish embryos reflecting the overall fitness of the intact organisms.     

Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii- environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene

The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned; however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatching enzyme is considered to be well adapted to the natural hatching environment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the products alternatively spliced from the LCE gene are generated by the mutations in intronic regions responsible for splicing.     

Induction of vitellogenesis by estradiol-17beta and development of enzyme-linked immunosorbant assays to quantify plasma vitellogenin levels in green turtles (Chelonia mydas)

Treatment of juvenile green turtles (Chelonia mydas) with estradiol-17beta resulted in the induction of a 200 kDa plasma protein, consistent with vitellogenin (Vtg). The N-terminal 15 amino acids of the anion exchange purified protein shared sequence homologies with vitellogenins of several vertebrate species. Rabbit antiserum raised against purified Vtg recognized the plasma protein as well as several yolk proteins. Monoclonal antibody (Mab) HL1248, produced by inoculating mice with turtle yolk granules, showed specificity for plasma Vtg as well as a set of yolk proteins 120, 82, 43 and 32 kDa in size. The N-terminal 22 amino acids of the 43 kDa yolk protein was similar to the lipovitellin I subunit of Vtg of several vertebrate species. The peptide mass map of the 82 kDa yolk protein shared enough ions with that of purified plasma Vtg to support the conclusion that this protein was derived from plasma Vtg. Taken together, these results validate the specificity of Mab HL1248 for Vtg. Using purified Vtg concentration standards, competition and antigen capture enzyme-linked immunosorbant assays (ELISAs) were shown to quantitatively detect Vtg in green turtle plasma. Pre-induced plasma of juvenile turtles had Vtg levels of 2-4 micrograms/ml whereas post-estradiol exposure samples had 38-40 mg/ml. The plasma Vtg concentration of a nesting female turtle was 4.6 mg/ml, approximately 20-fold higher than that of a non-nesting adult female. The antigen capture ELISA will be useful in population studies of this endangered species, to detect vitellogenesis in females that will nest in a given year and to detect inappropriate Vtg levels in turtles exposed to xenoestrogens.     

Characterization of the microtubule proteome during post-diapause development of Artemia franciscana

The microtubule proteome encompasses tubulin and a diverse group of proteins which associate with tubulin upon microtubule formation. These proteins either determine microtubule organization and function or their activity is influenced by microtubule association. To characterize the microtubule proteome in Artemia franciscana, tubulin assembly was induced with taxol in vitro after 0 and 12 h of post-diapause development. Proteins obtained by extraction of microtubules with 0.5 M NaCl were electrophoresed in two-dimensional gels and analyzed by mass spectrometry. Fifty-five proteins were identified with 10 of these occurring at both developmental stages, and multiple isoforms were observed for some proteins of the Artemia proteome. Their functions include roles in membrane transport, metabolism, chaperoning and protein synthesis, thus reflecting physiological properties of encysted Artemia such as stress resistance and the ability to rapidly initiate post-diapause development. For example, chaperones may protect tubulin during encystment and facilitate folding in metabolically active embryos. Additionally, the interaction of metabolic enzymes with microtubules funnels reaction intermediates, potentially enhancing efficiency within biochemical processes. This study represents the first systematic characterization of a crustacean microtubule proteome. Although it is difficult to be certain that all protein associations documented herein occur in vivo, the results suggest how protein-protein interactions contribute to cytoplasmic organization while implying how Artemia embryos resist stress and remain capable of development once diapause terminates.     

Matrix metalloproteinase-3 induction in rat brain astrocytes: focus on the role of two AP-1 elements

Embryos of the crustacean, Artemia franciscana, undergo alternative developmental pathways, producing either larvae or encysted embryos (cysts). The cysts enter diapause, characterized by exceptionally high resistance to environmental stress, a condition thought to involve the sHSP (small heat-shock protein), p26. Subtractive hybridization has revealed another sHSP, termed ArHsp21, in diapause-destined Artemia embryos. ArHsp21 shares sequence similarity with p26 and sHSPs from other organisms, especially in the alpha-crystallin domain. ArHsp21 is the product of a single gene and its synthesis occurred exclusively in diapause-destined embryos. Specifically, ArHsp21 mRNA appeared 2 days post-fertilization, followed 1 day later by the protein, and then increased until embryo release at day 5. No ArHsp21 protein was detected in embryos developing directly into larvae, although there was a small amount of mRNA at 3 days post-fertilization. The protein was degraded during post-diapause development and had disappeared completely from second instar larvae. ArHsp21 formed large oligomers in encysted embryos and transformed bacteria. When purified from bacteria, ArHsp21 functioned as a molecular chaperone in vitro, preventing heat-induced aggregation of citrate synthase and reduction-driven denaturation of insulin. Sequence characteristics, synthesis patterns and functional properties demonstrate clearly that ArHsp21 is an sHSP able to chaperone other proteins and contribute to stress tolerance during diapause. As such, ArHsp21 would augment p26 chaperone activity and it may also possess novel activities that benefit Artemia embryos exposed to stress.     

ArHsp22, a developmentally regulated small heat shock protein produced in diapause-destined Artemia embryos, is stress inducible in adults

Diapause embryos of the crustacean Artemia franciscana exhibit extreme stress tolerance, a property thought to involve molecular chaperones known as small heat shock proteins. To further explore this idea, the structure, function and synthesis of ArHsp22, an Artemia small heat shock protein, were characterized. ArHsp22 contains amino-terminal WXDPF motifs, an alpha-crystallin domain with a highly conserved arginine, and a carboxy-terminal I/VXI/V motif, all typical of small heat shock proteins. ArHsp22 formed large oligomers and exhibited molecular chaperone activity in vitro, protecting citrate synthase and insulin from denaturation. Quantitative PCR and immunoprobing of western blots revealed that ArHsp22 synthesis is restricted to diapause-destined Artemia embryos and that the protein is degraded during post-diapause development. ArHsp22 was observed in cyst nuclei, a location shared by p26 but not ArHsp21, which are two other diapause-specific Artemia small heat shock proteins. ArHsp22 production was enhanced by thermal stress, but only in adults, thus representing the first crustacean small heat shock protein whose synthesis is known to be both developmentally regulated and stress inducible. The results demonstrate that expression of the gene for ArHsp22 is modulated by multiple cues that vary with life history stage. Such findings are of importance in understanding diapause maintenance in Artemia embryos and the survival of adult animals experiencing environmental insult.     

Vitellogenin and its messenger RNA during ovarian development in the female blue crab, Callinectes sapidus: gene expression, synthesis, transport, and cleavage

Blue crab vitellogenin (VTG) cDNA encodes a precursor that, together with two other Brachyuran VTGs, forms a distinctive cluster within a phylogenetic tree of crustacean VTGs. Using quantitative RT-PCR, we found that VTG was primarily expressed in the hepatopancreas of a vitellogenic female, with minor expression in the ovary. VTG expression in the hepatopancreas correlated with ovarian growth, with a remarkable 8000-fold increase in expression from stage 3 to 4 of ovarian development. In contrast, the VTG levels in the hepatopancreas and hemolymph decreased in stage 4. Western blot analysis and N-terminal sequencing revealed that vitellin is composed of three subunits of approximately 78.5 kDa, 119.42 kDa, and 87.9 kDa. The processing pathway for VTG includes an initial hepatopancreatic cleavage of the primary precursor into approximately 78.5-kDa and 207.3-kDa subunits, both of which are found in the hemolymph. A second cleavage in the ovary splits the approximately 207.3-kDa subunit into approximately 119.4-kDa and approximately 87.9-kDa subunits. The hemolymph VTG profiles of mated and unmated females during ovarian development indicate that early vitellogenesis and ovarian development do not require mating, which may be essential for later stages, as VTG decreased to the basal level at stage 4 in the unmated group but remained high in the mated females. Our results encompass comprehensive overall temporal and spatial aspects of vitellogenesis, which may reflect the reproductive physiology of the female blue crab, e.g., single mating and anecdysis in adulthood.     

Sequence analysis of a fish vitellogenin cDNA with a large phosvitin domain

Vitellogenins (Vtg) are egg-yolk precursor proteins crucial for reproductive success in oviparous animals. We have cloned the first complete cichlid Vtg cDNA from the tilapia fish, Oreochromis aureus. This cDNA has the largest phosvitin (PV) domain amongst piscine Vtgs, being comparable to those of lamprey, Xenopus, and chicken. Thus, the size of PV is independent of the evolutionary advancement of a species. The closer interspecific relationship between O. aureus Vtg1 and Fundulus VtgII than the intraspecific relationship between Fundulus VtgI and II isoforms suggests that teleost ancestors had at least two Vtg isoforms. Contrary to the results of previous phylogenetic inference using Vtgs which indicate that insect lineage is most diverged and nematodes are closer to vertebrate lineage, our results show that nematodes and hexapods form two monophyletic sister groups. Another arthropod taxon, represented by a malacostracan crustacean, Penaeus japonicus, appears to be more closely related to the vertebrates than the hexapods.     

Dynamics of vitellogenin mRNA expression during vitellogenesis in the banana shrimp Penaeus (Fenneropenaeus) merguiensis using real-time PCR

An open reading frame (ORF) of vitellogenin (Vg) cDNA was amplified from the ovaries of the banana shrimp, Penaeus merguiensis. An examination of Vg-deduced amino acid sequence revealed the presence of cleavage sites at a consensus motif for subtilisin-like endoproteases prior to the N-terminal sequences of purified vitellin (Vt) subunits. A comparison of the primary structures of Vg molecules in decapod crustacean species revealed the existence of a common characteristic structure, and phylogenetic analysis reflected the current taxonomic classifications of crustaceans. A PCR product of 1.1 kb encoding the 3'-end of Vg cDNA was cloned from the hepatopancreas. Although its sequence was almost identical to that of the same region of the ovarian Vg, with only 18 nucleotide differences, analysis suggests that they have been subjected to natural selection, indicating that there may be two different, tissue-specific Vg genes in P. merguiensis. This is consistent with the different expression patterns of Vg mRNA, as determined by real-time PCR. Vg mRNA levels were maintained at low levels during the previtellogenic stage and they increased as vitellogenesis progressed to reach a peak at the early vitellogenic stage in the ovary or at the vitellogenic stage in the hepatopancreas, and thereafter, levels decreased. Expression of Vg mRNA was much higher in the ovary compared to the hepatopancreas at all stages of ovarian development, implying that the ovary is mainly responsible for Vt synthesis. These indicate that penaeids constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.