Marine Biotechnology最新文献

As an important aquaculture species and research model, Nile tilapia (Oreochromis niloticus) has not yet been systematically studied for the isolation, culture, and in vitro gene manipulation techniques of primary cells from various tissues. This study aimed to explore methods for isolating primary cells from various tissues, as well as developing in vitro gene manipulation techniques in Nile tilapia. Four different Nile tilapia tissues were enzymatically digested and separated using trypsin or collagenase. Collagenase (0.1%) was used for the digestion of the gonads, liver, and heart, while trypsin (0.25%) showed better adhesion efficiency for spleen tissue. Moreover, we assessed EGFP fluorescence intensity and cell survival rates following transfection with empty siRNA (siRNA-NC), lentivirus (LV-NC), and six adeno-associated virus (AAV-NC) serotypes (AAV2-NC, AAV5-NC, AAV6-NC, AAV8-NC, AAV9-NC, AAV-DJ-NC) in gonadal cells. The results demonstrated that cells transfected with siRNA-NC and LV-NC showed the highest levels of green fluorescent protein expression and survival rates in primary gonadal cells, compared to AAC-NC. Subsequently, we knocked down the Kdm6bb gene in Nile tilapia primary gonadal cells by transfecting them with LV-Kdm6bb and siRNA-Kdm6bb. qPCR and immunofluorescence analyses demonstrated a significant reduction in Kdm6bb mRNA levels following transfection with siRNA-Kdm6bb compared to siRNA-NC, and with LV-Kdm6bb compared to LV-NC. This study offers valuable tools for the validation of primary cell isolation and in vitro molecular regulatory mechanisms and functions in Nile tilapia.

Alternative splicing (AS) is an important post-transcriptional regulation, which can expand the functional diversity of gene products and is a mechanism for eukaryotes to cope with abiotic stress. However, there are few studies on AS events in rainbow trout under heat stress. In this study, RNA-Seq data were used to clarify the effect of selenium nanoparticles (SeNPs) on the AS events of rainbow trout head kidney under heat stress. The results showed that a total of 45,398 AS events were identified from 9804 genes, of which Skipped Exon (SE) was the most common type of AS event. Through the analysis of the differentially expressed genes (DEGs) in each group, we learned that DEGs were enriched in the spliceosome, and the relevant genes were significantly changed, which promoted the occurrence of AS. We found that lysine degradation, ubiquitin mediated proteolysis, RNA degradation, protein processing in endoplasmic reticulum processing and other pathways were significantly enriched after addition of SeNPs. In addition, some immune related signaling pathways, such as the mTOR signaling pathway, interact with each other to enhance the resistance of rainbow trout to heat stress. These results indicated that AS in head kidney of rainbow trout changed under heat stress and SeNPs played a key role in alleviating heat stress for rainbow trout.

LncRNAs are long non-coding RNAs that are widely recognized as crucial regulators of gene expression and metabolic control, involved in numerous dormancy-related processes. Aestivation is a common hypometabolism strategy of sea cucumber (Apostichopus japonicus) in response to high-temperature conditions and is typically characterized by the degradation of the intestine and respiratory tree. Although the aestivation process has been extensively studied in sea cucumbers, the role of lncRNAs in the context of aestivation states remains a conspicuous knowledge gap. Here, we identified and characterized 14,711 lncRNAs in A. japonicus and analyzed their differential expression patterns during the aestivation process in the intestine and respiratory tree. The results revealed the physiological differences, especially the metabolic processes, between the intestine and respiratory tree during the aestivation. The co-expression network of lncRNA-mRNA suggested the dominant role of lncRNA in regulating the differential response of the intestine and respiratory trees. Differentially co-expressed factors were significantly enriched in the deep-aestivation stage-specific modules. Conserved co-expressed factors included several transcription factors known to be involved in rhythm regulation, such as Klf2 and Egr1. Furthermore, a specific trans-acting lncRNA (lncrna.1393.1) was identified as a potential regulator of Klf2 and Egr1. Overall, the systematic identification, characterization, and expression analysis of lncRNAs in A. japonicus enhanced our knowledge of long non-coding regulation of aestivation in sea cucumber and provided new clues for understanding the common “toolkit” of dormancy regulatory mechanisms.

Autonomously replicating sequences (ARSs) are important accessories in episomal vectors that allow them to be replicated and stably maintained within transformants. Despite their importance, no information on ARSs in diatoms has been reported. Therefore, we attempted to identify ARS candidates in the model diatom, Phaeodactylum tricornutum, via chromatin immunoprecipitation sequencing. In this study, subunits of the origin recognition complex (ORC), ORC2 and ORC4, were used to screen for ARS candidates. ORC2 and ORC4 bound to 355 sites on the P. tricornutum genome, of which 69 were constantly screened after multiple attempts. The screened ARS candidates had an AT-richness of approximately 50% (44.39–52.92%) and did not have conserved sequences. In addition, ARS candidates were distributed randomly but had a dense distribution pattern at several sites. Their positions tended to overlap with those of the genetic region (73.91%). Compared to the ARSs of several other eukaryotic organisms, the characteristics of the screened ARS candidates are complex. Thus, our findings suggest that the diatom has a distinct and unique native ARSs.

Light sensitivity is important for marine benthic invertebrates, and it plays a vital role in the marine bivalves settling. Animal visual systems are enormously diverse; their development appears to be controlled by a set of conserved retinal determination genes (RDGs). Eyespots, as the simplest animal eyes, their appearance indicates the important effect on mussel larvae attachment. Nevertheless, the molecular mechanism of the eyespot’s development in Mytilus coruscus larvae is not clear. In this study, we identified 11 genes which play a regulatory role in the visual system (i.e. Pax1/9, Pax2/5/8, Pax6, Pax3/7, Six1/2, Six3/6, Six4/5, Dach, Eya, Brn and Tbx2) from transcriptome data and the whole genome sequence of M. coruscus. The results of chromosome localization showed that 11 genes were distributed on different chromosomes. Subcellular mapping revealed that all the proteins except Brn were located in the nucleus. Phylogeny and gene structure analyses revealed that the Pax members were divided into four subfamilies, the Six members were divided into three subfamilies and structures within the same subfamily were relatively conserved. Quantitative real-time PCR (qPCR) showed that Dach, Pax6, Pax3/7, Six1/2 and Six4/5 were expressed at high levels during the pediveliger stage. Moreover, Six1/2 and Six4/5 were highly expressed in mantle tissues. Subsequent overall in situ hybridization experiments in the planktonic larval stage revealed that Pax6, Six1/2 and Six4/5 detected signals in the region of the eyespot. Based on these analyses, we suggested that the development of vision in M. coruscus not only depended on the expression pattern of Pax6, but perhaps also related to Six1/2 and Six4/5 in the planktonic larval stage, while Six1/2 and Six4/5 were the dominant genes for visual function in the adult mussel. This study made a comprehensive analysis of the visual function of M. coruscus at the genome level, which helps us to understand the intrinsic mechanism of the visual system of marine bivalves, and also provides a molecular basis for improving the attachment and metamorphosis rate of M. coruscus larvae.

The Pacific oyster Crassostrea gigas is known to have an exceptional ability to accumulate zinc, which endows it with robust resistance to pathogens and makes it an excellent source of dietary zinc. ZIP1 has been identified as an important zinc uptake protein in other species, but its role in oysters remains unclear. In the present study, a ZIP1 homologue (CgZIP1-II) of the Zrt/Irt-like protein (ZIP) family was identified in C. gigas. The mRNA transcripts of CgZIP1-II were constitutively expressed in examined tissues of C. gigas, with higher levels in the hepatopancreas and gill. After zinc exposure, the mRNA transcripts of CgZIP1-II in the hepatopancreas showed a significant decline from 12 h to 14 d, while those in the gill significantly decreased at 72 h, followed by a recovery to basal level at 7 to 14 d. Immunocytochemical analysis revealed that the CgZIP1-II protein was mainly located at the plasma membrane of oyster hemocytes. Compared to the control cells, overexpression of CgZIP1-II in the transfected HEK293 cells resulted in a 2.44-fold (p < 0.05) increase in zinc content after incubation with 100 μM zinc for 24 h. Inhibition of endogenous CgZIP1-II expression with siRNAs led to a 42% reduction in zinc content in the hepatopancreas of oysters. Similarly, in vivo blocking of CgZIP1-II with anti-CgZIP1-II antibody caused a 43% decrease in zinc content in the hepatopancreas. These results collectively indicated that CgZIP1-II functioned as a zinc uptake transporter in C. gigas and played a certain role in zinc accumulation.

Variation in morphology-driven economic traits is a common issue hindering the development of the sea cucumber aquaculture industry. In this study, transcriptome comparisons, weighted gene correlation network analysis (WGCNA) and Pearson correlation coefficient (PCC) were first employed to identify key genes correlated with morphological variation in the sea cucumber Apostichopus japonicus, after which the relationship between identified key genes (relative expression and genotype) and economic trait phenotypes was investigated to screen potential biomarker targets for molecular-assisted breeding. The results showed that three genes (putative ficolin–2, fibrinogen c domain–containing protein 1, and angiopoietin–4) were closely associated with economic trait superiorities. Two single nucleotide polymorphisms (SNPs) were identified in the putative ficolin–2 gene as having a strong correlation with body weight and papilla number. The findings from this study will enrich breeding biomarker resources and benefit the development of molecular-assisted breeding techniques in sea cucumber aquaculture.

The spotted seal (Phoca largha) is the sole pinniped species that can reproduce in China and has been classified as the First-Grade State Protection animal. The conventional method for the protection and maintenance of the spotted seal population is the captive maintenance of the species in artificially controlled environments. Nevertheless, the efficacy of the captive strategy remains uncertain, with the potential to impact the health of spotted seals through alterations in gut microbiota. In this study, PacBio sequencing based on the full-length of the bacterial 16S rRNA gene was applied to faeces from captive and wild spotted seals, thereby providing a first reference for the gut microbiota profile of spotted seals at the species scale. The gut microbiota of captive spotted seals was found to be more diverse than that of the wild population. The gut microbiota of spotted seals exhibited notable variation due to captive breeding, with an enrichment of Firmicutes and a reduction in Proteobacteria. The results of the co-occurrence network analysis indicated that the gut microbiota of captive spotted seals exhibited a greater degree of complexity and stability in comparison to that observed in their wild counterparts. The analysis of community assembly mechanisms revealed an increased determinism for the gut microbiota of captive individuals, with a concomitant decrease in the contribution of drift. Furthermore, the results of the predicted functions indicated a reduction in stress responses and an enhanced ability to metabolise sugars in the gut microbiota of captive spotted seals. In conclusion, the results of this study provide evidence that the current captive breeding strategy is an effective approach for improving the gut microbiota of spotted seals. Furthermore, this study demonstrates the potential of monitoring the gut microbiota to assess the health of marine mammals and inform conservation strategies for endangered species.

The Pacific oyster (Crassostrea gigas) is a global aquaculture species of economic significance. Selective breeding programs have been conducted to produce multiple strains with fast growth as well as other desirable traits. However, due to the phenotypic plasticity of oysters, challenges existed for precise germplasm identification among selectively bred strains. In this work, we identified selection signatures of three fast-growing Pacific oyster strains originated from wild populations collected from China, Japan, and Korea, respectively, which were used for development of SNP-based molecular fingerprinting for precise identification of germplasm. We performed whole-genome resequencing of 59 oysters from three selectively bred strains and a wild population for genome-wide SNP analyses. Population structure analysis with these SNPs revealed significant genetic differentiation among the selectively bred strains. Based on the F_ST index, we identified 41, 49, and 36 strain-specific SNPs from the three selectively bred strains. Taking into account the “hitch-hiking effect” that occurs in the genome during positive selection, we identified two, three, and two molecular fingerprints for the three strains, respectively. We validated the molecular fingerprints of the China selectively bred strain (i.e., “Haida No. 1” variety) with a separate population of 42 oysters with diverse genetic background, demonstrating the accuracy of germplasm identification of over 96%. This work provides a reliable tool for precise germplasm identification of the "Haida No. 1" variety as well as other two selectively bred strains, which is valuable in germplasm conservation and breeding design in the C. gigas.

Octocorals, vital components of reef ecosystems, inhabit various marine environments across diverse climate zones, spanning from tropical shallows to frigid deep-sea regions. Certain octocoral species, notably Lobophytum and Sinularia, are particularly intriguing due to their production of diverse metabolites, warranting continuous investigation. Although octocorals played the roles in coral ecosystems, the studies are rare in comparison to scleractinian corals, especially in transcriptomic and genomic data. However, RNA extraction was massively interfered by the polysaccharides and secondary metabolites produced from octocoral holobiont. For this purpose, five lysis buffer systems and two extraction processes were examined for the RNA extraction efficiency in octocorals. We found CTAB/10%SDS as a new method for RNA extraction from six different octocoral genera. Furthermore, our new method is enable to extract RNA with good quality for downstream application such as quantitative PCR and RNA sequencing. Finally, comparative transcriptomic analysis between healthy octocorals and those dark-induced bleaching corals in Lobophytum hsiehi revealed extracellular matrix and immunity-related genes may play the important roles in coral-symbiodinium symbiosis. We believe that this study’s findings and the developed RNA extraction method will serve as valuable references for future research, particularly in octocorals.