Exploration of Molecular Mechanisms of Immunity in the Pacific Oyster (Crassostrea gigas) in Response to Vibrio alginolyticus Invasion.

Over the years, oysters have faced recurring mass mortality issues during the summer breeding season, with Vibrio infection emerging as a significant contributing factor. Tubules of gill filaments were confirmed to be in the hematopoietic position in Crassostrea gigas, which produce hemocytes with immune defense capabilities. Additionally, the epithelial cells of oyster gills produce immune effectors to defend against pathogens. In light of this, we performed a transcriptome analysis of gill tissues obtained from C. gigas infected with Vibrio alginolyticus for 12 h and 48 h. Through this analysis, we identified 1024 differentially expressed genes (DEGs) at 12 h post-injection and 1079 DEGs at 48 h post-injection. Enrichment analysis of these DEGs revealed a significant association with immune-related Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To further investigate the immune response, we constructed a protein-protein interaction (PPI) network using the DEGs enriched in immune-associated KEGG pathways. This network provided insights into the interactions and relationships among these genes, shedding light on the underlying mechanisms of the innate immune defense mechanism in oyster gills. To ensure the accuracy of our findings, we validated 16 key genes using quantitative RT-PCR. Overall, this study represents the first exploration of the innate immune defense mechanism in oyster gills using a PPI network approach. The findings provide valuable insights for future research on oyster pathogen control and the development of oysters with enhanced antimicrobial resistance.

Transcriptome profiling explores the immune defence mechanism of triploid Pacific oyster (Crassostrea gigas) blood against Vibrio alginolyticus based on protein interaction networks.

Triploid oysters have provided the oyster industry with many benefits, such as fast growth rates, meat quality improvement, and increased oyster production and economic benefits, since the first report on triploid oysters was published. The development of polyploid technology has remarkably increased the output of triploid oysters to meet the increasing demand of consumers for Crassostrea gigas in the past decades. At present, research on triploid oyster has mainly focused on breeding and growth, but studies on the immunity of triploid oysters are limited. According to recent reports, Vibrio alginolyticus is a highly virulent strain that can cause disease and death in shellfish, shrimp, as well as serious economic losses. V. alginolyticus may be a reason why oysters die during summer. Therefore, using V. alginolyticus to explore the resistance and immune defense mechanisms of triploid oysters against pathogens presents practical significance. Transcriptome analysis of gene expression was performed in triploid C. gigas at 12 and 48 h after infection with V. alginolyticus, and the respective 2257 and 191 differentially expressed genes (DEGs) were identified. The results of GO and KEGG enrichment analyses showed that multiple significantly enriched GO terms and KEGG signaling pathways are associated with immunity. A protein-protein interaction network was constructed to investigate the interaction relationship of immune-related genes. Finally, we verified the expression situation of 16 key genes using quantitative RT-PCR. This study is the first to use the PPI network in exploring the immune defense mechanism of triploid C. gigas blood to fill the gap in the immune mechanism of triploid oysters and other mollusks, and provide valuable reference for future triploid farming and pathogen prevention and control.

Sequencing-based network analysis provides a core set of genes for understanding hemolymph immune response mechanisms against Poly I:C stimulation in Amphioctopus fangsiao.

Aquatic viruses can spread rapidly and widely in seawater for their high infective ability. Polyinosinic-polycytidylic acid (Poly I:C), a viral dsRNA analog, is an immunostimulant that has been proved to activate various immune responses of immune cells in invertebrate. Hemolymph is a critical site that host immune response in invertebrates, and its transcriptome information obtained from Amphioctopus fangsiao stimulated by Poly I:C is crucial for understanding the antiviral molecular mechanisms of this species. In this study, we analyzed gene expression data in A. fangsiao hemolymph tissue within 24 h under Poly I:C stimulation and found 1082 and 299 differentially expressed genes (DEGs) at 6 and 24 h, respectively. Union set (1,369) DEGs were selected for subsequent analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were carried out for identifying DEGs related to immunity. Several significant immune-related terms and pathways, such as toll-like receptor signaling pathways term, inflammatory response term, TNF signaling pathway, and chemokine signaling pathway were identified. A protein-protein interaction (PPI) network was constructed for examining the relationships among immune-related genes. Finally, 12 hub genes, including EGFR, ACTG1, MAP2K1, and other nine hub genes, were identified based on the KEGG enrichment analysis and PPI network. The quantitative RT-PCR (qRT-PCR) was used to verify the expression profile of 12 hub genes. This research provides a reference for solving the problem of high mortality of A. fangsiao and other mollusks and provides a reference for the future production of some disease-resistant A. fangsiao.

Transcriptome analysis preliminary reveals the immune response mechanism of golden cuttlefish (Sepia esculenta) larvae exposed to Cd.

As a well-known marine metal element, Cd can significantly affect bivalve mollusk life processes such as growth and development. However, the effects of Cd on the molecular mechanisms of the economically important cephalopod species Sepia esculenta remain unclear. In this study, S. esculenta larval immunity exposed to Cd is explored based on RNA-Seq. The analyses of GO, KEGG, and protein-protein interaction (PPI) network of 1,471 differentially expressed genes (DEGs) reveal that multiple immune processes are affected by exposure such as inflammatory reaction and cell adhesion. Comprehensive analyses of KEGG signaling pathways and the PPI network are first used to explore Cd-exposed S. esculenta larval immunity, revealing the presence of 16 immune-related key and hub genes involved in exposure response. Results of gene and pathway functional analyses increase our understanding of Cd-exposed S. esculenta larval immunity and improve our overall understanding of mollusk immune functions.

Transcriptome profiling based on protein-protein networks provides a core set of genes for understanding blood immune response mechanisms against LPS stress in Amphioctopus fangsiao.

Gram-negative bacteria are significant pathogens in the ocean, posing serious threats to marine organisms. Lipopolysaccharide (LPS) is a characteristic chemical constituent in Gram-negative bacteria that can be recognized by the pattern recognition receptor (PRR) of immune cells. This system is often used to simulate the invasion of bacteria. Blood is a transport channel for immune cells, and its transcriptome information obtained from Amphioctopus fangsiao stimulated by LPS is essential for understanding the antibacterial biological mechanisms of this species. In this study, we analyzed the gene expression profiles of A. fangsiao blood within 24h under LPS stress and found 778 and 561 differentially expressed genes (DEGs) at 6 and 24h, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed to search for immune-related DEGs. The relationships among immune genes were examined by constructing a protein-protein interaction (PPI) network. Finally, 16 hub genes were identified based on the PPI network and KEGG enrichment analysis. The expression profiles of these genes were verified using quantitative RT-PCR (qRT-PCR). This research provides valuable resources for the healthy culture of A. fangsiao and helps us understand the molecular mechanisms of innate immunity.

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers.

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r=0.78) was better than that of the RAPD marker system (r=0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.

Improving seedless kelp (Saccharina japonica) during its domestication by hybridizing gametophytes and seedling-raising from sporophytes.

Dongfang no.7 (Saccharina japonica) was bred and maintained by hybridizing gametophytes, self-crossing the best individuals, selecting the best self-crossing line and seedling-raising from yearly reconstructed sporophytes. It increased the air dry yield by 43.2% in average over 2 widely farmed controls. Dongfang no.7 was seedling-raised from bulked sporophytes reconstructed from its representative gametophyte clones. Such strategy ensured it against variety contamination due to possible cross fertilization and occasional mixing and inbred depletion due to self-crossing number-limited sporophytes year after year. It derived from an intraspecific hybrid through 4 rounds of self-crossing and selection and retained a certain degree of genetic heterozygosity, thus being immune to inbred depletion due to purification of unknown detrimental alleles. Most importantly, it can be farmed in currently available system as the seedlings for large scale culture can be raised from reconstructed Dongfang no.7 sporophytes. Breeding and maintaining Dongfang no.7 provided a model that other varieties of kelp (S. japonica) and brown algae may follow during their domestication.

A census of nuclear cyanobacterial recruits in the plant kingdom.

The plastids and mitochondria of the eukaryotic cell are of endosymbiotic origin. These events occurred ~2 billion years ago and produced significant changes in the genomes of the host and the endosymbiont. Previous studies demonstrated that the invasion of land affected plastids and mitochondria differently and that the paths of mitochondrial integration differed between animals and plants. Other studies examined the reasons why a set of proteins remained encoded in the organelles and were not transferred to the nuclear genome. However, our understanding of the functional relations of the transferred genes is insufficient. In this paper, we report a high-throughput phylogenetic analysis to identify genes of cyanobacterial origin for plants of different levels of complexity: Arabidopsis thaliana, Chlamydomonas reinhardtii, Physcomitrella patens, Populus trichocarpa, Selaginella moellendorffii, Sorghum bicolor, Oryza sativa, and Ostreococcus tauri. Thus, a census of cyanobacterial gene recruits and a study of their function are presented to better understand the functional aspects of plastid symbiogenesis. From algae to angiosperms, the GO terms demonstrated a gradual expansion over functionally related genes in the nuclear genome, beginning with genes related to thylakoids and photosynthesis, followed by genes involved in metabolism, and finally with regulation-related genes, primarily in angiosperms. The results demonstrate that DNA is supplied to the nuclear genome on a permanent basis with no regard to function, and only what is needed is kept, which thereby expands on the GO space along the related genes.

Isolation and characterization of an endosperm-specific promoter from wheat (Triticum aestivum L.).

Genes coding for avenin-like proteins (ALP) represent a new family of wheat storage protein genes. To find a wheat endosperm-specific promoter, a 1644-bp fragment upstream of the ALP type-B gene (GenBank accession number JN622144) was isolated. The important promoter elements of the ALP type-B gene were ascertained through sequence analysis which revealed that this fragment contains the TATA and CAAT boxes, which are important elements in gene expression. A prolamin box containing an endosperm motif and a GCN4-like motif (GLM) is present at about 300 bp upstream of the translation start site. The promoter sequence has two ESP-like elements and one of them is followed by an RY motif with the nucleotides CATG overlapping. The RY motif is considered the core functional sequence in a promoter. In an attempt to confirm the promoter activity, a series of 5'-deletions of the promoter were fused with the beta-glucuronidase (GUS) gene, and the constructs were stably introduced into tobacco plants. GUS staining confirmed that the AVL type-B promoter is an endosperm-specific promoter in tobacco seeds. Quantitative analysis of GUS expression in transgenic plants showed that even the shortest 5'-deletion, i.e. a 290-bp promoter sequence within the prolamin box, was sufficient to drive GUS expression in the endosperm. The highest expression level was found in transgenic plants containing the 5'-deletion vector construct pALP-8. This suggests that the ESP-like element overlapping with the RY motif may play a crucial role in the regulatory function of the promoter.

Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.).

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and green fluorescent protein (gfp) as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL (targeting site) probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.