| Lateolabrax maculatus(spotted sea bass)is an important aquatic cultured fish species,and widely distributed in coastal areas of China.Due to long-term overfishing,the wild resources have been rapidly declined,and the germplasm has a tendency to degenerate,which seriously affects the genetic diversity of the fish.In the present research,a genome survey of L.maculatus was carried out using next-generation sequencing(NGS)technologies and followed de novo assembly.A total of 64.63 Gb clean data was obtained by Hiseq 2500 sequencing and an estimated genome size of L.maculatus was 534.46 Mb with GC content of 40.95%.67,748 unigenes were obtained by sequence assembly and gene prediction,of which 60,218 unigenes(88.89 %)were found to be orthologous to known protein sequences in Nr database.A total of 50,860 and 10,260 genes were annotated with GO function and COG database respectively.311,393 simple sequence repeats(SSR)were identified from the unigenes,in which the mono-(29,585,9.50 %),di-(144,548,46.42 %),and tri-(36,254,11.64%)nucleotide repeats contributed to 67.65 % of the SSRs,and sequence motif AC/TG(36.76 %)was the most abundant among the dinucleotide repeat motifs.We characterized eight microsatellite loci that were amplified for from 30 individuals collected from different areas of China and showed stable and repeatable polymorphism.All loci were displayed 4-8 alleles,of which the average observed and expected heterozygosity were 0.64 and 0.58 respectively.Genome survey sequencing of L.maculatus provides data support for further investigating physiological mechanism and population association.A total of 54,031 unigenes were obtained by transcriptome sequencing analysis from seven issues(brain,stomach,gills,liver,spleen and testis and ovary)in L.maculatus separately and a total of 16,644 SSR loci were identified from 12,256 unigenes.Among these,the AC/GT was the predominant dinucleotide repeat type(16.03 %).32,611(60.36%)unigenes were assigned Gene Ontology(GO)functional annotations which were divided into 3 categories(biological processes,cellular components and molecular functions)with 103 branches,among which “molecular function” has a largest amount annotation of unigenes.Some unigenes were assigned to 419 predicted KEGG pathways which counld be divided into 6 categories(metabolism,genetic information processing,environmental information processing,cellular processes,organismal systems and human diseases).Among these,“Signal transduction”(25 KEGG pathways)is a largest branch in environmental information processing,closely followed by “Infectious diseases”(24 KEGG pathways)in human diseases.Comparision results of RPKM values of different genes among seven tissues indicated that HPG,HPA and HPGH axis required hormones were expressed highly or specifically in brain;keratin,glial protein and epithelial cell adhesion molecule in gill;various digestive enzymes and immunoglobulin analogues in stomach;apolipoprotein,antifreeze protein and transferrin in liver;vasa,piwi,sex hormone receptors and cyclin in gonads.Through transcriptome sequencing technology,the results provide a rich source of data of L.maculatus to identify new genes,and make the first step for futher genomics researches.Alkaline phosphatases(Alps)performs its best functions under alkaline p H environments and has the physiological role of dephosphorylating compounds.The enzyme is found across a multitude of organisms,prokaryotes and eukaryotes alike,with the same general function but in different structural forms suitable to the environment they function in.In this study,a 1,874 bp intestinal alp c DNA sequence was cloned from L.maculatus and designated as Lm-alpi.It contained a 1,611 bp open reading frame which encoded a protein with 537 amino acids.Protein sequence alignment showed that Lm-alpi shared 29.82-79.85 % identity with its homologs.Alp I catalytic sites contained three metal ion sites(two Zn2+ and one Mg2+),referring to D73,H184,D348,H349,H352,H464,D389,and H390 residues,which are essential for enzymatic activity and conservation in different organisms.Two predicted disulfide bonds in Lm-Alpi were composed of four cysteines(C152–C214 and C499–C506),which were homologous to those of mammals.Immunohistochemical staining revealed that Lm-Alpi was mainly expressed on the mucosal surface of the gastrointestinal tract,including stomach,intestine,and gastric cecum.Lm-Alpi was mainly located on the plasma membrane of transiently transfected He La cells.The m RNA of Lm-alpi was mainly expressed in the intestine,and its expression levels gradually increased after LPS treatment and further increased by 1.81-fold after 48 h.After desalting culture,the relative m RNA expression level of Lm-alpi decreased at 30 and 50 days after hatching(DAH)and then returned to normal levels at 70 DAH.Further experiments demonstrated that the enzyme activity of Lm-Alpi exhibited an expression pattern similar to that of the m RNA expression of Lm-alpi after LPS treatment and desalting culture.This study provided valuable information on the Lm-Alpi functions associated with the mucosal immunity and salinity adaptation of L.maculatus.In order to study endocrine regulation in the digestive process of the spotted sea bass,gastrin(Gas)and gastrin receptor(cholecystokinin type-B receptor,Cckbr)of L.maculatus were cloned.Gastrins are a class of gastrointestinal hormones that regulate the secretion of gastric acid and digestive enzymes.Cckbrs specifically identify and combine with Gas,which are a class of isoform receptors of cholecystokinin in gastrin family.Gas and Cckbr c DNA sequence was cloned from L.maculatus and designated as Lm-gas and Lm-cckbr separately.333 bp Lm-gas c DNA contained 330 bp ORF,which encoded a protein with 110 amino acids.Lm-gas shared low similarity at animo acid level,but C-terminal structure domain “DFGRR” was conserved.1362 bp Lm-cckbr c DNA contained 1,359 bp ORF and encoded 453 amino acids.Protein sequence alignment showed that Lm-cckbr shared 48.72-89.45 % identity with its homologs.Phylogenetic tree analysis showed that Lm-cckbr had close relationship with gastrin receptor of other species.Immunohistochemical staining revealed that Lm-gas was mainly expressed on the mucosal surface of the stomach.The m RNA of Lm-gas and Lm-cckbr were mainly expressed in the brain and stomach,which showed that they may play neuromodulation role in physiological processes about feeding and digestion.Overall,this study provides a basis for further research into the acid secretion regulation function of Lm-gas and Lm-cckbr in L.maculatus. |
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