| Mandarin fish(Siniperca chuatsi)is an important carnivorous freshwater fish in China.In the process of artificial breeding,the lack of live bait limits the scale of mandarin fish farming.Moreover,the fish is susceptible to bacterial or viral infection in the process of high-density farming,such as infectious splenic kidney necrosis virus(ISKNV)causing explosive death of mandarin fish.Large-scale culture can be achieved by artificial diet culture,which can not only improve the economic efficiency but also minimize the ecological damage caused by the capture of bait fish.Therefore,in this study,The expression profiles of whole genes of mandarin fish in different tissues and developmental periods were analyzed based on weighted gene co-expression network analysis(WGCNA).Focusing on the digestive system,WGCNA and protein-protein interactions(PPIs)analysis of differentially expressed genes after domestication were analyzed.The Hub genes highly relevant to the digestive system were screened out.The sequence characteristics,expression,and functions of the candidate gene were further analyzed.In this study,after completing transcriptome sequencing of 17 tissues(spleen,gallbladder,skin,esophagus,stomach,pyloric caecum,intestine,testis,ovary,gill,pseudobranch,muscle,liver,posterior kidney,head kidney,myocardium,brain)and 9developmental stages(2-cell embryo,1K-cell embryo,morula,gastrula,pigment period,tail bud period,pharyngula period,1 day after hatching,5 days after hatching)of mandarin fish,a total of 270,230 unigenes were obtained.Based on WGCNA analysis,31,657 genes expressed in 26 tissues were divided into 23 modules,respectively.According to the correlation of modules and samples,the modules that are highly related to the digestive system including liver,stomach,intestine,pyloric caecum,esophagus,and gallbladder were screened out.Co-expression networks were constructed for genes within the modules,and KEGG enrichment analysis and identification of Hub genes were performed.The results showed that the genes in the module highly related to the liver were mainly enriched in the immune system,metabolism,and transport and catabolism related pathways.Hub genes were ceruloplasmin(cp),vitellogenin C(vtgc),C1inhibitor(c1in),complement component 9(c9),leukocyte cell-derived chemotaxin-2(lect2),and kallikrein B1(klkb1)and were highly expressed in the liver.The genes in the module highly related to the stomach were mainly enriched in the digestive system,metabolism,signal transduction,and signaling molecules and interaction related pathways.Hub genes were ghrelin(ghrl),gastric H~+/K~+ATPase alpha subunit(atp4a),gap junction beta-3 protein(gjb3),mucin 5AC(muc5ac),dual oxidase 2(duox2),and chitinase 2(chia2)and were highly expressed in the stomach.The genes in the module highly related to the intestine and pyloric caecum were mainly enriched in the digestive system and metabolism related pathways.Hub genes were solute carrier family 15member 1(slc15a1),cadherin-related family member 5(cdhr5),butyrophilin subfamily3 member A1(btn3a1),aminopeptidase N(anpep),solute carrier family 34 member 2(slc34a2),cadherin-related family member 2(cdhr2),and angiotensin-converting enzyme 2(ace2)and were highly expressed in the intestine and pyloric caecum.The genes in the module highly associated with the esophagus were mainly enriched in circulatory system,cellular community,digestive system,and immune system related pathways.Hub genes were myosin-binding protein C1(mybpc1),myosin regulatory light chain 2(myl2),and tropomyosin alpha-3 chain(tpm3)and were highly expressed in the esophagus.The genes in the module highly associated with the gallbladder were mainly enriched in signaling molecules and interaction,digestive system,signal transduction,and membrane transport related pathways.Hub genes were dystrophin(dyst),neuropeptide Y receptor Y2(npy2r),solute carrier family 13 member 1(slc13a1),and solute carrier family 39 member 4(slc39a4)and were highly expressed in the gallbladder.Performing KEGG enrichment analysis on the modules highly related to the digestive system and identifying the Hub genes in each module will help to understand the main functions and levels of the Hub genes in the digestive system.It has reference value for research on the metabolism,digestion and absorption,immunity,and molecular breeding of mandarin fish.The histomorphological changes in the digestive system and differentially expressed genes(DEGs)in the liver,stomach,and intestine of mandarin fish domesticated by artificial diet were analyzed.The skin of the domesticated mandarin fish was darker.The stomach and intestinal walls became thinner,the lumen became larger,and the muscle layer became thinner.The villi of the intestine became longer and more branched.The bile color became darker.No significant histomorphological changes were observed in the liver.There were 4,194,2,505,and 4,579 DEGs in the liver,stomach,and intestine between domesticated and control mandarin fish,respectively.KEGG enrichment analysis of DEGs showed that they were mainly enriched in the metabolism pathway.Significant enrichment of DEGs was also observed in protein digestion and absorption and antigen processing and presentation pathways.This suggests that artificial diet can significantly alter the expression levels of genes in the metabolism,digestion and absorption,and immunity related pathways in the digestive system of mandarin fish.Meanwhile,all the differentially expressed digestive enzyme genes was screened from the DEGs of the digestive system,which contained 12classes of digestive enzymes including lipase,cathepsin,nuclease,lysozyme,aminopeptidase,chitinase,trypsin,elastase,pepsin,chymotrypsin,maltase,and lactase.Among them,lipase and cathepsin were the most differentially expressed enzymes.These results suggest that the digestive system of mandarin fish is adapted to artificial diet.The DEGs were subjected to PPIs network analysis.The top ten degree-highest genes were selected as Hub genes,namely insulin(ins),heat shock protein family A member 5(hspa5),sterol regulatory element binding transcription factor 1(srebf1),heat shock protein 90 alpha family class A member 1(hsp90aa1),lect2,3-hydroxy-3-methylglutaryl-Co A reductase(hmgcr),calreticulin(calr),calnexin(canx),sterol regulatory element binding transcription factor 2(srebf2),and heat shock protein 90beta family member 1(hsp90b1).The differential expression levels of each Hub gene in the liver,stomach,and intestine after domestication were analyzed by combining WGCNA and DEGs of the digestive system.Among them,vtgc and lect2 were significantly upregulated in the liver.chia2 was significantly downregulated in the stomach.slc15a1,anpep,and slc34a2 were significantly upregulated in the intestine.These Hub genes may play an important role in the adaptation of the digestive system to artificial diet.Based on the WGCNA analysis of the digestive system and the PPIs analysis of DEGs in the digestive system after domestication,Siniperca chuatsi lect2(Sc-lect2)was significantly differentially expressed as a Hub gene.The m RNA sequence of Sc-lect2 is815 bp in length,including a complete open reading frame of 465 bp encoding 154amino acids.The spatiotemporal expression analysis of Sc-lect2 showed that it was highly expressed in the liver and extremely low in other tissues.The expression level of Sc-lect2 gradually increased with embryonic development from the tail bud period to 5days after hatching.The expression of Sc-lect2 was significantly increased by about 40-fold after fish infection with ISKNV.Sc-lect2 expression was significantly increased by about 17-and 7-fold after stimulation with lipopolysaccharide(LPS)and polyinosinic acid(Poly I:C),respectively.The expression level of Sc-lect2 increased about 8-fold after domestication with artificial diet.These results suggest that Sc-lect2 is highly expressed in the liver and that the expression level increases significantly when stimulated by different external conditions.Therefore,Sc-Lect2 may serve as a biomarker of stressful external stimuli in the mandarin fish liver.According to the WGCNA of digestive system and PPIs of DEGs,the top 10 genes most associated with Sc-lect2 were screened,which included supervillin(svil),sacsin(sacs),trafficking kinesin-binding protein 1(trak1),cathepsin K(ctsk),microfibril-associated glycoprotein 4(mfap4),serum/glucocorticoid regulated kinase 2(sgk2),angiopoietin-related protein 5(angptl5),selenoprotein P plasma 1(sepp1),serum amyloid A-like protein 1(saa1),and lysozyme C(lyz).Their differential expression levels were elevated after domestication except for lyz.The differential expression levels of this 10 genes after LPS and Poly I:C stimulation were also analyzed.Among them,sacs,ctsk,mfap4,sgk2,angptl5,sepp1,saa1 and lyz were still co-expressed with Sc-lect2,which are mainly immune and metabolic-related genes.The comprehensive results indicated that Sc-lect2 was involved in the regulation of immune and metabolism-related pathways.In this study,the molecular mechanism of regulation of Sc-lect2 gene expression was also investigated.Analysis from m RNA sequences predicted 25 mi RNAs targeting binding to Sc-lect2.q PCR analysis obtained that mi R-145-3p expression was negatively correlated with Sc-lect2.Moreover,plasmid construction,cotransfection,and dual-luciferase reporter gene assay experiments showed that wild-type Sc-lect2 expression was repressed by mi R-145-3p.This inhibitory effect disappeared when the seed region bound to the mi RNA was mutated,indicating that mi R-145-3p can directly target binding to Sc-lect2 and inhibit its expression.Analysis of the promoter sequence of Sc-lect2 revealed a Cp G island rich in CG sites in the-1004 to-1277 region.This Cp G island was analyzed for DNA methylation level via bisulfite sequencing.There was a significant difference in DNA methylation levels between the liver and stomach at the-1028 and-1068 loci.The transcription factors bound to the-1028 and-1068 sites were zinc finger protein 263(ZNF263)and transcription factor CP2-like protein 1(TFCP2L1),respectively.This suggests that these two sites and their bound transcription factors might be one of the factors affecting Sc-lect2 gene expression.In this study,combining WGCNA analysis and the PPIs analysis of DEGs in the digestive system screened Hub genes closely related to domestication.Sc-lect2 acts as a Hub gene and is significantly differentially expressed.By studying the expression and regulatory mechanism of Sc-lect2,it was shown that Sc-lect2 could be involved in fish immune response and metabolism-related pathways.Moreover,Sc-lect2 was highly expressed in the liver and was significantly increased under the changes of various physiological states.It may be used as a biomarker of liver stress to judge the physiological indicators of mandarin fish... |
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