| The swimming crab Portunus trituberculatus,which is an important marine economic animal widely distributed in China,belongs to Crustacea,Decapoda,Decapoda,Portunidae.P.trituberculatus is a species of aquatic crustaceans with wide salt and it can survive in a salinity of 13.7-47.7.With the reproductive migration habit,P.trituberculatus often hatch in the low-salt areas of the bay or estuary and overwinter in deep-sea high-salt areas.Salinity is one of the most important environmental factors affecting the growth and development of P.trituberculus.Ingestion,molting,growth,metabolism,immunity,etc.,of P.trituberculatus was affected greatly by salinity.Therefore,it is of great significance to study the mechanism of salinity adaptation for the breeding of improved salt-tolerant varieties of P.trituberculus.Therefore,to study the salinity adaptation mechanism plays an important role in promoting the cultivation of salt-tolerant species of P.trituberculatus.In order to analyze the salinity adaptation mechanism of the crab,the following research contents were designed:Firstly,the high-throughput expression profiles of salinity tolerance traits of P.trituberculatus were sequenced by next-generation sequencing technology.Analyze the salinity-adapted genes.Secondly,based on comparative transcriptome analysis,we found that the neuropeptide family genes were significantly differently expressed in the salinity tolerance traits of the crab after salinity stress.It plays an important role in adaptability of Low salt tolerance family.Therefore,we selected genes such as neuroparsin,crustacean cardioactive peptide,and crustacean cardioactive peptide,and obtained cDNA or genome full-length sequences by techniques such as RCAE and walking,and further analyzed salinity adaptation mechanism in qPCR,RNAi,and other techniques.The main research results are as follows:1.Analysis of gill expression profile of P.trituberculatus in low salinitiy A new generation of sequencing technology was used to sequence high-throughput expression profiling of P.trituberculatus tissues that were different in salinity tolerance traits.An average of 23,941,392 clean reads were obtained from 10 sequencing samples,which was 86.74%compared to the reference sequence.The 9475 differentially expressed genes,involving ion transporters,channel proteins,and neuropeptide amino acid metabolism,were explored using comparative transcriptomics methods.By comparing the differentially expressed genes between low salt tolerant families and low salt sensitive families,we found that ion transporter related genes,including Na~+/K~+-ATPase(up-regulation),CA(up-regulation),chitinase(down-regulation)and calcium reticulin(down-regulation)etc.,were significantly differentially expressed.In addition,heat shock protein and metabolism-related arginine kinase were significantly up-regulated.GO enrichment analysis showed that the differentially expressed genes were mainly enriched in GO entry such as biological regulation,cell process,metabolic process,single organism process,cell components,cell membrane,organelle,binding,catalytic activity,transport activity and so on.Enrichment analysis of KEGG signaling pathway showed that the differentially expressed genes were mainly concentrated in metabolic pathway,phagocytosis pathway,cancer pathway and ribosomal protein pathway.In addition,we selected 33 neuropeptide genes and related receptor genes from the transcriptome,and screened three neuropeptide genes(neuroparsin,crustacean cardioactive peptide,and crustacean hyperglycemic hormone)from the expression profile and significantly differentially expressed,we provide an experimental basis for further research.2.Expression analysis of neuroparsin gene under low salinity stress in P.trituberculatus In order to investigate the function of neuroparsin under low salinity stress in P.trituberculatus,neuroparsin gene was cloned by rapid amplification of cDNA ends(RACE).The PtNP gene was 1920 bp in size,and it included a 309 bp open reading frame(ORF)that encoded a 102 aa polypeptide of which the isoelectric point was 7.42 and the molecular mass was 10.8 kDa.PtNP contain 12-cysteine residue,which is a typical characteristic of the neuroparsin in decapoda.The homology and phylogenetic systematics analyzed revealed that the highest homology and similarity(reaching 89%)occurred between PtNP and the NP of Scylla paramamosain and P.trituberculatus cluster to Scylla paramamosian.The tissue expression analysis showed that PtNP genes were relatively large in the brain,followed by the gill and the eye,with very little or no distribution in the ovaries,muscles,heart and liver pancreas.The analysis of the expression pattern of PtNP gene in the process of low salt stress showed that low salt stress can significantly change the expression pattern of PtNP gene in brain,gill and eyestalk and the overall trend was upregulated.In brain,gill and eyestalk,the expression up to a maximum of 7.7 times,2.8 times and 2.6 times compared to the control respectively(P<0.05).PtNP Gene expression in the gills presents rising trend after eyestalk ablated and the expression of bilateral eyestalk ablated was significantly higher than the unilateral eyestalk ablated(P<0.05).The results of this study showed that the PtNP gene may play a role and regulated by neuroendocrine system in the salinity adaptation of P.trituberculatus.3.Cloning of crustacean cardioactive peptide and its functional verification in low salt adaptation RACE technology was used to clone the crustacean cardioactive peptide(CCAP)gene.The full-length of CCAP gene was 606 bp,including an open reading frame of 426 bp,a 5'UTR of 72 bp and a 3'UTR of 108 bp,with a poly A structure.Amino acid sequence analysis showed that CCAP gene encodes 141 amino acids with predicted molecular weight of 15.6 kDa and isoelectric point was 9.55.Homology analysis showed that the homology between CCAP of P.trituberculatus and Scylla Paramamosain and Callinectes sapidus was higher,85%and 82%,respectively.Phylogenetic analysis showed that the P.trituberculatus and C.sapidus were clustered first,followed by S.Paramamosain.Tissue expression analysis revealed that the relative expression of CCAP gene in thoracic ganglia was highest,followed by brain and eye stalk.By analyzing the expression pattern of CCAP gene during low salt stress,it was found that low salt could significantly change the expression pattern of CCAP in thoracic ganglia,and the expression level of experimental group was significantly higher than that of control group at 24,48,and 72 hours(P<0.05).)were 1.73,2.16,and 2.19 times the control group,respectively.In vitro injection of CCAP polypeptide could reduce the mortality of P.trituberculatus under low-salt conditions,and the activity of Na~+/K~+-ATPase and V-ATPase significantly increased.This experiment provided direct evidence that CCAP played a protective role in the low salinity of P.trituberculatus,implying that CCAP played a role in the regulation of osmotic pressure in P.trituberculatus and may have been associated with over-regulation of Na~+/K~+-ATPase and V-ATPase activity modulates osmotic pressure balance in P.trituberculatus.4.Full length cloning of crustacean hyperglycemic hormone gene DNA and its mechanism in low salt adaptation The Pt-CHH gene of P.trituberculatus consists of 4 exons and 3 introns spanning3849 bp in size.The primary gene transcript produced a cDNA encoded for the putative Pt-CHH2 from exons 1,2,3,4 and an alternative transcript encoded for a putative Pt-CHH1 peptide from exons 1,2 and 4.A promoter fragment of about 3kb was obtained by genomic walking.Bioinformatics analysis showed that the transcription initiation site was located at the 86 th nucleotide(0bp)upstream of the translation initiation codon(ATG),a TATA box was present at 31 bp upstream of the transcription start site,and the core promoter region was located at-40~+9bp.The putative binding sites for octamer binding protein(Oct-1),nuclear transcription factor(NF-kappaB),globin transcription factor(TBP),intracellular transcriptional activator(AP-1)and other binding sites were also found in the upstream region of Pt-CHH gene.Because of the expression in the gill may suggest its potential role in osmo-regulation.The results showed that the transcript level was changed significantly(P<0.05)in the experimental group,and up-regulated in the whole experiment group when crab were exposed to low salinity.After injecting dsRNA,the expression of Pt-CHH2 gene in gills decreased significantly,and the activity of Na~+/K~+-ATP and carbonic anhydrase decreased.The results showed that CHH gene played a role in the osmoregulation of P.trituberculatus and may regulate the osmotic pressure in vivo by regulating the activity of Na~+/K~+-ATPase and carbonic anhydrase. |
PDF Full Text Request