Screening,Cloning And Expression Analysis Of The Genes Related To Competitive Behavior In Fenneropenaeus Chinensis

Fenneropenaeus chinensis is an important cultured shrimp in China because of its high meat quality,large size and high economic value.It showed strong competitive behavior in the breeding which affects their growth,survival performance and group uniformity seriously.However,little information was available for the molecular mechanism of competition behavior in shrimp.Therefore,this study uses molecular biology methods and techniques to screen and obtain genes related to competitive strength,and will lay an important foundation for the theoretical basis and investigating the molecular mechanism of the competition behavior of F.chinensis.1.Screening differentially expressed genes of F.chinensis based on transcriptome under competitive environment.Eighteen individuals from the same family of F.chinensis in the 2017G12 generation with significant differences in competitive behavior were selected under different competitive stimulus environments.Three treatment groups were set up,that is to say,there are significant differences in competitiveness among individual mixed feeding group?MHL vs MHS?,individual independent feeding group?SCL vs SCS?and individual independent feeding group?SHL vs SHS?with significant differences in competitiveness.There were 94,092 Unigenes in SHL vs SHS group,which had significant differences in competitiveness.Among them,78.08%of Unigenes were annotated in Nr database and 77.04%of Unigenes were annotated in GO database.Compared with the other two groups,4,397 differentially expressed genes were obtained in MHL vs MHS,of which 4,087 were up-regulated and 312 were down-regulated.KEGG enrichment analysis showed that most of the signaling pathways involved by these differentially expressed genes were nerve signal transduction,suggesting that differences in nerve signal transduction velocities between individuals after receiving external stimuli could lead to differences in their competitiveness.The results laid an important foundation for the identification of candidate genes related to competition behavior of F.chinensis and the analysis of the molecular mechanism of competition behavior.2.Identification and characterization of the calcineurin?CN?in F.chinensis:its implications on competitive ability.Among the candidate genes associated with the competitive behavior of F.chinensis in the above comparative transcriptome analysis,including the calcineurin gene,which composes of a catalytic subunit?CNA?and a regulatory subunit?CNB?.Previous studies reported that CN played an important role in the central nervous system mediated by Ca²⁺/CaM.In addition,the CN genes was identified among the candidate genes that involved in the competitive behavior of F.chinensis from comparative transcriptome analysis in our previous study.In order to further prove the role of CN in the competition,in the present study,the full-length cDNA of CNA and CNB of F.chinensis?FcCN-A and FcCN-B?was cloned by RACE technology,and its expression in different tissues?nervous,heart,stomach,hepatopancreas,and intestine?between high competitive group?HCG?and low competitive group?LCG?in the ganglion of the 2018 G13 generation was analyzed by Real-time PCR.The results show that the full-length cDNA of FcCN-A is 3260 bp,containing 115bp of 5'untranslated region?UTR?,1566 bp of open reading frame?ORF?,and 1579 bp of 3'UTR;The results show that the full-length cDNA of FcCN-B is 2867 bp,containing95 bp of 5'UTR,540 bp of ORF and 2232 bp of 3'UTR.There are four conserved EF-hand Ca²⁺binding domains in the ORF.Protein homology analysis indicated that the amino acid sequence of FcCN-B has high homology with other species?72.2-93.4%?,among which FcCN-A has the highest homology with Macrobrachium rosenbergii and Eriocheir sinensis;FcCN-B is also the same,it has high homology with other species?78.8%-93.8%?,the highest of which is E.sinensis and Drosophila melanogaster.The phylogenetic relationship analysis showed that the vertebrate and invertebrates were independently clustered into two branches.Among them,based on FcCN-A analysis,F.chinensis was clustered with M.rosenbergii into an independent branches,and then it was clustered to E.sinensis.Based on the FcCN-B analysis,the F.chinensis and the E.sinensis were individually aggregated,and the clustering relationship with D.melanogaster was the closest.F.chinensis was clustered with E.sinensis into an independent branches,and then it was clustered to D.melanogaster.The results of Real-time PCR show that the expression of FcCN-A was significantly higher in the nerve ganglion of HCG than in LCG?P<0.01?,but its expression was significantly lower in heat and hepatopancreas of HCG than in LCG?P<0.01?.The results of Real-time PCR show that the expression of FcCN-B was significantly higher in the nerve ganglion of HCG than in LCG?P<0.01?,but its expression was significantly lower in heat of HCG than in LCG?P<0.01?.3.Identification and characterization of the caskin-2?CK?in F.chinensis:its implications on competitive ability.Among the candidate genes associated with the competitive behavior of F.chinensis in the above comparative transcriptome analysis,including the CK gene,CK is a scaffold protein that regulates cortical actin filaments and is enriched in mammalian synapses.In order to further prove the role of CK in the competition,in the present study,the full-length cDNA of CK of F.chinensis?FcCK?was cloned by RACE technology,and its expression in different tissues?nervous,heart,stomach,hepatopancreas,and intestine?between high competitive group?HCG?and low competitive group?LCG?in the ganglion of the 2018 G13 generation was analyzed by Real-time PCR.The results show that the full-length cDNA of FcCK is 4185 bp,containing 268 bp of5'UTR,2808 bp of ORF,and 1109 bp of 3'UTR.Protein homology analysis indicated that the amino acid sequence of FcCK has relatively low homology with other species?17.5-53%?,among which FcCK has the highest homology with Armadillidium vulgare;The phylogenetic relationship analysis showed that F.chinensis was clustered with A.vulgare into an independent branches,and then it was clustered to Onthophagus taurus and Diabrotica virgifera virgifera,which suggesting that FcCK might have similar functions as it did in A.vulgare.The results of Real-time PCR show that the expression of FcCK was significantly higher in the nerve ganglion of HCG than in LCG?P<0.01?.In this study,we selected candidate genes related to the competitive behavior of F.chinensis by comparative transcriptome analysis,and further cloned and characterized the calcineurin A?calcineurin B and caskin-1 genes,which proved that they may play a role in the competition behavior of F.chinensis.This study preliminarily proved that they may play a certain role in the competition behavior of F.chinensis,and will lay an important foundation for investigating the molecular mechanism of the competition behavior of F.chinensis.