Cloning And Expression Analysis Of Akt,Pi3k And STAT1 In Sebastes Schlegelii

Sebastes schlegelii is one of the important economic species of marine fish in the coastal areas of northern China.It is loved by consumers for its tender flesh,rich nutrition,resistance to adversity and high medicinal value.However,the expansion of the aquaculture industry and the destruction of the farming environment often lead to outbreaks of large-scale diseases,mostly bacterial diseases,such as Vibrio harveyi,Vibrio anguillarum.These pathogenic bacteria can cause skin ulcers and congestion in the body surface of fish,and in severe cases,can cause a large number of deaths,resulting in significant economic losses and seriously hindering the healthy development of the S.schlegelii aquaculture industry.Conducting research on the molecular mechanism of bacterial infection and immunity can help to clearly understand the response mechanism of the organism to bacterial infection,which is of great significance for the prevention and treatment of bacterial diseases in S.schlegelii.In this study,we investigated the immunoregulatory genes Akt,Pi3 k,and STAT1 in S.schlegelii.We cloned the coding region c DNA sequences of these genes and analyzed their sequence characteristics.The tissue expression of each gene was analyzed using quantitative fluorescence PCR technology.We also analyzed the expression patterns of the three genes after exposure to Micrococcus luteus and V.anguillarum,and preliminarily analyzed the role of S.schlegelii in responding to bacterial stress.Additionally,we recombinantly expressed and purified STAT1 to obtain the purified protein.The main research results are as follows.The open reading frame(ORF)of SsAkt is 1440 bp in length,encoding 479 amino acids.The predicted relative molecular weight of the protein is 55.80 k D,with a theoretical p I of 5.64.SMART analysis reveals that the SsAkt protein contains three characteristic conserved domains of the serine/threonine protein kinase family and two phosphorylation sites.Homologous comparison shows that SsAkt has a high homology with fish,with the highest similarity to Lates calcarifer,reaching 99.37%.Tissue expression analysis indicates that SsAkt is expressed in healthy tissues of S.schlegelii,including blood,gills,liver,muscle,kidney,spleen,intestine,brain,and heart,with higher relative expression levels in the kidney,brain,and blood.Expression results in response to bacterial stress in S.schlegelii show that after infection with M.luteus,the expression of SsAkt in the three tissues is significantly upregulated,showing an upward-downward trend.However,after infection with V.anguillarum,SsAkt exhibits different expression patterns in the three tissues: in the kidney,the expression of SsAkt first rises,then falls,then rises again and falls,while in the blood and liver,the expression of SsAkt first rises and then falls.The ORF length of SsPi3 k is 3123 bp,which encodes 1040 amino acids.The predicted relative molecular weight of the protein is 120.69 k D,and its theoretical p I is8.37.SMART analysis revealed that SsPi3 k contains five conserved domains.Homology analysis indicated that SsPi3 k shows high homology with fish,with the highest similarity(99.04%)observed with Sebastes umbrosus.Tissue expression analysis demonstrated that SsPi3 k is expressed in healthy tissues of S.schlegelii,including blood,gills,liver,muscle,kidney,spleen,intestine,brain,and heart.The relative expression levels were highest in the kidney,gills,and blood.Upon bacterial stress,SsPi3 k expression was upregulated in three tissues after M.luteus infection,following an upward and downward trend.After V.anguillarum infection,SsPi3 k showed different expression trends in the three tissues.SsPi3 k expression in the kidney and blood followed an upward and downward trend,whereas the expression trend in the liver followed an upward and downward pattern.The ORF of SsSTAT1 is 2298 bp long,encoding 765 amino acids.Its predicted relative molecular weight is 88.14 k D,and the theoretical isoelectric point(p I)is 5.82.SMART analysis shows that the SsSTAT1 protein contains five conserved domains.Homology comparison revealed that SsSTAT1 shares a high degree of similarity with fish,particularly with Lateolabrax japonicus,with a similarity score of 95.27%.Tissue expression analysis indicates that SsSTAT1 is expressed in healthy tissues(blood,gill,liver,muscle,kidney,spleen,intestine,brain,and heart)of S.schlegelii,with relatively higher expression levels in blood,liver,and gill.Expression analysis under bacterial stress showed that after infection with M.luteus,SsSTAT1 expression was significantly upregulated in liver and kidney,displaying an initial increase and then a decrease trend,whereas its expression in blood remained unchanged.After infection with V.anguillarum,SsSTAT1 exhibited different expression patterns in the three tissues tested.Specifically,in kidney and liver,its expression level initially increased,then decreased,and increased again before eventually decreasing,while in blood,its expression level initially increased and then decreased.Furthermore,after constructing the SsSTAT1 prokaryotic expression vector and inducing protein expression using IPTG,a clear band was observed between80-100 k Da in the SDS-PAGE analysis,which corresponded to the predicted molecular weight of 88.14 k Da,indicating successful induction of the target protein.The purified protein had a purity greater than 95%,and its concentration was determined to be0.83mg/ml.The study above demonstrates that SsAkt,SsPi3 k,and SsSTAT1 exhibit a response to external microbial stress on S.schlegelii and assume a pivotal function in countering the external microbial immune response.The research findings are instrumental in exploring the functionality of S.schlegelii’s antibacterial immune response and provide the groundwork for investigating its immune mechanism.