The Analysis Of The Structure,Evolution And Expression Analysis Of Miiuy Croaker Caspase Gene Family

Apoptosis is the process of cell death(PCD),which is controlled by its own genetic mechanism under certain physiological or pathological conditions under certain physiological or pathological conditions.Thus terminating the process of its life.In the field of immunology,the current research results show that there are three main signal transduction pathways,namely death receptor pathway,mitochondrial pathway and endoparasitic reticulum pathway.In these signal transduction pathways,four proteins are found to be involved in the mechanism of apoptosis,namely,apoptosis protease,cohesive protein,Bcl-2 and apoptosis inhibitory protein.Among them,cysteine aspartate proteinase(Caspase)is a protease system which directly leads to the disintegration of apoptosis cells,and is the core of the mechanism of apoptosis.Caspase enzyme is a group of enzymes that play a critical role in the process of cell apoptosis by the promoters or effectors of apoptosis.At present,14 kinds of enzymes have been discovered.According to the different positions and functions of the upstream or downstream of the apoptosis cascade reaction,they can fall into three types.The first class is the cell apoptosis promoter.The second is the apoptosis effector,and the third is the inflammatory medium.In this study,we mainly consider the sequence identification,evolutionary analysis and expression analysis of Caspase-2,-8 and downstream Caspase-6,-7 in the upstream of cascade reaction in the Caspase family.1.Analysis of expressed sequence,structure,and evolution of miiuy croaker Caspase cascade upstream geneThe upstream genes of Caspase cascade include Caspase-2,-8,-9 and-10.Caspase-2 and Caspase-8 genes were mainly studied in this study.The total length of Caspase-2 gene is 7412 bp and cDNA sequence is 1464 bp,which includes 5 ’end non translation region(UTR)6BP,3’-UTR 48 bp,open reading frame(ORF)length encoding 470 amino acid protein sequences.It makes a CARD domain and a CASc domain.The molecular weight of Caspase-2 is 53.1kDa,the theoretical locus(pI)6.4,including 11 exons and 10 introns,including 11 alpha helix and 10 beta fold structure,and the Caspase-2 gene of the fish has the QACRG of the conservative five peptide sequence of the Caspase family.The total length of Caspase-8 gene is 6413 bp,and the cDNA sequence is 2425 bp,which includes 5 ’-UTR 247 bp,3’-UTR 729 bp,CDS length 1449 bp encoding 483 amino acid proteins,with two DED domains and a CASc domain.The molecular weight of miiuy croaker Caspase-8 is 55.1kDa,the theory of site pI value is 5.5.It has 18 alpha helices and 6 beta folded structures,and has a conserved five peptide sequence.QACQG.Real-time PCR expression analysis showed that Caspase-2 and Caspase-8 genes were expressed in the liver,spleen,kidney,intestine,skin,gill,gill,heart and muscle tissue.The expression of Caspase-2 gene in the liver was the highest,monitored by muscle and skin,and the highest expression of Caspase-8 gene in skin,followed by muscle.The two genes are the lowest in the heart.In addition,three immune related tissues of the liver,kidney and spleen were selected to carry out the infection experiment.The expression of Caspase-2 in the liver reached the peak value in the24 h,the 6h in the spleen reached the maximum,and the 12 h in the kidney reached the maximum.Among them,the expression level of Caspase-2 in spleen at various time points after infection is at a high level.The expression level of Caspase-8 gene reached the highest value when liver and kidney were infected with 12 h,and reached the maximum value when the spleen was infected with 6h.2.The downstream gene sequence,structural and evolutionary analysis of miiuy croaker Caspase cascadeThe downstream effects of Caspase cascade include Caspase,Caspase-3,-6 and-7.In this paper,Caspase-6 and Caspase-7 are analyzed.The full-length cDNA of Caspase-6 gene of miiuy croaker was 4549 bp,including 8 exons and 7 introns.miiuy croaker Caspase-6 gene CDS length 906 bp encoding 302 amino acid protein sequences,only a CASc domain.The molecular weight of miiuy croaker Caspase-6 is33.8kDa,the theory of site 5.8.The conservative five peptide sequence was QACRG.The full-length cDNA of Caspase-7 gene of miiuy croaker was 1425 bp,including six exons and five introns.Miiuy croaker Caspase-7 gene CDS length 933 bp encoding311 amino acids protein sequence.The molecular weight of miiuy croaker Caspase-7is 35.1kDa,the theory of site 5.5.The conserved five peptide sequence QACRG.Caspase-6 expression analysis showed that Caspase-6 was highly expressed in the spleen.