|Other Abstract||The mandarin fish Siniperca chuatsi is an important species in China’s aquaculture. The culture of the mandarin fish has been threatened seriously by many kinds of diseases. It is thus important and necessary to develop some effective methods for the control of diseases. The understanding of immune system in this fish will provide a base for such development. The present study aimed to clone some apoptosis-related genes for the mandarin fish, with their apoptotic function analyzed in cell lines.
Three apoptosis related genes were cloned for the first time in the mandarin fish, i.e. TNF-related apoptosis-inducing ligand (TRAIL), a TRAIL decoy receptor (DcScTRAILR) and Mst3 and SOK1-related kinase (MASK) which are named as ScTRAIL, and ScDcTRAIL, and ScMASK. ScRAIL gene with the full-length cDNA of 1359 bp contains an open reading frame coding for a 283 aa protein; it’s full length genome has 9021 bp which contains six exons and five introns. The promoter region of ScTRAIL is 732 bp, which contains some putative transcription factor binding sites such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPalp, NF-kappaB and AP-1; however, it lacks typical TATA or CAAT box. The ScDcTRAILR gene with the full-length cDNA of 2494 bp contains an open reading frame coding for a 353 aa protein; its full-length genome sequence is 10437 bp which contains ten exons and nine introns. The promoter region of ScDcTRAILR is 804 bp, which contains some putative transcription factor binding sites such as C/EBPalp、Sp-1、Oct-1 and GATA-1, but it lacks typical TATA or CAAT box. The ScMASK gene with the full-length cDNA of 1743 bp contains an open reading frame coding for a 405 aa protein; its full-length genome sequence is 8561 bp, which contains nine exons and eight introns. The promoter region of ScMASK is 1526 bp, and lacks typical TATA or CAAT box, but it contains some putative transcription factor binding sites such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPalp, NF-kappaB and AP-1. Southern blotting analyses revealed that in mandarin fish genome, the ScTRAIL gene is present as a single copy; based on this result, the ScDcTRAILR and ScMASK genes were also found as single copy genes, as analyzed by real-time PCR.
The organ distribution of the three genes was analyzed both in the transcription and protein levels. By RT-PCR analysis, the transcription products of the cloned genes were investigated in different tissues of healthy mandarin fish, and the three genes had different patterns of tissue distribution. Western blotting analyses revealed that ScTRAIL protein existed broadly in examined tissues with the highest expression level in head kidney and gonad while lowest in brain. ScDcTRAILR protein had a relatively higher expression level in spleen, brain, heart and gill, while ScMASK protein was just detected in gill, liver and brain.
Furthermore, expression of the three genes was analyzed to mimic pathogen attacking by real-time quantitative PCR. Obvious changes in expression level of ScTRAIL were not observed when fish were stimulated with PolyI:C or LPS. ScDcTRAILR gene expression in LPS induced group was up-regulated in head kidney, and in PolyI:C stimulated group its expression level increased in head kidney and kidney, while decreased in liver and heart. The expression level of ScMASK in LPS stimulated group was up-regulated in head kidney and heart, and its expression level increased in liver and spleen when stimulated with PolyI:C.
Apoptosis effect of the three genes was investigated by over-expression of three genes in Hela cell line, respectively. Apoptosis were detected by several methods including fluorescence microscopy, agarose gel electrophoresis and flow cytometry. The existence of apoptotic bodies, DNA ladder and apoptosis peaks in the ScTRAIL and ScMASK transfected Hela cells indicated that these two genes induced apoptosis. However in the ScDcTRAILR gene transfected Hela cells, no apoptotic characters were observed, suggesting that this gene may not induce cell apoptosis and may be probably a decoy death receptor.|