Response Of Genes Involved In Th-cells Differentiation Of Trachinotus Ovatus To The Infection Of Photobacterium Damselae Subsp. Piscicida

We Used Trachinotus ovatus which is marine economic fish in southeast coast as the research material.After infection TOS1, we observe the count of bacteria in the body content changes over time in T.ovatus. The full length sequence of GATA-3 gene was studied using the gene cloning and RACE-PCR technology, and analyzed the characteristic of the sequence. The tissue distribution of T. Ovatus’ GATA-3,T-bet,Transferrin,Tumor necrosis factor- α were tested by Real-Time Quantitative PCR(RT-PCR).The expression profiles of genes involved in Th-cells differentiation of Trachinotus ovatus to the infection of Photobacterium damselae subsp. piscicida.The main results of this study were as follows:1 Cloning and prokaryotic expression of GATA-3from T. ovatus.We cloned GATA-3 c DNA in the T. ovatus. was 2347 bp in total length, within which a 1197 bp open reading frame(ORF) was identified. The ORF was found to encode a putative peptide comprising 398 amino acids(aa.). The putative molecule mass was43.1258 k D and the theoretical isoelectric point was 9.87. BLAST in Gen Bank database revealed that the polypeptide encoded by this c DNA was highly similar to certain fish GATA-3. Thomolgy analysis showed that GATA-3 of T. Ovatus exhibited the high identity(99%) with Lutjanus sanguineus,and the identity with other fish were 88% to 96%.Then,GATA-3 of T. Ovatus exhibited the identity with mammals was about 80%,and he identity with frog was 75%.2 T. Ovatus GATA-3 and T-bet tissue specific expressionResults of RT-PCR indicated that T.ovatus GATA-3 gene was expressed in the detected 11 organization,with highest expression levels in gill, followed by that in liver, kidney, skin and brain, again spleen and head kidney, and the lowest expression was in the stomach. T-bet gene was also expressed in various tissues.T-bet gene expression was highest in the kidney, the lowest expression in the brain. Besides, T-bet also had higher expression in kidney, spleen, gill, head kidney and liver.3 The number of bacteria in T. Ovatus vivo after infectedAfter infection TOS1, the number of bacteria continued to decrease in the kidney from 3 hours to 96 hours. In those times,the bacteria number descended significantly from 1.07 x 106 cfu/g to 2.3 x 104 cfu/g in 3-24 h.The bacterial number change was not big in 24-48 h and it was in the bacteria number-loss plateau.At the same time,the cumulative mortality of Trachinotus ovatus was 64.7% in 24 h.It was death peak in 12-24 h but the number of deaths decreased in 24-48 h.The death was no longer present after 48 h. As well as the bacteria number descended from 2.07 x 104 cfu/g(48 h) to 1.29 x 103 cfu/g(96 h).4 Response of genes involved in Th-cells differentiation of Trachinotus ovatus to the infection of Photobacterium damselae subsp. piscicidaAfter infected with TOS1,GATA-3 was expressed highest quantity in gill and kideny after 3h, in spleen after 72 h, in intestine and liver after 48 h, in head-kidney after 96 h.Among them, there was significant difference with the control group in the spleen, liver and kidney. IL-4 was expressed highest quantity in the kidney was after 24 h and in the head kidney after 48 h. However, T-bet expressed highest quantity in gill and kidney after 24 h, in spleen after 72 h, in intestine and liver after 48 h, in head-kidney after 96 h. Among them, there was significant difference with the control group in the liver and kidney.IL-12 was expressed highest in kidney after 12 h and in head-kidney after 48 h.IFN-γ was expressed highest quantity in kidney and head-kidney after 3h,TNF-α in kidney after 24 h and in head-kidney after 48 h was expressed highest.5 Expression analysis of T.ovatus transferrin and C-type lysozymeGene expression distribution result indicated that Transferrin is expressed in the liver and is a thousands times as abundant as other tissues. C-type lysozyme was expressed and reaches a peak in head-kidney, followed by kidney, and with relative low expression in stomach.In infection experimental, transferrin expression reached the peak in 6 h, and it obvious differ from the control(P < 0.05). In the head-kidney, it reached the highest expression quantity in 96h; C-type lysozyme expression in the kidney has been lower than the control group and there was also no significant change in head-kidney.