Molecular Cloning And Expression Analysis Of STAT1in Turbot, Scophthalmus Maximus, IRF9in Japanese Flounder, Paralichthys Olivaceus

Turbot (Scophthalmus maximus) and Japanese flouder (Paralichthys olivaceus) are important maricultured species in coastal areas of northern China, has been suffered from serious diseases, especially viral disease with the rapidly development in recent years. The viral diseases has led to great economic loss in the fish culture. Interferons (IFN) plays an important role in antiviral immune. Consequently, the aim of this study is to enrich the knowledge about the IFN system of turbot and Japanese flounder which have deep impact on control viral diseases of these commercially species.In this study, three cDNAs with different3’-untranslated region (UTR) encoding an STAT1(transcription factors signal transducers and activators of transcription1) were cloned from turbot, Scophthalmus maximus, by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The STAT1is involved in signal transduction of type Ⅰ and Ⅱ IFN. Sequence analysis reveals that they were generated by alternative polyadenylation. The predicted protein consists of750amino acid residues which shares the highest identity of58.8-92.7%to fish STATls and possesses a DNA-binding domain (DBD), a SH2domain and a transcriptional activation domain (TAD). Similarly, We use RT-PCR and RACE cloing the cDNA sequence of Japanese flounder IRF9, which is3305bp long containing272bp5’ UTR,1725bp3’-UTR and encodes a peptide of435amino acids. The predicted protein sequence shares33.7-72.0%identity,61.9-80.5%similarity to other fish IRF9s. Japanese flounder IRF9possesses a DNA-binding domain (DBD), an IRF association domain (IAD), nuclear localization signals (NLS) and a proline-rich domain (PRD).qPCR was undertaken to examine expression of turbot STAT1mRNA and Japanese flounder IRF9in different tissues tested including brain, gills, stomach, intestine, heart, head kidney, kidney, liver, spleen, gonad, muscle and skin. The results showed that turbot STAT1and Japanese flouder IRF9are conductive expression. The higher expression was detected in the intestine, head kidney, liver, spleen of turbot STAT1and in head kidney, kidney, liver and spleen of Japanese flounder IRF9.qPCR analysis was employed to investigate the two genes expression in response to poly Ⅰ:C and TRBIV challenges at0h,3h,6h,12h,1d,2d,3d,4d,5d,7d in turbot, poly Ⅰ:C challenges at0h,3h,6h,12h,1d,2d,3d,4d,5d,7d and LCDV challenges at0h,6h,12h,18h,1d,2d,3d,4d,5d,7d in Japanese flounder, respectively. The results exhibited that in an early phase of a7-daytime course, the poly Ⅰ:C was a quicker inducer in two genes. In both challenge cases, the earliest and strongest inductions were detected in the spleen of turbot and Japanese flounder. Uponchallenge with poly I:C, the expression peak arose at hour3post-injection in the spleen and hour6in the gills, head kidney and muscle with an approximate2.7-,3.9-,4.2-and4.4-fold increase,respectively. Additionally, a minor peak was observed in the gills and spleen at days5,3with anincrease of2.8-and3.5-fold, respectively. The expression peak of Japanese flounder IRF9arose athour3post-injection in the spleen, but very weak induction in the gills, head kidney and muscle,with an approximate8.6-,1.4-,1.3-and2-fold increase, respectively. Upon challenge with TRBIV,the maximum induction levels of turbot were3.4-,3.2-,4.5-and2.0-fold and appeared at day2,1,3and5post-infection in the gills, head kidney, spleen and muscle, respectively. Meanwhile, a minorpeak was observed in the head kidney and spleen at days5,3with an increase of2.9-and4.5-fold,respectively. In the case of LCDV challenge, Japanese flounder IRF9expression was also up-regulated, with a maximum increase of3.2-fold arising at day3in the spleen, but the induction inhead kideny and muscle was very weak, which maintained at a level similar to control in LCDVchallenge cases.We successfully cloned the cDNA of turbot STAT1and Japanese flounder IRF9and analysis thegene expression, which contribute to an better understanding of the IFN system in turbot andJapanese flouder and also help to develop effective and ecnomic strateges for protection turbot andJapanese flouder from viral diseases.