PKR Gene Cloning And Expression Study Of Interferon System Genes In Siniperca Chuatsi

Siniperca chuatsi, belonging to Perciformes, Sinipercinae, Siniperca,, commonly known as mandarin fish, fat salmon, etc., is a kind of freshwater benthic carnivorous fish. It is one of the chief economic freshwater fishes in China due to its fast grow speed and delcious flesh. But there are many pathogens for Siniperca chuatsi, and the disease caused by ISKNV (Infectious Spleen and Kidney Necrosis Virus) results in serious losses to the aquaculture industry. The disease is widely spread and highly lethal, leading to large economic loss in domestic area. So far there is no highly effective drug or treatment to this virus. And the specific immune system of Siniperca chuatsi is deficient, which result in the low efficiency of vaccine. Thus, identifying critical genes in the innate immunity networks will not only become the basis for disease-resistant breeding in the future but also facilitate further understanding on innate immune system of fish.Thus, the Siniperca chuatsi PKR gene was cloned by RACE.the stability of the three candidate internal reference genes of β-actin,18S rRNA, GAPDH in Siniperca chuatsi was estimated by geNorm, NormFinder and BestKeeper programs. The expression of PKR and other 6 immune-related genes under POLY(I:C) or LPS infection was analyesd in Siniperca chuatsi by qRT-PCR. the main research is structured as follows:1 Molecular cloning of PKR in Siniperca chuatsiThe primers were designed according to Siniperca chuatsi PKR gene cDNA sequence to obtain the whole sequence of it through RACE (rapid amplication of cDNA end) method of 5’and 3’end. The full length of Siniperca chuatsi PKR gene cDNA is 2791bp, with 66bp 5’-UTR(untranslated region), an open read frame(ORF) of 2097bp, encoding 698 amino acids probably,3’-UTR with length of 628bp, having obvious termination codon TGA and poly A tail. The protein of PKR owns 3 DSRMs (double-stranded RNA binding motif) and one protein kinase domain. Comparing Siniperca chuatsi PKR with those of fishes and mammals by ClustalW2, the results showed that the amino acid homology was between 30%～76%, while the highest one is Larimichthvs crocea.2 The stability of the three internal reference genes of β -actin、18S rRNA and GAPDH in Siniperca chuatsiTo obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. The stabilities of three commonly used internal referencel genes encoding 18S rRNA, (3-actin and GAPDH were integratedly assessed using the geNorm, NormFinder and BestKeeper programs in Siniperca chuatsi after qRT-PCR. The recommended ranking was 18S rRNA>β-actin >GAPDH in 10 different tissues of Siniperca chuatsi. The rank ordering of expression stability was β-actin>18S rRNA>GAPDH in different tissues of Siniperca chuatsi stimulated by POLY(I:C). The rank ordering of expression stability was also β-actin> 18S rRNA>GAPDH in different tissues of Siniperca chuatsi stimulated by LPS. The results indicated that 18S rRNA was the most suitable internal control gene in different tissues of Siniperca chuatsi, while β-actin was the most suitable one in Siniperca chuatsi stimulated by POLY(I:C) or LPS. These data laid the foundation for more precise results in qRT-PCR studies of gene expression in Siniperca chuatsi.3 The expression features of PKR and other Interferon system genes in Siniperca chuatsi stimulated by POLY(I:C) or LPSWe designed the Real-time primer according to Siniperca chuatsi PKR gene cDNA sequence. Six IFN system genes including Mx、Viperin、IRF-1、IRF-2a、IRF-2b、IRF-7 were chosen to analyse their expression features in different tissues of healthy Siniperca chuatsi and ones stimulated by POLY(I:C) or LPS.Among the 9 tissues including blood, brain, gill, kidney, heart, liver, muscle, intestine and spleen of healthy Siniperca chuatsi, genes were high expressed in liver, spleen, intestine gill and blood. The expression features of seven IFN system genes in different tissues of Siniperca chuatsi stimulated by POLY(I:C) or LPS were different,but were all up-regulated compared with Siniperca chuatsi injected with PBS. It demonstrated that all seven IFN system genes are related to the immune process against virus or bacterium in Siniperca chuatsi.