Cloning And Expression Analysis Of Three Genes Involved In PI3K/AKT Pathway From Ctenopharyngodon Idella

Grass carp(Ctenopharyngodon idella) is one fish of the highest produnction culturing in world. Farmed grass carp is vulnerable to bacterial infections, which cause heavy losses in the aquaculture industry every year. Preventing these diseases represents a great challenge because of the environmental concerns associated with chemical treatments and the emergence of multiple resistances to antibiotics. So selective breeding of high disease resistance is an effective way with prevention of grass carp disease.PI3K/AKT pathway is a signal pathway based on phosphorylation. PI3K/AKT pathway of grass carp was not studied by this time. In this paper, three genes were cloned involved in PI3K/AKT pathway. The physicochemical properties of these gene sequences were analyzed, amino acids predicted. Their gene expression was detected in four immune-associated organs during challenge with A. hydrophila. NFIL3 gene of grass carp was selected as further exploration. The influence of its overexpression was explored during the A. hydrophila’s invasion and obtained results as following:1. Full length sequences were obtained of AKT1, AKT3 and NFIL3 genes: 1554 bp, 2808 bp and 2018 bp with an ORF of 1155 bp, 1440 bp and 1407 bp, respectively. All the three predicted amino acid sequences had no signal peptide sites.2. BLAST results revealed that grass carp AKT1,AKT3 and NFIL3 were members of conservative protein family. Open reading frames of grass carp AKT3 and NFIL3 both shared 90% identity with zebra fish’s, while grass carp AKT1 shared lower consistency with zebra fish at 81%. NJ tree results showed predicted amino acids of three genes could cluster with other bony fish’s sequences which revealed the nearest relation between bony fish.3. Fluorescent quantitation results revealed that all of these genes were expressed in all the eight examined organs. During challenge with A. hydrophila, AKT1 gene was significantly up-regulated in liver; AKT3 was significantly up-regulated in liver and trunk kidney; NFIL3 was significantly up-regulated in trunk kidney and head kidney, which is the immune center organ of fish. The result of western blotting verified the up-regulations of NFIL3 protein in head kidney, trunk kidney and intestine.4. Through reconstruction of grass carp NFIL3 overexpression plasmid, the recombinant plasmid was transferred into CIK cells, and it was found that grass carp NFIL3 is mainly expressed in nucleus by subcellular localization.5. CIK cells were challenged with A. hydrophila, and it was found that overexpression of NFIL3 could effectively prevent bacteria invasion and proliferation.6. To explore the reason of resistance of bacteria by overexpression of NFIL3, with the Dual-Luciferase reporter gene detection system and fluorescence quantitative methods, it was found that overexpression of grass carp NFIL3 could significantly active NF-κB, up-regulated gene expression of IFN and IL10.