Molecular Cloning And Expression Of TLR7 Gene Under Viral Infection In Grass Carp, Ctenopharyngodon Idella

Grass carp (Ctenopharyngodon idella) is considered an important aquaculture resource in China with the largest yield. But the reovirus caused by GCRV (Grass carp reovirus) results in serious losses to the aquaculture industry. It is particularly important to study the expression and function of genes related to disease resistance. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become one of the most commonly used techniques for mRNA expression. An approoriate internal reference gene is necessary for the expression study of functional genes in the qRT-PCR. In this study, the stability of the four candidate internal reference genes ofβ-actin, 18S rRNA, GAPDH and EF1αin CIK was estimated under the infection of GCRV or the simulation of poly(I:C). Ctenopharyngodon idella Toll-like receptor 7 (CiTLR7) was cloned and the expression under the GCRV infection was determined in grass carp in vivo or in vitro.1. The stability of the four internal reference genes ofβ-actin,18S rRNA,GAPDH and EF1αin CIK cells.To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. The stabilities of four commonly used internal referencel genes encoding 18S rRNA,β-actin, EF1α, and GAPDH were integratedly assessed using the geNorm, NormFinder and BestKeeper programs in Ctenopharyngodon idella kidney (CIK) cells after qRT-PCR. The recommended ranking was EF1α>GAPDH>β-actin>18S rRNA in CIK cells infected by GCRV. The rank ordering of expression stability was EF1α>β-actin>18S rRNA>GAPDH in CIK cell (Ctenopharyngodon idella kidney) culture stimulated by poly(I:C). The results indicated that EF1αwas the most suitable in CIK cell culture infected by GCRV or stimulated by poly(I:C). These data laid the foundation for more precise results in qRT-PCR studies of gene expression in CIK cells.2. The molecular cloning and expression of CiTLR7 in grass carpTLR7 (Toll-like receptor 7) was played an essential role in the recognization of ssRNA and in triggering antiviral signaling pathways. TLR7 recognizes viral single-stranded RNA (ssRNA), and then induces signaling pathways leading to the production of interferon (IFN). In this study, we cloned and identified grass carp TLR7 (CiTLR7) gene, and examined the mRNA expression profiles in vivo and in vitro.The full length of CiTLR7 cDNA sequence is 3354 nt with the longest open reading frame (ORF) of 3156 nt encoding a peptide of 1051 amino acids. CiTLR7 gDNA is composed of two exons and one 940 nt intron. Tissue expression result showed that CiTLR7 mRNA was highly expressed in skin and spleen, and was moderately expressed in gill, heart and intestine. After grass carp reovirus (GCRV) injection, the expression of CiTLR7 mRNA was increased in most of the tested tissues by semi-quantitative RT-PCR detection. The expression of CiTLR7 was increased at 24 h time point and significantly up-regulated at 48 h time point after GCRV infection (P < 0.05) in liver, and then recovered to the original level at 72 h post-injection. The expression was significantly up-regulated at 12 h time point (P < 0.05) in spleen and reached the normal level at 48 h in spleen. The experiment results in vitro indicated that GCRV infection could inhibit the expression of CiTLR7, while the polyinosinic-polycytidylic acid sodium salt (poly(I:C)) stimulation could elevate the expression of CiTLR7.In summary, EF1αwas the most suitable in CIK cell culture infected by GCRV or stimulated by poly(I:C) among the four candidate internal reference genes ofβ-actin,18S rRNA,GAPDH and EF1α. CiTLR7 can make response to GCRV or poly(I:C) stimulation. This indicated that CiTLR7. This study was established an important foundation for the virus resistance mechanism to grass carp hemorrhage disease.