Studies On The Stability Of Four Candidate Internal Reference Genes And Molecular Cloning And Expression Of Two Immunization-associated Genes In Grass Carp, Ctenopharyngodon Idellus

Grass carp (Ctenopharyngodon idellus) is a one of the most cultured aquaculture species in China. However, grass carp reovirus (GCRV) which is considered to be the most pathogenic aquareovirus seriously threats the grass carp industry. In this study, we cloned two immunization-associated genes and studied the expression levels under different treatment in grass carp, which will give basic knowledge to understand the immune defense mechanisms and provide candidate genes in breeding for disease resistance in grass carp. This research composed of three experiments focus on selecting of suitable internal reference genes and cloning of MyD88 and TLR9 in grass carp. The main results are as follows:1. Studies on the stability of four candidate internal reference genesIn this study, the expression levels of 4 potential reference genes (18S rRNA,β-actin, GAPDH, EF1-α) were investigated in grass carp by SYBR green real-time RT-PCR under different experimental circumstance. (1) 4 potential reference genes from grass carp were selected to test for normalization of expression levels in 9 differenet tissue types (Gill, Head Kidney, Heart, Intestine, Liver, Muscle, Skin, Spleen, Trunk kidney). Their stability and suitability as reference genes were validated by geNorm,NormFinder and BestKeeper programs. The ranking of stability of 4 candidate genes is 18S rRNA>EF1-α>GAPDH>β-actin. So, 18S rRNA and EF1-αis selected as the reference genes in different tissues. (2) The stability of 4 potential reference genes were also checked on spleen tissue at 0h, 24h, 48h, 72h, 120h after injection of GCRV. The ranking of stability of 4 candidate genes is 18S rRNA>β-actin>GAPDH>EF1-α. So, 18S rRNA andβ-actin is selected as the reference genes during the time course studies after infected with GCRV. Intriguingly, 18S rRNA is recommended as the ideal reference genes both in different tissue types and during time course study after injection of GCRV.2. Molecular cloning and expression of grass carp MyD88The full length cDNA of MyD88 consists of 1204 nucleotides was cloned from grass carp based on homologous cloning and rapid amplification cDNA end (RACE) strategies. The 5'-untranslated region (UTR) is 249nt, the open reading frame (ORF) is 855nt which encodes 284 amino acid (aa). The 3'UTR is 99nt containing one"stop codon"and poly A tail. The grass carp MyD88 contains a DEATH domain and a TIR domain. Sequence analyses of MyD88 revealed the presence of three highly conserved regions among teleost fish and mammals of the family: Box1 (FDAFICYCQ), Box2 (RDVLPG), and Box3 (FWTRLA). Multiple sequence alignment shows that the deduced amino acid sequence of MyD88 shared high identity with those from other teleost species. The MyD88 transcription was broadly expressed in 9 different tissue types (Gill, Head Kidney, Heart, Intestine, Liver, Muscle, Skin, Spleen, Trunk kidney), strongly in spleen and weakly in head kidney and muscle confirmed by semi-quantitative RT-PCR analysis. The results confirmed by real-time RT-PCR also indicated the up-regulated levels of MyD88 expression in grass carp tissue infected with GCRV. In liver tissues, the expression of grass carp MyD88 was significantly up-regulated in the chanllenged group (P<0.05), reached a peak at 120h post-injection. In spleen tissues, the expression of grass carp MyD88 was significantly up-regulated at 12h and reached a peak at 24h,3. Molecular cloning and expression of grass carp TLR9The partial sequence of TLR9 was cloned from grass carp. The TLR9 transcription was weekly expressed in 9 different tissue types (Gill, Head Kidney, Heart, Intestine, Liver, Muscle, Skin, Trunk kidney) and highly expressed in spleen confirmed by semi-quantitative RT-PCR analysis.In conclusion, the stability of 4 reference genes was compared and confirmed by geNorm,NormFinder and BestKeeper programs. 18S rRNA is recommended as the ideal reference genes in different tissue types and during the time course study after injection of GCRV. The grass carp MyD88 shows highly conserved among fishes and plays important role in antivirus mechanism. Grass carp TLR9 also shows somewhat similarities with other fishes and weekly expressed in different tissues.