Cloning And Expression Of Three Toll-like Receptor Genes And Their Association With Resistance To Bacterial Hemorrhagic Disease In Grass Carp Ctenopharyngodon Idellus

Grass carp (Ctenopharyngodon idella) is impotant in fresh aquaculture fishes of China.While it is easy to be infected by many pathogens, limiting the development of grasscarp aquaculture. In this paper, we cloned the grass carp receptor TLR21, TLR20andTLR18genes, and characterized them. As well as the expression and linkage wereanalyzed to understand their biological function. By GCRV and Aeromonas hydrophilaliquid on grass carp, we study their expression change at different times and in differenttissues. We investigated the polymorphisms of receptor TLR21, in order to associatewith resistance to hemorrhagic disease in grass carp. The results are as follows:1. The full-length cDNA sequence of grass carp TLR21consists of3578bp, withthe open reading frame (ORF) of2958bp encoding985amino acid residues. Accordingto the SMART software, The putative TLR21protein contains a signal peptide sequence(24aa),17leucine-rich (LRR) motifs, a transmembrane region and a Toll/interleukin-1receptor (TIR) domain. The highest similarity is to zebrafish, with79%amino acididentity. The gene is expressed in a wide range of tested tissues, with the highestexpression in skin, followed by spleen and intestine. While the expressions in muscle,trunk kidney and heart were very week. After challenge with GCRV, TLR21geneexpression is significantly down-regulated in liver and spleen, while is significantlyup-regulated in liver and spleen after induction by Aeromonas hydrophila.2. The full-length cDNA sequence of grass carp TLR20consists of3197bp, Thelength of ORF is2835bp, encoding944amino acid residues. According to the SMARTsoftware, The putative TLR20protein contains a signal peptide sequence (19aa),6leucine-rich (LRR) motifs, a transmembrane region and a TIR domain. The highestsimilarity is to Cyprinus carpio, with78%amino acid identity. Real time PCR revealedthat the gene transcript is expressed in a wide range of tissues with the highestexpression in spleen, followed by head kidney and liver. The expressions in trunk kidney and heart were very week. Upon induction by Aquareovirus, grass carp TLR20expression is significantly down-regulated in liver, whereas is significantly up-regulatedin gill, spleen and kidney. After Aeromonas hydrophila infection, TLR20expression issignificantly up-regulated in liver at4h, significantly up-regulated in spleen at firstthree days and then significantly down-regulated at7d. The expressions in gill andkidney were at a high level at all the time points. These results suggest that TLR20playsan important role in Aquareovirus and A. hydrophila-related diseases. This work mayprovide the basis for further investigations into the immune system of grass carp andother teleost fishes.3. The TLR18cDNA fragment was obtained from transcriptome. Using RACEPCR to abtain the full-length cDNA of TLR18, we got a3600bp sequence. The lengthof ORF is2559bp, encoding852amino acid residues. According to the SMARTsoftware, The putative TLR18protein contains a signal peptide sequence (22aa),7leucine-rich (LRR) motifs, a transmembrane region and a TIR domain. The highestsimilarity is to zebrafish, with85%amino acid identity. Real time PCR revealed that theTLR18gene transcript is expressed in a wide range of tissues with the highestexpression in spleen, followed by gill and heart. While the expressions in liver, skin andbrain were very week. Upon induction by Aquareovirus, grass carp TLR18expression issignificantly up-regulated in liver at4h, while is abruptly significantly down-regulated.In spleen, it is significantly up-regulated at3d, then significantly down-regulated at7d. After Aeromonas hydrophila infection, TLR18expression is significantlydown-regulated in liver at4h, and significantly down-regulated in spleen at4h and7d.4. After the challenge with A. hydrophila, we constructed a resource populationconsisting of132infected individuals and143unfected individuals. Single nucleotidepolymorphisms were obtained through direct sequencing. Six SNPs were obtained ofTLR21gene and we investigated the association of these polymorphic locis withresistance trait.1470T-C SNP was significant difference of the genotypes frequency andallelic frequency between two groups (P<0.05).1518A-C SNP,2049G-A SNP and3823A-G SNP were highly significant difference of the genotypes frequency and allelicfrequency between two groups (P<0.01).