Identification、expression Pattern And Genetic Variation Of A Novel Incrna Gene Pouac22Specially Expressed In Poultry

Long non-coding RNAs (lncRNAs) are broadly defined as transcribed RNA molecules greater than200nt in length, between200nt and100kb, which belongs to noncoding RNA. lncRNA locates inside the nucleus or cytoplasm, widely transcribed in eukaryotic cells, but not having or rarely with functions of protein coding. Few studies about the characterization and expression of specific lncRNA loci for chickens have been reported by now. In this study, one expressed sequence tag (EST, CR406381.1) could not be aligned to any known genes in the species, Combination of techniques involving3’-rapid amplification of cDNA ends (RACE),5-RACE and reverse-transcription polymerase chain reaction (RT-PCR) were used to obtain the full-length cDNA of the EST and bioinformatic analysis was then conducted. Expression analysis was conducted by RT-PCR and quantitative RT-PCR (qPCR). In this study, the following three parts were mainly carried out.Test oneTo obtain the cDNA sequence of the unkown gene, the study was performed according to the manual of the SMARTTM RACE cDNA Amplification Kit (Clontech Laboratories Inc., USA). The results were as follows:a1197bp PCR product was obtained and sequenced in5’RACE. Four alternative splicing were generated in3’ RACE with the length of403bp,535bp,658bp and1310bp. By aligning and assembling the sequences of3’RACE and5’RACE products, the full-length cDNA of four splice variants was obtained, which were1827bp,1954bp,2077bp,2729bp long correspond to3’ RACE products (named as pouAC22-1, pouAC22-2, pouAC-3and pouAC-4respectively). The redults of bioinformatics analysis were as follows: pouAC22was a novel lncRNA mapped on the chicken chromosome22by bioinformatics analysis and specially expressed in poultry. Sequence comparison by performing Blast Search showed that pouAC22had homology sequence in turkey, duck, zebra finch, collared flycatcher and no homology sequence found in other species such as human, pig, mouse et al and highly conserved in3’UTR.80%、70%、43%(lower than80%) sequence identity were found in turkey, zebra finch, duck, which are much lower than the homologous proportion of coding gene. In collared flycatcher, highly homology sequence of pouAC22was only identified in3’UTR. Test twoTest two was performed to examine the exact expression trends of pouAC22in different growth stages and the expresdion difference among tissues and splice variants. The results of RT-PCR showed that the gene had different level in different tissues of chicken. The expression level of pouAC22was relative higher in brain, while the relative lower expression was detected in breast muscle. There were expression differences at different growth stages in the same organization by qPCR. Breast muscle was higher expression at17day of embryo; leg muscle express was higher expression in7day after hatching; Brain was higher expression in7day after hatching; The expression level of cerebellum was relative higher at17day of embryo and7day after hatching, which was identified with the results of RT-PCR. The different level of oil added in diet had no effect on the expression of pouAC22. The5%level of maize oil and lard oil has remarkable effect on the expression of pouAC22-4compared with5%linseed oil.Test threeGenomic DNA samples were extracted from Silkie, White Plymouth Rocks, Tibetan chicken, Princess chicken, Rhode island Red and F2of Gushi chickens crossed with Anka chickens, and then five SNPs was identified by clonal sequence and direct sequence, including C1800T, C1918T, A2020G, A2198G and A2217G. The assciation analysis of A2020G variant was conducted with chicken performance traits in F2population, which was also detected in F2of Gushi chickens crossed with Anka chickens,. The results showed that the mutation showed a significant association with weight at4weeks,6weeks, leg weight (P<0.05). Among them, highly significant association with weight at6weeks (P<0.01) was found in this study.