Cloning, Investigation of Nucleotide Sequence and Nucleotide and Protein Alignment of Mt-ND1 Gene in Khorasan Native Chickens

This research aim was to identify, isolate, and clone ND1 gene of native chicken genome of Khorasan and compare it with other genomes and protein sequences of this gene in order to examine the possible mutations. For this purpose, genomic DNA was extracted from the blood sample of Khorasan native chickens and after designing specific primers of this gene using reference nucleotide sequence and carrying out polymerase chain reaction (PCR) for gene amplification, the product of reaction was electrophoresed on the agarose gel. The resulting bands were cloned and sequenced into E. coli using the pTZ57R/T linear vector. The comparison between the sequenced fragment of nucleotide and the reference genome revealed three mutations and a 99%/100% similarity was found in length with complete mitochondrial genome sequence of Gallus gallus in the recorded poultry mitochondria with accession number X52392.1 (reference genome), GU261719.1, GU261715.1, GU261712.1, AP003318.1, KF826490.1 and HQ857210.1. The protein comparison showed that the resulted sequence was similar to the protein sequences related to gene ND1 of the poultry mitochondriain the database so that the most similarity (100%/100%) in length belonged to gene ND1 in Gallus gallus spadiceus with accession number NP 006515.1 and the least similarity belonged to species and genera of Criniferpiscator, Nyctiousgrandis, Cursoriustemminckii and Aceroswaldeni with 78%/100% similarity. The comparison of the sequence with the reference genome was also 99%/100% similar in length. The nucleotide comparison showed mutation in three places of which two primary mutations occurred in the tRNA area which were not entered in the ND1 translation. After changing the obtained nucleotide sequences to amino acid sequences, it was compared with the reference protein sequences. The result of this comparison showed the difference in placing of one amino acid. In amino acid 173, alanine, which is a polar amino acid, changed to threonin polar amino acid.