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To explore the structure characteristic of rpL14 gene of A. melanoleuca and the differences among other species reported, we have designed primers based on the known information of rpL14 gene. Then, the cDNA and the genomic sequence of rpL14 gene were cloned successfully from A. melanoleuca by RT-PCR and PCR strategy respectively, and followed by sequencing and analyzing. The result indicated that the length of cDNA fragment cloned from A. melanoleuca is 700 bp in size, containing an open reading frame of 636bp. Predicted protein was composed of 211 amino acids with an estimated molecular weight of 23.06 kD and PI of 11.70. The length of the genomic sequence is 3292 bp, which was found to have six exons and five introns. Alignment analysis indicates that rpL14 gene and deduced amino acid sequence are highly similarity with reported species, including Homo sapiens, Bos taurus, Rattus norvegicus, Mus musculus and Sus scrofa. The homologies for nucleotide sequences of A. melanoleuca rpL14 with this five species are 89.51%, 89.77%, 85.43%, 85.17% and 89.25%, while the homologies for amino acid sequences are 79.07%, 90.19 %, 86.45%, 84.79% and 94.37%. Topology predicted shows that six different patterns were found in the RPL14 protein of A. melanoleuca. The rpL14 gene of the A. melanoleuca was studied in this paper, which provided scientific information to enrich the rpL14 gene database.