Molecular characterization of a KIF3A-like kinesin gene in the testis of the Chinese fire-bellied newt Cynops orientalis

Abstract

KIF3A, the subunit within the kinesin-2 superfamily, is a typically N-terminal motor protein, which is involved in membranous organelle and intraflagellar transport. During spermatogenesis, KIF3A plays a critical role in the formation of flagella and cilia. KIF3A is also related to the left-right asymmetry, the signal pathway, DNA damage and tumorigenesis. We used RT-PCR and in situ hybridization to clone the kif3a gene, and we identified its function in the testis of the Chinese fire-bellied newt Cynops orientalis (termed as co-kif3a). The full-length sequence of co-kif3a was 2193bp, containing a 56bp 5'UTR, 2073bp ORF encoding a protein of 691 amino acids and a 64bp 3'UTR. The secondary structure analysis showed that co-KIF3A had three motor domains, representing the N-terminal motor domain (1-400 aa), α-helix domain (400-600 aa) and C-terminal tail domain (600-691 aa). The amino acid sequence of co-KIF3A shared an identity of 55.9%, 90.9%, 89.9%, 91.3% and 85.7% with its counterparts in Aedes aegypti, Mus musculus, Xenopus tropicalis, Homo sapiens and Danio rerio, respectively. The calculated molecular weight of the putative co-KIF3A was 79kDa and its estimated isoelectric point was 6.8. RT-PCR result showed that co-kif3a was expressed in several examined tissues, with a high level in the testis and low levels in liver, muscle and ovum. Kif3a was weakly expressed in the heart and spleen, and barely detected in the intestine. In situ hybridization analysis demonstrated that in early spermatid co-kif3a was expressed around the nuclear membrane. When the tail began to emerge in the middle spermatid, mRNA transcript was abundantly concentrated in the flagellum. The mRNA signal was still very strong along all the flagellum in late spermatid. In mature spermatid, the message was weak. Therefore, co-KIF3A probably plays a functional role in the spermiogenesis of C. orientalis.