Molecular Cloning And Characteration Of KIF3A-like Kinesin Gene (Co-KIF3A) In The Spermiogenesis Of The Chinese Fire-bellied Newt Cynops Orientalis

We used the Chinese fire-bellied newt Cynops orientalis as our experiment subjects to investigate the molecular cloning and characterization of KIF3A-like kinesin gene (co-kif3a) in its testis.Chinese fire-bellied newt, Cynops orientalis, belongs to the Amphibia,Caudata, Salamandridae. Since it locates in.the special position during the evolution-process of amphibian.Reproduction mechanism of animal is conserved to some extent, so spermatogenesis as the model system is helpful to compare reproduction evolution among different species.Kinesin has played a key role in the spermatogenesis of mammal.But in the amphibian,the research about the kinesin is blank.We employ degenerating primer PCR,RACE,RT-PCR and in situ hybridization to clone the kif3a gene and to identify 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 64 bp 3'UTR. The amino acid sequence of co-KIF3A shared identity of 55.9%,90.9%,89.9%,91.3% and 85.7% with its counterparts in Aedes aegypti, Mus musculus, Xenopus tropical is, Homo sapiens and Danio rerio respectively. The calculated molecular weight of the putative co-KIF3A was 79 kDa and its estimated aisoelectric point was 6.8. RT-PCR result showed that co-kif3a was expressed in many 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, 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 Cynopis Orientalis.