Molecular Cloning, Expression Pattern And Functional Analysis Of KIF3A/3B Gene (Ot-kif3a/3b) During Spermiogenesis Of Octopus Tankahkeei (Cephalopoda, Octopus)

Octopus tankahkeei, belonging to the phylum mollusk, class cephalopoda and order octopoda, is an economically important species in the Eastern China Sea. The supply of this fishery product is excessively dependent on catching of the wild population because artificial breeding and culturing technique is sill limited on a pilot scale due to a number of problems resulting from the poor understanding of the fundamentals of the fertilization biology and developmental biology in this species.As one of the most drastic cytological transformation processes, spermatogenesis refers to the period where progenitor cells sequentially undergo mitotic and meiotic divisions, giving rive to, after a dramatic remodeling process, a mature sperm capable of fertilizing an egg. The morphologic characteristics of spermatogenesis in Octopus tankahkeei has been comprehensively described, however, the molecular mechanism underlying these structural reorganizations remain to be addressed.Microtubule is one component of the cytoskeletons network in eukaryotic cells and is tied to multiple cellular events. Kinesin is a group of highly related molecular motors driving cellular transport of various cargoes along microtubules though ATP hydrolysis and having a saying in physiological regulations. KIF3A/3B is an N-terminal kinesin moving towards the plus end of microtubules with pivotal roles in intraflagellar transport therefore is essential for the formation of flagellum of spermatid during spermiogenesis in some rodentsBased on multiple protein sequences alignment of KIF3A/3B homologues and using a combined method of degenerate and RACE PCR, we are first to identify genes encoding highly homologues of rat KIF3A and KIF3B, belonging to Kinesin II subfamily, from the testis of Octopus tankahkeei and obtain their full-length cDNA, respectively. The 2732bp Ot-KIF3A nucleotides cDNA consists of a 2241 in-frame ORF encoding 746 continuous amino acids while the 2365bp KIF3B cDNA includes a 2208 bp ORF corresponding to 736 amino acids. Multiple alignments reveals that Ot-KIF3A share 69%,69%,69%,67% identity with that of human, mouse, rat and Danio reio; Ot-KIF3B shared an overall similarity of 68%, 68%, 69%, 68%, and 67% with that of Homo Sapien, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis. Tissue distribution analysis demonstrates the abundant expression in all the tissues examined. As histological and transmission electron microscopy analysis illustrated the morphological development during spermiogenesis, in situ hybridization showed an extremely similar temporal and spatial expression pattern of ot-kif3a transcripts with ot-kif3b. The messages appeared gradually in early spermtids, increased in intermediate spermatids and maximized at drastically remodeling and final spermatids, especially in the midpiece and flagellum of the tail, as well as in mature spermatozoa.In conclusion, the high identities of Ot-KIF3A and Ot-KIF3B in fundamental structures and the very similar expression pattern between ot-kif3a and ot-kif3b in the spermiogenesis of Octopus tankahkeei shed light on the their potential interactions in intracellular transport. Ot-KIF3A may form a conventional heterodimer with Ot-KIF3B, thus play a key role in the nuclear reshaping and flagellum formation by carrying different cargoes to the destination through intraflagellar transport and intramanchette transport and the regulation of timing and synchrony of spermatid differentiation. Moreover, Ot-KIF3A/3B may contribute to the aggregation of mitochondria to the midpiece of the spermatid.