CONSTRUCTION OF OOCYTE cDNA LIBRARIES OF GYNOGENETIC SILVER CRUCIAN CARP AND GONOCHORISTIC COLOR CRUCIAN CARP AND CLONING OF THEIR CYCLIN A1 cDNAs

Total RNAs were isolated from maturated oocytes of gynogenetic silver crucian carp (Carassius auratus gibelio) and gonochoristic color crucian carp (Carassius auratus color variety). After mRNA purification, double strand cDNAs were synthesized by using MMLV reverse transcriptase, inserted into λgtll Sfi-Not cloning vector, and packaged with packaging extracts. Therefore, the expressive cDNA libraries were constructed from both fish oocytes. The titration of libraries were 3.1×106 for silver crucian carp and 1.6×106 for color crucian carp respectively. A fast technique system for cloning full-length cDNA was also established through PCR-based method. Using the data from Gene Bank, we designed and synthesized conservative primers of cyclin A1 in carp, and cloned the full length cyclin A1 cDNAs of silver crucian carp and color crucian carp from their oocyte cDNA libraries. The length of cyclin A1 cDNAs is 1680 bp for silver crucian carp and 1627 bp for color crucian carp, and both have a same 70 bp of 5’ untranslated regions followed by a single open reading frame of 1173 bp. Both of the open reading frames are initiated from the consensus sequence ANNATG flanking translational start sites in vertebrates, and can be translated into proteins that consist of 391 amino acids. The differences mainly exist in 3’ untranslated regions, in which there are 373 bp for silver crucian carp, and there are 383 bp for color crucian carp. Pairwise comparison of the deduced amino acid sequences of the gynogenetic and gonochoristic crucian carp cyclin A1 with homologues from human, Xenopus and other fish revealed a high evolutionary conservation. Only 5 amino acid differences exist among three cyclin A1 of silver crucian carp, color crucian carp and goldfish, and they should be resulted from the variation of single nucleotide.