Partial CDNA Cloning, Comparative Analysis Of Transcriptional Level Of Cdc2 Gene In Ovaries With Different Ploidy Level Cyprinid Fish

The formation of allotetraploid hybrid population from red crucian carp (♀) ( Carassius auratus red variety )×common carp (♂) ( Cyprinus carpio L. ) is the first case in the world in which both the female and the male of allotetraploids are fertile. They provide important academic evidence and very good experimental materials for studying the mechanism of formation of fish multiploidy in the natural world. The triploid crucian carp, with obvious advantage in growth, is obtained by crossing the alloteraploid hybrids (♂) with the diploid Japanese crucian carp (♀) ( Carassius auratus cuvieri T. et S. ). Following activation by UV-irradiated sperms from scatter scale common carp and without the treatment for doubling the chromosome number, the eggs generated by the allotetraploid with 100 chromosomes, developed into diploid gynogenetic progeny (G₁) with 100 chromosomes. Two years later, the G₁ had grown up with mature eggs. Following same methods as the production of G₁, the eggs generated by the diploid gynogenetic progeny (G₁) with 100 chromosomes, developed into diploid gynogenetic progeny (G₂) with 100 chromosomes. The cytological research provides the evidence that the production of these diploid eggs from the diploid gynogens may have resulted from the pre-meiotic endoreduplication of chromosomes. So far, the G₃ have been produced, and the diploid gynogens provided precious experimental materials for studying the molecular mechanism of pre-meiotic endoreduplication.We have carried out a series of experiments on the cell cycle control gene-cdc2 with four kinds of cyprinid fish (allotetraploid hybrids, allotriploid crucian carp, red crucian carp, and diploid gynogen). The cdc2 was discovered firstly in yeast (S. pombe), and the subsequent studies substantiated that the G₂→M-phase transition was triggered in vertebrate cells by the cyclinB-Cdc2 complex (named MPF). The partial sequences of cdc2 cDNA for different ploidy level cyprinid fish were amplified by 3'-Race (Rapid Amplification of cDNA Ends) and the amplified fragments were sequenced. RT-PCR and Real-time PCR were used to detect the ode2 mRNA in the ovaries among these different ploidy level cyprinid fish.â‘ The partial sequences cloning of cdc2 cDNA.For these four different ploidy level cyprinid fish, the encoded amino acid sequences exhibited highly sequences homology (89.0-99.5%) with those reported for fish cdc2 (including goldfish, zebrafish, rainbow trout, Japanese rainbow trout, and anabas testudineus). Further more, the sequences homology in cyprinid fish (goldfish, zebrafish) was higher than that in the distant genetic relatives (rainbow trout, Japanese rainbow trout, and anabas testudineus). â‘¡Comparative analyses of cdc2 transcriptional level in the ovaries of different ploidy level (allotetraploid hybrids, allotriploid crucian carp and red crucian carp)The results of RT-PCR and Real-time PCR indicated that: cdc2 was expressed in all the ovaries of these three cyprinid fish. The relative quantities of cdc2 mRNA in red crucian carp, allotriploid crucian carp and allotetraploid hybrids were 10⁰, 10^0.87 and 10^1.11, respectively. Thus, we speculated that the transcriptional level of cdc2 mRNA was up-regulated as the ploidy increased, which may related to the function of p34^cdc2 condensing the chromosomes at early M-phase.â‘¢Comparative analyses of cdc2 transcriptional level in the ovaries between the diploid gynogens (G₃) and red crucian carpThe results of RT-PCR and Real-time PCR indicated that: the transcriptional level of cdc2 gene in the ovaries of G₃ is higher than that in the red crucian carp. These results provided some evidences to further study the molecular mechanism of endoreduplication.