Cyclin C Is Expressed Both Maternally And Zygotically During Zebrafish Embryo Development

Objective: We try to elucidate the probable mechanism of midblastula transition (MBT) in zebrafish through study the cyclin C gene which is expressed maternally and zygotically during zebrafish embryo development.Methods: (1) We got enough embryos for our experiments through fertilize in vitro. (2) Total RNA was extracted using TRIzol Reagent for the following experiments. (3) The complete cyclinc C gene of zebrafish was firstly cloned by us through RT-PCR, 3'/5' RACE, and also the sequences were analysed and compared with sequences of other species. (4) The probe which was labeled by DIG was prepared for Northern blot and in situ hybridization. (5 ) siRNA system was built using Dct gene as target gene, and we can use the system to study the function of cyclinC gene.Result: Initial cyclin C cDNA fragments were identified by RT-PCRusing a pair of primers which was designed by analyzing the homolog of cyclin C gene reported from other species, generating a 513 base pair (bp) DNA fragment. The remaining cDNA sequence of zebrafish cyclin C was cloned by the 3'/5' rapid amplification of cDNA ends (RACE). At last we got full-length cDNA sequence of zebrafish cyclin C that is 2950 bp in length. We confirmed the final ORF sequences and revealed some single nucleotide polymorphism (SNP) and a frame shifted error in published EST sequences. The 5'untranslated sequence around the putative initiation condon for ORF follows the Kozak's rule, indicationg that this initiation condon is capable of being efficiently used for translation. The 5'-noncoding sequence contains two in-frame termination codons at the nucleic acid number -66 and -45, respectively, indicating that this cDNA covers the entire coding region. The 1993-bp 3'untranslated region range from termination signal (TAG) to poly A tail contains one polyadenylation signal (AATAAA). The open reading frame potentially encodes a 283 amino acid protein with an estimated molecular weight of 33.1kD and predicted. Compared with known sequences of other species, it was found it was conservative gene. And it is expressed not only before midblastula transition (MBT) but also after midblastula transition (MBT), that is to say, it is expressed maternally and zygotically during zebrafish embryo development through in suit hybridization. After injection the siRNA of Dct gene into zebrafish embryos, it was found that the fuctionof Dct gene was inhibited and the phenotypes of embryo was changed. So we can say that siRNA is one of practical ways to study the function of gene. We can use it to further study the functions of cyclinC gene.Conclusion: Cyclin C is high conservative in the process of evolution. It was expressed both maternally and zygotically and much work was needed to do for elucidating its role during zebrafish embryo development. SiRNA is one of practical ways to study the function of gene.