Study On Cloning And Differential Expression Of Gene Dishevelled 3 In Goldfish

Dishevelled(DVL/DSH) protein was first discovered in Drosophila. Presently,three subtypes Dvl1, Dvl2 and Dvl3 have been found in the vertebrate genome,which encode protein DVL1, DVL2 and DVL3 respectively. All three isoforms have very conservative DIX, DEP and PDZ motif but with varied length. In Drosophila, scattered proteins(DVL/DSH) play important role in the homeostasis of intracellular β-catenin and the transcriptional regulation of certain genes involved in Wnt/ β-catenin signaling pathway. Recent progress on the study of Wnt signaling pathway suggested that, the DVL/DSH protein as a scaffolding protein coupling receptors to their downstream effectors, is essential for Wnt signaling pathway.Here, our study focused on gene Dvl3 in Carassius auratus. Firstly, we used RACE(rapid-amplification of cDNA ends) clone technology to obtain full-length cDNA of gene Dvl3, and then semi-quantitative RT-PCR and western blot were used to analyze the differential expression of Dvl3 in mRNA level and protein level respectively. The results showed that:(1) the full length of Dvl3 cDNA in Carassius auratus is 2063 bp. And the CDS region is 1878 bp, which encoded a protein composed of 626 amino acids. Sequence homology analysis indicated that it is61.3% similar to that of human, and 61.8% to Xenopus laevis, 64.5% to mouse,83.8 % to Danio rerio respectively. It can be seen that the homologous similarity ratios were high, indicating that the gene Dvl3 in Carassius auratus had a more conservative evolution.(2) Semi-quantitative RT-PCR results showed that the mRNA levels were different varied in different tissues. Gene Dvl3 had a highest expression level in ovary of Carassius auratus, followed by the brain. Heart, gill and kidney, while the m RNA levels in fins, muscle, and spermary were relatively lower.And the transcriptional level in liver was the lowest. In the 12 stages during embryonic development of Carassius auratus, the greatest mRNA expression level appeared in the heartbeat period, followed by the hatching larvae stage. The two cell stage, multiple cell stage, blastula stage, gastrula stage, neurula stage, optic rudiment stage, brain vesicle stage, muscle differentiation stage, and bodypigmentation stage all had higher expression levels, while the one in eye pigmentation stage was lowest. According to these results, we speculated that the gene Dvl3 had the existence of multiple functional roles in different tissues and embryonic development period in Carassius auratus.(3) We extracted the proteins from seven stages during embryonic development and seven different tissues in Carassius auratus. By semi-quantitative western blot analysis, we found that the highest expression level of DVL proteins in seven embryonic development stages in Carassius auratus was the blastula stage, and optic rudiment stage followed.Neurula stage, muscle differentiation stage, eye pigmentation stage and body pigmentation stage also had higher expression levels, while the heartbeat period had the lowest expression level. Ovary showed the highest expression level of protein in whole seven tissues of Carassius auratus, followed by the liver, whereas the expression levels were lower in brain, spermary, and gills, fins, the heart showed no expression. Thus it showed that the function of DVL3 proteins varied during embryonic development stages in Carassius auratus as well as in different tissues.However, the exact mechanism remained to be further explored. The results of our study provided clues for further investigation on specific regulatory mechanisms of gene Dvl3 in Wnt signal transduction pathway in lower vertebrates.