Cloning And Expression Of Wnt Signaling Pathway Related Genes And The Preliminary Analysis Of Mechanism To Cadmium Stress In Red Crucian Carp(Carassius Auratus Red Var.)

Wnt signaling plays an important role in organogenesis,cancer,and multicellular biological axis differentiation.In this paper,we mainly study the Gsk3b and Dvl2 genes in red crucian carp.We first obtained the full-length c DNA of Gsk3b and Dvl2 genes of red crucian carp by RACE(Rapid-amplification of c DNA ends)cloning technique,RT-PCR and Western Blot respectively,At the same time,the expression of Gsk3 and b-catenin was compared and analyzed with the different concentration of cadmium.The results showed that:(1)The full length of Gsk3b gene was 2195 bp,encoding 421 amino acids,the predicted molecular weight was about 46846.40,and the isoelectric point was 9.05.Amino acid homology,the similarity between the protein sequence and zebrafish was 98.5%,94.2% with that of mice,92.1% with Xenopus,and 94.7% for human.It can be seen that the similarity ratio is higher,indicating that the evolution of the red crucian carp Gsk3b gene has a high conservatism.(2)The results showed that the full length of Dvl2 gene was 3042 bp,encoding 754 amino acids,the predicted molecular weight was about82360.64 and the isoelectric point was 5.89.The homology similarity between the protein sequence and zebrafish was 95.3%,97.2% for carp,68.8% for mice,70.5% for Xenopus and 67.3% for humans.It is revealed that the Dvl2 gene of red crucian carp is not conserved in evolution.(3)After semi-quantitative RT-PCR analysis,it was proved that the expression of Dvl2 gene was the highest in the testes of red crucian carp,and the expression of Dvl2 was the highest in the brain,muscle,skin and ovary The expression level in the tissues is relatively high,and the expression in the liver and kidneys is relatively low.The expression of Gsk3b gene was the highest in the tissues of red crucian carp,and the expression level of brain and muscle was relatively high in the two tissues of testis and ovary,and the expression in liver,kidney and skin was relatively low.The expression of Dvl2 gene was the strongest in the somatosensory period,and the expression of Dvl2 gene was the highest during the period of muscle function.The expression of Dvl2 gene was higher in the period of muscle function,Time is low,the two cells did not express the basic period.The expression of Gsk3b was the strongest in the period of muscle growth,followed by the expression of blastocyst stage,blastocyst stage and somatic period,and the expression of Gsk3bwas lower in the period of pigmentation and body pigment.\(4)The embryonic development period of 8 and 8 different tissue proteins in red crucian carp were extracted.Semi-quantitative Western Blot technique was used to analyze the expression of Dvl2 and Gsk3bprotein in red crucian carp and showed some histological differences.Dvl2 and Gsk3 b were the lowest in the kidney of red crucian carp,followed by the liver.Gsk3 b protein in the brain expression of the highest,followed by muscle,in the kidney,liver and testicular basically no expression.And Dvl2 in the testis expression of the highest,followed by the eyes,the lowest expression in the kidney Dvl2 and Gsk3b were differentially expressed in different periods,both of which were the lowest in the two-cell period and the strongest in the period of membrane formation.The expression of Gsk3b was higher than that of Gsk3b,and the expression of Gsk3b was higher than that of Dvl2.The expression of Gsk3bwas similar in the blastocyst stage,somatic period and body chromatin stage.(5)By using thediploid red carp and riploid red carp that all have been treated with cadmium,testing the Gsk3 beta expression and beta catenin expression in m RNA level,found that the expression of Gsk3 was decreased and the expression of beta catenin was up-regulated.It can be seen that Gsk3 beta may exert specific biological function under Cadmium Stress.But the Gsk3 beta involved in two main signaling pathways,PI3K/AKT signaling path way and WNT/beta-catenin signaling pathway,so the participation of the specific function and mechanism still needs further research.These results provide an important basis for studying the regulation mechanism of Dvl2 and Gsk3 beta genes in Wnt signaling pathway and the molecular mechanism of cadmium stress response in lower vertebrates.