Smad Nuclear Interacting Protein, SNIP1, Mediates The Ecdysteroid Signal Transduction In Red Crayfish Procambarus Clarkia

Ecdysteroids has a critical role in the regulation of crustacean's sex differentiation, growth development and reproduction. However, the molecular mechanism is not clear.In research of ecdysone signaling pathway is significant to elucidate crustacean molting mechanism and to promote the development of shrimp aquaculture. Smad nuclear interacting protein(SNIP1) is a protein that interacts with Smad and contains a number of zinc finger proteins.SNIP1 has a specific DNA binding activity, and is involved in the regulation of multiple signaling pathways. Procambarus clarkii is also known as the red crayfish and freshwater crayfish. Procambarus clarkii has become an important aquaculture species for its delicious meat. The purpose of this study was to investigate the function SNIP1 in crayfish ecdysone signaling pathway and the main results are as follows:1? In this study, we cloned the ORF of SNIP1 in Procambarus clarkii by PCR method. The ORF of PC-SNIP1 was 867 bp and this gene encoded a polypeptide of 288 amino acids. Theoretical isoelectric point and molecular mass for the protein are 6.18 and 34.9 k Da, respectively. P.clarkii SNIP1 shared a highly conserved FHA domain of SNIP proteins at the C-terminus and an N-terminal nuclear localization signal(NLS). Phylogenic analysis showed that P.clarkii SNIP1 was highly homologous to that of invertebrates.2?The ORF of Pc-SNIP1 was ligated to p ET-28 a vector for protein expression. SDS–PAGE and western blot analysis demonstrated that a 35 KDa recombinant protein was successfully expressed in Escherichia coli cells. The induced cells were harvested and subjected to nickel affinity column chromatography to purify recombinant proteins. In order to obtain polyclonal antibody, the purified recombinant protein was used to immunize a male New Zealand rabbit, the titer(about1:9000) of the obtained antibody was measuring by ELISA.3?Proteins and total RNAs from muscle, heart, stomach, gut, gill and hepatopancreas were were extracted and analyzed by Western blotting and q RT-PCR. The result showed the expression level of Pc-SNIP1 was higher in heart.4?The expression of Pc-SNIP1 was significantly up-regulated in hepatopancreas after 20-hydroxyecdysone induction. Knockdown of Pc-SNIP1 gene by small interfering RNA(si RNA) transfection had significant influence on the expression of some ecdysteroid-responsive genes in hepatopancreas, which were confirmed by q RT-PCR and western blot.All together, these results suggest that Pc-SNIP1 is an ecdysteroid-responsive gene and is involved in the physiological process regulated by ecdysteroid in P. clarkii.