Molecular Cloning Of Kuruma Shrimp Marsupenaeus Japonicus Minichromosome Maintenance Protein 7 (MCM7) And The Exploration Of Its Regulation On Hemolymph Phagocytose

Minichromosome maintenance protein (MCM7) belongs to the MCM proteins, participating in the complex of MCMs as a licensing of cell replication cycle and the initiation factor of chromosome replication, is highly conserved in eukaryotes. Using RACE (Rapid Amplification of cDNA Ends) technology we cloned a full length cDNA sequence of 2307bp from the Kuruma shrimp, Marsupenaeus japonicus. Analyze showed the ORF of MCM7 is 1974bp, encoding a 658aa protein including a Poly(A) tail, UTR of 5’end and 3’end are 139bp and 191bp, respectively. Protein sequence BLAST showed a homologue over 75% and Neighbor-joining method of phylogenetic tree analyze indicated the high genomic conservation in vertebrates, sharing a closer evolutional relationship with fish and clawed toad.Tissue expression differ detection discovered the expression mainly took place in immunity organs as hemolymph and hepetopancraes, little higher in digestive gland and much lower in heart, muscle, gill, or gut. Further research discovered the over expression after white spot syndrome virus (WSSV) infection. The siRNA (short interfering RNA) was designed and synthesized in vitro for injection and treatment of the shrimps, successfully knocked down the expression level of MCM7. Real-time Q-PCR analyze showed that the expressions of multiple innate immunity related genes were affected, among them the expression of hemocyanin, proPO, TNF-a and energy transduction signal gene β-actin were up load, meanwhile the expression of cytoskeletal regulation proteins like myosin and Rho were decreased, indicating the expression of MCM7 can affect the hemolynph phagocyte. After inhibiting the expression of MCM7 through RNA interfering technology, the hemolymph phagocyte rate was found lowered, indicating a regulating function of MCM7. Meanwhile after the expression inhibition the copy number of WSSV was increased, revealing that the MCM7 regulated hemolymph phagocyte might be positive for control the replication of virus.This study successfully cloned the MCM7 in crusteans for the first time and revealed the role of MCM7 in hemolymph phagocyte progress, providing a novel clue for further research on innate immunity system of crusteans.