草蝦Kruppel-Like Factor蛋白質的功能性分析

Abstract

Sp1-like proteins and Kruppel-like factors (KLFs) are zinc-finger proteins and important to transcriptional regulation. One expressed sequence tag (EST, HPA-N-S01-EST0038) from shrimps is homologous to Sp1. In this study, a KLF from Penaeus monodon was cloned and characterized by 5’RACE. The full-length PmKLF encodes a polypeptide of 498 amino acids. Sequence analysis showed that PmKLF has a sequence that of similar to KLF11 in human, mouse and zebrafish. A phylogenetic analysis indicates that the zinc-finger DNA binding domain of PmKLF has high degree of sequences identities with KLF in other species. RT-PCR analysis indicates that PmKLF mRNA is expressed in midguts, muscles, hearts, nerves, gills, hepatopancreas and stomaches of shrimps. The antibody against PmKLF was raised using E. coli recombinant protein as an antigen. Immunoblotting using the antibody reveals that the molecular mass of PmKLF protein is about 60 kDa. Immunofluorescence analysis revealed that PmKLF is located in the nucleus as dots after overexpressing in a Spodoptera frugiperda cell line, Sf9. Moreover, analysis of PmKLF deletion mutants indicates that the zinc finger domain of PmKLF contains a nuclear localizaton signal (NLS). Localization of PmKLF in the nucleus is detected in the hemocytes from white spot syndrome virus (WSSV)-infected and WSSV-uninfected Litopenaeus vannamei. Moreover, it is well known that the immediate-early gene ie1 of WSSV displays high transcription activity in Sf9 cells. Cotransfection of PmKLF deletion mutants and the ie1 reporter plasmid into Sf9 cells suggested that the region between 166 and 381 amino acid residues of PmKLF might affect the ie1 transcription. Furthermore, inhibiting the expression of PmKLF by siRNA in WSSV-challenged shrimps prolongs the survival time. Taken together, this study identifies the first Kruppel-like factor in shrimps and reveals the importance of PmKLF to WSSV infection.