MOLECULAR CLONING, EXPRESSION ANALYSIS AND SUBCELLULAR LOCALIZATION OF TUMOR NECROSIS FACTOR RECEPTOR-ASSOCIATED FACTOR 6 IN KOREAN LAMPREY, LETHENTERON MORII

In order to investigate the role of TRAF6 in Lethenteron morii with signal pathway of TLR, the full-length cDNA of LmTRAF6 was cloned. The distribution of tissues (supraneural body, gill, intestine, kidney) and related gene expression in both juvenile and adult were studied with real-time quantitative PCR. Additionally, the change of gene expression in those tissues from adult L. morii was also examined after challenged by Pseudomonas aeruginosa. A recombinant plasmid pEGFP-TRAF6 was constructed by the double digestion method, and transformed into the HEK293T cells. Fluorescence observation was conducted after 48 hours, and the results were photographed. The results showed that the length of LmTRAF6 cDNA was 2751 bp, including 1785 bp open reading frame (ORF) and 594 amino acids. The structure of LmTRAF6 was highly similar to that in other mammal and fish species, containing one RING domain, one coiled-coil region, one MATH domain and two zinc fingers. Phylogenetic tree showed that LmTRAF6 was evolutionarily closer to that in mammals and fishes, but not to that in Drosophila melanogaster and Penaeus chinensis. LmTRAF6 was expressed in all tested tissues and their developmental stages. However, higher expression was detected in heart, skin, gill, liver of juvenile, as well as in kidney, gill, and muscle of adult. When adult L. morii was challenged by Pseudomonas aeruginosa, the expression of LmTRAF6 in gill, intestine and kidney reached to the peak after 24h. Subcellular localization showed that LmTRAF6 was expressed in both cytoplasm and nucleus.