Characterization and expression analysis of TNFR-associated factor 1 (TRAF1) in grass carp Ctenopharyngodon idella.
TNF receptor associated factor 1 (TRAF1) plays an important role in regulating the TNF signaling and protecting cells from apoptosis. In the present study, a TRAF1 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA is 2235bp, including a 250bp 5' UTR (untranslated region), a 1659bp open reading frame, and a 326bp 3' UTR. The polyadenylation signal (AATAAA, AATAA) and one mRNA instability motif (AUUUA) were found followed by a poly (A) tail in the 3' UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF1 (gcTRAF1). The putative amino acids of gcTRAF1 share 72% identity with the homologue in zebrafish. It is characterized by a zinc finger at the N-terminus and a TRAF domain (contains one TRAF-C and one TRAF-N) at the C-terminus. The identity of the TRAF domain among all the TRAF1 homologues in vertebrates varies from 52% to 58%, while the identities of TRAF-C were almost the same as 70%. The recombinant gcTRAF1 has been constructed successfully and expressed in Escherichia coli by using pET-32a expression vector. The polyclonal antibody for rabbit has been successfully obtained. The expression of gcTRAF1 in different organs was examined by real-time quantitative PCR and Western blotting, respectively. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of TRAF1 homologue molecule found in fish.
Xu
Z Y
ZY
State Key Laboratory of Freshwater Ecology and Biotechnology, and Laboratory of Fish Diseases, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, People's Republic of China.
Sun
B J
BJ
Chang
M X
MX
Nie
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique / Q000662:veterinary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D047990:TNF Receptor-Associated Factor 1 / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D011994:Recombinant Proteins; D047990:TNF Receptor-Associated Factor 1