title: Cloning and expression analysis of a heat shock protein 90 β isoform gene from the gills of Wuchang bream (Megalobrama amblycephala Yih) subjected to nitrite stress.
abstract: Heat shock protein 90 (HSP90), a highly conserved and multi-functional molecular chaperone, plays an essential role in cellular metabolism and stress response. In this study, HSP90 cDNA named MaHSP90 was cloned from Wuchang bream (Megalobrama amblycephala) gills by using rapid amplification of cDNA ends. The full-length MaHSP90 cDNA is 2674 bp and consists of a 3',5'-untranslated region and a 2250-bp open reading frame encoding a 750-amino acid long protein. Identity analysis revealed that the amino acid sequence of MaHSP90 is highly conserved. Homology analysis and structure comparison further indicated that MaHSP90 should be the β isoform member of the HSP90 family. MaHSP90 mRNA was ubiquitously expressed in the liver, heart, muscle, gill, intestine, kidney, and brain. The MaHSP90 mRNA levels under nitrite stress were analyzed using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR); the mRNA levels significantly increased at 3, 6, and 12 h after nitrite exposure in the gills and then stabilized between 24 and 48 h. Furthermore, a similar relationship between mRNA expression (qRT-PCR) and HSP90 protein levels (densitometric band analysis) was found. Transcriptional analysis of caspase-8 and caspase-9 expression in the gills of juvenile M. amblycephala after a 48-h exposure to nitrite suggested that MaHSP90 expression is related positively with nitrite-induced apoptosis. Fish exposed to nitrite also showed gill damage. Our results suggest that MaHSP90 mRNA is constitutively expressed in various tissues and inducible in the gills under nitrite stress, suggesting its important role in nitrite stress response.
authors.affiliation: Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China.
authors.forename: S M
authors.initials: SM
authors.lastname: Sun
authors.affiliation: Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China zhuj@ffrc.cn.
authors.forename: J
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China.
authors.forename: X P
authors.initials: XP
authors.lastname: Ge
authors.affiliation: Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China.
authors.forename: C F
authors.initials: CF
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China.
authors.forename: L H
authors.initials: LH
authors.lastname: Miao
authors.affiliation: Wuxi Fishery College Nanjing Agricultural University, Wuxi, China.
authors.forename: X J
authors.initials: XJ
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D003001:Cloning, Molecular; D003530:Cyprinidae / Q000378:metabolism*; D029941:Fish Proteins / Q000096:biosynthesis / Q000235:genetics*; D005880:Gills / Q000187:drug effects / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009573:Nitrites / Q000633:toxicity; D020033:Protein Isoforms; D013312:Stress, Physiological / Q000235:genetics
chemical_list: D029941:Fish Proteins; D018841:HSP90 Heat-Shock Proteins; D009573:Nitrites; D020033:Protein Isoforms; D053181:Caspase 8; D053453:Caspase 9