| In this study,the full-length c DNA of HSP70/HSC70 and ?6FAD/?9FAD in Trachinotus ovatus were cloned using homological cloning and rapid amplification of c DNA ends(RACE)techniques,and the sequences characterizations were also analyzed by bioinformatic methonds.Results showed that the full-length of HSP70 c DNA was 2196 bp,containing a 3?untranslated region(3?-UTR)of 276 bp,and an open reading frame of 1920 bp encoding 639 amino acids with an estimated molecular weight of 70 k Da and an estimated isoelectric point of 5.42;The full-length of HSC70 c DNA was 2298 bp,consisting of a 5?untranslated region(5?-UTR)of 101 bp,a 244bp3?-UTR and an open reading frame of 1953 bp encoding 650 amino acids with an estimated molecular weight of 71 k Da and an estimated isoelectric point of 5.18;The full-length of?6FAD c DNA was 1908 bp,consisting of a138 bp 5?-UTR,a 441bp3?-UTR and an open reading frame of 1329 bp encoding 442 amino acids with an estimated molecular weight of 52 k Da and an estimated isoelectric point of 8.88;The full-length of?9FAD c DNA was 2496 bp,consisting of a 94 bp 5?-UTR,a 1329bp3?-UTR and an open reading frame of 1002 bp encoding 334 amino acids with an estimated molecular weight of 38 k Da and an estimated isoelectric point of 9.13.Bioinformatics analysis showed that HSP70/HSC70 were Hydrophobic proteins which contained three signature sequences(IDLGTTYS,IFDLGGGTFDVSIL,IVLVG GSTRIPKIQK)and Eukaryotic cytoplasmic HSP70 feature motif EEVD.?6FAD/?9FAD also belong to Hydrophobic proteins,existing three conserved histidine cluster motifs of fatty acid desaturase family in the amino acid sequence,However,the three histidine-rich region are distinct between different FAD protein sequence.According to the homology and phylogenetic analysis,the deduced amino acid sequences of HSP70/HSC70 in Trachinotus ovatus were highly homologous with those from Tilapia(Oreochromis mossambicus)and Barramundi(Lates calcarifer),and ?6FAD/?9FAD of Trachinotus ovatus shared closet relationship with thecorresponding proteins of Barramundi(Lates calcarifer)and Gilthead seabream(Sparus aurata)respectively.The m RNA expression levels of HSP70/HSC70 and ?6FAD/?9FAD in different tissues of healthy Tachinotus ovatus were analyzed by fluorescent quantitative real-time PCR technology in this study,and ?-actin gene was served as the control gene.The results showed that the HSP70 expression was highest in muscle with significant differences compared with that in other tissues(p<0.05),and followed by liver,spleen,and skin(p>0.05),and the lowest expression was found in the gut;the HSC70 expression was highest in liver(p<0.05),followed by head kidney and skin,and the lowest expression was identified in the brain;the?6FAD expression was highest in liver with significant differences of other tissues(p<0.05),followed by brain and heat,with significant differences of other tissues(p<0.05),and were lower in others tissues,almost undetectable in stomach and muscle;the expression of ?9FAD also Likewise highest in liver with significant differences of other tissues(p<0.05),Foll owed by the brain and the muscles,and had a low expression level in the spleen and skin,the least expression was in intestine.The differential expression of HSP70/HSC70 and ?6FAD/?9FAD in tissues of fishes under different low temperature stress were analyzed by fluorescent quantitative real-time PCR technology based on the control genes of ?-actin.From the results,the HSP70 expression were undulates change in the range of 28 ?-13 ?when lower the temperature,and reached the peak at 25 ? with significant differences of other different temperature except 22?.At 19 ?,the wavy down to the minimum and slightly less before cooling(p<0.05).The expression of HSC70 increased gradually with decreasing temperature at the range of 28 ?-13 ?,which just slow rise at the beginning and significant increase in the later.And reached the peak at 13 ? with about 11.1 times before cooling(p<0.05).However,the expression level decreased slightly at 19 ?.The ?6 FAD expression was slowly lower at first and increased rapidly at the later with temperature decreasing at the range of 28 ?-13 ?,and reached the peak at 13? with significant differences of other different temperature(p<0.05);At the process of decreasing temperature,the expression of?9FAD as well as HSC70,and reached the peak at 13?with about 45.3 times before cooling(p<0.05).The same as HSC70,the expression level of ?9FAD decreased slightly at 19 ?.Analysis of temporal expression patterns under different low temperature showed that the HSP70 expression after first rise then decreasing overtime at 13? and 16?,respectively.However,change is more evident when 13?,it peaked within 48h(p<0.05),and declined to a minimum after 84h(p>0.05).It peaked within 36 h and declined to a minimum which below the initial level after 84 h at16?.When 19?,there are no significant change in the range of 0-84 h,and only declined to a minimum after 84 h.When 13?,the expression of HSC70 were Undulates change and reached the peak at 84 h.The expression level first increased and then decreased over time,and reached the peak at 36 h,and the down to a stable when at temporal expression patterns under 16?.There is no obvious change at 19?,and only within 24 h,it began to decline after a slight increase,until below the initial level.The temporal expression patterns of ?6 FAD under 13? was significant different from 16? and 19?.Gene expression of ?6FAD is in the rising trend over time at 13? and is in the decreasing over time by feedback adjustment way at16 ?and 19 ?.Under 13?,the ?9 FAD expression reached the maximum within 24 h,then decreased to the initial level(p>0.05).The temporal expression patterns of ?9FAD under16 ? is similar to 13?,however,when 19 ?,it declined rapidly at first then slowly within 12h. |
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