Response Characterization Of Some HSP And Caspase Genes Of Large Yellow Croaker To Temperature Stress

Large yellow croaker, Larimichthys crocea, is one of the most important mari-culturedspecies in China. In recent years, large yellow croaker has suffered from many severalenvironmental factors stress. Among which, temperature was the most important one. However,the impact of temperature on the growth, development and immunity of the animal was still poorunderstood.In this paper, some immune factors in blood were determined in large yellow croaker afterstressed at the temperature of10℃and35℃. Six genes including HSP27, HSP30, HSP47, andHSP90of HSP family and caspase-1and caspase-7of caspase family from large yellow croakerwere obtained by RT-PCR and RACE-PCR. Then, the genomic sequence of them were obtainedand analyzed. In addition, the spatial expression and temporal expression of these genes aftertemperature stress were detected by quantative real-time reverse PCR (qRT-PCR). Recombinantexpressions of several genes were performed in E. coli of BL21(DE3). The results were asfollows:(1) The activities of lysozyme were significantly decreased at6-12h comparing with thecontrol after10℃and35℃stress, and then recovered to the control at24h. The activities ofSOD in blood cells increased significantly (P<0.05). However, there was no significant changein the SOD activity in serum. And the albumin and immune globulin levels were signifciantlyhigher than those of the control group after the low and high temperature stress. All the datasuggested that there was a great impact on the non-specific immunity parameters after cold andheat stress of large yellow croaker.(2) The full length cDNA of HSP27was1227bp with an ORF of612bp coding203aminoacids, and HSP30had an851bp full-length with an ORF of624bp encoding207amino acids.There were the same HSP23_like superfamily domain in HSP27and HSP30. The HSP27of largeyellow croaker genomic sequence had three exons and two introns. Real-time PCR analysisrevealed that HSP27of large yellow croaker expressed highest in heart, next in muscle. Theexpression of HSP27increased significantly in heart and muscle after30℃heat stress. Thensolubility of recombination protein of HSP27was expressed in E. coli successfully, which madecondition for future study of HSP27protein.(3) The full cDNA length of HSP47was2027bp, containing an ORF of1218bp whichencoding405aa. Within the coding sequence, a typical SERPIN domain was found in HSP47of large yellow croaker. The HSP47gene is organized by six exons and five introns. A constituteexpression of HSP47was detected by real-time PCR in various organs of large yellow croaker,with a higher levels in spleen, heart, liver, gill. The expression levels were increased significantlyin liver, heart and muscle after heat stress (25℃,30℃)(P<0.05). The recombinant of largeyellow croaker HSP47was expressed in E. coli, which need further study on the function ofHSP47on protein level.(4) The full-length HSP90was2715bp with a2178bp ORF, encoding725amino acids. Thededuced protein contained a typical structure of the HSP90family and an ATP-binding domain.The large yellow croaker HSP90was organized by10introns and11exons. A basal expressionof HSP90was detected in organs analyzed, especially higher in liver and blood. A remarkableincrease was detected in muscle tissue analyzed after temperature stress except25℃(P<0.05).(5) The full-length caspase-1of large yellow croaker was1780bp with an open readingframe of1167bp encoding a polypeptide of388amino acids, which included a90-aa caspaserecruitment domain (CARD, residues1-90), a P20domain (residues137-265) with a highlyconservative catalytic active site of QACRG, as well as a P10domain (residues299-386).RT-PCR analysis demonstrated that Casapase-1expressed in all immune tissues analyzed and,unexpectedly, cold stress of fish decreased its mRNA levels in liver and heart, however, oppositeresults were found in fish went through hyperthermia stress of25℃and30℃. The recombinantprotein of caspase-1was expressed in E. coli.(6) We cloned a caspase-7-like gene from the bony fish large yellow croaker, containing afull-length of1392bp with a900bp ORF encoding299aa, which showed a conservative P20domain (residues69-185) with a highly conservative catalytic active site of QACRG, as well as aP10domain (residues205-297), however, there was no predomain in caspase-7. The caspase-7gene in large yellow croaker was organized with7exons and6introns. Real-time PCR analysisrevealed that caspase-7expressed in all analyzed tissues, especially high in muscle, head-kidneyand heart. And heat stress could increase expression of caspase-7in heart, while the expressionof caspase-7was decreased in liver after cold stress (P<0.05); anyhow, the significant declinewas discovered after both heat and cold stress (P<0.05).