| Heat shock responsive genes play important roles in initiating heat shock response when Organisms are exposed to thermal and hypoxia.In this study,five full length cDNA of heat shock and immune-related genes were cloned using degenerate primer PCR amplification,transcriptomic database searching and SMART-RACE technology from Haliotis diversicolor:HSF1(heat shock transcriptional factor 1),HSBP1(heat shock factor binding protein 1),HSP90(heat shock protein 90),AIF-1(allograft inflammatory factor 1),and PAP(Purple acid phosphatase).They were named as HdHSF1,HdHSBP1,HdHSP90,HdAIF-1,and HdPAP respectively.Using Real-time PCR to investigate and analyse their mRNA expression level under thermal and hypoxia.After HdHSF1 was inhibited using RNAi technology from cultured hemocytes,we detected mRNA expression level of other heat shock related genes.The results were as follows:(1)The whole cDNA of HdHSF1 was 2134 bp encoding 515 amino acid residues.qRT-PCR indicates HdHSF1 were expressed at the highest level in gills and at the lowest level in hemocytes.HdHSF1 was up-regulated significantly(p < 0.05)after thermal stress of 96 h and192 h in gills,and also up-regulated significantly in hemocytes at 28℃ 0 h,31℃ 0 h,and 31℃192 h(p < 0.05).Meanwhile,HdHSF1 was up-regulated significantly(p < 0.05)at 4 h,24 h and96 h under hypoxia stress in gills and was not up-regulated significantly(p < 0.05)until 96 h under hypoxia stress in hemocytes.When exposed to thermal plus hypoxia,the expression level of HdHSF1 in gill was higher than that of control group after 4 h,24 h and 96 h.While in hemocytes,HdHSF1 expression level was higher than the control group at 0 h.Furthermore,After HdHSF1 knockdown using RNAi technology,the expression level of HdHSF1 in experimental group was lower than those of blank control group and EGFP group in cultured hemocytes.(2)HdHSBP1 full-length cDNA sequence is 809 bp,encoding a protein of 75 aa.HdHSBP1 had the highest expression level in hemocytes.The expression level of HdHSBP1 was down-regulated significantly(p < 0.05)in hemocytes from the beginning of thermal stress.In gills,HdHSBP1 expression level was down-regulated significantly(p < 0.05)when abalones were exposure to thermal stress,then rose to the control level at 24 h.Both at 96 h and 192 h,HdHSBP1 mRNA expression levels were significantly lower(p < 0.05)than those of the control group.Furthermore,the expression level of HdHSBP1 mRNA was significantly reduced in the thermal stress group at all the time phases in hemocytes.The same was true for thermal stress,HdHSBP1 expression level of the hypoxia group was significantly lower than that of control group from 24 h to 192 h in gills(p < 0.05).Under the stress of thermal plus hypoxia,expression level of HdHSBP1 in gill was higher than the control group significantly after 4 h exposure.Meanwhile,expression level of at 0 h was lower than the control group and higher than the control group at 4 h in hemocytes.When expression of HdHSF1 was inhibited,the expression level of HdHSBP1 of experimental group was lower than the blank control group and EGFP group in cultured hemocytes.(3)The full-length cDNA sequence of HdHSP90 is 2592 bp,encoding a protein of 728 aa.HdHSP90 was detected at a higher expression level both in the digestive tract and the colleterial gland.After thermal stress,the HdHSP90 mRNA expression level was up-regulated significantly at 28℃ 0 h,31℃ 0 h,and 31℃ 192 h in gills and at 31℃ 0 h,and 31℃ 4 h in hemocytes(p< 0.05).HdHSP90 expression was up-regulated significantly at 4 h,24 h,and 96 h post hypoxia in the gills and at 192 h in hemocytes.Under thermal plus hypoxia stress,the expression level of HdHSP90 in gill was higher than the control group at 4 h significantly,while there was no difference in hemocytes.When the HdHSF1 was inhibited,the expression level of HdHSP90 of experimental group was lower than the blank control group and EGFP group.(4)The full cDNA length of HdAIF-1 was 942 bp,and the ORF encoded a sequence of 151 aa.The expression of HdAIF-1 was detected in 7 tissues,with the highest in hemocytes and followed by gill and hepatopancreas,while the digestive tract was the lowest.Under thermal stress,the expression level of HdAIF-1 was increased significantly,with the highest level when temperature rised to 31 ℃.However,the expression level of HdAIF-1 in hemocytes and hepatopancreas did not show significant difference between thermal group and control group from the first phase to the fourth,and its expression level was up-regulated significantly at 96 h in these two tissues.Under hypoxia stress,the expression level of HdAIF-1 in hemocytes was no significant difference between control and exposed groups.However,it was down-regulated at24 h and up-regulated in 192 h significantly in gill.(5)The full length cDNA sequence of PAP is 1215 bp,encoding a protein of 322 aa.Real-timePCR indicated that HdPAP was detected in all examined tissues,with the highest level in hemocytes and hepatopancreas.The expression of HdPAP was suppressed in different degrees in gill,hemolymph and hepatopancreas by thermal stress.The expression level of HdPAP in hemolymph of the hypoxic group was significantly lower than that of the control group at 4 h exposure,then rose to the same level of the control group at 24 h and to the level that was significantly higher than the control group at 96 h,and dropped to the same level of the control group at 192 h.Seven other heat shock-related genes from Haliotis diversicolor EST were obtained.they were HSP22(heat shock protein 22),HSP26(heat shock protein 26),HSP40(heat shock protein40),HSP60(heat shock protein 60),HSP70(heat shock protein 70),HSP105(heat shock protein105)and Sip1(Stress induced-phosphoprotein 1).We named them as HdHSP22,HdHSP26,HdHSP40,HdHSP60,HdHSP70,HdHSP105 and HdSip1.Under the condition of thermal,hypoxia and thermal plus hypoxia stress,expression level of these genes were up-regulated significantly.When HdHSF1 was inhibited,these genes were suppressed at different degrees in culture hemocytes. |
PDF Full Text Request