Characterization And Function Analysis Of Three Hsps Gene Under Different Acute Stresses In Black Tiger Shrimp, Penaeus Monodon

Considering the enormous economic losses induced by environmental stressors in the Penaeus monodon industry. However, heat shock proteins(Hsps) are a class of highly conserved proteins that play important roles in resistance to stress. In the present study, in order to understand the mechanisms of stress tolerance and disease resistance of P. monodon better, three Hsps of P. monodon(i.e. Pm Hsp60、Pm Hsp10 and Pm GRP94) were cloned and analysed.Partial sequences of Pm Hsp60, Pm Hsp10 and Pm GRP94 were obtained from the splicing transcriptome of P. monodon established in our laboratory. The full-length c DNA of three Pm Hsps were amplified through the rapid amplification of c DNA ends(RACE) approach. The tissue distributions of three Pm Hsps transcripts were analysed by quantitative real-time PCR(q RT-PCR). The m RNA expression profiles of Pm Hsp60, Pm Hsp10 and Pm GRP94 were detected in the gills and hepatopancreas of the shrimps under p H challenge, osmotic stress, and heavy metal exposure. In addition, recombinant proteins of Pm Hsps have been obtained through procaryotic expression system. The recombinant Pm Hsp60 and Pm Hsp10 proteins were purified through Ni-NTA affinity chromatography on the resin with the chelated nickel ions. The chaperone activity and ATPase activity assay were assayed after refolding. The results of the studies are listed as follows:(1) The 2352 bp full-length c DNA of Pm Hsp60 contained a 5′-untranslated region(UTR) of 129 bp, a 3′-UTR of 486 bp with a poly(A) tail, and an ORF of 1737 bp encoding 578 amino acids. The molecular weight of Pm Hsp60 was 60.99 k Da, and its theoretical isoelectric point(p I) was 5.48. Multiple sequence alignments indicated that Pm Hsp60 has a highly conserved cpn60-domain, a ring oligomerization interfaces and ATP/Mg binding sites. Tissue distribution of Pm Hsp60 shown that The highest expression of the Pm Hsp60 transcript was detected in the hepatopancreas and the lowest expression was noted in the gills. The Pm Hsp10 full-length c DNA was 744 bp, including a 309 bp ORF, a 102 bp 5′-UTR, and a 333 bp 3′-UTR. The ORF encoded a protein composed of 102 amino acids with a predicted molecular mass of 11.05 k Da and a theoretical p I of 7.8. Tissue distribution of Pm Hsp10 shown that the highest expression of the Pm Hsp60 transcript was detected in the hepatopancreas and the lowest expression was noted in the gills and lymph. The Pm GRP94 full-length c DNA was 2990 bp, including a 2388 bp ORF, a 75 bp 5′-UTR, and a 527 bp 3′-UTR. The ORF encoded a protein composed of 795 amino acids with a predicted molecular mass of 91.32 k Da and a theoretical p I of 4.72. Tissue distribution of Pm Pm GRP94 shown that the highest expression of the Pm Hsp60 transcript was detected in the hepatopancreas and the lowest expression was noted in the lymph.(2) In the experiments of environmental stress, the expression profiles of Pm Hsp60, Pm Hsp10 and Pm GRP94 were detected in the shrimps under p H challenge(p H7.0 and 9.0), osmotic stress(23‰ and 43‰), and heavy metal exposure(Cu, Zn and Cd).The expression level of Pm Hsp60 m RNA was up-regulated and reached peak values in gills 96 h after p H 7.0 exposure. Pm Hsp10 was induced at 6 h, and maximum m RNA expression levels were found at 6, 48 and 96 h after p H 7.0 challenge. In the hepatopancreas, the expression level of Pm Hsp60 m RNA was up-regulated at 12 and 48 h. No obvious variation in the expressions of Pm Hsp10 was observed after p H 7.0 challenge. The Pm Hsp60 and Pm Hsp10 genes were induced immediately by p H 9.0 stress. The relative expressions of Pm Hsp60 and Pm Hsp10 transcripts were rapidly up-regulated within 6 h after p H 9.0 exposure and declined at 12 h. Moreover, maximum expression levels were detected at 48 h in gills. In the hepatopancreas, Pm Hsp60 and Pm Hsp10 transcription levels were up-regulated compared with that of the control within 12 h under p H 9.0 stress and subsequently declined to the same levels at 24(Pm Hsp60) and 48 h(Pm Hsp10); peak values were observed at 96 h. In the salinity stress experiment, Pm Hsp10 m RNA expression reached maximum levels after 4 h of low-salinity exposure and declined after 8 h in the gills. In the hepatopancreas, Pm Hsp10 was induced at 4 h and reached maximum expression levels at 4, 8 and 32 h of low salinity stress. The Pm Hsp60 transcript was only slightly up-regulated under low salinity stress in both tissues. The Pm Hsp60 transcript level increased to maximum levelsat 32 h in the gills under high salinity. Moreover, maximum Pm Hsp10 expression level was 3.2-fold higher than that of the control at 16 h. In gills, the Pm Hsp60 transcript level significantly increased at 12 h and decreased 24 and 48 h after the shrimps were exposed to copper stress. This parameter reached maximum levels at 96 h. Pm Hsp10 expression level was slightly induced before 48 h and reached maximum levels at 96 h in the gills of shrimps under copper stress. In the hepatopancreas, Pm Hsps expression were maintained at levels similar to that of the control. Similar tendencies of the Pm Hsp transcripts in the gills and hepatopancreas were observed during zinc stress exposure. In gills, the maximal enhancement of Pm Hsp60 and Pm Hsp10 expression levels was reached at 12 h, In the hepatopancreas, up-regulation of Pm Hsp60 and Pm Hsp10 transcripts was detected at the beginning of zinc exposure. Maximum Pm Hsp60 and Pm Hsp10 m RNA expression levels were observed at 24 h. In the cadmium exposure experiment, the Pm Hsp60 transcript significantly increased at 24 h in the gills and reached maximum expression thereafter. Furthermore, the maximum m RNA expression level of Pm Hsp10 were observed at 12 h in the gills. In the hepatopancreas, the two Pm Hsp genes were induced slightly, and the highest expression levels of Pm Hsp60 and Pm Hsp10 were detected at 6 and 48 h, respectively. No obvious variation in the expressions of Pm GRP94 was observed before 12 h under p H 7.0 challenge. The maximum Pm GRP94 m RNA expression levels were observed at 24 h and decreased 48 and 96 h under p H 7.0 challenge. Pm GRP94 was induced at 6 h and reached the peak at 48 h, then decreased to a low level that also higer than contrl group at 96 h after p H 9.0 challenge. In the salinity stress experiment, no obvious variation in the expressions of Pm GRP94 was observed before 16 h and decreased the level lower than the control at 16 h and 32 h under low salinity stress. The Pm GRP94 transcript level increased to maximum levelsat 16 h and decreased at 32 h under high salinity. In the copper exposure, the maximum of Pm GRP94 expression was detected at 6 h then decreased after 12 h. The Pm GRP94 transcript was only slightly upregulated from 6 h to 96 h and upregulated to the peak at 96 h under cadmium exposure. Pm GRP94 was induced at 6 h and reached the peak at 24 h, then deareased to the control level under zinc exposure.(3) Pm Hsp60 revealed ATPase activity from 0 °C to 60 °C, and maximum activity was found at 37 °C. Pm Hsp60 ATPase activity increased at all temperature conditions when Pm Hsp10 was added to the reaction system. The maximum increase in activity was found over the temperature range of 37–42 °C, and the lowest increase in activity was detected at 60 °C. Heat-induced citrate synthase aggregation assay was performed to determine the chaperone activity of Pm Hsp60. At a 1:1 Hsp60/CS ratio(w/w), Hsp60 offered about 21% protection. The extent of protection increased as the Hsp60 concentration increased. Approximately 57% protection was observed at a 2:1 Hsp60/CS ratio. At a 4:1 ratio, over 60% protection was observed. Only approximately 38% protection was observed when the Hsp10/CS ratio was 4:1. However, over 76% protection was observed at a 4:4:1 Hsp60/Hsp10/CS ratio.