| Heat shock protein 70(HSP70)is one of the most widely distributed subfamily of heat shock proteins and is found in a variety of prokaryotic and eukaryotic organisms,It’s also the most common molecular chaperone that assists in folding of native proteins,and mediates the degradation or refolding of misfolded or aggregated proteins,and increases expression when an organism is exposed to external stimuli.The expression of these molecular chaperones could be increased when the organism is exposed to external stimuli to improve It’s tolerance to the hostile environment.To date,a total of five HSP70 family genes have been identified in crustaceans,but none is associated with the freshwater crustacean Sinopotamon henanense.On the orther hand,non-lethal heat shock(NLHS)to enhance the ability of crustaceans has been rarely reported,especially in benthic crab species living in the freshwater.They are susceptible to heavy metal contamination and have a high accumulation capacity to extreme heat and water pollution.We obtained part of the hepatopancreas transcriptome data of the crab ShHSP70 by screening the transcriptome data based on the previously obtained hepatopancreas data.To investigate how ShHSP70 improves the tolerance of the organism to environmental stress after NLHS treatment and whether it regulates apoptosis in Sinopotamon henanense tissues,this study was carried out based on the previous research and some of the ShHSP70 genes of Sinopotamon henanense,and focused on the ShHSP70 genes of Sinopotamon henanense.1.Full-length cloning,sequence analysis,and tissue distribution analysis of the ShHSP70 gene in S.henanense.2.Construction of a prokaryotic expression vector for the ShHSP70 gene of the S.henanense and in vitro expression and purification of r ShHSP70 recombinant fusion protein as well as ShHSP70 tissue cell localization.3.To analyze the role of the ShHSP70 gene in S.henanense after NLHS treatment in response to high temperature and cadmium exposure,and to analyze the mode of regulation of apoptosis by ShHSP70 gene after cadmium exposure by constructing RNAi interference and recombinant protein injection model.The results of the study were as follows:1.Full-length cloning,sequence analysis,and tissue distribution analysis of the ShHSP70 gene of Sinopotamon henanense.The full-length ShHSP70 gene encodes 2202 bases,including a 104-base 5’-UTR,a142-base 3’-UTR,and a 1956-base open reading frame,and the translated protein has 651 amino acids.S.henanense ShHSP70 has a typical HSP70 structure,containing three typical HSP70 family tag sequences IDLGTTYS,IFDLGGTFDVSIL,and IVLVGGSTRIPKIQK,as well as two typical HSP70 family functional domain nucleic acid binding structural domains and substrate binding structural domains.S.henanense ShHSP70 clustered evolutionarily with the rest of the crustaceans and showed the highest similarity to the Gecarcinus lateral.Tissue distribution showed that it was expressed in all eight tissues examined and was highly expressed in the reproductive organs.2.Construction of a prokaryotic expression vector for ShHSP70 and purification of the fusion protein as well as protein localization.The p ET32a-ShHSP70 prokaryotic expression vector was successfully constructed,and the target protein was highly soluble and expressed in large amounts in the supernatant after bacterial fragmentation,and a small portion had been expressed in the precipitate in the form of inclusion bodies,and it was consistent with the predicted protein molecular weight.After extensive induction,the target protein was purified by affinity chromatography through the Ni-NAT gravity column,and 50 m M imidazole buffer was eluted Most of the hemoproteins,250 m M imidazole buffer could obtain a large amount of target protein with high purity.Immunofluorescence results of tissue cells showed that ShHSP70 was mainly distributed in the cytoplasm of hepatopancreatic cells and sperm cells,mainly in the acrosome.3.Role of ShHSP70 in apoptosisAfter constructing RNAi interference and recombinant protein injection models by injecting siRNA sequences and recombinant proteins,the acute cadmium stress treatment of S.henanense was performed.q PCR results showed that there was an up-regulation trend of Bax,Bcl-2,caspase-3,and caspase-9 expression when the expression of the ShHSP70 gene,as well as protein,was low,indicating that high expression of ShHSP70 may help protect hepatopancreatic tissue cells from apoptosis.4.ShHSP70 gene in response to high temperature and cadmium exposure after NLHS treatmentThe q PCR results showed that the expression of caspase-3 and caspase-9 genes was lower in the hepatopancreas of S.henanense after chronic NLHS treatment compared to direct exposure to high temperature and cadmium stress,and the expression of apoptotic genes Bax,Bcl-2,caspase-3,and caspase-9 was higher when exposed to cadmium stress compared to The expression of apoptotic genes Bax,Bcl2,caspase-3,and caspase-9 was much higher when exposed to high temperature,and the ShHSP70 gene was highly significantly up-regulated when exposed to high temperature relative to the control and Cdtreated groups.It showed that the apoptosis of hepatopancreatic tissue could be alleviated by chronic NLHS treatment,and the apoptosis was also less severe when exposed to high temperature than when exposed to cadmium stress,which may be related to the high expression of ShHSP70 during chronic NLHS treatment and exposure to high temperature.Thus,the high expression of ShHSP70 in the hepatopancreas of Sinopotamon henanense after NLHS as well as after high-temperature exposure played a key role in improving the animals’ adaptation to the environment and in reducing the apoptosis induced by the organism during more severe stresses.The recombinant r ShHSP70 protein,synthesized in vitro by injection,alleviated apoptosis to some extent,associated with the involvement of the HSP70 protein in the regulation of the endogenous apoptotic pathway or in improving the free radical scavenging capacity and the total antioxidant capacity of the organism. |
