Studies On Molecular Mechanism Of Hsp90 And Cochaperone Cdc37 Under Cadmium Stress In Tiger Shrim,penaeus Monodon

Cadmium is a kind of toxic heavy metals,interfere with the normal functions by affecting various organs and systems of the black tiger shrimp(Penaeus monodon).Heat shock proteins(Hsps)play important roles in resistance to stress.Hsp90 is one of the most abundant cellular molecular chaperone,which can be combined with Cdc37 or other molecular chaperones into large multiprotein complexes.Therefore,the molecular mechanisms of Pm Hsp90 and cochaperone Pm Cdc37 defeating cadmium exposure in P.monodon was researched.In this study,Pm Cdc37 c DNA fragments were screened from the transcriptome of our laboratory.The full length of Pm Cdc37 was cloned by molecular biology techniques such as SMART-RACE.The full length of Pm Hsp90 have been cloned by our laboratory before.Recombinant proteins of the Pm Hsp90 and Pm Cdc37 were expressed in E.coli,purified by Ni-chelating affinity chromatography,and prepared antibody;To study the activity of Pm Hsp90 and Pm Cdc37 protein,the ATPase activity of Pm Hsp90 protein assay was carried out.To further explore the role played by Pm Hsp90 and Pm Cdc37 under cadmium exposure in vivo,si RNA interference(RNAi)technology was adopted to silence the expression of Pm Hsp90 and Pm Cdc37 in P.monodon,and a cumulative mortality rate assay was carried out after overexpression or inhibition the Pm Hsp90 and Pm Cdc37 under cadmium exposure.Finally,in order to study the relationship between Pm Hsp90,Pm Cdc37 and SOD,the enzyme activity of MDA and SOD in the hemolymph was detected after overexpression or inhibition Pm Hsp90 and Pm Cdc37 in shrimp.The result showed that Pm Hsp90 and Pm Cdc37 could influence the activity of SOD.To further study the mechanism of SOD under cadmium exposure.We successfully knockdown the expression of SOD in vivo through RNA interference technology.The cumulative mortality rate was calculated when the shrimp was challenged by cadmium after overexpression and inhibition the Pm SOD.A large amount of reactive oxygen will be induced after cdmium stress in P.monodon,which affects the stability and activity of protein kinases.Hsp90/Cdc37 can maintain the activity and function of protein kinases and enhance the shrimp body keep abalance to defence the cd stress.At present,the mechanism of Hsp90/Cdc37 against Cdmium stress is still not clearly in P.monodon.In this study,RACE technology,prokaryotic expression technology,RNA interference technology and protein overexpression technology were used.The research results could enrich the knowlege of the heat shock protein family in P.monodon,and can also provide some theoretical support for improving shrimp immunity.The main research is summarized as follows:(1)The Pm Cdc37 c DNA fragment was screened from splicing transcriptome of P.monodon established in our laboratory.The full length of Pm Cdc37 gene was cloned by SMART-RACE technique.The Pm Cdc37 c DNA sequence is 2,523 bp in length,contains 5-untranslated region(UTR)of 29 bp,3'UTR of 769 bp and 1,053 bp open reading frame which can encode 351 amino acids.The q RT-PCR analysis showed that Pm Cdc37 was ubiquitously expressed in all the tested shrimp tissues,and had the highest expressed in hemolymph and hepatopancreas.(2)The recombinant plasmid PET30a-Pm Hsp90 and PET21a-Pm Cdc37 were transformed into E.coli BL21(DE3)competent cells,and Fusion recombinant plasmids were expressed by an induction with 0.6 m M isopropyl ?-D-thiogalactoside(IPTG)when the OD600 of the bacteria reached 0.6.The protein purified by 6 x His Protein Purification Kit.Part of the purified protein is used for antibody preparation.Another part of the protein is used for detection the Pm Hsp90 ATPase activity.The result showed that Pm Hsp90 has ATPase activity,but the ATPase activity was reduced after added Pm Cdc37 protein.The result demonstrated the prokaryotic expressed proteins of Pm Hsp90 and Pm Cdc37 have protein activity in vitro,and Pm Cdc37 maybe interacts with Pm Hsp90 and Pm Cdc37 is also likely to bind the N-terminal of Pm Hsp90 just like the study result in higher animals.(3)The q RT-PCR analysis showed that the Pm Hsp90 and Pm Cdc37 expression level in the hemolymph and hepatopancreas were up-regulated after cadmium exposure,and the result of wb showed that the protein expression level in the hemolymph and hepatopancreas were aslo up-regulated after cadmium exposure.The q RT-PCR analysis showed that the Pm Hsp90 and Pm Cdc37 expression level in the hemolymph and hepatopancreas were reduced after injected si RNA,and the wb assay has same result.The result of cumulative mortality rate assay showed that the cumulative mortality rate assay of overexpression the Pm Hsp90 and Pm Cdc37 lower than the cumulative mortality rate assay of inhibition the Pm Hsp90 and Pm Cdc37.The result inferred that Pm Hsp90 and Pm Cdc37 may play important roles in the defence against cadmium stress in P.monodon.(4)In order to further explore that Pm Hsp90 and Pm Cdc37 may defeated cadmium stress through affecting the redox pathway.The activities of SOD and MDA were detected by SOD enemy activity kit or MDA enemy activity kit in hemolymph.The results showed that Pm Hsp90 and Pm Cdc37 could enhance the activities of SOD and MDA.To future study the relationship between SOD and cadmium stress,the m RNA expression level of Pm SOD was measured after cadmium stress.The result showed that the m RNA expression levels of Pm SOD in the hepatopancreas and hemolymph increased suddenly frist and then decreased.The Pm SOD of RNA interference and overexpression experiments was carried out to detect cumulative mortality of P.monodon under cadmium stress.The experimental results showed that Pm SOD also plays a defensive role against P.monodon against cadmium stress.