| Exogenous sulfide can generally induce metabolic injuries in most organismsand even cause death. However, organisms inhabiting intertidal zones, hydrothermalvents, and cold seeps, have adapted many mechanisms to avoid the hurt. Theoxidation of poisonous sulfide, especially the mitochondrial sulfide oxidation is themost important mechanism, in which the sulfide: quinine oxidoreductase (SQR) is thefirst and key enzyme. Urechis unicinctus which subjected to Echiuroidea, Echiurida,Xenopneusta, Urechidae, is a coastal benthic organism. Previous study has provedthat the worm can tolerate, metabolize, and utilize sulfide. In this study, we screenedthe sulfide metabolism-related genes from the body wall in the worm usingsuppression subtractive hybridization (SSH) and cDNA microarray techniques.Furthermore, we cloned a2505bp upstream sequence of sqr gene and searched manybind sites of transcript factors in the sequence by bioinformatics analysis. Based onthe prognostication, we selected and cloned transcript factor of Sp8cDNA, purifiedthe recombinant protein in vitro and verified that the Sp8could bind to the upstreamof SQR. The investigation will provide a new clue to understanding the sulfidemetabolic mechanism in molecular level and provide more insights into the transcriptregulation of SQR.SSH result from the body wall of the worm between the treated group (tester)exposed in50μM sulfide for24h and the control (driver, no sulfide exposure)indicated that3456monoclones were obtained. And then104differential expressiongenes were screened using cDNA microarray based on the SSH library above, inwhich76genes were upregulated in sulfide exposure while the other28weredownregulated. After sequenced successfully,82genes were arisen. These genes involve in metabolism, cellular process, biological regulation, response to stimulus,multicellular organismal process, localization, development, and cellular componentorganization. Besides, there contain a lot of unknown genes, which maybe play role insulfide metabolism. Eight genes from sequenced genes above were chosen to verifythe reliability of the results of cDNA microarray using real-time PCR technique. Theresults showed that5of8implied the good coincidence of cDNA microarray.In the second part of the thesis, we cloned the2505bp upstream sequence of sqrgene using genome walking. Through P-Match and Promoter Prediction softwareanalysis, the sequence contains bind sites of TATA box，CAAT box, and manytranscript factors such as Sp，AP-1，E2F，HSF，c-Myb，NF-1. Furthermore, a fulllength sequence of transcript factor of Sp8cDNA was cloned by homology strategyand RACE technique, and the full length of Sp8cDNA is1913bp, consisting of anopen reading frame of1521bp encoding a putative protein of506amino acids, whichhas a molecular mass of54.63kDa and a theoretical pI of9.03. Prokaryocyteexpression vector (pET28a-Sp8) was constructed by cloned U. unicinctus Sp8openreading frame cDNA into pET28a, and then transformed into E. coli BL21(DE3) toget the recombinant protein. The Sp8was expressed as inclusion body and got themaximum output after induced by1mM IPTG at37℃for4h. Recombinant proteinwas purified by Ni2+-NTA affinity chromatography after the inclusion body dissolvedin8mol/L urea. Rabbit antisera against the purified recombinant SQR were obtained,after that Chromatin Immunoprecipitation was used to analysis the relationshipbetween Sp8and upstream region of SQR. The results showed the Sp8could bindupstream region of SQR, which will provide valuable evidence for deepunderstanding the transcript regulation of SQR.Moreover, we constructed recombined plasmid consisting of different segmentsof the2505bp upstream region sequence using the modified EGFP-N1vector,furthermore a recombined plasmid with the2505bp sequence was constructed andtransfect into cultured mammal293FT cells via lentiviral system. A study on detectingthe activity of the upstream region sequence of SQR gene was attempted bytransforming or transfecting U. unicinctus embryos and larvae or293FT cells using the constructed recombined plasmid and lentiviral system mentioned above byelectroporation, lipofection and lentiviral system techniques. The results showed thatan obvious expression in mammal cells was detected when transfect using lentiviralsystem. The data will provide fundamental analysis of SQR transcript regulation. |
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