The Digital Gene Expression Profile Analysis In Response To Sulfide And Characteristics Of Sulfur Dioxygenase In Urechis Unicintus

Sulfide (sum of H2S, HS-and S2-) is a common toxin and can casuse several harms,even death to organisms. However, some invertebrates are able to tolerate, metabolizeand utilize sulfide. To explore the mechanism of sulfide metabolism at molecular level,the echiuran worm Urechis unicinctus, which inhabits a U-shaped burrow of coastalsediment in the Yellow Sea and the Bohai Sea of China, is employed in this study, andits transcriptome profile of the worm in response to sulfide was analyzed by digitalgene expression sequencing technique, and expression characteristics and function ofa key enzyme in mitochondria sulfide oxidation, sulfur dioxygenase (SDO) wereanalyzed by several techniques of molecular biology. The main findings were asfollows:In this study, four21bp-tag sequencing libraries from U. unicinctus exposed to50μM sulfide for0h,6h,24h and48h were constructed by Nla III and Mme I doubledigestion method. By Solex/Illumnia sequencing platform, a total of about16millioncDNA tags were obtained. After removing impurities, clean tag numbers in the fourlibraries were3909160(0h),3613737(6h),4057279(24h) and4027932(48h),and the numbers mapped to the gene of U. unicinctus reference transcriptome (NCBIassession number: SRA122323) were21121(0h),20526(6h),20129(24h) and20111(48h). Compared to transcriptome data of0h library, a total of1705(6h),1181(24h) and1494(48h) tags-mapped genes were determined as the DEGs(differentially expressed gene). For GO analysis, most DEGs involved in binding andcatalytic activities of molecular function category, cellular process and metabolicprocess of biological process category and cell part and intracellular of cellular partcategory. The DEGs affected176,166and176pathways in6h,24h and48h libraries, respectively. By pathway enriched analysis, oxidative phosphorylation,ribosome, proteasome, complement and coagulation cascades, and metabolicpathways were all significantly enriched in the6h,24h and48h libraries. Mostsignaling pathways such as ribosomes, the complement system and proteasome werefirst reported in sulfide metabolism. Furthermore, three key physiological actionsinvolving in energy metabolism, DNA damage response and inflammation induced bysulfide were analyzed, and discovered that glycolytic pathway and oxidativephosphorylation were inhibited when U. unicinctus exposed in the sulfide. Associatedwith the up-regulated expression of sulfide oxidation related genes in mitochondria, itwas suggested that mitochondrial sulfide oxidation is not only for the sulfidedetoxification but also is able to provided energy; In resistance of DNA damage, itwas analyzed that U. unicinctus maybe up-regulated the expression of the PIDD todecreased the DNA damage induced from sulfide, which could mediate NF-κBpathway to respair the DNA damage as well as apoptosis pathway to maintain theintegrity of genome. Moreover, activation of ERK pathway and Toll-like4pathwayas well as inhibition of complement and coagulation cascades when U. unicinctusexposed to sulfide might promote the inflammation response, which is helpful to thehomeostasis for itself and the survival of the worms. Data in this study has first beenreported in multicellular organisms on the expression profile in response to sulfide bydigital gene expression analysis at transcriptome level.The analysis results of the U. unicinctus SDO (sulfur dioxygenase) characteristicsshowed that its full-length is1976bp, coding293amio acids. The predicted aminoacid sequence contains the conserved amino acids of the mello-β-lactamasesuperfamily. The SDO recombinant was produced using an E. coli expression systemas inclusion body in vitro and the measured activity is0.80U mg protein-1afterrenatured. Furthermore, four sub-segments of the U. unicinctus SDO were expressedusing the E. coli expression system, and its functional domains was preliminarilyidentifided using the four recombinant sub-segment proteins by enzyme activity,kinetic parameters and GSH (glutathione) affinity measurements. The resultsindcatied that the metal ion in U. unicinctus SDO may bind in the metal I binding site (H113, H115, H169and D188), while the metal II binding site (D117, H118, H169and H229) without metal ion binding plays import role in maintenance of the enzymeactivity. And residues D117and H118in the metal II binding site were two key aminoacids in maintaince of SDO acitivity detected by the simultaneous mutations ofresidues D117E and H118A. Among the GSH sites R197, Y231, M279and I283,Y231is recognized as the most important amino acid residue.SDO polyclonal antibody was obtained using U. unicinctus SDO wild typerecombinant protein; and SDO was detected to be located in the mitochondria bywerstern blot and located in the epithelial tissue of different organs byimmunohistochemistry. By qRT-PCR, western blot and ELISA techniques, it wasshowed that SDO expressions in different organs of U. unicinstus at mRNA andprotein levels were different; and the SDO expressions were the highest in anal sac,followed by midgut, and significantly low in body wall and hindgut. However, thetotal SDO specific activities in the body wall and the midgut are higher than that inthe hindgut, but without significant difference among them.In order to explore the SDO adaption in response to sulfide, U. unicinctus wasexposed to different concentrations of sulfide in the lab condition. The expressioncharacteristics and activity changes of SDO in different organs were analyzed and theresults showed that the SDO in different organs had different adaptation to sulfide. Atlow concentration of sulfide (50μM), body wall first activated the sulfidedetoxification,but with prolonged exposure time, hindgut and body wall became themajor organs to detoxify sulfide, anal sac played a supporting role, but the ability ofmidgut to detoxify the sulfide was limited. At high concentration of sulfide (150μM),the total SDO specific activities in the organs except hindgut were elevated, but thetime for siginificant differences delayed; and body wall played the most importantrole in sulfide detoxification, followed by hindgut and anal sac, but the role of migutis not so obvious. Therefore, body wall, hindgut and anal sac played important role inthe sulfide detoxification while the midgut played limited role.