Screening And Identification Of The Gender-associated Differentially Expressed Genes And Analysis Of New Genes In Schistosoma Japonicum

Schistosomiasis caused by Schistosoma japonicum is an important zoonosis in China and southeast Asia. A virtually unique trait of all species of Schistosoma (phylum Platyhelminthes) is their evolution from hermaphrodite ancestors into sexually dimorphic species. Germ-cell differentiation is crucial to the developments of schistosome. Pairing between male and female worm of schistosome is necessary for female reproductive development and for the maintenance of her mature state. Adult female worm can produce a large number of eggs which cause the main pathological changes in the infected host and also are responsible for the dissemination of parasite. Based on above characteristics of schistosome, the screening and identification of gender-associated differentially expressed genes of S. japonicum were carried out, and several differentially expressed new genes were cloned and analyzed to probe the molecular mechanism of S. japonicum gender-associated gene development that may be considered new targets for vaccination.1. Construction and analysis of suppression subtractive hybridization libraries of S. japanicum female and male wormsIn this study, Poly A+RNA was isolated respectively from S. japonicum (Sj) female and male adult worms, and suppression subtractive hybridization(SSH) libraries of Sj female and male worms were constructed by using Clontech PCR-selectTM cDNA subtraction kit. The evaluation results indicated that the two SSH libraries were high quality, and were applicable to isolate Sj differentially expressed genes on the whole genome scale. The recombinant rates of female and male SSH libraries were 92% and 91% respectively. Some clones were selected randomly and sequenced, 64 female ESTs and 62 male ESTs were obtained. The nonredundancy rate of the female and male SSH libraries were 29.7% and 22.6% respectively. The homology search results suggest that there were 45 female and 48 male unigenes .There were egg shell protein ,ferrtin-1and female-specific 800 in female SSH library and actin, trypomysin in male SSH library. These genes were previously verified as gender-associated differentially expressed genes by other researchers. Identifying the more differentially expressed genes may uncover molecular mechanism of female and male sexual maturation, It was an important step in developing strategies for blocking the worm's reproductive life cycle, and their potential to induce host morbidity and ultimately preventing successful transmission.2. Analysis of the gender-associated gene differentially expression profile of S. japonicum using cDNA microarray techniqueFour pieces of S. japonicum gender-associated differentially expression cDNA microarrays were fabricated. Each cDNA microarray contained 1536 female and 1536 male cDNA clones originating from SSH libraries. Microarray hybridization results suggested that there were 953 female and 1014 male differentially expression gene clones.855 gene clones were selected and sequenced. The results of sequence analysis indicated that these gene clones represented 297unigenes composed of 137 female and 160 male unigenes. 247 genes were homologous to known ESTs in GenBank by the degree from 95% to 100%. Among 137 female unigenes,18 genes had known protein functions ,96 genes had unknown protein functions, 13 genes had known EST match, 8 genes represented unknown new genes of Schistosoma which had no database match. Among 160 male unigenes,30 genes had known protein functions, 109 genes had unknown protein functions, 15 genes had known EST match, 5 genes represented unknown new genes of Schistosoma which had no database match. Seven nonredundant differential expression gene clones were verified by Real-time PCR, which demonstrated that these clones were indeed differentially expressed, and confirmed the cDNA microarray results, thereby supporting the reliability of the system. Our results indicated that it is high efficient to isolate and identify differentially expressed genes by using SSH combined with cDNA microarray.Homology of sequenced ESTs were analyzed and functions of genes encoding protein were predicted. The results indicated that there were egg shell protein, ferrtin-1, RXR receptor, cathepsin B, cathepsin L, SOD, Glutathione reductase in female SSH library, and there were actin, tropomysin, myosin, collagen, Sj 22.6 kDa membrane- associated antigen, Sj 23 kDa membrane-associated protein, calpain,calponin,calmodulin,calreticulin,HSP60,HSP70,HSP90 etc. in male SSH library. The results suggested that genes from female SSH library mainly involved in the process of egg production, metabolism, transcription regulation and anti-oxidation, The genes from male SSH library were intimately involved in the structural organization including components of the muscular system, tegumental proteins and the underlying cytoskeleton, ion regulation, signal transduction, protein biosynthesis and catabolism. This apparent division of labour between the genders had an extensive transcriptional basis. The male ensured the survival of the egg-laying female by providing physical support and musculature to aid feeding, physical transportation within the vasculature. The female then concentrated energy expenditure on egg production.3. Cloning and analysis of gender-associated differentially expressed new genes of S. japonicumFull-length cDNA of the four new gender-associated differentially expressed genes were obtained by the RACE (rapid amplification of cDNA ends) technique. F2 was up-regulated in female worms, and cm3, cm4, m1 were up-regulated in male worms. With bioinformatics tools, protein encoded by these new genes were predicted in these, including their physical and chemical property, trans-membrane structure, antigen site, conserved domain, function site and homologous protein search. The results indicated that f2 contained conserved domains of ubiquitin and ribosomal protein S30 which may involve in protein biosynthesis and modification. Cm3 contained conserved domains of C2H2-type zinc-finger which may involve in nucleic acid binding, cm4 contained conserved domains of SPla/RYanodine receptor SPRY which mediate Ca2+-release from the sarcoplasmic or endoplasmic reticulum and the intracellular Ca2+ store.Investigation on biological function of these new genes will help to better understand the molecular mechanisms regulating schistosome sex developmental processes, and to establish a basis for exploiting effective vaccine candidate and new drug target.