Analysis Of Differentially Expressed Genes Among The Worms From Different Development Stage Of Schistosoma Japonicum

Schistosomiasis is a chronic debilitating disease that currently affects in human and livestock worldwide.At the beginning of this century, Brazil and China scientists made great progress in Schistosoma Genome Project (SGP) and deposite a lot of general information derived from Schistosoma japonicum and schistosoma manson adult worms and eggs. Our research focused on analysis gene expressed changes with biochemical and morphological alterations in Schistosom japonicum (S.japonicum) different life cycle stages using high throughput and high sensitivity approaches. It will contribute to understand host–parasite interaction and relationship to schistosomiasis pathogenesis, permitted a list of potential drug targets and vaccine candidates to be drawn up, and ultimately lead to improved control strategiesThe main results were summaried here:1. Analysis of early hepatic stage schistosomula expressed genes by subtractive expressed sequence tags libraryA early schistosomula suppression subtractive hybrization cDNA library was first constructed, using 7d schistosomula as tester and 42d adult worms as driver.PCR amplification revealed that the subtractive cDNA libraries contained approximated 90% recombinant clones, most insert about 500bp. 6000 positive clones were randomly selected and sequenced. In total 5747 clones with inserts length more than 100bp and had an average length of 571bp, mainly from 300-900bp. A total of 5474 raw ESTs were assembled into 1764 clusters contained 456 contigs and 1306 singletons by PHRED 100 which contain 11-8.8% of schistosoma genomic(schistosoma express gene is 15000-20000).Semi-quantitative RT-PCR analysis Tetraspanin,cytochrome C oxidase,NADH dehydrogenase,HSP90,Soluble vascular endothelial cell growth factor receptor and IBI protein express in 7d schistosomula and 42d adult worms.The result indictate these gene express higher in 7d schistosomula than 42d adult.Besides, there are 156 clusters in accord with the CHGC (Chinese National Human Genome Center) limited reported schistosomula specific clusters. All these data confirmed our subtractive library efficiency from the other side.65% clusters identity or homologe with known S.japonicum clusters compared with the S.japonicum database(http://function.chgc.sh.cn/sj-proteome/index.htm). 35% ,617 clusters had not been reported in S.japonicum database,about 4.1-3% of S.japonicum genomic.In this 617 clusters,only 92 clusters identity or homologe with known schistosoma manson(S.manson) clusters ,525clusters are new clusters compared with S.manson database(http://www.sanger.ac.uk/Projects/S_mansoni/). Then these 525 clusters that no homologe with schistosoma were submitted to BLASTX for homologous searching with the nr database in GenBank(http://www.ncib.nlm.nih.gov). There were 212 clusters left without any implication of known genes and protein, which probably meant that these clusters were the specific hepatic schistosomula genes.Preliminary function analysis indicate that the clusters come from early schistosomula subtractive cDNA library had impotrant biological function.Among the sequences, 7.5% of 1746 clusters had their Gene Ontology GO classification determined. According to the GO tree, Metabolic process (31%), Regulation of biological process (12%), Reproduction (13.4%) were three largest rates.175 sequences were classified into Glycolysis,Amino acid metabolism,Arachidonic acid metabolism and purine metabolism et al metabolism pathways and Cell Communication,Ubiquinone biosynthesis,Urea cycle and metabolism of amino groups,Oxidative phosphorylation,Pentose phosphate pathway,Carbon fixation,Proteasome,Ubiquitin mediated proteolysis et al 25 KEGG pathways. Interesting , we found gene chgc_new_contig406 homologe with dehydrogenase which is an important gene involved in antiparasite drug gamma-Hexachlorocyclohexane degradation pathway.On the other hand, S.japonicum as the parasite live in the host vascular, the identified gene soluble vascular endothelial cell growthor receptor maybe interact with the S.japonicum living environment.2. Analyze differentially expressed genes during different development stages of schistosoma japonicumTo identify the transcriptional profile of different development stages of S.japonicum, a microarry consist of 5000 S.japonicum cDNA clones (4000 from 7d schistosomula subtractive library, 1000 from 42d adult worms subtractive library)were customer premade, each clone repeat 3 times.The microarray was used to screen RNA from different development stage worms on 7d, 13d, 18d, 23d, 32d and 42d,42d♀, 42d♂triple. Self to self hybrization test confirm microarry platform. For each life cycle stage, the ratios of signal intensity were determined, normalised against the day 7 value, and classified as up- or down-regulated on the basis of a significant (P<0.005) two-fold change in signal using SAM software. The differential expression was validated in 18 randomly selected genes in 5 different development stage using quantitative RT-PCR.The result indicate that, compared to 7d worms, 34 cDNA clones represent 22 genes (2 genes unknow)differentially expressed in 13d worms,of which, 4 genes up regulated,18genes down-regulated; 238 cDNA clones represent 123 genes(9 genes unknow) differentially expressed in 18d worms,of which, 86 genes up regulated, 37 genes down-regulated; 244 cDNA clones represent 136 genes (7 genes unknow) differentially expressed in 23d worms, of which, 47 genes up regulated, 89 genes down-regulated; 186 cDNA clones represent 99 genes (7 genes unknow) differentially expressed in 32d worms, of which, 46 genes up regulated, 53 genes down-regulated; 158 cDNA clones represent 95 genes (4 genes unknow) differentially expressed in 42d worms, of which, 54 genes up regulated, 41 genes down-regulated; 671 cDNA clones represent 262 genes ( 31genes unknow) differentially expressed in 42d♀worms, of which, 54 genes up regulated, 208 genes down-regulated; 171 cDNA clones represent 115 genes (8 genes unknow) differentially expressed in 42d♂worms, of which, 94 genes up regulated, 21 genes down-regulated.The microarray approaches resulted in identifying many differentially expressed genes implicated in the metabolism, post-emnryonic development, sexual reproduction, protein binding, signal transduction and cell morphogenesis, suggesting the involvement of multi-biological processes in S.japonicum development.3. Cloning and Bioinformatics Analysis of Schistosoma japoncium fibrillin gene and COG8 geneTwo ESTs were choosed from 7d suppression subtractive hybridization library named SSH52.D11 and SSH33.A7,which had no homologe with known schistosoma gene. The complete sequence was cloned and sequenced after amplication with RACE technique with Genbank Accession number:1067183 and EU131676.1.Bioinformatics analysis show the SSH52.D11 had 2251bp length containing 2058bp complete ORF encoding 686AA with Mr 79519.9 and PI 5.22. Real time PCR comformed that the gene express in schistosomula higher than that in adult schistosoma.Amio acid function motif search showed that the gene had several EGF-like domain and EGF-like calcium-binding site that was the same with fibrillin family conserved charcater. Hence, we named the gene as sj-fibrillin. phylogenetic analysis homo(Genebank Accession number1335064), mus musculus(Genebank Accession number 118136302), Xenopus laevis(Genebank Accession number 83628282) et al 12 species indicates the protein belong fibrillin family and more like vertebrate in evolution which implied the schistosoma evolution may be affected by host. Structure protein fibrillin high express in schistosomula maybe associated with the schistosomula rapid growth and vigorous cell differentiation.Bioinformatics analysis show the SSH33.A7 had 2505bp length containing 2160bp complete ORF encoding 719AA with Mr 85160.4 and PI 5.22. Real time PCR comformed that the gene express in schistosomula higher than that in adult schistosoma.Amio acid sequence analysis show that it had Dor1 domain which conserved in Component of oligomeric golgi complex 8. Phylogenetic analysis homo sapiens(Genebank Accession number 21166361), mus musculus(Genebank Accession number 66392581),Danio retrio(Genebank Accession number 61806582 ) indicated it belong to COG protein family, named sj-COG8.COG protein is necessary during Drosophila spermatogenesis, is associate with gonad development.This protein high express in schistosomula may play an important role in schistosoma gonad development.