Identification And Analysis Of Differentially Expressed Genes In Haemocytes Of Variously Colored Abalone (Haliotis Diversicolor Reeve, 1846) Challenged With Bacteria

Abalones are one of the largest marine gastropod mollusks and they are a economically important seafood in aquaculture worldwide.However, bacterial epidemic infection has been reported in China and other countries in recent years and mass mortality in abalones causes significant economic losses.For this reason,a large scale screening of immune-related abalone genes is necessary.A better understanding of the immune responses in variously colored abalone(Haliotis diversicolor Reeve,1846) after bacterial infection is important to protect this animal from diseases in the farm.In order to find differentially expressed genes in bacteria-challenged abalones,a forward subtractive suppression hybridization(SSH) library with content of 1.37×10~6 cfu was constructed from haemocytes of H. diversicolor challenged with five bacterial species including two Gram negative bacteria(Escherichia coli and Vibrio parahaemeolyticus) and three Gram positive bacteria(Staphylococcus aureus,Micrococcus lysodeikticus and Staphylococcus epidermidis),and the potential ESTs or gene sequences were randomly screened on the middle sequencing scale(435 clones).A total of 435 clones in the SSH library were sequenced and 111 genes were recognized based on BLAST searches in NCBI and were categorized in association with different biological processes using AmiGO against the Gene Ontology database.Among seventy-one genes(64.0%) involved in different biological processes,24 genes(21.6%) were categorized as a group in association with cellular metabolic processes,of which 11 may involve in a cellular protein metabolic process(9.9%),8 were associated with a generation of precursor metabolites and energy(7.2%) and 5 with other cellular metabolic processes(4.5%);10 were related to cellular component organization and biogenesis(9.0%);4 were associated with other functions(3.6%);6 were listed as a signal transduction group (5.4%);8 were involved in a biological regulation group(7.2%),and the equal number of genes in immune system processes(7.2%) and response to stimuli(7.2%);3 were rRNA genes.In addition,40 genes(36.0%) were classified as an unknown-function gene group.Only 25 of the 111 genes (22.52%) had previously been reported in other gastropods,and most of the screened genes showed less similarity to known sequences based on BLASTn results,suggesting that at least 86 genes were found for the first time in H.diversicolor.Both semi-quantitative PCR and quantitative real-time PCR were used to validate the differential display expressions of genes which were screened from the SSH library.Fifty-two genes among 111(46.85%) were confirmed to be differentially expressed in the library,and 47 out of 111(42.34%) were up-regulated after bacterial challenge,while only five out of 111(4.5%) were up-regulated in the unchallenged controls.Among the 111 genes,55(49.55%) could not be confirmed to show differential expression patterns in either the bacteria-challenged or the unchallenged groups,and four(3.60%),often regarded as internal standard housekeeping genes,were obtained.In particular,30 genes(GlSTrs,CYP7A, Endoph,SOCS-2,Hypo2,PreP2;TroponinT,CDD,MEP and Unk29,AlInFa, MyosinLC,MMP1,MMPvar,Calp,Astacin-like,Hypo,Unk,Unk4,Unk5,Unk19, Unk24,Unk21,Unk28,Unk31,Unk32,Unk35,Unk39,HDr4CJ516, HDr2CJ15),encoding proteins involved in cellular metabolic processes; cellular component organization and biogenesis;signal transduction and biological regulation;immune defense and response to stimuli;other functions and unknown functions,were confirmed to be distinctly up-regulated in the bacteria-challenged group by both assays,and 17 genes (UbCoE,RPN2,ATPsynSub9,Profilin,TIMp,KLF,Thym-β,Caspase8, Ferritin,Tis11FPL,GSTisof,Hsp40,MnSoD,Unk2,Unk3,Unk17 and Unk34) were to some extent up-regulated in the bacteria-challenged group.In addition,five genes(FASCIN,AlDe,CuZnD,Unk11 and Unk7) were up-regulated in the unchallenged group.To exclude the possibility of contamination in abalone cDNAs from SSH by bacterial sequences,we designed 101 pair primers specific to the translatable genes indicated by SSH to amplify the genomic DNA of H. diversicolor.Specific genomic DNA fragments were obtained,especially for the genes showing no significant similarity.Cloned genomic DNA ofβ-thymosin suggested that two copies localed on chromosomal DNA.One copy is without intron,and the other is with two introns(477 bp and 2818 bp).Sixteen complete cDNAs of the genes screened from the SSH library were elucidated,including 8 genes of GlSTrs,Profilin,TIMp,PreP2,Thym-β, Ferritin,Calp and Hypo up-regulated in haemocytes of bacteria-challenged abalones during 36 h to 40 h.The differential time-respones relationship of 14 genes expression induced by bacteria in haemocytes of H. diversicolor were studied and the results of 8 genes including GlSTrs, CYP7A1,Hypo2,TIMp,CDD,AlInFa,MMPvar,Hypo showed to be distinctly up-regulated in haemocytes corresponding to time of bacterial challenge.In summary,this study represents the first large-scale investigation of immune-related genes in bacteria-challenged abalones.By SSH, differential expression patterns of genes were screened specifically related to bacterial stimulation and these genes were then categorized into 11 distinct functional groups.Eighty-six genes were for the first identified among 111 screened in H.diversicolor,of which 52 genes were confirmed to show the differential expression patterns using both semi-quantitative PCR and quantitative real-time PCR.A total of 30 genes were validated to be distinctly up-regulated in bacteria-challenged abalones,encoding proteins involved in cellular metabolic processes; cellular component organization and biogenesis;signal transduction and biological regulation;immune defense and response to stimuli;other functions and unknown functions.To our knowledge,this is the first report to unveil multiple up-regulated genes with differential expression patterns involving various biological processes in bacteria-challenged H.diversicolor.It is anticipated that these results will be useful in elucidating the immune mechanism in bacteria-challenged H.diversicolor. However,because it is not clear that the genes screened in this study take part in immune processes,further research may focus on revealing the biological functions of genes screened in the SSH that are up-regulated.We anticipate that this work will be helpful in facilitating future study of the immune response of H.diversicolor,and acquiring new insights into the mechanism involved.