Construction And Analysis Of Substracted Library In Ovary And Hepatopancreas Of Shrimp Fenneropenaeus Chinensis

Fenneropenaeus chinensis,is an economically important aquaculture species inChina.Its taxonomy belong to the phylum Arthropoda,class Crustacea,order Decapoda,family Penaeidae,genus Fenneropenaeus.In the present study,the developmentalstages of Chinese shrimp ovarian were determined into five stages:oogoniumproliferation stage,previtellogenesis stage,primary vitellogenic stage,secondaryvitellogenesis stage and declining stage by external observation,gonad index andparaffin.We performed suppression subtractive hybridization (SSH) experiment andconstructed the forward/reverse subtractive library of the ovarian and hepatopancreasfrom oogonium proliferation stage and primary vitellogenic stage and fromprevitellogenesis stage and declining stage.The sequencing results were comparedusing BLASTx homology on GenBank and their functional annotation was conductedvia the Gene Ontology.Then we targeted analysis of genes that are differentiallyexpressed during the development.SSH results were validated by the fluorescencequantitative PCR to study the gene expression abundance during the different stagesof the ovarian development in order to elucidate the relationship between theexpression features and functions of these genes,as well as the relationship betweenthese genes and ovarian development.SSH results show that differentially expressed genes of subtractive library werein accordance with the ovarian development.In the ovarian library,the expression levelof differentially expressed genes during the oogonium proliferation was higher inrelation with the cellular processes,especially the genes with an inferred function incell proliferation and differentiation (mitosis).The genes expressed in the stage ofprevitellogenesis stage,primary vitellogenic stage and declining stage were inferredwith the functions of protein synthesis,energy metabolism and signal transduction,which may play important roles in vitellogenesis and cortical stick formation.Using Real-time quantitative PCR to study the four differentially expressedgenes (peritrophin mRNA,cytochrome oxidase I mRNA,polehole-like protein mRNAand vitellogenin mRNA) changes in abundance during the different Ovariandevelopment stages.The results showed that the expression level of all the four genes is the highest at the primary vitellogenic stage,while the maximum value ofpolehole-like protein mRNA and vitellogenin mRNA during the primary vitellogenicstage was the minimum value during other stages of dozens of times,even to hundredsof times.Thus,he two genes elevated during the early stage may play roles in theprocess of ovarian maturation.The expression of polehole-like protein mRNA in thehepatopancreas is up to the maximum at the primary vitellogenic stage,while theexpression of vitellogenin mRNA in the ovarian is up to the maximum,implying thesetwo genes play important roles in the development of hepatopancreas and ovarian.