| The swimming crab,Portunus trituberculatus,distributes widely along the coasts of temperate western Pacific Ocean.As one of the most common edible crab species,it supports an important fishery in East Asian countries,and a large aquaculture industry in China(Dai et al.,1986).During the last decade,the farming of P.trituberculatus in China developed quickly,and the production has reached 118,836 t in 2014(China Fishery Statistical Yearbook,2015).However,due to rapid expansion of the crab farming and lack of reproduction-control technique,the high-quality seed production cannot meet the heavy demand,which became a constraint to the sustainability of this industry.Therefore,in recent years,increasing attentions have been paid to the reproductive mechanisms in this species.Over the past several years,many efforts have been made in the transcriptional regulation during gonadal development and gametogenesis in decapod crustaceans.However,limited information is currently available regarding the gene expression regulation at post-transcriptional levels.As a class of key post-transcriptional regulators,mi RNAs have gained considerable attention during recent years in the field of reproduction.This study shows the function of the genes in mi RNA biogenesis pathway and mi RNAs in gonadal development of Portunus trituberculatus.The main results are as follows.1.Molecular cloning of the genes in the mi RNA biogenesis pathway and their expression in gonadal development of the swimming crabIn this study,the full-length cDNA of Drosha,Exportin 5,Dicer 1,Dicer 2,AGO 1 and AGO 2 in the pathway of mi RNA/si RNA biogenesis are first cloned from the swimming crab using rapid amplification of cDNA ends(RACE),and then homology and phylogenetic tree analysis will be performed.The results show that Drosha,Dicer 1 and AGO 1 are highly conserved in evolution,they shares the highest similarity with the closely related species of each other(83%-99%).However Exportin 5,Dicer 2 and AGO 2 shares the lower similarity relatively(44%-51%).Quantitative real-time PCR(q RT-PCR)analysis shows that Drosha?Exportin 5?Dicer 1?Dicer 2?AGO 1 and AGO 2genes are expressed in all tested tissues,and most of these are highly expressed in ovary and hepatopancreas tissues,followed by testis tissues.At different periods of gonadal development,the expression of Drosha ?Exportin 5?Dicer 1and AGO 1 genes shared the same trend and both showed highest expression in stage II.Both Dicer 2 and AGO 2 are highly expressed in stage I.During ovarian development,the levels of Drosha and Exportin 5 genes are increased gradually from phase II to phase V.Our results suggested that Drosha?Exportin 5?Dicer 1?Dicer 2?AGO 1 and AGO 2 cooperatively regulate the gonadal development of P.trituberculatus which is mi RNA/si RNA dependent.2.Identification and comparative profiling of ovarian and testicular micro RNAs in the swimming crab Portunus trituberculatusThe present study provided a dataset for identification and characterization of mi RNAs in gonads of P.trituberculatus.A total of 188 known and 65 novel mi RNAs were identified from ovary and testis using Illumina sequencing.Several mi RNAs,such let-7?let-7c?let-7f,mir-2,mir-184 and mir-276,were abundantly expressed in both ovary and testis tissues,indicating their critical roles in gonadal development and functions.Expression analysis identified 75 mi RNAs with statistically differential expression between ovary and testis,and KEGG analysis indicated that the target genes of these mi RNAs were significantly enriched in signaling pathways associated with reproduction,such as Gn RH signaling pathway,ovarian steroidogenesis and Wnt signaling pathway.Further expression profiling of DE mi RNAs with highest foldchange revealed that these mi RNAs were expressed in a stage-specific manner during ovarian(mir-9,mir-125 and mir-27a)and testicular(mir-7,mir-87 and mir-2788)development. |
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