Identification And Expression Analysis Of Dicer Gene From Flounder (Paralichthys Olivaceus)

RNA interference (RNAi) is a powerful mechanism of posttranscriptional generegulation in eucaryon. Dicer is the key protein in the RNAiinitialperiod to producemature microRNAs (miRNAs) and small interfering RNAs (siRNAs) by cleavingdouble-stranded RNA (dsRNA), which mainly involved in invasive endogenous genesregulation. The Japanese flounder (Paralichthys olivaceus) is one kind of importantcommercial fish. In the flounder early larval development process, has a varied changedwhich involving eye migration, body color and lifestyle. Exogenous Thyroid hormone(TH) induced premature metamorphosis of flounder larvae, while the Thiourea hormone(TU) blocked metamorphosis of flounder larvae. We have analyzed the role in theflounder metamorphosis development, which including IGF-1, IGFBP-1and microRNA.However, the identification of flounder Dicer and the mechanism of Dicer regulatingthe flounder development have not been reported. So, we cloned Dicer of floundercDNA sequence, analyzed the structure of sequence, and detected the Dicer express inthe flounder. We also observed the effect of the thyroid hormone to the Dicer. In thepresent study, Lin-28binded especially the terminal loop of pri-and pre-let-7, therebyinterfering with cleavage by Drosha and Dicer, respectively. This research detected theexpression level of Lin-28b, pre-let-7d1and let-7d in the early development of Flounder.Silencing Dicer in the Flounder embryo cell, detected the expression of let-7d, Lin-28b,pre-let-7d1and thyroid hormone in the embryo cell.Based on the Race PCR, we cloned Dicer cDNA in the Flounder finally. Thecomplete cDNA consisted of a231nt5’-UTR, a663nt3’-UTR and an open readingframe (ORF) of5691nt. And the ORF encoded a polypeptide of1896amino acids bythe ORF Finder. We found the amino acids alignment of flounder was highlyconservative in six domains, which contained DEXD/H-box, DUF283, PAZ_dicer_like,two neighboring RNaseⅢ and dsRBD domain by the protein structrue analysis. BLASTanalysis showed the average homology of78%to other species. The highest homology of89%was found between the amino acid sequence of Oreochromis niloticus and thatof flounder. And it showed that the lowest identity of72%was found between theamino acid sequence of Xenopus laevis and that of flounder. A NJ phylogenetic tree ofDicer, which was used eighteen sequences of available animal Dicer proteins, wasconstructed to ascertain evolutionary relationship of Dicer in metazoan. It displayed thatthe flounder Dicer was located in the osteichthyes branch. Normally, the resultsindicated a step evolution relationship of Dicer from elegans to human.The mRNA of Dicer could be detected in all developmental stages andsignificantdifference. The highest expression level of Dicer was detected in blastulastage. The lowest expression level of Dicer was detected in unfertilized egg. Theexpression of Dicer in the E2was higher30times than that in the E1. However, theexpression of Dicer kept a lower level and was no significant difference from17dph to36dph. Dicer mRNA was detected in all investigated tissues and significantdifference.The highest level was observed in the brain, it was higher47times than that in themuscle, which was lowest level in the tissues. The levels of Dicer which are about equalamong spleen, gill and heart, are significantly higher than the levels which are onsignificant difference in stomach, kidney, intestine and liver.We detected the expression of Dicer in metamorphosis larvae sampled at17,23,29and36days after TH and TU treatment. At17d,23d and29d, the expression of Dicerwas no significant differences between TH and TU. Importantly, the level of Dicer inTH-treated at36d was significantly declined compared to the NC (untreated group).And the level of TH-treated and TU-treated in23d was also higher than that in otherstages. And the level of Dicer in untreated group was almost steady in every stage.This research detected the expression of Lin-28b, pre-let-7d1and let-7d in the earlydevelopment of Flounder. The results showed that Lin-28b was significant difference inFlounder all development. The highest expression level of Lin-28b was detected inblastula stage. However, the expression of Lin-28b kept a lower level and was nosignificant difference from14dph to36dph. On the contrary, the highest expressionlevel of let-7d was detected in29dph; the expression of let-7d kept a lower level andwas no significant difference from E1to E3. Otherwise, the highest expression level ofpre-let-7d1was detected in blastula stage.We detected the expression of Lin-28b, pre-let-7d1and let-7in metamorphosislarvae sampled at17,23,29and36days after TH and TU treatment. TH promoted the expression of Lin-28b, pre-let-7d1and let-7. Interestingly, TU also promoted theexpression of Lin-28b and pre-let-7d1at29dph and36dph. The expression level oflet-7d was restrained after TU treatment.In the Flounder embryo cell, the expression of Dicer was significantly declined.On this based, we detected the expression of Lin-28b, pre-let-7d1and let-7d. The resultshowed that Lin-28b and pre-let-7d1was not contacted with Dicer. TH-treated had nosignificantly changes that compared to the NC (untreated group). However，theexpression of TRα-A was significant difference compared to the NC.Our finding indicate that Dicer play a role in development and metabolism offlounder, providing basic information for further studying on the role of Dicer gene inthe P. olivaceus development.