Identification And Functional Study Of Non-coding RNA LncMPEG1 And MiR-67 In Pinctada Fucata Martensii

The shells and pearls of mollusks are typical biomineralization products.It is of great significance to explore the molecular mechanism of their formation for theoretical research on biomineralization or production practice of artificial pearl cultivation.Given the universality of non-coding RNAs involved in the regulation of biological metabolism.The purpose of this paper is to verify the regulation of long-noncoding RNA(Lnc MPEG1)and micro RNA(pma-mi R-67)in the formation of shell and pearl from Pinctada fucata martensii,and the interaction between Lnc MPEG1 and pma-mi R-67.The main findings are as follows:1.Identification and functional analysis of Lnc MPEG1In this study,the full length of Lnc MPEG1 was obtained by RACE technology with2,735bp.Bioinformatics analysis found that Lnc MPEG1 was encoded by three exons.The largest ORF is only 285 bp,encoding 94 amino acids.The total coding probability is about 0.0398,there is no encoding tag,indicated that Lnc MPEG1 may not have the potential to encode proteins.Lnc MPEG1 showed highly expression in mantle center than other tissues by q RT-PCR(P<0.05),followed by the mantle edge and pearl sac.In situ hybridization results showed that Lnc MPEG1 was mainly located in the outer epithelium of the middle fold of mantle edge,mantle pallial and mantle center.And it was located in both nucleus and cytoplasm.In addition,the expression of Lnc MPEG1 was significantly up-regulated at 12 h after shell notching(P<0.05),and remained relatively stable from12 h to 36 h,with the highest expression level at 48 h,which indicated that Lnc MPEG1may be involved in the process of shell notching reconstruction.Using ds RNA to decrease its expression,the formation of nacre and prismatic layer was obviously disturbed.These results indicated that Lnc MPEG1 could play important roles in the shell formation of P.f.martensii.2.Functional verification of pma-mi R-67The mature pma-mi R-67 was obtained by RNA-seq of mantle and pearl sac tissue of P.f.martensii.RNA secondary structure showed that Lnc MPEG1 could form a stem-loop structure to encode a new mi RNA,and its nucleic acid sequence was very similar to the reported mi R-67,so it was named pma-mi R-67.The phylogenetic analysis results showed that pma-mi R-67 clustered with mollusk species such as Lottia gigantea,Melibe leonine and Capitella teleta.It was highly similar to that in Melibe leonina named mle-mi R-67-3p(91%similarity)The expression level of pma-mi R-67 in the mantle center of P.f.martensii was significantly higher than that in other tissues(P<0.05).Using mimics of pma-mi R-67 for the in vivo overexpression,the expression level of pma-mi R-67 in the mantle pallial and mantal center of the experimental group were significantly increased(P<0.05),and the novel nacreous layer showed obvious disordered growth,which suggested that pma-mi R-67 was involved in the formation of nacre.Mi Randa software analysis found that pma-mi R-67 had binding sites with nine mineralization-related target genes,such as smad4 transcription factor and?-catenin of Wnt signaling pathway.3.pma-mi R-67 could be processed by Lnc MPEG1We used pc DNA3.1~+vector to overexpress Lnc MPEG1 in HEK293T cells.The expression levels of Lnc MPEG1 and pma-mi R-67 were significantly up-regulated.pc DNA3.1~+-Lnc MPEG1 vector and pma-mi R-67 complementary strand p GL3 vector were co-transfected into HEK293T cells,dual luciferase reporter vector system was used to detect the change of luciferase activity.The dual luciferase activity of the experimental group(pc DNA3.1~+-lnc MPEG1+p GL3-pma-mi R-67-sponge)was significantly down-regulated(P<0.05),indicated that cells overexpressed Lnc MPEG1 could generate pma-mi R-67.In addition,the expression level of pma-mi R-67 apparently inhibited in the Lnc MPEG1 decreased individual by RNAi.The above results indicate that pma-mi R-67could be processed by Lnc MPEG14.Cloning and expression analysis of Pm Ago2 geneAgo2 is an important molecule in the regulatory pathway of Lnc RNA and mi RNA.In this study,the full-length sequence of Ago2 in P.f.martensii(Pm Ago2)was 3,465 bp by RACE technique.The open reading frame was 2,679 bp and encoded 892 amino acid residues,with 5'UTR was 113 bp and 3'UTR was 673 bp.The predicted molecular weight was 101.1 k D and the theoretical isoelectric point is 9.48.Results of multiple sequence alignments showed that Pm Ago2 was highly conserved with vertebrates and other invertebrate Ago2,and with a highest similarity to Crassostrea virginica(88%).q RT-PCR result showed that Pm Ago2 expressed in various tissues with the highest expression in the mantle edge.We performed the western blotting experiment using vertebrate Ago2 antibody in mantle tissue extracted protein,the molecular weight of Ago2 was about 100 k D,which was consistent with the theoretical molecular weight.