The Studies Of Biomineralization Related Genes Based On Multi-omic Analyses In Pinctada Fucata Martensii

The mollusk shells consisted by calcite,aragonite or vaterite were the products of biomineralization,with morphological diversity.The nacreous layer?nacre?,as the main component of pearl is iridescent and resilient,and it provides superior strength and toughness;and the mechanism of shell formation nacre formation is attractive.The method of multiomic analyses improve the development of biomineralization of shell formation.The pearl oyster Pinctada fucata martensii is the main shellfish species used to product the marine pearl.In this study,on the basis of genome data of P.f.martensii,through performing the multi-omic analyses including genome,transcriptome and proteome,the vital shell matrix gene families and biocalcify-related metabolism and regulation factors were explored.And the important transcription factor PmRunt was discovered and its functions in nacre formation were detected.The main contents were as follows:1 Multi-omic analyses discovered the vital genes related to biomineralization?1?Genome evolution and exploration of biomineralization-related gene familiesNine species genome data including Capitella teleta,Helobdella robusta,P f martensii,Crassostrea gigas,Lottia gigantea,Homo sapiens,Danio rerio,Octopus bimaculoides and Aplysia californica,were used to cluster the gene families.Depended on that,the gene fimily expansion and contraction analyses were performed by CAFé.The results of evolution analyses show that calcium signal pathway and mineral absorption,the biosynthesis of Glycosaminoglycan?GAG?and Polymer glucosamine offered material basis.In these processes,the expansion of sulfotransferases is consistent with the abundance of GAGs in nacre.The expanded shell matrix protein families in both P.f.martensii and C.gigas are filtered.The results show that fibronection-like proteins?FN?are responsible for both shell formation;the expansion of tyrosinase?TYR?reveals unbalanced and lineage-specific expansion;VWA-containing proteins?VWAP?prefer to participate in nacre formation.The specific shell matrix protein families with amino acid preference and repeat motif belonging to RLCD?related low complexity domain?in P.f.martensii also play critical roles in shell formation.?2?Transcriptome analyses of eight tissues and twelve development stagesThe main results show belong.?1?The expression pattern of shell matrix proteins shows up-regulation at trochophorge stage?T?and Post-veliger stage?PV?,which is correspond to the formation of protoconch and dissoconch.?2?Mantle tissue is the main organ that contribute to shell formation.To some extent,haemolymph and adductor muscle also could contribute to shell formation.?3?The expanded and the lineage-specific families that pointed in genome evolution analyses including enzymes related to chitin biosynthase,sulfotransferases,FNs,TYRs,VWAPs and RLCDs,partly highly expressed in T stage and/or PV stage as well as highly expresses in mantle tissues and/or pearl sac,indicating the vital roles in protoconch and dissoconch formation.?4?The expression pattern of regulation factors at different development stages and in different tissues shows that Wnt,BMPs,VEGF,MAPK and osteoclast differentiation signal pathway and transcription factors such as Runt,SMAD,AP-1 could involve in the regulation of shell formation;And some hormones including estrogen,vitamin D3,thyroid hormones and ecdysone could participate in the dissoconch formation in PV stage.2 PmRunt and its related pathway regulate nacre formation?1?PmRunt regulates nacre formationIn this work,we clone runt-like transcriptional factor?designate as PmRunt?and CBF?designate as PmCBF?gene,which comprise the heterodimeric transcriptional factor in Pincatada martensii.PmRunt is identified with open reading frame-comprised nucleotides encoding545 amino acids,with a typical Runt domain.Phylogenetic analysis revealed that runt-like transcriptional factors?RDs?in vertebrates and invertebrates are separated into two branches.PmRunt and other RDs from mollusk cluster into one branch.Direct interaction between PmRunt and PmCBF is evidenced by yeast two-hybrid assay.Gene repression by RNA interference decreases the expression level of PmRunt,and subsequent observation of the inner surface of the nacre by SEM demonstrated disordered growth.RNA-seq results show that the expression level of some nacre matrix protein coding genes are down-regulated,such as carbonic anhydrase-like,TYR,VWAP and MG4.RO5-3335 is used to inhibit the interaction of PmRunt and PmCBF to decrease the interaction of PmRunt to DNA.The results show that the expression level of MG4s?Pma₃18.183 and Pma₁0000069?,VWAP?Pma₁0019836?,TYR2?Pma₁0029257?and Nacrein decrease.The luciferase activity of reporters containing promoter regions of VWAP?Pma₁0019836?and Nacrein were enhanced by PmRunt.Therefore,PmRunt could involve in nacre formation by regulating the expression of shell matrix protein coding genes.?2?MG4s reluated by PmRunt and its homologue genes?C1qDC?could participate innacre formationMG4 belongs to C1 q domain containing protein?C1qDC?family.We analyses the expression pattern of C1 qDCs that expanded in P.f.martensii and C.gigas.The results show that some of C1 qDCs highly expresses in mantle tissue in P.f.martensii.Gene repression by RNA interference decreases the expression level of Pma₃18.183,Pma₅76.501 and Pma₁0012374,and subsequent observation of the inner surface of the nacre by SEM demonstrated disordered growth.These findings indicated that C1 qDCs could affect nacre formation.?3?Ecdysone?EC?could regulate the expression of PmRunt and other biomineralization related genesIn this study,we clone the ecdysone receptor?EcR?and its potential binding protein retinoid X receptor?RXR?.The Elisa detection result shows that the concentration of EC are up-regulated at 2h and 36 h after shell damage.The mantle pallial fragment treated by EC with 40ng/L at different times could affect the mRNA expression of PmRunt and other biomineralization related genes,such as BMP2/7,VWAP,TYR2,KRMP and CHS,with different expression profiles.These results indicate that EC could be responsible for shell formation through regulating the expression of PmRunt and other biomineralization related genes.3 miRNAs could mediate the negative regulation of PmRunt and NF-?B pathwayIn this study,Pm-miR-183 obviously inhibited the relative luciferase activity of reporters containing the 3?-UTR of PmRunt.The expression level of PmRunt was repressed after Pm-miR-183 over-expression in mantle tissue,and subsequent observation of the inner surface of the nacre by SEM demonstrated disordered growth.This result indicates that Pm-miR-183 could regulated nacre formation via mediating expression of PmRunt.In this study,a novel miR-146 a is identified from P.f.martensii?designated as Pm-miR-146a?.Pm-miR-146 a ubiquitously expressed in all examined tissues,with the highest level in the mantle and lowest expression in the haemolymph.The over-expression of Pm-mi R-146 a in vivo could significantly inhibit the expression of macrophage migration inhibitory factor?MIF?,the potential target gene predicted by miRanda,while enforcin g Pm-miR-146 a involved in the down-regulation of NF-?B.Thus,we propose that Pm-miR-146 a plays a role of negative feedback regulation to the NF-?B signal by repressing the expression of MIF.