| Pearls are the only renewable jewelry formed by biomineralization.They are mainly composed of the pearlite secreted by the mantle or pearl sac of mollusks.Pinctada fucata martensii is one of the most important marine cultures in China.The recipient shellfish can form pearl sacs and secrete pearlite stably to form pearls through Nucleus implantation and bead cultivation.the mantle tissue of Pinctada fucata martensii has different functional specialization,which form shell with two kinds of calcium carbonate crystals: nacreous layer and prism layer.Therefore,it is a good material for studying the formation of nacre.In recent years,great progress has been made in the study of the formation of pearlite from organic matrix,but there are relatively few reports on how shellfish control the expression and secretion of organic matrix.Previous studies have shown that long non-coding RNA participate in the regulation of various life activities through a multi-level regulatory mechanism.Based on the fine genome map of Pinctada fucata martensii,the Pearl matrix protein group and the known mineralization related genes,the LncRNA expressed in the mantle and pearl sac tissue was identified by RNA-seq.The LncRNA related to pearl formation was screened,and the effect of one LncRNA(Lnc Pm-009195)on the formation of nacre was identified.The results of this experiment are as follows:(1)In this study,the high quality reading length of about 945 M was obtained by RNAseq.About 66.72% of the Clean reads were comparable to the reference genome of Pinctada fucata martensii.A total of 48544 LncRNA were identified,including 36088 linc RNA,4807 anti-sense LncRNA and 7659 intronic LncRNA.It was found that the ORF length and exon number of LncRNA in Pinctada fucata martensii were smaller than the known protein coding genes.Through co-location analysis and co-expression analysis,seven LncRNAs were screened out to regulate the expression of six known mineralized genes.The correlation analysis of gene expression showed that 53 LncRNAs were correlated with 12 high abundance matrix protein genes in nacre,among which 10 high abundance LncRNAs were correlated with at least 4 high abundance matrix protein genes in nacre.In addition,it was also found that 21 LncRNAs differentially expressed in the marginal and central regions of the mantle might be related to the regulation of nacre matrix protein gene expression.(2)LncPm-009195 was selected for cloning and functional analysis.The intermediate fragment of Lnc Pm-009195 is 559 nt in length,located in scaffold 1362 and belongs to intron LncRNA.Sequence analysis revealed that there were four ORFs in Lnc Pm-009195,the largest of which was only 114 nt,encoding 37 amino acids,indicating that Lnc Pm-009195 had no potential to encode Macromolecular proteins.The expression of Lnc Pm-009195 in different tissues was detected by q RT-PCR.It was found that the expression of Lnc Pm-009195 in mantle,especially in central region,was significantly higher than that in other tissues(P<0.05),also found expressed in the Pearl sac.In addition,the expression of Lnc Pm-009195 was positively correlated with that of fibrinogen-related protein gene and tubulin beta gene in the central membrane.In situ hybridization experiments showed that Lnc Pm-009195 was mainly located in the lateral epithelial cells in the pallial zone and the lateral epithelial cells in the central membrane area.RNAi experiments showed that the interference of Lnc Pm-009195 gene expression with ds RNA in the mantle of Pinctada fucata martensii caused the abnormal crystal structure of nacre.These results suggest that Lnc Pm-009195 may play a regulatory role in the formation of nacre. |
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