Molecular Basis Of Pearl Formation From Biomineralization Of Hyriopsis Cumingii Based On High-throughput Sequencing

In China's freshwater pearl culture industry,the phenomenon of high yield and low value is very serious.It is urgent to improve the size and quality of freshwater pearls,and thus improve the efficiency of Pearl culture.This is also in line with the requirements of aquaculture transformation and upgrading and green high quality development.The mantle film is the preferred beading site,with small space and thin tissue,which limits the cultivation of large-nucleus pearls.The visceral mass not only has the space conditions for cultivating large-scale nucleated pearls,but also has the biochemical conditions for cultivating pearls.In this paper,focusing on whether the visceral mass has the molecular conditions for cultivating high-quality large-scale nucleated pearls.Though multi-omics analysis,the molecular resources of Ca²⁺metabolism and matrix protein in the process of pearl cultivation of mantle and visceral mass were excavated from the level of mRNA and protein.In this paper,the differential expression of biomineralization-related genes and proteins in the process of culturing pearls in the mantle and visceral mass of Hyriopsis cumingii was compared.The important genes VDCC?voltage-dependent calcium channel protein?and CABP?calcium binding protein?in pearl formation of Hyriopsis cumingii were validated and analyzed.This study laid a theoretical foundation for improving the cultivation technology of large-scale and high-quality nucleated pearls in visceral mass.The research is as follows:1.Based on Illumina technology sequencing platform,the transcriptome Library of mantle was constructed by Paired-End method.After data filtering and splicing,257,457 Unigenes were obtained.Compared with Nr,Nt,Pfam,Swissprot,KO,GO and KOG databases,223,345 Unigenes were annotated,the annotation rate reached 86.7%.The gene library of pearl formation and growth-related calcium metabolism and matrix proteins was screened and constructed from the annotated Unigenes.There were 1235Ca²⁺metabolic genes,including 415 genes directly related to biomineralization;426matrix proteins,97 of which were directly related to the formation of shells and pearls.2.The difference of gene expression between mantle and visceral mass pearl-raising of Hyriopsis cumingii was analyzed by high-throughput RNA-seq technique and digital gene expression?DGE?.The results of data analysis showed that there were significant differences in gene expression between mantle and visceral mass.The highest number of differentially expressed genes was 20 days?20 thd?after nucleation,followed by 120 days?120 thd?after nucleation,and there were significant differences in gene expression without nucleation.The least number of differentially expressed genes was 50 days?50 thd?after nucleation.The differentially expressed genes of matrix proteins were the most in 120 thd and the least in 50 thd.Compared with the control group,the number of differentially expressed genes in mantle and visceral mass increased gradually,while that in visceral mass increased first and then decreased.The number and type of differentially expressed genes in Ca²⁺metabolism and matrix protein were different.3.Protein profiles of outer membrane of the mantle,inner membrane of the mantle,pearl sac and visceral mass of Hyriopsis cumingii were determined by iTRAQ technique.A total of 11522 peptide segments and 1871 proteins were identified.The results of data analysis showed that the total differential proteins and Ca²⁺metabolism-related differential proteins showed the same regularity.and the number of differential proteins between the visceral mass and the other three samples?outer membrane of the mantle,inner of the mantle and pearl sac?.The difference proteins between visceral mass and pearl sac were the least,and the difference proteins between outer mantle and inner mantle were also more.The KEGG database annotates 241 pathways.Metabolic pathways have the largest number of genes.Calcium signaling pathways,which are closely related to pearl formation,calmodulin?CAML?,VDAC?voltage-dependent anion channel?,CaN?Calcineurin?,CAMK?Calcium/calmodulin-dependent protein kinase?are up-regulated.Proteins related to calcium metabolism were screened from the annotated data,including calcium binding protein?CABP?,calmodulin?CAM?,cadherin,Ryanodine receptor?RYR?,voltage-dependent calcium channel protein?VDCC?,etc.,and matrix proteins related to calcium metabolism were screened,including maternal protein family 1?matrilin-1?,N66,and lectin-like protein 1?ASL1?.4.The screened genes VDCC and CABP related to calcium metabolism were validated and analyzed.The full-length cDNA of VDCC is 1453 bp,and ORF was 439-1053 bp,encoding 204 AAs.Phylogenetic tree analysis showed that the VDCC gene sequence of Hyriopsis cumingii had low homology with other aquatic organisms.Differential expression analysis showed that pearl culture had an effect on VDCC gene expression,showing an increasing trend,but the difference was not significant.The cDNA sequence of CABP was 564 bp.BLAST analysis showed that the gene fragment had the highest similarity with the CABP gene of Pseudocardium sybillae and Tapes philippinarum.Differential expression analysis showed that the expression of CABP gene was highest in mantle tissue and lowest in gill tissue.The expression level of other tissues increased except gill tissue,and the growth level of inner and outer mantle tissues reached a significant level?P<0.05?.The expression of CABP gene increased with the increase of Ca²⁺concentration in different concentrations of Ca²⁺incubation.The results of correlation analysis showed that the mRNA expression level of CABP gene was negatively correlated with extracellular Ca²⁺flux?R=-0.993;P<0.05?,and positively correlated with intracellular Ca²⁺fluorescence intensity?P<0.05?.Based on the above,the study concluded that visceral mass had molecular conditions for Pearl cultivation,but compared with mantle,the basic metabolism was different,and the molecular regulation pattern in pearl cultivation was different.