Analysis Of Transcriptome Sequencing And Differential Expression Genes Of Pearl Color In Hyriopsis Cumingii

The freshwater mussel Hyriopsis cumingii is an important aquaculture species,of which the pearl production accounted for more than 98%in the world.The pearl is natural organic gemstones,which can be used as drugs,accessories and can be used in the field of cosmetics.The biological characteristics of pearl in the bionics materials have attracted researchers' interest.Color is an important indicator affecting the pearl value and quality.Hence,study on the molecular regulation of pearl color of Hyriopsis cumingii will contribute to foster specific colors pearl.There is not much research on molecular biology of molecular mechanism of pearl color in Hyriopsis cumingii,therefore,mining a large number of genes related to pearl color in Hyriopsis cumingii is of great theoretical and practical significance.For screening candidate genes related to pearl color of Hyriopsis cumingii,this research has undertaken Illumina HiSeqTM 2500 transcriptome platform on ventral mantle tissue of Hyriopsis cumingii with purple and white narce color for high-throughput sequencing.Based on the transcriptome library of Hyriopsis cumingii,we have obtained a large number of Unigene sequences,developed lots of SSR makers,which will offer reference for related research of genetic relationship,population genetics and molecular mark assisted breeding analysis.The main results of the research were as follows:1.Sequencing and analysis of transcriptome of Hyriopsis cumingiiThe transcriptome of the Hyriopsis cumingiis 'ventral mantle tissue which the nacre colors were white and purple was sequenced by high-throughput sequencing technology in order to excavate candidate genes involved in pearl color produced by Hyriopsis cumingii.After quality control and assemble,these sequences acquired were blasted against NR,Swiss-Prot,COG,KOG,KEGG,GO and Pfam databases,and then cluster analysis were performed.In total,obtaining 89,529 Unigenes were acquired,and 2,644 genes were differentially expressed,among which 1,323 were up-regulated and 1,321 down-regulated.7,539 SSR makers and 1,759,869 SNP makers were filtrated throughout the transcriptomic data.The Unigenes GO function in the transcriptome library were divided into 50 branches;taking the KEGG database as a reference,it could be divided into 187 classes.There was differentially expressed analysis revealed that some genes were related to pigment(such as xanthommatin,melanin and pteridine)synthesis and transfer.And there were 6 cytochromeP450,3cytochromeb561 and 1 cytochrome b560 genes.They might play important roles in the formation of pearl color of Hyriopsis cumingii.These genes might play role in the formation of pearl color.2.The distribution characteristics of SSR makers and the development and utilization of primers in Hyriopsis cumingiiWe isolated 7,539 SSR loci from 89,529 Unigenes generated by high-throughput sequencing of the Hyriopsis cumingii transcriptome library.Primer designing was taken for these loci,among which 150 random primers were chosen for PCR amplification,102 of which have obtained clear,reproducible bands.30 mussels were used for 102 primers' polymorphism testing,and the result showed 40 loci were polymorphic.The number of alleles of 40 loci ranged from 2 to 15,with average number of alleles 6.63,the observed and expected heterozygosities ranged from 0.0333 to 0.9667 and from 0.3254 to 0.8977,respectively,and the average values were 0.5107 and 0.7054respectively.The average polymorphism information content was 0.6468.There remained 13 loci deviating from equilibrium through using the Hardy-Weinberg principle(?2 test)and sequential Bonferroni calibration.3.Expression analysis of genes related to pearl colorWe selected 5 color related genes from the transcriptome library:tyrosinase gene(HcTyrl),astacin metalloproteinases gene(HcAst),cytochrome P450 gene(HcCyt),tryptophan dioxygenase gene(HcTDO)and uroporphyrinogen decarboxylase gene(HcUROD).The mantle center(MC)of Hyriopsis cumingii mainly involved in nacre formation while the posterior mantle pallial(pMP)and anterior mantle pallial(aMP)mainly involved in prismatic layer and nacre formation.Tyrosinase encoded by HcTyr1 gene was involved in the synthesis of melanin.Real-time Q-PCR revealed that HcTyr1 gene expressed mainly in the pMP and aMP,low expression in MC and almost no expression in other tissues.HcTyrl s expression level of the pMP in purple mussels was significantly higher than that of white mussels(P<0.05),suggesting that HcTyrl gene might participate in color formation of nacre and prismatic layer in Hyriopsis cumingii and have great relevance with purple nacre color.Astacin metalloprotease encoded by HcAst gene was zinc-dependence.The HcAst gene was expressed in different tissues of Hy,riopsis cumingii.The expression of HcAst gene in aPM and pMP of purple mussels was significantly higher compared with corresponding tissues in white mussels(P<0.01),while HcAst gene expressed in MC of purple mussels was higher than white mussels(P<0.05),indicating that HcAst gene took part in purple nacre color formation of Hyriopsis cumingii.Cytchrome P450 encoded by HcCyt gene was a kind of cytochrome.The HcCyt gene was ubiquitously expressed in all tissues with the highest expression level in hepatopancreas.It was speculated that HcCyt gene might be involved in the metabolism of xenobiotics.In the purple mussels,the expression level of HcCyt gene in MC was significantly higher than white mussels(P<0.01),hence,inferring that HcCyt gene might partook in purple nacre color of Hyriopsis cumingii.Tryptophan dioxygenase that was encoded by HcTDO gene participated in the synthesis of xanthommatin.HcTDO gene was mainly expressed aMP and pMP tissues with very low expression in level in other tissues.HcTDO expression level of pMP in purple mussels was significantly higher than that of white mussels(P<0.01),indicating that HcTDO gene had great relevance with purple nacre color formation of Hyriopsis cumingii.Uroporphyrinogen decarboxylase which was encoded by HcUROD gene took part in heme synthesis.The HcUROD gene was expressed in all tissues of Hyriopsis cumingii.In the purple mussels,the expression of HcUROD gene in pMP was significantly higher than MC(P<0.01),which was the same with white mussels,and was higher than white mussels in mantle,suggesting that HcUROD gene was involved in color formation of prismatic and nacreous layer and had great relevance with purple nacre color.