| Silkworm, also named bombyx mori, is an important economic insect, whose cocoon is the main economic value. The B.mori silk gland begin to develop, then turn the mulberry into silk protein, finally, silk protein is secreted out. The whole process is related to the entire gene regulatory networks. Understanding differences in regulation of gene expression is a fundamental understanding of the difference of silk production between species. It is possible to improve the yield of silk production. In this study, we identify potential regulatory sites by FAIRE-seq and differences in gene expression by RNA-seq between the robust and high yield silk production strain 872 and low yield silk production strain Dz in the 3rd day of the fifth instar(5v3d). Hoping to find the regulatory sites which can be influencd the yield B.mori silk production.1. Identify potential regulatory sites by FAIRE-seqWe respectively identified 103,779 and 102,457 confidence peaks by FAIRE-seq data analysis in strain 872 and Dz, covering ~10% of the genome. It is probable that the large quantities of silk proteins were synthesized, needing regulatory sites of many genes were opened in this period. Difference peaks were respectively 11,947 and 10,600 in strain 872 and Dz, ~90% of common peaks totally. It shows small difference regulatory sites between species. The average FAIRE signal ±1kb around TSSs acrossed all genes analysis showed a strong nucleosome-free region ~125 bp upstream of the TSSs. Peaks length distribution statistical analysis found presented a continuous distribution, a large number of peaks was short than 500 bp and the longest peak was shorter than 3000 bp. Created a annotation of the regulatory sites, we found that a number of known regulatory sites localed in open chromatin regions, included regulatory elements of fib-H, fib-L, P25 and ser1 genes.Through research and analysis of FAIRE-seq experiments, we have successfully obtained open chromatin maps of silkworm in 5v3 d, which contained enhancers, promoters, and other important regulatory elements. We have also obtained the differentcandidate regulatory elements between the strain 872 and Dz in 5v3 d.2. Identification of the genes which may influence the yield of silk by RNA-seqBase on the Pearson correlation and map rate, we choose four biological duplicates in each samples. We found that, in the expression of top20 genes, many of them were silk protein genes, elongation factors which is necessary to translation process and ribosomal protein genes which were the minimal place of silk protein synthesis. Silk protein genes expression analysis found that the expression of silk protein gene expression levels in the straind 872 are higher than Dz. By analysis of the differential expression genes(DEGs), we identified 1657 DEGs. KEGG and GO enrichment analysis of DEGs showed that many genes involved in protein synthesis and secretion process. GO enrichment analysis found that there were many genes involved in the transport of protein and transcriptional regulatory of genes in molecular function category. Many of the DEGs enriched in purine metabolism, the protein processing in endoplasmic reticulum, ribosome and pyrimidine metabolism pathway. The Pathways DEGs enriched are closely related to the synthesis and secretion of silk proteins.3. Analyse the transcription regulatory elements combined RNA-seq and FAIRE-seq dataIn this part,We identified 131 DEGs which had differential peak/peaks in their promoter region. There were 40 genes of strain 872 expression levels up-regulated whose promoter region had differential peaks in strain 872, and 35 genes of Dz expression levels up-regulated whose promoter region had differential peaks in strain Dz. we defined them as the candidate of the positive regulatory regions(CPRRs); In contrast, there were 30 genes of strain 872 expression levels up-regulated whose promoter region had differential peaks in strain Dz and 29 genes of strain Dz expression levels up-regulated whose promoter region had differential peaks in strain 872. we defined them as the candidate negative regulatory regions(CNRRs). There was 3 genes whose upstream had different peaks in both strain.GO enrichment analysis found that genes were enriched in catabolic metabolism and biosynthesis. These genes may have important significance for synthesis and secretion of protein. The expression levels of genes influenced by candidate positive regulatory regions and negative regulatory regions, whose function published by KEGG enrichment analysis.(1) Genes related to the CNRRs, up-regulated genes of 872 were mainly enriched in basic metabolism pathways, like glucose metabolism and lipid metabolism pathways, and in Ribosomeã€Protein processing in endoplasmic reticulum pathway or protein transport pathway. In other hand, down-regulated genes of 872 mainly were related to signal pathways. Basic metabolism pathways may provide energy for synthetic and secretion of silk protein; At the same time, ribosome which is the smallest place of protein synthesis, The large number of genes in ribosome can effectively increase the synthesis of silk protein; The proteins which is in endoplasmic reticulum and related to protein transport were favor to silk protein synthesis.(2) In contrast, Influenced by CPRRs, up- and down-regulated genes of strain 872 were enriched in glucose metabolism and lipid metabolism pathways, and Ribosome 〠Protein processing in endoplasmic reticulum pathway or protein transport pathway. Besides, there are some additional upregulated genes associated with immune pathways in strain, such as lysosomes, NODlike receptor signaling and antigen processing and presentation. Possibly because the strain is the robust and high yield silk production line.4.The list of regulatory regions associated to yield silk production and motif analysisIn totally, we identified 40 candidate positive regulatory region and 29 negative led to genes upregulated of strain 872, motif predicted analysis found:(1) Through JASPAR online annotation software analysis, and GO enrichment analysis, 67.5% of CPRRs can bind the transcription factor upregulated gene expression. Many of them were the HOX transcription factor binding region, such as fkh. These factors have been reported that they can regulate the expression of silk protein genes. And Ubx plays an important role on silk gland development. A small number of CPRRs were annoted that they could regulate gene expression, but uncleared up-regulated or down-regulated.(2) 72.7% of the CNPRs can bind the transcription factor down-regulated gene expression. For example, the upstream of Notes α-1,2-glucosidase gene had CTCF inhibitory factor binding site opened in Dz, This may be related to the expression of glycoprotein P25 gene. What’t more, There are some important inhibitory factors, like gt and spl1, may affect the silk protein gene expression. A part of CNPRs were thought can regulate gene expression, but unclear up-regulated or down-regulated. Even,several of them is associated to up-egulated. |
