Study On The Function And Mechanism Of Histone Methyltransferase BmTrx To The Cocoon Silk Of Bombyx Mori

The silkworm is more than just a model organism in the Lepidoptera family.Its wide range of cocoon silk traits also makes it an important economic animal.However,traditional genetic breeding methods have reached a bottleneck.To address this,modern molecular biology combined with high-throughput sequencing has been used to identify cocoon silk-related genes.These genes are then verified through in vivo experiments.This is significant because it helps us analyze the molecular mechanisms of cocoon silk traits and silk gland development in the silkworm.Not only does this increase the industrial value of silkworm,but it also lays the foundation for developing efficient silk gland bioreactors in the future.Building on our group’s previous QTL mapping studies,we conducted an extensive investigation of the important gene,BmTrx,located within the QTL region.In this study,we utilized CRISPR/Cas9 genome editing technology to construct a BmTrx knockout mutant,Trx^ko,which displayed a significant phenotype of reduced cocoon shell weight.Through HE staining,we found that knockdown of BmTrx had a significant impact on silk gland development in silkworms,characterized by tumor-like protrusions,decreased silk protein content in cells,and abnormal protein deposition in the curved part of the posterior silk gland of the Trx^ko mutant.This suggests that BmTrx likely regulates the synthesis and secretion of cocoon silk by influencing silk gland development in silkworms.BmTrx also encodes a histone methyltransferase in silkworm genome that catalyzes histone H3K4me3,which is a conserved modification that is capable of participating in the regulation of gene expression.Therefore,we first used Western blot and immunofluorescence to verify that knockdown of BmTrx can reduce the level of histone H3K4me3 modification in the posterior filopodia of the mutant.CUT&Tag sequencing was then performed to verify the location of histone H3K4me3 modification in the genome of the posterior filament gland.GO enrichment analysis of those genes that lost H3K4me3 modification at the promoter revealed that DNA binding and transcription factor regulation were the most significant entries,and we subsequently investigated several reported transcription factors that can regulate silk traits in the callus of the silkworm,showing that SGF1,Awh,SGF3,and sage genes all showed reduced levels of H3K4me3 at the promoter.The differential expression of these genes was then verified by q RT-PCR.Because these genes are involved in the transcriptional regulation of the fibroin heavy chain,we then verified the downregulation of Fib-H and Fib-L gene expression in Trx^ko mutants using q RT-PCR.Since BmTrx has a DNA-binding protein domain,it is also able to act as a transcription factor to directly regulate gene expression.Therefore,we used RNA-seq to demonstrate the difference in expression patterns between Trx^ko mutants and Nistari,identified multiple differentially expressed genes,and found a massive and significant upregulation of spliceosome and ribosome biogenesis pathway genes by KEGG enrichment analysis.In addition,2 DNA methyltransferases were found to be up-regulated,and in subsequent WGBS sequencing,BmTrx was shown to affect DNA methylation by affecting DNA methyltransferases,and enrichment analysis again enriched to the spliceosome pathway and found higher levels of DNA methylation in some of these genes.We then combined alternative splicing analysis and q RT-PCR to demonstrate the presence of differential alternative splicing events.In addition,we integrated multi-omics data to demonstrate a positive correlation between H3K4me3 and gene expression.In summary,BmTrx,as a multifunctional gene,is able to participate in multiple pathways to regulate callus traits in the silkworm.