Identification And Functional Analysis Of Rumen Specific Long Noncoding RNA In Sheep

The rumen is a unique digestive organ of ruminants.Its keratinized squamous epithelium and other structures provide an environmental basis for digestion,absorption and microbial fermentation.Recent studies have shown that rumen development may originate from the esophagus,and it has recruited expressed genes from other organs during evolution,which has strengthened functions such as keratinization,ketone metabolism,and immunity.The genes related to rumen functions are partially known,but their expression regulation mechanisms,especially the regulation of long noncodingRNA(lncRNA)levels,need to be studied.lncRNA has an important regulatory effect on gene expression.Exploring the genetic mechanism of important economic traits of livestock from the perspective of lncRNA has important theoretical significance for breeding.This research takes the ruminant livestock--sheep(Ovis aries)as the research object,and uses the transcriptome sequencing data of 50 sheep tissues and a total of 832 samples to predict and analyze lncRNA.Then we screened the lncRNA specifically and highly expressed in the rumen,which we called rumen specific lncRNA.We predicted and analyzed the function of rumen specific lncRNA.Then we use the embryonic esophagus and rumen transcriptome and chromatin accessibility high-throughput sequencing technology(ATAC-seq)to compare and study the function of rumen specific lncRNA during development.The main results are as follows:(1)Using a variety of prediction methods,a total of 10006 high-confidence lncRNAs were identified in multiple sheep tissues.Among them,2045 lncRNAs have been annotated in the reference genome of sheep version 4.0,and 7961 lncRNAs are novel lncRNAs.Compared with mRNA,lncRNA has a shorter sequence,fewer exons,and low expression.Cluster analysis of lncRNA in different tissues shows that the results basically conform to the law of development and tissue differentiation.Through the correlation analysis of tissue-specific lncRNA,it is found that the reticulum,esophagus,and omasum are the closest to rumen lncRNA,and the correlation with keratin is relatively high.(2)Through tissue-specific expression analysis,343 rumen specific lncRNAs were screened.The co-expression network analysis of rumen specific lncRNA and all mRNA expressed in the rumen showed that rumen specific lncRNA mainly aggregated in three expression modules,one of which contained 42 lncRNAs and 492 coding genes,and GO analysis of coding genes showed significant enrichment.In cell epithelial development,cell epithelial differentiation,keratinocyte differentiation and other biological processes(P<0.05).This module contains 6core lncRNAs,whose expression is related to IVL,Cart14 and other epithelial cell differentiation-related coding genes.In addition,410 rumen specific lncRNA adjacent genes were screened,and the GO enrichment results showed that they were related to the migration function of epithelial cells.(3)The expression of 30 rumen specific lncRNAs was significantly up-regulated in the rumen relative to the esophagus during the embryonic period,and 11 of them belonged to the coexpression network of rumen epithelial cell differentiation modules,indicating that these lncRNAs involved in the differentiation process of rumen epithelium have begun to play a role in the embryonic stage.The newly discovered lncRNA MSTRG.39536.2 is a lncRNA that differs significantly from the rumen and esophagus during embryonic stage It is distributed in the differentiation module of rumen epithelial cells,and its neighboring genes are the key transcription factors of rumen epithelium and are highly expressed in rumen epithelial cells.(4)By using the embryonic rumen and esophagus ATAC-seq to study the open area of rumen chromatin,the results show that 8 rumen specific lncRNAs were located in the rumen specific opening and accessible peaks.Among them,LOC105611675 is a reported lncRNA.Its transcription start site is located on the rumen specific open and accessible peak,and is highly expressed in the rumen.At the same time,its adjacent gene GJB2 is also related to the differentiation of rumen epithelium.In summary,this study has enriched the sheep multi-tissue lncRNA database,and screened out a series of specifically expressed lncRNAs that may regulate rumen function.It also proves that sheep rumen specific lncRNA intensively participates in the regulation of the differentiation process of rumen epithelial cells,and this regulation may have occurred in the embryonic period,laying a theoretical foundation for the research on the function of rumen fermentation in ruminants.