Preliminary Analysis Of Differentially Expressed LncRNA And MRNA For Adaptation To Hypoxia In Tibetan Sheep

Tibetan sheep have formed a unique hypoxia adaptation mechanism in the process of selection and evolution of alpine and low-oxygen stress.Tibetan sheep’s blood characteristics,blood gas indexes and lung tissue structure changes are all studied on the adaptability of plateau animals.In recent years,the study of long non-coding RNA(lncRNA)regulating hypoxia adaptation in Tibetan sheep has become one of the popular areas.In order to reveal the mechanism of hypoxia adaptation in Tibetan sheep,we analyzed the physiological and biochemical characteristics,tissue structural characteristics and differentially expressed lncRNAs in Tibetan sheep.In this study,we select Holba sheep(HB),Awang sheep(AW)in high altitude areas of Qinghai Tibet Plateau(4452-4468 m),Ganjia sheep(GJ),Qilian white Tibetan sheep(WT)in medium altitude areas of Qinghai Tibet Plateau(3620-3851 m)and Hu sheep(HS)in low altitude in the Qinghai Tibet Plateau(-67 m)as the research objects.The serum physiological,biochemical and blood gas indexes of HB,AW,GJ,WT and HS were measured by automatic hematology analyzer,automatic biochemical analyzer and blood gas analyzer.The lung tissue structure was determined by histological method,and immunoglobulin was detected by kit;RNA was extracted from the liver and lung tissues of Tibetan sheep(Hb,AW,GJ,WT)and HS.The transcriptome was sequenced by Illumina Hiseq 2500 sequencing method.Taking |log2 FC| >1 and P<0.01 as the conditions for screening differentially expressed genes,the differentially expressed lncRNAs in the liver and lung tissues of Tibetan sheep(2high altitude and 2 medium altitude)and HS(low altitude)were analyzed,explore the signal pathways related to the adaptation to high altitude hypoxia.(1)With the increase of altitude,there were significant differences in serum physiology and biochemistry,blood gas indexes and immunoglobulin between Tibetan sheep and HS(P<0.01).The diameter of pulmonary terminal bronchioles increased significantly,the number of alveoli per unit area and the thickness of alveolar septum increased significantly between Tibetan sheep and HS(P<0.05).The results show that Tibetan sheep adapt to altitude cold and hypoxia stress by improve the ability and efficiency of carrying oxygen through hemoglobin,increase the oxygen exchange area of lung tissue and enhance immunity.(2)Completed the lncRNA sequencing of 15 samples of liver tissue from HB,AW,GJ,WT and HS,3 replicates of each breed.We obtained 187.90 GB Clean Data.The Clean Data of each sample reached 10.00 GB,Q30 base percentage above 85.01% respectively;the Clean Reads of each sample were sequenced with the specified reference genome,the average alignment rate was 82.21%.A total of 20,078 mRNAs were identified in liver tissue,and 2,728 new genes were discovered.1,153 new genes were functionally annotated.Differentially expressed mRNAs,lncRNAs and their target genes were enriched to 13 pathways in KEGG among the four groups,which were mainly related to people disease,fat metabolism,cytokine-cytokine receptor interaction;according to the expression amount of genes in different samples,495 differentially expressed genes were identified,and their functions were annotated and enriched;6,249 lncRNAs were identified.79 lncRNAs were differentially expressed.q RT-PCR was used to verify 11 mRNAs,3 lncRNAs and 11 target genes related to hypoxic adaptation.The expression levels were consistent with the sequencing results.(3)Completed the lncRNA sequencing of 15 samples of lung tissue from HB,AW,GJ,WT and HS,3 replicates of each breed.We obtained 190.63 GB Clean Data.The Clean Data of each sample reached 10.46 GB,Q30 base percentage above 90.92% respectively;the Clean Reads of each sample were sequenced with the specified reference genome,the average alignment rate was 81.89%.A total of 13,319 mRNAs were identified in lung tissue,and 750 new genes were discovered.588 new genes were functionally annotated.Differentially expressed mRNAs,lncRNAs and their target genes were enriched to 5 pathways in KEGG among the four groups,which were mainly related to amino acid and fatty acid metabolism;according to the expression amount of genes in different samples,1,233 differentially expressed genes were identified,and their functions were annotated and enriched;1,798 lncRNAs were identified.151 lncRNAs were differentially expressed.q RT-PCR was used to verify 7mRNAs,4 lncRNAs and 2 target genes related to hypoxic adaptation.The expression levels were consistent with the sequencing results.Through the study of serum physiology and biochemistry,blood gas index,immunoglobulin and lung tissue structure,this study showed that Tibetan sheep not only formed the unique physiological and biochemical characteristics and lung tissue structure of effective uptake,transportation and utilization of oxygen,but also enhanced humoral immune response.It shows that the lung tissue of Tibetan sheep adapts to high altitude hypoxia with important morphological and histological characteristics and immune characteristics.Using the Illumina Hiseq 2500 sequencing and bioinformatics analysis methods,the hypoxia adaptability of two tissues(liver and lung)between four Tibetan sheep breeds living at different altitudes on the Qinghai Tibet Plateau and low altitude HS were analyzed.The differentially expressed mRNA,lncRNA and their target genes related to hypoxia adaptability were screened,and their expression levels were verified by q RT-PCR.The study provides a theoretical basis for revealing the molecular mechanism related to high altitude hypoxia adaptation of Tibetan sheep,and further provides guidance and reference for studying the genes related to high altitude hypoxia adaptation.It also provides a reference for the prevention and treatment of adverse reactions caused by high altitude hypoxia in humans and other animals.