Identification And Characterization Of Long Non-coding RNAs In Goat Perirenal Fat

In the process of goat development,the fetus from the maternal fetus to the outside of the body,the changes in the external environment have a certain impact on the body temperature,while the brown adipose tissue(BAT)can produce non-thrilling heat to the fetus one week before birth.Brown adipose tissue(BAT)plays the role in the thermogenesis and energy storage.However,knowledge of cellular transition from BAT to WAT in goat is limited.The perirenal fat is BAT in newborn,and the perirenal fat changes transition from BAT to WAT with age.Long non-coding RNA(lncRNA)is a non-coding protein RNA of more than 200 nt,which can interact with transcription factors and proteins to participate in gene expression regulation.Therefore,the identification and analysis of lncRNA in different stages of goat perirenal fat can better understand the function of lncRNA fat regulation.In this study,the perirenal adipose tissue of different stages of the development of Chuanzhong black goats(D1,D30 and Y1)was used as the research material.The histological morphology of goat perirenal fat at different stages was first identified and passed through white,beige and brown fat cells.The marker gene was detected by q PCR to identify the type of adipose tissue.Subsequent,we used RNA-seq technology and lncRNA prediction software to identify lncRNAs that may play a role in three stages of goat perirenal fat.Further,the differential expression lncRNA was screened,and then the cis and trans target genes were predicted.Functional analysis was performed by GO analysis and KEGG analysis.Finally,the sequencing results of some lncRNAs were detected by q PCR.The main results are as follows:1.It was found by hematoxylin-eosin staining that the goat’s perirenal fat had a small number of lipid droplets and a small volume at the 1st day of birth.The nucleus was located in the center of BAT and surrounded by tiny lipid droplets,forming a multichamber structure.At 30 days after birth,it turns into white fat cells(WAT),the fat cells become larger,the nucleus moves to the edge,and the fat cells change from multichamber structure to single-chamber structure,from 30 days to 1 day after birth,white fat cell diameter Significantly increased with area,and the cells gradually increased.2.By immunohistochemistry,we observed UCP1 expression in goat perirenal fat at different stages.The uncoupled protein 1(UCP1)was at the highest level in 1 d and significantly down-regulated in 30 d,after which it became nearly undetectable in 1-year goats.The results show that UCP1 gradually decreases with the disappearance of BAT.3.Besides,LHX8,DIO2 and MPZL2 also reached its peak at birth,and then significantly increased in 30 d and 1 y.In addition,TCF21 in goat was at the lowest level in 1-day goats and then significantly increased to a peak in 30-day goats.In conclusion,BAT existed in goat perirenal fat at birth.LHX8,DIO2 and MPZL2 are suitable marker genes for BAT and TCF21 is a suitable marker gene for WAT in perirenal fat.4.A total of 12 samples,a total of 213.45 Gb Clean Data long-chain non-coding RNA analysis,each sample Clean Data reached 16.47 Gb,Q30 base percentage is not less than 93.76%.The Clean Reads of each sample were sequence-aligned with the designated reference genome,and the efficiency of the alignment was between 95.75%and 96.96%.5.we identified lncRNA in perirenal fat by deep RNA-sequencing using the Chuanzhong black goats.A total number of 21232 lncRNA from perirenal fat were identified,including 5393 intronic-lncRNA and 3546 antisense-lncRNA.In addition,the characteristics of the candidate lncRNA were analyzed,and it was found that the average length of lncRNA was 1700 bp,and the average lncRNA contained 2.51 exons.The result showed that lncRNA had fewer exons than m RNA,shorter length,and lower transcripts encoding potential protein.(Fold change ≥ 2.0,FDR < 0.05).6.in pairwise comparisons between the libraries of perirenal fat at the different development stages a total of 688 transcripts were differentially expressed,including257 up-regulated and 431 down-regulated.7.We predicted the cis and trans target genes of lncRNA,and classified them by GO and KEGG,and found many biological process items and signal pathways related to fat development,such as fatty acid elongation,biosynthesis of unsaturated fatty acid and AMPK signaling pathways.8.Then,we randomly selected 6 lncRNA,and detected the microarray data of those lncRNA by RT-q PCR.It was found that the RT-q PCR results showed consistent with RNA-seq sequencing results in expression at different developmental stages,indicating that the RNA-seq sequencing results were reliable.These results suggested that lncRNA and their target genes might participate in the biosynthesis of unsaturated fatty acid and provide valuable information for better understanding perirenal adipocyte.