| piRNAs are a type of small non-coding RNAs with a length of 26-44 nt that interact with PIWI family proteins different from mi RNAs and siRNAs.It is mainly distributed in the gonads,which are unevenly distributed on the chromosomes,generally clustered,mature The 2-OH of the3'end of piRNA is modified with methylation,and the 5'end has obvious uracil preference.piRNAs participate in heterochromatin formation,transcription and post-transcriptional silencing,inhibit transposons,protect the germline genome from DNA damage and mutations,and ensure the reliable transmission of genetic information from generation to generation.Xiang pig is a local small pig breed in Guizhou,which is an important economic animal.It is also an important experimental animal and model organism because of its small size,high physiological similarity with humans and other aspects.The number of fragrant litters is low but sexual maturity is early.The boar enters puberty at three months of age and reaches sexual maturity at six months of age.In this study,high-throughput sequencing and bioinformatics analysis were used to screen out the piRNA sequences at various developmental stages,and analyze their characteristics,chromosome distribution,origin,etc.,and perform GO enrichment analysis and KEGG pathway analysis on their source genes.At the same time,the piRNA cluster was also analyzed.The main results obtained in this study are as follows:1.Using high-throughput sequencing and bioinformatics analysis,100335 known piRNAs and 70514 candidate piRNA sequences were screened from 2 months(weaning),and 102013 known piRNAs were screened and 66667 candidate piRNA sequences from 3 months(puberty),125878 known piRNAs and 35008 candidate piRNA sequences were screened at 6 months of age(sexual maturity),and 95818 known piRNAs were screened at 12 months of age(body maturity)With 19074 candidate piRNA sequences,there were 16,289 expressed piRNAs of two or more periods.2.In this experiment,GO and KEGG analysis of gene-derived piRNAs were found,and it was found to be mainly enriched in cells,mainly involved in cellular processes and metabolic processes,and had molecular functions such as binding.KEGG enrichment analysis results show that these gene-derived piRNAs were enriched in Other types of O-glycan biosynthesis,Antigenprocessing and presentation,Thyroid hormone signaling pathway and other metabolic pathways,and enriched to metabolic pathways related to reproductive system development: Axon guidance,MAPK signaling pathway,etc.3.It was found that the expression levels of 2967 piRNA sequences were different in the four developmental stages.An in-depth analysis of the difference sequences found that the expression level of single differential piRNAs was roughly weaning higher than puberty,sexual maturity higher than body maturity,but lots of expression level of piRNAs in two and more periods were different that the level expression of piRNAs were lowest in weaning and which were highest in body maturity.4.In this study,a total of 758 piRNA clusters were obtained in four developmental periods,of which 198 clusters were expressed in two or more developmental periods,and the rest were unique clusters in each developmental stage.Through the analysis of the GO and KEGG pathways,three clusters were found related to reproduction and development,Clu-003(PAK6),Clu-033(HSPB1)and Clu-025(ECSIT),and two differential expression clusters related to spermatogenesis were also found,their target gene(CYP19A1,CCNO)proved that the piRNA cluster plays a role in testicular development.5.After verification by fluorescence quantitative PCR,selected randomly the number of six piRNA sequences were expressed in four developmental periods.The expression levels of piR-ssc-811833 during development were not different.The expression levels of piR-ssc-196195,piR-ssc-756757,piR-ssc-240587 and piR-ssc-347858 were the highest in body maturity,and the expression level of piR-ssc-756598 was the highest in puberty and the lowest in body maturity. |
PDF Full Text Request