The Identification And Validation Of Long Non-coding RNA In Komagataella Phaffii Based On RNA-seq

Pichia pastoris,as a widely used model strain,is of great significance for food,medicine and industrial production.Long non-coding RNA is a type of non-coding RNA with a length of more than 200 bp,which is ubiquitous in eukaryotes and has regulatory functions.In this study,the high-yield phospholipase A2 Pichia pastoris GS115 was used as the sample.The RNA-Seq technique was used to detect the transcriptomes of four samples,and non-coding RNA was predicted by bioinformatic technology.The results are as follows:By bioinformatics prediction,208 lncRNAs were obtained and the reliability of the data was verified by qRT-PCR.By comparing the length of mRNA and lncRNA,the length of the open reading frame,the transcriptional level and the number of exons.By comparing the transcriptional levels of lncRNAs in samples of methanol and glycerol and different protein expression,39 significantly different lncRNAs were obtained.Cis-acting regulation: the sense-antisense pairs prediction: lncRNA TCONS₀0002848,TCONS₀0003442,TCONS₀0004115 may act on their sense-mRNA.Enrichment of 10 k cis-acting target genes upstream and downstream of significantly different lncRNAs: There were 18 significantly different lncRNAs and 206 cis-acting target genes in the methanol and glycerol phases,which were enriched in pathways related to protein metabolism,cell repair,and energy metabolism.There were 30 significantly different lncRNAs in different exogenous protein expression samples,327 cis-regulatory target genes,related to protein synthesis,stress response,and cell cycle-related pathways.Trans-acting regulation: 18 significantly different lncRNAs in methanol and glycerol comparison group were targeted to 846 trans-regulated target genes,enriched in pathways related to macromolecular metabolism,stress response,and energy metabolism.30 significantly different lncRNAs in exogenous protein expression samples were targeted 757 trans-target genes,enriched in protein and energy synthesis,and stress response pathways.By changing the expression of lncRNA,the functions of the seven lncRNAs were verified.The results of fermentation showed that the knockout of lncRNA did not cause changes in its phenotype.The results of real-time quantitative PCR showed that after knocking out TCONS₀0004115 lncRNA,the expression of some predicted target genes changed significantly,and after overexpression,the difference of target genes between methanol induction phase and glycerol phase decreaseed.