Identification And Molecular Function Study Of LncRNAs And MiRNAs Related To Anthocyanin Synthesis In Radis

Radish(Raphanus sativus L.)is important a root vegetables crops.Anthocyanins give the plant rich color and antioxidant activity,which is beneficial to human health.Radish varieties rich in anthocyanidins are popular with consumers because of their bright colors and high nutritional value.Although some genes regulating radish anthocyanin biosynthesis have been reported,the molecular mechanism of radish anthocyanin regulation has not been fully elucidated.In this study,we screened the miRNAs,lncRNAs and their target genes which may be related to anthocyanin biosynthesis metabolism by lncRNA and miRNA sequencing.The main results are as follows:1.Screening of differential lncRNAs.The ‘YZH’ with the highest content of anthocyanin and the white-skinned and white-meat radish ‘WN07’ were screened and their lncRNAs were sequenced,25,672 reliable lncRNAs were identified,including4,763 antisense,20,178 lincRNAs and 731 sense overlapping.A total of 8,062 differential lncRNAs were identified from two different skin-colored radishes,with3,851 upregulated and 4,761 downregulated.16,773 differentially expressed mRNAs were identified,including 9,055 up-regulated transcripts and 7,718 down-regulated transcripts in sequencing samples,with the number of differentially expressed mRNAs being much greater than that of differentially expressed lncRNAs;The GO enrichment of lncRNA targets mainly occurs in nucleic acid metabolism,biological regulation,RNA metabolism,redox process,nucleic acid binding,DNA binding,oxidoreductase activity,nucleic acid binding transcription factor activity,sequence specific DNA binding transcription factor activity and RNA binding mRNA differential GO enrichment mainly occurs in biological processes,metabolic processes,monomer metabolism,cellular metabolism,redox processes,organic matter metabolism,primary metabolism,phosphate-containing compounds metabolism,catalytic activity and oxidoreductase activity;A total of 50,419 pairs of lncRNA-mRNA interaction pairs were identified by combined analysis of lncRNA and target genes,and eight differential lncRNAs and four genes related to anthocyanin biosynthesis were selected from the differential genes for RT-qPCR validation.The results showed that the expression trend of the differentially expressed lncRNAs and genes related to anthocyanin biosynthesis by RT-qPCR was consistent with that by sequencing.2.Screening for differential miRNAs.A total of 81 known miRNAs and 65 new miRNAs were identified from two radish small RNA sequencing data with different skin colors.44 differentially expressed miRNAs were screened,of which 21 miRNAs were up-regulated and 23 miRNAs were down-regulated.Most miRNAs have been predicted to regulate several target genes simultaneously and to cluster together to regulate one target gene.The RT-qPCR results of 10 differential miRNAs were basically consistent with the results of high-throughput sequencing.3.Functional verification of LINC15957.Overexpression of LINC15957 significantly increased anthocyanin accumulation and expression levels of genes related to anthocyanin biosynthesis in radish leaves,the accumulation of anthocyanin and the expression level of anthocyanin biosynthesis genes in silenced LINC15957 succulent roots were significantly decreased,and 5,772 differentially expressed genes were identified by transcriptome sequencing of overexpressed LINC15957 plants and controls.A total of 3,849 differentially expressed transcription factors were identified,among which MYB,bHLH,WD40,bZIP,ERF,WRKY and MATE were differentially expressed in overexpressed LINC15957 plants.KEGG enrichment analysis showed that these genes were significantly enriched in tyrosine,l-L-Phenylalanine,tryptophan,phenylpropanol and flavonoid biosynthesis.RT-qPCR analysis showed that eight DEGs were differentially expressed in LINC15957-overexpressing plants,which is consistent with the transcriptome data,indicating the reliability of the transcriptome sequencing results.These results suggest that LINC15957 is involved in the regulation of anthocyanin accumulation and provide abundant data for the study of genes involved in anthocyanin biosynthesis in radish.These results provide a reference for elucidating the molecular mechanism of lncRNA and miRNA regulating anthocyanin synthesis in radish.