Functional Study Of N~6-methyladenosine MRNA Methylation In Regulating Cotton Fiber Elongation

Cotton is an important source of natural textile fiber,and fiber length is one of the most important breeding targets.Cotton fibers,as one of the longest plant cells,are an ideal model for studying cell elongation.N6-methyladenosine(m6A)is the most abundant modification in eukaryotic mRNA and plays an important role in the development of different species.However,the role of m6A methylation in regulating fiber elongation has not been reported.In this study,m6A sequencing and transcriptome sequencing were used to compare and analyze the fiber of short fiber mutant Ligon lintless-2(Li2)and wild-type(WT)cotton,identifying the key gene GhMYB44 that affects cotton fiber elongation and verifying its function in the fiber elongation process.The main research contents are as follows:1.Compared with WT,the m6A level of Li2 fiber during elongation period(8 DPA)was significantly higher than that of the control;meanwhile,the expression of various m6A methyltransferases increased,the expression of m6A demethylases decreased significantly,and the expression of m6A binding proteins increased,preliminarily suggesting that m6A methylation modification is involved in the regulation of cotton fiber elongation in Li2.2.Based on the m6A-seq of Li2 and WT fiber tissues,a large number of genes were identified to be m6A methylated;m6A modification peaks were mainly enriched in stop codons,3’ untranslated regions(UTRs)and coding sequences(CDS),and the plantspecific sequence motif URUAY was identified.Most of the genes with m6A methylation peaks had a single peak(over 80%),and the differential m6A methylation peaks between Li2 and WT were further subjected to GO enrichment analysis,revealing that the relevant pathways involved in fiber elongation were significantly enriched.3.The correlation analysis between differential m6A methylation peaks and differentially expressed genes(DEGs)showed that the expression levels of highly and lowly methylated genes were higher than those of non-differential m6A-modified genes.447 differential target genes were identified,and GO enrichment analysis showed that these genes were involved in processes such as cytoskeleton and microtubule binding,further indicating the potential roIe of m6A modification in fiber elongation.4.GhMYB44 overexpression and RNAi genetic materials were obtained by genetic transformation of cotton CRI24.qPCR results showed that the expression levels of L1 and L3 overexpression strains were 6 times that of the control,and the decrease in gene expression of RNAi strains was about 70%,indicating that the transgenic materials could be used for further study of GhMYB44 gene function.5.It was found that m6A methylation affected the abundance of GhMYB44 mRNA by regulating mRNA stability.Phenotypic measurements showed that overexpression of GhMYB44 inhibited cotton fiber elongation,while silencing of GhMYB44 increased fiber length,indicating that GhMYB44 can negatively regulate cotton fiber elongation.In this study,a combination of m6A-seq and RNA-seq was used to measure the fiber of short fiber mutant Li2 and its wild-type control material during the elongation period(8 DPA),revealing that key genes in the fatty acid biosynthesis,cytoskeleton,microtubule binding,and other main pathways were regulated by m6A methylation to control fiber elongation.Genetic transformation of cotton receptor CRI24 obtained GhMYB44 overexpression and RNAi transgenic materials,with the expression of GhMYB44 in overexpression strains.