Identification Of RNA M~6A Writer Protein In Cotton(Gossypium Hirsutum) And Regulation Study Of Arabidopsis VIR Gene

N6-methyladenosine(m6A)is the most abundant internal post-transcriptional modification of all higher eukaryotic messenger RNA(mRNA),which can be involved in the regulation of mRNA metabolism and processing,including mRNA translation and stability,mRNA export from nuclear,nuclear retention and splicing.The m6A modification process is a dynamic reversible process that is co-regulated by methyltransferase("writer")and demethylase("eraser").The major components of the methyltransferase complex currently found in mammals include METTL3,METTL14,WTAP and KIAA1429,and the demethylases include FTO and ALKBH5.In addition,m6A modification sites can be recognized by binding proteins("reader")YTHDF1-3,YTHDC1,HNRNPA2B1.Although advances have been made in the elucidation of the methylation mechanism of m6 A mRNA in mammals,much of their knowledge in plants remains unknown.In this paper,six m6 A "writer" genes were isolated and identified from cotton,and their expression,protein-protein interaction patterns and their functions in cotton were studied.In addition,the function of homologous protein(VIR)of KIAA1429 in Arabidopsis was also analyzed.The main results obtained are summarized as follows:1.Identification and evolutionary relationship of cotton m6A "writer" genesAccording to the published tetraploid upland cotton genome database,we isolated and identified six putative genes encoding RNA methyltransferases in the cotton genome,of which three were from the A genome and three from the D genome.We named them GhMTA-A/D,GhMTB-A/D and GhFIP37-A/D.Phylogenetic analysis showed that the GhMTA-A/D,GhMTB-A/D and GhFIP37-A/D proteins in cotton were closer to that of cacao(Theobroma cacao).2.Expression patterns of cotton MTA,MTB and FIP37 genesRNA-seq data showed that GhFIP37-A/D,GhMTA-A/D,and GhMTB-A/D genes were preferentially expressed in pistil and petals of cotton,and their expression in stamens was relatively low.In the different stages of fiber development,GhFIP37-A/D,GhMTA-A/D,and GhMTB-A/D were expressed relatively higher at the stage of fiber initiation(-3DPA ovule to 3DPA ovule).At the same time,we used qRT-PCR to validate the expression patterns of these genes in cotton.The results showed that GhMTA,GhMTB and GhFIP37 were expressed in all tissues and fiber stages of cotton,and all of them were highly expressed in the shoot apices,suggesting that these genes may be involved in the regulation of the development of cotton shoot apical meristems.During the development of cotton fiber,the qRT-PCR results were consistent with the RNA-seq dates,both of them showed that the expression of GhMTA,GhMTB,and GhFIP37 in the fiber initiation phase were relatively high,and the expression at the stage of fiber elongation(10-15 DPA fiber)were relatively low,which speculated that the expression of these genes may be negatively related to fiber elongation.3.Subcellular localization of cotton MTA,MTB and FIP37 proteinsWe constructed the GhMTA-D::eGFP,GhMTB-A::eGFP and GhFIP37-A::eGFP fusion expression vectors and then transiently expressed tobacco.By using confocal microscopy to observe the tobacco leaf epidermal cells,we found that the green fluorescence signal mainly accumulated in the nucleus and showed scattered spots,indicating that the GhMTA,GhMTB,and GhFIP37 proteins were located in the nuclear speckles.4.Analysis of interaction patterns among cotton MTA,MTB,and FIP37 proteinsIn order to study the functional form of GhMTA,GhMTB and GhFIP37 proteins in plants,we studied the interaction patterns of these proteins.The resuls of yeast two-hybrid technique and BiFC assay showed that GhMTA-D could interact with GhMTB-A and GhFIP37-A proteins,indicating that GhMTA,GhMTB and GhFIP37 proteins of cotton could act through forming a complex.5.Functional analysis of cotton MTA,MTB and FIP37 proteinsIn order to study the biological functions of GhMTA,GhMTB and GhFIP37 proteins in cotton,virus-induced gene silencing(VIGS)experiments were used to study their function in cotton development.The results showed that the silence of MTA,MTB and FIP37 genes in cotton resulted in a reduction of m6A levels in total RNA of cotton leaves,and cotton showed leaf morphing,apical meristem abnormalities,and plant dwarf phenotypes.These results suggesting the m6A modification mediated by MTA,MTB and FIP37 play an important role in the vegetative growth of cotton.In order to study the function of these proteases in the development of cotton fibers,we constructed the RNAi vector of MTB and FIP37 gene and then transferred them into cotton by Agrobacterium tumefaciens-mediated transformation.After several months of subculture,we have obtained embryogenic callus and transgenic seedlings,which provid the transgenic cotton material for next work,and the related work is in progress.6.Arabidopsis VIR gene expression analysisTo predict the possible function of the AtVIR gene in Arabidopsis,we analyzed its expression.qRT-PCR analysis showed that the VIR gene was highly expressed in the first stem and inflorescence of Arahidopsis thaliana and was relatively low in rosette leaves.We also obtained 12 lines of AtVIRpromoter-GUS transgenic Arabidopsis plants.GUS staining results showed that the promoter of VIR gene has expression activity in Arabidopsis seedlings and all tissues,but the root and shoot apicals are relatively strong during the seedlings period.7.Phenotype analysis of AtVIR RNAi transgenic ArabidopsisIn order to study the physiological function of Arabidopsis VIR gene,we obtained 9 lines of AtVIR-RNAi transgenic Arabidopsis thaliana.Compared with the wild-type plants,these AtVIR-RNAi transgenic Arabidopsis all exhibited shorter primary root lengths,smaller cotyledons and leaves,and shorter plant.8.Regulation analysis of AtVIRIn order to study the regulation mechanism of VIR gene in Arabidopsis thaliana,we conducted yeast one-hybrid screening experiments to analyze the possible binding proteins of the AtVIR promoter.The results showed that Arabidopsis AP2/ERF transcription factors AIL6,BBM and PLT2 can all bind to AtVIR promoter.qRT-PCR analysis showed that AtVIR gene expression was significantly up-regulated in overexpressed AtAIL6 transgenic Arabidopsis,while AtVIR gene expression was significantly downregulated in ptll-4plt2-2 double mutants,implying that these transcription factors may positively regulate AtVIR genes expression.