Identification And Stress Resistance Functional Analysis Of DNA Methyltransferase Gene Family In Cotton

DNA methylation is an important epigenetic modification.DNA methylation involves processes such as gene silencing,inhibition of transposon elements,inactivation of X chromosomes,cell differentiation,and maintenance of embryonic development.DNA methyltransferase catalyzes key enzymes in DNA biomethylation.Previous studies have find the DNA methyltransferase being involved in response to abiotic stress but no systematic studies have been conducted in cotton.Our study was aimed to investigate theresponsive mechanism of DNA methyltransferase in cotton against abiotic stress and also to explore the stress resistant genes in cotton.In this study,51 DNA methyltransferase from A2,D5,AD1,and AD2 genomes have been identified and an evolutionary relationship among all of them was analyzed.At the same time,the GhDMT6 and GhDMT9 genes were cloned from Gossypium hirsutism L.,with gene silencing and overexpression vectorsfor their functional analysis.The main findings are as follows:1.Using PF00145 and IPR001525 as a reference domain,we have observed the DNA methyltransferase in A2 and D5 genomes /groups of diploid cotton and in AD1 and AD2 of tetraploid cotton.Gene no are 9,8,16 and 18.Subcellular localization predicts the location of most of DNA methyltransferase in cytoplasm and some of them were found in plasma membrane.GbDMT13 was predicted to found in periplasmic.2.DNA methyltransferase are divided into four subfamilies according to their Domains and gene structure: CMT,MET,DRM and DNMT2.Evolutionary relationship of the DNA methyltransferase in A2,D5,AD1,and AD2 were finding identical to those in the single genomes.DNMT2 is an ancient gene family,and homologous genes are identified in fungi,animals,and plants,and the structure is relatively conservative.3.Teal-time quantitative PCR analysis shows the expression of DNA methyltransferase being effected by cultivars,stress conditions,tissue parts and stress time,and the gene expression pattern changed with conditions.For A2(shixiya1)and D5 groups: on stress application DNA methyltransferase down-regulated genes were more distributed in the rhizome as compared to up-regulated genes in leaves.While for AD1 group(TM-1)and diploid Cotton has more different up-regulated genes in the rhizome,and more down-regulated genes in the leaves.4.After being silenced by GhDMT6,cotton seedlings were observed significantly less sensitive to salt and drought stress which could maintain normal growth and development.Overexpression of GhDMT6 in Arabidopsis thaliana seedlings have showed enhanced resistance.The cotton was transformed by constructing a knockout vector to obtain two positive plants.Expression of GhDMT9 was observed upregulated during drought and salt stress.After being silenced by GhDMT6,cotton seedlings were observed significantly less sensitive to salt and drought stress which could maintain normal growth and development.After TMGS cotton seedling VIGS silenced GhDMT9,the silenced plants were less able to resist drought and salt stress.Overexpression of GhDMT9 in Arabidopsis thaliana seedlings have showed enhanced resistance.Our study of cotton DNA methyltransferase gene family supported with Gene expression analysis and results of functional analysis exhibits that DNA methyltransferase can play an important role in cotton response to stress.This research has laid a foundation for further studies on cotton stress resistance mechanism.