Cloning And Functional Analysis Of DNA Demethylase(GhDME3)gene In Upland Cotton

Cotton is an important economic crop.Salt stress and drought stress seriously restrict the yield and fiber quality of cotton.DNA methylation is one of the earliest epigenetic modification pathways,which is a stable and reversible epigenetic marker.DNA active demethylation requires the catalysis and maintenance of DNA demethylase.DNA demethylase plays an important role in plant growth and development,as well as in the process of biotic and abiotic stress.Therefore,the study on the role of DNA demethylase gene in salt stress and drought stress of cotton can provide a new direction and thinking for cotton resistance breeding.In this study,DNA demethylase gene family members in cotton were identified at the genome-wide level,and GhDME3 gene encoding DNA demethylase gene in upland cotton was cloned,and gene expression characteristics and biological functions under stress were analyzed.1.There are 10 DNA demethylase gene family members in upland cotton identified by bioinformatics,named GhDME1-GhDME10.Analysis of gene structure and phylogenetic development showed that the DNA demethylase genes in upland cotton were divided into four subgroups: ROS1,DME,DML2 and DML3,and the genes in the same subgroup had similar numbers of exons and introns.The analysis of transcriptome data showed that all members of DNA demethylase gene family expressed in different tissues and organs,with the highest expression in leaves.The gene expression of DNA demethylase was induced by cold stress,heat stress,salt stress and drought stress.The expression of GhDME3 and GhDME4 was relatively high under four kinds of stress,and the expression of GhDME9 and GhDME10 was relatively low under four kinds of stress.2.The DNA demethylase GhDME3 gene of upland cotton is located on A04 chromosome.The length of cDNA is 5673 bp.The length of amino acid is 1890 aa.The molecular weight of protein is 210435.48 da.The theoretical isoelectric point is 6.515.The subcellular is located in the nucleus,and does not have transmembrane domain.The secondary structure of GhDME3 protein was predicted to contain ?-helix(29.37%),?-angle(2.75%),irregular curl(57.41%)and extended chain(10.48%).3.Shannong SF06,an upland cotton variety,was used as the material.Tissues and organs such as root,stem,cotyledon,hypocotyl and other tissues and organs as well as cotton leaves at different stages after salt treatment and drought treatment were selected to extract total RNA and reverse transcribe it into cDNA as the template.qRT-PCR was used to analyze the expression levels of DNA demethylase gene in different tissues and organs,and it was found that the dominant expression of GhDME3 gene was found in the leaves.In the process of salt treatment and drought treatment,with the increase of treatment time,the expression of GhDME3 gene increased,which may be related to salt stress and drought stress.4.Using virus-induced gene silencing(VIGS)technology and tobacco fragile crack virus vector pTRV,a pTRV-GhDME3 silencing system of GhDME3 gene in upland cotton was successfully established.The gene expression in cotton leaves was decreased after GhDME3 gene silencing by qRT-PCR.After salt stress and drought treatment,it was found that the tolerance of cotton plants to salt and drought was decreased,so it was speculated that decreasing the expression of GhDME3 gene increased the sensitivity of cotton to salt and drought.5.The recombinant plasmid pRI201-AN-GhDME3-GUS of GhDME3 gene was successfully constructed by using the overexpression vector pRI201-AN-GUS.The transgenic Arabidopsis thaliana with GhDME3 gene overexpression was obtained by using dip flower method.The results showed that the overexpression of GhDME3 gene enhanced the resistance of Arabidopsis to salt stress and drought stress.The overexpression of GhDME3 gene may improve the drought resistance and salt tolerance of Arabidopsis.