Identification Of Group Ⅲ WRKY Genes And The Functional Analysis Of GhWRKY53 In Gossypium Hirsutum L.

Cotton is an important cash crop that can produce natural fiber which are used in textile industry.The yield and quality of cotton fiber are the most critical factors determining the economic value of cotton.The differentiation and development of cotton fiber cells are very complex and regulated by various genes,among which transcription factors play a crucial role.WRKY is a ubiquitous transcription factor which plays a role in multiple plant life processes.However,so far,there are few reports about WRKY in cotton fiber development.Screening and functional verification of WRKY genes related to cotton fiber development will help us analyze the molecular mechanism of cotton fiber development and then provide an essential theoretical basis for improving cotton yield and fiber quality.We first used molecular biology and bioinformatics methods to analyze group Ⅲ WRKY genes in Gossypium hirsutum L.Then we selected the Gh WRKY53 which was highly expressed in the fiber initiation stage for cloning and functional identification.The main contents are as follows:1.Genome-wide analysis of group Ⅲ WRKY genes in G.hirsutum L.There are 21 group Ⅲ WRKY members in G.hirsutum L.Phylogenetic analysis showed that group Ⅲ WRKY genes in G.hirsutum L.were divided into three clades.This branching pattern also existed in group Ⅲ WRKY genes in Arabidopsis thaliana.Phylogenetic analysis,multiple alignment,conservative motif analysis,and gene structure analysis showed that the members clustered together in the phylogenetic tree had similar motif and gene structure characteristics.The group Ⅲ WRKY genes sequences in cotton were highly similar in sequences,with the conserved WRKY and C₂HC zinc finger domains and similar intron and exon numbers.We analyzed the expression patterns of group Ⅲ WRKY genes during the fiber development.We found that most of them were highly expressed during-3 DPA and 3 DPA.They might involve in the biological process of cotton fiber initiation.2.Cloning and functional identification of cotton fiber initiation-related gene Gh WRKY53.The DNA length of Gh WRKY53 was 3321 bp and contained 3 exons and 2 introns.The length of CDS was 1008bp and encoded 335 amino acids which had the typical structural characteristics of the group Ⅲ WRKY subfamily.The subcellular localization experiment of Gh WRKY53 showed that it was located in the nucleus,and the heterologous expression of Gh WRKY53 in A.thaliana could significantly increase the density of epidermal trichomes.Twelve proteins that interacted with Gh WRKY53 were screened from the cotton fiber c DNA library by yeast two-hybrid experiment.We verified the interaction between Gh WKY53 and Gh RAX3 by yeast two-hybrid experiment.In this study,we analyzed the group Ⅲ WRKY subfamily members in G.hirsutum L.in detail and explored the function of Gh WRKY53 in A.thaliana.These research results provide a new insight for further exploring Gh WRKY53 and group Ⅲ WRKY genes in G.hirsutum L.