Function Analysis Of GhWER In Cotton Fiber Development

Cotton,a kind of cash crops,plays an important role in the world.As the main production of cotton,Cotton fiber has a non-negligible contribution to the development of the textile industry.Cotton fiber development includes five stages,namely,fiber cells initiation,elongation,transformation,secondary wall thickening and dehydration,and each stage has its corresponding gene regulation network.The cell differentiation at the initial stage of fiber is directly related to the quality and yield of mature fiber,which is the focus of current research of cotton fiber.In order to analyze the regulatory network related to fiber initiation,a series of transcription factors(TFs)with high expression levels at fiber initiation stage were screened out in our laboratory,including GhWER,an R2R3-MYB TF.With CRISPR/Cas9 technology,GhWER was knocked out.Combining investigating phenotype of knockout lines and related molecular experimental techniques,the regulatory network of GhWER in fiber development was analyzed and the regulatory network related to fiber initiation was further improved.The main results are as follows.1.GhWER in upland cotton has the highest sequence similarity with At WER in Arabidopsis,and the similarity of the protein sequences reached 70% in R2R3-MYB domain.GhWER had been expressed highly in ovules at-1.5 DPA,and reached the highest level at-1 DPA,and then began to decrease gradually.In addition,according to expression profile data in our laboratory database,among all the homologous genes in upland cotton,the expression level of GhWER was the highest in ovules at fiber initiation stage.2.According to the GhWER genome sequence,a CRISPR/Cas9 construct with dual targets targeting the R2R3-MYB domain was developed and transformed into cotton Jin668.Total 13 T0 plants were obtained.The editing types of all transgenic plants in T0,T1 and T2 generations were detected by using Hi-TOM and Sanger sequencing.Among the two designed targets,at least one target of each individual plant was edited,and it could be stably inherited to the next generation.Compared with wild type,the fibers initiation and early elongation of GhWER knockout lines were inhibited,resulting in shorter fiber.3.TFs generally interact with other proteins to regulate gene expression,so at first,the self-activation domain of the GhWER was detected and was found located at amino acids 191～230,and then the truncated GhWERN2 protein with no self-activation was used for yeast two-hybrid experiments.Two interacting proteins,which were both bHLH TFs and highly expressed in the initiation stage of fiber development,GhDEL65 and GhbHLH121,were screened from the library of TFs related to fiber initiation.These results 2021 suggested that GhWER plays an important role in regulating fiber development as MYB/bHLH complex.4.In order to further study the downstream gene regulatory network of GhWER,the epidermis of-1.5 DPA ovule of GhWER knockout lines and the wild-type was collected for transcriptome sequencing(RNA-seq),and then the genes with expression changes greater than 2 folds were screened.A total of 241 differentially expressed genes(DEGs)were obtained.The DEGs were found mainly enriched in hormone signal transduction and stimulation response related pathways by GO and KEGG enrichment analysis.The ethylene(Eth)synthesis related gene ACS1,Eth receptor genes RAV1 and ETR2,Eth response related gene ERF017,auxin(IAA)response gene SAUR50,gibberellin(GA)regulatory genes SN2 and GASA14 were all down-regulated in GhWER knockout lines,which indicated that GhWER may positively regulate fiber initiation and early elongation by promoting the signal transduction of Eth,IAA and GA.