Study On The Role Of GhFP1 In Fiber Development Of Cotton (Gossypium Hirsutum) And Its Molecular Mechalism

Cotton is one of the most important economic crops in the world. Fiber is a single cell arises from epidermal cells on the outer integument of the ovules, and length is one of the important qualities of fiber. The bHLH (basic helix-loop-helix) proteins in Arabidopsis have been reported to function in cell elongation. Although 208 bHLH/HLH genes have been identified in the Graimondii genome, there are no reports about how cotton bHLH/HLH proteins regulate fiber development. In this study, several bHLH/HLH genes including GhFP1 had been cloned from fibers. The expression pattern and function of GhFP1 has been characterized. The main results are presented as following:1. Characterization of GhFP1 and its expression in cotton tissuesA cotton bHLH gene was cloned from fiber and named GhFP1. The transcript of GhFP1 could be detected in every cotton tissue. During fiber development, GhFP1 displayed a relatively high expression levels during 3-12 DPA fibers while a significant decrease was happened in 18DPA fibers. A 1061bp fragment upstream of the GhFP1 translation start codon was isolated by Genome Walking. GhFP1 promoter::GUS fusion vector was constructed and transformed into cotton. Although there were differences between the results of GUS staining and RT-PCR, GUS activity could also be detected in 3-20DPA fibers. These results implied that GhFP1 may be involved in the regulation of cotton fiber development.2. Overexpression of GhFP1 in Arabidopsis enhances the cell elongation and largement of trichomes on leaves of the transgenic plants35S::GhFP1 fusion vector was constructed and transformed into Arabidopsis. Difference between trichomes of GhFP1 overexpression plants and that of wild type was found when we observed the phenotype of transgenic plants. Statistical analysis indicated that GhFP1 overexpression increase the length of trichome significantly in Arabidopsis.3. GhFP1 promotes fiber elongation of cotton by modulating BR signalingIn order to analyze the function of GhFPl during fiber development, we constructed GhFP1 overexpression vectors which were derived by GhTUA9 promoter. Transgenic plants were generated by genetic transformation of cotton. The statistical analysis of length of mature fibers displayed that GhFP1 could enhance the fiber elongation and increase the length of fibers. The results of RT-PCR and ovule culture implied that the increase of fiber length may be due to the enhancement of BR signaling4. GhFP1 as a transcriptional activator regulates the expression of target genes in cotton fibersThe results of transactivation activity assay and subcellular localization displayed that GhFPl was a transcriptional activator. EMSA and ChIP assays indicated that GhFPl could bind to the E-box in the promoters of BR biosynthetic genes. The transcriptome analysis revealed that the expression of phytohormone-related genes and other transcription factors were affected by GhFP1 overexpression in fibers. These results implied that GhFP1 regulate fiber development through a complex mechanism5. Cotton bHLH/HLH proteins regulate fiber development through protein-protein interactionTo investigate the role of cotton bHLH/HLH in fiber development, we cloned other bHLH/HLH genes from 10DPA fiber cDNA. The different expression patterns of these genes implied that they may have different functions during fiber development. Yeast two-hybrid and Pull-down assays indicated that interactions exist between these cotton bHLH/HLH proteins. The results of EMSA showed that the homodimer of GhFPl could bind to DNA while the heterodimer of GhFPl and GhIBH2 or GhIBH3 could not. These results implied that the homodimerization or heterodimerization is important for GhHLH/bHLH proteins to function correctly.