Expression And Function Of A Novel MYB Gene In Cotton(Gossypium Hirsutum)

Cotton (Gossypium hirsutum) is a commercially important fibre crop worldwide, and has wide application in textile, paper, biofuel, and chemical industries. The fiber quality greatly determines its economic and commercial values. Cotton fibers represent an excellent model system of cellulose biosynthesis and cell biogenesis in plants. our understanding of the processes of cotton fiber growth and maturation is still rudimentary and no systematic study.MYB transcription factors are one of the largest transcription factors family in plant kingdom. The R2R3-MYB of this family function in a variety of plant-specific processes, as evidenced by their extensive functional characterization in Arabidopsis thaliana. R2R3-MYB transcription factors have a modular structure, with an N terminal DNA-binding domain (the MYB domain) and an activation or repression domain usually located at the C terminus. In contrast to the highly conserved MYB domain, the other regions of R2R3-MYB proteins are highly variable. Numerous R2R3-MYB proteins have been characterized by genetic approaches and found to be involved in the control of plant-specific processes including (i) primary and secondary metabolism,(ii) cell fate and identity,(iii) developmental processes and (iv) responses to biotic and abiotic stresses. Many MYB proteins involved in the control of secondary cell wall biosynthesis in Arabidopsis thaliana. Then we isolated and identified a novel MYB transcription factor from cotton EST libraries and designated as GhMYB103. The isolated GhMYB103gene is expressed predominantly in secondary wall biosynthesis stage cotton fibers, and its encoded protein is targeted to the nucleus and can activate transcription. In addition, GhMYB103showed high sequence similarity to AtMYB103、PtrMYB128and PtrMYB10which have been demonstrated to function as transcriptional activator of activating the secondary wall biosynthesis. So it is imply that GhMYB103transcription factor may be involved in secondary wall biosynthesis in cotton fiber. In this thesis, our studies mainly focused on the regulation mechanism of secondary cell wall deposition in sclerenchyma cells of Arabidopsis stems. and then, we can infer the function of the GhMYB103gene in cotton fiber. The main results are as follows:1. A cDNA encoding a novel MYB transcription factor was isolated from the cotton20DPA fiber cDNA library, and designated as GhMYB103. Sequence analysis revealed that GhMYB103ORF included975bp and encoded a protein with324amino acids. The N-terminal of which contained a highly conserved MYB domain including R2, R3subdomains, and C-terminal was transcription activation domain. Sequence comparison and phylogenetic analysis revealed that GhMYB103and PtrMYB128, PtrMYB10, AtMYB103are closely related, and these proteins share a66%,66%,51%identity respectively.2. Quantitative RT-PCR experimental results showed GhMYB103was predominantly expressed in secondary wall biosynthesis stage cotton fibers, the expression of the gene gradually increased with further development of fibre cells (12-20DPA). The results suggested that the expression of GhMYB103was developmentally regulated in fibre cells of cotton.3. GhMYB103::eGFP fusion expression vector was constructed and introduced into Arabidopsis by floral dip method. The green fluorescence distributed uniformly in the nuclei of roots cells in transgenic Arabidopsis using confocal laser scanning microscop. This indicated that the GhMYB103is a nuclear protein.4. To examine the trans-activation activity of GhMYB103, The pGBKT7-GhMYB103construct was transformed into the yeast strain AH109and Y187, and two reporter genes ADE2and lacZ were tested by streaking the AH109transformants on SD/-Trp/-Ade medium, and the flash-freezing filter assay of Y187transformants, respectively. The yeast harboring pGBKT7empty vector was used as the negative control. The pGBKT7-GhMYB103transformant cells were able to grow well on SD/-Trp/-Ade, whereas cells containing pGBKT7vector did not grow on the same medium. In addition, the transformant yeast harboring pGBKT7-GhMYB103turned blue at the presence of X-Gal, but the negative control didn’t. The results suggested that GhMYB103protein functions as an active transcriptional activator in cotton.5. Dominant repression of GhMYB103in model plant Arabidopsis caused a collapsed vessels morphology in xylem of bottom inflorescence stems. Overexpression of GhMYB103resulted in a more deposition of secondary walls in the cortical cells which were close to the interfascicular fibers and phloem fibres in the6-week-old stems of4CL1pro::MYB103transgenic lines, Moreover, Overexpression of GhMYB103by35S promoter also led to the secondary cell walls thickening of interfascicular fibers and xylary fibers. In addition, the expression of secondary wall associated genes, such as PALI, CesA4, CesA7, CesA8, were modestly upregulated by GhMYB103overexpression in Arabidopsis stems. These results suggested that GhMYB103involved in secondary wall biosynthesis in Arabidopsis stems. It was reported that arabidopsis xylem and cortical fibers of stems are preferred models for functional testing of putatively orthologous genes that may regulate cotton fiber secondary wall cellulose synthesis. and then, we inferred that the GhMYB103may participate in the process of secondary walls deposition in cotton fibers.