Study On Role Of GhKNL1in Fiber Development Of Cotton(Gossypium Hirsutum) And GhAGP31Involved In Response To Cold Stress

Cotton is the most important textile fiber crop in the world. Cotton fiber, a highly elongated and thickened single cell derived from the ovule epidermis, provides an excellent system for studies on cell elongation and cell wall cellulose biosynthesis. Cotton fiber cell wall is usually divided into two categories:the primary cell wall (PCW) and the secondary cell wall (SCW) during cotton fiber development. A series of complex regulations in PCW stage finally affect fiber cell intensity and length. Cellulose synthesis and SCW deposition in later phase determine cotton fiber yield and quality at last. Therefore, it is a vital significance for us to clone the genes related to SCW synthesis and study its molecular mechanism to improve the yield and quality of cotton fibers. In this study, a cotton KNOX gene and a gene encoding cell wall protein were insolated in cotton. The expression patterns and functions of these two genes have been characterized. The main results are as following:1. GhKNL1gene is highly expressed during the fiber development of SCW synthesisOne full-length cDNA encoding a Class Ⅱ knotted-like homeobox protein (KNOX) was isolated from cotton and designated as GhKNLl. The result of RT-PCR indicated that GhKNL1transcripts were accumulated at low level in elongating fibers and other tissues, but gradually reached the highest level as fiber further developed to SCW stage. The promoter fragment upstream of the GhKNL1was isolated and GhKNLlp::GUS fusion expression vector was constructed and transformed into Arabidopsis by Agrobacterium-mediated method. The expression of GUS gene driven by GhKNL1promoter was only detected in vascular systems of young and matured tissues. However, there was almost not detected in other tissues.The relationships between phytohormones and GhKNLl were further analyzed in vitro culture of cotton ovules. The expression level of GhKNLl gene was not regulated by ABA, KT and GA treatments. These results indicated that GhKNL1is a gene related to fiber development of SCW synthesis, and may be involved in the SCW synthesis, and does not response to phytohormones.2. GhKNL1protein is localized in cell nucleusTo evaluate subcellular localization of GhKNL1protein, a GhKNL1::eGFP chimeric gene was constructed and introduced into Arabidopsis thaliana. Transgenic plants were selected on a selective medium and observed under SP5Meta confocal laser scanning microscopy. The results indicated that GhKNL1was localized in the nucleus.3. Analysis the interactions of GhKNL1proteinThe result of transcription activation activity suggested that GhKNL1protein lacks the activity of transcriptional activation. It has reported that KNOX transcription factors can form into complex to have function. In order to determine the interactions of GhKNL1protein, yeast two-hybrid system was employed. The result indicated that GhKNL1can combine into homodimers. A cotton gene GhOFP4was predominantly expressed in the fiber development of SCW synthesis. The result of subcellular localization showed that GhOFP4protein is localized in cell nucleus. Yeast two-hybrid analysis showed that GhKNLl proteins can interact with GhOFP4in cells. These results indicated that GhKNL1protein could playe a role as heterodimer or heterodimers in the fiber development.4. GhKNL1-dominant repression and KNL1overexpression transgenic Arabidopsis have the same phenotype of thinner interfascicular fibers cell wall in the inflorescence stemTo further investigate KNL1functions in the regulation of secondary wall formation, the over expression and suppression function of KNL1vectors were constructed and introduced into Arabidopsis thaliana, and T2generation plants with different expression levels were used to further analyzed. Histological staining and TEM (Transmission Electron Microscope) revealed that there was a striking decrease in the secondary wall thickness of the interfascicular fibers of stem in transgenic plants compared with the wild type. The results of RT-PCR showed that the expression levels of SCW synthesis related genes were down-regulated in transgenic plants. In addition, complementation experiment suggested that overexpression of KNL1could rescue the irx phenotype of stems and seed coat mucilage in knat7mutant at some extent. These results suggested that the cotton gene KNL1negatively regulated SCW synthesis and was functionally conserved in regulation of SCW formation in Arabidopsis.5. Dominant repression of GhKNLl hinders fiber developmentIn order to investigate the function of GhKNLl protein, a large number of GhKNL1-dominant repression transgenic plants were generated by genetic transformation of cotton. The vegetative growth and flower development of these transgenic lines were similar with wild type. However, the bolls of transgenic plants were much smaller than those of wild type, and the yield of mature fibers was declined. The result of semi-thin sections showed that the fiber initials in transgenic cotton plants were delayed, and even a lot of5-20DPA fiber cells were abnormal and disordered, compared with those of wild type. Being consistent with the above phenotype, the expressions of the genes related to fiber elongation and secondary cell wall synthesis were down-regulated in transgenic fibers. These results indicate that cotton KNL1may play an important role in fiber cell elongation and secondary cell wall formation of cotton fiber cells.6. Molecular characterization of GhAGP31and its roles in root developmentA gene (including its cDNA), designated GhAGP31, encoding a non-classical AGP protein was isolated from cotton (Gossypium hirsutum). The deduced GhAGP31protein contains the conserved features of non-classical AGPs:a putative signal peptide, N-terminal histidine-rich stretch, middle repetitive proline-rich domain and a cysteine-containing ’PAC’ domain. GFP fluorescence assay demonstrated that GhAGP31protein was localised on cell walls. GhAGP31transcripts were mainly detected in roots, hypocotyls and ovules. In particular, expression of GhAGP31was decreased during the development of roots. Further study demonstrated that GhAGP31expression in cotton roots was remarkably up-regulated by cold stress. Expression of the GUS gene driven by the GhAGP31promoter was also dramatically enhanced in roots of transgenic Arabidopsis seedlings under cold treatment. Additionally, overexpression of GhAGP31in yeast and Arabidopsis significantly improved the freezing tolerance of yeast cells and cold tolerance of Arabidopsis seedlings. These data implied that GhAGP31protein may be involved in the response to cold stress during early root development of cotton.