| Cotton is not only the most important economic crop in the world, but also a principal raw material in textile industry and strategic materials. Cotton fiber is one of the powerful cell elongation, cell wall formation and cellulose biosynthesis research model because it is a long, easily isolated single cell with nearly pure cellulose. Cotton fibers are single-celled extentions differentiated from the ovular (seed) epidermal cells and with four distinct but overlapping developmental phases:initiation, elongation, secondary wall deposition and maturation. Among them, the stages of initiation, elongation and secondary wall deposition determine the amount, length and strength of the fiber respectively. Therefore, cloning and analysis of the function and molecular mechanism of cotton fiber development related genes not only has important theoretical significance, but also has vital application value to the improvement of cotton fiber yield and quality. We isolated 26 NAC genes from cotton and designated them as GhNACl-26. In this study, GhNAC1 transcription factor was functionally characterized in cotton. Also, a gene (GhMAPK17) encoding a mitogen-activated protein kinase was characterized by overexpression of this gene in Arabidopsis. The main results are as following:1. The expression analysis of transcription factor GhNACl during cotton fiber developmentGhNAC1 was homologous to Arabidopsis NST1, NST2 and SND1(NST3) and had similar genetic structures. The results of quantitative RT-PCR showed that in addition to having weakly expressed in hypocotyls, GhNAC1 was preferentially expressed during early stage of cotton fiber secondary wall thicking and its expression level was increased gradually with the prolonged development period and reached the maximum on 20 DPA (days post anthsis). GhNAC2 was preferentially expressed during cotton fiber elongation and secondary wall thicking and the expression level of GhNAC2 was similar with GhNAC1.2. The effects of GhNAC1 overexpression on secondary wall of cotton fiberIn order to investigate the function of GhNACl during cotton fiber development, we constructed vectors of GhNACl overexpression (GhNAC1OE) and RNA interference (RNAi), transformed them into cotton and got a number of transgenic cotton plants. The results of histological sections, observation and statistics, X-ray diffraction and GC-MS have been demonstrated that compared with the wild type, overexpression of GhNAC1 in cotton resulted in increment of lignin and cellulose amounts in xylem cells and ectopic deposition of them in cortex and pith cells in petioles, shortened fiber length, thickened secondary walls, the increment of crystalline cellulose in fibers and the changes of monosaccharide composition of fiber cell walls at different developmental stages. The phenotype and genetic analyses of RNAi transgenetic cotton plants are in progress.3. Analysis of GhNACl interacting proteinsThe results of subcellular localization and yeast transcription activation assay showed that GhNAC1 and GhNAC2 are both transcription factors. We used the GhNACl(NAC domain) as a bait protein to screen cotton fiber cDNA library and got several proteins which can interact with GhNAC1. In order to confirm the interactions between GhNACl and proteins of interest, yeast two-hybrid system was employed. The results revealed the ability of GhNACl with strong interactions to form homodimers and weak interactions between GhNACl and GhNAC2, E2 ubiquitin-conjugating enzyme. Furthermore, the pull-down assay gave the direct evidence that GhNAC1 can interact with each other in vitro.4. Analysis of degradation pathway of GhNACl proteinIt has been reported that the regulation of NAC transcription factors mainly included post-transcriptional control and post-translational control. miRNA are small regulatory RNAs that pair with target mRNAs, providing post-transcriptional repression of the targets. The methods used to control NAC activity also includes post-translational regulation by ubiquitin-mediated protein degradation and the modification of some enzymes. Under different treatment conditions, the transgenic Arabidopsis of GhNACl-eGFP showed different signal amount and intensity of GFP fluorescence, different mRNA and protein level of the transgene. In addition to these, the result of immunoprecipitation also indicated that GhNAC1 can be labeled with ubiquitin and was likely subjected to the ubiquitin-proteasome pathway-dependent proteolysis.5. Self-regulation of GhNAClWe used the obtained 35S::GhNACl and GhNAC1p::GUS transgenic Arabidopsis as the male and female parents for hybridization. The result of GUS staining of 2-week and 6-week F2 positive hybrid lines and GhNAC1p::GUS transgenic lines revealed that regardless of orthogonal or reciprocal cross, under the condition of GhNACl overexpression, the intensity and range of GUS signal were enhanced and increased. The analysis of real-time quantitative PCR showed that compared to transgenic lines, the expression level of GUS gene was higher in hybrid lines. Moreover, the result of electrophoretic mobility shift assay (EMSA) indicated that GhNACl protein can bind to the promoter of itself and lead to a positive self-regulation.6. Transcriptional regulation of downstream genes by GhNAClIn order to study the regulation of downstream genes by GhNACl, we isolated 12 promoters of theoretical downstream genes of GhNACl. According to the cis-acting elements which can be bound by SND1 in Arabidopsis, we mapped of the GhNAC1 binding sequence using EMSA and demonstrated that GhNACl can bind to an imperfect palindromic 13-bp consensus sequence designated as GhNAC1 binding site (GhNBS), in the promoters of its direct targets. Analysis of direct targets of GhNACl revealed that it directly activate the expression of not only downstream transcription factors, but also some non-transcription factor genes involved in secondary cell wall biosynthesis and cell wall modification. In addition, the expression levels of its downstream genes, especially related to cellulose biosynthesis were studied. The gene expression level of RNAi transgenic cotton plants and analysis of transcriptome are in progress.7. Molecular characterization of GhMPK17 and its role in transgenic ArabidopsisIn this study, a gene encoding a mitogen-activated protein kinase (MAPK) was isolated from cotton, and designated as GhMPKl 7. Quantitative RT-PCR analysis indicated that GhMPK17 expression was up-regulated in cotton under NaCl, mannitol and ABA treatments. The transgenic Arabidopsis plants expressing GhMPK17 gene showed higher seed germination, root elongation and cotyledon greening/expansion rates than those of the wild type on MS medium containing NaCl, mannitol and exogenous ABA, suggesting that overexpression of GhMPKl 7 in Arabidopsis increased plant ABA-insensitivity, and enhanced plant tolerance to salt and osmotic stresses. Furthermore, overexpression of GhMPK17 in Arabidopsis reduced H2O2 level and altered expression of ABA- and abiotic stress-related genes in the transgenic plants. Collectively, these data suggested that GhMPK17 gene may be involved in plant response to high salinity and osmotic stresses and ABA signaling. |
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