| Cotton is one of the most important economic crops in the world and the cotton fiber is the principal source of the textile industry. It will be of great significance to improve cotton fiber quality though isolating and identifying some key genes involved in cotton fiber development, and analyzing their expression profiles and regulation mechanism from the molecular level.NAC transcription factors are unique to plants and of wide distribution in the botanical world. They contain a conserved NAC domain about 150 amino acids in N-terminal ends and a highly variable transcription regulatory domain in C-terminal ends. Some studies have revealed that the NAC transcription factors participated in the regulation of plant growth and development, hormone level and responses to various kinds of stresses. Recently many researches have also showed that NAC transcription factors involved in the secondary cell wall development of inflorescence stems in Arabidopis. As the fiber strength mainly depends on the deposition secondeary cell wall, it is vital significance to isolate and identify some transcription factors special to cotton fiber and clarify their molecular mechanism in cotton fiber development.Several NAC genes (GhNAC1-26) have been isolated from cotton cDNA libraries in our laboratory, among of them GhNAC1 was predominantly expressed in cotton fiber, while the others were ubiquitiously expressed in different cotton tissues. In addition, GhNACl showed high sequence similarity to the NST1 and NST3 which have been demonstrated to function as master switches of activating the secondary wall biosynthetic program in inflorescence stems fibers of Arabidopsis. In this thesis, our studies are mainly focused on the genomic structure, activity of self-activication, subcellular localization and the regulation mechanism of secondary cell wall development to analyze the function of the GhNAC1 gene. About the other genes which were not special to cotton fiber, we mainly studied the expression profiles in the processes of stress response. The main results are as follows:1. GhNAC1 demonstrated the typical structrure characteristics of the plant NAC family transcription factor genesGhNAC1 cDNA sequence was isolated from the cotton 20dpa fiber cDNA library and then we acquired the genomic DNA sequence of GhNAC1 through PCR method.Sequence analysis revealed that GhNAC1 ORF included 1152bp and encoded a protein with 383 amino acids. The N-terminal of which contained a highly conserved NAC domain including A, B, C,D and E subdomains. and the C-terminal was rich in serme. Sequence comparison between cDNA and genomic sequence showed that the GhNAC1 genes contained three exons and two introns which were located in amino acid residue 90 and between amino acid residues 179 and 180 respectively. The NAC domain was located exactly in the first two exons, while the self-activation domain was in the third exton.2. The C-terminal region of GhNACl possesses trans-activation activityTo examine the trans-activation activity of GhNAC1, according to the characteristics of the genomic structure and amino acid sequence, yeast strain AH 109 was transformed with the fusion plasmids pGBKT7-GhNAC1 (Full), pGBKT7-GhNAC1 (N), pGBKT7-GhNAC1 (C) and the negative control pGBKT7, respectively, and then the positive clones were stroken in the selective medium SD/-Trp/-Ade and detected the x-gal activity. The results showed that the yeast cells containing pGBKT7-GhNAC1 (Full) or pGBKT7-GhNAC1 (C) grew well in the SD medium lacking Adenine, whereas cells containing pGBKT7-GhNAC1 (N) or pGBKT7 did not. Furthermore, in the presence of X-Gal, the yeast cells that grew well on the SD/-Trp/-Ade medium turned blue, indicating that another reporter gene, LacZ, was also activated. These results indicated that GhNAC1, like NTS1 and NST3 in arabidopsis, has transctivation activity and the transctivation daomain was located in the C-terminus.3. GhNAC1 is a nuclear-localized proteinGhNAC1-eGFP fusion expression vector were constructed and introduced into Arabidopsis by floral dip method. The green fluorescence distributed uniformly in the nuclei of hypocoty cells in transgenic Arabidopsis using confocal laser scanning microscop. This indicated that the GhNAC1 is a nuclear protein, and supports the idea that GhNAC1 functions as a transcription factor.4. Overexpression of GhNAC1 induces ectopic deposition of secondary walls in ArabidopsisTo investigate the role of GhNAC1 in plant development, we expressed GhNAC1 (NAC domain) and GhNAC1 (Full) driven by the cauliflower mosaic virus (CaMV) 35S promoter in wild type Arabidopsis, and some transgenic lines were obtained. RT-PCR analysis showed that they were expressed in plants of each transgenic line, respectively. It was found that about 20% of the seedlings with overexpression of GhNAC1 had severely curled leaves, a phenotype similar to that exhibited by Arabidopsis NST1 or NST3 overexpressors. To find out whether the curly leaf phenotype was due to ectopic deposition of secondary walls, we examined one of the secondary wall components, lignin. As a result. ectopic deposition of lignin was evident in the stem epidermis of GhNAC1 overexpressors by phloroglucinol staining. In addition, overexpression of GhNAC1 in Arabidopsis induced the expression of secondary wall-associated transcription factors and secondary wall biosynthetic genes. While the transgenic plants overexpressing GhNAC1 (NAC domain) did not show a visible phenotype like these.5. Interaction partners of GhNAC1In order to identify the interaction partners of GhNAC1 proteins in vivo, yeast two-hybrid analysis was performed using GhNAC1 (NAC domain) protein as a bait protein to screen cotton 10 dpa fiber cDNA library. Based on the results of sequencing, we speculated that GhNAC1 may interacte with two kinds of proteins:one is NAC family transcription factors, the other is ubiquitious protein. The results indicated that GhNAC1 proteins may form dimerization and regulated by ubiquitination pathway in vivo.6. Expression of GhNAC genes in cotton is induced by abiotic stressesTo determine whether GhNAC expression was induced by abiotic stresses and/or exogenous hormones, we performed real-time RT-PCR on roots of 5-day-old cotton seedlings under various treatments for 5h, including cold,100μMABA,20%PEG, 250mMNaC1. The experiment results showed that the effects of induced expression of GhNAC8, GhNAC10, GhNAC12, GhNAC16, GhNAC20 and GhNAC25 were obvious, while GhNAC 18 and GhNAC21 expressions were almost not induced by any of the four treatments. GhNAC8 expression was induced by PEG and ABA treatment and GhNAC10 expression was induced by H2O, ABA and NaCl. Likewise, H2O, ABA and NaCl also induced the expression of GhNACl2, especially, the expression of which was strongly induced by cold. In addition, the expression of GhNAC 16 gene was significantly increased by H?O and NaCl treatments, while the expression levels of GhNAC20 were upregulated after the Cold, PEG and NaCl. These results strongly suggest that the GhNACs genes may play important roles in the regulation of plant stresses.
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