| Cotton is one of the most important natural fiber crops in the world, and plays an important role in the national economy of China which is a major cotton producing and consuming country. Cotton fibers, the major product of cotton, are single-celled trichomes differentiated from the ovule epidermal cells. Due to the dependence of the output of production and efficiency on the yield as well as the quality of cotton fibers, improvement of the yield and the quality becomes the critical point of cotton breeding. However, the negative linkage between the yield and quality of cotton fibers limits the synchronous improvement of both of them by traditional methods. Gene engineering provides effective strategies such as transference of target genes to improve the yield and quality of cotton fibers. Therefore, it is significant to figure out the molecular basis for the development of cotton fibers. In addition, cloning the genes related to the yield and quality of cotton fibers, as well as studying the relationship between gene products, is of great theoretical and practical value for the improvement of both the yield and the quality of cotton fibers.Proteins of the 14-3-3 family are acid regulatory proteins that are highly conserved in all eukaryotes. They participate in various signal transduction and regulatory processes through binding to a wide range of target proteins, and play important roles in plant growth, development and signal transduction. In order to study the role of 14-3-3 proteins in cotton fiber development, Gh14-3-3 genes were isolated and characterized. The gene structures, expression patterns, as well as subcellular localizations, interactions and functions of the proteins encoded by Gh14-3-3 genes were further studied. The main results are as follows:1.Five cDNAs encoding 14-3-3 proteins were isolated from the cotton cDNA library, and designated as Gh14-3-3L, Gh14-3-3a, Gh14-3-3e, Gh14-3-3f and Gh14-3-3 g. Sequence comparison and phylogenetic analysis revealed that the five 14-3-3 proteins can be divided into two groups. Gh14-3-3L and Gh14-3-3a belong to theε-like group, and Gh14-3-3e and Gh14-3-3f belong to the non-εgroup. Four Gh14-3-3 genes were amplified from the cotton genome by PCR. Sequence comparison between the cDNAs and their corresponding genes revealed that all the introns in Gh14-3-3 genes were positioned in conserved residues. Gh14-3-3L and Gh14-3-3a which belong toε-like group contained five introns splitting their ORF into six exons. In contrast, Gh14-3-3e and Gh14-3-3f belonging to the non-εgroup contain three introns splitting their ORF into four exons.2. In order to investigate the subcellular localizations of Gh14-3-3 proteins, Gh14-3-3L:eGFP, Gh14-3-3a:eGFP and Gh14-3-3e:eGFP fusion expression vectors were constructed and introduced into cotton cells by transformation. The transformed cotton callus cells were observed under the confocal laser scanning microscopy. The results indicated that Gh14-3-3L was localized in the nucleis and cytoplasm, and Gh14-3-3a and Gh14-3-3e were localized in the cytoplasm.3. The results of RT-PCR indicated that the five Gh14-3-3 genes showed fiber-preferential expression patterns or exhibited its relatively high expression level in fibers. The expression levels of Gh14-3-3e and Gh14-3-3f were higher in ODPA ovules of wild type than those in fuzzless-fiberless mutant. The expression of the five Gh14-3-3 genes in fibers exhibit a developmentally regulated pattern, and their transcripts reached the highest levels in the elongation stage of fiber development. In addition, the expression levels of Gh14-3-3 genes were regulated by phytohormones such as IAA, GA3 and eBL. These results indicated that Gh14-3-3 genes might play a role in the differentiation, elongation and phtohormone response of cotton fiber development.4. The interactions between Gh14-3-3 proteins were analyzed by yeast two-hybrid. The results indicated that interactions were found in four cotton 14-3-3 protein pairs, but the interaction of the same 14-3-3 proteins was not detected in this study. The results indicated that each of the five Gh14-3-3 proteins might display isoform selectivity in the formation of heterodimers, and played a role as heterodimer in fiber development.5. To identify the interaction partners of Gh14-3-3 proteins, yeast two-hybrid analysis was performed using the three of the isolated Gh14-3-3 proteins (Gh14-3-3L,Gh14-3-3a,Gh14-3-3e) as bait to screen the two-hybrid library of cotton fiber cDNAs constructed on the prey vector. Each of the three 14-3-3 proteins was used in a separate screen.16 unique proteins were identified as target proteins of Gh14-3-3s. The targets of Gh14-3-3 proteins were related to various aspects of plant development, metabolism, and signal transduction. The results indicated that Gh14-3-3 proteins may function in the regulation of phytohormone response, plasma membrance and gene expression in fiber development.6. Overexpression of the Gh14-3-3e gene in Arabidopsis enhances the plant sensitivity to Brz. Gh14-3-3e was introduced into Arabidopsis plant, and several transgenic plants were obtained. RT-PCR and Western-blot analysis showed that Gh14-3-3e was expressed in plants of each transgenic line, respectively. Overexpression of Gh14-3-3e resulted in the seedlings displaying hypersensitivity to Brz in darkness. In addition, the expression level analysis of the BR-responsive genes in the transgenic lines is under way.7. In order to study the role of Gh14-3-3 proteins in cotton fiber development, overexpression and RNAi vectors of Gh14-3-3L and Gh14-3-3e were constructed and introduced into cotton by Agrobacterium-mediated transformation. Over 100 transgenic cotton plants were obtained. RT-PCR analysis showed that the expression level of Gh14-3-3 gene was higher in over-expression transgentic plants than that in wild type plants, and was knocked down in RNAi transgentic plants. The over-expression transgentic plants showed a drawf phenotype, and the branches were shorter, while the leaves were smaller than controls. The boll of over-expression transgentic plants drop within a few days after anthesis. Our results indicated that the expression levels of the genes related to fiber development were declined, resulting in slower initiation and elongation of fiber cells, compared with the wild type.
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