Study On The Role Of GhPRP5Gene In Fiber Development Of Cotton

Cotton is one of the most important economic crops in the world and the cotton fiber is the principal source of the textile industry. Cotton fibers are single-cell trichomes which are derived from the epidermis of the ovule. Fiber development consists of four overlapping stages:initiation, elongation, secondary cell wall thickening, and maturation. Cotton growth and yield are also affected by various abiotic and biotic factors. It is very crucial to elucidate the molecular mechanisms of cotton fiber development.Previously, five genes encoding proline-rich proteins were isolated from cotton cDNA libraries. One gene which was designated GhPRP5was fiber specific and was fiber developmental stage regulated. GhPRP5transcripts were most abundant in5dpa fibers and less abundant in10dpa fibers. Subcellular localization results indicated that GhPRP5was mainly localized at the plasma membrane. In this thesis, functions of GhPRP5were investigated, the main results are as follows.1. GhPRP5promoter is fiber specificCotton fiber-specific promoters can be used for genetic manipulation of fiber. A2217-bp fragment of GhPRP5promoter was isolated by Genome walking. Analysis of the promoter sequence of GhPRP5gene revealed hormone and/or stress response elements are present. The2217-bp fragment of GhPRP5promoter was cloned into pBI101vector upstream GUS gene, and was transferred into Arabidopsis and tobacco by Agrobacterium tumefaciens-mediated methods. In transgenic Arabidopsis, GUS expression was specifically localized in rosette leave trichomes. Similarly, strong GUS activities were detected in trichomes of leaves, petioles and stems of transgenic tobacco plants. Because trichomes in Arabidopsis and tobacco show a lot of structural and genetic similarities to cotton fibers. It can be concluded that this promoter is fiber specific.2. Overexpression of GhPRP5in Arabidopsis hinders plant growthTo evaluate the function of GhPRP5, we expressed GhPRP5in Arabidopsis. RT-PCR analysis showed that GhPRP5transcripts were highly abundant in the eight transgenic lines, while undetectable in wild type. Two homozygous transgenic lines (L7-2and L11-1) of the fourth generation (T4) with the highest expression of GhPRP5were selected for further phenotypic analysis. Germination rate did not show any difference between wild type and transgenic plants. However, the root length and leaf size were significantly different at seedling growth period. The root length of transgenic lines was significantly shorter and the leaf size of transgenic lines was much smaller than those of wild type. In addition, seeds collected from transgenic plants were much smaller than those from wild type, and the weight of one thousand seeds from transgenic plants were less than seeds from wild type. Epidermal cells were smaller in the leaves of the transgenic plants than those in the leaves of the wild type. Cross-sections of the leaf midvein also demonstrated that cells of transgenic lines were smaller than those of wild type, whereas the number of cells is similar in leaves of both transgenic plants and wild type. All of the above results clearly showed that overexpression of GhPRP5in Arabidopsis hindered cell growth and expansion, leading to the reduced cell size.3. Suppression of GhPRP5expression in cotton enhanced fiber elongationTo validate the function of GhPRP5in fiber development, GhPRP5RNAi vector was constructed and transformed into cotton. Twelve original (TO) transgenic cotton lines were generated. Real-time RT-PCR results showed GhPRP5expression levels in5dpa transgenic fibers from six lines in the first generation were ranged from10-40%of that in wild type, and this reduced expression was inherited in the second generation in six lines tested. We selected three representative transgenic lines (lines1,4, and8) for further phenotypic analysis. The results showed that the fiber length of transgenic cotton lines was longer than wild type. Interestingly, when the lint fibers were torn off, we found that the length of fuss fibers was also longer in transgenic lines.4. Transcriptional up-regulation or down-regulation of several fiber-related genes in GhPRP5-suppressed transgenic cottonThe cell wall-PM-cytoskeleton continuum is important for the perception of signals from the external environment and for coordinated cell growth and expansion during plant development. T2transgenic cotton plants were chosen to analyze the expression levels of these fiber-development related genes. The expression level of GhACTl showed little changes, but GhTUA9transcript level was up-regulated20-74%in transgenic lines, compared with wild type, whereas GhTUBl mRNA level was reduced dramatically (by down to50%). The results showed that GhFLA2mRNA level in transgenic lines is only about38-82%of that in wild type, and GhFLA14were markedly decreased (by almost80%) in transgenic plants. GhFLA4transcript level was increased significantly (by up to2-fold) and GhFLA15mRNA level was up-regulated about10-fold in the transgenic plants, compared with that of wild type. In addition, fiber specific GhPRP3mRNA level was increased about2-3folds in the transgenic lines, compared with wild type. The expression level of the expansin gene (GhEXP) was higher (with more than2-fold increase) in all the transgenic lines than that in wild type. The expression level of GhXET were increased dramatically by up to3folds in transgenic lines. All of these data showed that repression of GhPRP5affected expression of the genes implicated in cytoskeleton, AGPs, PRPs, expansin, and XTH, suggesting that the cell wall-PM-cytoskeleton continuum may play a crucial role in fiber development. 5. GhPRP5-interacting proteins identified by yeast two-hybrid assayTo identify the interacting protein with GhPRP5, we performed a yeast two-hybrid assay using GhPRP5as bait to screen the two-hybrid library of cotton10dpa fiber cDNAs constructed on the prey vector. The results showed that the identified eight proteins were divided into four classes. Five out of eight represent unknown proteins, one is a GCN5-related N-acetyltransferase, one belongs to proline-rich proteins, and one is an auxin-responsive family protein. Three selected proteins that we are interested in were checked for the presence of cDNA-AD fusion and confirmed in the one-to-one interaction analysis. The experiment clearly demonstrated that the selected three proteins could interact with GhPRP5. These results suggested that GhPRP5may form homodimers.6. Expression of the GhPRP5gene in Arabidopsis enhances susceptibility to ABA and salt stressConstitutive expression of GhPRP5in Arabidopsis thaliana under the control of the Cauliflower Mosaic Virus (CaMV)35S promoter enhanced plant sensitivity to salt and abscisic acid (ABA) during seed germination and vegetative growth. Salt stress and ABA significantly inhibited the germination of the transgenic lines overexpressing GhPRP5. When growing on media with NaCl, the GhPRP5-overexpressed plants displayed less cotyledon greening rate compared with the wild type. In contrst to the normal growth conditions, ABA inhibited the elongation of primary root more severely in the transgenic lines. Real-time RT-PCR technique was used to analyze the transcription of the stress-related genes RD29A, RD29B, and ABA-related genes ABI1in transgenic lines when subjected to salt stress and ABA treatment. These results demonstrated that the transgenic plants became more sensitive to ABA and salt stress.