Identification And Function Analvsis Of TCP Transcription Factors In Watermelon

The plant-specific TCP transcription factor family was known to play versatile functions in plant growth and development. For example, it has been found to involve in seed germination, plant branching and organ development in several model plants. As an important economic crop, watermelon is widely cultivated around the world and brings remarkable economical benefits. However, no systematical study about this family has been performed in watermelon to date. In this thesis, I identified and named all TCP members for the first time. A comprehensive analysis of CITCP sequences, classification, structures, evolution, chromosome location, conserved domains and spatio-temporal expression was also performed. Plant height is an important agronomic trait of watermelon. Dwarf watermelon plants were prone to be cultivated and managed because of their compact morphology. They were also suitable for intensive planting, which could increase the utilization of solar energy and land resources as well as save labor force and production cost. However, most dwarf types watermelon cannot be used in agricultural industry because of bad fruit quality. Thus, study on the molecular mechanism of shortened stem can provide theoretical support for introducing dwarf trait to high-quality watermelon cultivars. On the other hand, leaf senescence caused by grafting or stress can not only result in a decline in photosynthetic capacity, but also affect the yield and quality of watermelon fruit. Some TCP proteins have been indicated to involve in plant height development and leaf senescence. Here, I perform a research on the functions of CITCP genes in regulating plant height and leaf senescence of watermelon. The main results are as follows:1. Identification of the number, chromosome distribution, gene structure and the classification of subfamilies of TCP family in watermelonA total of 27 TCP encoding genes distributed across 9 chromosomes were identified in watermelon and they were named CITCPla-ClTCP21 based on sequence homology and the naming rule of Arabidopsis. The analysis on gene structure showed that only three ClTCP genes contain 1-2 introns among the whole family. All of ClTCP genes were confirmed to contain a non-canonical basic-Helix-Loop-Helix (bHLH) domain composed of 55 or 59 amino acids, also called TCP domain. They could be distinguished into two subfamilies according to the differences in TCP domain and Class Ⅱ could be further classified into two branches CYC/TB1 and CIN. The analysis of conserved motif structure showed that R domain is absent in all Class Ⅰ proteins and present in CYC/TB1 proteins. While the miR319 site is only present in a subset of the CIN-like genes. These findings provided useful information for the function predication of TCP transcription factors in watermelon.2. Detection of expression characteristics of CITCP genes in different growth stages and tissuesThe spatio-temporal expression profiles of CITCP genes were analysed using semi-quantitative RT-PCR. The results indicated that all genes exhibited relative higher expression level in shoot apical meristem. Furthermore, most CIN genes showed higher expression level in seeds, cotyledons and leaves and most CYC/TB1 genes in internode and shoot apical meristem, while TCP genes in Class Ⅰ seemed to be constitutively expressed, which might be involved in the regulation of each growth stage. These differences in expression level provide important guidance for the function analysis of CITCP genes in different tissues.3. ClTCP14a and CITCP15 genes play an important role in the regulation of watermelon plant heightIt was found in this study that plant growth regulators GA and CCC exhibit positive and negative effect on plant height, respectively. Meanwhile, the expression levels of ClTCP14a and ClTCP15 were promoted by GA and inhibited by CCC. In addition, ectopic expression of either ClTCP14a or CITCP15 in Arabidopsis tcpl4 tcpl5 double mutant, which showed decreased plant height, reverted its height to the wild type level. In ClTCP14a and ClTCP15 overexpression Arabidopsis plants, the expression levels of AtKO1 and AtGID1a, which were associated with GA biosynthesis and reception, were both up-regulated and the expression level of GA2Ox3 associated with GA degradation was down-regulated. Indicating that ClTCP14a and CITCP15 could positively regulate plant height via GA-related pathway.4. CITCPla,2a,16,20a, and 21 genes might play a significant role in regulating leaf senescenceQuantitative RT-PCR analysis showed that the expression levels of ClTCP1a and ClTCP16 were induced during age-triggered and dark-induced leaf senescence, while ClTCP2a, ClTCP20a and ClTCP21 were repressed. Suggesting that ClTCP1a and CITCP16 may accelerate leaf senescence and ClTCP2a, ClTCP20a, ClTCP21 may delay leaf senescence. And it has been verified that ClTCP1a positively regulates leaf senescence in Arabidopsis.