Identification And Expression Analysis Of Genes Associated With The Bud Dormancy And Its Release Of Tea Plant (Camellia Sinenesis)

As a perennial plant, tea plant (Camellia sinensis) growth may be influenced by different environment stress, such as low temperature, short daylight during each growth cycle. In winter, its leaf buds need to enter dormancy and this help them to safely pass low temperature. Following the temperature rise and daylight becomes longer, the dormant buds transform to normal growth. In this study, suppression subtractive hybridization was used to construct two pairs of double-barreled cDNA-SSH libraries of bud dormancy and bud break of the two varieties, the special early sprouting cultivar, Longjing 43 and the late sprouting cultivar, Zhenghe Dabaicha. Some dormancy-related or bud break-related genes were isolated, and 3 genes were cloned through RACE method. Gene expression patterns of the 3 genes and some auxin-related genes during the time of bud dormancy and bud break were analyzed through real-time quantitative PCR. It is very helpful to explore the molecular mechanism of bud dormancy in tea plant.1. After screening, the positive clones were sequenced, 1287 unigenes were obtained, which can be classified to 208 contigs and 1079 singletons. The libraries of dormant buds exhibited higher sequence redundancy compare to the ones of sprouting buds no matter species or quantities. And each library has its specific dominant gene expression. In the sprouting bud libraries, the expression of genes related to ribosomal protein, histone, and chlorophyll a/b-binding showed relative higher abundance. While in the dormant bud libraries, the expression of genes related to dormant-related ESTs of tea plant, dehydrin and cold-induced proteins were richness. Based on TAIR gene ontology (GO) classification system, the differential gene expression profiles of dormant and sprouting buds were established. According to the GO results, we found that there were significant differences on gene expression between dormant and sprouting buds. The ratio of genes which associated with the stress response in dormant buds was higher than sprouting buds. But the ratios of genes which associated with growth, cell proliferation, electron transport or energy metabolism in sprouting buds were higher than dormant buds.2. The full length cDNA sequence of the auxin-repressed protein gene (CsARP1) was cloned from dormant buds by SMART RACE-PCR. Homologous alignment showed that the deduced amino acids of CsARP1 contained auxin-repressed domain characteristic and it is hydrophilic protein without signal peptide and transmembrane domains. The results of real-time quantitative PCR showed that the CsARP1 gene was expressed in a higher level in dormant buds than sprouting buds. It suggests that the expression of CsARP1 gene is correlated to the bud dormancy transition.3. The full length cDNA sequences of the cyclin (CsCYC1) and cyclin-dependent kinase (CsCDK1) were cloned from sprouting buds by SMART RACE-PCR. The pupative cyclin of tea plant contained the characteristic cyclin family signature sequence and conservative cyclin-box dormain. The pupative CDK of tea plant contained the characteristic CDK family signature sequence and conservative dormains. The cyclin and CDK of tea plant are all hydrophilic proteins without signal peptides and transmembrane domains. And they have cyclin and CDK typical three dimensional structures, respectively. The results of real-time quantitative PCR showed that the two genes were expressed in a low lever in dormant stages, and then their expression lever increased quickly before sprouting, and after bud break, their expression lever decreased, but it was higher than dormant buds. The results suggested that CsCYC1 and CsCDK1 were correlated to the bud dormancy release.The expression parterns of some auxin-realted genes, including auxin response factors (ARFs), primary auxin responsive genes, auxin-repressed protein genes, auxin-regulated genes, auxin influx carrier gene and auxin-binding protein (ABP) gene, were studied in different stages of tea plant bud dormancy and its release. The results showed that (1) the expression levels of five in six ARF homologous EST fragments were up-regulated in the deep dormancy and down-regulated after sprouted, the level of the last one showed the opposite expression levels. ARF genes were involved in the complex process of tea bud dormancy and its release. (2) There were significant differences among the expression patterns of 4 ESTs which are homologous with primary auxin responsive genes. EST which is homologous with SAURs was up-regulated in the dormancy stage but down-regulated after dormancy release. On the contrary, one of 3 ESTs which are homologous with GH3 was up-regulated in sprouting stage while down-regulated in dormant stages and after sprouting stages. Another two ESTs expressed the highest expression levels in the deep dormancy. One of them showed down-regulated trends in the later dormancy stage and growth stages while the other one showed a small expression peak which lower than that in the deep dormancy. The results indicated primary auxin responsive genes were also involved in the complex process of tea bud dormancy and its release. (3) Expression levels of ARP genes were higher in dormancy stage than that in the post-germination stage. (4) The CsGLP1 gene which was homogenous with ABPs has a lower expression level in the dormancy stage but maintain rising trend after germination. (5) The EST which is homogenous with auxin influx carrier gene had the highest expression levels in the deep dormant stage but maintain lower levels in other stages. (6) Two auxin-regulated ESTs showed different expression patterns. The expression levels of auxin-induced EST were higher in the dormant stages than that in the sprouting stages, while the levels of the other auxin-repressed EST were higher in the sprouting stage than that in the dormant stages. These results indicated auxin promote the dormancy of tea axillary buds.