Cloning Of The B-type Gene CsTM6 Of The Tea Tree MADS-box Family And Its Function With CsDEF

Tea plant (Camellia sinensis (L.) O. Kuntze) as a perennial evergreen woody plants, is an important economic plant leaf. Annual growth of tea by the vegetative and reproductive growth composition, flowering is the reproductive growth of tea tree, that is a necessary stage in the formation of fruit tea. Tea tree blooms will compete for nutrients, resulting in the decline in tea production and quality. Therefore, the control of tea reproductive growth, promote vegetative growth is the key to improving the yield and quality of tea.How reasonable regulation tea reproductive growth and vegetative growth, increase yield and quality of tea from fundamentally which are problems to be solved of tea researchers. Based on flower development ABCDE model, B class genes were involved in sexual differentiation and functionality development. Therefore, it is necessary to clone B class genes and dissect the molecular mechanisms of them in the development of tea plant. Clarify the regulatory mechanisms of transcription and molecular levels on tea flower development, for the future use of biological technology to control the reproductive growth of tea tree to provide important theoretical basis.In this study, we isolated a B class MADS-box genes, CsTM6, from the representative of Shaanxi province tea plant "Camellia sinesis cv. Ziyangzhong" by PCR-based homology cloning and RACE. Bioinformatics prediction and phylogenetic analysis were employed for this genes. Moreover, we preliminary dissected the function molecular mechanism of CsTM6 and CsDEF gene in the flower development of tea plant by performing real-time PCR, ectopic expression in Arabidopsis, subcellular localization, BiFC, yeast two hybrid and transactivation ability assay.1. We isolted a cDNA sequences with 630bp open reading frame by PCR-based homology cloning and 3’RACE from differentiated flower buds of "Camellia sinesis cv. Ziyang zhong". It was homologous to TM6 gene of Camellia japonica, Camellia oleifera by BLASTN alignment, and contained the conserved MADS domains, K domain and paleoAP3 motif. Therefore, it was named after CsTM6. Then CsTM6 was submitted to GenBank under the accession number of ALK82292.2. Bioinformatics analysis demonstrated that CsTM6 was B class genes and encoded a MIKC-type MADS-box transcription factor possessing a similar function mechanism with other MADS-box transcription factor by predicting conserved domain, subcellular localization, hydrophobicity/hydrophilicity. secondary structure tertiary and quaternary structure, and homology alignment. Moreover, phylogenetic tree showed that CsTM6 gene be accurately classified to TM6 evolutionary.with the evolutionary tree of B class gene AP3/DEF evolution system of 18 species of affinity is consistent with the traditional plant classification, divided into CsTM6 genes and highest CjTM6 and CoTM6 similarity, which are in complete accord with expected results.3. The real-time PCR showed that CsTM6 and CsDEF have different levels of expression in tissues and organs in the tea, but two different modes of expression. CsTM6 in high amount of expression in the petals, stamens, and fruit, followed by expression of the carpel, expression in sepals than flower buds and shoots in a slightly higher expression levels; CsDEF are expressed in different tissues and the abundance of different expression, its high expression in petals, followed by expression of sepals, stamens, fruit and flower buds, carpel and shoots lowest expression. Showed that the CsTM6 and CsDEF gene expression pattern have distinct species specificity. As floral organ identity genes, CsTM6 and CsDEF high expression of common regulation in the second and three wheels of flower organs petals and stamens development, in addition, the two genes in fruits high expression also hinted that they may have to participate in the growth of tea tree fruit development.4. Ectopic expression of Arabidopsis results show,35S::CsTM6 and 35S::CsDEF transgenic Arabidopsis plants phenotypic differences, CsTM6 performance ectopic expression in Arabidopsis as petals transformed into stamens, similar results also appear in the petunia and tomato; CsDEF ectopic expression of in Arabidopsis caused sepals into petals,the same results also appear in gerbera, white petal-like sepals edge appeared organized and middle of the green sepals like tissue, such as the petals and sepals bloom.Indicates that during the development of flower, CsTM6, CsDEF conserved gene function and sequence differences CsTM6 and CsDEF genes may lead to functional differentiation.5. Subcellular localization analysis and transcriptional activity assay were employed and verified CsTM6 and CsDEF were nuclear localization proteins, as a transcription factor, however, did not possess transactivation ability alone. And the same time, yeast two hybrid showed CsTM6 and CsDEF are associated with CsGLO1 interact to form heterodimers,CsDsDEF also with CsAG interact to form heterodimers; BiFC analysis indicated CsTM6 and CsDEF can interact with CsGLO1、CsGLO2 and CsAG to form heterodimers. The results of two protein interaction model differences, possibly due to CsTM6 and CsDEF gene coding sequence differences, and indicated the possible need to be combined with other transcription factors get involved in the transcriptional regulation of the activity of flower development regulation.