Cloning Of CsGLO1 And CsGLO2 In The MADS-box Family Of C Tea And Its Function With C Gene

Tea plant (Camellia sinensis (L.) O. Kuntze) is a kind of economically important woody crop for leaves consuming. It will take about one and a half year from bud formation to seed maturation, namely "co-occurence of flower and fruit", which consumes a great deal of nutrient and will inevitably disturb leaf development. Therefore, controlling reproductive growth and promoting vegetative growth is a key that raises the yield and qualities of tea.Belonged to core eudicots, tea plant are composed of four types of organs arranged in concentric whorls from outside to inside:sepals, petals, stamens and carpels. As important floral organs, stamens and carpels are the morphological bases of sexual reproduction and fruit development. Based on flower development ABCDE model, B and C class genes were involved in sexual differentiation and functionality development. Therefore, it is necessary to clone B and C class genes and dissect the molecular mechanisms of them in the development of tea plant. Results expected not only provide a scientific basis for the genetics research of sexual reproduction, but also pave a theoretical foundation for inhibiting "co-occurence of flower and fruit" and promoting leaf development from the reproduction organs differentiation using breeding and cultivation.In this study, we isolated two B class MADS-box genes, CsGLO1 and CsGLO2, from differentiated flower buds of 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 those two genes. Moreover, we preliminary dissected the function molecular mechanism of CsGLO1, CsGLO2 and CsAG gene in the flower development of tea plant by performing real-time PCR, ectopic expression in Arabidopsis, subcellular localization, yeast two hybrid and transactivation ability assay.1. We isolated two cDNA sequences with 630bp and 627bp open reading frame by PCR-based homology cloning and 3’RACE from differentiated flower buds of "Camellia sinesis cv. Ziyang zhong". They were homologous to PI/GLO gene of Camellia japonica, Camellia oleifera, Actinidia chinensis, Arabidopsis thaliana and Antirrhinum majus by BLASTN alignment, and contained the conserved MADS domains, K domain and PI motif. Therefore, they were named after CsGLO1 and CsGLO2, respectively. Then CsGLO1 and CsGLO2 were submitted to GenBank under the accession number of KJ630568 and KJ630569, respectively.2. Bioinformatics analysis demonstrated that CsGLO1 and CsGLO2 were B class genes and both 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 CsGLO1 and CsGLO2 are belonged to basal asterids PI subfamily and the similitude of PI/GLO homologous genes are positive correlated with relationship of species, which demonstrated that PI/GLO transcription factor functions by forming a similar high structure based on conserved sequence and motif.3. The real-time PCR showed that CsGLO1 and CsGLO2 were predominantly expressed in the petal, stamen and fruit, and CsAG was abundantly expressed in the stamen, carpel and fruit. It supported that there was a evident species specificity in terms of the expression pattern of CsGLO1, CsGLO2 and CsAG, as flower organ identity genes, both CsGLO1 and CsGLO2 might exert their role as a function unit to bear the complete GLO-function in the development of petal, and stamen combined with high expression level CsAG, which specified carpel identity in the fourth whorl organ. Besides, those three genes may be involved in the development of fruit in the tea plant.4. Flowers of Arabidopsis plants of both 35S::CsGL01 and 35S::CsGLO2 showed partial first whorl conversion of sepals to petals. Sepals in the first whorl were mosaic organs composed of petal-like white tissue on the margins and green sepal-like tissue in the middle and opened significantly.35S::CsAG plants showed flowering early, poor vegetative growth, and curling and yellowing rosette leaves. And the same time, stigmatic papillae and ovules developed on the first whorl-converted carpeloid sepals and petals were partially or entirely absent and converted into stamens. These results supported that those three genes had a conserved function in the development of flower. Besides, the sequence difference between CsGLO1 and CsGLO2 can not lead to absolute subfunctionalization. And both CsGLO1 and CsGLO2 might redundantly function as a unit in the development of tea plant.5. Subcellular localization analysis and transcriptional activity assay were employed and verified CsGLO1, CsGLO2 and CsAG were nuclear localization proteins, as a transcription factor, however, did not possess transactivation ability alone. And the same time, yeast two hybrid showed there is protein interaction between each other. All these results hinted that CsGLO1, CsGLO2 and CsAG perhaps as one subunit of tetramer contains a protein with an activation domain then the entire tetramer might function to active transcription in the regulation of flower development. By forming heterodimers, both CsGLO1 and CsGLO2 might function as a unit in the development of tea plant. Besides, it reflected that these two genes possessed a similar function.