The Cloning Of Tea Plant CsFD Gene And Its Function With CsFT Gene In Floral Transformation

Tea plant(Camellia sinensis(L.)O.Kuntze)is a perennial evergreen economic crops.At present,the theoretical research of tea plants is mainly concentrated in the mechanism of tea inclusion and the synthesis of effective components,the self incompatibility and molecular mechanism of flower development.There will be a phenomenon that is the fruits and the flowers of tea appear at the same time during the reproductive period,will compete with vegetative growth of leaves.And flowering is the first step of tea from vegetative growth to reproductive growth.Therefore,it is necessary to elucidate the molecular mechanism of floral transition of tea tea and grasp the flowering time.This study will provide theoretical basis for improving tea or tea flower yield and quality,promoting the development of tea industry.In the process of flowering,flowering time genes FT(FLOWERING LOCUS T)and FD(FLOWERING LOCUS D)play the important roles,not only can accept and integrate the signals of flower induction pathways,and the signals can be transmitted to the flower meristem identify gene and flower organ identify gene.In this study,the full-length cDNA sequence of CsFD gene was successfully obtained from the differentiating flower buds of the representative of Shaanxi province tea plant "Camellia sinesis cv.Ziyangzhong" by RT-PCR and RACE,and analysis its sequence.Secondly,the bioinformatics analysis and phylogenetic analysis were employed for CsFT and CsFD genes.In addition,explored the function of CsFT and CsFD gene by quantitative real-time PCR,yeast two hybrid system and transgenic Arabidopsis.The main conclusions are as follows:1.We isolated three fragments from early flower buds of tea plant "Camellia sinesis cv.Ziyangzhong" by RT-PCR and RACE,respectively,169 bp conservative fragment,342 bp 3'terminal and 659 bp 5' terminal,then obtein the 959 bp length cDNA sequence by sequence assembly.Using BLAST and phylogenetic analysis found that the sequence has similar structure with FD homologous genes of Actinidia chinensis,Arabidopsis thaliana,Vitis vinifera and other species,and has a conserved bZIP domain:N-X7-R-X9-L-X6-L-X6-L,at the same time,the sequence also has three conserved motifs respectively,A,LSL and SAP,so was named CsFD,GeneBank accession number is MF362998.The CsFD gene has 959 bp full length of cDNA,and the length of CDS is 567 bp,which encodes 189 amino acids.2.Bioinformatics analysis showed that CsFD and CsFT proteins belong to PEBP and bZIP transcription factors,respectively.They are hydrophilic proteins and phosphorylated sites are mainly Serine.Phylogenetic analysis showed that tea CsFD,grape VvFD and kiwifruit AcFD were first grouped into one class,and bunched Solanaceae plant FD proteins together,then with Cruciferae FD protein as a branch;CsFT,Oil-tea CoFTand kiwi FT protein were clustered into one clade,secondly,bunched Rosaceae plants and Solanaceae plant FD proteins together,finally,with monocot FT homologous proteins clustered into one clade.It shows that the conservatism between CsFT and CsFD homologous proteins is higher in the Ericales,and the similitude of FT and FD homologous proteins are positive correlated with relationship of species.3.Ectopic expression of Arabidopsis thaliana showed that the average flowering time of CsFT transgenic Arabidopsis thaliana was advanced 5.87 days,and the average flowering time of CsFD transgenic Arabidopsis was earlier 2.08d than wild Arabidopsis thaliana under long day conditions,indicating that the CsFT and CsFD genes of tea plants could promote the flowering of plants.that its possible molecular mechanism is tea CsFD and CsFT has high expression in the early floral buds,then form protein by transcription and translation,CsFD and CsFT protein interaction,promoting the expression of downstream genes SOC1 and AP1 to control the flowering time by real-time quantitative PCR,the yeast two hybrid experiment and further discuss.In addition,there was also a weak interaction between CsFD and CsFD by the yeast two hybrid experiments.4.Transgenic experiments found that the number of transgenic Arabidopsis rosette leaves decreased,and the leaves are thin,showed CsFT and CsFD genes not only promote flowering,but also inhibit vegetative growth.In addition,in the time of flowering,the number of apical buds of CsFD transgenic plants has a significant increasing,the number of the main stem of CsFT transgenic plants are markedly increased,it showed that CsFD and CsFT gene were able to control the floral meristem and number of main stem to increase the number of flowers.