Subcellular Localization Of CsE1? And CsCML21 And Cloning And Expression Of The Promoters From The Pollen Of Camellia Sinensis

Ca²⁺ is a crucial second messenger as mediating responses to various abiotic and biotic environmental stimuli and regulating several developmental processes in plants.Tea plant?Camellia sinensis?L.?O.Kuntze?is an economic woody crop grown in different agro-climatic zones of the world with warm and wettish climates.However,cold stress always results in low yield,poor quality and delay to market of tea,which seriously influences the production and economic benefits of tea.The promoter region of CsCML21 dominates the transcription and expression of the gene,and involves in calcium-dependent signaling pathways.?1?In order to investigate the induction mechanism of the regulatory elements in the promoter region responses by Ca²⁺,we analyze the motifs and alter an ABRE cis elements related with calcium.We use a reverse genetics approach based on gain and loss of function mutants by SOEing-PCR to research on the physiological role of the promoter and motif.We apply the transient expression method using co-cultivation of Arabidopsis seedlings with Agrobacterium rhizogenes to visualize fluorescent fusion proteins which encoded by the gene downstream of the promoter in epidermal cells of acrospires.Statistically,function of the promoter and,particularly,motifs is identified by quantitation and analysis of the GFP.A model is presumed to describe the progress and function of a promoter contacts conspicuously and closely with the function of the gene or the protein as conclusion.?2?In this paper,full-length cDNA is identified for designing the gene-special primers in TAIL-PCR to clone the promoter of CsE1?.By sequencing and bioinformatics analyzing,we observe two CAAT-box,two TATA-box,two GATA-box,one LTR and one G-box locate in the 336 bp promoter region.After constructing and transferring the transient expression vectors into the onion epidermal cells,subcellular localization of CsE1?-GFP fusion proteins is verified in mitochondria,while the promoter can activate downstream gene expressing in entire cell.This experiment may provide useful information for the subsequent stable expression of the gene and regulation of the promoter in transgenic Arabidopsis in further study,and contribute to understand the molecular mechanism of cold resistance in pollen of Camellia sinensis.