Characterization Of Calcineurin B-Like Gene OsCBL6 From Rice (Oryza Sativa L.)

Intracellular signal molecule has been named as the secondary messenger in plant cell. Calcium is a ubiquitous secondary messenger in plants and play crucial roles in plant growth and development and responses to internal and external stimulation.The production and transduction of calcium signals are realized through the change of calcium concentration in cytoplasm.The hormones,light,mechanical wounding,abiotic stresses and pathogen elicitors may induce calcium transient,calcium oscillation or calcium wave. These Ca²⁺ signals is deciphered by various intracellular Ca²⁺ sensors which relays and enlarges Ca²⁺ signals into a wide variety of biological responses.In plant cells many Ca²⁺ sensors have been identified,including calmodulin(CaM) and calmodulin-related proteins, Ca²⁺-dependent protein kinases(CDPKs),and calcineurin B-like proteins(CBLs).Among them,CBLs proteins have been implicated as one type of the most important Ca²⁺ sensors in plant-specific calcium signaling.This thesis aims to clone and functional analysis of OsCBL6,a member of CBLs gene families,related in plant environmental stresses.In order to investigate the cellular function of OsCBL6 gene(accession number: DQ201200 in GenBank),Arabidopsis thaliana AtCBL1 was used as a query probe to search japonica rice genome database through BLAST algorithm program.By the specific primers and RT-PCR reaction the full length ORF of OsCBL6 gene were amplified.The 35S::OsCBL6-GFP fused gene was transformed into the onion epidermal cells by Agrobacterium tumefaciens.The results showed GFP fluorescence exclusively in cytoplasm. It was constructed a plant expression vectors carrying rice OsCBL6 gene under the regulation of cauliflower mosaic virus 35s promoter.The tabacco leaves were infected by Agrobacterium tumefaciens EHA105 with pBI121-OsCBL6 and deriving 10 transgenic plants.RT-PCR identification proved that the OsCBL6 was integrated into the genomes of thetabacco.There was marked difference in phenotype and tolerance to NaCl and PEG6000 stresses between T₀ transgenic plants and nontransgenic plants.The prokaryotic expression vector pGEX-4T-2-OsCBL6 was constructed and transformed into BL21(DE3) cells.The expression of OsCBL6 in E.coli was helpful to short lag phase in growth and increase tolarence to salt and osmotic stress.