Studies Of Two Members Of CBLs Gene Family From Hordeum Brevisubulatum

CBLs (calcineurin B-like proteins), a class of plant-specific Ca²⁺ sensor, which were originally identified in A. thaliana, were named as they are similar to CNB and NCS in animals. CBLs play important roles in plant's specific growth and development processes and stress responses with its interaction protein CIPK (CBL-interacting protein kinase) . Research on the CBLs is currently focused in Arabidopsis and rice, but there was very little study on the other plants .Shortsubulate barley (Hordeum brevisubulatum (Trin.) Link) is one of the most salt-tolerant halophytes in Triticeae, which has evolved the mechanism of adaptation to salinity during long term of evolution. It is valuable for research and application.Cloning CBLs in Shortsubulate barley not only shows theoretical meanings, but also benefits to practical application, providing potential candidate genes for improving stresses torlerance by genetic transformation in plant. Here, we isolated two genes in Shortsubulate barley and name them HbCBL1 and HbCBL2. We analyzed the response of the genes to drought stresses, subcellular location and their function, the main progresses are as follows:1. Clone and sequence analysis of HbCBLs: RT-PCR was used to clone full length of HbCBL1 and HbCBL2. Using software analysis,we know that both HbCBL1 and HbCBL2 has CBL speicific Ca²⁺ binding site-EF hands, HbCBL1 has a N-myristoylation site at its N terminal domain.2. Fussion express vectors were constructed and transformed into onion epidermal cells by particle bombardment, respectively. These vectors were also transformed into Arabidopsis by floral dip.Confocal observations showed that HbCBL1 accumulated in plasma membrane as well as in nucleus and HbCBL2 was accumulated in tonoplast.3. Using Nouthern blot to analysis the expression of HbCBLs while under drought stess. Without any stress, HbCBL1 and HbCBL2 have a small amout of background espression, and expression in roots is more strong than in the leaf. By the treatment of PEG6000 and mannitol, espressions of HbCBL1 and HbCBL 2 started to diminish as the processing time went by.4. In order to explore the biological function of HbCBLs, several overexpression vectors were constructed and transformed into wild type and mutant Arabidopsis. We obtained several transgenic plants and now they have been selected to the T2 generation.