Functional Analysis Of CBL Family Gene NsylCBL10 Innicotiana Sylvestris

The calcineurin B-like protein(CBL) represents a unique group of calcium sensors and was well known to modulate abiotic stress. Although research progress has been made in Arabidopsis, Poplar and Oryza as well as the other species, the CBL family of tobacco is still unclear. A new CBL family gene Nsyl CBL10 was cloned from Nicotiana sylvestris using homologous cloing strategy. The sequences of amino acid and genome as well as the expression patterns of Nsyl CBL10 were analyzed. Furthermore, experiments have been done to verify the function of Nsyl CBL10 involved in salt stress signaling pathway. The results were as follows:(1) Nsyl CBL10( Genbank number: KF667488.1) was cloned from N.sylvestris using homologous cloning strategy. The full length sequence of CDS was 783 bp and encodes a protein with 260 amino acids. The phylogenetic tree showed that Nsyl CBL10 has associated with At CBL10 and Pt CBL10 were clustered into numberⅠsubgroup. A 3857 bp genomic DNA sequence of Nsyl CBL10 was contains 9 exons and 8 introns.(2) p GDG-Nsyl CBL10 and p GDR-Nsyl CBL10 were constructed which CBL10 were fused to C-terminal of GFP or Ds Red2. Subcellular localization assays showed that Nsyl CBL10 was predominantly localized to the plasma membrane, cytoplasm and the nucleus.(3) The q RT-PCR analysis illustrated that Nsyl CBL10 expressed in main root, stem, leaf, petal, sepal and axillary bud in almost equal amount with little variation except lateral root(the lowest). Nsyl CBL10 is predominantly expressed in all parts of N.sylvestris.(4) The expression levels of Nsyl CBL10 were up-regulated under high salt, high temperature and drought stress, but down-regulated under low K+ stress. These results demonstrated that Nsyl CBL10 might play a role in abiotic stress signaling pathways.(5) Phenotype assays under high salt stress demonstrated that Nsyl CBL10 not only can rescue salt sensitivity of Arabidopsis cbl10 mutant but also enhanced the Na Cl hypersensitivity to high salt conditions. The distribution assay of Na+ and K+ indicated that Nsyl CBL10-OE can maintain less intracellular Na+ in roots and lower Na+/K+ ratio under high salt stress, which may result in better maintenance of ionic balance and reduce harmful ion accumulation in plant cells.(6) The promoter of Nsyl CBL10 was cloned. It contains some cis-elements of stress-respondence. p BI101-CBL10 pro vector was constructed which was in-frame fused to the reporter gene GUS. And it was transformed into tobacco and got the transgenic plants.