Functional Analysis Of NsylCBL10 And NsylCBL5 In Nicotiana Sylvestris

The leaf quality of tobacco,an important commercial crop in China,is easily affected by biotic and abiotic stresses.The calcium sensor Calcineurin B-like protein?CBL?,cooperating with CBL-interacting protein kinase?CIPK?,occupies an essential position in stress-responsive mechanisms in plants.Therefore,the identification and functional analysis of CBL-CIPK pathways in tobacco will contribute to clarify the defence mechanism of tobacco under stresses,which will further help to stable the leaf quality by guiding breeding.This study aims at conducting a deeper functional analysis of NsylCBL10 and NsylCBL5,two genes cloned from Nicotiana sylvestris,by expressing them in Arabidopsis thaliana.Moreover,NsylCBL10 and NsylCBL5 promoters are also analyzed by cis-elements prediction and GUS??-Glucuronidase?staining assay.Under the condition of 100?M K⁺,the roots of NsylCBL10-overexpressing lines and NsylCBL10-complementary lines are more sensitive to NH₄⁺,which exhibited a shorter root phenotype under a relatively low NH₄⁺concentration?20 mM NH₄⁺?.Besides,there is no distinctive phenotypic difference between wild type and NsylCBL10-overexpressing lines,NsylCBL10-complementary lines and cbl10 when treated with 85?M¹25?M Cd²⁺.NsylCBL10 promoter was predicted to contain various cis-acting regulatory elements,including tissue-specific elements,light-responsive elements,phytohormone responsive elements and stress-responsive elements.GUS staining assay showed that the GUS,driven by NsylCBL10 promoter,mainly expressed in cotyledon node-root transition section and root tips during germination stage,while the active region expanded to cotyledons during cotyledon stage.Besides,GUS in adult plants was detected to express in leaves during cross-seedling stage,as well as in style,mature anther,root tips and lateral root base.Additionally,GUS staining assay showed that the expression of GUS,driven by NsylCBL10 promoter,was down-regulated under light condition while up-regulated in dark.NsylCBL5,a homologous gene of AtCBL5,was cloned from N.sylvestris and predicted to contain8 exons and 7 introns.The subcellular location assay indicated that NsylCBL5 mainly localizes in the plasma membrane and cell nucleus.Besides,the phenotype experiments showed that there is no phenotype difference between cbl5 mutant and WT when treated with 150 mM Na⁺,while NsylCBL5-overexpressing lines exhibited a salt-tolerance phenotype.NsylCBL5 promoter in a length of 2698 bp was cloned from N.sylvestris and was predicted to contain stress-responsive elements,light-responsive elements,tissue-specific elements and phytohormone responsive elements.Above results showed that the overexpression of NsylCBL10 enhanced root sensitive to NH₄⁺under a low K⁺condition in Arabidopsis,while had no effect on its high Cd²⁺-tolerance ability.NsylCBL10 promoter,an inducible promoter whose activity dynamically change in different developmental stages,has the drive activity in many tissues of tobacco such as the meristematic tissue with a high proliferative activity and floral organs and its expression in hypocotyl may be regulated by light signal.The overexpression of NsylCBL5 enhance the salt-tolerance of Arabidopsis.