Cloning and Bioinformatics Analysis of Ds CBL4 Gene from Dianthus spiculifolius Schur

摘要

Dianthus spiculifolius Schur,as an emerging ornamental plant,has extensive applications and economic values.In this study,the DsCBL4 gene was successfully cloned,and its tissue-specific expression,expression patterns under various abiotic stresses,subcellular localization,and bioinformatics analysis of the encoded amino acid sequence were conducted.The results showed that the coding region of the DsCBL4 gene was 675 bp long,encoding 224 amino acids.It had high homology with the amino acids encoded by Amaranthus tricolor,Chenopodium quinoa and Spinacia oleracea.The predicted relative molecular mass of DsCBL4 was25.61 ku,with an isoelectric point of 4.58,and it had phosphorylation sites,belonging to an unstable hydrophilic protein.Its secondary structure included α-helices,irregular coils and extended chains.The tertiary structure prediction revealed that DsCBL4 had four EFhand calcium-binding domains necessary for Ca²⁺ binding in plant calmodulin-like proteins and the FPSF motif for calcineurin B-like protein（CBL）-interacting protein kinase（CIPK） activation.The expression level of the DsCBL4 gene showed tissue specificity,with the highest expression in roots.It was induced by drought,low temperature,combined drought and low temperature,salt stress,nitrogen stress,phosphorus stress,calcium ion stress,high temperature stress,and abscisic acid（ABA） stress.Both transient infection in Nicotiana tabacum L.and stable expression in transgenic Arabidopsis thaliana showed that the DsCBL4 protein was localized to the cell membrane.These results suggested that DsCBL4 might be involved in the abiotic stress response of Dianthus spiculifolius through the calcium signaling pathway,providing a theoretical basis for understanding its molecular mechanism.This study provided an important reference for further exploring the role of the DsCBLs gene family in plant stress resistance.

关键词

Dianthus spiculifolius Schur / CBL4 gene / gene cloning / expression analysis / structural analysis