| In the course of cultivation,crops are often threatened by various diseases and harsh environment.Drought,high temperatures,cold and salt stress exert a great influence on the yield and quality of crops.Calcium,as a ubiquitous secondary messenger,responds to a variety of stress stimuli.The proteins that decode calcium signals in plants can be divided into four categories,the most important of which are calcium-dependent protein kinases(CPKs).CPKs are a class of serine / threonine protein kinases that are distributed in plants and some protozoans.Increasing studies have shown that CPKs play a key role in plant stress response and immunity to pathogens.Canola(Brassica napus L)is an important oilseed crop in cruciferae.In this study,a calcium-dependent protein kinase BnaCPK2,was identified to be located at the endoplasmic reticulum.Transient expression of constitutively activated form of BnaCPK2,BnaCPK2 VK and full-length form BnaCPK2 FL in Nicotiana benthamiana,caused a significant accumulation of ROS and accompanied by significant changes in physiological indexes of cell program death.The relative conductivity,contents of MDA and anthocyanin in BnaCPK2 VK increased significantly.At the same time,over-expression of Bna CPK2 VK and BnaCPK2 FL in N.benthamiana also resulted in fragmentation of genomic DNA.Through real-time quantitative RT-PCR,we also found that the expression of BnaCPK2 VK and BnaCPK2 FL caused significant up-regulation of ROS-and defense-related genes in N.benthamiana.In order to explore the relationship between BnaCPK2 and BnaRBOHs,we discovered the interaction between BnaCPK2 and BnaRBOHD by bimolecular fluorescence complementation(BIFC)assay.Subsequently,with the support of a split ubiquitin yeast hybrid system(mbSUS),we further identified the physical interaction between BnaCPK2 and BnaRBOHD.This study has laid the foundation for further study of BnaCPK2 and other CPKs in oilseed rape. |
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