CDNA Cloning,Expression Analysis And Identification Of Interacting CBL Proteins Of Seven CIPKs In Rapeseed(Brassica Napus L.)

Calcineurin B-like interacting protein kinase(CIPK)is a kind of serine/threonine protein kinase widely existing in plants.It interacts with upstream CBLs and forms a CBL-CIPK complex,which plays an important role in the transmission of calcium signal.Calcium signaling plays an important role in regulating plant growth,development and abiotic and biological stress signaling pathways.Although there are many reports on CIPK in Arabidopsis thaliana and rice,there are few reports on CIPK in many other crop plants.Rapeseed(Brassica napus L.)genome is relatively large and genome sequencing was finished in 2014,so there are still some CIPK genes that have not been cloned or studied.Rapeseed as an important oil crop in China has important economic value and practical value,the yield and quality of rapeseed are often affected by environment stresses and pests.Mining stress-resistant gene resources in rapeseed at molecular level is of great significance in exploring and improving plant stress resistance.In this study,rapeseed was used as the experimental material.Firstly,c DNA sequences of seven CIPK genes were cloned by RT-PCR.The structural characteristics and phylogenetic tree of the predicted CIPK proteins were analyzed by sequence alignment and evolutionary tree analysis.The expression profiles of selected genes Bna CIPK2,Bna CIPK13,Bna CIPK16 and Bna CIPK18 under hormone abscisic acid(ABA),salicylic acid(SA),jasmonic acid(JA),heat,cold,hydrogen peroxide,dehydration,salt and methyl viologen(MV)treatments were analyzed by q RT-PCR.Subcellular localizations of some Bna CIPK proteins were analyzed by utilizing green fluorescent protein(GFP).The yeast two-hybrid(Y2H)assay explored the interactions between these seven Bna CIPKs and calcineurin B-like proteins(CBLs),which were validated by bimolecular fluorescence complementation(Bi FC)technique.The results showed that these seven Bna CIPK gene-encoded proteins contained conserved kinase domains and NAF motifs.These seven CIPKs are more closely related to orthologous proteins in Arabidopsis in evolutionary relationship.It was found that JA,cold and polyethylene glycol(PEG)8000 significantly induced the expression of Bna CIPK2,the expression of Bna CIPK2 and Bna CIPK13 was inhibited by methyl viologen treatment for 24h;SA,heat,cold,hydrogen peroxide and PEG8000 could induce up-regulation of Bna CIPK16 expression;the expression of Bna CIPK13 was significantly decreased by heat and salt treatments,but was significantly increased by hydrogen peroxide treatment for 1 h,and then decreased;the expression of Bna CIPK18 was up-regulated under ABA treatment.Interactions between Bna CIPK2 and Bna CBL4,Bna CIPK16 and Bna CBL3,Bna CIPK18 and Bna CBL4,Bna CIPK20 and Bna CBL1,Bna CBL9 were found in Y2 H.These interactions were further verified through Bi FC.In addition,interactions between Bna CIPK26 and multiple ABA-responsive element binding factors(ABFs)as well as interaction between Bna CIPK26 and Bna HAB1,a member of PP2C(protein phosphatase,type 2C)family were identified.These results indicate that different CIPK genes differ in expression patterns and therefore may participate in different stress responses.These results laid a foundation for elucidating the functions and regulatory mechanisms of these seven newly cloned CIPKs genes in rapeseed.