Identification And Functional Analysis Of HbCBLs From Hordeum Brevisubulatum

CBLs(calcineurin B-like proteins),a class of plant-specific Ca2+ sensors,which were identified in Arabidopsis thaliana,were named because they are similar to CNB and NCS in animals.CBLs act as calcium sensors and play important roles in binding with calcium,signaling,and regulating adaptability of plants.In recent years,researches on CBL-CIPK signal system have made progress in the model plants.A variety of stress-responsive CBL-CIPK pathways have been identified.However,the mechanisms of CBL-CIPK response to drought stress and CBL-CIPK regulates K+/Na+ balance under high salinity stress are still not clear.Currently,researches about CBLs are mainly conducted on Arabidopsis and rice,there was very little study on other plants.Shortsubulate barley(Hordeum brevisubulatum(Trin.)Link)is one of the most salt-tolerant halophytes in Triticeae,which has evolved the mechanism of adaptation to salinity during long term of evolution.It is valuable for research and application.Cloning CBLs in Shortsubulate barley not only shows theoretical meanings,but also benefits to practical application,providing potential candidate genes for improving stresses torlerance by genetic transformation in plant.Here,we isolated five genes in Shortsubulate barley and name them HbCBL3?HbCBL4?HbCBL8?HbCBL9 and HbCBL10.We analyzed the response of a part of genes to stresses,subcellular location and their function,the main progresses are as follows:(1)Cloning and sequence analysis of HbCBLs:RT-PCR was used to obtain full length cDNA of HbCBL3?HbCBL4?HbCBL9 and HbCBL10,part of EST sequence of HbCBL8.HbCBL3?HbCBL4?HbCBL8?HbCBL9 and HbCBL10 have CBL speicific Ca2+ binding motif,namely EF hands;HbCBL4 and HbCBL9 have a N-myristoylation motif at its N-terminus.(2)Expression analysis of HbCBLs under different stresses:Under the 350 mM NaCl stress for 6 hours,the expression of HbCBL4 and HbCBL10 in wild barley root dramatically increased;under the cold(4 ?)stress for 12 hours,the expression of HbCBL4 in root also significantly rose;However,the expression of other genes under high salinity,and simulated drought and low temperature stress has no obvious changes compared to that of the control,indicating that HbCBL4 and HbCBL10 may response to adverse stresses in wild barley.(3)Subcellular location of HbCBLs:the expression vector contaning HbCBL4 fused with GFP was constructed and transformated into Arabidopsis protoplast by PEG-mediation,Confocal observations indicate that HbCBL4 is located at the plasma membrane.(4)In order to analyze biological functions of HbCBLs,the over-expression vectors were constructed and transformated into wild-type and mutant of Arabidopsis,by Agrobacterium-mediated method.Physiological tests showed the transgenic mutant lines improved salt tolerance under NaCl stress with the different concentration.