Functional Characterization Of Wheat Stress-related Genes TaCIPK2,TaCIPK10 And TaCIPK27

Plants have to face a variety of abiotic stresses such as drought,high salt,extreme temperatures and ultraviolet radiation during their growth and development processes.Under those stress conditions,the vegetative and reproductive growth of plants were greatly affected,resulting in the decreasing in the yield and quality of plants(crops).As an important second messenger,calcium ion Ca2+ plays important roles in stress signalling in plant.Calcineurin-B like protein(CBL),unique Ca2+ sensor of plants,can sense various Ca2+ signals evoked by abiotic stressed.After combining with Ca2+,CBL can interact and activate its interacting protein kinase(CIPK),resulting in target molecules phosphorylation,and then initiate a series of stress response in plant.Wheat is an important crop worldwide.Our previous systematical analysis reveled 79 CIPK genes at least from wheat genome database.The 22 TaCIPK genes have been cloned from Chinese spring wheat and the biological functions of some TaCIPK genes have been clarified.In this study,we focused on functional analyses of three TaCIPK family members,including TaCIPK2,TaCIPK10 and TaCIPK27.The main research results are as follows:1)The expression of TaCIPK2 can be up-regulated by PEG,H2O2 and ABA treatments,but its expression was down-regulated under GA treatment and no expression changes were observed by cold and salt treatments.The transcriptional expression results suggested that TaCIPK2 could specifically respond to drought stress and play a role in response to ABA and GA treatments.The expression of TaCIPK10 and TaCIPK27 were up-regulated under PEG,H2O2,NaCl and ABA treatments but not by cold.Among those treatments,the up-regulated expression levels were higher under ABA treatment.While the expression levels of TaCIPK10 were higher under NaCl and PEG treatments.The results indicated that both TaCIPK10 and TaCIPK27 genes were able to respond to a variety of stress treatments,but the expression levels of TaCIPK10 and TaCIPK27 genes were different under the same stress treatment,suggesting that TaCIPK10 and TaCIPK27 might be involved in different signaling pathways in response to these stress treatments.2)The subcellular localization analysis showed that TaCIPK2 was localized in the nucleus,cytoplasm and cell membrane after transiently expressed in onion epidermal cells.In order to clarify the biological function of TaCIPK2,transgenic tobacco over-expressing TaCIPK2 was generation.Three stable transgenic tobacco lines were selected and used for further analysis.When exogenous ABA and sodium tungstate were added to the culture media,the seed germination of TaCIPK2 transgenic tobacco seeds were more sensitive to exogenous ABA,indicating that TaCIPK2 may play a positive regulatory role in the ABA signaling pathway.The expression analysis results of some related genes also showed that TaCIPK2 was involved in ABA response.At the same time,TaCIPK2 transgenic tobacco can also respond to GA treatment.The expression analysis of some GA related genes again proved that the TaCIPK2 played a role in GA signaling.Under drought stress treatment,TaCIPK2 could improve the drought resistance of transgenic tobacco plants by improving the clearance of ROS in tobacco leaves and regulating expression of some stress response genes,under drought stress condition.3)Yeast-two hybrid analysis results showed that TaCIPK27 could interact with AtCBLl,AtCBL3,AtCBL4,AtCBL5 and AtCBL9.Therefore,TaCIPK27 over-expression transgenic Arabidopsis was generated for further functional study.Phenotypic analysis showed that TaCIPK27 could enhance the drought tolerance of transgenic Arabidopsis plant under drought treatment but showed a more sensitive phenotype under salt stress treatment.Results of experimental analyses including seed germination rate,root length,physiological indexes and stress related genes expression,all illustrated that TaCIPK27 can improve the drought resistance in transgenic Arabidopsis plants.Besides,TaCIPK27 could effectively remove the accumulation of intracellular ROS.TaCIPK27 could also increase ABA content,and activate the SnRK2 activity to regulate the downstream stress related gene expression levels.While more s The salt sensitive of TaCIPK27 transgenic Arabidopsis plants was due to unable to clear the oxidative damage of ROS in time,and can not increase the expression levels of salt related genes,and resulting in losing the ability of keeping Na+ and K+ balance in plant cells.Under the exogenous ABA treatment,lower seed germination rate,shorter root length and faster stomatal closure were observed in TaCIPK27 transgenic Arabidopsis plants,resulting in TaCIPK27 transgenic Arabidopsis plants more sensitive to ABA.4)Over-expression of TaCIPK10 in Arabidopsis plants could increase the tolerance of transgenic plants to drought and salt stress.The phenotype analysis showed that TaCIPK10 transgenic Arabidopsis plants had a higher survival rate comparing to the control under drought and salt treatments.The root length,fresh weight of the Arabidopsis seedlings,physiological indexes and the transcriptional expression levels of drought and salt related genes showed that TaCIPK10 played an important role in improving the drought and salt tolerances in Arabidopsis plants by reducing accumulation of intracellular ROS and upregulating expression of some stress responsive genes.5)In order to confirm the function of TaCIPK10 in wheat,we also over-expressed TaCIPK10 in model wheat cultivar Chinese Spring.The T3 TaCIPK10 transgenic wheat plants were treated with the mannitol and salt solutions.TaCIPK10 overexpressing wheat plants showed higher survival rate and lower ion leakage than wild type wheat,indicating that TaCIPK10 can improve wheat drought and salt resistance.The physiological indexes also showed that TaCIPK10 increased the resistance to abiotic stress in wheat,which was mainly due to the effective removal of ROS and the decrease of oxidative damage in cells.In addition,TaCIPK10 could improve the stress tolerance of transgenic wheat by increasing the expression of some stress related genes,which was similar to that of TaCIPK10 transgenic Arabidopsis.