Identification Of Abiotic Stress-related StCIPK Genes And Functional Analysis Of StCIPK02 And StCIPK10 Genes In Potato

Ca²⁺,as a second messenger,not only receives and integrates the signals triggered by biotic and abiotic stresses,but also regulates a variety of different biochemical reactions.Known as one of major Ca²⁺sensors,calcineurin B-like proteins（CBL）can interact with its specific binding protein CBL-interacting protein kinases（CIPK）to form CBL-CIPK modules which are responsible for regulating plant growth,development and stress response by phosphorylating or interacting with downstream proteins.As one of the most nutrient-rich foods,potato（Solanum tuberosum L.）is the most widely planted crops around the world.However,the yield and quality of potato can be significantly affected by various abiotic stresses,especially drought and salt.Up to now,there remains a lack of clarity on the function of StCIPK in potato growth and response abiotic stress,which prompts the thorough identification and analysis of the StCIPK gene family in potato genome in this research to explore the StCIPK genes of function and molecular mechanism under drought salt and osmotic stress and their specific interaction system.The main findings are detailed as follows:1.In this study,a total of 27 StCIPK genes were identified using the Hidden Markov Model（HMM）and BLAST search in potato.According to the phylogenetic analysis,all StCIPK could be classed into five groups:A,B,C,D and E.In addition,the intron-rich StCIPK were classed into group A.There were six duplication events detected among StCIPK genes,including one tandem duplication and five segmental duplications.The stress-related cis-acting elements in the promoter regions of most StCIPK genes were identified.2.The expression levels of StCIPK genes in different tissues of potato under abiotic stress and hormone treatments were examined using RT-q PCR to reveal the variation in StCIPK expression levels between different tissues.It was discovered that a vast majority of StCIPK genes were evidently up-regulated in the petals and stamen.In addition,StCIPK18 exhibited higher expression levels in the stem and root than in other tissues.The expression patterns of StCIPK genes varied significantly under abiotic stress and hormone treatments,which lead to the speculation that StCIPK genes could play a more significant role in potato growth and abiotic stress tolerance.3.The drought and salt stress-related StCIPK02 was cloned in potato.Both the over-expression vector p CAM35S-StCIPK02 and interference expression vector p BI121-ami RStCIPK02 were transformed into potato by the mediated agrobacterium tumefaciens.StCIPK02 was identified as a negative regulator of potato tolerance to drought as well as a positive regulator of tolerance to salt.In addition,potato not only displayed a greater sensitivity to ABA by interfering the expression of StCIPK02,but also induced the stomatal closure of potato leaves under drought stress.4.StCIPK02 specifically interacted with StCBL01,StCBL04,StCBL07,StCBL09and StCBL11,but weakly interacted with StCBL02 and StCBL03 by Yeast-two hybrid technique.StCIPK02 was expressed in the cytomembrane and cell nucleus,which indicates the possibility that StCIPK02 could interact with various protein kinases and play different roles in the cytomembrane and cell nucleus.However,StCBL11 was expressed exclusively in the cytomembrane.As suggested by the analysis of subcellular co-location,BIFC and luciferase reporter system,StCIPK02 could be located on cytomembrane by StCBL11 and interact with each other to form StCBL11-StCIPK02module,playing different roles in potato response abiotic stress.5.The drought and osmotic stress-related StCIPK10 was cloned in potato.The over-expression vector p CAM35S-StCIPK10 and interference expression vector p BI121-ami RStCIPK10 were transformed into potato by the mediated agrobacterium tumefaciens.It was discovered that the over-expression of StCIPK10 not only improved the drought and osmotic tolerance and sensitivity to ABA of transgenic potato plants but also induced the stomatal closure of potato leaves under drought stress,which implies that StCIPK10 could be located between ABA signaling pathway and calcium signaling pathway,regulating both root growth and stomatal closure in potato.6.The specific patterns of interaction were found between StCIPK10 and StCBL01,StCBL04,StCBL07,StCBL09 and StCBL11,there was weak interaction between StCIPK10 and StCBL02 and StCBL03 by yeast-two hybrid technique.As indicated by the analysis of subcellular co-location,BIFC and luciferase reporter system,StCIPK10 could be located on cytomembrane by StCBL11 and interact with each other to form StCBL11-StCIPK10 modules which play different roles in potato response abiotic stress.To sum up,it was confirmed in this study that StCIPK02 was involved in the regulation of potato drought and salt tolerance,while StCIPK10 was involved in regulation of potato drought and osmotic tolerance.Therefore,this study provides new gene resources for improving the quality and stress-resistance traits of potato,which lays a foundation for potato CBL-CIPK signal path.