Identification Of CBL-CIPK Interact With Rboh D In Cucurbita Moschata

The secondary salinization of greenhouse soil has become prevalent as China’s vegetable cultivation area keeps expanding.Considering the poor salt tolerance of Cucumber,it is often grafted on salt-tolerant pumpkins rootstocks.In our previous lab studies,we have found that pumpkin rootstocks can improve cucumber salinity tolerance by controlling the early H₂O₂signal burst of rootstock roots,enhancing the Na⁺efflux ability of roots,and promoting early leaf stomatal closure.The respiratory burst oxidase homolog（Rboh）mediates the production of H₂O₂and plays a vital role in plant growth and development during adverse environmental conditions.The N-terminus of Rboh protein contains an EF domain that can bind Ca²⁺and a potential phosphorylation site.CIPK is an essential kinase in calcium signal transduction.In our previous studies,we have identified all members of the CIPK gene family in Cucurbita moschata.The differential expression of multiple CmoCIPK genes under salinity stress indicates their involvement in Cucurbita moschata salinity tolerance.Therefore,this research focused on screening CmoCIPK protein members that interact with the key CmoRboh protein in response to salt stress.The results obtained are as follows:1.Expression profiles of CmoRbohs gene families were analyzed in different tissues under salinity stress with transcriptomic studies and found that CmoRboh D1and CmoRboh D2 were up-regulated in roots by salt stress.Online databases were used to compare the protein sequences and transmembrane domains of CmoRboh D1and CmoRboh D2,which play important functions in response to salt stress.The results showed that CmoRboh D1 and CmoRboh D2 are typical Rboh proteins,having4 transmembrane domains,with an amino acid sequence similarity of 90.74%.2.Using the yeast two-hybrid system,29 CmoCIPK proteins were tested for their interaction with the N-terminal of CmoRboh D1 and CmoRboh D2.CmoCIPK1,which belongs to the At CIPK11 homologous gene,was found interact with CmoRboh D1 and Rboh D2.LCI and Co-IP experiments were used to verify the interaction between CmoCIPK1 and CmoRboh D1 and CmoRboh D2.Subcellular localization experiments showed that CmoCIPK1,like CmoRboh D1 and CmoRboh D2,is located on the plasma membrane.3.Using the yeast two-hybrid system,further 9 CmoCBL proteins that interact with CmoCIPK1 were selected as CmoCBL1,CmoCBL2,CmoCBL3,CmoCBL7,CmoCBL8,CmoCBL9,and LCI experiment was performed for further verification of these results.4.Expression profiles of CmoCIPKs and CmoCBLs gene families were analyzed in different tissues under salinity stress with transcriptomic studies and found that CmoCIPK1 expression was the highest in rootstock stems,followed by roots.ultimately our results indicate that it may play a role in salt stress induction and signal transmission.All 6 CmoCBLs interacting with CmoCIPK1 have different expression levels under salinity stress and can be divided into four categories.However,these results need to be further studied.5.The CmoRboh D1 and CmoRboh D2 proteins were successfully induced and purified in the E.coli.Online predictions show that CmoRboh D1 and CmoRboh D2proteins have more phosphorylation sites.Through phosphorylation experiments in vitro and LC-MS/MS technology,predicting the phosphorylation sites of CmoRboh D1 and CmoRboh D2 will be the most important work.