Identification And Expression Pattern Analysis Of CBL And CIPK Gene Families In Pumpkin

Calcium(Ca²⁺)serves as the second messenger in plants.The changes of calcium ionic concentration are induced by stresses.Calcineurin B-like protein（CBLs）,as one of the calcium receptors,can sense these changes and specifically bind to the downstream target protein which are called CBL-interacting protein kinase（CIPKs）.Then they constitute the CBL-CIPK signaling system and activate a cascade of downstream reactions through signal transduction,thus mediating a multitude of plants responses to stimuli.Pumpkins（Cucurbita moschata）are an allotetraploid,which are different from diploid cucumbers and melons in Cucurbitaceae.There have been many studies on the CBLs and CIPKs gene families and gene functions in model crops such as Arabidopsis and rice.In2017,the pumpkin genome was released.Currently,there are few studies on the CBLs and CIPKs gene families in pumpkin.In this study,we identified the whole genome CBLs and CIPKs gene families of pumpkin,analyzed their expression patterns,and studied the genes responsing to salt stress.The main research results are as follows:1.8 CmCBLs and 30 CmCIPKs were identified in the whole pumpkin genome by bioinformatics analysis.CBLs and CIPKs gene families were highly conserved in gene structure,conserved motif and conserved domain analyses.Phylogenetic tree analysis showed that CmCBL1,CmCBL2,CmCBL7,CmCBL8 clustered in the same evolutionary branch as AtCBL1 and AtCBL9;the clusters of CmCIPK7,CmCIPK21 and CmCIPK9,CmCIPK26 and AtCIPK9 and AtCIPK23 were in the same evolutionary branch;only 1CmCIPK3 clustered in the same evolutionary branch with AtCIPK3 and AtCIPK26;the members of the same evolutionary branch above are highly consistent with the corresponding Arabidopsis protein sequences.Multiple sequence alignment analysis showed that the absolute conserved sites of pumpkin CBLs and CIPKs proteins were different from those of Arabidopsis.Evolutionary analysis showed that CmCBL1,CmCBL2,CmCBL7 and CmCBL8 were very conserved in the evolutionary process.The prediction results of cis-acting elements showed that there was no necessary relationship between the types and quantities of cis-acting elements in the same evolutionary branch.2.Transcriptome data showed that there were some highly expressed genes,such as CmCBL1,CmCBL7 in CBLs and CIPKs gene families in pumpkin.The transcriptome results of pumpkin root tip treated with 75 mM NaCl stress for 24 hours showed that the highly expressed genes were CmCBL1,CmCBL8,CmCBL5 and CmCIPK20,CmCIPK21,CmCIPK2;the transcriptome results of vein and mesophyll in pumpkin treated with 100mM NaCl stress for 24 hours showed that the high expression genes were CmCBL7,CmCIPK16,CmCIPK24,CmCIPK29;the transcriptome results of pumpkin treated with 75mM NaCl stress for 24 hours showed that the high expression genes were CmCBL1,CmCBL4,CmCBL8,CmCIPK16 and CmCIPK18.In pumpkin,the potential CIPK downstream gene of AKT1 was highly expressed in roots and RBOHF-1 was highly expressed in roots.3.The result of qRT-PCR analysis showed that CmCBL1,CmCBL2 and CmCBL3 were highly expressed in CBLs gene family under four kinds of stress,and other genes were also induced to different degrees.After 24 h treatment,CmCBL3 in leaves and CmCBL1 in roots were significantly up-regulated under NaCl stress;CmCBL3 in leaves and CmCBL2 in roots were significantly up-regulated under sorbitol stress;CmCBL3 in leaves and CmCBL2,CmCBL3 in roots were significantly up-regulated under ABA stress;CmCBL2 in leaves and roots were significantly up-regulated under H₂O₂ stress.4.The results of subcellular localization showed that CmCBL1,CmCBL2,CmCBL7and CmCBL8 were located in cell membrane and nucleus.Combined with the results of systematic evolutionary tree,the identity of protein sequence and evolutionary analysis,the genes of CmCIPK3,CmCIPK9,CmCIPK26,CmCIPK7 and CmCIPK21 related to ABA and ion transport were found.The homologous genes of AtCIPK3 and AtCIPK26 in pumpkin were CmCIPK3;The homologous genes of AtCIPK9 were CmCIPK9 and CmCIPK26;The homologous genes of AtCIPK23 were CmCIPK7 and CmCIPK21.The yeast two-hybrid results showed that CmCBL1 interacted with CmCIPK9,CmCIPK26,CmCIPK21 and CmCIPK7;CmCBL2 interacted with CmCIPK3,CmCIPK9,CmCIPK26,CmCIPK21 and CmCIPK7;CmCBL7 interacted with CmCIPK9,CmCIPK26,CmCIPK7;CmCBL8 interacted with CmCIPK3,CmCIPK9,CmCIPK26.These above results indicate that the interaction between CBL protein and CIPK protein have both similarity and specificity,and the mechanism remains to be further studied.