Identification Of Pumpkin ERF Gene And Screening Of CmHKT1 Interactive Protein Under Salt Stress

Pumpkin has strong resistance against abiotic stresses because of its strong root system,therefore,it is used as rootstock for watermelons and other cucurbitaceous vegetables.According to results of previous research of our group,grafting cucumbers onto salt-tolerant pumpkin rootstock effectively limited Na~+transport to the shoot and significantly improved the salt tolerance of cucumber.Cm HKT1 is a Na~+transporters,its main function is to unload Na~+from the xylem.However,the molecular regulatory mechanism of Cm HKT1 involved in the response to salt stress is still unknown.Ethylene responsive factor(ERF)is one of the plant-specific transcription factor families and plays an important role in the regulation of salt stress.Until now,several members of the ERF family have been identified in the whole genomes of many plants,but there have been no reports in pumpkins.Therefore,the research work of this paper was carried out from two aspects:identification of pumpkin ERF gene family and expression profile analysis under salt stress,screening and functional verification of pumpkin Cm HKT1 interaction protein.The results of this study are as following:1. A total of 131 ERF family members were identified in the whole genome of pumpkin using bioinformatics methods.According to the phylogenetic tree clustering relationship,the pumpkin ERF members were divided into 12 groups.Pumpkin ERF family contain 3?-sheets and 1?-helix,And the number,type and arrangement order of motifs in each ERF group were consistent,which further illustrated the reliability of grouping;2. About 17%of the 131 ERF members of pumpkin originated from scattered replication,and 81%originated from segmental duplication.It can be inferred that along with the most recent genome-wide replication event of pumpkin(30MYA,Ks?0.1-0.7),the ERF gene family was expanded.And,purification selection is the main way to drive the evolution of pumpkin ERF gene family.3. The expression levels of most ERF members in the root and hypocotyl were significantly higher than other tissues under salt stress.And the expression patterns of ERF genes in the same group were different,indicating that the ERF genes in the same group played different roles in the response to pumpkin salt stress.Under salt treatment,the number of ERF differential genes in pumpkin roots was the highest.ERF transcription factors can directly or indirectly participate in multiple stress regulation pathways,and some stress response genes were regulated by the CBF subfamily.Cmo Ch01G014870.1 and Cmo Ch16G001580.1(CBF)were up-regulated in the root,and both genes belong to the group III,indicating that group III members play an important role in the salt tolerance regulation of pumpkin roots.4. Through the screening of the membrane protein library,the interaction protein Cm CNIH1(Cmo Ch07G013500.1)with Cm HKT1 was obtained,which is a member of the CNIH family.It participated in the intracellular membrane vesicle transport process mediated by COP?and COPI vesicles,by means of interact with transmembrane proteins to realize the correct transport of transmembrane proteins.Both point-to-point verification and firefly luciferase complement verification indicated that there was an interaction relation between the Cm HKT1 and Cm CNIH1.Cm CNIH1 is a transmembrane protein containing three transmembrane domains,it was localized on the plasma membrane and there was a physical space to bind to Cm HKT1.5. We used the Agrobacterium rhizogenes-mediated CRISPR/Cas9 gene editing to study the function of Cm CNIH1 gene.The results showed that the Na~+concentration of the xylem and shoot of the Cm CNIH1 editing pumpkin root was significantly higher than the wild type,indicating that the salt tolerance of the editing root was lower than that of the wild type.In addition,Cm CNIH1 did not affect the expression of Cm HKT1gene after being edited.Based on these results,we suppose that after the pumpkin Cm CNIH1 was edited,the Cm HKT1 protein cannot be transported to the plasma membrane resulting an increased Na~+concentration in the shoot,therefore,plant salt tolerance was decreased,but further it needs the results of subcellular localization experiment of the Cm HKT1 on pumpkin editing plant.