The Primary Functional Analysis Of RhSKP1,A Core Component Of E3 Ubiquitin Ligase In Rose Senescence

Rosa(Rosa hybrida)is one of the most ornamental and economic flowers in the world.The value of rose depends on the ornamental quality of rose flowers.Generally,there is a long transportation distance between the place of sale and the place of origin of rose.Because long-distance transportation can cause ethylene accumulation and water loss stress,the cut rose flowers will wilt abnormally and be earlier senescent,thus resulting in up to one third of the loss.Therefore,understanding the mechanism of rose senescence,slowing down the senescence and improving the quality of rose flowers are the important problems to be solved in rose production industry.Our previous study found that Rh SKP1,the core component of E3 ubiquitin ligase SCF complex,may be involved in the regulation of rose flower senescence.However,there is no report on the regulation of flower senescence by SKP1.This study preliminarily analyzed the function of Rh SKP1 in rose flower senescence.(1)The expression level of Rh SKP1 in different opening stages of rose flowers was detected by fluorescence quantitative PCR.It was found that the expression level of Rh SKP1 was lower in the 0-2 stage in the early opening stage of rose flowers,and began to rise in the3-4 stage.The expression level of Rh SKP1 was the highest in the 5 stage.It was speculated that Rh SKP1 indeed responded to and participated in the regulation of rose flower senescence.(2)The ORF region of Rh SKP1 gene was cloned.The gene structure was analyzed by comparing with other homologous SKP1 genes in other species and phylogenetic tree analysis.The location vector of Rh SKP1 was constructed and was transformed into Agrobacterium and injected into tobacco leaves.The results of confocal laser scanning showed that Rh SKP1 was located in the nucleus and cytoplasm,which was consistent with other known SKP1.(3)The VIGS(virus induced silencing)vector of Rh SKP1 was constructed to infect rose petals(disc).Compared with the control,silencing Rh SKP1 delayed the senescence of rose petals.The overexpression vector of Rh SKP1 was constructed and transformed into Arabidopsis thaliana to obtain T3 generation positive plants.The 1500 bp-length promoter of Rh SKP1 was cloned and co-injected into tobacco with Rh NAC100 gene to analyze the promoter activity.The preliminary analysis showed that Rh NAC100 inhibited the promoter activity of Rh SKP1.(4)The possible binding site of NAC was found by analyzing Rh SKP1 promoter.The probe containing the possible binding site of NAC gene was synthesized and the protein of Rh NAC100 was purified using prokaryotic expression system.EMSA analysis in vitro showed that Rh SKP1 was the direct downstream gene of Rh NAC100.In vivo part of the binding verification will be carried out in subsequent experiments.In conclusion,it is preliminarily speculated that Rh SKP1 may be the direct downstream gene of Rh NAC100 to regulate the senescence of rose flowers.This study will lay a foundation for further understanding the regulation mechanism of rose flower senescence affected by Rh SKP1.