Study Of Interactions Between VAMP7 Proteins And OLE4 And Development Of Three-protein Interaction Detection System In Yeast Based On MbSUS

Rapeseed(Brassica napus L.)is one of the most important oilcrops.It is important to increase the oil production from rapeseed.Rapeseed is is closely related to the model plant Arabidopsis for they both belong to Crucifers.Thus the Arabidopsis oil production related genes can be used to investigate the lipid biosynthesis mechanism in rapeseed,which will help to find potential genetic transformation sites in rapeseed.Rapeseed oil is mainly stored in the oil body of seeds.Oleosin(OLE)is the most important structural protein in oil bodies.In Arabidopsis,OLE affects seed development and oil content.SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor)proteins participate in the cell membrane transport through mediating membrane fusion.Our preliminary experiment found that SNARE proteins,such as VAMP721,interact with cytoplasmic proteins.Many of these interacting proteins play an important role in the metabolic process,suggesting the role of SNARE proteins in the biosynthesis of important metabolites.Whether SNARE proteins affect lipid metabolism through interaction with OLE is worth studying.We firstly amplified the sequence of OLE from Arabidopsis,and investigate its interaction with the VAMP7 proteins by the yeast mating-based Split-Ubiquitin System(mb SUS).We found VAMP712,VAMP721,VAMP724,and VAMP726 interacted with OLE4 protein.Because multiple SNARE proteins interact with OLE4,they may compete with each other.To further investigate the mechanism,based on the mb SUS,we established a 2in1-Bridge SUS system which can be used todetect the effect of the third protein on the interaction between the bait and prey protein.