Interaction Protein And Function Anaiysis Of Polyphenol Oxidase In Nelumbo Nucifera

The Lotus root(Nelumbo nucifera Gaertn.)is a large amount of aquatic vegetables in China.It is used for both medicine and food.Six out of ten parts of the whole plant are used for medicine.Nelumbo nucifera has high edible and medicinal value.Polyphenol oxidase is an antioxidant enzyme widely present in plants.It is also an important cause of postharvest browning of fruit and vegetable agricultural products such as Nelumbo nucifera.In the early stage,the research team screened for the protein interaction between Nelumbo nucifera polyphenol oxidase(Nn PPO1),aureus synthase(Nn AUS),and cysteine desulfurase(Nn Cys)through the method of yeast two-hybrid,it is not clear.On this basis,this study further screened and verified the interaction between Nn PPO1 and its protein,and clarified their tissue-specific expression and subcellular localization,and gradually explored the mechanism of action of Nn PPO1 in Nelumbo nucifera to accurately inhibit its activity.Laying the foundation is of great significance for controlling the browning of Nelumbo nucifera after harvest and extending its industrial chain.The main test results are as follows:1.In this experiment,the gene sequences of enzymes such as ascorbate peroxidase,catalase,superoxide dismutase,cysteine peroxidase and peroxidase were connected to the p GADT7 vector respectively.The constructed JDPPO1-BD vector(the vector constructed by Nn PPO1 with the N-terminal leader peptide removed)co-transformed yeast to screen for interacting proteins.The results showed that there is an interaction between Nn PPO1 and Nn CAT1.The yeast system has no self-activating effect and is non-toxic to yeast.2.The CDS regions of Nn PPO1 and Nn AUS,Nn Cys,and Nn CAT1 were connected to Bi FC carriers 35S-SPYNE(R)173 and 35S-SPYCE(M),respectively,and the fluorescence signal was observed under confocal after tobacco transformation.The results showed that Nn PPO1 and Nn CAT1 had strong interaction on the cell membrane and nucleus,Nn PPO1 and Nn Cys had interaction on the cell membrane,and there was a weak interaction between Nn PPO1 and Nn AUS.3.Nn PPO1 was truncated.Three prey vectors were constructed with tyrosinase domain,PPO1?KFDV domain,and PPO1?DWL domain.They were co-transformed into yeast with Nn AUS-AD,Nn Cys-AD,and Nn CAT1-AD vectors.The Y2 H test found that the interaction sites of Nn PPO1 with Nn AUS,Nn Cys,and Nn CAT1 are all in the conserved domain tyrosinase domain.Nn AUS,Nn Cys,and Nn CAT1 were truncated with their respectivedomains to construct bait vectors and JDPPO1-BD co-transformed yeast.The results show that the site where Nn AUS exerts interaction is also the tyrosinase domain.Nn Cys,Nn CAT1 truncated and JDPPO1-BD no protein interaction.A series of vectors were constructed by truncating the tyrosinase domains of Nn PPO1 and Nn AUS to find specific functional sites for interaction.The results show that Nn PPO1 interacts with Nn AUS and Nn CAT1 proteins,mainly N-glycosylation sites and N-myristoylation sites on the Nn PPO1 protein sequence play a role in protein interactions.The functional site where Nn AUS interacts with Nn PPO1 is its Cu A domain and its first 30 aa parts.AD-Nn Cys interacts with the series of vectors constructed by the truncated tyrosinase domain of Nn PPO1.4.Subcellular localization found that Nn AUS and Nn CAT1 are located on the nucleus and cell membrane.Nn Cys protein is located on the cell membrane,nucleus and cytoplasm.Tissue-specific expression results show that Nn PPO1,Nn AUS,Nn Cys,and Nn CAT1 are expressed in four genes of lotus root seedling.The expression patterns of Nn PPO1,Nn CAT1 and Nn Cys are basically the same,Both expressed the most in the leaves,and the lowest in the shoot tips.Nn AUS has the highest expression in roots,with a small amount of expression in leaves and lotus roots,and only a small amount in leaf petioles and shoot tips.Analysis of the physical and chemical properties,promoters and secondary and tertiary structures of Nn AUS,Nn CAT1,Nn Cys shows that these proteins are very similar to Nn PPO1 in function and structure.