Screening And Verification Of Polyphenol Oxidase Interacting Protein In Lotus

Lotus root is the thick rhizome of perennial plant which belongs to the Nelumbonaceae family.It is not only rich in nutrients,but also has good health care functions.Chinese lotus roots’varieties are diversity,they have rich resources and broad prospects for development.However,since the fresh lotus roots are prone to browning and not easy to store that limit the production and sale of the products to some extent.Studies have shown that polyphenol oxidase(PPO)is a kind of oxidoreductase that commonly found in fruits and vegetables,which can cause Enzymatic Browning of tissues,and the activity of PPO is related to plant resistance to diseases and insect pests.At present,there are fewer molecular studies on PPO in lotus root,the specific process of PPO causing the browning of lotus root and the role of PPO played by participating in stress resistance are still unclear.Therefore,based on the previous laboratory research,lotus roots were used as the materials in this experiment.We have selected the Yeast Two-Hybrid method to screen the protein interacting with lotus root PPO and use the yeast rotation verification method to verify the interaction.By speculating the pathway of PPO interaction with these proteins and the signal regulation methods etc,in order to use the interaction protein to regulate the activity of PPO,selectively reduce or increase the expression of PPO in different periods of plants.To lay a foundation for solving the problem of lotus root browning and enhancing plant stress resistance.The main results of this experiments are as follows:1.According to the PPO gene sequence published in the NCBI database(GenBank:FJ999635.1),we have constructed the bait expression vectors pGBKT7-PPO and pGBKT7-JD-PPO(remove guide peptide)of lotus root.The bait protein toxicity and self-activation test results indicated that the bait protein PPO was non-toxic to yeast Y2H Gold and had no self-activating effect.The bait protein could not activate the GAL4 system of this yeast and could be used for cDNA library screening.2.The Yeast Two-Hybrid(Y2H)technique was used to screen the protein interacting with PPO from the lotus root cDNA library.The number of colonies screened was 1.96×10~6cfu/mL,indicating that the yeast has a good hybridization state.After three rounds of screening,48 blue positive clones were initially screened on QDO/X/A.By BLAST alignment sequencing results on NCBI,28 candidate proteins interacting with PPO were finally obtained.The functions of these selected genes were analyzed and found to include disease resistance,antioxidant,electron transport,developmental regulation,phenolic acid synthesis,ethylene synthesis and some functionally unknown proteins.3.Some key genes which need to be verified were amplified by PCR,reconstructed the prey expression vector and transferred them into Y2H Gold yeast together with the bait plasmid pGBKT7-JD-PPO.Perform rotary yeast validation to further verify interaction with PPO.The results showed that four of the candidate proteins:Aureusidin Synthase-like,Cysteine Desulfurase,NADH dehydrogenase,GLK1 transcription factor showed interacted with PPO in yeast Y2H Gold.4.The lotus root PPO was fused with the reporter gene GFP to construct the subcellular localization vector p35S-PPO-eGFP,and the recombinant plasmid p35S-PPO-eGFP was transferred into GV3101 Agrobacterium by freeze-thaw method.PPO was transiently expressed in Nicotiana Benthamiana leaves using Agrobacterium-mediated methods,and observed fluorescence signals in leaf epidermis under a laser confocal microscope.We analysised the Image founded that PPO in the lotus root leaves is located in the chloroplast and PPO in the lotus root may be located in the plastid.5.Using the biological website to analyze the physical and chemical properties of PPO,the relative molecular mass of PPO is about 67.2 kD.PPO is hydrophilic protein and has no transmembrane region.A phylogenetic tree was constructed for the PPO gene family of lotus root.It was found that PPO of lotus root had the closest relationship with Aquilegia coerulea in Ranunculaceae,and was closer to Morus notabilis,Malus domestica and pyrus pyrifolia than other plants.