The Function Analysis Of PmPPO Gene Under Drought Stress In Prunus Mira

Prunus mira Koehne,a genus of peach in the rosaceae family,grows mainly in Tibet and other regions of China and has excellent characteristics such as drought tolerance,cold tolerance,and high genetic diversity and so on.Polyphenol oxidase（PPO）is a class of copper-containing oxidoreductases that can catalyze the oxidation of phenolic substrates and is widely distributed in plants.In this study,PmPPO was obtained by gene cloning technology,physicochemical properties and subcellular localization of PmPPO were analyzed by bioinformatics.Proteins interacting with PmPPO were identified by screening the yeast library of Prunus mira combined with a dual luciferase complementation technique,the response mechanism of PmPPO to drought and exogenous abscisic acid in Prunus mira were analyzed,the PmPPO transgenic Arabidopsis thaliana were obtained,and the functions of PmPPO in Prunus mira were investigated through the measurement of relevant physiological indicators and changes of gene expression under drought stress.The main findings obtained were as follows:1.The open reading frame of PmPPO from Prunus mira was cloned and obtained as 1770 bp,encoding 589 amino acids,with a relative molecular mass of 65.45 k Da and an isoelectric point of 7.63.The PmPPO protein had three functional structural domains,and the amino acid sequence comparison results showed that the structural domains were relatively conserved in the evolutionary process.The phylogenetic tree showed that PmPPO of Prunus mira and Pp PPO of Prunus persica were most closely related to each other.The subcellular results showed that the PmPPO was mainly localized in the chloroplast,nucleus and cell membrane.2.Using p GBKT7-PmPPO as a decoy protein,the Prunus mira yeast library was screened to initially obtain the reciprocal proteins PmRGLG2 and PmCht2,which were further validated using yeast two-hybrid technique and dual luciferase complementation technique,The results demonstrated that PmPPO interacted with PmRad23d,PmRGLG2 and PmCht2 respectively.3.The results of quantitative real-time fluorescence PCR（q RT-PCR）showed that PmPPO was expressed in all tissues of Prunus mira,with higher expression in young roots and leaves,the expression of PmPPO was significantly tissue-specific.PmPPO was responsive to natural drought,PEG6000 and exogenous ABA.4.The primary root length of transgenic Arabidopsis thaliana after PEG6000 treatment was higher than that of wild type（WT）,and it was more sensitive to exogenous ABA.With drought stress,the relative conductivity,chlorophyll content,anthocyanin content and PPO,POD,CAT,SOD enzymatic activity of the leaves of transgenic Arabidopsis thaliana were higher than those of WT.The O₂·^-,H₂O₂,and MDA contents of the leaves were lower than those of WT,and the results of DAB and NBT staining were consistent with them.The relative expression of At DFR,At CAT2,At RGLG1,At RGLG3,At LDOX,At RD29A and At PYL4 genes in transgenic Arabidopsis thaliana were higher than that in WT under drought.The results suggested that PmPPO positively regulated the drought resistance of Arabidopsis thaliana.5.The p ET-15b-PmPPO prokaryotic expression vector was constructed to optimize the protein induction and purification conditions and the study results showed that the recombinant protein was expressed in large amounts under the condition of 37℃induction temperature,2 h induction time and 0.25 m M/L IPTG.The target protein was mainly concentrated in the precipitation.The recombinant protein of His-tag was purified using 50 m M/L imidazole elution to obtain a large amount of target protein.