| Mulberry leaves(Morus alba L.)are important in the sericulture economy,which have been widely planted in Sichuang,Zhejiang,Jiangsu and Shandong provinces.As a traditional Chinese medicine,mulberry leaf has the functions of clearing heat,moistening the lung,and improving vision etc.Pharmacological studies showed that mulberry leaves could decrease blood glucose,inhibit bacteria,resist inflammation,prevent decrepitude and resist fatigue.UV-B is one of the high-energy particles from ultraviolet rays and high level UV-B radiation has strong damage effects on the growth and development of plants.Our previous study showed that the subcellular structures of mulberry leaves was damaged after 15 min UV-B irradiation with 36 h dark incubation(UV+D).Proteomic analysis indicated that proteins related to primary and secondary metabolism significantly altered under UV+D stress.Endoplasmic reticulum(ER)is a continuous membrane system that participates in the protein synthesis,modification,processing and transportation in cells,presents as the center of protein quality control.The stress response mechanism of the ER can alleviate the damages caused by adverse environmental conditions in plants.However,there are few reports on the isolation and purification of plant ER and we have no systematic and universal methods to use.In this study,we investigated and optimized the isolation and purification methods of ER from mulberry leaves.Based on this,we analyzed the ER from mulberry leaves under UV+D stress by proteomics.The main results are as follows.(1)Isolation and purification of ER from mulberry leaves.The methods of sucrose density gradient centrifugation,differential centrifugation combined with calcium chloride precipitation and differential centrifugation(with ultracentrifugation)were used to isolate ER from mulberry leaves.Western blot and enzymatic activity analysis showed that sucrose density gradient centrifugation couldn't remove the nucleus from ER component;differential centrifugation combined with calcium chloride precipitation couldn't effectively enrich ER;differential centrifugation could enrich ER effectively and remove nucleus,chloroplast,mitochondria and cytoplasm pollution in certain degree.Then we optimized the experimental details of the differential centrifugation(with ultracentrifugation)method.Results showed that using fresh leaves instead of frozen leaves could avoid the pollution of nucleus;grinding samples with buffer instead of using liquid nitrogen could ensure the extraction efficiency;using horizontal rotor instead of fixed-angle rotor could avoid large amounts of chloroplast pollution.(2)Proteomic analysis of ER proteins purified from mulberry leaves under UV+D stress.Using TMT-based proteomics,3948 quantifiable proteins were identified.297 ER proteins were selected by three subcellular localization prediction software,of which 200 proteins were changed(p<0.05).Bioinformatic analysis indicated:P450 enzymes were enriched,PDIs were significantly reduced and peroxisome membrane protein 11C was significantly increased.UGGT and CNX/CRT cycle belonging to the N-glycan biosynthesis pathway and ?-mannosidase related proteins were decreased.Heat shock proteins(HSP70,HSP90 and HSP40),protein targeting related protein BAP31 and protein output related protein SEC63 were increased;number of decreased ribosomal proteins was more than that of increased ones;nuclear envelope related proteins,EFl-?1 and EF1-? were increased significantly.These results suggest that mulberry leaves activated the redox system and ERQC system,enhanced the protein biosynthesis function and inhibited the protein N-terminal glycosylation to resist UV+D stress.This study provides a reference for ER purification of other plants through the purification of ER from mulberry leaves.The proteomics of ER from mulberry leaves under UV+D stress further explains the UV+D resistance mechanism of mulberry leaves and provides new ideas for resistance mechanism studies of other plants. |
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