The Establishment Of Rice Protein Sample Library RiceS-A300 And Preliminary Functional Study Of MPK7

Background:Rice is one of the most important food crops as it is the staple food for more than half of population of the world.Rice is also a model organism for basic research.Among the important crops,the whole genome sequencing of rice is the first one to be finished,and it is also the top one to carry out transcriptomic investigation.Various biotic and abiotic stresses are encountered during the life cycle of rice.In order to cope with these stresses,rice has developed different strategies.Protein is the major player of the function of the gene,thus the understanding of state of each protein involved in each pathway under different temporal and spatial conditions is an challenge for biologists.Based on the concept developed in human protein research,we proposed and launched the RiceAb project,which aims to generate and use of each protein-specific antibody to comply Western blot（WB）analysis of representative protein samples,eventually,a rice protein expression profiling database（RiceP）will be accumulated.During the process,we realized that the representative rice protein samples（RiceS）is the bottleneck.Objectives:The aim of the current project is establish RiceS-A300,a library of protein samples for stress treatment of rice seedlings.Coomassie blue stained SDS-PAGE and BCA method will be used to monitor the quality of proteins extracted from various stresses on rice seedlings.WB technology will be used to detect HSP82,an internal control protein in rice identified in our laboratory.The expression profiling of HSP82 and MPK 7 protein under different stress conditions will be investigated.Materials and Methods:The seeds of Geng rice（Oryza sativa.subsp.Geng）variety TP309 were soaked in water for 3 days at 30℃,and then cultured either in soil mixture（soil and vermiculite 1:1）or aquaculture condition at 30℃with photoperiod L12 h/D12h for 5 days.Then the seedlings were treated by cold（4℃）,heat（44℃and 48℃）,submerge,constant light,constant dark,PEG 6000（20%）and NaCl(0.2 mol·L^-1),while the seedlings cultured at 30℃with photoperiod L12 h/D12 h was used as control.Rice seedlings were monitored,photographed and samples were collected at different time points,the plant height and fresh weight were measured under different stressed conditions.To establish a library of protein samples total proteins were extracted from rice seedlings.The BCA method was used to determine the concentration of the extracted protein.SDS-PAGE separated proteins were stained with coomassie blue to evaluate the quality of protein sample.Western blot（WB）was carried out to investigate the expression patterns of reference protein HSP82 and MPK7.Results:Using rice seedlings as material,we investigated the shoot height,root length,and fresh weight under various abiotic stresses,including cold,heat,PEG,submerge,NaCl,constant light and constant dark.We collected about 300 protein samples at different time points and extracted total proteins.After SDS-PAGE separation,quantitative analysis and evaluation were performed and establish the first version of the rice protein sample library RiceS-A300.Furthermore,the expression characteristic of HSP82,an internal reference protein,were analyzed using WB.The analysis of the expression of MPK7 in rice at different stress conditions.It was found that submerge treatment promotes the elongation of plant height,but other stresses suppressed plant height,4℃and constant dark reduced the fresh weight,low or high temperature stresses increased the total protein content,in contrast,constant dark reduced the total protein content.Other stresses have little effects on the total protein content.The expression of MPK7 varies greatly at different stress conditions.Conclusions:This study proposed a process for the construction and evaluation of the protein sample library,and the first version of the library（RiceS-A300）derived from rice seedlings under different stressed conditions was established.On the basis of RiceS-A300,the expression patterns of reference protein HSP82 and MPK7 was investigated using WB and it was found that HSP82 was specifically induced under heat stress.