水稻蛋白质样品资源库RiceS-A300的建立与应用  

The Establishment and Application of Rice Protein Sample Library RiceS-A300

作　　者：张剑硕 马金姣 张彤 陈悦 魏健[1] 张柳 史佳楠[1] 徐珊 燕高伟 杜铁民 窦世娟[1] 李莉云[1] 刘丽娟[1] 刘国振[1] ZHANG JianShuo;MA JinJiao;ZHANG Tong;CHEN Yue;WEI Jian;ZHANG Liu;SHI JiaNan;XU Shan;YAN GaoWei;DU TieMin;DOU ShiJuan;LI LiYun;LIU LiJuan;LIU GuoZhen(College of Life Sciences,Hebei Agricultural University,Baoding 071001,Hebei)

机构地区：[1]河北农业大学生命科学学院,河北保定071001

出　　处：《中国农业科学》2018年第19期3625-3638,共14页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31171528)

摘　　要：【目的】建立水稻幼苗胁迫处理的蛋白质样品资源库RiceS-A300,应用RiceS-A300调查内参蛋白质HSP82的表达特征。【方法】粳稻TP309种子在30℃条件下浸泡3 d露白,土培用蛭石土(土壤与蛭石1:1)培养,30℃、光周期L12 h/D12 h培养5 d,进行冷(4℃)、热(44℃、48℃)、淹和恒光、恒暗处理,水培播种在纱网上,以30℃、光周期L12 h/D12 h培养5 d,进行PEG 6000(20%)和NaCl(0.2 mol·L^(-1))处理。以30℃、光周期L12 h/D12 h非胁迫处理为对照。在不同时间点观察、拍照并取材,测定各种处理条件下的株高和鲜重,提取总蛋白质建立水稻幼苗胁迫处理的蛋白质样品资源库,SDS-PAGE分离总蛋白质后进行考染,以此评价蛋白质质量。通过免疫印迹(WB)技术分析样品中内参蛋白质HSP82的表达特征。【结果】调查各种胁迫处理过程中9个时间点的幼苗表型、株高、鲜重和总蛋白质含量,发现:冷(4℃)、热(44℃、48℃)温度胁迫在2 d内即表现出对株高的抑制作用,淹胁迫能促进株高伸长,恒暗和恒光处理均能抑制株高,恒暗处理使幼苗变黄,PEG与盐胁迫也能明显抑制株高的伸长,且盐胁迫在5 d后可见明显叶尖干枯。对鲜重的调查结果表明,冷(4℃)、热(44℃、48℃)温度胁迫、PEG胁迫和盐胁迫均会降低幼苗鲜重,淹胁迫对鲜重的影响较小,恒暗处理会降低鲜重而恒光处理对鲜重影响不明显。3种温度胁迫都使总蛋白质含量提高,恒暗胁迫则降低总蛋白质含量,其他胁迫对总蛋白质含量影响不大。累计收集近300份蛋白质样品,建立了水稻蛋白质样品资源库(RiceS-A300),每份样品体积为2 mL,可供200次WB分析,由于试验所采用的条件比较规范且容易控制,可根据需要重复扩大样品的规模,预期不同的实验室采用相同的条件获得的蛋白质样品也能获得可重复的试验结果,利用RiceS-A300调查HSP82蛋白质的表达特征,显示热胁迫明显提高了HSP82的表�【Objective】The aim of the study is establish a protein sample library namely RiceS-A300,which was collected from rice seedlings treated by abiotic stressed conditions and investigate the expression patterns of the reference protein HSP82 by Western blot analysis using the library of RiceS-A300.【Method】The seeds of japonica rice 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/D12 h 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.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.【Result】The phenotype,plant height,fresh weight and total protein content of rice seedlings at 9 time points under various stressed conditions were investigated.It was found that cold(4),hot(44 and 48℃℃℃)stresses demonstrated the inhibitory effect of the elongation of plant height in 2 d,while the submerge treatment promotes the elongation of plant height.Constant dark and constant light inhibited the elongation of plant height,the seedlings became yellow under the constant dark treatment.PEG and NaCl treatments had an inhibitory effect on the plant height also,and the leaf tip was dry under NaCl stress at 5 d.The results for fresh weight investigation showed that cold(4℃),hot(44℃and 48℃),PEG and NaCl stresses reduced the fresh weight of the seedlings,while the submerge treatment had littl

关 键 词：水稻 蛋白质样品资源库 内参蛋白质 免疫印迹 热激蛋白 

分 类 号：S511[农业科学—作物学]