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

Abstract: 【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 little effect on the fresh weight, constant dark treatment reduced the fresh weight but constant light treatment had no obvious effect. The three kinds of temperature stresses increased the total protein content, and constant dark stress decreased the total protein content, the other stresses had little effect on the total protein content. Collectively, a total of approximately 300 protein samples are collected，the rice protein sample library (RiceS-A300) was established. The total volume of each sample was 2 mL, enough for about 200 WBs. As standard operational protocol was used for sample collection and protein isolation, the scale of sample can be enlarged. It is expecting that repeatable results can be achieved from different laboratories using similar treatment conditions. RiceS-A300 was used to investigate the expression patterns of HSP82 protein, the results showed that heat stress increased the expression of HSP82 significantly, constant light, PEG and NaCl stresses increased the expression of HSP82 slightly, cold stress, submerge and constant dark had little effect on the abundance of HSP82 protein.【Conclusion】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 was investigated using WB and it was found that HSP82 was specifically induced under heat stress.

Key words: rice, protein sample library, reference protein, western blot, heat shock protein