水稻120kDa光敏色素的提纯

PURIFICATION OF 120-KILODALTON PHYTOCHROME FROM ORYZA SATIVA L

采用改进的 Grimm 等方法,从晚粳“农垦58”水稻(Oryza sativa L.subsp.japonica,var.nongken 58)6天龄黄化苗中提取光敏色素。经液氮深冻的胚芽鞘在含有乙二醇和苯甲基磺酰氟化物等、pH 为8.45的 Tris 缓冲液中匀浆。对聚乙烯亚胺沉淀后的提取液照射红光,经硫酸铵盐析后、通过羟基磷灰石柱层析梯度洗脱。合并的含有光敏色素的洗脱液再经硫酸铵盐析和三次不同体积、不同浓度的磷酸缓冲液洗涤。最后溶于 HEPES 缓冲液的天然光敏色素(120kDa)经 SDS-PAG 电泳检测为一条带,并具有经典的纯净光敏色素的吸收光谱,产率达12%。

The procedures of Grimm and Rüdiger for the purification of 120 kDa phytochrome from oat seedlings were modified to isolate native phytochrome from etiotated rice(Oryza sa- tiva L.subsp,japonica,var.nongken 58)seedlings.Approximately 1kg of 6d old seedlings (the first 2 days at 33℃,the last 4 days at 27℃ in darkness)were frozen in liquid nitrogen and then homogenized in a modified Waring blendor with an extraction buffer,at final pH 8.45(4℃).After polyethylenimine precipitation,phytochrome in extract was converted to P_(?) by irradiation of the resulting supernatant for 10 min with red light.The step of ammonium sulfate precipitation was followed by resuspending of resultant pellet in buffer B with the ra- tio of 10 ml per phytochrome unit.The pellet precipitated with ammonium sulfate at 42& sa- turation from combined phytochrome cont ning fractions after hydroxyapatite chromatography was washed with 10 mmol/l phosphate buffer in 0.8ml instead of 0.65ml per phytochrome unit. Then it was washed successively with 200mmol/l and 100mmol/l phosphate b(?)ffer(0.85ml per phytochrome unit).Native phytochrome(120kDa)in 12% yield was dissolved in 2mmol/l EHPES buffer(2.2ml per phytochrome unit,pH7.8,containing 5mmol/l EDTA and 14 mmol/l 2-mercaptoethanol)was proved to be pure in SDS-polyacrylamide electrophoresis and showed typical absorption spectrum as that of native oat phytochrome.