HPLC法测定孕穗期水稻剑叶中多种内源激素的含量

Determination of Multiple Endogenous Hormone in Rice Flag Leaves during Booting Stage by HPLC

以孕穗期水稻剑叶为材料,建立了HPLC法,对水稻中的吲哚乙酸(IAA)、脱落酸(ABA)、赤霉素(GA)、玉米素(Z)和水杨酸(SA)等5种植物内源激素进行分离和测定。以甲醇为提取溶剂,再经SPE C18小柱过柱子除杂。HPLC分离采用C18(5μm×4.6×250 nm)反向色谱柱,最终采用PDA检测;以甲醇-水(0.5%乙酸)为流动相,流速为0.9 m L/min,进样量为20μL,在波长254 nm、272 nm和236 nm下同时检测。洗脱梯度为:0~4 min,A:20%~30%;4~10 min,A:30%~48%;10~20 min,A:48%~80%;20~30 min,A:80%;30~35 min,A:80%~20%;35~40 min,A:20%。结果表明,5种激素峰形良好、尖锐且保留时间稳定,方法的线性范围为3.125~1000 mg/kg,决定系数R2>0.9996,加标回收率达45.73%~116.70%。同时,本研究采用建立好的方法对不同胁迫处理下孕穗期水稻剑叶中内源激素含量进行了分离测定,效果良好。

High performance liquid chromatography(HPLC)method was used to separate and determine indole acetic acid(IAA),abscisic acid(ABA),gibberellic acid(GA),zeatin(Z)and salicylic acid(SA)in rice.Using methanol as the extraction solvent,the column was removed by SPE C18column.HPLC separation was performed on a C18(5μm×4.6×250 nm)reverse phase column,which was finally detected by PDA;methanol-water(0.5%acetic acid)was used as the mobile phase,and the flow rate was 0.9 m L/min.The injection volume was 20 u L;simultaneous detection was performed at wavelengths of 254 nm,272 nm and 236 nm.The elution gradient is:0~4 min,A:20%~30%;4~10 min,A:30%~48%;10~20 min,A:48%~80%;20~30 min,A:80%;30~35 min,A:80%~20%;35~40 min,A:20%.The results showed that the peaks of the five hormones were sharp and stable,and the retention time was stable.The linear range of the method was 3.125~1000 mg/kg,the coefficient of determination was high(R2>0.9996),the recovery rate was 45.73%~116.70%.In this study,the content of endogenous hormones in flag leaves of rice at the booting stage under different stress treatments were defermined by the established method with good results.