高分子量麦谷蛋白亚基HPCE高效分离及图谱鉴定

摘要

高分子量麦谷蛋白亚基(HMW-GS)是决定小麦加工品质的重要因素.高效毛细管电泳技术(HPCE)以其用样少、速度快、精确度高等特点被越来越多地应用到分离鉴定中来.目前小麦 HMW-GS 的 HPCE 研究尚处于起步阶段, 对标准鉴定图谱的研究甚少, 且已有分离体系在连续多次试验中的分离速度及分辨率仍有提升空间.本研究通过SDS-PAGE结合分子标记的方法鉴定了不同小麦品种的HMW-GS, 以"中国春"小麦为标样确立了HPCE高效分离体系, 体系条件为75 mmol L–1IDA+0.05% HPMC+15% ACN, pH 2.5, 电泳参数为毛细管内径25 μm, PDA检测波长200 nm, 分离电压20 kV, 运行温度30°C.通过混合进样方式对不同类型亚基进行HPCE分析, 建立了18个亚基的标准图谱, 亚基迁移顺序为 1Dy12→1Dy10→1By9→1By8→1By18→1By16→1By20→1Bx17→1Bx20→1Bx13→1Bx6→1Bx7→1Ax2*→1Ax1→1Dx5→1Dx4→1Dx3→1Dx2, 标准出峰时间依次为9.39、9.69、10.30、11.70、11.89、12.09、12.22、12.36、12.62、12.83、13.08、13.18、13.50、13.73、14.04、14.24、14.46和14.73 min, RSD＜0.2%.以1Bx17为分界线, 9.39 min到12.36 min为y型亚基区域, 12.36 min到14.76 min为x型亚基区域.结合亚基迁移顺序、标准出峰时间及图谱特点可对小麦相关 HMW-GS 进行 HPCE 快速鉴定.本研究获得的分离体系及鉴定图谱可用于小麦HMW-GS定性分析和种质资源筛选.%High molecular weight glutenin subunit (HMW-GS) is important for processing quality of wheat. High performance capillary electrophoresis (HPCE) is increasingly used in the work of separation and identification due to its advantages of small sample, rapidness and high precision. However, this technique has been seldom used in wheat HMW-GS study and its separation system needs improvement in analytical speed and discernibility. On the basis of HMW-GS identification with SDS-PAGE and molecular markers, an high-efficiency separation system of HPCE was set up using Chinese Spring as the standard. The system components (pH 2.5) were 75 mmol L–1IDA, 0.05% HPMC, and 15% ACN. The electrophoresis parameters were 25 μm of inner diameter of the capillary, 200 nm of detection wavelength, 20 kV of separation voltage, and 30°C of operating temperature. Using mixed injection method, the standard spectrums were obtained for 18 subunits. Their migration order was 1Dy12→1Dy10→1By9→1By8→1By18→1By16→1By20→1Bx17→1Bx20→1Bx13→1Bx6→1Bx7→1Ax2*→1Ax1→1Dx5→1Dx4→1Dx3→1Dx2, and standard peak time of these subunits was 9.39, 9.69, 10.30, 11.70, 11.89, 12.09, 12.22, 12.36, 12.62, 12.83, 13.08, 13.18, 13.50, 13.73, 14.04, 14.24, 14.46, and 14.73 min, respectively. The relative standard deviation was smaller than 0.2%. The y-type and x-type subunits appeared in the phases of 9.39–12.36 min and 12.36–14.76 min, respectively, between which there was 1Bx17 as the boundary. These results indicate that the HPCE separation system is applicable in rapid identification of HMW-GS in wheat germplasm resources when we simultaneously consider migration order, standard peak time, and HPCE spectrum.