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为建立小麦种子高分子量麦谷蛋白亚基(HMW-GS)高通量检测方法,本研究改进了传统聚丙烯酰氨凝胶电泳(SDS-PAGE)中的样品制备和电泳分析方法,提高了检测效率。具体方法为:在小麦种子无胚端切取其体积的1/4~1/5,置于单孔容积为200μL的96孔PCR扩增板中,并在每个微孔中放入1粒直径2 mm的钢珠,并加入浸泡缓冲液静置1~2 h,再加入提取缓冲液,经高通量研磨器(Retsch Tissue Lyser)研磨后,在60℃的烘箱静置30 min。对微孔板离心和上清液预处理后在DYCZ-30C高通量垂直版电泳槽进行电泳分离,获得清晰的HMW-GS分离图谱。该方法简易、快速、成本低,适用于小麦标记辅助选择和资源评价中的大规模HMW-GS组成的检测。
In order to establish a high-throughput detection method for high molecular weight glutenin subunits (HMW-GS) of wheat seeds, this study improved the sample preparation and electrophoresis analysis methods in traditional polyacrylamide gel electrophoresis (SDS-PAGE) effectiveness. The specific method is as follows: one fourth to one fifth of the volume of the wheat seed is excised and placed in a 96-well PCR amplification plate having a single-hole volume of 200 μL, and one particle diameter 2 mm steel balls were added and soaked in the buffer solution for 1 ~ 2 h. The extraction buffer was added and ground by a high-throughput mill (Retsch Tissue Lyser) and then allowed to stand in an oven at 60 ° C for 30 min. After the microplate was centrifuged and the supernatant pretreated, the DYCZ-30C high-throughput vertical electrophoresis tank was separated by electrophoresis to obtain a clear HMW-GS chromatogram. The method is simple, rapid and low cost and is suitable for the detection of large-scale HMW-GS composition in wheat marker-assisted selection and resource evaluation.