National Institute of Agricultural Science, RDA, Jeonju 54874, Korea.
Division of Horticultural Biotechnology, Hankyong National University, Anseong 17579, Korea.
Molecules. 2021 Oct 13;26(20):6174. doi: 10.3390/molecules26206174.
High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7 and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.
高分子量麦谷蛋白亚基（HMW-GS）仅占小麦总贮藏蛋白的 10%，但在小麦面粉加工品质中起着重要作用。因此，鉴定与良好加工品质相关的 HMW-GS 等位基因可为小麦育种者提供重要信息。为了快速、准确和重现性地鉴定 HMW-GS，我们建立了一种优化的反相超高效液相色谱（RP-UPLC）方法。使用 ACQUITY UPLC Protein BEH C 柱和逐步 ACN 梯度优化分离参数，并在 16 个标准小麦品种中比较分析了 22 个亚基的分离模式和保留时间（RT）。所有 HMW-GS 蛋白在大约 5.5 分钟内得到很好的分离，所有分析在 12 分钟内完成。我们根据 RT 区分了 16 个亚基，尽管 1Bx 中的三个亚基（1Bx7/1Bx7 和 1Bx17）和 1By 中的三个亚基（1By8*、1By9 和 1By15）具有重叠的 RT，但通过 SDS-PAGE 可以区分。为了区分在蛋白丰度上存在差异的 1Bx7 和 1Bx7，RP-UPLC 与 DNA 连接标记的 PCR 分析相结合。该优化方法成功应用于鉴定面包小麦种质资源（1787 条品系）中的 HMW-GS 等位基因。该方案是小麦育种中选择携带有利 HMW-GS 等位基因的品系的合适选择。
