Hauvermale Amber L, Matzke Courtney, Bohaliga Gamila, Pumphrey Mike O, Steber Camille M, McCubbin Andrew G
Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA.
School of Biological Sciences, Washington State University, Pullman, WA 99164, USA.
Plants (Basel). 2023 Nov 8;12(22):3798. doi: 10.3390/plants12223798.
Accurate, rapid testing platforms are essential for early detection and mitigation of late maturity α-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated α-amylase levels and negatively impact flour quality, resulting in substantial economic losses. The Hagberg-Perten Falling Number (FN) method is the industry standard for measuring α-amylase activity in wheatmeal. However, FN does not directly detect α-amylase and has major limitations. Developing α-amylase immunoassays would potentially enable early, accurate detection regardless of testing environment. With this goal, we assessed an expression of α-amylase isoforms during seed development. Transcripts of three of the four isoforms were detected in developing and mature grain. These were cloned and used to develop expression lines expressing single isoforms. After assessing amino acid conservation between isoforms, we identified peptide sequences specific to a single isoform (TaAMY1) or that were conserved in all isoforms, to develop monoclonal antibodies with targeted specificities. Three monoclonal antibodies were developed, anti-TaAMY1-A, anti-TaAMY1-B, and anti-TaAMY1-C. All three detected endogenous α-amylase(s). Anti-TaAMY1-A was specific for TaAMY1, whereas anti-TaAMY1-C detected TaAMY1, 2, and 4. Thus, confirming that they possessed the intended specificities. All three antibodies were shown to be compatible for use with immuno-pulldown and immuno-assay applications.
准确、快速的检测平台对于小麦晚期成熟α-淀粉酶（LMA）和收获前发芽（PHS）的早期检测和缓解至关重要。这些情况的特征是α-淀粉酶水平升高，会对面粉质量产生负面影响，从而导致重大经济损失。哈格贝格-佩尔滕降落数值（FN）法是测定小麦粉中α-淀粉酶活性的行业标准。然而，FN不能直接检测α-淀粉酶，且有重大局限性。开发α-淀粉酶免疫测定法可能会实现无论测试环境如何都能进行早期、准确的检测。出于这个目标，我们评估了种子发育过程中α-淀粉酶同工型的表达。在发育中的和成熟的籽粒中检测到了四种同工型中的三种的转录本。将这些转录本进行克隆，并用于构建表达单一同工型的表达系。在评估了同工型之间的氨基酸保守性之后，我们鉴定出了特定于单一同工型（TaAMY1）或在所有同工型中都保守的肽序列，以开发具有靶向特异性的单克隆抗体。开发了三种单克隆抗体，抗TaAMY1-A、抗TaAMY1-B和抗TaAMY1-C。这三种抗体都检测到了内源性α-淀粉酶。抗TaAMY1-A对TaAMY1具有特异性，而抗TaAMY1-C检测到TaAMY1、2和4。因此，证实它们具有预期的特异性。结果表明，这三种抗体都适用于免疫沉淀和免疫测定应用。
