阐明小麦细胞 壁防御机制 的 #946;-1,3-葡聚糖酶和过氧化物酶的物理化学特性
1Carbohydrates and Enzymology Laboratory (CHEM-LAB), Department of Plant Sciences,University of the Free State, 2Faculty of Natural and Agricultural Sciences, Department of Plant Science, Botany, Plant Biochemistry and Physiology,University of the Free State, 3Agricultural Research Council – Small Grain Institute
Summary
本方案描述了用于研究和表征小麦植物中细胞壁相关酶（主要是 β-1,3-葡聚糖酶和过氧化物酶）的程序。在小麦-RWA 相互作用期间，它们的活性水平增加，并通过细胞壁加固参与植物防御反应，从而阻止蚜虫取食。
Abstract
受俄罗斯小麦蚜虫 （RWA） 侵染的小麦植株会诱导一连串防御反应，包括超敏反应 （HR） 和发病机制相关 （PR） 蛋白的诱导，例如 β-1,3-葡聚糖酶和过氧化物酶 （POD）。本研究旨在表征细胞壁相关 POD 和 β-1,3-葡聚糖酶的物理化学性质，并确定它们在 RWASA2-小麦相互作用过程中对细胞壁修饰的协同作用。在温室条件下对易感 Tugela、中度抗性 Tugela-Dn1 和抗性 Tugela-Dn5 栽培品种进行预发芽和种植，种植后 14 天施肥，每 3 天灌溉一次。植物在 3 叶期被 20 个相同 RWASA2 克隆的孤雌个体侵染，并在侵染后 1 至 14 天收获叶片。使用真空过滤提取细胞间洗涤液 （IWF） 并储存在 -20 °C。 将叶残留物粉碎成粉末，用于细胞壁成分。使用 5 mM 愈创木酚底物和 H2O2 测定 POD 活性和表征，监测 470 nm 处吸光度的变化。通过使用 β-1,3-葡聚糖和 β-1,3-1,4-葡聚糖底物用 DNS 试剂测量水解物中的总还原糖，测量 540 nm 处的吸光度，并使用葡萄糖标准曲线，证明了 β-1,3-葡聚糖酶活性、pH 值和温度最佳条件。在 pH 4 至 9 之间测定最适 pH 值，在 25 至 50 °C 之间测定最佳温度，在 30 °C 至 70 °C 之间测定热稳定性。在 25 °C 和 40 °C 下使用可得然胶和大麦 β-1,3-1,4-葡聚糖酶底物测定 β-1,3-3-葡聚糖酶底物特异性。此外，使用海带二糖到海带五糖测定 β-1,3-葡聚糖酶的作用模式。用薄层色谱 （TLC） 定性分析寡糖水解产物模式，并用 HPLC 定量分析。本研究中提出的方法展示了一种用 RWA 感染小麦、从细胞壁区域提取过氧化物酶和 β-1,3-葡聚糖酶及其综合生化表征的稳健方法。
Introduction
俄罗斯小麦蚜虫 （RWA） 侵扰小麦和大麦，导致产量严重损失或谷物质量下降。小麦通过诱导多种防御反应来响应侵染，包括增加抗性品种的 β-1,3-葡聚糖酶和过氧化物酶活性水平，而易感品种在侵染早期降低这些酶的活性 1,2,3,4。β-1,3-葡聚糖酶和 POD 在小麦植株中的关键功能包括调节抗性品种中的胼胝质积累以及在 RWA 侵染期间细胞壁和质外体区域的活性氧 （ROS） 淬灭 1,3,5,6,7。Mafa 等人6 证明，RWASA2 侵染后抗性小麦品种中 POD 活性的增加与木质素含量的增加之间存在很强的相关性。此外，木质素含量的增加表明受侵染的抗性小麦品种的细胞壁得到加强，导致 RWA 取食减少。
大多数研究小组在小麦/大麦-RWA 相互作用过程中提取并研究了质外体 β-1,3-葡聚糖酶和 POD;此外，这些研究中的大多数声称这些酶会影响受 RWA 侵染的小麦植株的细胞壁，而没有测量细胞壁区域中酶的存在。只有少数研究使用显微技术表明 β-1,3-葡聚糖酶活性水平与胼胝质调节 7,8,9 有关，或提取主要细胞壁成分以证明 POD 活性与抗性细胞壁修饰之间的相关性 6,10。缺乏探测 β-1,3-葡聚糖酶和 POD 与细胞壁的结合表明需要开发允许研究人员直接测量细胞壁结合酶的方法。
目前的方法提出，在提取细胞壁结合酶之前，有必要从叶组织中去除质外体液。质外体液的提取程序必须从叶组织中进行两次，用于提取细胞壁结合酶。这个过程减少了质外体酶与细胞壁区域中发现的质外体酶的污染和混淆。因此，在本研究中，我们提取了细胞壁结合的 POD、β-1,3-葡聚糖酶和 MLG 特异性 β-葡聚糖酶，并进行了它们的生化表征。
Protocol
该研究是在自由州大学环境和生物安全研究伦理委员会 （UFS-ESD2022/0131/22） 的批准和许可下进行的。此处的试剂和设备的详细信息列在 材料表中。 1. 植物生长条件 在单独的培养皿中发芽每个小麦品种的 250 粒种子，即易感的 Tugela、中度抗性的 Tugela-Dn1 和抗性的 Tugela-Dn5。 在每个培养皿中加入 5 mL 蒸馏水，用石蜡膜密封，…
Representative Results
小麦品种的四个生物学重复品种 （Tugela、Tugela-Dn1 和 Tugela-Dn5） 在 3 叶生长阶段被 RWASA2 侵染。侵染后，以 1 、 2 、 3 、 7 和 14 dpi 收获叶子。对照处理未被 RWASA2 侵染，使实验结果与未受到胁迫的小麦植株相当。实验一式四份进行，结果以平均值表示。 使用 BSA 作为蛋白质标准品对 RWASA2 感染和对照提取物的蛋白质浓度进行定量。这在测定 β-1,3-葡聚糖酶和 POD 的…
Discussion
小麦和大麦是经常受到蚜虫物种侵扰的谷类作物，包括俄罗斯小麦蚜虫 （Diuraphis noxia）7,24。抗性小麦植物诱导 POD 和 β-1,3-葡聚糖酶活性的上调，作为整个侵染期间的防御反应，通过调节胼胝质和木质素积累来改变细胞壁 6,25,26,27。值得注意的是，大…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
M. Mafa 获得了 NRF-Thuthuka 的资助（参考编号：TTK2204102938）。S.N. Zondo 的硕士学位获得了国家研究基金会研究生奖学金。作者感谢农业研究委员会 – 小谷物 （ARC-SG） 研究所提供本研究中使用的种子。本材料中表达的任何意见、发现和建议均为作者的观点、发现和建议，因此，资助者对此不承担任何责任。
Materials
| 10 kDa Centrifuge concentrating membrane device | Sigma-Aldrich | R1NB84206 | For research use only. Not for use in Diagnostic procedures. For concentration and purification of biological solutions. |
| 2 g Laminaribiose | Megazyme (Wicklow, Ireland) | O-LAM2 | High purity laminaribiose for use in research, biochemical enzyme assays and in vitro diagnostic analysis. |
| 3 g Laminaritriose | Megazyme (Wicklow, Ireland) | O-LAM3 | High purity laminaritriose for use in research, biochemical enzyme assays and in vitro diagnostic analysis. |
| 3,5 Dinitro salicylic acid | Sigma-Aldrich | D0550 | Used in colorimetric determination of reducing sugars |
| 4 g Laminaritetraose | Megazyme (Wicklow, Ireland) | O-LAM4 | High purity laminaritetraose for use in research, biochemical enzyme assays and in vitro diagnostic analysis. |
| 5 g Laminaripentaose | Megazyme (Wicklow, Ireland) | O-LAM5 | High purity laminaripentaose for use in research, biochemical enzyme assays and in vitro diagnostic analysis. |
| 95% Absolute ethanol | Sigma-Aldrich | 107017 | Ethanol absolute for analysis |
| acetic acid | Sigma-Aldrich | B00063 | Acetc acid glacial 100% for analysis (contains acetic acid) |
| Azo-CM-Cellulose | Megazyme (Wicklow, Ireland) | S-ACMC | The polysaccharide is dyed with Remazolbrilliant Blue R to an extent of approx. one dye molecule per 20 sugar residues. |
| Beta glucan (barley) | Megazyme (Wicklow, Ireland) | G6513 | A powdered substrate, less soluble in water. Used in determining β-1,3-glucanase activity. |
| Bio-Rad Protein Assay Dye | Bio-Rad Laboratories, South africa | 500-0006 | Colorimetric assay dye, concentrate, for use with Bio-Rad Protein Assay Kits I and II |
| Bovine serum albumin (BSA) | Gibco Europe | 810-1018 | For Laboratory use only |
| Citrate acid | Sigma-Aldrich | C0759 | For Life Science research only. Not for use in diagnostic procedures. |
| CM-curdlan | Megazyme (Wicklow, Ireland) | P-CMCUR | Powdered substrate for determining β-1,3-glucanase activity. Insoluble in water. |
| D-Glucose | Sigma-Aldrich | G8270 | For Life Science research only. Not for use in diagnostic procedures. |
| Guaiacol | Sigma-Aldrich | G5502 | Oxidation indicator. Used for determining peroxidase activity. |
| Hydrogen peroxide | BDH Laboratory Supplies, England | 10366 | Powerful oxidising agent. |
| Mikskaar Professional Substarte | Mikskaar (Estonia) | NI | Peat moss-based seedling substrate. |
| Multifeed fertiliser (5.2.4 (43)) | Multifeed Classic | B1908248 | A water soluble fertiliser for young developing plants and seedlings with a high phosphorus (P) requirement to ensure optimum root development. |
| Naphthol | Merck, Germany | N2780 | Undergoes hydrogenations in the presence of a catalyst. |
| Phenol | Sigma-Aldrich | 33517 | Light sensitive. For R&D use only. Not for drug, household, or other uses. SDS available |
| Potassium sodium tartrate tetrahydrate (Rochelle salt) | Sigma-Aldrich | S2377 | used in the preparation of 3,5-dinitrosalicylic acid solution used in the determination of the reducing sugar. |
| Silica plate (TLC Silica gel 60 F254) | Sigma-Aldrich | 60778-25EA | Silica gel matrix, with fluorescent indicator 254 nm |
| Sodium hydroxide | Sigma-Aldrich | S8045 | For R&D use only. Not for drug, household, or other uses. |
| Sodium metabisulfite | Sigma-Aldrich | 31448 | Added as an antioxidant during the preparation of 3,5-dinitrosalicylic acid solutions. |
| Sodium phosphate dibasic heptahydrate | Sigma-Aldrich | S9390 | Used as a buffer solution in biological research to keep the pH constant. |
| Sodium phosphate monobasic heptahydrate | Sigma-Aldrich | 71500 | An inorganic compound, which is soluble in water. Used as a reagent in the development of silicate-based grouts. |
| Statistical analysis software | TIBCO Statistica | version 13.1 | |
| Sulfuric acid | Merck, Darmstadt, Germany | 30743 | Sulfuric acid 95-97% for analysis of Hg, ACS reagent. |
| Tris-HCl | Sigma-Aldrich | 10812846001 | Buffering agent in incubation mixtures. It has also been used as a component of lysis and TE (Tris-EDTA) buffer. For life science research only. Not for use in diagnostic procedures. |
| UV–Visible Spectrophotometer | GENESYS 120 | ||
| NI = not identified. |
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Zondo, S. N., Mohase, L., Tolmay, V., Mafa, M. S. Elucidating #946;-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation. J. Vis. Exp. (209), e66903, doi:10.3791/66903 (2024).