Cat assay kit a007 1 1

After homogenizing in cold physiological saline solution, testis samples were centrifuged at 500 g for 15 min to obtain supernatants for the biochemical analysis of oxidative stress markers. As described in a previous study [22 (link)], glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) were analyzed using commercial kits, according to the procedure provided by the manufacturer (GPx assay kit, A005-1-2; SOD assay kit, A001-3-2; CAT assay kit, A007-1-1; Nanjing Jiancheng Biological Engineering Research Institute Co. Ltd., Nanjing, China). Malondialdehdye (MDA) levels were measured using a colorimetric method (MDA assay kit, A003-1-2, Nanjing Jiancheng Biological Engineering Research Institute Co. Ltd.). The protein concentration of all samples was determined using the Bradford method [23 (link)].

qRT-PCR was performed with three independent biological replicates using AceQ® qPCR SYBR® Green Master Mix (Vazyme, Nanjing, China) in a 20 μL volume on a qTOWER3G Real-time System (Analytik Jena AG, Germany). The qRT-PCR primers are listed in Supplementary Table 2. EF1α was used as an internal control for normalization of the data. Relative expression was calculated using the 2^–△△CT method.
The activities of the main disease-resistance enzymes, including ROS, SOD, POD, and CAT, were determined using the ROS Assay Kit E004–1-1, SOD Assay Kit A001–3-2, POD Assay Kit A084–3 and CAT Assay Kit A007–1-1 (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with the protocols provided by the manufacturer. The leaves we collected are random and they’re mixed together to extract total RNA. The leaves were collected at 10:00 AM on the 0, 1, 3 and 5 day after inoculation, and the collected samples were immediately used for analysis. All the treatment groups were carried out at the same time, and the whole experiment was repeated three times.