Chitinase assay kit

The POD and CAT activities of APL01 and Holly were measured using the Micro Peroxidase Assay Kit (Solarbio, Beijing, China) and Catalase Assay Kit (Solarbio), respectively. Ultraviolet (UV) spectrophotometry was also used to measure the activity of these two enzymes. For more details on POD activity assay methods, please refer to Wang et al. [84 (link)] and, for CAT activity assay methods, please refer to Zhang et al. [85 (link)]. The chitinase activity of APL01 and Holly was measured by visible spectrophotometry using the Chitinase Assay Kit (Solarbio), following the manufacturer’s instructions. The activity of POD, CAT, and chitinase enzymes in each variety was measured before, as well as 7 and 13 days after inoculation with aphids. Three plants per variety were selected for each treatment (before inoculation, 7 days after inoculation, and 13 days after inoculation with aphids). The first fully developed leaf at the top of each plant was collected and mixed to create a biological sample. Each treatment group consisted of three biological samples.

The sub-cuticular tissue was collected at 48 h after WSSV infection and homogenized in PBS. After centrifugation at 12, 000 × g for 10 min, the supernatant was collected, and the protein concentration was determined as described above. Chitinase activity was determined using a Chitinase Assay Kit (Solarbio, Beijing, China; BC0825) by monitoring its ability to convert chitin to N-acetyl glucosamine. Generally, the reaction mixture (200 μL) consisted of 40 μL of sodium phosphate buffer (50 mM, pH 7.0), 80 μL of colloidal chitin (0.5%), and 80 μL of the test sample. The mixture was incubated at 37°C for 1 h. After terminating the reaction by boiling for 5 min, the mixture was centrifuged at 12, 000 × g for 10 min. The resultant supernatant (100 μL) was mixed with 20 μL of 3,5-dinitrosalicylic acid and incubated for 5 min at 100°C. The absorbance at 585 nm was monitored using a Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). A standard curve generated using gradient-diluted N-acetyl glucosamine was used to determine the amount of reducing sugar. One unit (U) represents the chitinase activity necessary to convey the colloidal chitin to 1 μg of reducing sugar for 1 h at 37°C. Chitinase activity is expressed as U per mg of sample protein.

After 48 h of stress treatment, a 1 mL sample was taken from each flask for measurement of chitinase activity using the Solarbio Chitinase Assay Kit (Cat#BC0820) following the manufacturer’s instructions. The remaining 49 mL of T. pseudonana cells in each flask were collected by centrifugation at 2850 g, washed once with fresh medium, and flash-frozen in liquid nitrogen. The T. pseudonana cell pellets were homogenized by vortexing in TRIzol reagent (Invitrogen, Waltham, MA, USA), then centrifuged at 9700 g. The supernatants were used for RNA extraction with chloroform and isopropanol. RNA pellets were resuspended in diethyl pyrocarbonate-treated water, followed by elimination of genomic DNA and synthesis of complementary DNA (cDNA) with a PrimeScript RT reagent kit (Takara, Japan). The synthesized cDNA was then used for qRT–PCR experiments.
The transcriptional profiles of 14 randomly selected T. pseudonana chitinase genes were obtained by qRT–PCR using TB Green™ Premix Ex Taq™ II (Takara, Japan) and a Thermal Cycle Dice™ Real Time System (Takara, Japan). Gene-specific qRT–PCR primers were designed using the NCBI Primer-BLAST website [70 (link)]. Information on primers is provided in Table S9. The beta tubulin gene (TUB3) was used as the reference gene for normalizing the expression of the T. pseudonana chitinase genes [71 (link)].