Real-time quantitative PCR (RT-qPCR) was used to quantify the expression levels of CNM-related enzymes in rice seedlings after SCN− exposure. Total RNA was extracted from both the root and shoot of all rice samples by using an Ultrapure RNA Kit (CWBio, Taizhou, China). DNase I (CWBio, Taizhou, China) was used to remove genomic DNA contamination, if any, from RNA extract. Then, the total RNA was purified by an RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany). Each sample was prepared in four independent biological replicates.
A total of 40 genes encoding enzymes or proteins activated in the CNM pathways were searched from the databases, including RGAP (http://rice.plantbiology.msu.edu/analyses_search_blast.shtml), NCBI (https://www.ncbi.nlm.nih.gov/), and RAPDB (http://rapdb.dna.affrc.go.jp/). Expression of genes was assayed after SCN− exposure by RT-qPCR analysis, including PEPC (Osppc1, Osppc2a, Osppc3, and Osppc4), ERS (OsERS1, OsERS2, and OsERS3), TPS (OsTPS1, OsTPS4, OsTPS5, OsTPS8, and OsTPS9), SPS (OsSPS1, OsSPS2, OsSPS4, OsSPS5, and OsSPS6), NR (OsNIA1, OsNIA2, and OsNR1), NiR (OsNiR1, OsNiR2, and OsNiR3), GS (OsGS1;1, OsGS1;2, OsGS1;3, and OsGS2), NADP-ICDH (OsICDH1, OsICDH2, OsICDH3, and OsICDH4), NAD-IDH (OsIDHc;2, OsIDHc;1, OsIDHa, and OsIDH1), and GDH (OsGDH1, OsGDH2, OsGDH3, and OsGDH4). All genes primer sequences are listed in Table S1. RT-qPCR cycling conditions were as follows: 1) denaturation at 95°C for 10 s, 2) annealing at 58°C for 30 s, and 3) extension at 72°C for 32 s. This cycle was imitated 40 times. The RT-qPCR analysis was executed using the 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) and SYBR green chemistry. Rice GAPDH (glyceraldehyde-3-phosphate dehydrogenase, LOC_Os08g03290.1) was selected as the housekeeping gene (Yang et al., 2021 (link)). The standard 2−ΔΔCT method was used to calculate the relative expression of each of the targeted genes (Schmittgen and Livak, 2008 (link)). All values were represented as cumulative means ± standard deviation of four independent replicates.
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