2.6. Validation of Gene Expression via qRT-PCR

To verify the accuracy of the RNA-Seq data and assay the expression levels of the SMV-CP gene, qRT-PCR assays were conducted using gene-specific primers. Semi-quantitative RT-PCR was also used to detect the expression of viral coat protein genes according to Zhang et al., 2019 [40]. Total RNA from the same treated samples was extracted from V1 leaves in soybeans. Reverse transcription was performed using a reverse transcription kit from Beijing Tsingke Biotech Co., Ltd. (Goldenstar™ RT6 cDNA Synthesis Kit Ver.2). In addition, 2×RealStar Fast SYBR qPCR Mix (Tsingke Biotech Co., Ltd., Beijing, China) was used, and an Eppendorf Mastercycler ep realplex (Eppendorf, Hamburg, Germany) instrument was used for the qRT-PCR experiment. Each treatment contained three independent biological replicates and three technical replicates. The expression level of the soybean β-actin gene was used as an internal reference. The fold change value of gene expression was calculated using the 2^−∆∆Ct method. The sequences of specific primers are listed in Tables S2–S4.