*4.6. Gene Expressions Analysis.*

The isolated RNA was then used to synthesize first strand cDNAs using an oligo dT primer and the cDNA synthesis kit (Shengong Ltd, Shanghai, China). The resulting cDNA samples were individually diluted to 100 ng/µL prior to qPCR using gene specific primers (Table S2). For each PCR reaction, three biological replicates with three technical replicates were each used. qPCR reactions were 40 cycles of 95 ◦C for 5 s, 60 ◦C for 15 s, and 72 ◦C for 34 s using a SYBR Green Master (Shengong Ltd, Shanghai, China). The relative gene expression levels were determined using the comparative ∆∆*Ct* method [71]. The expression level of the *C. songorica GADPH* gene was used as an internal control. Two-way analysis of variance and Duncan's multiple range test (DMRT) were used for multiple mean comparisons. SPSS (IBM Corp. 2013, IBM SPSS Statistics for Windows, Version 21.0, Armonk, NY, USA) was used to determine the significant differences between means (*p* < 0.005).
