Icycler iq5 rt qpcr system

Two pairs of RT-qPCR primers (Table S1) was synthesized to amplify a 136 bp and a 221 bp fragment of the CHS ORFand the Actin2 gene (used as internal reference), respectively, from the cDNA samples of the three replicates of each time point of the inducing treatments^{47 (link)}. The amplification reaction was conducted by using SsoFastEvaGreenSupermix (Bio-Rad, USA) in Bio-Rad iCycler iQ5 RT-qPCR System. The 2^−ΔΔCT method of the CFX Manger™ software version 2.0 (Bio-Rad, USA) was used to normalize the expression differentiation between the internal reference and the CHS gene^{48 (link)}. The relation expression levels under the inducing conditions were calculated by their expression level to those under the non-inducing control.
As described by Liu et al.^{49 (link)}, six arbitrary degenerate primers (AD) and three nested primers (Table S1) complementary to the coding sequences of the CHS genes were synthesized, and used to amplify the promoter sequences of the CHS genes from the genomic DNA samples by TAIL-PCR. The products of the second and third rounds of amplification were separated by 1.2% argarose gel electrophoresis, purified and sequenced as above. The sequenced results were used for prediction of cis-affecting elements by Plant CARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

A pair of precise primers (5′- GCATGCCAAGGTGCAAAGAA-3′/5′- GCCTATGTCGCTTCTCCCTC-3′) was created to amplify a 172 bp segment of the ArTPS gene. A second set of precise primers (5′-CGGGCATTCACGAGACCAC-3′/5′-AATAGACCCTCCAATCCAGACACT-3′) was created to amplify a 221 bp Actin2 gene fragment that was used as an internal reference [39 (link)]. In the Bio-Rad iCycler iQ5 RT-qPCR System, the amplification reaction was carried out using SsoFast EvaGreen Supermix (Bio-Rad, USA). The two-step RT-qPCR temperature cycle was as follows: 39 cycles of 95 °C for 5 s, followed by 30 s at 56 °C. So that a melting curve could be constructed and utilized to distinguish between specific and non-specific amplicons, the temperature was then raised by 0.5 °C/s to 95 °C. The internal reference and TPS gene expression were normalized using the 2^−ΔΔCT method of the CFX Manger^TM software version 2.0 (Bio-Rad, USA) [40 (link)]. The IBM-SPSS software (http://www-01.ibm.com/software/analytics/spss/ accessed on 15 April 2023) was used to evaluate the statistics.