2.6 Enzyme assay for CsGES
Enzyme activity assays were carried out in 1-mL reaction buffer (pH 7.2, 0.1 M PBS, 10 mM MgCl2, 1 mM MnCl2, 100 mM KCI, and 1 mM DTT, 10% glycerol (v/v)) containing 50–100 µg of crude recombinant protein and 5 µg of FPP and GPP substrate in a 20-mL glass tube (Martin et al., 2010a). The reactions were incubated at 30°C for 1 h and then at 42°C for 15 min (Zhou et al., 2017), and the volatiles were collected by SPME. At least three biological replicates (different preparations) were carried out. The reaction products were identified using GC-MS per the method described above. Enzyme activity products, geraniol, (Z)-β-ocimene, and (E)-β-ocimene were identified using comparison standards.
2.7 Gene suppression of CsGES inC. sinensis using AsODNs
Functional assays of CsGES(CsTPS1 , CsTPS1-AS , and CsTPS1/1 -AS ) in tea plants were carried out by suppressing the expression of CsGES inC. sinensis following a previously described method (Zhao et al., 2020b). Candidate sequences (Table S1) of the antisense oligonucleotide (AsODN) with complementarity to the segment of target gens (CsTPS1 , CsTPS1-AS , and CsTPS1/1 -AS ) were selected using Soligo software (http://sfold.wadsworth.org/cgi-bin/index.pl), respectively. AsODNs were synthesized by TSINGKE Biological Technology Co., Ltd. (Anhui, China). The target gene in the tea leaves was silenced using AsODN following a previously described method (Zhao et al., 2020a; Zhao et al., 2020b). Briefly, 1 mL of 40 µM AsODN- CsTPS1/1 -AS solution (to suppress bothCsTPS1 and CsTPS1 -AS ) and AsODN-CsTPS1solution (to suppress CsTPS1 ), or AsODN-CsTPS1 -ASsolution (to suppress CsTPS1 -AS ) was injected into whole tea leaves. The sense oligonucleotides (sODN) were injected into tea leaves as a control treatment. At least six experimental replicates were conducted for each treatment. After treatment, the tea leaves were harvested, immediately frozen in liquid nitrogen, and then kept at –80°C before analysis. The content of geraniol in the treated tea was detected as described above.