Clone and quantification expression analysis of LaTPS7,LaTPS8 and LaCYP71D582
Glandular
trichomes are tissues located in metabolism-specific sites (Tissieret al . 2017). Total RNAs were extracted from glandular trichomes
of flowers and leaves in different developmental stages and reverse
transcribed using oligo d(T) primers following the manufacturer’s
directions
(TSINGKE,
China). After obtaining cDNA, target genes were amplified using cloning
primer and Phanta Max Polymerase (Vazyme, China). To clone full-length
open reading frames (ORFs), we used a PCR program at the following
settings:
95°C
for 5 min; 35 cycles at
95°C
for 2 min; 58°C for 30 s and 72°C for 2 min; and final extension at 72°C
for 5 min. The qRT-PCR was conducted using 2 × T5 Fast qPCR Mix (SYBR
Green II) (TSINGKE, China) on an Mx3000P system (Agilent Stratagene),
according to the manufacturer’s instructions and the following settings:
95°C for 1 min, followed by 40 cycles of 10 s at 95°C, then 5 s at 55°C,
and 15 s at 72°C. Leaf trichomes at the blossoming stage were used as
controls. Normalised expression values
(2-△△CT)
were calculated according to the expression of 18S rRNA used as
reference gene. The primers used in this part of our study are shown in
table S1.