Nanodrop2000 ultra microscope photometer

Total RNA was extracted from cells at the exponential growth phase using an Easy-Pure Plant RNA kit (Transgen, ER301, Beijing, China). RNA quality was assessed using a NanoDrop2000 Ultra Microscope Photometer (Thermo, Massachusetts, USA). First-strand cDNA was prepared using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, AE311, Beijing, China) strictly following the manufacturer’s instructions. Gene expression patterns were analysed by qRT-PCR with SYBR Green Master (Roche, 4913914001, Basel, Switzerland) using an ABI QuantStudio 6 Flex Detection Device (Thermo, Massachusetts, USA) as recommended by the manual. Three biological replicates and three technical replicates were included. The CrePP2A gene (PP2A, Cre11.g477300) served as an internal control, and relative expression levels of target genes were calculated via the 2^−ΔΔCt method. Primers used for qRT-PCR analysis are listed in Supplementary Table S1.

The total RNA was isolated during the phase of exponential growth using the SteadyPure Quick RNA Extraction Kit (Accurate Biology, AG21023, Changsha, China). Subsequently, an assessment of RNA integrity was conducted employing the NanoDrop2000 Ultra Microscope Photometer (Thermo, Waltham, MA, USA). The synthesis of first-strand cDNA was executed utilizing the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, R211, Nanjing, China), strictly adhering to the instructions by the manufacturer. To analyze patterns of gene expression, quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the SYBR Green Master kit (Roche, 4913914001, Basel, Switzerland) in accordance with guidelines by the manual on an ABI QuantStudio 6 Flex Detection Device (Thermo, Waltham, MA, USA). Each experimental condition comprised three distinct biological repetitions and an additional three technical repetitions. The α-tubulin gene (TUA1, CHLRE_03g190950v5) was employed as an internal reference for normalization, while the relative expression levels of the target genes were deduced using the 2 −∆∆Ct method.

Plasmid DNA was extracted using a Plasmid MiniPrep Kit (Transgen, EM101), and genomic DNA was extracted with a Genomic DNA Kit (Transgen, EE101) following the manufacturer’s instructions. DNA quality was assessed using a NanoDrop2000 Ultra Microscope Photometer (Thermo, United States). PCR amplification was performed with Taq DNA Polymerase (Transgen, AP101) based on a standard three-step program (94°C 3 min; 30 cycles of 94°C 5 s, 60°C 30 s, 72°C 10 s/kb, 72°C 5 min).