Rnapure plant kit

Total RNA for real-time PCR was extracted from flowers and other vegetative tissues of R. delavayi using RNA pure Plant Kit (CWBIO, China). Synthesis of first strand cDNA from 1 μg total RNA was performed using oligo (dT) and M-MLV reverse transcriptase (Takara, Japan). Then, real-time detection was conducted by using BioRad CFX96 Real-Time PCR System (BIO-RAD, Hercules, CA, USA) and TransStart^® Green qPCR SuperMix (TRANSGEN, China) with primers designed based on sequence information from transcriptome analysis performed previously (Supplementary Table 1). Amplification of RdActin under the identical conditions was carried out to normalize the levels of Rd3GTs as follows: 60 s at 95°C, followed by 40 cycles of 5 s at 95°C and 60 s at 60°C. Each sample was carried out in three biological replicates, and the relative expression levels of target genes were calculated by formula 2^−ΔΔCt. Meanwhile, the specific amplification of each gene was confirmed by melting curve analysis and agarose gel electrophoresis.

Based on the transcriptome data of different tissues of O. japonica measured before, a total of five sequences of the CHI gene from O. japonica were identified through blastn alignment with reference genes of proximal species and Arabidopsis CHI protein sequence. Then comparative analysis was conducted by using the following database: national center for biotechnology information (NCBI) and the results indicated that the sequence of unigene (OjCHI) (Unigene35425_All) showed the highest similarity to CHI genes from other plants. Therefore, the specific primers (OjCHIF1 and OjCHIR1) were designed from sequence information of OjCHI gene. To isolate the total RNA, flowers of O. japonica at stage 4 were ground into powder and extracted by RNA pure Plant Kit (CWBIO, China). Then the cDNA was synthesized from 1.0 μg total RNA using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, China). Subsequently, for obtaining the full-length sequence of OjCHI, its complete open reading frame (ORF) generated by RT-PCR was cloned into pMD18-T vector and sequenced by Sangon Biotech (Shanghai, China). All the primers used in this work are listed in Supplementary Table S1.

Real-time PCR was performed to detect the expression pattern of Whirly genes according to a previous study (Liu et al., 2022 (link)). The total RNA was isolated using RNApure Plant Kit (CWBIO), and the first-strand cDNA was synthesized from 1 μg of total RNA using Prescript III RT ProMix (CISTRO). The real-time PCR was performed using gene-specific primers (Supplementary Table S1) with 2× Ultra SYBR Green qPCR Mix (CISTRO), and the TaActin gene was selected as a reference control. The real-time PCR cycling parameters were 95°C for 30 s, followed by 45 cycles at 95°C for 5 s and 60°C for 30 s, with a melting curve analysis. All reactions were performed on three technical and biological replicates. The relative expression levels of target genes were calculated using the 2^−△△CT method (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted from plant tissue samples using RNApure plant kit (Cwbio, Beijing, China) and then used to synthesize cDNA using the PrimeScript^TMRT reagent kit (TaKaRa, Beijing, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the TB Green Premix Ex Tap^TM Ⅱ kit (TaKaRa, Beijing, China), targeting AChE1, AChE2, and AChT genes. Actin was also included as an internal amplification control. The reaction was performed using a CFX96 system (Bio-Rad, Hercules, CA, USA) under the conditions described by the reagent’s instructions. The primer sequences for target genes are outlined in Table 4. All mRNA levels were normalized by the Actin level [49 (link)]. Gene expression was relatively quantitated by the 2^−ΔΔCT method.

Total RNA was extracted from youngest fully expanded trifoliate leaves at 20 DAE using RNApure Plant Kit (CWBIO). The RNA was reverse transcribed to cDNA with M-MLV reverse transcriptase kit (Takara). Quantitative RT-PCR (qRT-PCR) was performed on a Roche LightCycler 480 system (Roche) using 2× Ultra SYBR Green qPCR Mix (CISTRO). Tubulin was used as an internal control. The primers are listed in Datasets S1.

Total RNA was extracted by RNApure Plant Kit (CWBIO) according to the manufacturer’s protocol. cDNA was reverse-transcribed using PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa). SYBR Premix Ex Taq II (TaKaRa) was used for qPCR on a ABI StepOne Plus real-time system (Life Technologies). qRT-PCR was performed in triplicate and data were collected and analyzed with ABI STEPONETM software version 2.1 [121 (link)]. Various gene specific signal was normalized relative to ACTIN2 gene (At3G18780) expression. The primer sequences were listed as follows:

The tender roots at the bud stage under two treatments for 12 h were used as samples for RNA-Seq analysis, and each treatment had three biological replicates. These samples were rapidly frozen in liquid nitrogen. And then RNA was extracted by RNApure Plant Kit (CW0559, CWBIO, Beijing, China) following the manufacturer’s instructions. The RNA was measured in 1% agarose gel electrophoresis and NanoDrop instrumentation (OneC, Thermo, Waltham, MA, USA) using the methods of Zhang [20 (link)], which determined whether the quality of the RNA was qualified. The qualified RNA of these samples underwent RNA-Seq by Bio-maker (Beijing, China), while those with no reference genome were analyzed by Biocloud (https://international.biocloud.net/, accessed on 1 January 2022). UniGene was analyzed by Trinity software [32 (link)], while the differentially expressed genes (DEGs) were assessed by DESeq2 software [33 (link)]. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used for gene and pathway enrichment analyses [34 (link)].