Ta blunt zero cloning kit

Total RNA was isolated from the young leaves of 4-week-old seedlings of 4036G and 4036Y using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The cDNAs were subsequently used for Illumina library construction and sequencing with an Illumina Hi-Seq 4000 sequencer by Biomarker Technologies Co., Ltd. The transcripts of chloroplast genes were identified by referencing the cabbage chloroplast genome [37 (link)]. RNA editing site analysis was performed as previously reported [13 (link)].
Specific markers for RNA editing sites were designed based on the cabbage chloroplast reference genome (Supplementary Table S1). RNA editing-specific markers were then used to amplify the cDNAs from the young leaves of 4-week-old seedlings of 4036G and 4036Y. PCR products were then cloned into a T-vector using the TA/Blunt-Zero Cloning Kit (Vazyme, Nanjing, China). Fifty positive clones were selected from each sample for sequencing and RNA editing efficiency analysis.

To amplify the Class 1 integron, forward (5′-ATCATCGTCGTAGAGACGTCGG-3′) and reverse primers (5′-GTCAAGGTTCTGGACCAGTTGC-3′) were used as described previously by Rosser et al. [36 (link)]. The primer pair for 16SrRNA (forward 5’-CGGTGAATACGTTCYCGG-3’ and reverse primer 5’-GGTACCTTGTTACGACTT-3) was used as described by Gaze et al. [37 (link)]. The PCR reactions were conducted in a reaction volume of 50 µL, and the amplified fragments were separated using gel electrophoresis for visualization. The different bands were cut from the gel and extracted using a gel extraction kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The DNA fragments were cloned into a blunt cloning vector using a TA/Blunt-Zero Cloning Kit (Vazyme, Nanjing, China), according to the manufacturer’s instructions, and accordingly transferred into E. coli DH_5α competent cells. These cells were then cultured overnight on LA medium containing ampicillin at a concentration of 100 µg/mL. The positive colonies were selected after overnight incubation, and the plasmids were extracted from the culture using a plasmid extraction kit (Vazyme, Nanjing, China). These recombinant plasmids were Sanger sequenced via a universal sequencing primer at AuGct Biotechnology Co. Ltd. (Beijing, China).

The Tn5 transposon mutant library was constructed as previously described (He et al., 2021 (link)). Briefly, a fragment of the kanamycin resistance gene (Kan) was amplified from plasmid pBBR1MCS-2 by PCR using primers MEKANA-F/R containing Tn5 mosaic end (ME) sequences. The PCR product was ligated into the pCE2 vector using a TA/Blunt-Zero Cloning Kit (Vazyme Biotech, Nanjing, China). After confirmation by sequencing, the Kan-ME fragment was transformed into B. arboris using an EZ-Tn5™ Transposase Kit (Lucigen, Middleton, WI, United States). Putative mutants were selected using 500 μg/ml kanamycin.