Plasmid miniprep kit

Transformation and conjugation experiments were performed to acquire purified single plasmids as described previously (Jiang et al., 2010 (link), 2015 (link); Shen et al., 2016 (link); Zheng et al., 2018 (link)). The bla_KPC−2-carrying plasmids were extracted with a Plasmid Miniprep Kit (Transgen Biotech, China) from the transconjugants. Multilocus PCR was performed to evaluate the relationships of the 6 untypeable plasmids discovered in this study. In addition to the repA and bla_KPC genes, primers (Table 1) were designed to target the backbone genes taxA, virB5, virB11, and repB and the mobile elements Tn1721-TnpA and Tn1721-TnpR. Amplicons were analyzed by electrophoresis and sequencing. Meanwhile, plasmids were digested with EcoRI and subjected to restriction fragment length polymorphism (RFLP) by electrophoresis on 1% agarose (Sangon, China) gels in 1 × TAE buffer as reported before (Ho et al., 2012 (link)).

Total RNA was extracted using a Total RNA Kit (TIANGEN, China), and purity and integrity of the RNA were measured. Total RNA from the bulb of C. yanhusuo was reverse transcribed into cDNA using Superscript™ IV (Invitrogen, USA). ​According to the transcriptome data obtained in the laboratory, five CyCYP719As subfamily genes were screened, and specific primers were designed based on their open reading frames. Spe I restriction site was selected to design primers, as shown in Additional file 1: Table S1. PrimeSTAR^® HS DNA Polymerase (Takara, Japan) was utilized for amplification, and the target fragment was obtained by purifying the PCR product using the Quick Gel Extraction Kit (TransGen, China). The target fragment was attached to the eukaryotic expression vector pESC-His by seamless stitching [40 (link)]. Plasmid was transformed into E. coli Trans1 T1 and the positive clone was confirmed through PCR detection and DNA sequencing. Recombinant plasmid pESC-His-CyCYP719As was obtained from the strains using the Plasmid Miniprep Kit (TransGen, China).