Escherichia coli strain top10

The GenBank accession numbers of Ochrobactrum sp. M231 MPH and OPHC2 are ACC63894 and CAE53631, respectively. Escherichia coli strain Top10 and the plasmid pGEM-T were purchased from Promega Corp. (Madison, WI, USA). E. coli strain BL21(DE3) and the expression plasmid pET-30a(+) were purchased from Novagen (Darmstadt, Germany). P. pastoris strain GS115 and the vector pPIC9 were purchased from Invitrogen (Carlsbad, CA).
E. coli cells were cultured aerobically at 37°C in Luria-Bertani medium. Minimal dextrose (MD) medium, buffered complex glycerol medium (BMGY), yeast extract peptone dextrose medium (YPD), and buffered complex menthol medium (BMMY) were prepared according to the manufacturer’s instructions (Invitrogen).

Total RNA was extracted from Yanshan-1 leaves using E.Z.N.A. total RNA Kit I (OMEGA). The first cDNA was amplified by the SMART MMLV Reverse Transcriptase (TaKaRa). Specific primers were designed according to the DEG VIT_14s0066g01090 MYB gene with the forward primer: 5′-atggataaaaaaccctgcaattctcag-3′ and the reverse primer: 5′-ttaatctccattaagtagctgcatag-3′. The VyMYB24 gene was cloned using PrimeSTAR^® HS DNA Polymerase (Takara, Beijing, China). PCR was performed in 30 cycles of 98°C for 10 s and 60°C for 10 s, followed by 72°C for 10 min. The obtained product was cloned into pGEM-T Easy Vector System (Promega, USA) at 4°C overnight, transformed into Escherichia coli strain TOP10 (Tiangen, Beijing, China), screened on the LB plate, and then sequenced by Sangon Biotech Co., Ltd (Shanghai, China).