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Çó·ÒëÒ»¶ÎÎÄ×Ö Assessment of the stability of chlorinated alkaloid production in subsequent subcultures. Ten root tips from hairy roots transformed with pCAMRebH/RebF and pCAMPyrH/RebF/STRvm were subcultured every 3 weeks in Gamborg¡¯s B5 solid medium (half-strength basal salts, full-strength vitamins, 30 g l21 sucrose, 6g l21 agar, pH 5.7). and grown at 26 uC in the dark. Alkaloids were isolated from 21-day-old hairy roots and analysed as described above (Supplementary Figs 11¨C14). Verification of expression of RebH, RebF and PyrH, STRvm enzymes by realtime RT¨CPCR. Real-time RT¨CPCR was used to assess the expression levels of RebH and RebF. Expression levels in hairy roots infected with A. rhizogenes lacking the pCAMRebH/RebF construct were compared with expression levels in hairy roots harbouring pCAMRebH/RebF. Messenger RNA from transformed hairy roots was isolated and purified from contaminant DNA using a Qiagen RNeasy Plant Mini Kit and Rnase-free DnaseI, respectively. The resulting mRNA was then reverse-transcribed to cDNA using a Qiagen QuantiTect Reverse transcription kit and then subjected to PCR with specific primers (see below), a Qiagen SYBR Green PCR kit and a Biorad DNA Engine Opticon 2 system. The threshold cycle (CT) was determined as the cycle with a signal higher than that of the background plus 10 s.d. Catharanthus roseus 40S ribosomal protein S9 (Rps9), encoded by a house-keeping gene, was used to adjust the amount of the totalmRNAin all samples. Real-time RT¨CPCR was performed in triplicate and the data are pictured as the relative expression levels of rebH and rebF mRNA in transgenic hairy roots as well as hairy roots lacking the pCAMBIA plasmid (Supplementary Fig. 23). |
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