High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

We developed transgenic Nicotiana plumbaginifolia hairy roots with sucrose-inducible minimal promoter (Spo(min))-beta-glucuronidase (GUS) gene-fused constructs with signal sequences for sorting to cytosol, apoplast, and ER, and we analyzed the GUS activities of hairy roots after sucrose treatment. Induced GUS activities by Spo(min) were about 10 times higher than those by the CaMV 35S promoter. GUS activities in hairy roots induced with a Spo(min)-UTR-GUS construct after 6% sucrose treatment were higher than those with either Spo(min)-UTR-GUS construct after 10% sucrose treatment, Spo(min)-apoplast or ER-GUS constructs after sucrose treatment. High GUS activities in hairy roots with Spo(min) constructs were induced during 20 wk by culturing hairy roots in LS liquid medium containing 6% sucrose in 300 ml conical flasks. Hairy roots in Linsmaier and Skoog (LS) liquid medium showed the increase of fresh weight (FW) for 16 wk. Total yield (mu g) of GUS by the hairy roots with Spo(min)-UTR-GUS construct using 5 l culture container was calculated to about 6742 mu g after 8-wk culture. Productive efficiencies of GUS were over 3.0% of total soluble protein after 1-2 wk 6% sucrose treatment. Productive efficiencies of GUS in transgenic N. plumbaginifolia hairy roots were higher than that in transgenic N. plumbaginifolia leaves, stems, and roots. And growth speeds of transgenic N. plumbaginifolia hairy roots were faster than transgenic N. plumbaginifolia plants. These results showed that N. plumbaginifolia hairy roots with Spo(min)-heterologous construct systems and sucrose treatment would be useful tools to develop protein production system.

Automated summary

The GUS activities of transgenic Nicotiana plumbaginifolia hairy roots induced by sucrose were analyzed. GUS activities induced by Spo(min) were about 10 times higher than those induced by the CaMV 35S promoter. The highest GUS activities were observed in hairy roots with Spo(min)-UTR-GUS construct after 6% sucrose treatment. Hairy roots cultured in LS medium containing 6% sucrose for 20 weeks showed high GUS activities.