High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants

Abstract

In this study, we report the obtaining of carrot plants expressing human interferon alpha-2b via Agrobacterium-mediated transformation using two vector constructs containing the sequence coding for interferon gene fused with Nicotiana plumbagenifolia calreticulin apoplast targeting signal driven by 35S CaMV promoter and root-specific Mll promoter. The human interferon alpha-2b gene was correctly translated in carrot plants according to Western blot analysis. The recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus replication in established piglet testicular cells. The results demonstrated the higher activity of interferon accumulated in carrot plants for young leaves (up to 50.7 × 10(3) IU/g FW) compared to the mature ones probably due to the degradation-susceptible nature of this protein. The taproot-expressing system could have also provided the sufficient protein amounts (up to 16.5 × 10(3) IU/g FW) and could possibly be used for generating interferon alpha-2b protein in planta for preventing and curing infectious diseases.

Antiviral activity of extracts of transgenic chicory and lettuce plants with the human interferon α2b gene

“…Such a result might be expected, since there is the ifn α2b gene under the control of root specific promoter Mll in vector pCB161 used for transformation [32]. However, previously, using the same vector, it was revealed that the active α2b inter feron was synthesized not only in carrot roots, but also in the carrot leaves [12]. Perhaps, the effect of the Mll promoter is species specific.…”

“…Genetic transformation using bacteria of the Agro bacterium genus is often applied to obtain transgenic plants, including those with IFN genes [12,29], since these bacteria have a natural ability to transfer their own DNA into plant cells (in nature, this is the forma tion of "crown galls" when infected by A. tumefaciens or "hairy" roots when infected by A. rhizogenes). This genetic transformation method is quite simple, requires no special equipment, and can be used for the transformation of different plant species [30].…”

“…Lettuce and chicory transgenic plants were obtained through the transformation of leaf explants using the bacteria A. rhizogenes (vector pCB161 [12]) and A. tumefaciens (vector pCB124 [22]). The pCB161 and pCB124 vectors had the ifn α2b target gene under the control of sugar beet root specific promoter Mll or the 35S promoter of cauliflower mosaic virus, respec tively, as well as the nptII selective gene (neomycin phosphotransferase II) under the control of the nopa lin synthase promoter and calreticulin signal [23] before the coding part of the ifn α2b gene.…”

“…The creation of recombinant technologies led to attempts to transfer human IFN genes into plant cells not only as factors of antiviral plant protection, but also as an alternative to the synthesis of this protein in bacterial systems. So, for this purpose, tobacco [8], potato [9], lettuce [10], rice [11], carrot [12], and duckweed [13] plants were used; at that, the recombi nant IFNs synthesized in plants possessed biological activity [14]. Studies have shown that plant cells are quite efficient bioreactors for the production of not only different IFN subtypes, but also of other recom binant proteins, including viral and bacterial proteins, some hormones, etc.…”

Plant biotechnology in support of the Millennium Goals

“…These are the subject of the article by Zhang et al (2011) which focuses on the example of tomato, while Sato et al (2011) address the problem of chilling tolerance in rice. The problem of expressing pharmacologically active peptides in plants is addressed in the study by Luchakivskaya et al (2011) and Soria-Guerra et al (2011). Finally, Subramanyam et al (2011) and Basnayake et al (2011) present progress towards the transformation of banana and diverse sugarcane cultivars, respectively.…”