Role of RNAi technology in solanaceous vegetables
Authors: Hanuman Ram and G. S. Jat
Indian Agricultural Research Institute, New Delhi-110 012


Gene silencing is a eukaryotic genome defense system against viruses and mobile DNA elements that works by processing double-stranded RNA (dsRNA) into short interfering RNA (siRNA). The discovery of RNA interference (RNAi) added a new dimension in the regulation of gene expression. Introduction of a piece of double stranded RNA (dsRNA) into the cytosol initiates the phenomenon of RNAi, in turn activating a pathway culminating in the degradation of the targeted gene transcript (Agrawal et al., 2003; Kuznetsov, 2003; Arenz and Schepers, 2003). RNA interference (RNAi) refers to a multi-step process, including the introduction of double-stranded RNA (dsRNA) into a cell, cleavage of dsRNA into short interfering RNA (siRNA) 21– 26 nt in size, formation of RNA-induced silencing complex (RISC), degradation of complementary mRNA, and suppression of target gene expression. RNAi technology has become a powerful tool for the study of the functions of individual genes in a range of organisms.

To enhance coloration in tomato fruit, many attempts have been made (Davidovich-Rikanati et al., 2007) using genetic engineering, for example, to transfer genes encoding carotenoid biosynthesis enzymes (Fraser et al., 2002) or related transcription factors (Bovy et al., 2002) to increase carotenoid content. Abscisic acid (ABA) plays important roles during tomato fruit ripening. To study the regulation of carotenoid biosynthesis by ABA, the SlNCED1 gene encoding 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the ABA biosynthesis, was suppressed in tomato plants by transformation with an RNA interference (RNAi) construct driven by a fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20–50% of that in control fruit. This significant reduction in NCED activity led to the carbon that normally channels to free ABA as well as the ABA metabolite accumulation during ripening to be partially blocked. Therefore, this ‘backlogged’ carbon transformed into the carotenoid pathway in the RNAi lines resulted in increased assimilation and accumulation of upstream compounds in the pathway, chiefly lycopene and b-carotene. Fruit of all RNAi lines displayed deep red coloration compared with the pink colour of control fruit.

Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Patatins encoded by a multi-gene family are one of the major storage glycoproteins and represent up to 40% of the total soluble protein in potato tubers. Patatins are approximately 40–45 kDa in size and are the major storage protein of potato tubers (Bauw et al, 2006). The CaMV 35S promoter-driven patatin hpRNAi vector was transformed into the potato cultivar Desiree by Agrobacterium-mediated transformation. Ten transgenic potato lines bearing patatin hpRNA were generated. Dependent upon the patatin hpRNAi line, patatins decreased by approximately 99% at both the protein and mRNA levels. More importantly, patatin-knockdown potato tubers appear to be an ideal host for the production of human therapeutic glycoproteins, because they eventually allow fast, easy purification of recombinant proteins, with less contamination from potato glycoprotein patatins.


References:

Agrawal, N., Dasaradhi, P. V. N., Mohommed, A., Malhotra P., Bhatnagar, R. K. and Mukherjee, S. K. (2003). RNA Interference: Biology, Mechanism, and Applications. Microbiology and Molecular Biology Reviews, 67: 657-685.

Arenz, C. and Schepers, U. (2003). RNA interference: from an ancient mechanism to a state of the art therapeutic application? Naturwissenschaften, 90: 345-359

Bauw, G., Nielsen, H. V., Emmersen, J., Nielsen, K. L., Jorgensen, M., Welinder, K. G. (2006). Patatins, Kunitz protease inhibitors and other major proteins in tuber of potato cv. Kuras. FEBS J, 273: 3569-3584.

Bovy, A., Vosa, R. and Kempera, M. (2002). High-flavonol tomatoes resulting from heterologous expression of the maize transcription factor genes LC and CI. The Plant Cell, 14: 2509-2526.

Davidovich-Rikanati, R., Sitrit, Y. and Tadmor, Y. (2007). Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway. Nature Biotechnology, 25: 899-901.

Fraser, P. D., Romer, S., Shipton, C. A., Mills, P. B., Kiano, J. W., Misawa, N., Drake, R. G., Schuch, W. and Bramley, P. M. (2002). Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruitspecific manner. Proceedings of the National Academy of Sciences,USA, 99: 1092-1097.

Kuznetsov, V. V., (2003). RNA Interference. An Approach to Produce Knockout Organism and Cell Lines. Biochemistry (Moscow), 68: 1301-1317.



About Author / Additional Info:
Ph D Scholar, Division of Vegetable Science, IARI, New Delhi - 110012