Functional characterization of a stilbene synthase gene using a transient expression system in planta.
The expression and functionality of a resveratrol synthase (RS) gene from peanut (Arachis hypogaea) was studied using an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana leaves. Functional analysis of RS was demonstrated by tracking its expression during 96 h. To measure the transcripts levels of RS transgene, real-time qRT-PCR was used and revealed that the highest level of transcripts was at 48 h post-transfection. Western blot analyses showed that RS protein was accumulated to the highest levels at 72 h post-transfection. Finally, HPLC and mass spectrometry analyses revealed the production of trans-piceid (resveratrol glucoside) as the major stilbenoid compound confirming the functional activity of the RS enzyme in planta. No activity of RS transgene was detected in negative controls. This strategy showed advantages over conventional systems because it does not require establishment of cell cultures, feeding with appropriate substrates or generation of stable transgenic plants. This transient system proved to be a rapid and direct approach to perform functional analysis of stilbene synthases, such as resveratrol synthase. Furthermore, this approach can be useful to study the metabolic effects of over-expressing or silencing specific genes within a short period of time.
Condori
Jose
J
Arkansas Biosciences Institute, Arkansas State University, AR 72467, USA.
Medrano
Giuliana
G
Sivakumar
Ganapathy
G
Nair
Vipin
V
Cramer
Carole
C
Medina-Bolivar
Fabricio
F
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D010367:Arachis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005960:Glucosides / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D018749:RNA, Plant / Q000378:metabolism; D013267:Stilbenes / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D005960:Glucosides; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D013267:Stilbenes; D000217:Acyltransferases; C022674:stilbene synthase; C058229:polydatin