Open AccessA multisite gateway‐based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits
Author(s) -
Estornell Leandro Hueso,
Orzáez Diego,
LópezPeña Lucas,
Pineda Benito,
Antón María Teresa,
Moreno Vicente,
Granell Antonio
Publication year - 2009
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/j.1467-7652.2009.00402.x
Subject(s) - promoter , biology , gene , gene silencing , cloning (programming) , genetics , small hairpin rna , multiple cloning site , reporter gene , gene expression , computational biology , rna interference , expression vector , rna , recombinant dna , computer science , programming language
Summary A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple‐gateway format, a recombination‐based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato‐ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ‐specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical difficulties of current ligase‐dependent cloning techniques in the construction of fruit expression cassettes. The pENFRUIT vector collection is available for the research community in a plasmid repository, facilitating its accessibility.