Intein-mediated protein assembly in transgenic wheat: Production of active barnase and acetolactate synthase from split genes

Abstract

Engineering traits by the assembly of non-functional gene products is a promising tool for modern plant biotechnology. In this article, we describe the establishment of male sterility and herbicide resistance in wheat (Triticum aestivum) by complementing inactive precursor protein fragments through a split intein system. N- and C-terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from the Synechocystis sp. gene DnaB and delivered into the wheat genome via biolistic particle bombardment. Both barnase fragments were expressed under the control of a tapetum-specific promoter. High efficiency of the split barnase system was achieved by introducing GGGGS linkers between the fusion domains of the assembled protein. Depending on the vector version that was transformed, up to 51% of primary transformed plants produced sterile pollen. In the F1 progeny, the male-sterile phenotype segregated with both barnase gene fragments. Expression of the cytotoxic barnase in the tapetum did not apparently affect the vegetative phenotype and remained stable under increased temperatures. In addition, the reconstitution of sulphonylurea resistance was achieved by DnaE intein-mediated assembly of a mutated acetolactate synthase (ALS) protein from rice. The impacts of the technical advances revealed in this study on the concepts for trait control, transgene containment and hybrid breeding are discussed. © 2009 Blackwell Publishing Ltd.