|Beck Von Bodman, Susanne - UNIV OF ILLINOIS|
|Farrand, Stephen - UNIV OF ILLINOIS|
Submitted to: Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 24, 1995
Publication Date: N/A
Interpretive Summary: Many important plant characteristics are the product of coordinate expression of several genes. Current genetic engineering techniques for introducing genes into plant chromosomes work well for single genes, but not nearly so well when it is necessary to introduce several genes at one time. In this paper, we report the development of a strategy to introduce multiple genes into plant chromosomes at one time in a controlled manner. To demonstrate this strategy, two genes from a soilborne bacterium were combined with a gene from a common plant virus. The three genes were then inserted into the chromosomes of tobacco cells. Even though each of the genes had a different function, each was shown to be expressed and produce a functional protein product. Once the genes for complex agronomic traits such as height, yield or drought tolerance are identified, these could be linked together and introduced into the chromosomes of target plants at one time using this genetic engineering strategy.
Technical Abstract: We engineered an expression unit composed of three eukaryotic genes driven by a single plant-active promoter and demonstrated functional expression in plants. The individual genes were linked through spacer sequences encoding specific heptapeptide cleavage recognition sites for NIa protease of tobacco vein mottling virus (TVMV). The NIa gene itself was included as the second gene of the multi-gene unit. The first and third genes, obtained from the TR region of pTi15955, encoded enzymatic functions associated with the mannityl opine biosynthetic pathway. The mannityl opine conjugase gene (mas2) was the first unit of the construct and provided the native plant-active promoter and 5' untranslated regulatory sequence. The third gene (masl), encoding the mannityl opine reductase, furnished the native 3' untranslated region. Cis-processing of the polyprotein by the NIa protease domain was demonstrated in vitro using rabbit reticulocyte lysate and wheat germ cell-free translation systems. Tobacco plants transformed with the multi-gene unit produced detectable levels of mannopine, mannopinic acid, and their biosynthetic intermediates, deoxyfructosyl-glutamate and deoxyfructosyl-glutamine. This indicates that the polygene construct results in a set of functional enzymatic activities that constitute a complete metabolic pathway.