Methods and materials for high throughput testing of transgene combinations

摘要

High throughput methods are described for identifying combinations of transgenes that can be used to improve a phenotypic feature in an organism. Large populations of organisms (e.g., plants) containing different combinations of transgenes as well as different promoter-coding sequence combinations can be assessed using the methods.

主权项

1. A method for identifying a combination of genetic elements responsible for a desirable phenotype of a plant, wherein said method comprises:
(a) selecting at least four target transgenes, wherein each target transgene of said at least four target transgenes comprises a regulatory region comprising an upstream activating sequence operably linked to a nucleotide sequence to be expressed, whereby said upstream activating sequence of each of said at least four target transgenes is the same or different; (b) selecting at least two activator transgenes, wherein each activator transgene comprises a plant promoter operably linked to a sequence encoding a chimeric polypeptide comprising a DNA binding domain fused to a transcription activation domain, wherein each DNA binding domain of said at least two chimeric polypeptides binds to the upstream activating sequence comprised within said regulatory region of at least one of said at least four target transgenes; (c) obtaining a first parental plant and a second parental plant, wherein each of said at least four target transgenes and said at least two activator transgenes (i) is individually present in either said first parental plant or said second parental plant, (ii) is unlinked from each of the other of said at least four target transgenes and said at least two activator transgenes that are present within the same said first parental plant or said second parental plant, and (iii) is in a hemizygous state within said first parental plant or said second parental plant; (d) sexually crossing said first parental plant and said second parental plant to produce a population of progeny plants, whereby said population of progeny plants has different combinations of said at least four target transgenes and said at least two activator transgenes; (e) comparing different plants of said population of progeny plants to one another and selecting at least one progeny plant from said population as having said desirable phenotype to obtain a selected progeny plant; and (f) determining which target transgenes and activator transgenes are present within said selected progeny plant, thereby identifying a combination of genetic elements responsible for said desirable phenotype of a plant.