Modified upstream open reading frames for modulating npq relaxation
AssigneeThe Board of Trustees of the University of Illinois
InventorsBenjamin HAAS, Steven James BURGESS
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Abstract
The present disclosure relates to genetically modified plants including one or more edited open reading frames (uORFs) in endogenous nucleotide sequences encoding photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP), or violaxanthin de-epoxidase (VDE). The present disclosure further relates to methods of producing the genetically modified plants, as well as to isolated constructs and expression cassettes for use in producing the genetically modified plants. In addition, the present disclosure relates to transient screening methods to study gene expression.
Claims25
A genetically modified plant comprising one or more edited endogenous nucleotide sequences encoding a photosystem II subunit S (PsbS) polypeptide, a zeaxanthin epoxidase (ZEP) polypeptide, or a violaxanthin de-epoxidase (VDE) polypeptide, wherein the one or more edited nucleotide sequences are edited at one or more upstream open reading frames (uORFs) in a transcript leader sequence (TLS).
An expression vector or isolated DNA molecule comprising one or more gene editing components that target one or more uORF sequences of an endogenous PsbS gene, wherein the uORF sequence is edited by the one or more gene editing components to remove uORF translational repression;
- comprising one or more gene editing components that target one or more uORF sequences of an endogenous VDE gene, wherein the uORF sequence is edited by the one or more gene editing components to remove uORF translational repression;
- and/or comprising one or more gene editing component that target one or more uORF sequences of an endogenous ZEP gene, wherein the uORF sequence is edited by the one or more gene editing components to remove uORF translational repression, optionally
- wherein the one or more gene editing components are selected from the group consisting of a ribonucleoprotein complex that targets the one or more uORF sequences;
- a vector comprising a TALEN protein encoding sequence, wherein the TALEN protein targets the one or more uORF sequences;
- a vector comprising a ZFN protein encoding sequence, wherein the ZFN protein targets the one or more uORF sequences;
- an oligonucleotide donor (ODN), wherein the ODN targets the one or more uORF sequences;
- and a vector comprising a CRISPR/Cas enzyme encoding sequence and a targeting sequence, wherein the targeting sequence targets the one or more uORF sequences.
A transient screening method to measure gene expression, comprising:
- a) providing one or more dual-fluorescence expression vectors comprising a test insert to produce one or more test vectors and at least one dual-fluorescence expression vector comprising a control insert to produce at least one control vector, wherein the dual-fluorescence expression vectors comprise a first fluorescent reporter and a second fluorescent reporter;
- b) introducing the one or more test vectors into a first leaf tissue and introducing the at least one control vector into a second leaf tissue, wherein the first leaf tissue and the second leaf tissue are of the same plant variety;
- d) excising leaf portions from the first leaf tissue and from the second leaf tissue 3 days post injection;
- e) placing the leaf portions abaxially in a multiwell plate;
- and
- f) measuring fluorescence on a monochromator-based plate reader in two separate channels, wherein a first channel is used to obtain a signal from the first fluorescent reporter and a second channel is used to obtain a signal from the second fluorescent reporter.
Dependencies are inferred from the claim text.