Citrus derived transcription and translation regulatory elements for tissue specific expression
Inventors
Belknap, William R. • McCue, Kent F. • Thilmony, Roger L. • Stover, Eddie W. • Thomson, James G.
Assignees
US Department of Agriculture USDA
Publication Number
US-10047369-B2
Publication Date
2018-08-14
Expiration Date
2036-12-16
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Abstract
Expression vectors and expression cassettes containing a tissue specific 5′ transcription regulatory element, optionally linked to a translation regulatory element, optionally linked to an intron transcription regulatory element, operably linked to a heterologous polynucleotide encoding a protein or RNA of interest are described. The 5′ transcription regulatory elements control expression in root tissue cells, phloem tissue cells, or fruit and/or abscission zone tissue cells. These sequences are obtained from citrus plants. Methods of use of these expression vectors and expression cassettes are described, as well as genetically altered plants and parts thereof that contain these expression vectors and/or cassettes.
Core Innovation
This invention relates to transcription regulatory elements and translation regulatory elements obtained from citrus plants, which are useful for tissue-specific transcription and translation of heterologous polynucleotides encoding a protein or RNA of interest in dicot plants. Expression vectors and cassettes containing these regulatory elements are described, wherein the 5′ transcription regulatory elements control expression specifically in root tissue cells, phloem tissue cells, or fruit and/or abscission zone tissue cells.
The problem being solved is the need for novel promoters with beneficial expression characteristics, particularly promoters capable of directing strong expression of heterologous genes specifically in root tissue cells, phloem tissue cells, or fruit and/or abscission zone tissue cells in genetically altered plants. Existing promoters often lack this tissue specificity or strong expression in these critical tissues.
The invention provides expression vectors and expression cassettes where the tissue specific 5′ transcription regulatory element from citrus is operably linked to a heterologous polynucleotide of interest, optionally including a translation regulatory element and/or an intron transcription regulatory element. These regulatory elements direct transcription and translation in a tissue specific manner, and methods are described for creating genetically altered plants containing these constructs that express the polynucleotide of interest specifically in the tissues noted.
Claims Coverage
The patent contains one independent claim that defines an expression cassette comprising specific transcription regulatory elements operably linked to a heterologous polynucleotide of interest, along with dependent claims extending features such as inclusion of translation and intron regulatory elements, use in genetically altered plants and cells, and methods of use.
Tissue-specific 5′ transcription regulatory element
An expression cassette comprising a 5′ tissue specific transcription regulatory element comprising nucleotides 1-2484 of SEQ ID NO. 26 operably linked to a heterologous polynucleotide of interest.
Intron transcription regulatory element operably linked in the expression cassette
An intron transcription regulatory element comprising nucleotides 2588-2647 of SEQ ID NO: 26 operably linked 3′ to the 5′ transcription regulatory element and 5′ to the heterologous polynucleotide of interest.
Translation regulatory element operably linked in the expression cassette
A translation regulatory element comprising nucleotides 2485-2541 of SEQ ID NO: 26 operably linked between the 5′ transcription regulatory element and the heterologous polynucleotide of interest.
Combined 5′ transcription regulatory element, translation regulatory element, and intron transcription regulatory element
An expression cassette containing the sequence comprising nucleotides 1-2647 of SEQ ID NO: 26, operably linked to a heterologous polynucleotide of interest, optionally including a linker sequence.
Genetically altered plant containing the expression cassette
A genetically altered plant, seed, pollen, germplasm, or cell comprising the expression cassette described above.
Method for expressing heterologous polynucleotide using expression vector
A method comprising providing an expression vector with the 5′ transcription regulatory element (nt 1-2484 of SEQ ID NO: 26) operably linked to a heterologous polynucleotide of interest, introducing the vector into plant cells to generate altered cells, and growing genetically altered plants expressing the heterologous polynucleotide, optionally including the translation regulatory element and intron transcription regulatory element as above.
The claims cover expression cassettes containing citrus-derived tissue specific transcription regulatory elements, optionally linked to translation and intron transcription regulatory elements, operably linked to heterologous polynucleotides. They further cover genetically altered plants and cells containing these cassettes, and methods of using these vectors to achieve tissue-specific expression of heterologous genes.
Stated Advantages
Allows strong, tissue-specific expression of heterologous polynucleotides in root, phloem, or fruit and/or abscission zone tissue cells of genetically altered plants.
Provides novel promoters and regulatory elements derived from citrus plants that address the need for improved tissue specificity and expression levels in important plant tissues.
Enables more precise genetic modifications for agriculturally relevant traits by targeting expression to specific tissue types critical for plant growth or fruit production.
Documented Applications
Expression of heterologous proteins or RNAs in root tissues to promote beneficial rhizosphere microbes, increase water or nutrient uptake, enhance nitrogen fixation, support bioremediation, inhibit soil-borne pests, or facilitate allelochemical production.
Modification in fruit and abscission zone tissues to control abscission and reduce pre-harvest drop, enable inducible fruit drop, enhance nutraceutical accumulation, improve fruit cosmetics, increase post-harvest fruit quality, or develop trap crops for pest control.
Phloem-specific expression to suppress phloem pathogens causing critical plant diseases, control flowering timing, modulate dwarfing traits, or enhance photosynthate transport efficiency.
Use of expression cassettes and vectors containing these regulatory elements to produce genetically altered dicot plants and plant parts expressing heterologous genes in a tissue-specific manner.
Transformation of Arabidopsis, tomato, and citrus plants with expression cassettes containing these regulatory elements to demonstrate tissue-specific GUS expression.
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