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Recombinant DNA constructs and methods for modulating expression of a target gene

A technology of constructs and target genes, applied in the direction of recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of no reports

Active Publication Date: 2011-08-10
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No similar natural antisense transcript microRNA ("nat-miRNA") pathway has been reported (Lu et al. (2008) Proc. Natl. Acad. Sci. USA, 105:4951-4956)

Method used

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  • Recombinant DNA constructs and methods for modulating expression of a target gene
  • Recombinant DNA constructs and methods for modulating expression of a target gene
  • Recombinant DNA constructs and methods for modulating expression of a target gene

Examples

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Embodiment 1

[0162] This example illustrates the preparation and use of "cleavage blocker" recombinant DNA constructs containing DNA processed into RNA containing single-stranded RNA, the single-stranded RNA and the transcript of at least one target gene A hybrid fragment of RNA that binds to form at least partially double-stranded RNA that renders the transcript resistant to cleavage by RNase III ribonucleases in or near the hybrid fragment; wherein the binding of the single-stranded RNA to the transcript ( and eventual formation of hybrid fragments) inhibits double-stranded RNA-mediated repression of target genes. More specifically, this example describes constructs for the production of artificial or genetically engineered miRNAs or cleavage blockers in plants, and which cleavage blockers inhibit miRNA-mediated miRNA-mediated transformation of the Arabidopsis GL1 gene in transformed plant cells. The use of induced inhibition.

[0163] Target gene: The Arabidopsis GLABROUS1 (GL1) gene...

Embodiment 2

[0176] This example illustrates an alternative "cleavage blocker" recombinant DNA construct with a modification at the position corresponding to the 5' end of the mature miRNA that naturally binds to the recognition site of the target gene, i.e., a "cleavage blocker" produced transgenic in plants. 5'-modified cleavage blockers", and methods of using the cleavage blockers to inhibit miRNA-mediated suppression of target genes in transformed plant cells.

[0177] In one example, DNA encoding an artificial miRNA (miRGL1) precursor (SEQ ID NO: 6) was modified by a single nucleotide change (changing the 5' end of mature miRGL1 from U to C), resulting in a 5'- Modified cleavage blocker precursor sequence AATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCCACTTCATGCAAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTTTACAGGATCGTCC CGATGAACAATGACGGTGGAG CCACATGATGCAGCTATGTTTGCTAT GGTCGCCCTTGTTGGACTGTCCAACTCCTACTGATTGCGGATGCACTTGCCACAAATGAAAATCAAAGCGAGGGGAAAAGAATGTAGAGTGTGACTACGATTGCATGCATGTGATTTAGGTAATTAAG...

Embodiment 3

[0184] This example provides an embodiment of a target gene identified as a "verified miRNA target" (ie, containing a verified miRNA recognition site). The recombinant DNA constructs of the present invention can be used to modulate the expression of such target genes and can be used to prepare non-naturally transgenic plant cells, plant tissues and plants (particularly non-naturally transgenic crops) with increased yield or other desirable traits.

[0185] Prediction of recognition sites is accomplished using methods known in the art, eg, the sequence complementarity rules described by Zhang (2005) Nucleic Acids Res., 33:W701-704 and Rhoades et al. (2002) Cell, 110:513-520. One method of experimentally confirming predicted miRNA recognition sites is a technique known as RNA ligase-mediated rapid amplification of the 5' end of cDNA ("5'RLM-RACE" or "5'RACE"), which identifies miRNA cleavage patterns; see eg Kasschau et al. (2003) Dev. Cell, 4:205-217 and Llave et al. (2002) Sci...

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Abstract

This invention provides recombinant DNA constructs and methods for manipulating expression of a target gene that is regulated by a small RNA, by interfering with the binding of the small RNA to its target gene. More specifically, this invention discloses recombinant DNA constructs encoding cleavage blockers, 5-modified cleavage blockers, and translational inhibitors useful for modulating expression of a target gene and methods for their use. Further disclosed are miRNA targets useful for designing recombinant DNA constructs including miRNA-unresponsive transgenes, miRNA decoys, cleavage blockers, 5-modified cleavage blockers, and translational inhibitors, as well as methods for their use, and transgenic eukaryotic cells and organisms containing such constructs.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTING [0002] This application claims the benefit of priority to US Provisional Patent Application 61 / 077,244, filed July 1, 2008, which is incorporated herein by reference in its entirety. Sequence Listing contained in a file entitled "38-21_55745_A.txt" created on June 27, 2008 and filed electronically with US Provisional Patent Application 61 / 077,244 on July 1, 2008 (using operation 2574 kilobytes for system MS-Windows statistics) is incorporated herein by reference in its entirety. The Sequence Listing (2611 kilobytes using the operating system MS-Windows) contained in a file titled "38-21_55745_B.txt" created on June 24, 2009 is filed with and incorporated herein by reference . technical field [0003] The present invention discloses recombinant DNA constructs having DNA that will be processed into RNA that provides RNAse III cleavage resistance to target gene transcripts. This RNA acts as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N5/10
CPCC12N15/8218C12N15/8216C12N15/8261C12N15/111C12N2310/141Y02A40/146
Inventor S·I·埃瓦舒塔B·E·威金斯张远记
Owner MONSANTO TECH LLC
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