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Recombinant DNA constructs and methods for controlling gene expression

a technology of dna and constructs, applied in the field of molecular constructs and methods for controlling gene expression, can solve the problem of low efficiency of anti-sense gene suppression, and achieve the effects of suppressing the expression of a target rna, reducing damage to a plant, and reducing the accumulation of mature mirna

Inactive Publication Date: 2006-09-07
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for reducing damage to plants by pests or pathogens by introducing a recombinant DNA construct containing a transcribable DNA sequence that transcribes to an RNA aptamer capable of binding to a ligand. The ligand can be an endogenous molecule of the pest or pathogen, or a molecule of the plant's digestive tract lining. The RNA aptamer can be embedded in an intron, and the intron can include a promoter element or a regulatory RNA that is allosterically affected by the binding state of the RNA aptamer. The invention also provides recombinant DNA constructs for suppression of at least one target gene, and methods for gene suppression and gene expression in plants.

Problems solved by technology

The efficiency of anti-sense gene suppression is typically low.

Method used

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  • Recombinant DNA constructs and methods for controlling gene expression
  • Recombinant DNA constructs and methods for controlling gene expression
  • Recombinant DNA constructs and methods for controlling gene expression

Examples

Experimental program
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example 1

[0266] This example illustrates the construction and use of vectors designed for double-stranded RNAi suppression or for anti-sense suppression of a luciferase gene. The gene suppression experiments used were similar to a dual luciferase assay described by Horstmann et al. (2004) BMC Biotechnol., 4:13, which is incorporated by reference herein. A prior art vector, “vector 1A”, designed for double-stranded RNAi suppression of a luciferase gene was constructed as depicted in FIG. 1A with an RNAi transcription unit with a polyadenylation site including (a) a chimeric promoter including an enhanced CaMV35S promoter linked to an enhancer element (an intron from heat shock protein 70 of Zea mays, Pe35S-Hsp intron), (b) an inverted repeat of DNA coding for firefly luciferase (LUC) with anti-sense oriented DNA followed by a sense oriented DNA, and (c) a 3′UTR DNA from Agrobacterium tumefaciens nopaline synthase gene (3′NOS) which provides a polyadenylation (polyA) site. Elements of the plas...

example 2

[0271] This example further illustrates the construction and use of vectors designed for double-stranded RNAi suppression or for anti-sense suppression of a luciferase gene. The gene suppression experiments used were similar to a dual luciferase assay described by Horstmann et al. (2004) BMC Biotechnol., 4:13. The vectors illustrated in FIG. 2 were constructed. Vector 2A (FIG. 2A), a control vector not encoding anti-sense or double-stranded RNA for the target gene (firefly luciferase), consisted of (a) the CaMV e35S-Hsp 70 intron chimeric promoter as described in Example 1 and Table 1, (b) an inverted repeat of DNA coding for beta-glucuronidase (GUS) (uidA) with anti-sense oriented DNA followed by a sense oriented DNA, and (c) a 3′UTR DNA from Agrobacterium tumefaciens nopaline synthase gene (3′NOS) as described in Example 1 and Table 1, which provides a polyadenylation (polyA) site. Vector 2B (FIG. 2B), a prior art vector designed for double-stranded RNAi suppression of a luciferas...

example 3

[0273] This example describes transformation of a crop plant (maize) with an enhanced anti-sense construct. A plasmid for binary vector Agrobacterium-mediated transformation of maize is constructed including the elements shown in FIG. 4. Specifically, the plasmid includes an nptlI gene as an antibiotic selectable marker and a recombinant DNA construct for enhanced anti-sense gene suppression, consisting of a CaMV35S promoter operably linked to transcribable DNA consisting of about 300 base pairs of a green fluorescent protein (gfp) gene in an anti-sense orientation, wherein a functional polyadenylation site is absent in this transcribable DNA. The plasmid also includes left T-DNA border (LB) and right T-DNA border (RB) elements. A control plasmid for RNAi suppression of green fluorescent protein (GFP) is constructed by adding to the enhanced anti-sense construct shown in FIG. 4 a repeat of the gfp DNA in the sense orientation followed by a 3′ NOS element including a functional polya...

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Abstract

The present invention provides molecular constructs and methods for use thereof, including constructs including heterologous miRNA recognition sites, constructs for gene suppression including a gene suppression element embedded within an intron flanked on one or on both sides by non-protein-coding sequence, constructs containing engineered miRNA or miRNA precursors, and constructs for suppression of production of mature microRNA in a cell. Also provided are transgenic plant cells, plants, and seeds containing such constructs, and methods for their use. The invention further provides transgenic plant cells, plants, and seeds containing recombinant DNA for the ligand-controlled expression of a target sequence, which may be endogenous or exogenous. Also disclosed are novel miRNAs and miRNA precursors from crop plants including maize and soy.

Description

[0001] This application claims the benefit of priority of U.S. Provisional. Patent Applications 60 / 638,256, which was filed on 21 Dec. 2004, 60 / 639,094, which was filed on 24 Dec. 2004, 60 / 701,124, which was filed on 19 Jul. 2005, 60 / 711,834, which was filed on 26 Aug. 2005, 60 / 720,005, which was filed on 24 Sep. 2005, 60 / 726,106, which was filed on 13 Oct. 2005, and 60 / 736,525, which was filed on 14 November 2005, all of which are incorporated by reference in their entirety herein. The sequence listings contained in the files “53429A.ST25.txt” (file size of 15 kilobytes recorded on 21 Dec. 2004, and filed with provisional application 60 / 638,256 on 21 Dec. 2004), “38-21(53709)B.ST25.txt” (file size of 4 kilobytes, recorded on 23 Dec. 2004, and filed with provisional application 60 / 639,094 on 24 Dec. 2004), “38-15(53429)B.rpt” (file size of 7 kilobytes, recorded on 19 Jul. 2005, filed with provisional application 60 / 701,124 on 19 Jul. 2005), “38-15(54068)A.rpt” (file size of 6 kiloby...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01H1/00C12N15/82
CPCC12N15/8216C12N15/8286C12N15/8218C12N15/8217C12N15/113C12N2310/113Y02A40/146
Inventor LUTFIYYA, LINDAGUO, LIANGHUANG, SHIHSHIEHMALVAR, THOMASLUETHY, MICHAELMILLER, PHILIPGILBERTSON, LARRYALLEN, EDWARDSHEISEL, SARAKOVALIC, DAVIDROBERTS, JAMESHOUMARD, NANCYFRIZZI, ALESSANDRAZHANG, YUANJI
Owner MONSANTO TECH LLC
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