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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: 2007-04-12
HUANG SHIHSHIEH +13
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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 RNA aptamer that can bind to a ligand and reduce the expression of a target sequence. The RNA aptamer can be controlled by the ligand and can be embedded in an intron. The invention also provides a recombinant DNA construct for suppression of at least one target gene in a plant cell, which can be used to genetically modify plants for improved resistance to pests and pathogens. The invention also provides a method for suppressing production of mature microRNA in a cell by targeting the suppression of a microRNA precursor. Overall, the invention provides a way to protect plants from damage and improve their resistance to pests and pathogens."

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 nptII 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

PRIORITY CLAIMS AND INCORPORATION OF SEQUENCE LISTINGS [0001] This is a divisional application of U.S. patent application Ser. No. 11 / 303,745, filed 15 Dec. 2005, which claims the benefit of priority to 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 Nov. 2005, incorporated by reference in their entirety herein. The sequence listing that is contained in the file named “38-15(53429)100.rpt” which is 97 kilobytes (measured in operating system MS-Windows), created on 29 Sep. 2006, and located in computer readable form on a compact disk (CD-R), is filed herewith and incorporated herein by reference. The sequence listing contained in the file named “38-15(53429)C.rpt”, which is 97 kilobytes (meas...

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 HUANG, SHIHSHIEHMALVAR, THOMASLUETHY, MICHAEL H.MILLER, PHILIP W.GILBERTSON, LARRY A.ALLEN, EDWARDSHEISEL, SARA E.KOVALIC, DAVID K.ROBERTS, JAMES K.HOUMARD, NANCYFRIZZ, ALESSANDRAZHANG, YUANJIGUO, LIANGLUTFIYYA, LINDA
Owner HUANG SHIHSHIEH
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