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Down-regulation and silencing of allergen genes in transgenic peanut plants

Inactive Publication Date: 2005-05-26
DODO HORTENSE +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0029] Also provided is a method wherein the recipient peanut plant cell is transformed with a DNA construct comprising a peanut allergen gene, or fragment thereof, that is the Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7 or any other peanut allergen gene. The invention further provides a method wherein the recipient peanut cell is transformed with a DNA construct comprising more than one peanut allergen gene.
[0030] Also provided is a method wherein homologous sequence region between two or more peanut allergen genes is used to down-re

Problems solved by technology

Food allergy is a serious health problem, and can be life threatening.
Hypersensitive responses to peanut allergens can be fatal.
Contact with the slightest amount of peanut protein can be life threatening to particularly sensitive individuals.
However, some patients may rapidly develop severe angiodema, swelling of the face, bronchospasm and anaphylaxis, following exposure.
Peanut, peanut butter, and peanut flour retain their allergenicity through processing, and crude peanut oil may also be contaminated with these proteins.
Currently, no treatment exists for food allergies.
However, this is difficult to do, as it requires diligent reading of labels and ingredient listings.
Because dining out is prevalent in the current American lifestyle, the social stigma associated with refraining from taking part in restaurant or party meals by allergic individuals because of the potential threat for accidentally ingesting peanut, makes the strict avoidance of peanut unlikely and unrealistic (Heiner and Navin, 1975, J. Allergy Clin.

Method used

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  • Down-regulation and silencing of allergen genes in transgenic peanut plants
  • Down-regulation and silencing of allergen genes in transgenic peanut plants
  • Down-regulation and silencing of allergen genes in transgenic peanut plants

Examples

Experimental program
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Effect test

example 1

Isolation and Characterization of the Genomic Clones Encoding the Peanut Allergen Genes

[0169] a) Library Screening

[0170] To identify the genomic clone of the gene coding for the peanut allergen Ara hII, a peanut genomic library constructed in a Lambda Fix II vector (Stratagene Inc, La Jolla, Calif.) was screened with an 80 base pair oligonucleotide probe. The probe sequence (5′ ctagtagccctcgccttttcctcctcgctgcccacgcatctgcgaggcagcagtgggaactccaaggagacagaaga tg-3′) (SEQ ID NO: 7) corresponds to nucleotide eleven to ninety-one of a published Ara h2 cDNA sequence (GeneBank accession L77197).

[0171] Twenty picomoles of the probe was end-labeled with radioactive adenosine 5′-triphosphate, tetra (triethylammonium), salt [gamma 32P] (32P) as described by Ausubel et al. (Ausubel F, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. Short Protocols in Molecular Biology. 3rd ed.: John Wiley & Sons, Inc.; 1995) Fresh Echerichia Coli (E. Coli) VCS 257 (300 μL of 1×1010 cells / mL)...

example 2

Construction strategy of peanut allergen gene plasmids

[0204] Peanut allergen gene plasmids were constructed using expression cassettes containing antisense, and / or sense orientation of allergen genes linked to 35S / Ara h2 promoter and nos terminator, as shown in FIG. 8. Five types of constructs were used for the transformation of peanut tissue. The plasmid constructs pBI426, modified pBI426, and pCB13, were used in biolistic transformation. pBI434modified pBI434 were used in Agrobacterium-mediated transformation The Ara h2 promoter is shown in FIG. 9.

[0205] PCB13 is used in co-bombardments with modified pBI426 which contains peanut allergen fragments (Ara h transgenes), to select transgenic plants. pCB13 contains the 35S promoter, hygromycin gene, and the nos terminator. This cassette is cloned into pUC 19.

[0206] The plasmid pBI426 (Dalta et al., 1991 Gene 101: 239-246) contains a fusion gene (GUS fused to nptII) cloned between XbaI and SacI, and driven by the 35S promoter. Its al...

example 3

Methodology Used in Peanut Tissue Culture, Transformation and Regeneration

[0211] Peanut Varieties

[0212] The most widely cultivated peanut cultivars in the USA, ‘Florunner’, ‘New Mexico Valencia’, ‘Georgia Green’, and ‘Georgia Red’ can be used in the present method, although a person skilled in the art will realize that the method is applicable to other peanut varieties.

[0213] Genetic Constructs

[0214] Efficient strategies to down-regulate genes in transgenic plants utilize antisense RNA, co-suppression, or double-stranded RNA. All three methods are being used to down-regulate peanut allergens. Transformation vectors used are pUC 18 for biolistic transformation, and modified versions of pBI434 (Dalta et al, 1991), a binary vector for transformation using Agrobacterium tumefaciens. Transformation vectors carry the transgenes, flanked by a (the Arah2 promoter, shown in FIG. 9, or the 35S promoter) and the nopaline synthase terminator. Transgenes are portions of the open reading fram...

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Abstract

An allergen-free transgenic peanut seed is produced by recombinant methods. Peanut plants are transformed with multiple copies of each of the allergen genes, or fragments thereof, to suppress gene expression and allergen protein production. Alternatively, peanut plants are transformed with peanut allergen antisense genes introduced into the peanut genome as antisense fragments, sense fragments, or combinations of both antisense and sense fragments. Peanut transgenes are under the control of the 35S promoter, or the promoter of the Ara h2 gene to produce antisense RNAs, sense RNAs, and double-stranded RNAs for suppressing allergen protein production in peanut plants. A full length genomic clone for allergen Ara h2 is isolated and sequenced. The ORF is 622 nucleotides long. The predicted encoded protein is 207 amino acids long and includes a putative transit peptide of 21 residues. One polyadenilation signal is identified at position 951. Six additional stop codons are observed. A promoter region was revealed containing a putative TATA box located at position−72. Homologous regions were identified between Ara h2, h6, and h7, and between Ara h3 and h4, and between Ara h1P41B and Ara h1P17. The homologous regions will be used for the screening of peanut genomic library to isolate all peanut allergen genes and for down-regulation and silencing of multiple peanut allergen genes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. application Ser. No. 09 / 715,036, filed Nov. 20, 2000, now allowed, which claims priority to U.S. Application No. 60 / 167,255, filed Nov. 19, 1999. [0002] This invention was made with Government support under Grant No. 96-02658 awarded by the United States Department of Agriculture Cooperative States Research Education and Extension Services (USDA / CSREES) Capacity Building Program. The Government has certain rights in the invention.FIELD OF THE INVENTION [0003] This invention relates to transgenic peanut cells, peanut seeds and peanut products with reduced or undetectable quantities of one or more peanut allergen proteins. The invention also relates to isolated DNA sequences coding for peanut allergen proteins, as well as antisense, genes corresponding to each of the allergen protein genes. Furthermore, the invention relates to recombinant methods of reducing, and eliminating one or more of the pea...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/82
CPCC07K14/415C12N15/8251C12N15/8242
Inventor DODO, HORTENSEARNTZEN, CHARLESVIQUEZ, OLGAKONAN, KOFFI
Owner DODO HORTENSE
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