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Methods for the modulation of oleosin expression in plants

A technology for oleosin and plants, applied in the field of plant genetic engineering construction, can solve the problems of not being able to specifically inhibit specific genes, unclear oleosin gene expression, and impracticality of T-DNA Agrobacterium insertion mutagenesis

Inactive Publication Date: 2007-11-28
SEMBIOSYS GENETICS INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although these Arabidopsis mutants showed ablation of oleosin gene expression, the methods used to produce these plant lines were random and could neither be used to specifically suppress specific genes nor be used to produce genes with specific Plant lines with different ranges of expression levels of the oleosin gene
T-DNA Agrobacterium insertional mutagenesis is also becoming increasingly impractical when using cereal crops with larger genome scales
[0006] Chaudhary S. (2002, Ph.D.Thesis.University of Calgary.Molecularbiology of flax(Linum usitatissimum L)seed oleosin genes.) speculated that an antisense gene knockout strategy could be used to suppress the level of endogenously present oleosin, however Details are lacking to demonstrate how these methods were used to achieve gene suppression of said oleosin, or even to demonstrate whether oleosin suppression is achievable
[0007] Therefore, considering the shortcomings of the prior art, it is not clear how to suppress oleosin gene expression in plants other than by using the T-DNA Agrobacterium insertion method
Furthermore, it is unclear whether and how inhibition of oleosin gene expression serves to regulate seed lipid and protein composition in plant seeds

Method used

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  • Methods for the modulation of oleosin expression in plants
  • Methods for the modulation of oleosin expression in plants
  • Methods for the modulation of oleosin expression in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0269] Example 1: Construction of an oleosin suppression cassette

[0270] Antisense box

[0271] Use the forward primer NTD (5’-TATT) containing restriction sites HindIIII and NcoI (underlined) AAGCTTCCATGG CCGATACTGCTAGAGG-3') (SEQ ID NO: 92) and the reverse primer CTR (5'-AGCCAT) containing the restriction site SpeI (underlined) ACTAGT AGTGTGTTGACCACCACGAG-3') (SEQ ID NO: 93), and Atol1 cDNA (SEQ ID NO: 94) was used as a template to amplify Atol1 cDNA. The PCR product was purified and inserted into the vector pSBS2090 controlled by the phaseolin promoter / terminator (Slightom et al., 1983 Proc. Natl. Acad. Sci. U.S.A. 80:1897-1901). The vector was previously digested with restriction enzyme SwaI (Figure 3b). Insert the PCR product in cis or trans orientation because the enzyme SwaI can produce blunt ends. The plasmid containing Atol1 cDNA in trans orientation was screened by NcoI. The vector containing Atol1 cDNA in the trans-direction digested by NcoI released a 551 bp DNA fra...

Embodiment 2

[0277] Example 2: Transformation of Agrobacterium and Arabidopsis

[0278] By electroporation, the binary vectors pSBS3000-antisense sequence, pSBS3000-hairpin and pSBS3000-hairpin+intron were inserted into Agrobacterium EHA101 (Hood, EE et al., 1986. Journal of Bacteriology 168: 1291-1301). ). Using spectinomycin resistance ("SpecR" in Figure 5e), the transformed Agrobacterium line containing the binary vector was selected. One Agrobacterium line was selected for each construct.

[0279] The Arabidopsis ecotype C24 was used for transformation. In a 4-inch pot, five seeds were planted on the surface of the soil mixture (two-thirds of Redi soil and one-third of perlite, pH=6.7). Let the seedling grow to the 6-8 leaf rosette stage, about 2.5cm in diameter. These seedlings were transplanted into a 4-inch pot containing the soil mixture described above, and covered with a screen material with five meshes of 1 cm diameter, one in the corner and one in the center. The pot was placed on ...

Embodiment 3

[0283] Example 3: Separation of oil bodies

[0284] The accumulation of Atol1 in the seeds recovered from the selected strain was analyzed by SDS-PAGE of the oil body part. Using the method reported by van Rooijen Moloney ((1995) Biotechnology (N.Y.) 13, 72-77), the oil bodies of these seeds were obtained by the following modifications. Briefly, in a 1.7 ml microcentrifuge tube with 0.4 ml of oil body extraction buffer (50 mM Tris-HCl with 0.4 M sucrose and 0.5 M NaCl, pH 7.5), mill 10 to 20 mg of dry mature seeds. The extract was centrifuged at 10,000 g for 15 minutes at room temperature (RT). After centrifugation, the lipid layer containing oil bodies was removed from the water phase and transferred to another microcentrifuge tube. The oil body is suspended in 0.4ml of high stringency urea buffer (100mM sodium carbonate buffer containing 8M urea, pH8.0). The sample was centrifuged at 10,000 g for 15 minutes at 4°C, and the undernatant was removed. Finally, the oil bodies are sus...

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Abstract

Methods for modulating oleosin expression levels in plants are provided. In particular, it relates to methods for preparing seed-derived products from seeds in which the nutrient stores of the seeds, particularly the content of seed lipids and proteins, have been altered. In particular, the present invention provides methods for the preparation of seed-derived products from seeds wherein the nutrients stored in the seeds are altered by modulating, more particularly inhibiting, oleosin gene expression.

Description

Invention field [0001] The invention relates to a method for constructing plant genetic engineering. More specifically, the present invention relates to a method for regulating the expression level of oleosin in plants. Background of the invention [0002] Plant seeds represent an important source of nutrients for humans and animals. For example, plant seed protein represents the main component of animal feed, and plant seed oil is used to produce vegetable oil that has been widely used for human consumption. [0003] In seeds, the water-insoluble oil fraction is stored in various discontinuous subcellular structures known in the art, such as those with a diameter range of 0.5 to 2.0 microns (Tzen, 1993 Plant Physiol. 101: 267-276) Oil bodies, oleosomes, liposomes or round spheres (Huang, 1992 Ann. Rev. Plant Mol. Biol. 43: 177-200). In addition to the oil mixture (triacylglycerol) chemically called glycerides of fatty acids, oil bodies also include phospholipids and many related...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00C11B1/00
CPCA23D9/00C12N15/8247C12N15/8251
Inventor R·M·西罗托M·M·莫洛尼
Owner SEMBIOSYS GENETICS INC
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