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Method for compositions for modifying levels of secondary metabolic compounds in plants

A technology for plants and plant cells, applied in the field of genetic constructs and vectors, modified seed powder, seed powder with reduced or changed secondary metabolite content, and plant seeds, which can solve the problem of affecting the expression of related genes, without providing secondary Metabolites, harmful effects, etc.

Inactive Publication Date: 2001-04-25
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the use of antisense RNA or co-suppression strategies may not provide commercially acceptable reduced levels or specificity of secondary metabolites
In addition, the inhibition of genes encoding key enzyme activities may also affect the expression of related genes
Thus, little progress has been made in causing reductions in phenolic compounds that are believed to impair nutrition, or in reducing lignin content by antisense RNA or co-suppression without deleterious side effects.
[0014] A second effort to alter metabolic pathways that was not successful in producing the desired results was the modification of glucosinolate biosynthesis in canola
[0016] However, it appears that the enzymes used to obtain this modification also act on primary metabolites (the amino acid tryptophan and the mineral sulfur), and thus any significant changes in these compounds in plant cells would be expected to have deleterious effects
Therefore, the proposed method cannot specifically target secondary metabolic pathways

Method used

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  • Method for compositions for modifying levels of secondary metabolic compounds in plants
  • Method for compositions for modifying levels of secondary metabolic compounds in plants
  • Method for compositions for modifying levels of secondary metabolic compounds in plants

Examples

Experimental program
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Embodiment 1

[0207] The following examples serve to illustrate this method, but in no way limit the scope of the invention. Example 1: General method used to identify products of the phenylpropanoid pathway: Determination of the content of sinapine, the end product of the phenylpropanoid pathway, in the seeds of cruciferous plants

[0208] It will be apparent that all of the exemplified methods can be adapted by the skilled artisan for the determination of products in the phenylalanoid pathway with reference to numerous publications in analytical chemistry. In the first method as an example, a simple assay for the identification of sinapine in plant tissue, namely the seed tissue of cruciferous plants, was adapted from a published method (Chapple C.C.S., T. Vogt, B. E. Eills and C. R. Somerville, 1992, The Plant Cell 4: 1413-1424). Thin-layer chromatography (TLC) protocols were standardized for the assessment of the product sinapine in the phenylalanine pathway. This method uses the isol...

Embodiment 2

[0215] Plant material can also be extracted with a chloroform:methanol:formic acid (CMF) mixture in a ratio of 5:12:3, respectively. Typically, 2 μl of CMF is added to 1 mg of sample, the sample is crushed, left overnight (16 hours) at room temperature (21-23° C.), and then centrifuged at approximately 12,000×g for 5 minutes. The supernatant was collected, and the pellet was thoroughly mixed with another volume of CMF added as before, left for 20 minutes, and centrifuged as above. The supernatants were pooled and extracted with chloroform-water (CW) as previously described, and the aqueous fraction was analyzed as above. Example 2: Identification of the localization of product synthesis of the phenylpropanoid pathway in specific plant tissues.

[0216] In this example, the developmental timing of product synthesis of the phenylalanoid pathway was determined. This particular example exemplifies sinapine synthesis and accumulation in cruciferous plant seed tissues. In this ex...

Embodiment 3

[0217] The portion of the pod wall from which the seeds were detached contained no sinapine, as did the younger seeds on the samples from 7-, 14-, and 16-DAP. This analysis indicated the onset of sinapine accumulation at approximately 18 DAP, however, because this method uses UV fluorescence detection, even small amounts present in younger seeds were not detectable. Figure 3 shows an example of TLC analysis of samples up to 28 DAP, the second panel shows an example up to seed maturity. Since, the above results are not quantitative, extracts of whole seeds obtained with methanol solution were analyzed by HPLC as described in Example 1. The results presented in Figure 4 demonstrate a rapid sequential growth from 20DAP sinapine synthesis to maturity. The amount of sinapine in mature seeds is about 0.8% dry weight of seeds, since oil will make up about 45% of seeds, which will convert to about 1.5% of defatted seed meal. Example 3: Determination of Temporal and Spatial Aspects o...

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Abstract

The present invention provides a method of making a genetically transformed plant which has an altered content of at least one product of a secondary metabolic pathway. The method consists of introducing into a plant cell capable of being transformed and regenerated to a whole plant a DNA expression cassette. The expression cassette includes DNA sequences required for transformation and selection in plant cells. It also includes a DNA sequence that, under the control of a promoter active in plant cells, encodes a protein capable of modifying the utilization of a substrate in the secondary metabolic pathway. The substrate is not a primary metabolite of the group selected from glucose, amino acids, common fatty acids and nucleotides. A plant or plant tissues including seeds can then be recovered having an altered content of at least one product of the secondary metabolic pathway. The invention also provides for feed products derived from the plants and seeds obtained according to the method.

Description

[0001] This application is the U.S. serial application No. 09 / 012,453 is a continuation-in-part and claims priority from provisional application 60 / 072,156. field of invention [0002] The present invention provides methods and compositions for altering compounds produced by secondary metabolic pathways in plants. The present invention also provides plant cells with altered secondary metabolite content and plant seeds with altered secondary metabolite content. In one embodiment, the content of detrophic secondary metabolites is varied in the plants, plant cells and plant seeds according to the invention. In another embodiment, in the plants, plant cells and plant seeds according to the invention, the content of phenylpropanoid and products found in the sugar alcohol secondary metabolic pathway is altered. The invention further provides genetic constructs and vectors useful for altering the content of secondary metabolites in plant cells and seeds. The present invention also...

Claims

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Application Information

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IPC IPC(8): A01H5/00A01H5/10C12N5/10C12N15/09C12N15/53C12N15/55C12N15/60C12N15/82
CPCC12N15/8255C12N15/8243C12N15/8242C12N15/52
Inventor W·A·凯勒R·S·S·达特拉董金琢F·乔治斯A·A·K·胡萨恩G·塞尔瓦拉
Owner NAT RES COUNCIL OF CANADA
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