Process for converting storage reserves of dicot seeds into compositions comprising one or more gene products

A technology of dicotyledonous plants and gene products, applied in the field of germination seed-derived compositions, can solve problems such as unsolved problems

Inactive Publication Date: 2003-04-02
UNICROP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] While malting is known and is a method of producing polyhydroxyalkanoates using the plant's respiratory pathway, the application of stored nutrients in dicots to produce proteinaceous products such as structural proteins and enzymes has not been addressed

Method used

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  • Process for converting storage reserves of dicot seeds into compositions comprising one or more gene products
  • Process for converting storage reserves of dicot seeds into compositions comprising one or more gene products
  • Process for converting storage reserves of dicot seeds into compositions comprising one or more gene products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] GUS Gene Expression in Germinated Seeds of Rapeseed in Example 1

[0100] GUS expression was confirmed by histochemical assay. GUS expression was regulated with 4 different promoters. The promoters used were soybean heat shock promoter, Vigna mungo endopeptidase promoter, tobacco pr-promoter and CaMV 35S promoter. Using the total plant DNA as a template, the promoter sequence was obtained by PCR.

[0101] The promoter was ligated to the GUS gene using NcoI enzyme. The promoter-GUS construct was cloned into the plant transformation vector pGPTV-hpt (Becker et al. 1992, Plant Mol. Biol. 20: 1195-97). Rapeseed rape was transformed according to the method introduced by Kuvshinov, V. et al. (Plant Cell Reports 18: 773-777, 1999). The transgenic plants were grown in the greenhouse until they produced seeds. The transgenic seeds were germinated and used for histochemical GUS assays.

[0102] The results are shown in the accompanying drawings.

[0103] Figure 1A The lef...

Embodiment 2

[0111] Example 2 Preparation of novel rbcS promoter for expression system a. Isolation of novel rbcS promoter

[0112] Sequencing of Rbc cDNA clones from 4-day-old transgenic rapeseed cotyledons. Clones were divided into two groups based on the similarity of the sequence in the 3' UTR region.

[0113] SDS-page analysis of rapeseed seedlings. Rapeseed seeds were allowed to germinate continuously over 1-7 days. Grind 5 pairs of cotyledons in liquid nitrogen and resuspend in lysis-loading buffer (50 mM Tris HCl pH 8.5, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 2% mercaptoethanol (2-ME)) middle. Following the method of Laemmli (1970), samples were boiled in a water bath for 10 minutes and analyzed on 15% SDS-PAGE gels. Estimated initiation of Rbc gene expression 4 days after imbibition. b. RNA-purification of rapeseed cotyledons

[0114] RNA from 4-day-old cotyledons was purified using Qiagen RNeasy Kit. c. cDNA-synthesis from total RNA of rape cotyledons

[0115] 2 μg ...

Embodiment 3

[0123] Example 3 Comparing different constitutive promoters and inducible promoters of tobacco and rapeseed transgene (GUS) overexpression

[0124] plant

Promoter

Specific activity (nmol MU / min / mg can

soluble protein)

tobacco

B. rubisco type I

7

Rapeseed

heat shock

44

Rapeseed

heat shock amylase

5

Rapeseed

35S

28

[0125] The promoter activity of the germinated seeds was monitored during the germination period of 7 consecutive days and an exponential increase in the specific activity of eg the rubisco promoter was detected. The rubisco promoter specific activity was 0.25 on the 4th day, 0.5 on the 5th day, 1.6 on the 6th day, and 7 on the 7th day. Chloroplast development inhibitors for analysis of rubisco protein and mRNA levels in germinating seeds

[0126] Analysis of natural rape seedlings showed that rubisco small subunit protein reached the highest concentration on ...

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PUM

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Abstract

The present invention is related to a process based on a source-sink principle, for producing products of interest from crushed or uncrushed germinating dicot seeds comprising an expression system, which is induced or can be induced during germination. The product is either a seed derived composition comprising one or more gene products. Alternatively, it is a product of interest obtained by placing the composition in contact with a substrate, containing a substance capable of being transformed by the seed derived composition as such, dried or in down-stream processed form.

Description

[0001] Technical Field of the Invention [0002] The present invention relates to a method based on the source-sink principle for the conversion of stored nutrients of transgenic dicotyledonous plant seeds into compositions comprising one or more gene products of interest. Also disclosed are germinated seed-derived compositions comprising at least one gene product in cotyledons (including seedlings) or in the medium surrounding the germinated seeds or seedlings. Background of the invention [0003] The production methods of isolating and purifying therapeutically active mammalian proteins from various sources in mammals due to their increased risk of contamination are now largely replaced by recombinant DNA techniques which allow new production methods of large quantities of industrially desired mammalian proteins. Recombinant DNA technology offers a wide variety of host and expression systems to choose from. When a mammalian protein is the desired protein of interest, eukary...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H3/00C12N9/88C12N15/09C12N15/82
CPCC12N15/8216C12N15/8243C12N9/88C12N15/8222C12N15/8234C12N15/8257C12N15/8237C12N15/82
Inventor K·科伊伍V·库夫施诺夫A·卡纳瓦E·佩胡
Owner UNICROP LTD
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