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Expression of sedoheptulose 1,7-bisphosphatase in transgenic plants

A technology of sedum heptulose and diphosphatase, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve problems such as chlorosis, reduced carbon assimilation level, and reduced sugar content

Inactive Publication Date: 2002-07-24
MONSANTO TECH LLC
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0006] It has been reported that more than 80% of the SBPase enzyme activity was removed in tobacco plants using antisense technology, resulting in chlorosis, reduced growth rate, and reduced levels of carbon assimilation (Harrison et al., 1998)
Reduced quantum efficiency of photosystem II was also observed, which resulted in lower sugar content in leaves

Method used

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  • Expression of sedoheptulose 1,7-bisphosphatase in transgenic plants
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  • Expression of sedoheptulose 1,7-bisphosphatase in transgenic plants

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Embodiment Embodiment 1

[0094] The cDNA clone of embodiment embodiment 1SBPase and excessive expression to produce antibody

[0095] To isolate the gene region encoding the mature SBPase protein (without CTP), RT-PCR reactions were performed. One microgram of common wheat (Triticum aestivum) CV OSLO leaf RNA was mixed with 100 pmol random hexamer primers (BRL / Life Technologies Inc., Gaithersburg, MD) or with 100 pmol oligo dT primers (Promega, Madison, WI), in Heat at 75°C for 5 minutes, then cool on ice. Using Superscript II TM Reverse transcriptase (BRL / Life Technologies Inc, Gaithersburg, MD), according to the manufacturer's protocol, was used for first-strand cDNA synthesis. The terminated reverse transcription reaction was diluted 1:7. Three microliters of the diluted first-strand synthesis product was mixed in 100 μL with: 100 μM of each dNTP, 50 pmol with homology to the 5' end of the gene, designed to create an NdeI cleavage site for subcloning Gene-specific primer (5...

Embodiment 3

[0099] To test the expression of wheat SBPase subunits and their assembly into active enzymes, a vector containing the following elements was constructed: the CaMV E35S promoter, the coding sequence of the complete SBPase protein including CTP, the NOS 3' termination signal, and Bacillus selection for ampicillin resistance. The SBPase gene was isolated from pMON47200 as a XbaI / BamHI fragment. The SBPase gene was ligated into XbaI / BamHI linearized pMON999 (Fig. 6) in the region with CaMV E35S, NOS 3', resulting in pMON47203 (Fig. 7). The DNA constructs were electroporated into maize protoplasts according to the method of Sheen et al. (1991). Embodiment 4 transforms the analysis of maize protoplast

Embodiment 4

[0099] To test the expression of wheat SBPase subunits and their assembly into active enzymes, a vector containing the following elements was constructed: the CaMV E35S promoter, the coding sequence of the complete SBPase protein including CTP, the NOS 3' termination signal, and Bacillus selection for ampicillin resistance. The SBPase gene was isolated from pMON47200 as a XbaI / BamHI fragment. The SBPase gene was ligated into XbaI / BamHI linearized pMON999 (Fig. 6) in the region with CaMV E35S, NOS 3', resulting in pMON47203 (Fig. 7). The DNA constructs were electroporated into maize protoplasts according to the method of Sheen et al. (1991). Embodiment 4 transforms the analysis of maize protoplast

[0100] Transform the precipitated protoplast samples with pMON47203 (SBPase) in 0.18 mL of extraction buffer (50 mM HEPES pH 7.5, 1 mM fructose diphosphate, 1 mM sedoheptulose-1,7-diphosphate, 10 mM MgCl 2 , 10mM MnCl 2 , 10mM DTT, 1% polyvinylpolypyrrolidone, 10% glycerol and C...

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Abstract

Sedoheptulose 1,7-bisphosphatase (SBPase) is an enzyme catalyzing the reaction converting sedoheptulose 1,7-bisphosphate into sedoheptulose 7-phosphate. This enzyme is located in the chloroplast in leaves and stems. Overexpression of SBPase in transgenic plants is provided to improve plant yield by increasing leaf starch biosynthetic ability in particular and sucrose production in general. Deregulated variants of the enzymes are also provided.

Description

[0001] This application claims priority to US Provisional Application Serial No. 60 / 133,964, filed May 13,1999. field of invention [0002] The present invention relates to the expression of Sedoheptulose-1,7-bisphosphatase (SBPase) in transgenic plants to increase or improve crop growth and development, yield, vigor and partitioning of products of carbon assimilation. Transgenic plants expressing SBPase have improved carbon assimilation, carbon export and carbon storage in plant source and sink organs, resulting in improved crop growth, yield and quality. Background of the invention [0003] Recent advances in genetic engineering have provided the necessary tools for transforming plants to contain exogenous (often referred to as "heterologous" or "xenogeneic") genes or modified endogenous genes. Introduction of such a gene in plants would advantageously improve existing pathways in plant tissues or introduce new pathways to alter le...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/82
CPCC12N15/8245C12N9/16
Inventor R·L·斯陶布P·W·米勒
Owner MONSANTO TECH LLC
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