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Transgenic plant or plants with a naturally high water content overproducing at least two amino acids of the aspartate family

A technology of transgenic plants and aspartate kinase, which is applied in the fields of plant products, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2000-01-19
AVEBE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The AK coding sequence can also be regulated by such a promoter, but the restriction is that the expression system provided for the AK coding sequence is stronger than that provided for the DHPS coding sequence

Method used

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  • Transgenic plant or plants with a naturally high water content overproducing at least two amino acids of the aspartate family
  • Transgenic plant or plants with a naturally high water content overproducing at least two amino acids of the aspartate family
  • Transgenic plant or plants with a naturally high water content overproducing at least two amino acids of the aspartate family

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The construction of the chimeric double gene construct of embodiment 1 expressing AK and DHPS

[0060] DNA isolation, subcloning, restriction analysis and DNA sequence analysis were performed using standard methods (Sambrook et al, 1989; Ausubel et al, 1994).

[0061] A chimeric gene containing the lysC gene was constructed by fusing the enhanced CaMV35S promoter to the chimeric lysC gene. This chimeric lysC construct contains a DNA segment of a mutated lysC allele fused at the 5' end to an omega DNA sequence derived from the envelope protein of tobacco mosaic virus (Gallie et al, 1989). Downstream of the lysC sequence, a termination signal derived from the octopine synthase gene of Agrobacterium tumefaciens was inserted (Greve et al, 1983). A DNA fragment (Fluhr et al, 1986) containing the coding sequence for the pea rbcS-3A transit peptide was inserted between the omega DNA and the lysC coding sequence. The chimeric lysC gene construct was cloned in pBluescript (Str...

Embodiment 2

[0064] Embodiment 2, chimeric gene is introduced in the potato

[0065] 2.1 Transformation of potato plants

[0066]The binary vector pAAP50 was used for freeze-thaw transformation of Agrobacterium tumefaciens strain AGLO (Hofgen and Willmitzer 1988). The transformed AGLO was subsequently used for inoculation of diploid potato (Solanum tuberosum, var. Kardal) stem explants as described by Visser (1991). After regeneration of stems and roots on kanamycin-containing medium, plants were placed in soil and transferred to the greenhouse. Plants regenerated (on kanamycin free medium) from stem explants treated with Agrobacterium strain AGLO without the binary vector served as controls.

[0067] 2.2 Tuber formation in vitro

[0068] To induce tuber formation in vitro, excised nodes (about 4-5 cm long) of transformed potato plantlets were placed vertically on solid Murashige and Skoog (1962) medium supplemented with 10% (w / v) sucrose, 5 μM BAP middle. Cultures were maintained at ...

Embodiment 3

[0069] Example 3 Analysis of free lysine, threonine and methionine content in transgenic potato plants

[0070] Tissue (0.5-1.0 g) was homogenized with a mortar and pestle in 2 ml of 50 mM Pi buffer (pH 7.0) containing 1 mM dithiothreitol. Norleucine was added as an internal standard. The free amino acid was partially purified by extraction with 5 ml of a water:chloroform:methanol mixture (3:5:12). The aqueous phase was collected and extracted two more times. After concentration to 3 ml by lyophilization, 20 μl of the sample was used for 6-aminoquinolyl-N-hydroxysuccinimide carbamate (AQC) derivatization reaction. Derivatized amino acids were analyzed by DHPS on a reverse phase C18 column according to the manufacturer's instructions (WatersAccQ.Tag system)

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Abstract

The present invention provides a double gene construct which includes a nucleic acid sequence with a code having active enzyme of aspartokinase (AK) and a nucleic sequence with a code having active enzyme of dihydropyridine dicarboxylic acid synthase. The construct can differently express two genes in plant or part of plant, and can generate lysine and threonine with a level more than five times of the level of wild amino acid.

Description

Summary of the invention [0001] The invention provides a chimeric double gene construct comprising a nucleic acid sequence encoding an enzyme with aspartokinase (AK) activity and a nucleic acid sequence encoding an enzyme with dihydrodipicolinate synthase (DHPS) activity. The nucleic acid sequence of the enzyme. Differential expression of the two genes by this construct resulted in a five-fold increase in the levels of lysine and threonine in the plant or parts thereof compared to the wild-type level of each amino acid. This construct also increased methionine levels. This expression can now be done without the previously occurring problems associated with high accumulation of lysine or combined expression of AK and DHPS genes in plants. Expression regulation should be such that expression occurs in such a way that lysine and threonine are produced as much as possible within limits that do not harm the plant, ie do not negatively distort the phenotype compared to wild-type p...

Claims

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Application Information

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IPC IPC(8): A01H5/00C12N15/29C12N15/52C12N15/82
Inventor 英格丽德・玛丽亚・范德梅尔奥斯卡・弗雷德里克・约瑟夫・福斯特彼得・马丁・布吕纳贝尔赫约翰内斯・彼得・马里纳斯・桑德斯阿德里安斯・约翰内斯・范图内恩
Owner AVEBE
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