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Compositions and methods for enhancing amino acid levels in plants

An amino acid, plant technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, plant genetic improvement, etc., can solve problems such as increased costs

Active Publication Date: 2011-05-25
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This supplementation of food and feed results in a substantial increase in the costs associated with these foods

Method used

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  • Compositions and methods for enhancing amino acid levels in plants
  • Compositions and methods for enhancing amino acid levels in plants
  • Compositions and methods for enhancing amino acid levels in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0237] Example 1: Isolation and cloning of the aspartokinase gene

[0238] The full-length coding sequence of the wild-type aspartokinase gene isolated from 30 ng of X. burgdorferi genomic DNA was amplified by PCR using oligonucleotide primers SEQ ID NO: 11 and SEQ ID NO: 12. Use Expand TM High-fidelity PCR kit (Boehringer Mannheim, Germany), PCR was performed in a total volume of reaction mixture of 50 μl. PCR conditions were as follows: 1 cycle of 4 minutes at 95°C; 26 cycles of 1 minute at 95°C, annealing at 56°C for 1 minute, and extension at 72°C for 2 minutes; 1 cycle. The resulting product was digested with appropriate restriction enzymes (NdeI and XhoI), gel-purified, and ligated into the corresponding sites in pET30a (Novagen), resulting in plasmid pMON81662 ( image 3 ; XbAK). The integrity of the gene sequence was confirmed by DNA sequence analysis.

Embodiment 2

[0239] Example 2: Identification of novel aspartokinase genes encoding variants with desirable enzymatic properties

[0240] This example illustrates the identification of an AK gene encoding an enzyme with desirable enzymatic properties, ie, insensitivity to end-product inhibition by aspartic acid family amino acids and good kinetic properties. In order to characterize the AK variants, using recombinant E. coli lysC and E. coli T352I lysC (SEQ ID NO: 17) gene products as controls, a recombinant expression system and AK enzyme analysis were established. The E. coli lysC gene product is known to be sensitive to feedback inhibition by Lys, whereas the E. coli T352 IlysC gene product is known to be insensitive to lysine. Expression in plants of the E. coli T352I lysC gene product with aspartokinase activity resulted in a 6-7% increase in threonine content in seeds (Karchi, et al., THE PLANT J.3:721-727 (1993 ); Galili, et al., European Patent Application No. 0485970). The lysC ...

Embodiment 3

[0293] Example 3: Construction of Soybean Transformation Vectors Containing Feedback-Insensitive Aspartokinase Coding Sequences and Transformation into Soybean Plants

[0294] Six soybean transformation vectors (listed in Table 3) containing wild-type and mutant alleles of the X. burgdorferi AK gene were constructed and transformed into soybean.

[0295] Table 3. Soybean Transformation Vectors Containing Aspartokinase Coding Sequences

[0296] pMON number

coding region

pMON101817

CTP1-Pathogenus burgdorferi AK

pMON101818

CTP1-Pathogenus burgdorferi AK E257K

pMON101819

CTP1-Pathogenus burgdorferi AK T359I

pMON101820

CTP1-Pathogenus burgdorferi AK T359I

pMON101821

CTP1 - Pathogen burgdorferi AK T359I - non-native

pMON101822

CTP1 - Pathogen burgdorferi AK T359I - non-native

[0297] The coding sequence for CTP1 (SEQ ID NO: 13) was incorporated at the N-terminus of the bacterial protein to target...

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Abstract

Threonine is an essential amino acid for humans and in the animal feed industry where its levels in feed rations can significantly impact the cost of production of important meat sources, such as swine and poultry. Threonine as well as essential amino acids lysine and methionine are all synthesized via the aspartate family pathway. Aspartate kinase (AK) is the first enzyme in the pathway, and catalyzes the ATP-dependent phosphorylation of aspartate to form ss-aspartyl phosphate. AK constitutes the main regulatory step controlling the metabolic flux through the pathway, and is subject to end product inhibition by Lys and / or Thr. The current invention provides a method to produce a transgenic high free threonine soybean via the overexpression of feedback-resistant AK enzymes in developing soybean plants and seeds. These modifications provide a method to enhance both plant nitrogen metabolism and crop growth performance.

Description

[0001] References to related applications [0002] This application claims priority to US Provisional Application No. 61 / 077,043, filed June 30, 2008. [0003] Introduction of Sequence Listing [0004] Contains 35KB files created on June 9, 2009 (on Microsoft Windows The sequence listing of the file named MONS211WOSequencelisting.txt included in Statistics in 2010) includes 23 nucleotide sequences, which are fully incorporated herein by reference. field of invention [0005] The present invention relates to the field of plant molecular biology and plant genetic engineering and polynucleotide molecules for gene expression in plants. In particular, the present invention relates to the genetic modification of gene sequences for producing increased levels of amino acids in plants for food and / or feed applications. The present invention also discloses methods of producing and using the deregulated genes. Background of the invention [0006] Monogastric animals, including hum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/82
CPCC12N9/1217C12N15/8254C12N15/8251
Inventor J·H·克劳利B·S·葛德曼J·黄齐群刚W·D·拉普
Owner MONSANTO TECH LLC
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