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Mateirals and methods for alteration of enzyme and acetyl coA levels in plants

A technology of acetyl-CoA and plants, applied in the field of acetyl-CoA synthetase

Inactive Publication Date: 2001-11-14
IOWA STATE UNIV RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current research indicates that the production of genetically modified bioplastics may be limited by the supply of acetyl-CoA (Nawrath et al., PNAS USA 91:12760-12764 (1994))

Method used

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  • Mateirals and methods for alteration of enzyme and acetyl coA levels in plants
  • Mateirals and methods for alteration of enzyme and acetyl coA levels in plants
  • Mateirals and methods for alteration of enzyme and acetyl coA levels in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] This example describes the cloning of ACS cDNA from Arabidopsis and comparison of its sequence with that of E. coli and S. cerevisiae.

[0131] The deduced amino acid sequences of the E. coli and S. cerevisiae ACS genes were searched using the program BLAST against the dbEST database (National Library of Medicine, National Institute of Health, Bethesda, Maryland). One Arabidopsisthaliana cDNA clone was identified as a possible ACS gene. This clone, 220J9T7 (accession number N38599), was obtained from the Arabidopsis Research Center for Biology (ARBC) at Ohio State University.

[0132] Plastid DNA of this clone was prepared and sequenced by the Biotechnology Instrumentation Facility of Iowa State University. Analysis of the sequence data found that this clone (J9) encoded approximately 40% of the ACSC-terminal portion. Repeated searches of dbEST failed to reveal any longer cloned sequences.

[0133] The remainder of the ACS gene was isolated using cRACE (Maruyama et a...

Embodiment 2

[0139] This example describes the production of polyclonal antibodies to Arabidopsis ACS.

[0140] PCR primers were designed to allow the coding region of clone J9 to be PCR amplified as a DNA fragment terminated by a restriction site suitable for cloning into the E. coli protein expression plasmid pMALC-2 (New England Biolabs, Beverly, MA). The expression vector clone J9-NT1 / pMALC-2 was constructed according to the instructions provided by the vector supplier. When transcribed into E. coli and induced with isopropylthio-β-galactoside (IPTG), this clone results in the production of a recombinant chimeric fusion protein consisting of the C-terminal portion of the Arabidopsis ACS in conjunction with E. coli maltose protein (MBP) fusion. The recombinant protein was purified from one liter of transformed E. coli culture and purified using amylose columns from New England Biolabs according to the manufacturer's protocol. Purified proteins were analyzed by electrophoresis for puri...

Embodiment 3

[0142] This example describes the cloning of the E1[alpha], E1[beta] and E3 subunit cDNAs of pPDH, and compares the cDNA sequence encoding the E1[beta] subunit with the red algae pPDH sequence and the Arabidopsis mtPDH sequence.

[0143] The deduced amino acid sequences of the E1α and E1β subunits of Porphyra PDH were searched against the dbEST database using the program BLAST. A clone identified as likely to be pPDH was obtained from ABRC at Ohio State University.

[0144] Arabidopsis EST clones 232D14T7 (N65567) and 232D13T7 (N65566) showed a significant degree of homology to the PDH Elα subunit of Porphyra. Both clones contain the same nucleotide sequence, encoding all amino acids except the first 50-70 amino acids of the pPDHE1α subunit. Clone 232D14T7 (D14) was selected for sequencing. The DNA sequence of this clone comprises SEQ ID NO: 3 and the deduced amino acid sequence comprises SEQ ID NO: 4 (see Figure 2 and Sequence Listing).

[0145] Arabidopsis EST clones 163C...

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Abstract

The invention provides acetyl-CoA synthase (ACS), plastid pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehydes Dehydrogenase (ALDH), specifically the nucleic acid sequence and amino acid sequence of ALDH-2 and ALDH-4. The invention also provides a recombinant vector, which contains a nucleic acid sequence encoding one of the above-mentioned enzymes, its antisense sequence or its ribozyme. In cells transformed by such a vector, the antibodies of these enzymes are changed at the enzyme level. A plant cell, a plant tissue, a plant organ or a plant, and methods of producing such plant cells, plant tissue, plant organs or plants. Ideally, changes in enzyme levels result in changes in acetyl-CoA levels in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector, including encoding pyruvate decarboxylase (PDC), the E1α subunit of pPDH, the E1β subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde Antisense sequence of the nucleic acid sequence of dehydrogenase (ALDH), or a ribozyme that can cleave RNA molecules encoding PDC, E1αpPDH, E1βpPDH, E2pPDH, mtPDH or ALDH.

Description

[0001] Government funding [0002] This invention was funded in part by National Science Foundation Grant No. IBN-9696154 and Department of Energy Basic Agreement No. DE-FC05-920R22072 to the Plant Biotechnology Research Consortium. Accordingly, the US Government has certain rights in this invention. [0003] invention technical field [0004] The present invention relates to acetyl-CoA synthetase (ACS), plastid pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase Nucleic acid and amino acid sequences of hydrogenase (ALDH). The present invention also relates to a recombinant vector comprising (i) a nucleic acid sequence encoding one of the above enzymes, (ii) its antisense sequence or (iii) its ribozyme, cells transformed with such a vector, antibodies to these enzymes , a plant cell, a plant tissue, a plant organ or a plant that has been ...

Claims

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Application Information

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IPC IPC(8): A01H1/00C12N5/04C12N9/00C12N9/02C12N9/10C12N9/16C12N9/88C12N15/113C12N15/82
CPCC12N9/93C12N15/8243C12N9/88C12N9/1029C12N9/0051C12N9/16C12N9/0008C12N15/1137
Inventor B·J·尼古劳E·S·维特勒D·J·奥利弗R·贝阿尔P·S·施纳贝柯金闪J·L·约翰逊C·C·奥尔雷德B·法特兰德I·吕策格温翠蓉
Owner IOWA STATE UNIV RES FOUND
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