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Means for improving agrobiological traits in a plant by providing a plant cell comprising in its chloroplasts enzymatic activities for converting glycolate into malate

A plant cell, malate synthase technology, applied in the field of yield transgenic plants or plant cells, can solve the problems of weakened photorespiration, endogenous enzyme patterns and changes in the content of UV protectants

Inactive Publication Date: 2011-04-13
UNIV OF COLOGNE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[21] have shown that the overexpression of C4 cycle genes in C3 plants (potato and tobacco) leads to weakened photorespiration and changes in endogenous enzyme patterns and UV protectant content

Method used

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  • Means for improving agrobiological traits in a plant by providing a plant cell comprising in its chloroplasts enzymatic activities for converting glycolate into malate
  • Means for improving agrobiological traits in a plant by providing a plant cell comprising in its chloroplasts enzymatic activities for converting glycolate into malate
  • Means for improving agrobiological traits in a plant by providing a plant cell comprising in its chloroplasts enzymatic activities for converting glycolate into malate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1: PCR Amplification of Transgene

[0134] The cDNAs corresponding to glycolate oxidase 2 (GO, At3g14420) in Arabidopsis leaves and malate synthase (MS, X56948) in pumpkin cotyledon and E. coli catalase (KatE; M55161) genomic sequence and cloned into pGEMT-Easy (Promega, Mannheim, Germany) or pCR-Blunt II-TOPO (Invitrogen). For the case of GO and MS, the nucleotides encoding the last amino acids (A / SRL) were omitted, which were estimated to represent peroxisome targeting signals (31, 32). The following primer combinations were used: GO forward primer: (5'-TACAATTGGAGATCACTAACGTTACCGAGT-3' SEQ ID NO: 9) and GO reverse primer: (5'-TGGGACACTCCACGTCCTTAGTCTAGACTAGTA-3' SEQ ID NO: 10); MS forward primer: (5'-ACACCGGTCGCTGGGAATGTATTCTGAATCGGCA-3'SEQ ID NO: 11) and MS reverse primer: (5'-CACATAGGCATACATCATCCAGGTGAGTCGACGTT-3'SEQ ID NO: 12); KatE forward primer: (5'-ACACCGGTCGCAACATAACGAAAAGAACCCA-3'SEQ ID NO : 13) and KatE reverse primer: (5'-ACGTCGACTCAGGCAGGAATTTTG...

Embodiment 2

[0135] Embodiment 2: Construction of binary vector

[0136] To direct expression in Arabidopsis, DNA encoding the plastid precursors of these enzymes was cloned into a modified form of the binary vector pGreenII [33, 34]. DNA encoding these enzymes was cloned into binary vectors using different strategies. For MS and KatE, the CaMV35S promoter was excised from the vector and expression was directed by the tomato-rbcS3C promoter. The selectable marker genes nosKan (kanamycin resistance), nosHyg (hygromycin resistance) and nosBAR 18 (BASTA resistance) were cloned into the StuI-site in the basic pGreenII vector. GO was cloned into pGreenII 35S-nosKan vector, MS was cloned into pGreenII rbcS3C-nosHyg vector, and KatE was cloned into pGreenII rbcS3C-nosBAR vector, and the resulting plasmids were called 35S:GO, rbcS3C:MS and rbcS3C:KatE.

Embodiment 3

[0137] Example 3: Transformation of Arabidopsis thaliana and selection of transformants

[0138] The binary vectors 35S:GO, rbcS3C:MS, and rbcS3C:KatE were electroporated into Agrobacterium tumefaciens GV3101 carrying the helper plasmid pSoup for transformation of Arabidopsis plants (Ecotype Columbia, Col-0) by vacuum invasion [35 ]. Transformed seeds were selected for resistance to kanamycin, hygromycin or BASTA. Leaf material was collected from selected plants and DNA was extracted for PCR analysis. Plants containing the transgene are capable of self-pollinating. The transgenic lines underwent two more rounds of screening and characterization using PCR and enzyme activity detection.

[0139] Generally, plants are grown under the conditions of 8 hours of light / 16 hours of darkness, and the photosynthetically active photon flux density (PPFD) is 100 or 600 μmol quantum m-2s-1 (normal and increased light intensity, respectively), and the temperature is adjusted to 22°C duri...

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Abstract

The present invention is concerned with the improvement of aghcobiological traits in plants. More specifically, it relates to a plant cell comprising in its chloroplasts enzymatic activities for converting glycolate into malate. Preferably, said plant cell comprises a first polypeptide having glycolat oxidase activity, a second polypeptide having malate synthase activity and a third polypeptide having catalase activity or a first polypeptide having glycolat dehydrogenase activity and a second polypeptide having malate synthase activity. Also encompassed is a plant comprising said plant cells as well as seeds obtainable from the said plants. The present invention, furthermore, relates to a method for producing a transgenic plant or a plant cell having an increased water use efficiency or increased yield. The present invention contemplates polynucleotides comprising a combination of nucleic acids encoding the aforementioned polypeptides as well as vectors comprising the polynucleotides and uses thereof.

Description

field of invention [0001] The present invention relates to the improvement of the agrobiological properties of plants. In particular, the invention relates to plant cells comprising in their chloroplasts enzymatic activity to convert glycolic acid to malic acid. Preferably, the plant cell comprises: a first polypeptide having glycolate oxidase activity, a second polypeptide having malate synthase activity, and a third polypeptide having catalase activity; or having glycolate dehydrogenase An active first polypeptide and a second polypeptide having malate synthase activity. The invention also relates to plants comprising the above-mentioned plant cells, and seeds obtainable from such plants. The present invention further relates to methods of producing transgenic plants or plant cells with increased water use efficiency or increased yield. The present invention relates to polynucleotides comprising nucleic acid combinations encoding the above polypeptides, vectors comprising...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N9/08C12N9/10C12N15/82C12N5/04A01H5/00A01H5/10
CPCC12N15/8243C12N9/0065C12N15/8261C12N9/0006C12N9/1025Y02A40/146
Inventor V·G·莫里诺U-I·弗卢格
Owner UNIV OF COLOGNE
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