Detailed Molecular Characterisation of the Transgenic Potato Line, AppA6, Modified with the Apple (Malus domestica) Polygalacturonase Inhibiting Protein 1 (pgip1) Gene
Current safety assessment of genetically modified crops requires detailed information about the insertion of the transgene and the effect of its expression on the biochemistry and physiology of the host plant. Whilst the intended effect of the transformation can be verified through phenotypic screen...
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Published in | Potato research Vol. 59; no. 2; pp. 129 - 147 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
Dordrecht
Springer Netherlands
01.06.2016
Springer Nature B.V |
Subjects | |
Online Access | Get full text |
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Summary: | Current safety assessment of genetically modified crops requires detailed information about the insertion of the transgene and the effect of its expression on the biochemistry and physiology of the host plant. Whilst the intended effect of the transformation can be verified through phenotypic screening, molecular approaches are required to observe unintended effects. We investigated the molecular details of the integration of a
polygalacturonase inhibiting protein 1
gene from
Malus domestica
(
Mdpgip1
), overexpressed in
Solanum tuberosum
(cv BP1) for enhanced resistance against
Verticillium
wilt. Genome walking studies of the selected AppA6 transformant revealed that the T-DNA containing the
Mdpgip1
transgene under control of the CaMV 35S promoter was inserted into the genome without any non-T-DNA sequences from the pCAMBIA2300 vector. Sequence data indicate that the insertion of the
Mdpgip1
transgene was in a gene-rich region of chromosome 1, adjacent to the photosystem
Q
B
gene but without disruption of structural genes. Transcriptome-based cDNA-representational difference analysis revealed the distinctive expression of
Mdpgip1
in the transgenic AppA6 line, verifying the intended effect. Protein extracts from the transgenic plants inhibited the activities of
Verticillium dahliae
polygalacturonases in in vitro studies, showing that the transgene is expressed to produce an active PGIP defense protein. cDNA-AFLP fingerprinting revealed genes that were differentially expressed, including genes encoding tryptophan/tyrosine permease, Ef-Tu domain and SKP1-like 1A proteins. qRT-PCR indicated that the
Mdpgip1
transgene insertion resulted in increased expression in the AppA6 transgenic of the xyloglucan endotransglycosylase (
xth
) gene and an endogenous
Stpgip1
gene. These unintended changes were either caused by the constitutive expression of the
Mdpgip1
transgene or transformation-related somaclonal variation. The results indicate that the stable, single copy integration of the
Mdpgip1
gene in the AppA6 transgenic line did not disrupt any structural genes but caused unintended effects that affected gene expression compared to the parental counterpart under the non-stressed experimental conditions investigated. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0014-3065 1871-4528 |
DOI: | 10.1007/s11540-016-9316-x |