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Optimisation of transgene action at the post-transcriptional level: high quality parthenocarpic fruits in industrial tomatoes.

Pandolfini T, Rotino GL, Camerini S, Defez R, Spena A - BMC Biotechnol. (2002)

Bottom Line: An optimal expressivity of parthenocarpy correlates with a reduced transgene mRNA steady state level in DefH9-RI-iaaM flower buds in comparison to DefH9-iaaM flower buds.By using an auxin biosynthesis transgene downregulated at the post-transcriptional level, an optimal expressivity of parthenocarpy has been achieved in a genetic background not suitable for the original transgene.Thus, the method allows the generation of a wider range of expressivity of the desired trait in transgenic plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento Scientifico Tecnologico, University of Verona, Verona, Italy. pandolfini@sci.univr.it

ABSTRACT

Background: Genetic engineering of parthenocarpy confers to horticultural plants the ability to produce fruits under environmental conditions that curtail fruit productivity and quality. The DefH9-iaaM transgene, whose predicted action is to confer auxin synthesis specifically in the placenta, ovules and derived tissues, has been shown to confer parthenocarpy to several plant species (tobacco, eggplant, tomato) and varieties.

Results: UC82 tomato plants, a typical cultivar used by the processing industry, transgenic for the DefH9-iaaM gene produce parthenocarpic fruits that are malformed. UC82 plants transgenic for the DefH9-RI-iaaM, a DefH9-iaaM derivative gene modified in its 5'ULR by replacing 53 nucleotides immediately upstream of the AUG initiation codon with an 87 nucleotides-long sequence derived from the rolA intron sequence, produce parthenocarpic fruits of high quality. In an in vitro translation system, the iaaM mRNA, modified in its 5'ULR is translated 3-4 times less efficiently than the original transcript. An optimal expressivity of parthenocarpy correlates with a reduced transgene mRNA steady state level in DefH9-RI-iaaM flower buds in comparison to DefH9-iaaM flower buds. Consistent with the known function of the iaaM gene, flower buds transgenic for the DefH9-RI-iaaM gene contain ten times more IAA than control untransformed flower buds, but five times less than DefH9-iaaM flower buds.

Conclusions: By using an auxin biosynthesis transgene downregulated at the post-transcriptional level, an optimal expressivity of parthenocarpy has been achieved in a genetic background not suitable for the original transgene. Thus, the method allows the generation of a wider range of expressivity of the desired trait in transgenic plants.

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RT-PCR analysis of tomato transgenic floral buds. Analysis was performed with single strand cDNA synthesised from mRNA extracted from young flower buds of UC82 plants transformed with either DefH9-iaaM (panel b, lanes 1,2,3,4) or DefH9-RI-iaaM (panel a, lanes C3, C5, C6, C9, C10, C11; panel b, S3, S4, S5 and S6) gene. Either 0.05 fg (C3, C5, C6, C9, C10, C11 DefH9-RI-iaaM transgenic lines) or 0.2 fg (S3, S4, S5, S6 DefH9-RI-iaaM and 1, 2, 3, 4 DefH9-iaaM transgenic lines) of a 600 bp DefH9 cDNA fragment were used as internal standard in the PCR, giving an amplicon of 351 bp. The chimeric fragments are amplicons of 161 and 195 bp respectively, corresponding to the 5' end of the DefH9-iaaM and DefH9-RI-iaaM mRNAs.
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Figure 3: RT-PCR analysis of tomato transgenic floral buds. Analysis was performed with single strand cDNA synthesised from mRNA extracted from young flower buds of UC82 plants transformed with either DefH9-iaaM (panel b, lanes 1,2,3,4) or DefH9-RI-iaaM (panel a, lanes C3, C5, C6, C9, C10, C11; panel b, S3, S4, S5 and S6) gene. Either 0.05 fg (C3, C5, C6, C9, C10, C11 DefH9-RI-iaaM transgenic lines) or 0.2 fg (S3, S4, S5, S6 DefH9-RI-iaaM and 1, 2, 3, 4 DefH9-iaaM transgenic lines) of a 600 bp DefH9 cDNA fragment were used as internal standard in the PCR, giving an amplicon of 351 bp. The chimeric fragments are amplicons of 161 and 195 bp respectively, corresponding to the 5' end of the DefH9-iaaM and DefH9-RI-iaaM mRNAs.

Mentions: The transgenic state of four independent transgenic plants analysed shows that the copy number of the DefH9-iaaM gene ranged from one copy (plant 3; Fig. 2a, Lane 3) to six copies (plant 1; Fig. 2a, Lane 1). RT-PCR analysis performed with mRNA extracted from flower buds (0,5–1 cm long) shows that the transgene is expressed in all four independent plant clones (Fig. 3b, lanes 1,2,3,4). The steady state level of DefH9-iaaM mRNA estimated by competitive (Fig. 3) and real time RT-PCR (not shown) is, on the average, 1 × 10-7 of the total mRNA population, ranging from approximately 3 × 10-7 (transgenic event 1) to 5 × 10-8 (transgenic event 3) in the four independent transgenic events analysed.


Optimisation of transgene action at the post-transcriptional level: high quality parthenocarpic fruits in industrial tomatoes.

Pandolfini T, Rotino GL, Camerini S, Defez R, Spena A - BMC Biotechnol. (2002)

RT-PCR analysis of tomato transgenic floral buds. Analysis was performed with single strand cDNA synthesised from mRNA extracted from young flower buds of UC82 plants transformed with either DefH9-iaaM (panel b, lanes 1,2,3,4) or DefH9-RI-iaaM (panel a, lanes C3, C5, C6, C9, C10, C11; panel b, S3, S4, S5 and S6) gene. Either 0.05 fg (C3, C5, C6, C9, C10, C11 DefH9-RI-iaaM transgenic lines) or 0.2 fg (S3, S4, S5, S6 DefH9-RI-iaaM and 1, 2, 3, 4 DefH9-iaaM transgenic lines) of a 600 bp DefH9 cDNA fragment were used as internal standard in the PCR, giving an amplicon of 351 bp. The chimeric fragments are amplicons of 161 and 195 bp respectively, corresponding to the 5' end of the DefH9-iaaM and DefH9-RI-iaaM mRNAs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC65046&req=5

Figure 3: RT-PCR analysis of tomato transgenic floral buds. Analysis was performed with single strand cDNA synthesised from mRNA extracted from young flower buds of UC82 plants transformed with either DefH9-iaaM (panel b, lanes 1,2,3,4) or DefH9-RI-iaaM (panel a, lanes C3, C5, C6, C9, C10, C11; panel b, S3, S4, S5 and S6) gene. Either 0.05 fg (C3, C5, C6, C9, C10, C11 DefH9-RI-iaaM transgenic lines) or 0.2 fg (S3, S4, S5, S6 DefH9-RI-iaaM and 1, 2, 3, 4 DefH9-iaaM transgenic lines) of a 600 bp DefH9 cDNA fragment were used as internal standard in the PCR, giving an amplicon of 351 bp. The chimeric fragments are amplicons of 161 and 195 bp respectively, corresponding to the 5' end of the DefH9-iaaM and DefH9-RI-iaaM mRNAs.
Mentions: The transgenic state of four independent transgenic plants analysed shows that the copy number of the DefH9-iaaM gene ranged from one copy (plant 3; Fig. 2a, Lane 3) to six copies (plant 1; Fig. 2a, Lane 1). RT-PCR analysis performed with mRNA extracted from flower buds (0,5–1 cm long) shows that the transgene is expressed in all four independent plant clones (Fig. 3b, lanes 1,2,3,4). The steady state level of DefH9-iaaM mRNA estimated by competitive (Fig. 3) and real time RT-PCR (not shown) is, on the average, 1 × 10-7 of the total mRNA population, ranging from approximately 3 × 10-7 (transgenic event 1) to 5 × 10-8 (transgenic event 3) in the four independent transgenic events analysed.

Bottom Line: An optimal expressivity of parthenocarpy correlates with a reduced transgene mRNA steady state level in DefH9-RI-iaaM flower buds in comparison to DefH9-iaaM flower buds.By using an auxin biosynthesis transgene downregulated at the post-transcriptional level, an optimal expressivity of parthenocarpy has been achieved in a genetic background not suitable for the original transgene.Thus, the method allows the generation of a wider range of expressivity of the desired trait in transgenic plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento Scientifico Tecnologico, University of Verona, Verona, Italy. pandolfini@sci.univr.it

ABSTRACT

Background: Genetic engineering of parthenocarpy confers to horticultural plants the ability to produce fruits under environmental conditions that curtail fruit productivity and quality. The DefH9-iaaM transgene, whose predicted action is to confer auxin synthesis specifically in the placenta, ovules and derived tissues, has been shown to confer parthenocarpy to several plant species (tobacco, eggplant, tomato) and varieties.

Results: UC82 tomato plants, a typical cultivar used by the processing industry, transgenic for the DefH9-iaaM gene produce parthenocarpic fruits that are malformed. UC82 plants transgenic for the DefH9-RI-iaaM, a DefH9-iaaM derivative gene modified in its 5'ULR by replacing 53 nucleotides immediately upstream of the AUG initiation codon with an 87 nucleotides-long sequence derived from the rolA intron sequence, produce parthenocarpic fruits of high quality. In an in vitro translation system, the iaaM mRNA, modified in its 5'ULR is translated 3-4 times less efficiently than the original transcript. An optimal expressivity of parthenocarpy correlates with a reduced transgene mRNA steady state level in DefH9-RI-iaaM flower buds in comparison to DefH9-iaaM flower buds. Consistent with the known function of the iaaM gene, flower buds transgenic for the DefH9-RI-iaaM gene contain ten times more IAA than control untransformed flower buds, but five times less than DefH9-iaaM flower buds.

Conclusions: By using an auxin biosynthesis transgene downregulated at the post-transcriptional level, an optimal expressivity of parthenocarpy has been achieved in a genetic background not suitable for the original transgene. Thus, the method allows the generation of a wider range of expressivity of the desired trait in transgenic plants.

Show MeSH
Related in: MedlinePlus