Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi
| Autor: | Jan Kreuze, Dennis Solorzano, Ida Bartolini, Maria Lupe Roman, Wilmer J. Cuellar, Jeanette Orbegozo, Marc Ghislain |
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| Jazyk: | angličtina |
| Předmět: |
0106 biological sciences
0301 basic medicine DNA Bacterial Inverted repeat Genetic Vectors Cre recombinase Genetically modified crops Biology 01 natural sciences Marker gene 03 medical and health sciences Viral Envelope Proteins Genetics Northern blot Gene Solanum tuberosum Original Paper Integrases Inverted Repeat Sequences fungi Potato leaf-roll virus resistance food and beverages Plants Genetically Modified Virology Luteoviridae Transformation (genetics) 030104 developmental biology Marker free RNAi Cre-loxP RNA Interference Animal Science and Zoology Cre-Lox recombination Agronomy and Crop Science 010606 plant biology & botany Biotechnology |
| Zdroj: | Transgenic Research |
| ISSN: | 0962-8819 |
| DOI: | 10.1007/s11248-016-9976-y |
| Popis: | An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB. Electronic supplementary material The online version of this article (doi:10.1007/s11248-016-9976-y) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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