Gene for resistance to plant disease
A plant resistance technology, applied in the field of genes for resistance to plant diseases, can solve the problems of reduced plant adaptability, low yield performance, and complexity
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Embodiment 1
[0207] The introduction of resistance-conferring genes into sugar beet subspecies by CRISPR-mediated homologous recombination is shown in Example 1.
[0208] An alternative approach consists in increasing the expression of the nucleic acid molecules according to the invention in plants. This can be achieved by modifying the native promoter, preferably by gene editing or site-directed mutagenesis (which is mediated by a site-directed nuclease) and optionally a repair model. Examples of such nucleases are listed above. The expression of the nucleic acid molecule according to the invention can likewise be increased by fusing the nucleic acid molecule to a heterologous promoter which exhibits a higher activity compared to the native promoter, especially after infection with Cercospora. Fusions can also be performed by site-directed nucleases and repair models, as well as by direct insertion after a double-strand break.
[0209] As mentioned above, the method for increasing Cerco...
Embodiment
[0288] Example 1: Introduction of resistance-conferring genes into sugar beet subspecies by CRISPR-mediated homologous recombination
[0289] crRNA design and selection:
[0290] With the aid of the CRISPR RGEN tool, suitable crRNAs for the induction of Cpf1-mediated double-strand breaks were designed (Park J., Bae S. and Kim J.-S. Cas-Designer: A web-based tool for choice of CRISPR -Cas9 target sites Bioinformatics 31, 4014-4016(2015); Bae S., Park J. and Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches forpotential off-target sites of Cas9 RNA-guided endonucleases Bioinformatics 30, 1473-1475 (2014)). For this purpose, a suitable protospacer sequence was found within the genomic DNA sequence having a length of 500-1300 bp, flanking the 5'-end and 3'-end of the Cercospora resistance gene from sea beet. To ensure the functionality of the endonuclease Cpf1 of Lachnospiraceae bacterium ND2006 (Lb), a protospacer sequence with a length of 24 nt was selected,...
Embodiment 2
[0315] Example 2: Introduction of resistance-conferring genes as transgenes in sugar beet subspecies by genetic transformation
[0316] The transgenic approach to generate Cercospora-resistant plants is not only useful for surrogate verification of LRR genes as resistance-conferring genes, but also as a means of generating transgenic resistant genes that confer resistance to novel Cercospora species or increase resistance in existing Cercospora species. means of sexual affairs.
[0317] The binary vector pZFN-nptII-LRR was generated by the following standard cloning procedure: Within the T-DNA of this vector, the cDNA of the resistance gene according to SEQ ID NO 2 was cloned together with its native promoter sequence. T-DNA also includes the neomycin phosphotransferase II (nptII) gene, which confers resistance to aminoglycoside antibiotics such as kanamycin or paromomycin. These antibiotic resistances are used to select for transgenic plant cells and tissues. The NOS promot...
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