Method for producing mutant plants
A plant, non-synonymous mutation technology, applied in botany equipment and methods, plant products, plant gene improvement, etc., can solve the problems of plant fitness and reduced mutation dose
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Embodiment 1
[0357] Example 1 - Low mutation density protocol
[0358] In order to induce mutations, according to the details provided by Kleinhofs et al. (1978) and those provided in US 7,838,053, with the following changes, grains collected from barley plants were treated with the mutagen sodium azide (NaN 3 ): the concentration of sodium azide for mutagenesis was reduced to 0.3 mM for 2 hours.
[0359] This method induces point mutations in the genomic DNA (gDNA) of the barley grain, typically conferring randomly distributed codons for amino acid substitutions or translation termination of protein-coding DNA, i.e., resulting in protein in the protein encoded by the mutagenized DNA, respectively Variations and truncations. However, the methods of the present invention can also be used to produce cereals with point mutations in the gDNA of non-protein coding regions, such as promoters, terminators and introns.
[0360] 15kg of grain was mutagenized and planted in 30 plots of 7.5m each ...
Embodiment 2
[0361] Example 2 - RNase H2-dependent PCR (rhPCR) to detect mutants in mutant libraries
[0362]RNase H2-dependent PCR (rhPCR) (Dobosy et al., 2011) can improve the specificity of PCR amplification by utilizing so-called "blocking primers". These primers contain a single ribonucleotide residue and a 3' blocking moiety that prevents primer extension during PCR. However, primers can be activated by RNase H2, which then allows them to extend during PCR amplification. RNase H2 cleavage occurs only when the blocking primer binds to a perfect match. Even mismatches with only nucleotide changes can significantly inhibit cleavage. This means that blocking primers can be designed to specifically block the amplification of non-specific or unwanted amplicons, providing a very sensitive tool to detect rare specific nucleotide substitutions. Fluorophore-labeled probes are complementary to the 5' ends of the rhPCR primers, enabling the quantification of successful primer extension during...
Embodiment 3
[0367] Example 3 - Direct Sequencing of Low Mutagenic Barley Plants
[0368] We sequenced a library (cv.Quench) containing 6000 barley plants. Grains were mutated for 2 hours using 0.3 mM sodium azide. A library of 6000 individual M3 spikes was constructed. One kernel per ear was used for DNA extraction.
[0369] We have amplified a 790 bp fragment of the gene of interest in order to analyze the 645 bp exon 3 of this gene. Amplicons of all 6000 lines were analyzed for polymorphism. We have identified 3 individual mutants within 645bp for 6000 lines. This corresponds to 3 / (645 x 6000) = 1 mutant per 1.29 million bp, which corresponds to about 24 non-synonymous mutations in the coding region.
[0370] The three mutants correspond to the following mutation sites:
[0371] 1. Nucleotide G989>A (cDNA) corresponds to amino acid change S330>N (protein)
[0372] 2. Nucleotides C1091>T (cDNA) correspond to amino acid changes P364>L (protein)
[0373] 3. Nucleotide C858>T (cDNA)...
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