Polynucleotides and methods for making plants resistant to fungal pathogens
A technology of nucleotides and plants, applied in botany equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve problems such as different reactions and exact effects
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[0117] In the preparation of expression cassettes, the various DNA fragments can be manipulated to provide the DNA sequence in the correct orientation and, where appropriate, in the correct reading frame. For this purpose, adapters or linkers may be used to join the DNA fragments, or other manipulations may be included to provide convenient restriction sites, remove excess DNA, remove restriction sites, and the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re-substitutions, eg transitions and transversions may be involved.
[0118] The expression cassette may also contain a selectable marker gene for selection of transformed cells. Selectable marker genes are used to select transformed cells or tissues. Marker genes include those encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes that confer resistance to herbicidal compounds, such as glufosinate ...
Embodiment 1
[0186] Fine mapping of the Rcg1 locus in specific regions of 4L
[0187] In order to map and clone the genes responsible for the resistance to Cg of the maize line MP305, lines have previously been generated that differ as little as possible from each other genetically, with the exception that the loci responsible for the resistance phenotype are present. Such lines are called near-isogenic lines. For this purpose, DE811 has been crossed with MP305 and the progeny have been backcrossed 3 times with the sensitive line DE811, at each backcross resistance to Cg and additional DE811 characteristics were selected (Weldekidan and Hawk, (1993), Maydica , 38:189-192). The resulting line was designated DE811ASR(BC3) (Weldekidan and Hawk, (1993) supra). This line was used as a starting point for fine mapping of the Rcg1 locus. It first had to know roughly where it was located in the maize genome. The locus on the long arm of chromosome 4 has previously been mapped by Jung et al. (...
Embodiment 2
[0198] Isolation of BAC clones from resistant lines and identification of candidate genes in the region of the Rcg1 locus
[0199] To isolate the gene responsible for the phenotype conferred by the Rcg1 locus, BACs containing the region between the FLP 8 and FLP 27 markers were isolated from a BAC library prepared from the resistant line DE811ASR (BC5). The library was prepared using standard techniques for preparing genomic DNA (Zhang et al. (1995) Plant Journal 7:175-184), then partially digested with HindIII, and the size-selected fragments ligated into a modified form of the commercially available vector pCC1BAC TM (Epicentre, Madison, USA). Transform into EPI300 according to the vendor's instructions (Epicentre, Madison, USA) TM After E. coli cells, 125,184 recombinant clones were arrayed in 326 384-well microtiter plates. These clones were then gridded onto nylon filters (Hybond N+, Amersham Biosciences, Piscataway, USA).
[0200]Overlapping oligonucleotide probes (...
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