Rice retrotransposon gene LOC_Os11g45295 and encoding protein and application thereof
A technology of transgenic plants and proteins, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of easy mutation of pathogenic varieties, narrow resistance spectrum, difficult to use, etc.
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Embodiment 1
[0071] Example 1. Method for knocking out rice retrotransposon gene LOC_Os11g45295 based on pYLCRISPR / Cas9 system
[0072] 1. Sequence analysis and target sequence screening of rice retrotransposon gene LOC_Os11g45295
[0073] The nucleotide sequence of the rice retrotransposon gene LOC_Os11g45295 is shown in sequence 2, and the amino acid sequence of the protein encoded by it is sequence 1 in the sequence listing. Sequence analysis shows that the gene includes 4 exons in total, which are sequence 2 1-26 (first exon), 1308-1642 (second exon), 2610-2709 ( Exon 3), positions 4359-4737 (exon 4).
[0074] The sequence on the second exon of the rice retrotransposon gene LOC_Os11g45295 was used as the target sequence of LOC_Os11g45295-T1 and LOC_Os11g45295-T2 in the targeted knockout method of the rice retrotransposon gene LOC_Os11g45295 based on the pYLCRISPR / Cas9 system.
[0075]After extensive screening, it was determined that the pYLCRISPR / Cas9 technology was used to target th...
Embodiment 2
[0106] Example 2. Application of pYLCRISPR / Cas9 technology-based targeted knockout method in rice varieties
[0107] 1. Targeted knockout of LOC_Os11g45295 gene by pYLCRISPR / Cas9 technology
[0108] The recombinant Agrobacterium EH105-Cas9-LOC_Os11g45295 was used to infect the callus induced by mature embryos of the rice variety Nipponbare (hereinafter referred to as wild-type rice), and the obtained rice transformed plants were respectively named NIP-Cas9-LOC_Os11g45295; the specific methods of the experiment are as follows:
[0109] 1. Inoculate the recombinant Agrobacterium obtained in Example 1 into YEB liquid medium (containing 50 μg / ml kanamycin and 20 μg / ml rifampicin), and culture it with shaking at 28° C. and 200 rpm until the OD600 is 0.6-0.8 ; Centrifuge at 5000rpm and 4°C for 5min, and resuspend the bacterial pellet with AAM liquid medium (acetosyringone concentration: 200μM / L, pH 5.2) until the OD600 is 0.6-0.8.
[0110] 2. Remove the glumes from the mature seeds...
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