Rice disease-resistant related gene OsDR2
A genome and rice blast fungus technology, applied in the field of plant biology, can solve problems such as disease resistance and improvement of rice varieties that have not been well used
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Embodiment 1
[0034] Example 1: Isolation and cloning of the OsDR2 gene
[0035] 1. Prediction of homologous gene structure with OsDR2 gene
[0036] In the present invention, the sequence (1634bp) of the above-mentioned cDNA clone EI5P11 is used as a template, and the public nucleotide database GenBank (http: / / www.ncbi .nlm.nih.gov), found that a segment (from 31,101 to 29,570 bp) of a 136,107bp sequence (GenBank accession number: AP003846) from the rice variety Nipponbare in the database was located on rice chromosome 7 and 27 of the EI5P11 sequence The homology at 1555 reaches 96% (E value = 0), suggesting that AP003846 contains genes homologous to OsDR2 ( image 3 ).
[0037] The genes included in the sequence of AP003846 were analyzed using GENSCAN (http: / / genes.mit.edu / GENSCAN.html) gene structure prediction software. Analysis results showed that the coding sequence of the gene homologous to the EI5P11 cDNA sequence was located at 31,751 to 29,840 bp of the AP003846 sequence ( Figur...
Embodiment 2
[0043] Embodiment 2: OsDR2 gene structure analysis
[0044] 1. Extraction of total RNA
[0045] Total RNA was extracted using TRIzol Reagent (purchased from Invitrogen, USA). The operation method was carried out according to the kit instructions provided by the company.
[0046] 2. Determination of gene introns
[0047] Using the gene prediction software GENSCAN (http: / / genes.mit.edu / GENSCAN.html) to analyze the genomic DNA sequence (2212bp) containing the OsDR2 gene segment, the results show that there is an intron in the coding segment of the OsDR2 gene ( Image 6 ). The present invention designs a pair of PCR primers according to the DNA sequences on both sides of the predicted intron: 5P11F2 (5'-GTTCATCGACGAGATGACCA-3', located at 118 to 137bp of the sequence shown in SEQ ID NO: 1 in the sequence table) and 5P11GSP2 (5 '-GTCGTAC GGCCGGTTCTTGA-3', located at 704 to 685 bp of the sequence shown in SEQ ID NO: 1 in the Sequence Listing). Genomic DNA and RNA reverse trans...
Embodiment 3
[0048] Embodiment 3: Analysis of OsDR2 gene coding product
[0049] According to the results of BLAST analysis, the protein encoded by the OsDR2 gene has a high homology with a class of auxin-responsive proteins. For example, the amino acid identity of the coding product of the OsDR2 gene and the coding product of the soybean GH3 gene (Hagen et al., Auxin-induced expression of the soybean GH3 promoter in transgenic tobacco plants, 1991, Plant Mol. Biol. 17: 567-579) is 67% ( E value = 0), and the coding product of tobacco cDNA Nt-gh3 (Roux and Perrot-Rechenmann, Isolation by differential display and characterization of a tobacco coauxin-responsive cDNA Nt-gh3 related to GH3, 1997, FEBS Lett.419: 131 -136) has an amino acid identity of 75% (E value = 0), and the coding product of the Arabidopsis DFL1 gene (Nakazawa et al., DFL1, an auxin-responsiveGH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light respo...
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