Oryza minuta bacterial blight-resisting major gene Xa3/Xa26-3 and application thereof in improving disease resistance of paddy rice
A technology for leaf blight resistance and leaf blight, which is applied in the field of plant genetic engineering and can solve problems such as yield and quality decline
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Embodiment 1
[0024] Example 1: Isolation and clone Xa3 / Xa26-3 gene from Oryza sativa and gene structure analysis
[0025] 1. Identification of large fragments of DNA carrying homologous sequences of Xa3 / Xa26 genes
[0026]The researchers of the present invention first use the specific PCR primers RKb-3'race2 (5'-TGGTCAAATACCGGAAGGAG-3') and RKb-2R (5'-CAGTCCACCACATGGACAAG-3') of the Xa3 / Xa26 gene and the Xa3 / Xa26 family member MRKa gene Specific PCR primers RKa-11L (5'-TTGGCTTGAACGGCTTAACT-3') and RKa-1R (5'-AAGATGAAATATGCTCGGTGGT-3') amplified Xa3 from rice variety Minghui 63 (a rice variety popularized in China) / Xa26 gene and MRKa DNA fragments, the length of each amplification is about 1kb ( figure 2 ). The two PCR amplification products were mixed as probes to screen the genomic BAC (bacterial artificial chromosome) library of Oryzaminuta (Ammiraju et al., 2006), and 7 positive BAC clones were identified. These 7 positive BAC clones (Ammiraju et al., 2006) were donated by Professo...
Embodiment 2
[0037] Example 2: Functional verification of Xa3 / Xa26-3 gene
[0038] 1. Construction of genetic transformation vector
[0039] The carrier used in the present invention is pCAMBIA1301 ( Figure 5 ), which is a commonly used rice genetic transformation vector (Sun et al., 2004). Reclaim the 13.7kb fragment that comprises the coding region of Xa3 / Xa26-3 gene, promoter and tail sequence ( Figure 4 ). At the same time, the genetic transformation vector pCAMBIA1301 was digested with restriction endonuclease SmaI; after digestion, dephosphorylated with SAP (shrimp alkaline phosphatase); extracted and purified with chloroform:isoamyl alcohol (volume ratio 24:1) Digestion product. The ligation reaction was performed with the recovered fragment containing the Xa3 / Xa26-3 gene and the purified vector. The positive clone was verified by enzyme digestion, and the obtained recombinant plasmid was named D103O.
[0040] 2. Genetic Transformation and T 0 Genetic Transformation Plant A...
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