Oryza officinalis anti-Xanthomonas oryzae major gene Xa3/Xa26-2 and application for improving disease resistance of rice thereof
A leaf blight resistance and leaf blight technology, applied in the field of genetic engineering, can solve problems such as yield and quality decline
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Embodiment 1
[0023] Example 1: Isolation and clone Xa3 / Xa26-2 gene from medicinal wild rice and analysis of gene structure
[0024] 1. Identification of large fragments of DNA carrying homologous sequences of Xa3 / Xa26 genes
[0025]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 The specific PCR primers RKa-11L (5'-TTGGCTTGAACGGCTTAACT-3') and RKa-1R (5'-AAGATGAAATATGCTCGGTGGT-3') amplified the DNA fragments of Xa3 / Xa26 gene and MRKa from Minghui 63, and amplified Each about 1kb in length ( figure 2 ). The two PCR amplification products were mixed as probes to screen the genomic BAC (bacterial artificial chromosome) library of medicinal wild rice (Oryza officinalis) (Ammiraju et al., 2006), and 9 positive BAC clones were identified. The nine positive BAC clones (Ammiraju et al., 2006) were donated by Professor Rod Win...
Embodiment 2
[0036] Example 2: Functional verification of Xa3 / Xa26-2 gene
[0037] 1. Construction of genetic transformation vector
[0038] 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 11.9kb fragment that comprises the coding region of Xa3 / Xa26-2 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. A ligation reaction was performed with the recovered fragment containing the Xa3 / Xa26-2 gene and the purified vector. The positive clone was verified by enzyme digestion, and the obtained recombinant plasmid was named D101O.
[0039] 2. Genetic Transformation and T 0 Genetic Transformation Plant Ana...
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