Application of rice bacterial leaf blight resistance gene Xa2 in improving rice disease resistance
A leaf blight resistance and gene technology, applied in the field of plant genetic engineering, can solve the problem that the main disease resistance genes are not found to be alleles and other issues
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Embodiment 1
[0021] Example 1: Identification of Xa2 Candidate Genes
[0022] To further analyze the Xa2 gene, the RGAf in rice varieties IRBB2 (International Rice Research Institute), IR24 (International Rice Research Institute), Zhonghua 11 (Institute of Crop Science, Chinese Academy of Agricultural Sciences) and Mudanjiang 8 (Heilongjiang University) were sequenced, respectively, After sequencing, the sequences were named as: RGAf-BB2, RGAf-IR24, RGAf-ZH11, RGAf-MDJ8. Homology analysis was performed on the sequences RGAf-BB2, RGAf-IR24, RGAf-ZH11, RGAf-MDJ8, RGAf-BB1, RGAf-BB14 and RGAf-Nip. After comparison, it was found that the genome sequences of RGAf-BB2 and RGAf-ZCL were similar to those of RGAf- The genome sequences of BB1 and RGAf-BB14 are very similar ( figure 1 ).
[0023] Further analysis of the differences in the RGAf sequences of different rice varieties. RGAf-MDJ8 also encodes a complete NB-LRR protein, which has a sequence identity of 99.9% with RGAf-Nip; both RGAf-IR2...
Embodiment 2
[0024] Embodiment 2: Structure and coding product analysis of Xa2 candidate gene
[0025] 1. Gene end sequence analysis
[0026] The total RNA of rice IRBB2 was extracted according to the conventional method, and then Micro-FastTrack of Invitrogen Company was used TM2.0 Kit (Invitrogen K1520-02) to extract mRNA, and then use Invitrogen's GeneRacer Core Kit kit to determine Xa2 in rice family IRBB2 by 5'-RACE (rapid amplification of cDNA end) and 3'-RACE analysis methods The 5' and 3' end sequences of the candidate genes were operated according to the instructions of the kit provided by Invitrogen. The primers for the first round of 5'-RACE amplification are Xa2-1R (5'-CAAAGCTGATGTCCCGTTTCTTAGATGTGC-3') and UPM (included in the kit); the second round of amplification primers is Xa2-2R (5'-ATGCCTTGGACCGTTTCTTG- 3') and NUP (included in the kit); primers for the first round of 3'-RACE amplification are Xa2-1F (5'-CAGGGCTATGAGCTATGCCC-3') and UPM; primers for the second round of...
Embodiment 3
[0032] Example 3: Functional Verification and Application of Xa2 Gene
[0033] The invention adopts the genetic transformation method mediated by Agrobacterium, and further verifies the function of the gene by transferring the Xa2 gene into the susceptible rice variety Zhonghua 11 (Institute of Crop Science, Chinese Academy of Agricultural Sciences).
[0034] 1. Construction of genetic transformation vector
[0035] The used starting carrier of the present invention is the commonly used carrier pCAMBIA1301 ( Figure 5 , Cao et al., 2007). According to the results of genome sequencing, design primers (5'- GGTACC GGCATAAGAAATCATAGCAGTGG-3',5'-CC AAGCTT GCATGCCTGCAGGTCGACGGTGTAAGGCCTCCATTCTAACAG-3'), which included HindIII and BamHI linkers, amplified the genome of bacterial blight-resistant rice IRBB14. The total volume of the PCR reaction system is 50 μl: 1 μl DNA, 5 μl 10×buffer, 5 μl 10 mM dNTPs, 1 μl each of 10 μM primers, 0.5 μl high-fidelity Taq enzyme; 37.5 μl steri...
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