Application of rice bacterial leaf blight resistance gene Xa14 in improving rice disease resistance
A technology of leaf blight resistance and 1.xa14, applied in the field of plant genetic engineering, can solve problems such as yield reduction and rice withering
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Embodiment 1
[0024] Embodiment 1: Positional cloning of Xa14 gene
[0025] 1. Fine mapping of dominant gene Xa14
[0026] Xa14-containing F 2 group. In order to identify the dominant and recessive nature of the gene, F 2 315 individuals were randomly selected from the population to form a random population. According to the lesion length investigated after inoculation, it was determined that Xa14 was a dominant disease resistance gene ( figure 1). PCR and polyacrylamide gel electrophoresis, as well as SSR and SNP molecular markers were used to detect the separation of molecular markers of each F2 individual plant. According to F 2 The genetic linkage map of molecular markers in rice chromosomes was constructed using JoinMap3.0 software. According to the previous research results, using PCR and polyacrylamide gel electrophoresis, RM17487 (5′-CGGAGCATGTGGAGAGGAACTCG-3′ / 5′-GGAGAGGGCAAGGGCTTCTTCG-3′) and RM17524 (5′-TCCAGCTAGTTTGGTTGCATACG-3′ / 5 '-GGACAATGAACTACAGTAGCACACG-3') molecular...
Embodiment 2
[0034] Embodiment 2: Structure and coding product analysis of Xa14 candidate gene
[0035] 1. Gene end sequence analysis
[0036] The total RNA of rice IRBB14 was extracted according to the conventional method, and then Micro-FastTrack of Invitrogen Company was used TM 2.0 Kit (Invitrogen K1520-02) to extract mRNA, and then use Invitrogen's GeneRacer Core Kit kit to determine Xa14 in rice family IRBB14 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 Xa14-1R (5'-CAAAGCTGATGTCCCGTTCCTTAGATGTGC-3') and UPM (included in the kit); the second round of amplification primers is Xa14-2R (5'-ATGCCTTGGACCGTTTCTTG- 3') and NUP (included in the kit); primers for the first round of 3'-RACE amplification are Xa14-1F (5'-CAGGGCTATGAGCTATGCCC-3') and UPM; primers for the second...
Embodiment 3
[0042] Example 3: Functional Verification and Application of Xa14 Gene
[0043] The present invention adopts the genetic transformation method mediated by Agrobacterium, and further verifies the function of the gene by transferring the Xa14 gene into the susceptible rice variety IR24 (International Rice Research Institute).
[0044] 1. Construction of genetic transformation vector
[0045] 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 10mMdNTPs, 1 μl each of 10 μM primers, 0.5 μl high-fidelity Taq enzyme; 37.5 μl sterilized double-distilled water...
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