Gene for regulating and controlling rice disease resistance and cell death and application thereof
A cell death, gene technology, applied in the field of plant molecular genetics
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Embodiment 1
[0029] Embodiment 1, the acquisition and phenotype of rice spl5 mutant
[0030] The rice spl5 mutant was screened from the mutagenized mutant library by γ-ray irradiation (Iwata et al., 1978) on the seeds of Oryza sativa L. ssp. Japonica Norin8. Under the environment required for the normal growth of rice, reddish-brown necrotic spots appeared spontaneously on the leaves of this mutant from the tillering stage, which increased with the growth of the plant until almost covering the entire leaf at the heading stage. At the same time, the spl5 mutant was significantly Improved resistance to rice diseases such as rice blast and bacterial blight, indicating that the normal SPL5 gene has the function of negatively regulating rice cell death and disease resistance, and the gene mutation can activate the plant's cell necrosis and disease resistance defense response .
Embodiment 2
[0031] Embodiment 2, fine mapping of rice spl5 gene
[0032] Using the rice spl5 mutant and the wild type indica rice (Oryza sativa L.ssp.Indica) Zhefu 802 for multiple backcrosses, the spl5 mutant gene was transferred into Zhefu 802, and the spl5 mutant with the genetic background of Zhefu 802 was obtained . Then use the obtained indica rice spl5 (Zhefu 802) mutant to cross with wild type indica 93-11, the F 1 All generation plants were wild-type, and in F 1 F 2 In the population, the segregation ratio of the wild type and the mutant type is about 3:1, which conforms to the law of Mendelian genetics, indicating that the rice-like disease spl5 mutant is controlled by a single recessive gene.
[0033] The spl5 mutant was first identified by Iwata et al. in Japan, and the spl5 gene was initially located on the short arm of chromosome 7 near the centromere by using morphological markers (Iwata, 1978). The inventor crossed F from spl5 mutant (Zhefu 802) and rice 9311 2 100 mu...
Embodiment 3
[0044] Example 3, Genome Sequencing and Determination of SPL5 Candidate Genes
[0045]Primers were designed according to the genome sequence of Nipponbare, and conventional PCR amplification was performed. The size of the PCR product was 1000-1500 bp, and the ends of adjacent PCR fragments overlapped each other. The PCR contig formed covered the entire 15.1kb sequence. Each PCR product was sequenced (commissioned by Invitrogen Company), and the 15.1kb sequence of the rice spl5 mutant was sequenced. It was found that in this interval, the spl5 mutant except for a base G (guanine) was missing, and the rest of the sequence was similar to that of the wild-type japonica rice Nipponbare totally agree. At the same time, the original parent of the rice spl5 mutant, the corresponding site of the japonica rice Norin8 genome, was amplified by PCR and sequenced. The results showed that the site was consistent with the sequence of the wild-type Nipponbare, and no base deletion occurred. T...
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