Protein phd11, and coding gene thereof and application of protein phd11 and coding gene in breeding of male sterile line of corn
A technology that encodes genes and proteins, applied in the biological field, can solve problems such as inaccurate identification, increased seed production costs, and loose linkage
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Embodiment 1
[0071] Example 1, map-based cloning of the Phd11 gene associated with maize male sterility
[0072] 1. Phenotype identification of maize mutant phd11-d
[0073] 1. Observe the phenotypes of the maize mutant phd11-d (hereinafter referred to as the mutant) and the maize inbred line B73 (hereinafter referred to as the wild type).
[0074] The results showed that there was no significant difference in the vegetative growth status between the maize mutant phd11-d and the maize inbred line B73 (see figure 1 middle A and B); at the pollen stage, the anthers of the maize mutant phd11-d could not dehisce naturally (see figure 1 Middle C and D); by microdissection, it was found that the anthers of the maize mutant phd11-d were severely atrophied (see figure 1 Middle E and F).
[0075] 2. Identification of anther fertility
[0076]The anthers of maize mutant phd11-d or maize inbred line B73 were taken, crushed (in order to obtain pollen grains), and stained with 1% I2-KI solution...
Embodiment 2
[0100] Embodiment 2, maize Phd11 gene allelic test
[0101] In order to further prove and confirm that the phenotype of the maize mutant phd11-d is caused by the mutation of the Phd11 gene, the inventors of the present invention have studied the phd11-mu mutant. The full name of the phd11-mu mutant is phd11-mu (Mu1081314::Mu), which is also a male sterile line.
[0102] 1. Observe the phenotypes of the phd11-mu mutant and the maize inbred line B73.
[0103] The results showed that there was no significant difference in the vegetative growth status between the phd11-mu mutant and the maize inbred line B73 (hereinafter referred to as the wild type) (see Figure 4 middle A and B); during the pollen stage, the anthers of the phd11-mu mutant cannot dehisce naturally (see Figure 4 Middle C and F); by microdissection, severe atrophy of the anthers of the phd11-mu mutant was found (see Figure 4 Middle D and G).
[0104] 2. Identification of anther fertility
[0105] The anthers...
Embodiment 3
[0112] Embodiment 3, the expression analysis of maize Phd11 gene in each organ
[0113] 1. Extract maize inbred line B73 tissue (root, stem, leaf, leaf ear, tassel, anther of 1.0-1.5mm, anther of 1.5-2.0mm, anther of 2.0-2.5mm, anther of 2.5-3.0mm, Anthers larger than 3.0 mm, grains 3 days after pollination, grains 6 days after pollination, or grains 9 days after pollination) were reverse transcribed to obtain cDNA of maize inbred line B73 tissue.
[0114] 2. Using the cDNA of the maize inbred line B73 tissue as a template, real-time fluorescent quantitative PCR was used to detect the relative expression level of the PHD11 gene (maize Action was used as an internal reference).
[0115] The primers used to detect PHD11 gene were 5'-TCTCTGTCGCTGCGCCGGTTG-3' and 5'-GGAGCCACCAACACGACCA-3'.
[0116] The primers used to detect maize Action were 5'-GAGATGCCTGATGGTCAGGTCA-3' and 5'-AGTTGTACGTGGCCTCATGGAC-3'.
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