Gene Rf5 for restoring corn C-type cytoplasmic fertility and application thereof
A maize and cytoplasmic technology, applied in the field of genes, can solve problems such as timeliness limitations, and achieve the effects of improving production efficiency, reducing breeding costs, and shortening breeding cycles
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Embodiment 1
[0083] Example 1: Obtaining the Sequence of Maize Type C Cytoplasmic Restorer Gene Rf5
[0084] The restorer gene Rf5 derived from Fengke 1 was self-crossed after 5 consecutive generations of backcrossing, and finally the Rf5 gene was introduced into the background of the inbred line Mo17 to obtain a restorer line, which was named 6233 and recorded as Cms-6233. Using the sterile line Cms-CMo17 (rf4rf4rf5rf5) and the restorer line Cms-C6233 (rf4rf4Rf5Rf5) as experimental materials, the BC1 segregation population of Rf5 was constructed. On the basis of preliminary positioning of the restorer gene Rf5 with 1,500 isolated populations, the scale of the isolated population was expanded to 32,000 strains, and molecular markers such as SSR and Indel were developed with the help of B73 genome information, and the restored gene Rf5 was finely mapped ( figure 1 ), the restorer gene Rf5 was positioned between the molecular markers chr2.09-36 and chr2.09-6+1, with a physical distance of 82...
Embodiment 2
[0097] Example 2: Rf5 corn transgenic experiment and fertility analysis of transgenic materials
[0098] In this example, the tetrad anther of the restorer line Cms-6233Rf5Rf5 was used as the material, and the full-length cDNA of the Rf5 gene was amplified by reverse transcription.
[0099] (1) Construction and transformation of Rf5 overexpression transgene vector
[0100] According to the cDNA sequence of the Rf5 gene involved primers:
[0101] p3300U-BamH1-F: 5'-cgcggatccATGCCGTCATGTGCCCGCAT-3'
[0102] p3300U-Sacl-R:5'-cgagctcTCACGGACTGGCCTCTGCAA-3'
[0103] Using the full-length cDNA of the Rf5 gene as a template, the full-length sequence of Rf5 was amplified, and the PCR product was digested with BamhI and SacI to recover the target fragment, and connected to the p3300U vector recovered by double-digestion with the same two enzymes to construct into the overexpression vector p3300U-Rf5 ( figure 2 ). The vector was introduced into maize transformation recipient inbre...
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