Building method and breeding method for rice male-fertile controllable line
A technology of male fertility and construction method, which is applied in the field of plant genetic engineering, can solve the problems such as the lack of a genetic engineering hybrid seed production system, and achieve the effect of large degree of freedom, complete sterility and stable sterility
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Embodiment 1
[0030] Example 1 Positional cloning and functional verification of rice male sterility mutant gene and its functional fertility gene
[0031] A japonica rice variety 02428 was mutagenized with cobalt 60-γ rays, and a single-locus recessive sporophytic male sterile mutant was obtained, named msl12 ( figure 2 ). Will msl12 F 2 targeting groups. With the PCR-based insertion / deletion polymorphism molecular markers listed in Table 1, about 10500 strains of F 2 individual will msl12 Mapped to a 43 kb interval on chromosome 6 ( image 3 A). There are 2 predicted genes in this interval (gene number Os06g006700 and Os06g006800 ) 。 DNA sequencing analysis showed that, compared with the genome sequence of the original parent, msl12 of Os06g006800 There is no variation, while in Os06g006700 A 1479 bp fragment including 1 exon and 2 introns was missing ( image 3 B, SEQ ID No. 1). The present invention named the wild-type (functional) gene as OsABCG15 (SEQ ID N...
Embodiment 2
[0035] Example 2 Isolation of chemically inducible gene promoters from rice
[0036] In the present invention, a mixed aqueous solution of 0.05% propiconazole and 0.05% myclobutanazole is applied to rice leaves, and after 24 hours, the anthers from the meiosis stage to the microspore stage of the treatment and the control are taken, and RNA is extracted. Using the rice genome-wide expression chip (OsAffx) hybridization (completed by Boao Biological Co., Ltd.) screening, it was found that the expression level of a gene represented by a gene probe (OsAffx.13615.1.S1_at) was very low in non-treated samples, while in Expression levels in treated samples were substantially higher than in non-treated controls. Further verification by quantitative RT-PCR confirmed that its background expression was low, while propiconazole or myclobutanil could increase its expression level, and the common expression level of these two inducers was higher ( Figure 4 ).
Embodiment 3
[0037] Example 3 Construction of Rice Male Fertility Controlled Line
[0038] With specific primer 5'-CTTGA GGCCGGCC TAAATTATTTTTTTCCAATATTTA-3’ (underlined Fse I cut point) and TCA GGTACC GATCGGTGATCCCTCTCCAA-3’ (underlined Kpn I cutting point) from rice genome PCR amplification described promoter sequence; With 5 '-GATC GGTACC TGATTAAAAGGAGAGA AG-3’ (underlined Kpn I cut point) and 5'-GGA GGATCC AGACCTCGCGCA-3' (underlined as Bam H I cutting point) PCR amplification of wild type OsABCG15 Near full-length cDNA coding sequence; with 5'-CTCT GGATCC TCCTCGCCATGGC-3' (underlined as Bam HI cutting point) and 5'-CCTA CTCGAG CTCCCTGCAGGTGAGGA-3' (underlined as xho I cutting point) PCR amplification OsABCG15 A small part of the cDNA coding sequence and the 3' terminator sequence. Cut out the cohesive ends of these 3 fragments with the indicated restriction endonucleases, and connect them into a recombinant gene expression cassette structure ( Figure ...
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