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 Locational cloning and functional verification of rice male sterility mutant genes and their functional fertility genes
[0031] Cobalt 60-γ rays were used to mutate the rice japonica variety 02428, and a single-locus recessive sporophyte male sterile mutant was obtained, which was named msl12 ( figure 2 ). will msl12 Cross with indica rice variety Huang Huazhan to obtain F 2 Target groups. Using PCR-based insertion / deletion polymorphism molecular markers listed in Table 1, about 10,500 strains of F 2 Individual will msl12 Located in a 43 kb interval on chromosome 6 ( image 3 A). There are 2 predictive genes (gene number Os06g006700 with Os06g006800 ) 。 DNA sequencing analysis showed that compared with the genome sequence of the original parent, msl12 of Os06g006800 No mutation, but in Os06g006700 A fragment of 1479 bp including 1 exon and 2 introns was deleted ( image 3 B, SEQ ID No. 1). The present invention names the wild-type (functional) gene a...
Embodiment 2
[0035] Example 2 Isolation of chemically inducible gene promoter from rice
[0036] In the present invention, a mixed aqueous solution of 0.05% propiconazole and 0.05% myclobutanil is applied to rice leaves. After 24 hours, the anthers from the meiotic stage to the microspore stage of the treatment and control are taken to extract RNA. Using rice whole genome expression chip (OsAffx) hybridization (completed by Boao Biological Co., Ltd.), it was found that the expression level of the gene represented by 1 gene probe (OsAffx.13615.1.S1_at) in untreated samples was very low, while The expression level of treated samples was much higher than that of non-treated controls. It was further verified by quantitative RT-PCR that its background expression was low, while propiconazole or myclobutanil could increase its expression level, and the expression level shared by these two inducers was higher ( Figure 4 ).
Embodiment 3
[0037] Example 3 Construction of Controllable Rice Male Fertility Line
[0038] With specific primer 5’-CTTGA GGCCGGCC TAAATTATTATTTTTCCAATATTTA-3’ (underlined Fse I cut point) and TCA GGTACC GATCGGTGATCCCTCCTCAA-3’ (underlined Kpn I cut point) PCR amplifies the promoter sequence from rice genome; 5’-GATC GGTACC TGATTAAAAGGAGAGA AG-3’ (underlined Kpn I cut point) and 5’-GGA GGATCC AGACCTCGCGCA-3’ (underlined Bam H I cut point) PCR amplified wild type OsABCG15 Near-full-length cDNA coding sequence; with 5’-CTCT GGATCC TCCTCGCCATGGC-3’ (underlined Bam Hi cut point) and 5’-CCTA CTCGAG CTCCCTGCAGGTGAGGA-3’ (underlined Xho I cut point) PCR amplification OsABCG15 A small part of the cDNA coding sequence and 3'terminator sequence. Cut out the sticky ends of these 3 fragments with the restriction endonuclease shown, and ligate them into a recombinant gene expression cassette structure ( Figure 5 ), its DNA sequence is shown in SEQ ID No.6. This expression cassette se...
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