Separation and cloning of alleles Sc-j and Sc-i of pollen fertility gene loca Sc of paddy rice hybrids, and application in seed breeding
A technology of alleles and fertility genes, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve problems such as hindering utilization, achieve the effect of improving fertility and overcoming Sc hybrid sterility
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Embodiment 1
[0031] Example 1: sc Fine-positioned cloning and sequence structure analysis of
[0032] The inventor's previous studies have sc Mapped to the approximate region on rice chromosome 3 (Yang et al. 2004. Chinese Science Bulletin 49, 1718-1721). The present invention uses as figure 2 The map-based cloning method shown further clones sc Gene. According to the publicly available rice genome sequence database (http: / / www.ncbi.nlm.nih.gov), multiple molecular markers were developed in this region (Table 1). With T65 ( Sc-j / Sc-j genotype) and its near isogenic line E5 ( Sc-i / Sc-i ) to hybridize to produce F 1 and F 2 population, after linkage analysis 8689 strains of F 2 individual, put sc finely mapped to a 8.6 kb interval in japonica rice ( figure 2 A). According to the rice genome sequence database GenBank (http: / / www.ncbi.nlm.nih.gov), there is one annotation numbered in the 8.6 kb interval of the mapped Japonica rice Os03g0247300 The gene of the present inven...
Embodiment 2
[0036] Example 2: sc Functional verification of genes
[0037] Amplified from Japonica Rice by RT-PCR Sc-j cDNA fragment (located at 514-1284 bp of the coding region), ligated in reverse to maize Ubiquitin The gene promoter is connected to construct a binary vector for antisense gene expression. Transform T65 with this vector to obtain a transformant. Transgenic hemizygous (one chromosome has a transgene and the other homologous chromosome has no transgene) Sc-j The expression level decreased ( image 3 A), roughly half of the pollen was sterile ( image 3 B). prove Sc-j It is the pollen gamete sports gene.
Embodiment 3
[0038] Example 3: sc Mechanism Analysis of Hybrid Sterility
[0039] With primers P3 and P8 (amplified all sc alleles) for quantitative RT-PCR, for T65, E5, hybrid F 1 The expression analysis of Sc-j It is mainly expressed specifically in pollen in the early stage of dinuclei, and the expression level is low. Sc-i The repeated gene was expressed at different developmental stages from anther microspore mother cell to dinuclear early pollen, and the expression level was high ( Figure 4 A). by Sc-j Quantitative RT-PCR expression analysis with specific primers (P3, P4) showed that F 1 of Sc-j The expression level of each copy is only equivalent to about 1 / 5 of its parental T65 ( Figure 4 ), indicating that in the hybrid Sc-j Inhibition of expression results in pollen abortion.
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