Rice male fertility-related protein, encoding gene and application thereof
A technology of male fertility and related proteins, applied in application, genetic engineering, plant gene improvement, etc., can solve problems such as undiscovered functional analysis and incompletely clear molecular basis
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Embodiment 1
[0064] The phenotype of embodiment 1VR1 gene mutation
[0065] A sterile mutant in the rice material XF1 (purchased from the Resource Center of the Rice Research Institute of Sichuan Agricultural University) was found to be sterile in all male organs through forward and reverse hybridization and cytological observation. Compared with the wild type, the mutant has elongated filaments, shriveled anthers, white or light yellow, no pollen grain coloring in the anther wall of the mutant, and belongs to the typical "pollenless sterile" type ( figure 1). Further using techniques such as paraffin section, it was found that the mutant microspores had a tendency to vacuolate in the late tetrad stage, but microspore adhesion occurred in the early stage of vacuolation, and then formed condensates, which failed to undergo effective vacuoleization, and later condensed The substance gradually degrades to form a cavity. Therefore, the pollen development disorder of the mutant is mainly mani...
Embodiment 2
[0066] Map-based cloning of embodiment 2VR1 gene mutation
[0067] 1. Map-based cloning of VR1 gene mutation
[0068] Using M63 as the male parent to cross the mutant, construct a segregated population, and finely locate the gene in an interval of about 48 KB on the fourth chromosome of rice ( image 3 ), the interval includes 8 predicted genes. Sequence comparison analysis of the fine-mapping interval showed that in this region of a BHLH gene ATG, the T at position -640 was changed to G, and the T at position -730 was changed to C, which led to a sharp down-regulation of the gene transcription level ( Figure 4 ), thereby causing a sterile phenotype in rice. Through gene prediction and transgenic methods, introducing the wild-type gene VR1 into the mutant can restore the normal fertile phenotype ( Figure 5 ), confirming the function of the gene.
[0069] The sequence of the wild-type VR1 gene is shown in SEQ ID NO.1, which encodes a transcription factor, and its sequence...
Embodiment 3
[0081] Embodiment 3VR1 gene rice transgenic test
[0082] In this example, the expression vector PHB (Mao et al., 2005, PNAS 102:12270-12275, commercially available) was used as the rice transgene vector. The vector encodes a bacterial origin of replication (ori), a kanamycin resistance gene (Kan r ), hygromycin resistance gene (Hyg r ), herbicide resistance gene (Bar r ), 2×35S promoter, NOS gene termination signal sequence and multiple cloning site (MCS) for connecting target fragments. The VR1 gene or its fragment can be inserted into the restriction endonuclease site to construct a transgenic plasmid.
[0083] 1. Construction of complementary expression vector for VR1 gene
[0084] In this example, pBS-P was first restricted with EcoRI and BamhI VR1 and PHB-35S, recovered pBS-P VR1 The promoter fragment of about 2.5K VR1 gene after enzyme digestion was ligated with the recovered PHB after digestion and digestion with about 12K vector backbone (2×35S promoter was remo...
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