Rice male and female fertility-related protein, encoding gene and application thereof
A technology related to proteins and coding genes, applied to rice male and female fertility-related proteins, coding genes and their application fields, can solve undiscovered problems
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Embodiment 1
[0061] The phenotype of embodiment 1MFS1 gene mutation
[0062] A sterile mutant in the rice material Zhonghua No. 9 (purchased from the Resource Center of the Rice Research Institute of Sichuan Agricultural University) was found to be sterile in both male and female organs through forward and reverse hybridization and cytological observation ( figure 1), and further using techniques such as paraffin section, it was found that the root cause of its sterility was the abnormal meiosis of mutant pollen mother cells ( figure 2 ) and abnormalities in embryo sac structure ( image 3 ).
Embodiment 2
[0063] Map-position cloning of embodiment 2MFS1 gene mutation
[0064] 1. Map-based cloning of MFS1 gene mutation
[0065] Using G630 etc. as the female parent to hybridize with the heterozygote containing the mutant gene, construct a segregated population, locate the gene, and realize fine-mapping interval marker encryption and sequence comparison analysis show that a fragment deletion of about 20 KB is generated in this region of the mutant, It resulted in the loss of expression products of 3 predicted genes (orf1, orf2 and orf3) ( Figure 4 ), thereby causing a sterile phenotype in rice. Through gene prediction and transgenic methods, introducing one of the normal wild-type genes MFS1 into the mutant can restore the normal fertile phenotype ( Figure 5 ), and the introduction of the other 2 genes will not restore the fertility.
[0066] The sequence of the wild-type MFS1 gene is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.2. The full-len...
Embodiment 3
[0079] Embodiment 3 MFS1 gene rice transgenic test
[0080] 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 MFS1 gene or its fragment can be inserted into the restriction endonuclease site to construct a transgenic plasmid.
[0081] 1. Construction of MFS1 gene complementation expression vector
[0082] In this example, pBS-MFS1 and PHB were firstly digested with PstI and SacI restriction enzymes, and the about 1.8K full-length cDNA fragment digested by pBS-MFS1 was ligated into the PstI and SacI sites of PHB, and the recombinant Enzyme digestion and sequencing identification.
[0083] ...
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