Submitted In Partial Fulfillment Of Requirements For The Degree Of Master

Rice (Oryza sativa) is one of the most important food crops in the word and the fertility is important to rice yields. Male and female sterility phenomenon occasionally occurring in plant, which is bad for the plant themselves but it offer a good chance for us to understand the mechanism of plant development. Research concern fertility is thus important for its theoretical significance and application value. In the recent years, With the swift development of genetics and experiment methods in molecuLar biology, many genes had been cloned that provided the theory foundation to further elucidate the complex molecuLar mechanism involved in the rice fertility gene.We reported the male and female sterile mutant, MFS-Z9, was generated from a T2 generation transgenic line of normal Japonica rice, Zhonghua 9. In the previous study,we analysed its genetic effect, morphological cytology and chromosome localization. In this study, we carried out the fine mapping and candidate analysis for the mutant.Main resuLts are as follows:1 By employing a map-based clone strategy, the mutant gene was located to a 85 kb interval on the short arm of chromosome 2, flancking the marker RM12585 and DI014.2 Within the 85 Kb physical interval, totally 8 putative genes were predicted by TIGR RICE. LOC_Os02g08430 is a hypothetical protein; LOC_Os02g08440 is a OsWRKY71-Superfamily of TFs having WRKY and zinc finger domains; LOC_Os02g08450 is an En/Spm sub-class and a transposon protein; LOC_Os02g08460 is hypothetical a protein; LOC_Os02g08470 is a conserved hypothetical protein; LOC_Os02g08480 is an ubiquitin fusion degradation protein; LOC_Os02g08490 is a chaperone protein clpB1; and LOC_Os02g08500 is a two-component response reguLator.3 Clone and sequencing resuLts of the mapped region indicated that no changes were found between MFS-Z9 and the wild-type rice in the locus of LOC_Os02g08430,LOC_Os02g08440. LOC_Os02g08450,LOC_Os02g08460 and LOC_Os02g08470.However, no PCR products were generated from the mutant in the locus of LOC_Os02g08480,LOC_Os02g08490 and LOC_Os02g08500, though expected products were got from the wild-type rice.432 markers flanking the three gene region were further generated to check the difference of the mutant and the wild-type rice. PCR resuLts showed that all these makers were absent in the mutant plant while normally in the wild-type rice. The same pattern were shared in the F2 popuLation, which suggested approximately 20KB genomic segement were deleted from the mutant genome. Another evidence is that all the three genes were missing expressed in the mutant, while in the wild-type rice, the expressions were detectable.5 To clearly define the gene function, the CDNA and the promoter of two candidates were amplified by PCR with the primers designed according to this gene sequences. Several vectors, for complementation, over-expression, anti-sense and RNAi, were finally constructed. All the the recombinant plasmids were identified by enzymes digestion and direct sequencing.6 The transgenic plants were obtained by agrobacterium-mediated transformation of plant transgenic technology.35 hygromycin B resistant plantlets, which was transformed with the complementary vector of ORF1, were generated. Other vector transformations are present preparing.