| Male-sterile mutations provide an ideal source material for a range of genetic and molecular biological studies of reproductive biology. In order to well apprehend the molecular mechanism of pollen development, all genes involved in this course developmental stage must be identified and characteried.Many studies on the hybrid semi-sterility between indica and japonica have been reported and many hypothesizes have been put forward, but the molecular mechanism of semi-sterility has been unknown. It is difficult to study this problem due to this hybrid semi-sterility causing by indica-japonica interaction. The hybrid genetic foundation is complex, so hybrid semi-sterility in different varietal combinations may be different sterility loci. If we can use a stable genetic semi-sterile cultivar to study the semi-sterile molecular mechanism, it would be a reference for overcoming the semi-sterility in practices.w207-2 is a stable mutant, and mutants are favorable materials for studying gene expression and function analysis, at the same time, analyzing mutants is also the development aspect for researching the functional genomes. The entire rice genome has been sequenced and publicly considered to be the genome of the model plant in grass, at the same time, there is synteny between rice and other grass genome. Therefore, there is an important use for reference to other grass plants through disclosing the biology basis in molecular levels.The main results were as follows:1. Through the morphological and cellular observation, we found the main character of the semi-sterility mutant is as follows:w207-2 is almost normal in vegetative and floral development. The most obvious difference between the mutant and wild-type is the fertility. Under normal conditions, the spikelet fertility of w207-2 is 30-40%, and the pollen fertility is about 50%, but the female gamete fertility was normal. We examined the wildtype and w207-2 pollens using scanning electron microscopy and transmission electron microscopy and found the sterile pollens in w207-2 have irregular shape and most of them are shrunken, which have no accumulations of starch granules, lack intine and have abnormal extine. Their tectum and foot layer is much plumper than the normal ones, and the columella tend to degenerate. We counted the total number of pollen grains per anther. On average, the pssl mutant has～78%total pollen grains equivalent to wild-type and the fertile pollen grains is equivalent to～40%of wild-type. Others, we found the anther of w207-2 was almost indehiscent, and the anther indehiscence of w207-2 was found to reduce the number of pollen grains dropped onto stigma per spikelet, thus reduced and induced the spikelet semi-sterility of w207-2. The pollen semi-sterility may be one of the causes that make the anther indehiscent.2. The anther transverse sections demonstrated that w207-2 anther wall development is normal, and the tapetum and the middle layers can degenerate normally. The DAPI staining revealed that most of the sterile pollen aborted in the uninucleate stage, but the abnormalities can be found ever since pollen meiosis stage:The w207-2 forms univalent at metaphaseⅠ, delayed chromosomes and chromosomal bridge can be found at anaphaseⅠ, delayed chromosome also can be found at anaphaseⅡ, and forms micronucleus at tetrad stage. These results indicate that parts of the chromosomes have abnormalities in chromosome pairing and chromosome movement, this is different from other meiosis mutants. So w207-2 is a good material for the study of the molecular mechanism of rice meiosis.3. According to pollen fertility,2100 recessive homozygous semi-sterile plants were selected from a large w207-2/Dular F2 population to identify the recombination between the pssl locus and the tightly linked molecular markers. Finally, pss1was mapped to the region between one CAPS L2 and one dCAPS L3 marker with the genetic distances of 0.02 cM, respectively. Both L2 and L3 markers were located on a same PAC clone P0470F10 with physical size of about 28 kb. Five open reading frames can be predicted by a sequence analysis of this fragment. We sequenced the entire region and found a single nucleotide convert in the coding region of the KINESIN1-LIKE gene, which caused change of a conserved amino acid in the KINESIN-1 protein family. So we presumed this gene should be candidate of PSS1, and carried on the complementary test by transgenic technology.4. We get the full-length cDNA of OsKINESIN-1 by the RACE and RT-PCR technology. We also analyzed the expression pattern by RT-PCR and GUS staining of the OsKINESIN1 promoter-GUS transgenic plants, we found that this gene is widely expressed in various organs, but the expression level is higher in panicle, and there is a obvious up regulate period during anther development, then the expression level goes down, the peak appears near the meiosis stage. Transient expression in onion epidermal cells showed the OsKINESINl-EYFP fusion protein is exclusively localized to nuclear. We expressed this protein by an E coli. expression system, and found this protein can bind to microtubule in vitro. |
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