Clonging Of The Rice Photoperiod-Sensitive Genic Male Sterile Ge Ne Pms3and Functional Analysis Of Wide Compatibility Gene S5

Rice is one of the most important food crops, and how to improve the rice production more effectively is one of the important issues. The discovery and use of rice photoperiod-sensitive genic male sterile (PSMS) line (Nongken58S) is a breakthrough in rice breeding. Genetic studies have identified several loci controlling PSMS using different parental crosses involving this rice mutant. It was determined that pms3on chromosome12was the original mutation that changed Nongken58to Nongken58S. In this study, we cloned pms3and conducted in-depth study of its function. Comparative sequencing analysis showed that there is only a single-nucleotide polymorphism (SNP) between Nongken58and Nongken58S in pms3region. According to RT-PCR and RACE methods, we determined three transcripts in the pms3region which we named them Transcript-1to3respectively. We over-expressed Transcript-1in Nongken58S and found that the male fertility of positive transgenic plants can be recovered to normal under nature long-day conditions. Expression analysis showed that the expression level of Transcript-1was much higher under long-days than short-days in both Nongken58and Nongken58S. Comparative expression analysis in young panicles showed that the expression level of Transcript-1in Nongken58under long-day conditions was significantly higher than the other three treatments:Nongken58under short-day and Nongken58S under both long-day and short-day conditions in all four developmental stages. Combined the result of genetic transformation, we deduced that Transcript-1is the pms3gene we were searching for. We further analyzed the DNA methylation level of pms3region. The result showed that there was a contrasting pattern of DNA methylation between the putative promoter and transcribed region of pms3. In the promoter region, the methylation level of CG contexts in Nongken58S was much higher than that in Nongken58. We speculated that decreased the expression of pms3in Nongken58S is due to the heavier DNA methylation in the promoter of pms3. To investigate the molecular mechanism of pms3, we made several deletion constructs driven by the rice actin promoter and delivered to Nongken58S. The result showed that pms3encodes a long non-coding RNA. Also we found that the52bp region containing SNP is essential for the function of pms3. We predicted the RNA secondary structure of pms3and found that there was a predicted stem-loop structure in this region which seems to produce small RNAs. We also found some small RNAs do exist in this region in the rice small RNA database. We then verified them using the stem-loop RT-PCR method. In the pms3locus, we also found other transcripts which overlapped with pms3in sense or antisense. AK111270is one of them, and it located upstream pms3with110bp overlapped. Over-expressed AK111270in Nongken58S delayed the fertility restoration under nature short-day conditions. Expression analysis showed that the expression of pms3was down-regulated in AK111270over-expressed plants. We detected the DNA methylation level of the promoter region of pms3in AK111270over-expressed plants and found that all three contexts (CG, CNG, CNN) were heavier methylated. Further analysis showed that the level of small RNAs produced from this region also increased significantly. We speculated that the over-expression of AK111270, part of which has homologous to the promoter region of pms3, caused RNA-mediated DNA methylation (RdDM) and thus decreased expression of the pms3. Transcript-3was another transcript which located antisense with pms3. The pollen fertility was not influenced when overxpressing Transcript-3. However, its specific expression pattern implied that there might be some specific function of Transcript-3. As all these transcripts were structurally overlapped, their function also might be closely associated with each other.Hybrids between indica and japonica varieties demonstrate stronger hybrid vigor, which is another way to improve rice production. However, the partial sterility observed in the hybrids blocks the utility. A special group of rice germplasm, referred to as wide compatibility varieties, is able to produce highly fertile hybrids when crossed to both indica and japonica. S5is a major locus for indica-japonica hybrid sterility and wide compatibility. In this study, we isolated and cloned the rice wide compatibility gene S5, and characterized the function and molecular mechanism of this gene. S5encodes an aspartic protease (AP). Comparative sequencing analysis showed that there are two amino acids differences in the coding region between japonica and indica. The phenylalanine (Phe) and alanine (Ala) in indica were substituted to leucine (Leu) and valine (Val) in japonica respectively. To assess whether S5is an AP, an in vitro protease assay of the S5protein obtained by expressing the cDNA of S5in Escherichia coli was conducted, which showed that the activity of S5was low but detectable in acidic pH, the highest at pH3.0, and greatly reduced with the addition of pepstatin A. We also tested the autoproteolytic activity of S5, which showed that the protein could cleave itself especially at acidic conditions. Yeast two-hybrid assay showed that S5might form dimmers. We also tried to find the substrates of S5using the proteomics approach and obtained several possible target proteins.