Functional Analysis And Molecular Mechanism Of The Rice Wide Compatibility Gene S5-n And Photoperiod-Sensitive Genic Male Sterile Gene Pms3 And Comprehensive Sequence And Expression Profile Analysis Of Hsp20 Gene Family In Rice

Hybrids between indica and japonica varieties demonstrate stronger hybrid vigor,but 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.S5 is a major locus for indica-japonica hybrid sterility and wide compatibility,and was previously located on chromosome 6.The objective of our study was to clone S5,which was referred to as a wide compatibility gene, and characterize the function and molecular mechanism of this gene.In this study,we cloned S5 and obtained the full length cDNA sequence of the different alleles by RACE in three cultivars.The S5 gene is comprised of three exons and two introns.The indica(S5-i) and japonica(S5-j) alleles differ by two nucleotides.The wide compatibility gene(S5-n) has a large deletion in the N-terminus of the predicted S5 protein,causing subcellular mislocalization of the protein,and thus is presumably nonfunctional.BLASTp analysis showed that S5 encodes an aspartic protease.We searched a dataset of whole-genome expression arrays spanning the entire life cycle and the result showed that expression of S5 was extremely low throughout the life cycle.We also analyzed the expression of S5 in the developing panicles during different stages in comparison with the leaves harvested at the corresponding stages.S5 expression was not detectable in leaves,whereas variable levels of expression were observed in the developing panicles in all three genotypes, Balilla,Nanjing 11,and 02428.Real-time PCR was used to compare the S5 expression levels in different parents and their hybrids in certain stage.The analysis revealed significant differences in S5 expression among the parents as well as among the hybrids. Real-time PCR was also performed using developing panicles at stage 6 to compare the S5 expression levels in 55 cultivars.The result showed that the S5 expression levels were not relevant to different varieties.We examined the embryo fertility in 13 hybrids,and the analysis revealed that the embryo fertility was positive relevant to spikelet fertility,but not relevant to the S5 expression levels.These results showed that S5 affected the indica-japonica grain production by conditioning embryo-sac fertility and the expression level of S5 was not essential in this progress.Photoperiod-sensitive genie male sterility(PSGMS) rice Nongken 58S,which under long-day condition is sterile while under short-day condition is fertile,is a very important germplasm to develop the "two-line" hybrid in rice breeding program.Previous studies have delimited pms3 to a 28.4kb DNA fragment containing only one SNP.The objective of our study was to determine the pms3 candidate and characterize the function and molecular mechanism of it.Sequence analysis of this fragment in database predicted the presence of Gene M.RT-PCR was performed to confirm the cDNAs by sequencing. Further analysis by RT-PCR showed that the four transcripts of Gene M expressed differently in different conditions in 58S and 58N.Primers were designed randomly near the SNP locus and an expression EST was found crossing the SNP,which was named as Gene SNP.We obtained the full length cDNA sequence of the Gene SNP by RACE. Interestingly,the 5’ fragment of Gene SNP was overlapped with the 3’ end of GENE M3, one of the transcripts in Gene M.Real-time PCR was performed to exam the expression level of Gene SNP and GENE M3 in panicles in different developmental stages,and the result showed that Gene SNP was highly expressed in stage 6 in panicle.In situ hybridization showed that Gene SNP and GENE M3 were expressed in young panicles. These results may be helpful for identifying the candidate genes of pms3 and provide a basal approach for the mechanism of pms3.The Hsp20 genes represent the most abundant small heat shock proteins in plants. Hsp20 gene family has been shown to be involved in preventing heat shock and promoting resistance to environmental stressors,but very little is known about this gene family in rice.Here,we report the identification and characterization of 39 OsHsp20 genes in rice,describing the gene structure,gene expression,genome localization,and phylogenetic relationship of each member.We have used RT-PCR to perform a characterization of the normal and heat shock-induced expression of selective OsHsp20 genes.A dataset of whole-genome expression arrays spanning the entire life cycle involving 25 stages in 3 rice cultivars has revealed that 36 OsHsp20 genes were expressed in at least one of the experimental stages studied.Among these,transcripts for OsHsp20 were accumulated differentially during vegetative and reproductive developmental stages and preferentially down-regulated in Shanyou 63.Meanwhile,OsHsp20 genes were identified as showing prominent heterosis in family-level.Our results suggest that the expression patterns of the OsHsp20 genes are diversified not only in development but also in variety level.