| Organ size is an important external feature of rice,and organ size is mainly determined by cell proliferation and cell expansion.In our previous study,a short anther mutant designated as san1 with significantly reduced plant stature and anther length was obtained from screening an EMS-induced mutant library of N ipponbare cell suspension culture.In this study,a combined strategies including genetics,molecular biology,and cytology were used to characterize san1 mutant,and the main results were as follows:1.Phenotypic observation showed that the plant stature of san1 mutant is much smaller than that of N ipponbare.Lengths of all elongated internodes and panicles of san1 are shorter than those of the wild type,and another prominent phenotype of san1 is that its anther length is significantly shorter than the wild type.Meanwhile,the seed setting rate was also significantly decreased in san1 mutant compared to wild type,with approximately 30% of the wild type seed setting rate.2.Genetic analysis indicated that the short anther character was caused by a pair of recessive mutant gene.And the mutant gene san1 was mapped using two segregation populations derived from the crosses between san1 and Annong(AN)or Huanghuazhan(HHZ).Map-based cloning of showed that san1 was mapped into the region flanked by InDel markers 429470 and 429581 in the long arm of rice chromosome 4,and the interval was 111 kb in physical length.3.According to the annotation of Rice Genome Annotation Project,18 ORFs were predicted in the mapped genomic region between molecular markers 429470 and 429581.Sequence analysis of the target region showed that two nucleotide changes was found in the locus of LOC_Os4g48760.1 in san1 mutant,which lead to a nonsense mutation and a missense mutation,respectively.The wild type SAN1 gene encodes a LRR receptor-like protein kinase(LRR-RLK)and its RLK domain was changed in san1 mutant.4.A complementary vector carrying SAN1 gene from N ipponbare was constructed and introduced into san1 mutant.And the phenotypic observation of the regenerated transgenic rice plants showed that the mutant phenotype was recovered,which indicated the mutation of SAN1 caused the mutanted phenotype of san1.5.Phylogenetic analysis showed that SAN1 is clustered into the same clade with some proteins in monocotyledonous plants,and was more diverged from the proteins in dicotyledonous plants.According to the results of rice gene expressio n chip data from Rice XPro and qRT-PCR analysis,san1 was highly expressed in younger tissues and organs,suggesting that san1 may play importants roles mainly in the early development of organs in rice.6.Phenotypic observation showed that the lenghtes of the elongated internodes and panicles of san1 was all decreased compared to those of wild type plants,which indicated san1 was a dn type rice dwarf mutant.Semi-thin sectioning observation of internodes showed that the cell number was decreased in the san1 mutant,however,the cell size was similar to that of wild type.The results suggested that SAN1 gene may play important role in the regulation of cell division.7.Observation of reproductive orga ns showed that the san1 exhibited curly anthers and the mature anthe dehiscence was incomplet in san1 mutant,which might result in insufficient pollen amount during anthesis.Meanwhile,some of pistils have three or four plumous stigmas,and enlarged ovaries were also observed,which might affect the pollination.Semi-thin section observation of anther cross-section showed that the development of 1-2 pollen sacs were arrested in some anthers,which might be the cytological cause for the curly anthers of san1 mutant.8.Treatment with low concentration of IAA showed that the root growth was promoted in both N ipponbare and san1 mutant seedlings.However,the relative growth rate of roots of san1 mutant was significantly lower than that of wild type.These results suggested that the sensitivity to IAA was decreased in san1 mutant compared with wild type. |
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