Phenotypic Identification And Map Cloning Of Male Sterile Mutant Dms1 In Rice(Oryza Sativa L.)

Recessive genic male sterile genes are of great value in basic and applied research.Therefore,it is of great significance to study the function and regulation mode of related genes.In this study,a rice male sterile mutant dms1（defective microspore 1）was obtained from the mutant library of EMS（Ethyl methane sulfonate）.The mutant phenotype of dms1 was identified and analyzed by means of somatoscope observation,scanning electron microscope observation,semi-thin section observation,ultra-thin section transmission electron microscope observation,fluorescence white detection of pollen inner wall chitin,observation of meiosis of microspore mother cells and so on.The candidate genes were identified by genetic analysis and fine mapping,and alternative splicing was found in the mutants by c DNA sequencing.The functional verification of DMS1 protein was carried out by subcellular localization,RT-PCR,m RNA in situ hybridization analysis,meiosis-related gene expression analysis,yeast transcriptional activation activity detection,yeast two-hybrid verification interaction protein and other experimental methods,which established a solid foundation for the follow-up study of the regulatory mechanism of the protein.1.Morphological analysis of dms1Compared with the wild type,there was no significant difference in the vegetative growth stage of the mutant dms1.In the reproductive growth stage,the plant became shorter,the anther was smaller and the color was lighter.I₂-KI staining showed that the mutant could not produce dynamic normal pollen grains,and the panicle was erect in the mature stage and could not bear fruit at last;the pollination of Xinong 1B could bear fruit,indicating that dms1 is a female fertile and male sterile mutant.2.Cytological analysis of dms1Scanning electron microscope observation showed that the dms1 anther became shorter compared with the wild type,the exine horny layer and waxy layer of the anther became less,the inner wall of the anther had no obvious difference,the pollen germination pore cover disappeared,the peripheral ring structure was abnormally expanded,and the pollen grains collapsed and became smaller.The observation of semi-thin section showed that the cytoplasmic staining of microspore mother cells during dms1 meiosis was irregular and the density was low;the microspore cells in stage 9 of anther development were shrinking and irregular in shape;the size and shape of microspores in stage 10 were inconsistent and irregular,and the cytoplasmic staining of tapetum was irregular and low density;the pollen was not filled with starch at mature stage.Transmission electron microscope showed that the cytoplasm of microspore of dms1 was missing and the density of tapetum cytoplasm was low in stage 10.Fluorescence white experiment showed that the development of pollen inner wall of dms1 was abnormal.The observation of meiosis behavior of microspore mother cell showed that dms1 could not form normal diad and tetrad,indicating that the male gamete meiosis of mutant dms1 is abnormal.3.Genetic analysis of dms1The normal fertile F₁ progeny was produced by crossing wild type Jinhui 10 and dms1,and the F₁ progeny was selfed to produce F₂ population.The phenomenon of character segregation was found in F₂ population.Through statistics and chi-square test,it was found that the character segregation complied with the Mendelian genetic segregation ratio at 3:1(χ²=1.629<χ²_0.05,1=3.841),indicating that the male sterility of mutant dms1 is controlled by a pair of invisible nuclear genes.4.Fine mapping of genes and determination of candidate genesThe DMS1 was located between the polymorphic SSR marker primers W6 and W8,and the physical distance was 123kb by using the map-based cloning method.There are20 annotated genes in the interval.After gene sequencing comparison,it was found that there was a base substitution at the end of the 13th intron of ORF7.c DNA sequencing found that the mutation led to the abnormal termination of splicing and the phenomenon of alternative splicing occurred.Finally,ORF7 was identified as the candidate gene DMS1.5.Transcriptional level identification of DMS1RT-PCR and m RNA in situ hybridization experiments showed that DMS1 was specifically expressed in microspore mother cells during meiosis,and the expression of meiosis-related genes CRC1,Os BVF1/P31^comet,Os DMC1B,PAIR1,PAIR2 and ZEP1were down-regulated in different degrees,suggesting that DMS1 protein is involved in the meiosis regulatory network.6.Functional identification of DMS1 proteinDMS1 encodes a Coiled-coil domain,subcellular localization results show that DMS1 is a nuclear localized protein,yeast self-activation test show that it has strong transcriptional activation activity,and the activity disappeare after truncation.The interaction between DMS1 protein and meiotic genes Os BVF1/P31^comet,PAIR1 and ZEP1 was found in yeast two-hybrid,which indicating that DMS1 protein is involved in the regulation of meiotic network and provided evidence for the involvement of Coiled-coil domain in rice meiosis.